A LABORATORY MANUAL OF 
PHYSIOLOGICAL CHEMISTRY A LABORATORY MANUAL 
OF 
PHYSIOLOGICAL CHEMISTRY 
Prepared for the use of the Students of Northwestern 
Medical School 
BY 
[$&*** I 
CHESTER j! FARMER 
n\ 
Professor of Chemistry 
Northwestern Medical School 
With the cooperation of Staff Members 
Student's Name TABLE OF CONTENTS 
Method of Weighing 1 
Preparation of Standard Solutions .... 5 
Kjeldahl Method for Nitrogen ..... 10 
The Law of Mass Action 12 
Indicators 16 
The Hydrogen Ion Exponent 21 
The Gas Chain Method 23 
Lipins 28 
Carbohydrates 42 
Colloids 64 
Proteins 71 
Salivary Digestion 100 
Gastric Digestion 101 
Pancreatic Digestion 105 
Urine Analysis 107 
Bone 134 
Milk 135 
Blood 137 
Bile 141 
COPYRIGHTED—1923 
BY 
Chester J. Farmer n METHOD OF WEIGHING 
1. —Determine the zero-point of the balance by setting it in 
motion without any load on the pans. Observe and write down 
the turning points, or extreme positions, of the pointer on the 
scale, of an uneven number of swings, say five or seven. These 
readings must be taken with the balance case closed. Disregard 
the first two readings as they are inaccurate. In order to give 
the same algebraic sign to all of the observed readings, it is best 
to number the divisions on the scale from left to right: 0 to 20. 
In other words, call the long line in the center 10 (the one over 
which the pointer usually stands), then 9, 8, 7, etc.; 2, 1, 0 to the 
left and the 10 ascending numbers, 11, 12, 19, 20, to the right. 
Average the left swings and the right swings separately, then get 
a mean by averaging the two average values. Take this as the 
zero point without load (see example). Assume it is 10.99. 
2. —Now determine the sensitiveness of the balance for the 
object to be weighed. That is, you are about to determine how 
many scale divisions the pointer traverses or is displaced by 
changing the weight 1 milligram. This enables you to calculate 
the fourth decimal place of your weight. To do this, place a 
clean crucible on the left pan, add weights to the right pan until 
equilibrium is established as nearly as possible when using only 
the milligram division on the beam or milligram weights in the 
box, as the smallest denomination. Suppose with this first point 
of rest a mean scale reading of 12.35 with a weight of 22.052 
grams is obtained. Since the weight is a little light, add 1 milli- 
gram on the beam with the rider or from the box, making the 
weight 22.053 gms., and assume a mean scale position of 9.71. 
The sensitiveness is therefore 12.34-9.71 — 2.64 scale divi- 
sions. In case you add too much weight at first, the process will, 
of course, be just reversed. 
As the zero-point of the balance was at 10.99 and the point 
of rest with a load of 22.052 grams was 12.35, it follows that 
the object was heavier than the weights in the scale pan. In 
fact, the excess of weight of the object was sufficient to move 
the pointer 12.35- 10.99 = 1.36 scale divisions. 
3 The above data enables us to calculate the true weight of 
the object to the fourth decimal place, as follows: 2.64 scale 
divisions correspond to 1 mg., then 1.36 scale divisions corre- 
spond to that fraction of a milligram which must be added to 
22.052 gms., to obtain the true weight of the crucible in air. (If 
the first weight had been heavier, the reasoning is the same, 
except that the fractional part of the milligram should be sub- 
tracted.) That is: 
1 :36:2.64 ::x:l 
x = 0.5 mg. 
Therefore the true weight of the crucible in air is 22.052 plus 
0.0005, which is 22.0525 gms. 
The students’ record of weighing for the above case should 
be kept in the following manner: 
Zero Point 
1st Point of Rest 
2nd Point of Rest 
22.052 gms. 
22.053 
gms. 
Left Right 
Left Right 
Left 
Right 
4.2 17.6 
5.8 18.7 
3.5 
15.8 
4.6 17.1 
6.2 18.3 
3.8 
15.4 
5.1 
6.6 
4.2 
Sum 
13.9 34.7 
18.6 37.0 
11.5 
31.2 
Mean 
4.63 17.35 
6.2 18.5 
3.83 
15.6 
Sum of 
means. .21.98 
24.7 
9.71 
Means average . 10.99 
12.35 
19.43 
Sensitiveness--12.35—9.71 =2.64 scale divisions. 
Weight deflection from zero point = 12.35 — 10.99 = 1.36 div. 
Fractional weight: = =0.54 mgs. 
Weight of crucible in air = 22.052 plus 0.0005=22.0525 gins. 
The Use of Analytical Weights 
The student is to ask for the box of weights at the store 
room window, giving the number of his balance. He must sign 
a loan slip, and is charged with the set until returned. The 
weights will be inspected immediately and, if found complete 
and undamaged, the slip will be returned. The weights must 
always be handled with forceps provided in the box, never with 
the fingers or crucible tongs. 
4 The large weights range from 1 to 50 grams, while the 
small flat weights covered by a glass plate are fractional parts 
of the gram. The fractional weights are marked either in frac- 
tions of a gram or as multiples of a milligram, thus a weight 
marked 0.1 and 100 are the same, the first system calls it one- 
tenth of a gram or a decigram, while the latter system indicates 
the fact that it is also one hundred milligrams. 
Again a fractional weight may be marked 0.01 (one centi- 
gram) or 10 (ten milligrams). 
The best method of obtaining weights less than 10 milli- 
grams is by the use of the bent wire called a “rider,” which slides 
along the right arm of the balance beam. The graduations vary, 
but in all cases the figures indicate milligrams. A little study 
will enable the student to properly evaluate the smaller lines 
between milligram markings on the beam. A few balances have 
only six milligram divisions on the beam. They require the 
use of a five-milligram weight (placed on the weight pan) in 
conjunction with the rider to obtain values between 5 and 10 
milligrams. 
For the sake of accuracy, learn to count up your weights 
from the empty box, at first writing down each one removed in 
the note book. For example, suppose you remove the following 
weights: 10-gm., 5-gm., 2-gm., 1-gm., 0.1-gm., 0.05-gm., and 
have moved the rider to a point indicated on the beam by figure 
7. Record weights as above, check from the empty box to see if 
correct, then as you remove them from the scale pan, draw a 
pencil mark through the weight recorded. By so doing, you will 
often find a mistake before a determination has been spoiled, 
or much subsequent work depending upon this one weight lost, 
as in weighing out material for a standard solution. 
The weight obtained by using the above individual weights 
should be recorded as 18.3570 grams. The fourth decimal place 
is obtained as directed above under the method of “swings.” 
Acidimetry and Alkalimetry 
3.—Preparation of N/2 Oxalic Acid Solution. Grind in 
a clean mortar about 10 grams of purest obtainable oxalic acid 
crystals. Spread on a 4-inch watch crystal and expose to the 
air for 15 minutes. At the end of this time transfer to a wide- 
mouth, glass-stoppered bottle. This treatment is to assure the 
5 acid of a definite amount of water of crystallization. It should 
correspond to the formula C2H204-2H20. 
From this material prepare 250 c. c. of N/2 oxalic acid, as 
follows: Carefully clean and dry a small beaker of 150 cc. 
capacity. Place it on the left pan of the balance and accurately 
obtain its weight to the fourth decimal. The amount of oxalic 
acid required for the solution is 7.8780 gm. Why this weight? 
Having obtained the weight of the beaker, add to it the required 
weight for the oxalic acid, and reset the weights on the balance 
to this figure. 
Carefully add to the beaker sufficient oxalic acid to exactly 
counterbalance this weight. (Accurate to the fourth decimal.) 
Record your weighings. Be careful not to spill oxalic acid on 
the pan or in the balance case. Remove the beaker from the 
pan, add 50 cc. of distilled water and gently warm, using a wire 
gauze, over a very low flame. 
Clean the 250 cc. volumetric flask with cleaning fluid, wash 
out thoroughly with tap water, finally rinse twice with distilled 
water. Have a funnel which will fit conveniently into the neck 
of the volumetric flask. 
Now transfer, using a stirring rod, and without the loss of 
a drop, the solution in the beaker to the flask. Add more dis- 
tilled water, warm as before, transfer to the flask. Repeat until 
all of the material has been brought into solution and transferred 
to the flask. Finally rinse the beaker very thoroughly into the 
flask, using small portions of distilled water and repeating the 
washing 4 to 5 times. The funnel is rinsed and removed. Cau- 
tion: Watch the volume of fluid in the flask. It must now be 
at least 1 inch below the graduation on the neck. 
The flask has a capacity of 250 cc. only at a given temper- 
ature. This is indicated just below the figure showing its 
capacity. To bring the flask to this temperature, insert a clean 
thermometer, then immerse the bowl of the flask in water using 
ice if necessary, or warm water as required. The flask should 
be gently rotated and its contents brought to the desired tem- 
perature. 
Now remove the thermometer from the volumetric flask, 
6 directing a small stream of water from the wash bottle on to it 
as it is being withdrawn, so that the rinsings return to the flask. 
Remove when rinsed. Caution—Do not fill above graduation on 
neck! 
The remaining volume of water is added from the wash 
bottle, or by means of a pipette until the meniscus rests exactly 
on the calibration mark. Any droplets in the neck above this 
point are removed by a roll of filter paper. The flask now 
should be stoppered, and inverted occasionally until diffusion 
is complete. At a later time after the solution has thoroughly 
diffused, it must be transferred to clean dry bottle. The label 
should show the name, concentration, and date on which the 
solution was prepared. 
4.—Preparation of an N/2 Sodium Hydroxide Solution 
The strong sodium hydroxide solution in the laboratory con- 
tains about 40% NaOH. From it prepare about 1200 cc. of a 
solution so diluted as to be a little stronger than half normal. 
Stopper and mix thoroughly. Allow the solution to stand at least 
three hours with occasional shaking. 
Determine the strength of the solution so prepared by titrat- 
ing it several times against the standard N/2 oxalic acid. To 
titrate this solution, pipette out 25 cc. of standard oxalic acid 
solution into a 200 cc. beaker. Add 25 cc. distilled water and 
one drop of phenolphthalein as an indicator. Carefully rinse 
a clean rubber-tipped burette with small portions of the alkali, 
and then fill with the same solution. Care should be taken to 
expel any air from the rubber tip of the burette before the alkali 
is drawn down to the graduated scale. Read the burette and 
record the volume. Carefully allow the alkali to flow into the 
oxalic acid contained in the beaker, constantly stirring with a 
glass rod. The alkali should be added slowly at first, and later 
a very few drops at a time. The first faint pink color permanent 
for 15 seconds should be taken as the end point. Read the 
burette and subtract the first reading. This gives the number of 
cc. of the alkali solution which are equivalent to 25 cc. of an 
exactly N/2 oxalic acid solution. Repeat the titration until 
checks are obtained, then from the results of the determinations, 
calculate how much of this solution must be used for the prepa- 
ration of 1000 cc. of an exactly half normal sodium hydroxide 
7 solution. (Consult the instructor.) Prepare this by suitable 
dilution of the standardized sodium hydroxide solution, and after 
thorough diffusion has taken place, check the correctness of your 
solution against the standard oxalic acid solution. If correct, 
transfer the liquid to a clean, dry bottle. Stopper and preserve 
in a properly labeled bottle. 
5.—Exercise 
By means of the N/2 NaOH solution, determine the con- 
centration of the unknown HC1 solutions furnished, using alizarin 
red as an indicator. Report the result to the instructor before 
proceeding with the next experiment. 
6.—Preparation of N/2 Hydrochloric Acid 
The concentrated hydrochloric acid of the laboratory con- 
tains about 35% HC1. First make a rough dilution of the 
required amount to 1200 cc. From this prepare 1000 cc. of an 
N/2 HC1 in the same manner as the N/2 NaOH solution was 
prepared from the concentrated sodium hydroxide, using the 
N/2 NaOH solution as a standard and alizarin red as indicator. 
If correct, transfer the N/2 HC1 solution to a clean, dry bottle. 
Label and preserve. 
7.—Preparation of N/10 Solutions 
Prepare 1000 cc. of N/10 HC1 and 1000 cc. N/10 NaOPI 
from the N/2 standard solutions, and determine by titration 
whether they are equivalent. 
8.—Determination of Nitrogen in Ammonium Sulphate 
Ammonium sulphate for analytical work should be specially 
prepared1, dried for one-half hour at 110 degrees C., and then 
allowed to stand in a dessicator over sulphuric acid until used. 
a.—Preparation of a Standard Ammonium Sulphate 
Solution 
Carefully clean and dry a small beaker of 100-150 cc. 
capacity. Place it on the left-hand pan of the balance, and 
accurately obtain its weight. Place in this beaker a sufficient 
amount of ammonium sulphate to weigh between 2.75 and 3.000 
grams. Whatever the weight is, be sure to record it accurately 
to the fourth decimal place. 
1Folin and Farmer. Jour. Bio. Chem. 11, 406, 1912, 
8 Clean the 500 cc. volumetric flask with cleaning fluid, wash 
out thoroughly with tap water, then rinse twice with small 
amounts of distilled water. Have a clean funnel also, which will 
fit conveniently into the neck of the volumetric flask. 
Fill the beaker containing the ammonium sulphate two-thirds 
full of distilled water. Place on the wire gauze over a low flame, 
and stir gently until dissolved. Do not lose a drop. 
Now transfer it carefully to the volumetric flask, without 
loss, pouring from the beaker by means of a stirring rod. Rinse 
the beaker and stirring rod 4 or 5 times with portions of dis- 
tilled water. 
To the flask add 1 to 2 cc. of concentrated hydrochloric 
acid, then fill the flask up to the neck with distilled water. 
The flask has a capacity of 500 cc. only at a given tem- 
perature, namely: 15 degrees, or 20 degrees C. Note the tem- 
perature at which it was calibrated and proceed as directed in 
the preparation of the N/2 Oxalic Acid (page 6). While the 
ammonium sulphate is dissolving, it is a good plan to have the 
wash bottle immersed in a dish of cold water until the tempera- 
ture required by the volumetric flask is obtained. 
After the ammonium sulphate solution has stood sufficiently 
long to be thoroughly diffused, it should be transferred to a clean, 
dry bottle, and properly labelled. The label should not fail to 
show the concentration of the solution as well as the date on 
which it was prepared. This solution will be used in b below, 
and also later as directed on page 113. 
b.—Distillation of Ammonia 
Procure two clean 500-700 cc. Florence flasks. 
Pipette 25 cc. N/10 hydrochloric acid into each. 
Add 25 cc. distilled water. 
2 drops of alizarin red. 
Flasks so prepared can be depended upon to receive 30 
milligrams of nitrogen. Place each flask on the shelf under the 
ammonia still. Insert the delivery tube, being careful that the 
end dips into the receiving acid. 
Pipette 25 cc. of the standard ammonium sulphate solution 
prepared above, into each of two 800 cc. Kjeldahl flasks. Add 
a small pinch of powdered pumice, 400 cc. of distilled water 
9 and two drops of alizarin red. Now place the flask upon the 
ring of the still. Just before stoppering add 2 to 5 cc. of con- 
centrated sodium hydroxide, in such a manner that the alkali, 
when poured from the graduated cylinder, strikes against the 
neck of the flask 2 inches below that portion touched by the rub- 
ber stopper. The alkali, following along the inner wall of the 
flask, sinks to the bottom, and should be thoroughly mixed, after 
the stopper is inserted, by gentle rotation. Caution—Press stop- 
per into neck of flask during mixing. Immediately light the 
burner below, regulate the flame so that gentle boiling ensues, 
and distill over about 300 cc. The delivery tube should be 
withdrawn into the neck of the flask while the last 100 cc. are 
coming over. Suction movements in the delivery tube can be 
reduced by tipping the receiving flask at such an angle that the 
acid just seals the opening of the tube. If in doubt, consult an 
instructor. 
The distillation of ammonia is complete, when a drop of 
distillate, falling on a strip of neutral litmus paper, produces a 
color of no greater intensity than that produced by distilled 
water. 
When the distillation is complete, disconnect the delivery 
tube and rinse into the flask by means of the wash bottle. Back 
titrate with N/10 sodium hydroxide and, from the data obtained, 
calculate the number of grams of nitrogen (or milligrams) in 
the ammonium sulphate taken. Check your results against the 
theoretical quantity, allowing an error of not over 0.5 milligram 
of nitrogen. Report your results to the instructor. 
9.—Kjeldahl Determination of Nitrogen 
Small test tubes containing uric acid have been placed in 
the drawer of each balance. This tube must be carefully wiped 
with filter paper and then placed upon the scale pan of the 
balance. Whenever the tube is to be handled, use filter paper 
to prevent adherence of grease from fingers. Record the weight 
of this tube to the fourth decimal. 
Remove the tube from the scale pan and, while holding it 
over the neck of a clean 800 cc. Kjeldahl flask, remove the 
stopper. Carefully drop 50 to 60 milligrams of uric acid into 
the flask, then stopper the tube and re-weigh. The difference in 
weight should be recorded as the charge taken. Repeat this 
process with a second Kjeldahl flask. 
10 To each flask add 15 cc. concentrated sulphuric acid (from 
a cylinder), using this acid to rinse down any uric acid adhering 
to the neck of the flask. Add 2 cc. of 5% copper sulphate, 
and finally a few small pieces of broken glass. 
Place the flasks on the digestion rack in the hood, and light 
the burner. Allow the mixture to reach the boiling point then 
turn down the flame until boiling is just perceptible. Avoid too 
vigorous boiling, otherwise a loss of nitrogen will occur. 
After one-half hour digestion, extinguish the flame and allow 
to cool. When cold, consult the instructor as to the manner of 
adding water to the acid in the flask. After the water has been 
added, add 2 drops of alizarin red, and then proceed to distill 
over the ammonia as directed under experiment 8-b. 
Notice: The amount of alkali required for this distillation 
is greater than that indicated in the procedure above. Why? 
To calculate the alkali required, assume that 1 cc. of concen- 
trated sulphuric acid is neutralized by 2.5 cc. of concentrated 
sodium hydroxide. See also note, page 108. 
Calculate the nitrogen content of the uric acid taken, and 
check against the theoretical figure. Report your results to the 
instructor. 
11 10.—The Law of Mass Action (Concentration Law) 
Briefly stated, the Law of Mass Action reads: The rate 
of chemical change is proportional to the molecular concentra- 
tion of the reacting substances. The rate of change is also under 
the influence of a “constant,” which is characteristic of the chem- 
ical nature of the reacting substances and also of the temper- 
ature. 
The following exercise is designed to illustrate: 
(a) The change in speed of reaction with change in con- 
centration of components (plotting of curve). 
(b) The condition of equilibrium, i. e., where the speeds 
of reaction on the two sides have become identical. 
(c) The manner in which the equilibrium constant can be 
calculated; and, finally, 
(d) The effect of changing the concentration of one com- 
ponent upon the point of equilibrium of the reaction. 
The reaction to be studied is that of the esterification of 
ethyl alcohol by acetic acid. To be successful, these directions 
must be followed minutely. 
Equation, C2H5OH-f-CH3COOH CH3COOC2H5-f H20. 
Start, 1 mole -f- 1 mole O + O 
Equilibrium, 1/3 mole -f- 1/3 mole 2/3 mole -(- 2/3 mole. 
Procedure: Select a clean and dry 250 cc. Florence flask. 
Draw a reflux condenser from the stock room, and arrange flask 
and condenser, by means of suitable clamps, for refluxing. Place 
an evaporating dish, to serve as a water bath under the flask 
and support by a ring. The micro-burner should be used for 
heating. 
When the apparatus is ready and known to be tight, take 
the flask to the stock room and get 29.26 cc. of absolute alcohol 
(half molar quantity). To this add from a Mohr pipette exactly 
0.5 cc. of concentrated sulphuric acid. Add another 0.5 cc. of 
the acid to a small (150 cc.) clean beaker and set aside. The 
acid serves only to catalyse the reaction and influences the reac- 
tion otherwise to no appreciable extent. 
Clean two pipettes, a 25 cc. and a Mohr; have both dry, 
Borrow a second Mohr pipette, having it likewise clean and dry. 
Place on the table a burette filled with N/2 sodium hydroxide. 
Place a second clean 150 cc. beaker on the table. 
12 When all is ready, pipette exactly 28.35 cc. of glacial acetic 
acid into the Florence flask containing the alcohol. (Use the 
25 cc. pipette and the 3.35 cc. from the Mohr pipette.) 
Cautiously mix the components by gently rotating the flask, 
then immediately withdraw exactly 2 cc. of the mixture by 
means of the second Mohr pipette. Place this portion in the 
clean beaker; note the time by the watch (hour and minutes), 
then immediately attach flask to condenser and heat very gently. 
Caution. The temperature of the water bath is to be main- 
tained at 70° C. by the thermometer. Note also that only two 
cubic centimeters of the mixture are withdrawn. Any excess 
in pipette should be immediately returned to the flask. Add 1 
drop of phenolphthalein to mixture in beaker and titrate imme- 
diately with N/2 sodium hydroxide. 
To the beaker containing the 0.5 cc. of concentrated sulphuric 
acid add 25 cc. of distilled water. Add 1 to 2 drops of phenolph- 
thalein and titrate with N/2 sodium hydroxide. Divide the num- 
ber of cc. of alkali required by 29 and subtract the quotient so 
obtained from each titration of 2 cc. of reaction mixture. This 
gives the correction figure for the sulphuric acid. After 10 
minutes’ heating, rapidly disconnect condenser, and withdraw 
another 2 cc. portion of the reaction mixture for titration in a 
similar manner as previously described. Stopper the flask (the 
condenser is never disconnected longer than necessary to remove 
2 cc. sample, then continue heating. Repeat until 4 or 5 such 
titrations have been taken. 
Allow the apparatus to remain connected with flame extin- 
guished and cooling water turned ofif until another laboratory 
period if equilibrium has not been reached. Continue the experi- 
ment, making titrations, until two successive tests give the same 
titratable acidity. Record all values as shown in the table below. 
When equilibrium is obtained, calculate the amount of react- 
ing mixture which has been removed. Subtract this from the 
initial volume of mixture. Add a definite calculated amount of 
distilled water, then heat for 10 minutes, noting change in equili- 
brium. Remember, when calculating, the proportional decrease 
in titration due to dilution by water added! Continue until a 
new point of equilibrium is established, and check agreement 
with theoretical as deduced from reaction constant found. 
13 Record data as below: 
Time 
Minutes 
0.00 
Cc. N/2 NaOH 
Correction 
(Factor for 
Acid) 
Cc. N/2 NaOH 
(Corrected) 
10.00 
20. 
30. 
40. 
60. 
90. 
Etc. 
At Equilibrium 
After Addn. of 
Water 
0.00 
10.00 
20.00 
30.00 
At Equilibrium 
14 Work out the following: 
1. Plot the curve for the speed of reaction, putting time 
in hours along abscissa, and molecular change in concentration 
along ordinate. 
2. Work out the value of the equilibrium constant “K.” 
3. Using the value found for K, add a definite molecular 
quantity of water, and calculate the molecular concentration of 
acid when equilibrium is again established. Check this value 
with that obtained by titration. 
15 INDICATORS 
The term “indicator” cannot be precisely defined. As used 
in acidimetry, an indicator is a weak organic acid, or the salt of 
such an acid, which manifests by a color change, differences 
in the reaction (hydrogen ion concentration) of the substance 
in which it is placed. The color change accompanying the change 
in reaction is frequently due to an intramolecular combination 
of a quinone group with a salt of phenol or analine, etc. 
The student must clearly understand at the outset, that most 
indicators do not change color at the point of exact neutrality. 
The hydrogen ion concentration at which the change takes place 
is indicated in the tables on pages 544, Plate A, and 546, of 
Mathew’s Physiological Chemistry, 3rd edition. 
Strong and Weak Acids 
When an acid is added to water, the acid is dissociated into 
+ 
the cation (H) and a corresponding anion. The completeness 
of dissociation at ordinary dilution is made the basis upon which 
its strength is judged. Acetic acid is dissociated but little, while 
hydrochloric acid is almost completely dissociated in N/10 solu- 
tion. 
A strong acid will remove the base from the salt of a weaker 
one. It is therefore possible to “neutralize an acid” by means 
of a salt as indicated in the following experiments. 
11.—Replacement of Weak Acid by Strong Acid in Salts 
a. Place 1 to 2 cc. of saturated sodium carbonate solution 
in a 250 cc. beaker. 
Add 25 cc. distilled water. 
Add 1 drop of methyl orange. 
Add dilute sulphuric acid (1:4) until neutralized. 
Notice the formation of gas. Explain and write equation. 
b. Repeat the above experiment, placing 1 cc. of concen- 
trated hydrochloric acid in the beaker, using congo-red as an 
indicator, and adding saturated sodium acetate solution. Explain 
and write the equation. 
12.—Point of Change of Indicators 
Select 10 clean test tubes of uniform size. Place the stand- 
ard buffer solutions of pH values ranging from pH 1 to pH 10 in 
16 these tubes. To each tube, add 1 drop of methyl orange solution 
(caution, previously diluted to one-fourth strength) and note 
point of change. Record observations. 
Repeat the experiment with alizarin red, and phenolphthalcin 
(diluted with 95% alcohol). Record. 
The standard buffer solutions used are described on pages 
543 and 544 of Mathews Physiological Chemistry, 3rd edition. 
A more accurate set of standard buffer solutions is described 
by Clark and Lubs, Journal of Biological Chemistry, 25,479, 
1916. 
Preparation of Solutions of Definite pH value: 
A. Stock Solutions— 
1. Hydrochloric Acid—N/10, N/100, N/1000. 
2. M/15 Potassium Dihydrogen Phosphate (KH2P04). 
9.078 gms. KH2P04 per liter. 
Designated as Primary Phosphate. 
3. M/15 Disodium Phosphate (Na2HP04). 
11.876 gms. Na2HP04-2 H20 per liter. 
Designated as Secondary Phosphate. 
4. M/10 Secondary Sodium Citrate. 
Dissolve 21.008 gms. Citric Acid (Crystals) in 200.00 
cc. N/10 NaOH. 
Dilute to 1000 cc. 
Designated as Citrate. 
5. 0.2 M. Alkaline Borate. 
Dissolve 12.404 gms. Boric Acid in 100.00 cc. N/l 
NaOH. 
Dilute to 1000 cc. 
Designated as Borate. 
B. Preparation of Solutions of Known pH.— 
Solution 
Call pH 
Preparation 
pH—1.04 
“i” 
10 cc. 
N/10 HC1. (Students’ Acid.) 
pH—2.02 
“2” 
10 cc. 
N/100 HC1. (Students’ Acid.) 
pH—3.01 
“3” 
10 cc. 
N/1000 HC1. (Students’ Acid.) 
pH—3.95 
5.50 cc. 
Citrate plus 4.5 cc. N/10 HC1. 
pH—4.96 
“5” 
10 cc. 
Citrate. 
pH—5.97 
“6” 
6 cc. 
Citrate plus 4 cc. N/10 NaOH. 
pH—6.98 
tcjij 
6 cc. 
Secondary Phosphate plus 4 cc. of 
Primary Phosphate. 
17 pH—8.04 
“8” 
9.75 cc. 
Secondary Phosphate plus 0.25 cc. 
Primary Phosphate. 
pH—9.01 
“9” 
8.5 cc. 
Borate plus 1.5 cc. N/10 HC1. 
pH—9.97 
“10” 
6 cc. 
Borate plus 4 cc. N/10 NaOH. 
Reference: 
Sorensen, S.P.L. Biochem. Zeitschrift. 21, 167, 1909. 
Michaelis, L. Die Wasserstoffionenkonzentration. Ber- 
lin. 1914. 
The student is advised to consult the original literature 
respective to the theory underlying, and preparation of buffer 
solutions. 
13.—Titration Experiments 
Perform the following titrations, writing up your conclu- 
sions : 
Place in 
Beaker 
Add 
Titrate 
with 
Cc. with 
Methyl 
Orange 
Cc. with 
Alizarin 
Red 
Cc. with 
Phenol- 
phthalein 
lOcc.N/10 
HC1 
15 cc. 
Dist. H20 
N/lONaOH 
lOcc.M/10 
h3po4 
15 cc. 
Dist. H20 
N/lONaOH 
IOcc.M/IO 
NaH2P04 
15 cc. 
Dist. H20 
N/lONaOH 
lOcc. M/30 
Citric Acid 
15 cc. 
Dist. H20 
N/lONaOH 
IOcc.M/IO 
NaHCOg 
15 cc. 
Dist. H20 
N/10HC1 
10cc.N/10 
NH4OH 
15 cc. 
Dist. H20 
N/10 HC1 
If possible make a table showing the conditions governing 
the use of indicators (vide-Mathews, p. 868). 
18 14.—Titratable Acidity and Reaction 
Although two acids of like normality may neutralize the 
same quantity of alkali, their respective strengths (hydrogen ion 
concentration) will vary with their degrees of electrolytic disso- 
ciation. The following experiment is intended to make this 
relationship clear: 
A. Equivalence of titration—because of same normality. 
1. Pipette 10 cc. of N/10 hydrochloric acid into a 
beaker. 
Add 1 drop of phenolphthalein. 
Titrate with N/10 sodium hydroxide. 
Record the cubic centimeters required in the table 
below. 
2. Repeat the above titration using 10 cc. of N/10 
acetic acid, instead of the hydrochloric acid. Re- 
cord the amount of alkali in the table 1. 
B. Difference in strength—because of unequal degree of 
dissociation. 
1. Select five clean test tubes of equal size. Into these, 
pipette respectively 10 cc. of the acids mentioned 
in the headings of the following table. To each 
tube, add 3 drops of a 0.1% solution of Methyl 
Violet 6B, and record the observed color in table 2. 
Table 1. 
Titration 
10 cc. N/10 HC1 
10 cc. N/10 CH, COOH 
Cc. N/10 NaOH 
with Phenol- 
phthalein 
19 Conclusions: 
Table 2. 
Titration 
10 cc. N/l 
ch3 cooh 
10 cc. N/10 
HC1 
10 cc. N/100 
HC1 
10 cc. N/1000 
HC1 
Color with 
3 drops 0.1% 
Methyl Vio- 
let 6B 
pH 
Conclusions: 
20 THE DETERMINATION OF THE HYDROGEN ION 
EXPONENT pH, BY THE GAS CHAIN 
METHOD 
In 1909 Sorensen1 proposed to indicate the reaction of a 
solution by the character pH, which he called the hydrogen ion 
exponent. It is the Brigg’s logarithm of the reciprocal value of 
the normality of the solution, in terms of the hydrogen ion con- 
centration. 
The definition as well as the relationship between the hydro- 
+ 
gen ion concentration [H] and the hydrogen ion exponent will 
be made clear by the following examples: 
Examples— 
+ 
In N/10 HC1 the [H] is 0.091 N 
then 0.091 N = 9.1 X 10~2 
9.1 =10* 
log 9.1 =X log 10 = X 
X = log 9.1 
in the log book 9.1 =0.96 
then 9.1 X 10"2 = 10-9G X 10“2 = 10"1-04 
therefore pH = 1.04 
The above may be expressed by the following formula: 
pH - log = log 1 — log 0.091 
= 0.00+ (—2 + 0.96) 
= 1.04 
The student is advised to consult the table in Mathew’s, page 
544, 3rd edition, and, for practice, calculate pH values from 
+ 
[H] values. 
Ex.— Concentration Hydrogen Ion Exponent 
+ 
[H] X N. pH 
Given 5.76 X 10“4 ? 
1S<3rensen, Biochem. Zeitsch. 21, 159, 1909. 
21 From your observations in a previous experiment fill in the 
proper pH values. 
From this experiment, what is the explanation of “b,” 
page 19? 
Write one page summarizing your knowledge of indicators. 
Include specific statements drawn from the experiments in this 
section. 
This means that log 5.76 is an exponent of 10 
log 5.76 = 0.76042 = 100-76042 
Then 100-76 X 10~4= 10-3-24 
Therefore pH = 3.24. 
To change hydrogen ion exponent to concentration, proceed 
in the following manner: 
Example— 
Hydrogen Ion Exponent Concentration 
+ 
pH [H] X N. 
Given: 5.05 ? 
This means that pH 5.05 is 10-5 05 
Then 10-5 03 = 10~6 X 10+0 95 
In the log book Mantissa 0.95 corresponds to number 8.9125, 
therefore pH 5.05 == 8.9 X 10"® N. 
The following diagram, Eig. 1, will help the student to 
understand the method of recording an alkaline solution in terms 
of hydrogen ion concentration. 
ACIDITY 
Figure 1. Reaction Chart. 
NEUTRALITY 
ALKALINITY 
22 The Gas Chain Apparatus 
The simple gas chain apparatus1 used by students in this 
laboratory is shown in Figure 2. 
It consists of 
a two-cell iidi- 
son storage 
battery ii, tne 
terminals 
ot which are 
attached to the 
high-resistance 
wire a-b, thus 
allowing a uni- 
form drop in 
potential The 
negative wire 
continues t o 
the hydrogen 
electrode. A 
sliding contact 
(c) taps off 
any d e s i r e d 
fraction of the 
potential, 
which is indi- 
cated in milli- 
volts by the 
millivolt meter 
shunted across 
the circuit. G 
is a simple 
g a 1 v a n o - 
meter to indi- 
cate the nul 
point, when 
key k is press- 
ed. A satur- 
ated calomel 
electrode is at- 
tached at the 
end of the 
positive wire, 
while the neg- 
ative wire ter- 
minates in the 
platinum wire 
of the hydro- 
gen electrode. 
Figure 2. The Gas Chain Apparatus. 
1 The student is advised to read—Hildebrand, Jour. Am. Chem. Soc. 35, 846, 
1913. 
For the preparation of the Calomel electrode see Findlay, Practical Physical 
Chemistry. For the Hydrogen electrode, see Bovie, Jour. Med. Research, 33, 295, 
1915. 
23 In using the apparatus the determination is made briefly as 
follows: The solution to be tested is placed in the beaker. The 
platinum wire of the hydrogen electrode, previously covered with 
platinum black, is immersed as shown. The capillary tubing 
from the saturated calomel electrode, which is filled with satu- 
rated potassium chloride solution and prevented from syphoning 
by the closed, ungreased stop-cock, is then dipped into the liquid. 
Hydrogen gas is now bubbled through the solution for a 
minute or two, until the electrode is saturated. A bubble should 
remain around the upper part of the platinum wire in the neck 
of the jacket tube after the flow has been shut off to keep the 
electrode completely saturated. The gas is washed by passing 
through an alkaline pyrogallic acid solution to remove traces of 
oxygen if present, then through a saturated mercuric chloride 
solution, to remove sulphides, and finally through distilled water. 
After the apparatus has stood for a minute or two the gal- 
vanometer is watched while the key is quickly closed and imme- 
diately released. If the needle moves, the circuits are not in 
balance. Sliding contact c is now adjusted and the key again 
closed as above. The contact is moved and the above process 
repeated until the circuits are in balance. 
The combined voltage of the calomel electrode and that 
generated by the ions about the hydrogen electrode has now 
just balanced the imposed potential of the battery current, conse- 
quently its value is indicated by the millivolt meter. Note: The 
mercury at the bottom of the calomel electrode is always the 
positive element, while the platinum wire of the hydrogen elec- 
trode is the negative. These, when joined by the capillary tube 
containing potassium chloride solution, form a small battery. 
The circuits are so connected that the current from this “bat- 
tery” opposes in direction of flow that supplied from the storage 
cells. 
Calculations 
The hydrogen ion exponent pH is calculated according to 
the following equation: 
1 E. M. F. (Obs. in volts)—E. (Cal. Elec, in volts) 
pH=logj—1 = 0.0001983 T 
Note: T is the absolute temperature. 
24 The values for E are given below in millivolts. 
Temperature 
N/10 Cal. Elect. 
Satd. Cal. Elect. 
15° C. 
252.5 
16° C. 
251.7 
17° C. 
250.9 
18° C. 
337.7 
250.3 
19° C. 
249.5 
20° C. 
337.5 
248.8 
22° C. 
247.5 
24° C. 
246.8 
25° C. 
246.3 
30° C. 
336.4 
.... 
37° C. 
235.5 
38° C. 
335.5 
235.0 
40° C. 
333.9 
.... 
50° C. 
332.6 
60° C. 
329.0 
If all voltages are recorded in millivolts, the expression for 
the temperature correction (0.0001983 T) has the following 
value at the temperature indicated in Centigrade degrees: 
15° C 57.1 
16° C... 57.3 
17° C 57.5 
18° C 57.7 
19° C 57.9 
20° C 58.1 
21° C 58.3 
22° C 58.5 
23° C 58.7 
24° C 58.9 
25° C 59.1 
26° C 59.3 
27° C 59.5 
28° C 59.7 
29° C 59.9 
30° C 60.07 
31° C 60.27 
32° C 60.47 
33° C 60.66 
34° C 60.86 
35° C 61.06 
36° C 61.25 
37° C 61.45 
38° C 61.64 
39° C 61.85 
40° C 62.05 
Testing of Calomel Electrodes: Before using a new elec- 
trode its E. M. F. should be tested against a hydrogen electrode, 
using a standard fluid of known value. The best solution for 
25 this purpose is the “Standard Acetate Mixture,” recommended 
by Michaelis. It is prepared as follows: 
Normal sodium hydroxide 50 Ccm. 
Normal acetic acid 100 Ccm. 
Distilled water 350 Ccm. 
When testing with the above solution, values agreeing within 
+ 0.5 millivolt with those recorded below should be obtained: 
Temperature 
Against N/10 Elect.. 
Against Sat’d. Elect. 
15° 
C. 
602.5 millivolts 
517 millivolts 
16° 
C. 
17° 
c. 
517 millivolts 
18° 
c. 
604.5 millivolts 
517 millivolts 
19° 
c. 
518 millivolts 
20° 
c. 
21° 
c. 
607.5 millivolts 
518 millivolts 
22° 
c. 
518 millivolts 
23° 
c. 
519 millivolts 
24° 
c. 
38° 
c. 
520 millivolts 
The above potentials should be obtained after 1 hour, and 
will remain constant for at least 24 hours. At first a lower volt- 
age is found due to the rapid diffusion occurring. 
The student is required to become familiar with the theory 
of the gas chain apparatus, and then do the following experi- 
ments : 
15.—Determination of pH Value 
1. Determine the pH value of your N/10 HC1 solution. 
How does it compare with the value given on page 21 ? 
2. Determine the pH value of the N/l CH3COOH solu- 
tion used in experiment 14, page 19. Now fill in the value 
required in table 2, page 20. 
16.—Electrometric Titration 
1. Place 10 cc. of N/2 HC1 in a beaker. Add sufficient 
water to submerge the hydrogen electrode to the required depth. 
Place your N/2 NaOH in the burette. Determine the pH value 
26 of the acid, then add about 1 cc. (recording the exact amount) 
of the N/2 NaOH and again determine the pH value. Repeat 
this process until 9 cc. have been added. Now add 0.25 cc. por- 
tions until 2 cc. more have been added, then finish by adding 2 cc. 
portions until a total of 16 cc. have been added. 
Obtain a piece of plotting paper. Plot pH values along 
the ordinate and cubic centimeters of alkali along the abscissa. 
Note the point at which your titration curve cuts the pH 7 line. 
The cubic centimeters of alkali equivalent to this on the abscissa 
is the quantity required for the neutralization of the acid. 
2. Repeat the above titration using 5 cc. of 0.5 molar phos- 
phoric acid, adding the alkali slowly (about 0.25-0.5 cc. por- 
tions), when the voltage is rapidly rising. Add a total of 16 cc. 
of N/2 NaOH. 
Plot your curve and compare with that previously obtained 
for hydrochloric acid. 
3. Repeat the titration, using 10 cc. N/2 acetic acid, adding 
a total of 16 cc. N/2 NaOH. Plot and compare the curve with 
those previously obtained. 
4. Write about a page, stating how you would prepare a 
normal phosphoic acid solution. Be specific in stating your 
reasons. 
27 LIPINS 
The term Lipin is used here to indicate a group of sub- 
stances which may be extracted from plant or animal tissue by 
alcohol and ether (Rosenbloom, 1911). Some of these sub- 
stances are true fats, that is, esters of glycerol with higher fatty 
acids. Others, as for example, the essential oils, waxes, and 
sterols, “bear no resemblance whatever to fat, except the acci- 
dental one of being soluble in the ordinary fat solvents.” 
(Mac Lean, 1918.) 
The following is a rough classification of the material 
extractable from tissue by alcohol or ether: 
I.—Fats 
A. Neutral Fats. The esters of glycerol with higher fatty 
acids frequently accompanied by fatty acids. The latter are 
formed through the action of lipases in the tissues upon neutral 
fats. 
II.—The Fat-Like Bodies 
B. The Phosphatides (Thudichum). These substances con- 
tain fatty acid, nitrogen and phosphorus. 
1. Monamino-mono-phosphatides, N :P: :1:1, 
Examples, Lecithin and Kephalin. 
2. Diamino-mono-phosphatides, N:P::2:1, 
Example, Sphingomyelin. 
3. Monamino-diphosphatides, N :P : :1 :2, 
Example, Cuorin. 
C. The Cerebrosides (Thudichum). These substances con- 
tain fatty acid, nitrogen, and a carbohydrate group. They do not 
contain phosphorus. The carbohydrate is lactose. 
Examples, Phrenosin, and kerasin. 
III.—Unrelated Substances 
D. Waxes. These substances are esters of sterols (mono- 
hydric alcohols, solid frequently and often terpenes) and higher 
fatty acids. Examples, Sperm oil, bees wax. 
E. Sterols. These substances are alcohols, mostly terpenes, 
which are solid at room temperatures . They are therefore chem- 
ically unrelated to true fat. Examples, cholesterol, and phytos- 
terol. 
28 17.—Solubility of Neutral Fat 
A. Put a small piece of beef suet in each of four test tubes. 
Add 5 cc. water, alcohol, ether, and choloroform to the respec- 
tive tubes. Shake. Do you notice solubility in any of the sol- 
vents? 
B. Put a small piece of beef tallow in each of four test 
tubes. Add solvents as directed above. Record result. 
Note: Suet is natural tissue fat as it occurs in the animal. 
Tallow, on the other hand, is the fat from tissue, tried out by 
heat, therefore devoid of cellular structure. 
18.—Emulsification of Fats 
When a substance goes into solution, the result is a fluid 
of perfect homogenity. Immiscible fluids like oil and water, 
when mixed with soap, proteins, gums or the physiological fluid 
bile, may, by agitation, become more or less permanently sub- 
divided into minute particles. This state is known as an emulsion. 
One of the characteristic properties of this state is the develop- 
ment of an extremely great surface area, as compared with the 
mass of particles. The following experiments are intended to 
bring out the above-mentioned facts. 
29 Prepare a series of test tubes containing the following: 
Tube 
Oil Added 
Other Addition 
State 
Shaking 
After 
30-min. 
Standing 
1 
3 drops neutral 
cotton seed oil 
5 cc. water 
2 
3 drops neutral 
oil 
5 cc. of 0.25% 
Na2C03 
3 
3 drops rancid 
oil 
5 cc. water 
4 
3 drops rancid 
oil 
5 cc. of 0.25% 
Na2COs 
5 
3 drops neutral 
oil 
5 cc. of 2% egg 
albumen 
6 
3 drops neutral 
oil 
5 cc. of 10% 
gum arabic 
7 
3 drops neutral 
oil 
4 cc. water plus 
1 cc. bile 
Shake all tubes thoroughly and equally. Note the appear- 
ance immediately after shaking and again after 30 minutes’ 
standing. Why do the particles in an emulsion not cohere, and 
form stratified layers? Examine one of the emulsions under the 
microscope. 
19.—Fat Crystals 
Fat from different sources may be made to crystalize in 
definite and characteristic form which may assist in its identi- 
fication. Usually fat crystals are transparent, and therefore over- 
looked. The addition of Sudan III to the solution of fat, makes 
their detection extremely easy. 
Place a piece of fresh, dry tallow (beef, mutton, etc.) about 
the size of a pea in a dry test-tube. Add dry chloroform drop 
by drop until the tallow is dissolved. Assist the solution of the 
tallow by holding the tube near, but not in a flame. When dis- 
solved, allow to cool to room temperature and add a few drops 
more chloroform if the fat separates out. 
30 To this saturated solution of fat, add three volumes of chlor- 
oform and enough Sudan III from the point of a knife blade 
to color the solution fairly heavily. Do not add so much dye- 
stuff that particles remain undissolved. Note: To save the dye- 
stuff, a sufficient amount has been added to the chloroform placed 
on the shelf for this experiment. 
Clean a microscope slide and also a square cover slip. By 
means of a pipette, place 3 drops of the fat solution in the cen- 
ter of the slide. Cover immediately with the cover slip, and 
watch for the formation of characteristic crystals at the edge of 
the cover slip as the solvent slowly evaporates. 
Larger crystals may be obtained if one gram of tallow is 
dissolved in 10 cc. chloroform and stained as directed above. 
The “bunching” of the crystals varies somewhat with the con- 
centration of the fat. 
Sketch the crystal obtained, and, if possible, make prepara- 
tions from three or four different fats and also from fatty acids. 
Sketch and label. 
20.—The Iodine Number—Wij’s Method 
Certain fats and oils contain varying amounts of the esters 
of unsaturated fatty acids. It is possible to cause iodine to 
enter the molecule at these points of unsaturation (double 
bonds). The iodine number serves as an index of the relative 
degree of unsaturation of fats and oils. 
Start simultaneously the determination of the iodine num- 
ber of two fats (beef tallow and some one of the oils). 
Weigh out about 0.3 gm. of oil or about 1 gm. of beef tallow 
into a 250 cc. Florence flask, and dissolve in 10 cc. chloroform. 
Add 25 cc. Wij’s iodine solution by means of a pipette; stopper, 
and put in a dark place for one-half hour. Add 15 cc. of 10% 
potassium iodide and dilute with 100 cc. of water, then titrate 
the excess of iodine (partly in solution in water and partly in 
the chloroform) with N/10 Sodium thiosulphate, running the 
latter into the flask until after repeated shaking, both the chloro- 
form and the watery solution are but faintly straw colored. 
Then add 1 cc. of 1% starch solution and continue the titration 
to the disappearance of the blue color. 
While waiting for the absorption of the iodine to take place, 
the value of the iodine solution may be determined in terms 
31 of N/10 sodium thiosulphate, by adding to 25 cc. of Wij’s solu- 
tion, 15 cc. of 10% potassium iodide and titrating as above. 
The difference between the two values represents the amount of 
iodine absorbed by the fat, and is calculated in terms of grams 
of iodine per ioo grams of fat. 
Example: Charge is 0.3 gm. cottonseed oil. 
Back titration is 35 cc. N/10 Na2S203. 
Wij’s solution, 25 cc. equals 60 cc. N/10 
Na2S2Os. 
Therefore, 60-35 equals 25 cc. N/10 Iodine. 
Since 1 cc. N/10 Iodine—0.0127 gm. we have, 
25X0-0127 = 0.317 gm. Iodine absorbed. 
Therefore X 0.317 = 105.6. 
Iodine number is 105.6. 
Wij’s Iodine Solution 
Dissolve 13 grams of iodine in 1 liter of glacial acetic acid. 
Titrate the iodine content of the solution, then pass washed 
and dried chlorine gas into the solution until the titration num- 
ber is doubled. A very distinct change in color of the solution 
indicates when this has taken place. 
The relative value of the Wij’s iodine solution to N/10 
sodium thiosulphate will be given by the instructor. 
Using oleic acid as an example, write structurally the reac- 
tion which would take place in this determination. 
Write the structural formulae of three other fatty acids 
which would have a high iodine number. 
21.—The Saponification Number (Kottstorfer Value) 
The saponification number is the number of milligrams of 
potassium hydroxide required to completely saponify one gram 
of fat or wax. Since during saponification, the neutral glycer- 
ides or other esters (waxes) of fatty acids are decomposed, 
yielding the potassium salt of the fatty acid (soap) and the 
corresponding alcohol (glycerol from neutral fats), it is apparent 
that the lower the molecular weight of the fatty acids involved, 
the greater the saponification number. 
32 Procedure: Procure two clean, dry Erlenmeyer flasks of 
200-250 cc. capacity. Into one of these weigh 1.5 to 2 gms. of 
purified and filtered fat. Pipette into each 25 cc. of special alco- 
holic potassium hydroxide solution. To each attach an efficient 
condenser, and heat for one-half hour on the boiling water bath. 
(The heat should be so regulated that the alcohol is just sim- 
mering.) Caution'. Avoid loss of alcohol through volatilization 
from condenser. The flasks are gently shaken in a rotatory 
direction at frequent intervals to thoroughly mix the contents. 
After one-half hour, remove the flasks from the water bath, 
cool to room temperature, add 1 cc. of 1% phenolphthalein, and 
then titrate each with N/2 hydrochloric acid. 
From the titration of the blank, subtract the titer of the 
flask to which fat was added. The difference corresponds to 
the quantity of N/2 potassium hydroxide solution required to 
saponify the weight of fat taken. This, when calculated to milli- 
grams of potassium hydroxide per gram of fat, is the saponifi- 
cation number. 
Potassium hydroxide in alcoholic solution turns dark brown 
on standing and especially if exposed to the light unless the 
alcohol used as solvent has been previously purified. The most 
satisfactory method we have found is to mix the ordinary 95% 
alcohol with commercial granular caustic soda; about 200 grams 
to 5 gallons of alcohol; and allow the mixture to stand in the 
light for several weeks. The alcohol is then distilled off. The 
distillate is neutral, and when used in preparing an alcoholic 
solution of potassium hydroxide, the reagent remains clear prac- 
tically indefinitely. 
To prepare the special potassium hydroxide solution, weigh 
out 40 grams of “Potassium Hydroxide C. P. by alcohol,” dis- 
solve in the purified alcohol, finally making the volume up to 1 
liter. If a precipitate of carbonate is noticed, it is removed by 
filtering the reagent through glass wool. 
Problem: Calculate the saponification numbers of ethyl 
acetate and of glyceryl tributyrate, respectively. 
22.—Fat in Milk (Babcock’s Method) 
The physician is frequently called upon to modify cow’s 
milk for infant feeding, as well as to analyze breast milk to 
determine the cause of malnutrition in infancy. Since the fat 
33 content in both cases is of primary importance, the following 
methods are given. 
A. Cow’s Milk. 
Procure a Babcock bottle, graduated to 10% butter fats. 
Into this pipette 17.6 cc. (18 grams) of milk. Add 17.5 cc. of 
commercial sulphuric acid (sp. gr. 1.82-1.84). Mix by rotation 
until all curds are dissolved. The mixture becomes hot and 
dark colored. 
The bottle is now placed in the bucket of a Babcock cen- 
trifuge, and counter-balanced with a duplicate test bottle, or 
by other suitable means. 
Revolve in the centrifuge for 5 mins, at a rate of 800 to 
1,000 revolutions per min. Stop the machine, remove the bottle 
then add boiling water until the liquid comes just to (but not 
into) the graduated neck. Return to the centrifuge, and revolve 
for 2 mins. Again stop the centrifuge, remove the bottle, add 
boiling water until the graduated neck is nearly filled. Return 
to the centrifuge and revolve again for 2 minutes. The bottle 
is calibrated to give directly the percentage of fat in the sample 
by noting upper and lower readings of fat column, provided the 
temperature is 60° C. It is therefore best to immerse the bottle 
nearly to the top of the neck in a beaker of water heated to 60° 
C., for two minutes before making the readings. 
B.—Human Milk 
For human milk, where usually the amount available is 
limited, a special bottle which requires but 5 cc. is used. This 
bottle fits the cup of an ordinary 15 cc. centrifuge tube used for 
urine sediments. 
Procedure: Fill the special bottle with milk to the 5 cc. 
mark. Add commercial sulphuric acid until the body of the 
bottle is filled. Rotate until curds are dissolved. Fill the neck 
of the bottle with a mixture consisting of equal parts of con- 
centrated hydrochloric acid and amyl alcohol. 
The tube is next placed in the cup of the centrifuge, coun- 
ter-balanced, then rotated for 2 minutes. The percentage of fat 
is now read by observing its location in the graduated neck of 
the bottle. 
34 23.—Refractive Index of Butter Using Zeiss Butero-Refrac- 
tometer 
The determination of the refractive index of fat or oil serves 
its greatest purpose in the detection of adulterants. It also assists 
materially in identification of pure fats and oils. 
The work in the laboratory is intended to be upon butter. 
Any other fat or oil having a refractive index within the range 
of the instrument (1.4220 to 1.4895) may be substituted. 
Open the movable half of the prism block of the refrac- 
tometer and clean all surfaces thoroughly and carefully with a 
piece of fine linen cloth moistened with a little alcohol or benzine. 
See that the temperature of the heating system is constant 
and a few degrees above the melting point of the fat under inves- 
tigation. Holding the instrument so that the open half of the 
prism is horizontal, carefully applying a drop or two of the clear 
(filtered, if necessary) melted fat or oil to its optical surface. 
Close the prism faces and lock by turning the thumb-screw at 
the top of the movable half of the prism. 
Adjust the mirror so that the critical line separating the 
bright left-hand field from the dark right is clearly visible when 
looked at through the telescope. If the critical line is not dis- 
tinct probably the space between the prism faces is not uniformly 
filled by the oil or fat. Finally adjust the movable part of the 
ocular so that the scale becomes clearly visible. 
Allow a current of water at a proper and uniform tempera- 
ture to flow through the instrument for some time previous to 
taking the reading. Notice the critical line. If hazy at first, it 
should, after a minute, attain a fixed position and reach maxi- 
mum distinctness. 
When this is attained, notice whether the critical line is 
colorless or colored, and if the latter, what color. Now note 
the exact location of the critical line on the centesimal scale, 
estimating to tenths of a division by means of the milled scale 
head on the side of the instrument. Immediately read the tem- 
perature recorded by the thermometer. 
This instrument is primarily intended for use with butter- 
fats, which, if pure, give a colorless critical line.- A red colored 
line indicates low dispersive power, while a blue line is given 
by a substance of relatively high dispersion. Oleomargarine 
shows a greater dispersion, and therefore a blue colored line. 
35 After the instrument has been read, a table should be con- 
sulted for temperature correction and also for transposing the 
scale readings to indices of refraction (nD). Such tables may 
be found in most books on food analysis, e. g., Leach—“Food 
Inspection and Analysis,” 3rd Ed., P. 105. 
Use of Special Thermometer. Instead of employing the 
ordinary thermometer a special instrument has been devised for 
work with butter and lard. It has two scales, the left for butter, 
the right for lard, each of which indicates at once the highest 
allowable reading for the pure fat, corresponding to the tempera- 
ture at which the observation is being made. 
If the scale reading of the instrument as observed through 
the telescope, differs materially from the reading of the special 
thermometer, the fat under examination is undoubtedly adulter- 
ated, or in the case of butter, a higher reading indicates oleo- 
margarine. The special thermometer thus indicates the highest 
possible number for pure butter. 
24.—Saponification of Neutral Fat by Pancreatic Lipase 
Dilute 1 part of 16% cream with three parts of water. 
Pipette 10 cc. of this fat suspension into each of two small flasks 
(100-150 cc.) or beakers. Pipette a third portion of 10 cc. into 
a 250 cc. Erlenmeyer flask. Saponify this sample as directed 
under Exp. 21 above, using only 10 cc. special alcoholic potash, 
recording your value in cc. of N/2 alkali required by the 10 cc. 
portion. 
Caution : Be sure that the diluted cream suspension (emul- 
sion) is uniform as each sample is pipetted out! 
To one of the small flasks, add 2 drops of chloroform, and 
2 cc. of distilled water. Cover the top with a tinfoil cap. Label 
with your name and the word “control.” To the other flask 
is added 2 drops of chloroform, 1 cc. of distilled water and 1 cc. 
of freshly prepared 10% pancreatin suspension (Fairchild’s Hol- 
adin). 
Cover this flask with a tinfoil cap, and label properly. Place 
both flasks in the incubator at 40° C. 
At the next laboratory period, not less than two days, 
remove the flasks, record any difference in appearance, then add 
1 cc. of 1% phenolphthalein and 10 cc. of 95% alcohol to each. 
36 Carefully titrate each flask with half normal sodium hydroxide. 
Notice the appearance of soap suds on one of the flasks you 
titrate. Explain, Subtract the titre of the “control” from that 
of the flask to which pancreatin was added. The difference 
gives the amount of alkali required to neutralize the fatty acids 
formed by the hydrolytic cleavage of the butter fats. (The 
cream is assumed to be fairly free from protein. No allowance 
is made for amino acids.) From this value, and that obtained 
by saponification with alcoholic potash, calculate the approxi- 
mate percentage digestion effected by the pancreatic lipase. 
25.—Saponification of Tallow. Preparation of a Soap 
Preparation of a Fatty Acid. Detection of Glycerol. 
Place 20 grams of tallow in a 6-inch metal dish. Add 30 cc. 
of 10 per cent sodium hydroxide (in 70 per cent alcohol) solu- 
tion, and heat upon the steam bath until dry. Add 150 cc. of 
boiling water to the dish and stir until all of the soap is dissolved. 
Divide the solution into two equal parts, and proceed with each, 
as follows: 
A. Preparation of a Soap. Heat one portion of the soap 
solution to boiling. Add about 10 grams of sodium chloride, 
stir until dissolved, then allow the separated soap curd to collect 
in a cake as the solution cools. When cold, remove the soap. 
Try its solubility in water, alcohol, etc. To a portion of soap 
dissolved in water add a few cubic centimeters of 2% calcium 
chloride solution. Examine the curd; observe its greasy feel. 
Write the equation for the saponification of the tallow (stear- 
in). Write an explanation of the action of the sodium chloride 
upon the soap solution, using equations wherever possible. What 
soaps are soluble? What is the cause of the difficulty encoun- 
tered in using soaps in a limestone country? 
B. Preparation and Properties of Fatty Acid. Heat the 
second portion of the soap solution to boiling. Add 5 drops of 
methyl orange, and then with constant stirring, add dilute (1 :3) 
sulfuric acid until a distinct acidity is obtained, and the liberated 
fatty acid floats on the surface as an oily layer. Filter the hot 
(but not boiling) mixture through a large dry filter paper, col- 
lecting and saving the filtrate for part “C” below. Wash the 
fatty acid on the paper with a small amount of hot water, then 
allow to cool. When cold, note its physical appearance; try its 
37 solubility in water, alcohol, ether, etc. Dissolve a little fatty 
acid in alcohol, add a drop of phenolphthalein, and try to titrate, 
with N/10 NaOH. What is formed? Dissolve another portion 
of fatty acid in alcohol and add to it a dilute (1%) sodium car- 
bonate solution. What is formed? Write equations. Compare 
the above with the behavior of neutral fat and soaps when treated 
in a similar manner. If in doubt try each reaction. (Note: 
When neutral fat is to be dissolved in alcohol, the tube should 
be stood in warm water, never held in a flame.) 
C. Isolation and Detection of Glycerol. To the filtrate 
from the preparation of fatty acid (“B” above) add 20% sodium 
carbonate solution, drop by drop while stirring until any acid 
present is just neutralized. Pour the solution into a 6-inch dish 
and evaporate on the steam bath to dryness. When dry, extract 
the residue with two 25 cc. portions of 95% alcohol. Combine 
the alcoholic extracts, pour into a small evaporating dish and 
evaporate off the alcohol, placing the dish upon the top of the 
steam bath. The material remaining should be crude glycerol. 
Perform the following tests with the glycerol: Cautiously 
note taste. Note solubility of a drop in ether. 
Borax Bead Test: Moisten a platinum loop with glycerol. 
Touch it to a little powdered borax, allowing some to adhere. 
Fuse in a bunsen flame, noticing the characteristic green color 
imparted to the flame. This color is due to the formation of a 
glycerol ester of boric acid. 
Acrolein Test: Moisten about 2 grams of potassium bisul- 
phate, contained in a small metal spoon, with 3 or 4 drops of the 
crude glycerol. Heat the spoon over a bunsen flame until dense 
white fumes are emitted. Remove from the flame and cautiously 
note the characteristic sharp stinging odor. Note also effect 
upon the eyes. 
Glycerol loses hydrogen and hydroxyl groups equivalent to 
two molecules of water during the above heating. Rearrange- 
ment of the molecule occurs, forming acrylic aldehyde (acro- 
lein). Write structural formulae indicating the reaction. 
Repeat the above tests with any neutral fat. Explain. Do 
fatty acids or soaps respond to this test? Do waxes? If in 
doubt, try each. 
38 26.—Exercise 
Identification of Neutral Fat, Fatty Acid, and Soap, when 
occurring in a mixture. 
The solubilities and reactions given above should enable the 
student, after a little thought, to identify fat, fatty acid, or soap, 
when occurring separately or in a mixture containing any two 
or all three. 
Make out a scheme of procedure for this purpose, and then 
obtain two unknowns from the instructor. Report your findings 
on a slip of paper, dated and signed. 
27.—Lecithin Preparation from Egg Yolk 
(Procedure from Manual of W. Lee Lewis.) 
1. Place 2 freshly separated egg yolks in a 300 cc. Erlen- 
meyer flask. Stopper and shake. 
2. Add 100 cc. of 95% alcohol, then stopper and shake for 
two minutes. Now stopper flask with cork, through which passes 
a Afoot glass tube (8-10 cm. in dia.). 
3. Boil gently for 45 mins, on the steam bath. 
4. Decant alcohol through dry filter paper into a dry 500 
cc. flask. Stopper. 
5. Add to residue in flask 100 cc. 95% alcohol, repeat 
extraction, and decant as above. (Nos. 2, 3 and 4.) 
6. Add to residue in flask 100 cc. ether, then extract, and 
repeat a second time, exactly as directed above (No. 3, No. 4). 
(Caution—Place flask on cool part of steam bath top—ether 
must not be allowed to boil out.) 
7. Evaporate united alcohol-ether extracts in a large evapo- 
rating dish on the water-bath until solvent is gone. Caution— 
avoid flames! 
The residue in the evaporating dish contains the egg fat 
together with impure lecithin and cholesterol. 
8. Extract the residue, by means of a glass stirring rod, 
with 3 portions of cold ether, using 20 cc. each time. (Filter if 
decantation is difficult.) 
9. Add acetone to clear ethereal solution, little by little, 
until a maximum precipitate occurs. This is impure lecithin. 
10. Allow precipitate to settle, decant and save supernatant 
liquid, collect lecithin on a glass rod; transfer to a watch glass. 
39 11. Dry in a dessicator. (Keep lecithin in dessicator until 
collected.) 
12. Perform the following tests: 
Physical and Chemical Properties 
a. Emulsification. Shake a small piece of lecithin with 5 
cc. of water. Note the emulsion formed. Note myeline move- 
ments when a drop on a slide is examined under the microscope. 
b. Presence of phosphorus. Fuse another small portion 
with three parts of fusion mixture (one part of potassium nitrate 
and two parts of sodium carbonate) in the spoon. When the 
hot mixture is white, allow to cool, dissolve in a small amount of 
water and test for phosphorus by means of nitric acid and 
ammonium molybdate solution. If the yellow precipitate does 
not form at once, warm and rub the sides of the test tube with 
a glass rod. What is the precipitate? 
c. Saponification Test. Heat another fraction of your 
preparation with 15 cc. of sodium alcoholate in a flask for 10 
minutes; evaporate to dryness. What is the residue? Dissolve 
in some hot water, acidify with sulphuric acid and allow to 
stand over night. Notice the separation of fatty acids. 
d. Glycerine. Fuse half of the remainder of your prepara- 
tion with two parts of potassium bisulphate. 
e. Test for Nitrogen. Form a paste from the remainder 
of the lecithin with three parts of slaked lime and a few drops 
of water in the bottom of a small beaker. Cover the beaker with 
a watch glass on the lower side of which a moist piece of red 
litmus paper has been made to adhere. Now heat the beaker 
and contents very gently. Notice the change in color of the 
litmus. Explain. 
28.—Cholesterol Preparation from Egg Yolk. 
(Procedure from the manual of W. Lee Lewis.) 
1. Evaporate ether-acetone solution from the previous 
experiment to dryness on the water bath. (Use evaporating 
dish.) 
2. Saponify fats by adding 50 cc. of N/l alcoholic sodium 
hydroxide, which dissolves residue. Cover evaporating dish 
loosely with a watch glass, and heat on the water bath until all 
of the alcohol has evaporated. (Use top of water bath.) 
40 3. Transfer dry residue to a mortar and grind finely. 
4. Extract with three 15 cc. portions of cold ether, triturat- 
ing thoroughly. 
5. Filter ethereal extracts, into a small beaker. 
6. Allow slow spontaneous evaporation of the ether to occur 
on the desk, until crystals are formed. 
7. Filter crystals from mother liquid (after latter is nearly 
evaporated). 
8. Recrystallize from 95% alcohol. Filter and dry. 
9. Sketch crystals and perform the following tests. 
a. Evaporate a few of the crystals with concentrated nitric 
acid to dryness. Observe the color of the residue and add a few 
drops of ammonia. Notice the change of color from yellow to 
brick red. 
b. Salkowski’s reaction. Dissolve a crystal or two in one 
cc. of chloroform and add an equal volume of cone, sulphuric 
acid. Notice violet coloration of chloroform due to certain 
hydrocarbons formed by dehydration. 
c. Schiff’s reaction. Evaporate a few crystals of your 
preparation to dryness with two volume of concentrated sul- 
phuric acid and one volume of ferric chloride solution. Notice 
violet color. 
41 CARBOHYDRATES 
Carbohydrates are aldehyde or ketone derivatives of polyhy- 
dric alcohols; or polymeric forms of these, yielding active alde- 
hydo-alcohols, or keto-alcohols upon hydrolysis. 
The simple carbohydrates contain one carbonyl group and 
one or more hydroxyl groups. It is essential that one of the 
hydroxyl groups be linked to a carbon, which is in direct union 
with a carbonyl group. The characteristic group of these com- 
H 
i 
pounds is therefore -C-C- 
i ii 
O 
H 
Classification: 
Carbohydrates 
1. Monosaccharides (2-7 carbon atoms). 
2. Disaccharides (12 carbon atoms). 
3. Polysaccharides (18 carbon atoms and above). 
For a complete classification of carbohydrates the student 
is expected to refer to Mathews, p. 19. 
29.—Elementary Composition— 
Tests for Carbon and Hydrogen. 
Place a little sugar in the bottom of a clean, dry test tube, 
and heat gently over the micro burner. Notice the changes that 
are taking place. Into the mouth of the test tube thrust a stirring 
rod moistened with barium hydrate solution. What is formed? 
Notice drops of moisture collecting on the sides of the test tube. 
To what are they due? 
30.—Molisch-Udransky Test 
Place 5 cc. of concentrated sulphuric acid in a test tube. 
Incline the tube and slowly pour down its inner side 5 cc. of 0.5% 
glucose solution, to which 3 drops of Molish’s reagent (1% alpha 
napthol in alcohol) has been added. The sugar solution should 
stratify over the sulphuric acid. A reddish-violet zone is pro- 
duced at the point of contact. This reaction is due to the forma- 
tion of furfurol from the carbohydrate by the action of con- 
centrated sulphuric acid. The furfurol then condenses with 
alpha napthol. This test is given by all bodies containing a car- 
42 bohydrate group, whether free or in combination, as in certain 
proteins. It is therefore not specific for the class of compounds 
we designate by the name “carbohydrates.” 
Monoses or Monosaccharides 
All monosaccharides and certain disaccharides give the fol- 
lowing four characteristic tests: 
1. Formation of silver mirror—by warming a tube contain- 
ing ammoniacal silver nitrate to which a monosaccharide has 
been added. 
2. Formation of caramel and resins—by warming a mono- 
saccharide to which an alkali has been added. 
3. Reduction of an alkaline copper solution—when an alka- 
line copper solution to which a monosaccharide has been added, 
is heated; reduction is evidenced by the formation of a yellowish- 
red suboxide of copper. 
4. Formation of an osazone—when monosaccharides are 
heated with an excess of freshly recrystallized phenyl-hydrazine, 
in the presence of a dilute acid, characteristic yellow needle- 
shaped crystals are formed. The melting point of the crystals 
serves to identify the mono-saccharide. (This test is given by 
all reducing carbohydrate.) 
31.—Silver Mirror 
Procure a small test tube. Have it perfectly clean inside, 
then add 5 cc. of an ammoniacal silver nitrate solution. Add 
8-10 drops of 0.5% glucose solution and place the tube in a 
beaker of boiling water. A bright silver mirror should be formed, 
indicating reduction of the reagent by the glucose. 
32.—Moore’s Test 
To 5 cc. of dextrose solution in a test tube, add 5 cc. of 5% 
sodium hydroxide solution, and warm. The solution becomes 
first yellow, then orange, yellowish brown, and lastly, dark brown. 
Notice a faint odor of caramel. This odor is more pronounced 
upon acidification. 
33.—Trommer’s Test 
To 5 cc. of a 1% copper sulphate solution in a test tube, 
add an equal volume of 5% sodium hydroxide solution. Note 
the formation of a precipitate and its disappearance. Boil. Note 
the result and compare with the following: 
43 Trommer’s test is based upon the property that dextrose 
possesses of reducing cupric hydroxide in alkaline solution to 
cuprous oxide. Treat 5 cc. of 0.5% dextrose solution with not 
more than 2 cc. of 5% NaOH. Copper sulphate solution is now 
added, drop by drop, until a very small amount of the hydroxide 
remains undissolved in the liquid. Warm the solution. A yellow 
hydrated oxide or red sub-oxide separates out even below the 
boiling temperature. If too little copper salt has been added, the 
test will be yellowish brown in color, as in Moore’s test. An 
excess of copper salt should also be avoided, since upon boiling 
the excess of hydroxide is converted into a dark brown hydrate, 
which interferes with the test. To prevent these difficulties, the 
so-called Fehling’s solution is employed. 
34.—Fehling’s Test—Qualitative 
Mix 10 cc. of the Fehling’s copper solution with 10 cc. of 
the Fehling’s alkaline solution in a test tube, and heat to boiling. 
The solution should remain clear. Divide the solution into three 
parts. To one portion add a few drops of glucose solution. Note 
the results. Continue the addition of the glucose, drop by drop, 
boiling between the addition of each drop, until no more blue 
color remains. Pour the contents of the test tube into an evapo- 
rating dish, and allow it to remain in the air for some time. What 
is the effect of atmospheric oxygen upon the solution? 
To the second portion add a few drops of glucose solution, 
but without heating. 
To the third portion add a few drops of diabetic urine, and 
heat. 
35.—Quantitative Determination of Sugar—Fehling’s 
Method. 
Pipette out 10 cc. of the Fehling’s copper solution into a 250 
cc. flask. Add 10 cc. of the Fehling’s alkaline solution and dilute 
with 50 to 60 cc. of water. Heat the solution to boiling. Place 
the glucose solution in a burette and add the solution drop by drop, 
boiling after each addition to the Fehling’s solution. Continue 
until no blue remains. To determine the absence of blue, the 
meniscus of the liquid should be examined against a light back- 
ground. If a volume of less than 5 cc. of glucose is required, 
dilute the solution so that at least this amount shall be used, and 
repeat the test, this time running in nearly the required volume 
44 at first. Toward the end point, add the solution drop by drop as 
before. Calculate the percentage of sugar in the solution, con- 
sidering that the Fehling’s solution has been reduced by 0.05 
grams of glucose; that is to say, the volume necessary to com- 
pletely precipitate all of the copper must contain this amount of 
dextrose. Report the percentage of sugar in the unknown speci- 
mens furnished, to the instructor before proceeding. Note: Fehl- 
ing’s solution is prepared in the following manner: Solution A 
(copper sulphate solution)—17.325 grams of purest obtainable 
copper sulphate are weighed out and dissolved in water, and made 
up to 500 cc. 
(10 cc. equals 0.050 grams of glucose.) 
Solution B (alkaline tartrate solution)—86 grams of Roch- 
elle salts and 25-30 grams of potassium hydroxide are dissolved 
in water and made up to 500 cc. 
These solutions must be preserved separately. Before using, 
10 cc. of each of the solutions are mixed, diluted and boiled as 
described above. The tartrate holds the excess of cupric hydrox- 
ide in solution, and an excess of the reagent does not, therefore, 
interfere in the performance of the test. 
36.—Benedict’s Test—Qualitative 
Several copper reduction tests for carbohydrates have been 
originated within the last twelve years, claiming superiority to 
the original Fehling’s test. Part of this contention is based upon 
the fact that small amounts of sugar present in a fluid may be 
decomposed by the caustic alkalinity of the Fehling’s solution. 
A second factor to be considered is that uric acid and creatinine 
present in urine have also a reducing action upon Fehling’s solu- 
tion. 
The chief objections to the use of Fehling’s solution from 
the practical standpoint is not due to any inherent fault of the 
method, but largely to the inability of the person conducting the 
estimation to determine readily the disappearance of the last trace 
of blue color, indicating unreduced copper. 
The operator will be well repaid if he takes the trouble to 
master the required technic. It is briefly as follows: Find the 
clear hair line, representing surface reflection, which may always 
be observed at about 1/16 to 1/4 inch below the surface of the 
hot liquid (contained in a 250 cc. Florence flask), when lowered 
45 slowly from a point just above to a point just below the level of 
the eye. A blue color indicates that more sugar solution should 
be added. When colorless or water clear, the end point is 
obtained, while a yellow or yellow green color indicates that an 
excess of sugar has been added. 
Benedict’s test was invented to banish all the faults of Fehl- 
ing’s method. It is said to be more suitable for urine work on 
account of not being reduced by uric acid or creatinin. Other 
advantages are recited in many text books. 
Qualitative Test 
Place 5 cc. of Benedict’s solution (qualitative) in a test tube. 
Add 3 to 4 drops of 0.5% glucose solution. Boil vigorously 
for 1 to 2 minutes, then place in the rack to cool. The tube 
should contain a large bulk of red, yellow, or green precipitate, 
depending upon the concentration of the sugar. 
Note—Benedict’s Qualitative Solution. 
Weigh out 173 grams of sodium citrate, add to a beaker con- 
taining 600 cc. of boiling water. Stir until dissolved. Now 
weigh out 90 grams of anhydrous sodium carbonate, and add to 
the citrate solution, little at a time and with stirring. If neces- 
sary filter through paper. Make the volume of these solutions 
up to 850 cc. when cold. 
Weigh out 17.3 grams of crystalline copper sulphate. Dis- 
solve in 100 cc. hot water, and when cold dilute to 150 cc. 
Now pour slowly and with stirring, the copper sulphate solu- 
tion into the carbonate-citrate solution. When mixed, the solu- 
tion should remain clear indefinitely. 
37.—Quantitative Determination of Sugar — Benedict’s 
Method. 
Pipette 25 cc. of Benedict’s quantitative solution into a 250 
cc. Florence flask. Add 8-10 grams of solid sodium carbonate, 
and heat the flask to boiling. The carbonate should be dissolved 
in a minute or two. The fluid in the flask must be kept gently 
boiling during the entire addition of the sugar solution. With 
the flask gently boiling run in the sugar solution (or urine) from 
a burette, slowly, until the flask is opaque, from the formation 
of a white precipitate. The blue color will now be rapidly disap- 
pearing. Now add the sugar solution, cautiously, a few drops 
at a time until the last trace of blue has disappeared. If the 
46 volume of sugar solution required is less than 10 cc. dilute it 
with water so that from 10 to 15 cc. will be required. Repeat 
the determination with the diluted solution thereby obtaining 
greater accuracy. 
25 cc. of Benedict’s solution equals 0.05 gm. glucose. 
25 cc. of Benedict’s solution equals 0.053 gm. fructose. 
25 cc. of Benedict’s solution equals 0.074 gm. maltose. 
25 cc. of Benedict’s solution equals 0.0676 gm. lactose. 
Note: Benedict’s quantitative solution is prepared as fol- 
lows : 
Weigh out and dissolve in 500 cc. of hot water, 200 grams 
of sodium citrate, 100 grams of crystalline sodium carbonate and 
125 grams of sodium or potassium sulphocyanide. When dis- 
solved, allow to cool, then transfer to a 1,000 cc. volumetric 
flask. 
Weigh out exactly 18.0000 grams of purest obtainable copper 
sulphate crystals. Dissolve with the aid of heat in 100 cc. 
of distilled water. 
When both solutions are cold, pour the copper sulphate 
solution without loss into the alkaline citrate solution. The 
volumetric flask should be almost continuously shaken during 
the addition of the copper sulphate solution and but little of the 
latter should be added at a time. 
Now add 5 cc. of a 5% solution of potassium ferrocyanide to 
the mixture in the flask, then fill to the 1000 cc. mark by thor- 
oughly rinsing the beakers used for dissolving the copper sul- 
phate solution, and the alkaline citrate solution into the flask. 
Thoroughly mix the solution. 
Benedict’s solution possesses the advantages of having a 
white end-point. The reduced copper, instead of settling out 
as a reddish cuprous oxide, as in Fehling’s solution, unites with 
the thiocyanate, forming the colorless precipitate. The addition 
of the small amount of potassium ferrocyanide is to prevent 
decomposition of the cuprous oxide. 
Each student is required to obtain checks with a 0.5% glu- 
cose solution, and to do and report one unknown. 
Formation of Osazones—Reaction with a Phenyl-hydrazine. 
All reducing sugars react with either phenyl-hydrazine or a 
substituted phenyl-hydrazine to form crystalline compounds. 
47 Certain sugars are capable of forming easily identifiable com- 
pounds with but one molecule of phenyl-hydrazine. These com- 
pounds are called hydrazones. If an excess of phenyl-hydrazine 
is present, then three molecules will react with each molecule 
of reducing sugar. The product in this case is an osazone. This 
reaction is also given with certain substituted phenyl-hydrazines. 
It is important to remember that those monosaccharrides 
sharing a common enolic form give the same identical osazone. 
As sugars are identified by the shape of crystal and especially by 
the melting point of the osazone, the ability to form enols must 
not be overlooked. 
To obtain satisfactory results, if the base phenyl-hydrazine 
is used, it must be colorless, or at most, of a very light yellow. 
Dark-colored samples are contaminated by an oxidation product, 
and require distillation in vacuo (15 mm.), followed by further 
purification. In this laboratory we find it more convenient to 
use phenyl-hydrazine hydrochloride. The commercial samples 
are invariably impure and should be purified by recrystallization 
from hot alcohol. The material should be filtered upon a Buchner 
funnel as soon as dissolved. The filtrate cooled, and the sepa- 
rated platelets filtered on a second Buchner, preferably in very 
subdued light. The mother liquor should be sucked out as thor- 
oughly as possible, then the platelets removed and air dried in 
a dark place. When dry the platelets are glistening and pearly 
white. Keep in a stoppered brown glass bottle. 
38.—Preparation of Phenylglucosazone 
Place in a test tube 0.2 gram purified phenyl-hydrazine 
hydrochloride, and 0.3 gram crystalline sodium acetate. Add 
0.1 gram glucose (or 10 cc. of a 1% solution) and water to a 
total volume of 20 cc. 
Place the tube in a hot water bath and maintain just at the 
boiling point for \ l/2 hours, remove the flame and allow the tube 
to cool slowly in the water hath. Note and record in this and 
subsequent work tbe appearance of crystals if in the hot solu- 
tion or only upon cooling. Remove a few of the crystals to a 
microscope slide, examine and sketch. 
If time permits, the crystals of monosaccharides may be 
filtered off and recrystallized from hot alcohol or pyridine, dried, 
and their melting point determined. 
48 Osazones of disaccharides may be purified by recrystalliza- 
tion from hot water. 
The phenylosazones of the common sugars, have the follow- 
ing melting points: 
Phenylosazone of d-Arabinose, 160° C. 
Phenylosazone of d-Glucose, 208° C. 
Phenylosazone of d-Mannose, 208° C. 
Phenylosazone of d-Galactose, 193° C. 
Phenylosazone of Maltose, 206° C. 
Phenylosazone of Lactose, 200° C. 
In determining melting points, the heating should be rapid, 
to the point at which melting is first observed. Immediately 
withdraw the heat and note temperature and completeness of 
fusion, with or without decomposition (gas development). 
39.—Phenylosazone from Urine 
Under certain conditions it is of the utmost importance to 
be able to distinguish between a pentose (usually d-l-arabinose), 
glucose, or lactose in urine. Pentoses are found occasionally in 
urines, usually of people addicted to the morphine habit; glucose 
in urines of diabetics and others committing a dietary indiscre- 
tion ; lactose, often in the urine of women during certain stages of 
pregnancy, or after child birth. Urines to be tested must be free 
from albumen. 
The technic as outlined by Emil Fischer is given below: 
To a tube containing 10 cc. of urine add 0.2 gram purified 
phenylhydrazine hydrochloride. Add 0.4 gram crystalline sodium 
acetate. Heat for U/i hours in a gently boiling water bath. 
The amorphous precipitate is the crude and impure osazone. 
Filter this off; dissolve in a little hot alcohol (50%) ; filter again 
if necessary; add a little water, then as the alcohol evaporates, 
characteristic yellow crystals appear. Examine under the micro- 
scope, and after drying, take the melting point, 
ides will be referred to again in proper order. 
Note: The student is expected to make osazones of glu- 
cose, arabinose, maltose, and lactose at this time. He is expected 
to practice the technic required for identifying glucose or lac- 
tose when 0.1 gram is added to 10 cc. of urine. The disacchar- 
49 ides will be referred to again in proper order. 
40.—Nylander’s Test—Reduction of a bismuth salt. 
To 10 cc. of 0.5% glucose solution add 2 cc. of Nylander’s 
reagent. Place the tube in a boiling water bath for 5 minutes. A 
black precipitate of metallic bismuth separates out. Note: 
Nylander’s reagent is prepared by dissolving 50 grams of Rochelle 
salts together with 20 grams of bismuth subnitrate in 1 liter of 
8% sodium hydroxide. 
This test is not effected by urates or creatinine, but is by 
albumin. It is also useful in distinguishing homogentisic acid, 
found in alkaptonuria from glucose. 
The student should repeat this test with arabinose, and also 
with the disaccharides. 
41.—Barfoed’s Test—Reduction of Copper Acetate in Dilute 
Acetic Acid Solutions. 
Put 5 cc. of Barfoed’s reagent in a test tube. Add 1 cc. of 
0.5% glucose and heat the tube from 3 to 4 minutes in the boiling 
water bath. Examine for the presence of a precipitate. Repeat 
this test with the disaccharides. Is this test specific for a mono- 
saccharide? 
Note: To prepare Barfoed’s reagent, dissolve 45 grams of 
neutral copper acetate crystals in 900 cc. of distilled water. 
Filter if not clear. Add 1.5 cc. of 50% acetic acid, and dilute 
to 1 liter. 
42.—Fermentation by Brewer’s Yeast 
Fill a small test tube full of 0.5% glucose solution. Add 
% to % cake of Fleishmann’s compressed yeast. Rub the yeast 
to a fine suspension by means of a glass rod, then transfer to a 
fermentation bulb, stopper and invert, or use a Durham Fermen- 
tation tube, plugged with cotton. Place in a suitable beaker. 
Allow the apparatus to incubate in the warm room until the next 
period, then examine for evidence of fermentation. Note the 
volume of gas formed, and test a clear filtered portion of the 
remaining liquid for alcohol by means of the iodoform test. 
Run simultaneously a control containing only yeast and dis- 
tilled water. Note: To perform the iodoform test, aerate 5-10 
cc. filtered fermentation mixture, which is slightly warmed by 
being placed in a water bath, into 3-4 cc. of a mixture of iodine 
solution, just decolorized by the addition of 40% NaOH. For 
further details consult the instructor. 
50 This test should be applied to all monosaccharides (includ- 
ing a pentose) and also to disaccharides as indicated below. 
Note: To avoid confusion, it should be remembered that brew- 
ers’ yeast contains an enzyme capable of hydrolysing certain 
disaccharides to monosaccharides. These then ferment. Pure 
yeast ferments only four monosaccharides. Which are they? 
Prove it by experiment. Conjugated glucuronic acids (occurring 
in urine after taking menthol, turpentine, etc.) reduce copper 
and bismuth solutions, but do not ferment. 
43.—Detection of a Pentose—Bial’s Orcin Test. 
Place 5 cc. of Bial’s reagent in a test tube. Heat just to 
the boiling point, then remove from flame. Now add drop by 
drop, the pentose solution. Not over 25 drops should be added. 
If pentose is present, the solution turns a beautiful green (Ham- 
marsten) or violet blue (Abderhalden). 
This test is not influenced by glucuronic acids in urine 
because the solution is not boiled. Abderhalden claims that a 
spectroscopic examination of the fluid is unnecessary. Note: 
Bial’s Reagent—Dissolve 1 gram of orcin in 500 cc. of 30% 
hydrochloric acid. Add 25 drops of a 62.9% (crystalline) ferric 
chloride solution (German “liquor ferri”). 
44.—Pentose—Other Reactions 
Repeat test 30, 31, 34, 36, 38, 40, and 41, with a 0.5% 
arabinose solution. 
45.—Detection of Fructose—Seliwanoff’s Test 
Place 5 cc. of Seliwanoff’s reagent in a test tube. Add a 
few drops of a 1% fructose solution, then heat the solution just 
to boiling. A red-colored fluid and a red precipitate indicates 
fructose. The precipitate dissolves upon the addition of alcohol. 
Note: Seliwanoff’s Reagent: Dissolve 0.05 gram of resor- 
cin in 100 cc. of concentrated hydrochloric acid. To this solu- 
tion add 100 cc. of distilled water. 
If glucose is present instead of fructose, the solution may 
color if boiled, but no precipitate is usually obtained. 
46.—Fructose—Other Reactions 
Repeat tests 30, 31, 34, 36, 38, 40, 41, 42, and 43 with a 1% 
fructose solution. 
51 POLARIMETRY 
Optical Activity 
Certain substances, among which are various solids, liquids, 
vapors and solutions, have the ability, when placed in the path 
of polarized light, of rotating the plane of polarization. These 
substances are said to be optically active. The rotation varies 
both in amount and in sense of rotation. If the rotation, relative 
to the direction of propagation, is to the right, the substance 
is said to be dextro-rotatory and a plus mark is placed in front 
of the figure indicating the amount in angular degrees. If to the 
left, the substance is laevo-rotatory and is indicated by a minus 
sign preceding the angular degrees of rotation. Substances not 
rotating plane-polarized light are said to be inactive. 
Plane-polarized light (vibrations proceeding in a single 
plane) is readily obtained by the so-called “Nicol” prism. This 
consists of a prism of Iceland spar, the natural angles of which 
are ground down until 68 and 90 degrees, respectively, are 
obtained. The rhombohedron is then cut into two 90° angles. 
After polishing, the wedges are cemented together by Canada 
balsam, thus restoring the rhombohedron. 
Mono-chromatic light is admitted through the end of the 
prism. It is refracted into two rays. The extraordinary ray 
is refracted but little and consequently emerges in a single plane 
from the opposite face of the prism. The ordinary ray is 
refracted to such an extent that upon reaching the film of Canada 
balsam cementing the two halves of the prism together, it is 
reflected to the side of the prism and completely absorbed by 
the black varnish covering its wall. 
Monochromatic light must be used with the polariscope. 
Such light is obtained by vaporizing a sodium salt in a Bunsen 
flame, and has a wave length of 0.00005893 cm. This is the 
average for the values of the two “D” lines. A mercury vapor 
lamp is often used, because the wave length of the green light 
obtained is shorter than that of the sodium light. Its value is 
0.0000546074 cm. With a mercury vapor lamp, the observed 
rotation is greater than with sodium light. This is due to the 
shorter wave length of the former. 
The polarimeter or polariscope is an instrument designed 
52 to measure the rotatory polarization of a substance. For general 
use, the rotation is measured in degrees of arc. 
The design of each polarimeter should be explained by the 
laboratory instructor. In general, the simplest polarimeter con- 
sists of two Nicol prisms—the first (near the source of light) is 
called the “polarizer” and plane-polarizes the entering light. The 
second Nicol prism is mounted in a headpiece capable of rota- 
tion around the optical axis, and is provided with a graduated 
scale and vernier. A space is left for placing the fluid to be 
examined in the path of light between the prisms. Adequate 
auxiliary lenses and other optical parts to increase the sensitive- 
ness of the apparatus are provided. (Consult any book on 
physics.) 
The field of view of a simple polariscope consisting of but 
two prisms is dark when the prisms are crossed. This is taken 
as the zero point of the apparatus. If an optically active sub- 
tance is now placed between the prisms, the beam of light is 
rotated and the field appears brighter. By rotating the analyzer 
until the light is again extinguished, one can measure the amount 
of rotation due to the optically active substance. 
To increase the sensitivity of the apparatus, the field of 
observation is divided into halves (Laurent half-shadow). A 
plate, one-half of which is glass, while the other half is quartz, 
cut parallel to its axis, is placed just back of the polarizer. The 
thickness of the quartz plate is such as to retard the sodium light 
exactly one-half wave length. This causes the two halves of the 
field to appear unequally illuminated except when the principal 
plane of the analyzing Nicol is parallel to the axis of the quartz. 
At this point the two halves appear uniformly illuminated. By 
changing the position of the polarizer with respect to the quartz 
plate the sensitiveness of the apparatus can be varied. The zero 
point is that position of uniform field illumination; the dark 
change being taken on account of greater sensitiveness. 
Triple shade polariscopes have auxiliary Nicol prisms. These 
increase the sensitiveness and ease with which the apparatus can 
be read. Other types of instruments using quartz wedges are 
described in any book on the subject. 
Many organic substances, like sugars, proteins, etc., contain 
within their structure one or more asymetric carbon atoms, i. e., 
53 a carbon atom to which is attached no two similar elements or 
groups of elements. These asymetric carbon atoms have the 
power of rotating the plane of polarized light to a greater or 
less extent, depending upon the number of molecules through 
which the polarized light passes. 
The specific rotation of a substance is the rotation expressed 
in degrees which is given by 1 gram of a pure substance dissolved 
in 1 cc. of water, and viewed through a tube one decimeter in 
length. Under these conditions, we have unity of volume, unity 
of mass, and a unit length, therefore, unity of concentration. 
20 
The specific rotation ( oc) may be calculated by means of the 
D 
following formula: 
20 a 
(oc) = 
D c x 1 
in which: 
D is sodium light. 
a is observed rotation in degrees. 
c is grams of substance dissolved in 1 cc. of fluid. 
1 is length of the tube in decimeters. 
After determining the specific rotation of any optically active 
substance we may determine either the concentration or the per- 
cent of the substance in an unknown solution. To determine 
the percentage, use the following formula: 
a x 100 
Per cent — 
20 
( oc ) x 1 
D 
The following specific rotations are taken from Cole: 
20 20 
( a ) O ) 
D Hg 
Glucose —[-52.5 +62. 
Lactose hydrate +52.4 +61.9 
Lactose anhydride +55.2 +65.2 
54 Maltose +138.0 +163.0 
Sucrose +66.5 +78.5 
Fructose —93.8 —110.8 
d-galactose +81.0 +95.7 
1-xylose +19.0 +22.4 
Invert sugar —20.6 —24.6 
47.—Determination of Specific Rotation of Cane Sugar 
Weigh out exactly 10 grams of pure cane sugar and dissolve 
in water. Transfer to a 100 cc. volumetric flask, and make to 
volume at 20° C. Polarize immediately, then set aside until the 
next laboratory period and polarize again. The second reading 
multiplied by 10 should give a figure closely agreeing with that 
given in the table if a decimeter tube is used 
After determining the specific rotation, pipette 50 cc. of the 
sugar solution into a beaker—add 0.5 cc. of acid and boil for 
fifteen minutes, supplying the water lost by evaporation, and read 
again. What is happening to the cane sugar? What is the mean- 
ing of the term “invert sugar”? 
Determine the percentage in two unknowns and report to the 
instructor. 
Add about 1 gram of glucose to 100 cc. of urine. Determine 
the percentage of glucose by means of the polariscope and com- 
pare the result with that obtained by Fehling’s or Benedict’s 
method. 
Disaccharides. 
Disaccharides should be looked upon as glucosides of mono- 
saccharides. Maltose is glucose- oc -glucoside. Lactose is glu- 
cose-yd-galactoside. If the reducing group of one component is 
free, then the disaccharides will exhibit corresponding reactions 
(lactose and maltose). If both active groups are involved in the 
condensation, the resultant molecule is relatively inert (sucrose). 
48.—Maltose—Preparation from Starch 
Mix 25 grams of starch with 30 cc. of cold water until a 
smooth paste is obtained. Pour this slowly and with stirring into 
350 cc. of boiling water in a 500 cc. beaker. Continue the boiling 
for one or two minutes until a smooth paste is obtained. Allow 
to cool to 60°-70° C. Place in a water bath at this temperature. 
55 and stir in a teaspoonful of malt. Maintain at this temperature 
until the mixture becomes thin and watery. Remove from the 
water bath, carefully drying the outside of the beaker, and heat 
to boiling again . What is the object of this heating? Boil for 
one or two minutes. Cool to 60° C. and add another teaspoonful 
of malt and keep at this temperature by means of the water bath 
until the sample no longer gives any color with iodine solution. 
Remove from the water bath, dry the beaker, boil, filter, and 
taste. Allow to cool. Pipette out 10 cc. of the solution into a 
100 cc. volumetric flask. Add water up to the mark. Make a 
quantitative determination for sugar. Remove 50 cc. of the 
solution just titrated, add 5 drops of concentrated hydrochloric 
acid and boil for fifteen minutes. Cool. Neutralize the acid 
by means of sodium hydroxide (using litmus paper) and make 
up to 50 cc. volume. Determine once more the reducing power 
of the solution and compare with that of the above. Explain. 
What is the reducing power of maltose compared with that of 
glucose? 
t 
If fresh malt is not available, this experiment may be car- 
ried out by adding 0.5 gram of Taka-Diastase (Park-Davis & 
Co.) in place of the spoonful of malt. 
49.—Maltose—Sugar Reactions 
Reactions numbered 31, 34, 38, 40, 41, 42, and 43 are to be 
performed with the maltose solution just prepared, and com- 
pared with those obtained when using other sugars. 
50.—Lactose—Preparation from Milk 
Dilute 300 cc. of skimmed milk with 500 cc. of water. Add 
acetic acid cautiously—drop by drop—with constant stirring until 
the precipitation of casein is evidenced by curd formaton. Now 
cautiously continue the addition of a 25% acetic acid solution 
until no further precipitate is formed when a drop of dilute acid 
is added to a filtered test portion. The floculent precipitate settles 
readily. An excess of acetic acid is avoided, to prevent redis- 
solving the casein. Filter the solution, catching the filtrate in a 
large beaker. Save the precipitate and place in the receptacle 
provided. 
Heat the filtrate to boiling. Coagulation of the albumin 
occurs, which, when complete, is removed from the solution by 
filtration through a plaited filter paper. Evaporate the filtrate 
56 upon the steam bath until a thin syrup is obtained. The solu- 
tion is now allowed to evaporate spontaneously until crystals of 
lactose separate out. Do not carry evaporation so far that crys- 
tals cannot be separated from the mother liquor! Separate the 
crystals by filtration (through cloth), then if desired (although 
not necessary), purify by recrystallization from water. 
Rub a few crystals between the fingers. Why is milk sugar 
sometimes called “sand sugar’’? Is milk sugar sweet? Taste 
it. With the remainder of the preparation, make a solution of 
approximately 1 per cent. 
51.—Lactose—Sugar Reactions 
Reactions numbered 31, 34, 38, 40, 41, 42, and 43 are to be 
performed with the solution prepared in the last experiment, and 
compared with those obtained when using other sugars. 
52.—Lactose1—Mucic Acid Test 
Measure 100 cc. of 1 per cent solution of lactose into a suit- 
able evaporating dish. Add 20 cc. of concentrated nitric acid, 
and place on the steam bath. Note the brown fumes. The lac- 
tose is oxidized to mucic acid, COOH-(CHOH)4-COOH. 
After the reaction is complete, continue the evaporation 
until a final volume of 20 cc. remain. Transfer the solution to a 
small beaker, rinsing the evaporating dish with a small quantity 
of distilled water. If the beaker is put in a cool place for 24 
hours, white crystals of mucic acid should appear. Add a little 
water to the beaker, then separate the crystals from the mother 
liquor by filtration. Wash and examine under the microscope. 
The crystals should be sharply pointed prisms with oblique angles. 
Melting point, 213°-215° C. 
Note—The mucic acid test is not dependable enough for use 
when distinguishing between lactose and glucose in urine. 
53.—Lactose in Urine—Cole’s Test 
To 1 gram of good charcoal (Merck’s purified blood char- 
coal, washed with hot hydrochloric acid until free from phos- 
phates, then washed free from chlorides) in a large test tube, 
add 25 cc. of the suspected urine. The contents of the tube 
should be mixed by shaking, boiled for a few seconds, cooled 
thoroughly under the tap, and vigorously shaken for 10 minutes. 
The material is filtered by suction, and the charcoal drained as 
thoroughly as possible. Transfer the charcoal to a small evapor- 
57 ating dish, freeing it from the filter paper, then add 10 cc. of dis- 
tilled water and 1 cc. of glacial acetic acid. Stir the charcoal with 
a glass rod and then transfer the mixture to a large test tube. 
Heat to boiling for about 10 seconds, filter while hot, collecting 
the filtrate in a test tube containuing 0.2 gram of purified phenyl- 
hydrazine hydrochloride and 0.3 gram of sodium acetate. 
Proceed to form the osazone as directed in experiment 38. 
Identify the crystals. If necessary, recrystallize the osazone by 
dissolving the precipitate in 4 to 5 cc. of hot water, filtering sev- 
eral times through a small paper, then cool. The solution must 
be heated after each filtration to prevent the crystals from form- 
ing in the paper during filtration. 
Note: The lactose is absorbed by the blood charcoal. Each 
charcoal should be carefully tested with solutions of known lac- 
tose content before being considered suitable for the above test. 
Mathews distinguishes between lactose and glucose by their 
respective fermentability with ordinary yeast. He first estimates 
the total reducing power of a solution or urine by Benedict’s 
method. Then one-half cake of yeast is shaken in a large tube 
with 20 cc. of urine or solution until uniformly mixed. If glu- 
cose is present, it will be completely fermented in 50 minutes, 
providing its concentration is not over 6 per cent, if the tube is 
placed in a beaker of water maintained at 42° C., and the yeast 
kept in suspension by inverting the tube at frequent intervals. 
The unfermented lactose is then determined in the solution after 
filtration, by Benedict’s method. Subtracting this value from 
that previously obtained gives the percentage of glucose. The 
lactose may be still further identified by preparation of its osa- 
zone. 
54.—Sucrose—(Cane Sugar)—Sugar Reactions 
Reactions numbered 31, 34, 38, 40, 41, 42, and 43 are to 
be performed with a 1% sucrose solution and contrasted with 
those obtained when using other sugars. To what is the differ- 
ence in reactivity due? Review experiment 47 at this time. 
55.—Sucrose—Hydrolysis or “Inversion” 
To 3 cc. of a 1% sucrose solution add 2 or 3 drops of con- 
centrated hydrochloric acid. Boil for a few seconds. Cool 
under the tap. To the solution add y2 cc. of 20% copper sul- 
phate, 3 drops of glycerol, and 3 cc. of 20% sodium hydroxide. 
58 Boil. Explain the reduction. What is the glycerol added for? 
56.—Problem 
Identify the sugar or sugars present in two unknowns. 
Report your results to the instructor, stating reasons for your 
opinion as to the identity of the sugars. 
Polysaccharides 
Polysaccharides are formed by condensation from mono- 
saccharide molecules (water being eliminated). 
Two groups are commonly encountered, namely: 
1. The Pentosans (C5H804)n which, upon hydrolysis, yield 
the pentoses. Example of this group are various gums, as gum 
arabic, and cherry tree gum. 
2. The Hexosans (C6H10O5)n which, upon hydrolysis, 
yield glucose. Starch, cellulose, dextrin and glycogen are repre- 
sentatives of this class. 
57.—Starch—Microscopic Appearance and Solubility 
Sketch the granules of the following starches: Potato, corn, 
wheat, and rice. Note their shape, the concentric markings, if 
any, and the hilum. Add a drop of dilute iodine solution to one 
of the varieties and re-examine. Record the result. 
Solubility of starch: Add a little powdered starch to cold 
water. Filter, and add to the filtrate a drop of iodine solution. 
What are your conclusions ? 
Gelatinization (Cole) : Shake some dry starch with a little 
20% sodium carbonate solution. No change is effected. Repeat, 
substituting 20% sodium hydroxide. Note the appearance; add 
a few drops of iodine solution. Why do you not obtain a color? 
Add glacial acetic acid. What happens? Why? 
58.—Preparation of a Starch Paste 
(a) Amylopectin or starch cellulose, forming about 60% of 
the grain, serves to enclose it. It is insoluble in cold water, swells 
(imbibition) without dissolving in hot water. A similar gelatin- 
ization occurs when starch is treated with cold sodium hydroxide. 
While acids hydrolyse amylopectin to glucose, amylases 
(diastases) convert it into maltose and a dextrin which is very 
resistant to further hydrolysis. The opalescence of a starch 
paste is due to amylopectin'. 
Note: Starch consists of two substances— 
59 (b) Amylose, or granulose, the material within the grain, 
is protected by the external layer of amylopectin. Amylose is 
soluble in cold water, is completely hydrolysed to maltose by 
amylases. It gives the characteristic blue color when treated with 
iodine solution. 
Starch Paste: Rub 2 grams of starch to a thin paste with 
a little water and pour the mixture slowly with constant stirring 
into 200 cc. of boiling water. Use the paste so obtained in the 
following experiments. 
59.—Starch—Properties and Precipitation Reactions 
(a) To some of the starch paste in a test tube add a drop 
of dilute iodine solution. Note the result. Place the tube in a 
beaker of water and heat gradually. What change takes place? 
Allow the solution to cool and observe the result. 
(b) Add a few drops of a solution of tannic acid to a few 
cc. of the starch solution. A yellow precipitate, soluble upon the 
application of heat, is obtained. 
(c) To a few cubic centimeters of starch paste in the test 
tube, add alcohol until a definite precipitation occurs. 
(d) To a few cubic centimeters of cold starch paste, add 
an equal volume of saturated ammonium sulphate solution. 
Shake the tube, allow to stand for 5 minutes, then filter. Test 
the precipitate and filtrate with dilute iodine solution. Is the 
precipitation complete? 
60.—Starch—Hydrolysis by Acid and Enzyme 
(a) Test a portion of starch solution with Fehling’s solu- 
tion and record the result. 
(b) To 25 cc. of starch solution in a flask add 4 or 5 drops 
of concentrated hydrochloric acid, or a few drops of dilute sul- 
phuric acid, and boil for fifteen minutes. What change do you 
observe in the appearance of the starch solution? Neutralize 
some of this solution with sodium hydroxide solution, and apply 
Fehling’s test. Report the result. (If no reduction takes place, 
continue the boiling for another fifteen minutes and repeat the 
test.) What sugar is formed? 
(c) To 5 cc. of starch solution in a test tube, add 3 cc. of 
saliva and place in a water bath at 40° C. After twenty minutes, 
test for reducing sugars by Fehling’s and by Barfoed's tests. 
What variety of sugar do you find present? 
60 61.—Dextrin—from Starch 
The two following procedures, together with the note, are 
taken from Cole’s Physiological Chemistry. 
(a) Erythro-dextrin: Measure 2 cc. of starch paste into a 
test tube, add 6 drops of concentrated hydrochloric acid from a 
pipette, then heat to boiling and maintain the boiling for one 
minute by the watch. Cool thoroughly under the tap. Add first 
one drop, and then another drop of dilute iodine solution. A red 
or violet color is produced, indicating the conversion of starch 
into erythro-dextrin by acid hydrolysis. 
(b) Reduction test: (Probably due to the glucose formed 
simultaneously with erythro-dextrin.) Boil 2 cc. of starch paste 
with 6 drops of concentrated hydrochloric acid as before. Cool. 
Add 3 drops of glycerol and 8 drops of 20% copper sulphate. 
Add 20% sodium hydroxide until a grey precipitate is produced. 
Now add another 2 cc. of the sodium hydroxide and boil for a 
minute. A slight reduction is obtained. 
Note: If 12 drops of hydrochloric acid be added and the 
mixture boiled for 1 minute, it will generally be found that only 
a yellow color is produced with iodine and that the amount of 
glucose formed is not sufficient to reduce 9 drops of copper 
sulphate. At this stage a considerable proportion of the carbohy- 
drate is in the form of achroo-dextrin. It is important to note 
that complete hydrolysis of starch by acids is relatively slow 
compared to that of sucrose and the other disaccharides. 
62.—Dextrin—Properties 
Using a 1% aqueous solution of dextrin, perform the fol- 
lowing tests: No. 59 b, c, d, and No. 60 a, b, c. Contrast in 
each case with starch. 
Glycogen 
Glycogen is so resistant to the action of boiling alkali that 
this reagent may be used for its preparation from muscle or 
liver tissue. Pure glycogen is colorless, dissolves in water to an 
opalescent solution. It may be precipitated from its solution by 
addition of an equal volume of 95% alcohol or by completely 
saturating its solution with (solid) ammonium sulphate. 
It is relatively inactive, as indicated by the absence of reduc- 
ing power. It gives a characteristic port-wine color with dilute 
61 iodine solution. Yeast cannot ferment glycogen, while diastatic 
enzymes, except those occurring in the liver, hydrolyse is to 
maltose. The glycogenase of the liver hydrolyses it to glucose. 
Its solution is dextrototatory. 
63.—Glycogen—Preparation from Animal Tissue 
Oysters, scollops, or the liver of a recently killed animal 
serves well as material for this preparation. If dogs are avail- 
able, and especially if needed for other work, their livers may 
be used most satisfactorily, especially if the animals are fed upon 
boiled potatoes about six hours before being killed. 
Each student should obtain 30 grams of fresh material, and 
immediately, after grinding as finely as possible, drop it into a 
flask containing 200 cc. of boiling water, which is slightly acidi- 
fied with acetic acid. As soon as the protein has coagulated, the 
material is filtered off, using a plug of cotton in the funnel, and 
catching the filtrate in a flask. Place the residue in a mortar, 
and after adding coarse sand, grind thoroughly. Return the 
material to the filtrate, and boil for a few minutes. Filter hot 
through a cloth or plug of cotton in a funnel, collecting the 
opalescent fluid in a flask. Press the fluid out of the residue as 
thoroughly as possible, and rinse with a little water. 
Evaporate the filtrate to a volume of 75 to 100 cc. Add to 
the filtrate alternately a few drops of hydrochloric acid and 
potassio-mercuric iodide until a precipitate of proteins ceases to 
form. This may be determined most conveniently by filtering off 
a small portion of the liquid from time to time, and adding to 
the clear filtrate a drop of hydrochloric acid and a drop of potas- 
sio-mercuric iodide. When the precipitation of proteins is com- 
plete, filter and to the milky filtrate add double its volume of 
alcohol; the glycogen will precipitate as a white powder. Filter 
this off, wash with 66% alcohol and dissolve in water. With 
this solution perform the following tests. 
64.—Glycogen : Properties—Reactions—Digestions— 
Hydrolysis 
(a) Add a drop of iodine solution to a portion of the gly- 
cogen solution. The liquid will assume a dark red color. This 
color disappears, with the exception of the color due to iodine, 
upon gentle heating, and reappears upon cooling. (Compare 
with dextrin.) 
62 (b) Test with Fehling’s solution and report the result. 
(c) Add basic lead acetate to some of the solution. (Com- 
pare with dextrin.) 
(d) Add some saliva to portions of the glycogen solution 
and heat the mixture for twenty minutes in a water bath at 40° C. 
Remove and divide into two portions. Test one with iodine solu- 
tion for glycogen and the other with Fehling’s solution for sugar. 
Report the result. 
(e) Boil a portion of the glycogen solution with a few 
drops of dilute sulphuric acid. Neutralize with sodium hydroxide 
and test with Fehling’s solution. Report the result. 
63 COLLOIDS 
Graham in 1861 showed that potassium hydroxide, potas- 
sium sulphate, magnesium sulphate, sugar and alcohol diffuse 
more rapidly in water than hydrous silicic acid, hydrous alumina, 
starch, dextrin, gums, albumin, and gelatin. The latter sub- 
stances usually occur in non-crystalline or gelatinous condition, 
and therefore, as they resembled glue in this particular, were 
called colloids by him. Substances of the first group, crystallize 
readily in a definite way, therefore those substances capable of 
ready diffusion usually resemble them in their ability to crystal- 
lize and were grouped together as a class called crystalloids. 
Today the distinction between colloids and crystalloids indi- 
cated above has been largely dropped. We now speak of a col- 
loidal state instead of a colloidal substance. This indicates that 
any substance in a sufficiently fine state of subdivision or “dis- 
persion,” as it is called, may constitute one phase of a colloidal 
system. The subdivided phase is called the internal or dispersed 
phase; while the enveloping phase is called the external or con- 
tinuous phase. 
The student should consult the following texts for further 
information upon this subject: Holmes—Laboratory Manual 
of Colloidal Chemistry; Bancroft—Applied Colloidal Chemistry; 
and Loeb—Proteins and the Theory of Colloidal Behavior. 
65.—Preparation of a Mastic Suspension 
Prepare a saturated alcoholic solution of gum mastic. To 
a beaker containing 100 cc. of distilled water add 1 cc. of the 
mastic solution, drop by drop, with stirring. Filter off the 
coursely suspended particles. The suspension is electro-negative 
in character. To demonstrate the effect of electric charges of 
opposite as well as like nature, perform the following experi- 
ment : 
Place 10 cc. of mastic suspension in each of four test 
tubes. Number them 1, 2, 3, and 4. 
To 1 add 5 cc. N/10 HC1. 
To 2 add 5 cc. N/10 NaOH. 
To 3 add 5 cc. 6% NaCl. 
To 4 add 5 cc. 10% sugar solution. -f- 
1. Hydrochloric acid is abundant in positive (H) ions. 
64 These have what effect upon the negatively charged mastic solu- 
tion? 
2. Sodium hydroxide is characterized by the negative 
(OH) ions. Note the stabilizing effect of the ion with like 
charge. Now add a 6% sodium chloride solution to this solu- 
tion. Note the quantity added and compare with the result 
obtained in tube “3.” 
3. Sodium chloride dissociates into sodium and chlorine 
ions. Which ion is responsible for the effect observed? 
4. Sugar is a non-electrolyte. What is the nature of the 
precipitations observed above? 
66.—Arsenic Sulphide Suspension 
If hydrogen sulphide be passed into a solution of arsenious 
oxide, dissolved by the aid of sodium carbonate, a beautiful 
orange-colored solution of colloidal arsenic sulphide is obtained. 
This is also an electro-negative suspensoid. 
With a solution of colloidal arsenic sulphide repeat the 
experiments given under No. 65. 
67.—Colloidal Iron—“Dialysed Iron” 
If a solution of ferric chloride be dialysed against distilled 
water, a slow decomposition occurs whereby a part of the 
chloride is changed over to ferric hydroxide. This mixture of 
ferric chloride and ferric hydroxide is known as “dialysed iron.” 
It is electro-positive in nature. 
Repeat experiment No. 65 using dialysed iron. Add the 
sodium chloride to tube No. 1, instead of tube No. 2 as directed. 
Explain fully the results obtained. 
68.—Emulsion Colloids 
Many proteins, gums, starches, etc., go into a colloidal state 
in solution. Only those properties exhibited by gelatin and casein 
can be considered here. The student should carefully consider 
the presence or absence of an electric charge, as well as its 
character in all cases where emulsoids are encountered. See 
Loeb—Proteins and the Theory of Colloidal Behavior. 
69.—Geletin—Swelling or “Imbibition’ 
Place 5 grams of gelatin in a beaker and just cover with 
65 water. Note what happens upon standing. Explain. Save the 
material for the next experiment. 
70.—Reversibility of the “Sol” and “Gel” State 
To the gelatin in the beaker in experiment No. 68, add just 
enough water to get it into solution when heated in a boiling 
water bath. This represents the sol state. 
Remove the gelatin from the water bath and allow the beaker 
to cool. What do you observe? What is the relation of gelatin 
to water in the gel phase? Explain. Save the material. 
71.—Rfversibilf and Irreversible Colloids 
To the gelatin solution used in experiment No. 70, add suf- 
ficient warm water to make a total volume of 100 cc. Use when 
required in experiments below. 
(a) Measure 10 cc. of this solution into an evaporating 
dish and place on the steam bath. Evaporate to dryness. Add 
water and determine whether the gelatin returns to its former 
state of solution. (Reversible Colloid.) 
(b) Measure 10 cc. of arsenic sulphide solution into an 
evaporating dish and add water and compare the solubility of 
the residue with that of the gelatin. (Irreversibility.) 
72.—Salting out of Emulsoid 
To 25 cc. of a 2% solution of egg albumin, add solid ammon- 
ium sulphate until the solution is completely saturated. What 
happens? Filter off the precipitate. Add distilled water. Is it 
possible to redissolve the albumin? What is the difference 
between the precipitation here and that occurring in tube No. 3, 
experiment No. 65? 
Iso-Electric Point of Emulsoids 
Many proteins may be dissolved in dilute acid and in dilute 
alkali solutions. In the former they adsorb the hydrogen ion 
and become positively charged. In the latter the adsorption of 
hydroxyl ions imparts a negative charge to them. There is, 
however, a definite point of reaction for each colloid, attained 
by the adsorption of the proper amount of positive or negative 
ions to render the colloid neutral or without charge. The reac- 
tion of the solution at this time indicates what is known as the 
iso-electric point. This is the point (or reaction) at which pre- 
cipitation occurs. 
66 73.—Determination of Iso-Electric Point of Casein (Cole) 
Into a 50 cc. measuring flask place 0.3 gm. of purified casein. 
Add about 25 cc. of distilled water, previously warmed to about 
40° C. Add exactly 5 cc. of normal sodium hydroxide. Agitate 
until the casein dissolves, taking care to prevent frothing. Rapid- 
ly add 5 cc. of normal acetic acid, mix, cool, and make up to 50 
cc. with distilled water. A faintly opalescent solution of casein 
in 0.1 N. sodium acetate is thus obtained. 
Make up the following series of tubes, using clean, dry test 
tubes: 
Tube number 123456789 
-Ccm. casein in 0.1 
N. sodium acetate 111111111 
Ccm. dist. water 8.38 7.75 8.75 8.50 &00 7.00 5.00 1.00 7.40 
Ccm. 0.01 N. 
acetic acid 0.62 1.25 
Ccm. 0.1 N. 
acetic acid 0.25 0.50 1.00 2.00 4.00 8.00 .. . 
Ccm. normal acetic 
acid 1-60 
Place the casein solution in the tubes first, then the water, 
and mix. Now add the acetic acid to the first tube and shake 
immediately. Then add the acid to the second tube and shake 
this, and so on. Examine the tubes at intervals and record 
observations as below. 
0=no change. -)-=opalescence. X—precipitate. 
Tube No. 
1 
2 
3 
4 
5 
6 
7 
8 
9 
On mixing 
After 10 minutes 
After 20 minutes 
H-ion conc’n 
pH value 
(Indicate intensity by one or more characters) 
67 Calculate the approximate hydrogen ion concentration for 
each solution from the ratio of concentration of acetic acid and 
sodium acetate molecules as indicated in the following formula: 
K x (acetic acid in mols. per liter) 
ax (sodium acetate in mols. per liter) 
H is Hydrogen ions in grams per liter. 
K is dissociation constant of acetic acid, 
is 1.85X10-5 
a is dissociation constant of sodium acetate, 
is 0.87 for 0.01 N. 
Example: Tube No. 1. 
0.62 cc. acetic acid of N x 10~2 is diluted to 10 cc. There- 
fore the concentration is 0.62 x 10~3. Put this value in the 
equation: 
(1.85 x 10-5) x (0.62 x 10-3) 
H= 0.87 x 10-2 
H = 1.32 x 10~6 
To convert hydrogen ion concentration to the hydrogen ion 
exponent proceed as directed on page 21. Fill both values into 
the table above. Indicate clearly what the iso-electric point of 
casein is. 
74.—The Influence of the Charge and Valence upon Pre- 
cipitation (Cole) 
Prepare a solution of casein in 0.1 N. sodium acetate as indi- 
cated in the last experiment. 
To 2 cc. add 17.5 cc. of distilled water, and then 0.5 cc. of 
0.1 N. acetic acid and mix quickly. A solution of casein is thus 
obtained, alkaline to the iso-electric point, and therefore carry- 
ing a negative charge. Divide the solution into four equal parts 
and place them into four clean tubes labelled -1, -2, -3, and -4. 
To another 2 cc. of the original solution of casein add 10 cc. of 
distilled water and 8 cc. of 0.1 N. acetic acid and mix quickly. 
An acid solution of casein is thus obtained. Divide into four 
parts, and place into four clean tubes labelled +1, +2, -[-3, 
and -f-4. 
68 To the tubes marked 1 add 3 drops of normal KC1 (7.45%). 
To the tubes marked 2 add 1 drop of normal BaCl2 (10.40 
per cent). 
To the tubes marked 3 add 1 drop of normal KoS04 (8.7%). 
Mix the contents of each tube and place them all in a water 
bath at 35°C. Examine and record results after 15 minutes, 
using the same characters as in the previous experiment. 
Tube 
Negative 
Positive 
1. 
2. 
3. 
4. 
Warm the tubes to 60° C. to still further increase the effect 
of the precipitating ion. 
Study carefully the effect of charge on emulsoid, together 
with the charge and valence of precipitating ion. Distinguish 
clearly the difference between the results obtained here and those 
in experiment No. 72. 
Write up your results fully, mentioning each tube. 
75.—Mutual Precipitation Suspensoids of Opposite Charge 
To 5 cc. of colloidal arsenic sulphide in a test tube add 
5 cc. of dialysed iron solution. What happens? Explain. 
76.—Protective Action of an Emulsoid for a Suspensoid 
(a) To 5 cc. of colloidal arsenic sulphide solution add 10 
drops of a 1% gelatin solution. Now add 6% sodium chloride 
solution, noting amount required to cause precipitation. Com- 
pare with experiment No. 66. Explain fully. 
(b) Place 10 cc. of colloidal gold solution (prepared as 
for use in the Lange test) in each of two small evaporating 
dishes. Mark one G, and to this add 1 cc. of 1% gelatin. Evapo- 
69 rate both solutions to dryness. Notice any difference in color. 
The courser particles of gold have a blue color. Add 10 cc. of 
distilled water to each and try to re-dissolve. Can you get the 
original solution back in either case? Why? Explain fully. 
77.—Decrease in Agglomeration of Colloidal Precipitate by 
Strongly Absorbed Substance 
Place 10 cc. of milk in each of two small beakers. Label 
them 1 and 2. To beaker 1 add 25 cc. of water. To beaker 2 
add 25 cc. of 20% cane sugar solution. Add 10% acetic acid 
drop by drop to each, stirring vigorously, until first evidence of 
coagulation is noticed. Record the amount in each case. Is it 
different? Now notice the relative degree of fineness of the 
curds. Allow each beaker to stand for 5 minutes, then re-ex- 
amine. Do you notice any change? 
70 Mulder in 1839 signified the importance of the substance 
of which flesh and all living cellular material is composed, by 
applying to it the term protein (from the Greek meaning “pre- 
eminence” or “of first importance”). This material; either of 
animal or plant origin; when carefully freed from fats, car- 
bohydrates, extractives, and mineral salts, has a fairly uniform 
composition. It always contains the four essential elements, 
carbon, nitrogen, hydrogen and oxygen, and often in addition, 
smaller amounts of sulphur and phosphorus. In certain pro- 
teins, iron, copper or iodine in small quantities may be present. 
The uniformity of composition of protein, especially in 
respect to the four named elements is striking. For the pur- 
pose of illustration, the following figures give some idea of 
the general composition of protein material as a class: Carbon, 
50-52%; hydrogen, about 7%; nitrogen, 15-16%; sulphur, 0- 
3%; phosphorus, 0-3%, and 23-25% oxygen. 
The colloidal nature of many proteins when in solution 
is clearly indicated in the precipitation reactions given below. 
The student should clearly distinguish between the phenomena 
of coagulation, precipitation, and “salting out” of proteins from 
their solution. The latter term is used here to indicate an ag- 
glomeration of the protein material without adsorption, and is 
to be contrasted with precipitation. 
Proteins are composed of amino acids, linked together from 
amino to carboxyl group. The proteins may be unlinked by 
the action of certain proteolytic enzymes, as well as by acid and 
alkali hydrolysis. Fragments of the protein molecule are 
formed, varying in size and nature. The completeness of dis- 
ruption of the molecule by an enzyme depends upon three fac- 
tors : the nature of the protein; the particular enzyme used; 
and finally upon the length of time through which it acts. The 
rate of decomposition of proteins by acids depends upon the 
strength of the latter, and the completeness upon the time of 
action. Other factors such as temperature also effect the rate 
of the hydrolysis. 
The color reactions, with the exception of the biuret test, 
indicate either the presence of a specific amino acid in the 
PROTEINS 
71 molecule, or of a very limited number of amino acids which 
possess certain structural resemblance. The student is cautioned 
not to report the presence of protein in his solutions upon the 
basis of a single color reaction. 
The study of protein chemistry is much simplified if the 
protein classification is kept clearly in mind.' For that purpose 
it is given below in abbreviated form: 
Protein Classification According to the American Society of 
Biochemists 
Three classes are recognized. Several subdivisions occur 
under each class. 
I. Simple Proteins. Naturally occurring proteins. Yield only 
alpha amino acid or their derivatives upon hydrolysis. 
Solubility in 
Coagulability 
Name 
Water 
Dil. Salt 
Dil. Acid 
Dil. Alkali 
Alcohol 
Heat 
Example 
Albumins 
+ 
+ 
+ 
Egg 
Albumin 
+ 
Serum 
Globulin 
+ 
globulin, 
Edestin. 
Glutelins 
— 
— 
+ 
+ 
+ 
Glutenin 
Gliadin, 
Prolamines 
80% 
Hordein, 
Zein. 
Elastin, 
Albuminoids 
— 
— 
— 
— 
Keratin, 
Insoluble 
Collagen. 
in 
Histones 
Histones 
+ 
+ 
Ammonia 
of Bird’s 
Corpuscle 
Soluble 
Sturine, 
in 
— 
Salmin, 
Protamines 
Ammonia 
Clupein. 
Note: Histones and protamines are basic in nature. Histones have 
less basic nitrogen than the protamines, and usually contain sulphur. 
72 IT. Conjugated Proteins.—Compounds of simple proteins and 
non-protein group. The non-protein group is usually acid 
in nature. 
A. Chromoproteins or Hemoglobins: Compounds of pro- 
tein and a pigment bearing molecule. Example: 
Hemoglobin, hemocyanin, etc. 
B. Glycoproteins (glucoproteins) : Compounds of pro- 
tein and a group which contains a carbohydrate 
molecule. Example: mucin. 
C. Phosphoproteins: Compounds of protein and a group 
containing phosphoric acid other than nucleic acid. 
Examples: Casein and vitellin. 
D. Nucleoproteins: Compounds of protein and a nucleic 
acid. (Note: Although these proteins contain phos- 
phorus, its combination is different from that of the 
phosphoproteins. In the same way, the carbohydrate 
molecule present here is in different combination 
from that of the glycoproteins.) Examples: Nu- 
clein; nucleohistone; (the chromatin material). 
III. Derived Proteins.—Members of this group are products 
of enzyme action or the resultant of chemical or physical 
changes upon native proteins. This accounts for the modi- 
fication of their properties. 
A. Primary Derizred Proteins.—Those products which are 
modified the least. 
a. Proteans.—The first products formed when the pro- 
tein is subject to the action of enzymes, acids, 
or in some cases, water. They are insoluble in 
water. Examples: Edestan, myosan. 
b. Metaproteins.—Products obtained by a little longer 
continued action of acid or alkalies. Properties: 
weakly acid or basic; insoluble in water. Ex- 
amples are acid or alkali albuminate. 
c. Coagulated proteins.—Proteins rendered insoluble 
by the action of alcohol or heat. Examples: Egg 
albumin to which alcohol has been added—or 
heat coagulated. 
73 1). Secondary Derived Protein.—Highly modified. 
a. Proteoses.—Hydrolytic cleavage products of pro- 
teins. They are soluble in water. Non-coag- 
ulable by heat. Precipitahle when their solutions 
are completely saturated with ammonium sul- 
phate. 
b. Peptones.—Hydrolytic cleavage products of pro- 
teins. Soluble in water. Non-coagulable by 
heat. Not precipitahle by saturating their solu- 
tions with ammonium sulphate. Usually diffu- 
sible—give biuret reaction. 
c. Peptides.—Compounds of amino acids of known 
composition. May or may not give a positive 
biuret test. Not coagulable by heat. 
78.—Elementary Composition of Proteins 
Detection of Nitrogen hy Lassajigne’s Method. Also Test 
for Sulphur and Phosphorus. 
(a.) Place a little dry powdered protein (a mixture of 
casein and dry egg albumin) into a dry cheap test tube. Add a 
piece of metallic sodium the size of a pea. Then gradually heat 
to redness, maintaining at this temperature for a short time. Al- 
low to cool. The heated end of the tube is broken into an evap- 
orating dish containing 2 to 3 cc. of water. Cover immediately 
with a moist filter paper. After five minutes cautiously add 25 
cc. of water. Stir well and filter the solution into a test tube. 
Divide the filtrate into three portions. This solution contains 
sodium cyanide, sodium sulphide and a sodium phosphorus com- 
pound, together with some sodic hydrate. 
(b.) To test for nitrogen add to one portion a few drops 
of a fresh ferrous sulphate solution and then a drop or two of 
ferric chloride solution. Acidify slightly with hydrochloric acid. 
The hydrochloric acid dissolves the precipitated ferrous and fer- 
ric hydroxides. If sodium cyanide is present there remains a 
blue precipitate, due to the formation of Prussian Blue. 
(c). Test another portion for phosphorus in the follow- 
ing manner: Acidify a small portion with nitric acid and add 
twice its volume of ammonium molybdate solution (NH4),Mo04. 
74 Heat gently for a minute. Allow the solution to stand if no pre- 
cipitate forms at once. The yellow precipitate is ammonium 
phospho-molybdate of a complicated and somewhat variable 
composition. (Approximately (NH^gPO^lOMoOg). 
(d.) To another portion test for the presence of sulphur 
by adding 5 drops of concentrated sulphuric acid to the liquid 
the test tube (or sufficient to make it strongly acid), and sus- 
pend a piece of filter paper previously moistened with lead ace- 
tate solution over the mouth of the tube, heat gently if neces- 
sary. The lead acetate turns dark. This is due to the formation 
of lead sulphide by the hydrogen sulphide, which is liberated 
from the liquid in the test tube. 
Sulphur in Protein 
Sulphur is believed to be present in two different forms in 
the protein molecule. The first form, which is present in great- 
est amount, is loosely combined with carbon and hydrogen. Sul- 
phur in this form is variously termed “unoxidized, loosely com- 
bined, mercapton, and lead blackening sulphur.” The second 
form is combined in a more stable manner with carbon and oxy- 
gen and is known as oxidized or acid sulphur. The protamines 
are the only class of sulphur-free proteins. 
Test for Loosely Combined Sulphur 
(e.) Put a little protein (wool) into a test tube with a 
few cc. of strong sodium hydrate solution. Add 2 or 3 drops 
of lead acetate solution and boil. Loosely combined sulphur is 
indicated by a darkening of the solution, the color deepening to 
black if sufficient sulphur is present. Acidify the solution with 
sulphuric acid and note the characteristic odor which is evolved 
from the solution. Explain the chemistry of this reaction. 
Test for Total Sulphur in the Molecule 
(f.) Boil some protein with 10 cc. of strong nitric acid in 
an evaporating dish in a hood, taking care not to char the or- 
ganic matter. When a clear solution is obtained add 10 cc. of 
strong hydrochloric acid and evaporate to one-third of its vol- 
ume. Add 25 cc. of water and test the solution for sulphates 
by means of barium chloride and for phosphorus as in the above 
experiment, (c). 
75 (g.) Test protein for carbon and hydrogen, using the 
methods employed under carbohydrates. 
Note: The protein color reactions and precipitation tests 
can best be illustrated in the laboratory by the use of a dilute 
solution of egg albumin. As egg white contains about 12% 
of albumin, it is best to dilute it with five volumes of distilled 
water. The egg white should 'be thoroughly agitated with the 
water, preferably by shaking in a corked bottle until uniform. 
It must be filtered through a plug of absorbent cotton until clear 
or at least nearly so. The filtrate is then suitable for use. 
79.—Color Reactions 
A. Millons Reaction: 
Place 5 cc. of egg albumin solution in a test tube and add 
a few drops of Millon's reagent. A white precipitate forms, 
which upon heating should turn red. 
Note: The color reaction is due to the presence of the 
hydroxyl-phenyl group (C6H4OH) in the protein molecule. Cer- 
tain non-proteins (compounds) such as tyrosine, carbolic acid 
and thymol respond to this reaction. Inasmuch as tyrosine con- 
tains the. only hydroxyl-phenyl group which has been positively 
proved to be present in the protein molecule, it (is evident that the 
proteins which respond to Millon’s reaction must contain this 
tyrosine complex. On account of the nature of the reagent this 
test is extremely unsatisfactory in the presence of certain inor- 
ganic salts, e.g. sodium chloride. 
B. Xanthoproteic Reaction: 
To 2 to 3 cc. of egg albumin solution in a test tube add con- 
centrated nitric acid. A white precipitate forms, which upon 
heating turns yellow and finally dissolves, imparting a yellow 
color to the solution. Cool the solution and carefully add an 
excess of ammonium or sodium hydroxide. The color deepens 
into an orange. 
(Note: This reaction is due to the presence in the protein 
molecule of the phenyl group which, with nitric acid, forms cer- 
tain nitro derivatives. The complexes in the protein molecule 
responsible for this reaction are tyrosine, phenvlaline and trypto- 
phane.) 
76 C. Hopkins-Cole Reaction: 
To 1 or 2 cc. of egg albumin solution in a test tube add 3 
cc. of glyoxylic acid solution (CHO-COOH-H20). Carefully 
pour 5 cc. of concentrated sulphuric act’d down the side of the 
inclined test tube At the junction of the two liquids a reddish- 
violet color is formed. This is due to the presence of the tryp- 
tophane group. This test is not given in the presence of oxi- 
dizing agents. 
(Note: Glyoxylic acid or “reduced oxalic acid” is pre- 
pared by treating half a liter of a saturated solution of oxalic 
acid with 40 grams of 2% sodium amalgam in a tall cylinder. 
When all the hydrogen has been evolved, the solution contains 
oxalic acid, sodium binoxalate and glyoxylic acid. It is kept 
in a closed bottle containing chloroform. Benedict recommends 
reducing the oxalic acid by means of powdered magnesium. 10 
grams of powdered magnesium are suspended in 100-150 cc. of 
water in a lj4 liter pyrex flask. Have a large dish of cold water 
available in which the flask may be placed for cooling. Now 
add 250 cc. cold saturated oxalic acid; slowly, and while rotating 
the flask. Keep the temperature down by setting the flask into 
the cold water. When the reaction is complete, filter of¥ the 
precipitate of magnesium oxalate; acidify the filtrate with acetic 
acid and dilute with distilled water to 1 liter. Preserve as indi- 
cated above.) 
D. Biuret Test: 
To 2 to 3 cc. of egg albumin solution in a test tube, add 
an equal volume of concentrated (40%) potassium or sodium 
hydroxide solution, mix thoroughly, and add, drop by drop, a 
very dilute (1%) copper sulphate solution, until a purplish-vio- 
let or pinkish-violet color is produced. 
(Note: The depth of the color depends upon the nature 
of the protein. Proteoses and peptones give a decided pink 
color, while gelatin gives a color which is not far removed from 
blue. All substances containing any of the following amino 
groups: -CONH2: -CSNHa -C(NH)NH2; or -CH2NH2; re- 
peated at least twice in the molecule directly linked together, or 
77 when linked by means of a third carbon atom or nitrogen atom 
will give the biuret test. This test is given by such compounds 
as oxamide, malonamide and biuret, representing some of the 
types of linkages referred to above.) 
The biuret reaction alone is therefore no proof as to the 
protein nature of a substance. Even substances like urobilin 
give a color hardly distinguishable from that given by proteins 
with this test. Proteins can be modified by treatment with 
nitrous acid, and rendered incapable of giving the reaction. 
No protein reaction is in itself characteristic, and there- 
fore, in testing for proteins, one reaction is not sufficient. A 
number of precipitation tests and color reactions must be em- 
ployed. 
Repeat the biuret test, after adding to the albumin solution 
some ammonium sulphate. How is the color modified? Repeat 
the test with the ammonium sulphate, adding a large excess of 
40% alkali. What is the practical bearing of this experiment? 
E. Liebermann's Reaction: 
Place 5 cc. of concentrated hydrochloric acid in a test tube, 
and add a little dry egg albumin or a small pjece of the white 
of a hard boiled egg. Boil the mixture until a pinkish-violet 
color results. If the protein is boiled with alcohol, then washed 
with ether previous to making the test, a deeper blue color is 
obtained. This test is indicative of tryptophane in the protein 
molecule. 
F. Molisch's Reaction: 
Place 2.5 cc. of egg albumin in a test tube. Dilute with 
an equal volume of water. Add 3 to 4 drops of Molish reagent, 
(1% solution of alpha napthol in alcohol) and mix. Incline 
the test tube and allow 5 cc. of concentrated sulphuric acid to 
run down the side of the tube and stratify under the albumin 
solution. A violet ring, forming at the junction of the fluids 
should be observed. This reaction is dependent upon the pres- 
ence of a carbohydrate group in the molecule. In certain pro- 
teins this group is glucosamine. Compare with Experiment 30, 
page 42, carbohydrates. 
Influence of Ammonium Sulphate Upon the Biuket Test 
78 G. Sulphur Reaction: 
The test for lead blackening sulphur, page 104,-e, under 
“Elementary Composition,” above, indicates the presence of 
cystein or cysfin in the protein molecule. 
80.—Precipitation Tests 
(a.) “Salting Out” by Neutral Salts. 
1. Precipitation by Ammonium Sulphate. 
Add some solid ammonium sulphate to 10 cc. 
of the albumin solution in a test tube, shaking fre- 
quently, until the solution is thoroughly saturated. 
Allow to stand for a while, shaking occasionally. 
Then filter, and test the filtrate for albumin by 
means of the heat test. Test the solubility of the 
precipitate remaining on the filter paper. 
2. Precipitating Action of Magnesium Sulphate. 
Perform a similar experiment, using solid 
magnesium sulphate instead of ammonium sul- 
phate. To a portion of the filtrate add 1 or 2 
drops of acetic acid. Heat. 
(Note: Magnesium sulphate does not pre- 
cipitate albumin upon saturation of its solution. 
A closely related protein called globulin may be 
distinguished from albumin by reason of its be- 
havior with this salt.) 
(b.) Precipitation by Mineral Acids. 
Nitric Acid Test 
Put 5 cc. of the albumin solution into a test tube and intro- 
duce 5 cc. of concentrated nitric acid (very carefully by means 
of a pipette) into the bottom of the test tube. This forms a 
layer under the albumin solution at the junction of which a whr'te 
ring is seen. Determine the lowest protein concentration at 
which this test is unmistakable. Allow ten minutes if the re- 
action is slow in appeaning. This test is clinically known as 
“Heller’s Ring Test,’’ when applied to urine analysis. Sulphuric 
acid, hydrochloric acid, and meta-phosphoric acid are also capa- 
ble of precipitating protein. Orthophosphoric aoid will not cause 
precipitation. Why? 
79 (c.) Precipitation by Alcohol. 
Add an excess of alcohol (1 or 2 volumes) to 5 cc. of the 
albumin solution. 
(d.) Coagulation Test. 
Heat 5 cc. of albumin solution in a test tube to boiling. 
Note the degree of coagulation. Now add from 1 to 3 drops 
of 0.5% acetic acid and boil again. Complete coagulation should 
occur. 
(e.) Precipitation by Metallic Salts. 
1. To 5 cc. of albumin in a test tube add 5% mercuric 
nitrate solution drop by drop, until a definite pre- 
cipitate is obtained. To the solution containing 
the precipitate, add saturated sodium chloride, a 
few drops at a time. What happens? Is there 
any difference in degree of dissociation of mer- 
curic nitrate and mercuric chloride? Explain. 
2. Repeat the above experiment with a 5% ferric chlor- 
ide solution. Omit the addition of sodium chlor- 
ide. 
3. Repeat with solutions of any lead, silver and copper 
salts which are in the laboratory. Record your 
results. 
(f.) Precipitation bv “Alkaloidal Reagents.” 
Most of these reagents acts upon protein in acid solutions 
(peptones are exceptions in some cases). The protein under 
these conditions possesses a positive charge. The large nega- 
tively charged cation of the precipitating reagent is believed 
to be adsorbed by the protein, precipitation resulting. 
1. Tannic Acid. 
Make 5 cc. of the albumin solution acid with dilute 
acetic acid, then add a few drops of a tannic acid solu- 
tion. 
2. Picric Acid Test (Esbach’s Reagent). 
Add to a portion of the albumin solution a few 
drops of Esbach’s reagent. Esbach’s reagent is a dilute 
solution of picric acid containing some citric acid. De- 
80 termine the lowest protein concentration at which this 
test is unmistakable. 
3. Acetic Acid and Potassium Ferrocyanide. 
Make a portion of the albumin solution strongly 
acid with acetic acid, and add a few drops of potassium 
ferrocyanide solution. A white flocculent precipitate 
is formed (not with peptones). 
4. Phospho-tungstic Acid. 
Acidify 5 cc. of the albumin solution with dilute 
hydrochloric acid, and add a few drops of the phospho- 
tungstic acid reagent. This is a very delicate test. 
5. Potassio-mercuric-iodide (Brack’s Reagent). 
Acidulate 5 cc. of the albumin solution with dilute 
hydrochloric acid, and add a few drops of potassio- 
mercuric-iodide solution. 
Note.—Brack's reagent is prepared by dissolving 
50 grams of potassium iodide in 500 cc. of distilled 
water. To this solution add 120 grams of mercuric 
iodide, filtering if necessary to remove any undissolved 
material. The solution is usually diluted to 1 liter, 
but may be used without dilution if advisable. 
6. Sulphosalicylic Acid. 
To 5 cc. of albumin solution add a few drops of a 
20 per cent, solution of sulphosalicylic acid. 
Note: To prepare sulphosalicylic acid, dissolve 
13 grams of salicylic acid in 20 grams of warm sul- 
phuric acid. When dissolved, cool, and pour into 67 
cc. of distilled water. 
7. Metaphosphoric Acid. 
To 5 cc. of albumin solution add drop by drop a 
25 per cent, solution of metaphosphoric acid. This so- 
lution must be freshly prepared, and without the aid of 
heat. 
81 81.—Properties of Individual Proteins 
Simple Proteins 
(Substances yielding only alpha-amino acids or their derivatives 
on hydrolysis) 
(a.) Albumins. Coagulation by Heat. 
1. Heat a little of the solution in a test tube. Compare 
the coagulation so obtained with that obtained when 
the solution is diluted: 
(a) 10 times with distilled water. 
(b) 20 times with distilled water. 
(c) 20 times with 0.1% salt solution. 
(d) 20 times with 0.01% acetic acid. 
(e) 20 times with equal volumes of 0.1% salt solu- 
tion, and 0.01% acetic acid solution. From 
the above tests, what do you conclude are the 
necessary factors for coagulation? 
2. Ascertain the temperature of coagulation of albumin as 
follows: Faintly acidify a portion of the solution 
with a few drops of 0.5% acetic acid. Filter if nec- 
essary. Place the solution in a test tube, insert a 
thermometer by means of a cork, and suspend the 
tube in a large beaker of water. Heat the beaker 
slowly with a small flame, and observe the point at 
which the albumin becomes cloudy. 
3. Include all tests in Experiment 80 above as part of 
this division also. 
(b.) Globulins. 
Globulins are simple proteins and are especially predomi- 
nant in the vegetable kingdom. They are closely related to 
the albumens, and consequently react to all the general precipi- 
tants and color tests. Globulins are insoluble in pure water, 
thus differing from albumins. They are soluble in neutral salt 
solutions, which are not too concentrated. Most globulins are 
precipitated from their solutions by saturation with solid sodium 
chloride or solid magnesium sulphate. Ammonium sulphate at 
half saturation precipitates globulins. As a class, they are less 
82 stable than the albumins. Upon repeated precipitation, globu- 
lins dissolve with greater difficulty. The following tests serve 
to distinguish globulins from other proteins. These tests are 
made upon blood serum, which contains an albumin as well as 
globulin. At least two and possibly more globulins are present 
in blood serum. Euglobulin is fractionated out most easily, 
being precipitated by ammonium sulphate when the concentra- 
tion of the salt has reached 28 to 36 per cent. The second frac- 
tion called pseudoglobulin comes out between 36 and 44 per 
cent of saturation. Each fraction probably represents a number 
of globulins rather than a single definite compound. 
1. Uoagulation by Heat. 
Globulins, like albumins, are coagulated by heat. 
They are distinguished from other proteins and from 
one another by the temperature at which they coagu- 
late. To test this upon serum globulin, proceed as 
follows: To 10 cc. of serum, add an equal volume of 
a saturated solution of ammonium sulphate, thus ob- 
taining a half saturated solution. The bulky precipi- 
tate is globulin. Filter off the precipitate, wash two or 
three times with a half saturated ammonium sulphate 
solution, and dissolve in 20 cc. of distilled water. This 
will/ give you a clear solution of globulin. As stated 
above, the pure globulin does not dissolve in water, 
but in this case, the presence of salts, used for pre- 
cipitation and held mechanically in the precipitate, is 
sufficient to cause it to dissolve. Test the temperature 
of coagulation of this solution with the same apparatus 
that you used for egg albumin. What is the difference? 
2. Action of Carbon Dioxide. 
Dilute 5 cc. of serum with 45 cc. of ice cold water. 
Place the mixture in a cylinder, surrounded by ice 
i water, and pass through it a stream of carbon dioxide. 
A flocculent precipitate is obtained. Continue the car- 
bon dioxide for some time and notice its effect upon 
the precipitate. Explain to the instructor. 
83 3. Precipitation by Dialysis. 
Pour 20 cc. of serum into a parchment dializing 
tube, previously soaked in distilled water. Suspend 
the tube with its contents in a large vessel of distilled 
water, changing the water at frequent intervals, (one 
hour at first). After 24 hours, examine the serum in 
the dializing tube. The flocculent precipitate is globu- 
lin. Explain to the instructor the cause of this pre- 
cipitation. 
4. Precipitation of Euglobulin by Dilution. 
Slightly acidify 5 cc. of serum with dilute (0.5%) 
acetic acid. Pour the serum, drop by drop, into a 
graduate filled with distilled water. What takes place? 
Exolain. Allow to stand a few minutes. 
5. Precipitation by Magnesium Sulphate. 
Saturate 5 cc. of serum with solid magnesium sul- 
phate. A heavy precipitate will be formed. Compare 
this with the action of the same salt on a solution of 
egg albumin. The globulin is so completely precipi- 
tated by this salt that the method may be used for its 
quantitative estimation. 
6. Edestin. 
The preparation of this vegetable globulin is given 
on page 94. 
(c.) Glutelins and Prolamines. 
Osborne lias shown that seeds of cereals, after extracting 
with water and with a dilute solution of a neutral salt, still con- 
tain protein. One of these proteins may be removed by 80 per 
cent alcohol. He designates this group as prolamines, because 
upon hydrolysis of these compounds, especially large amounts of 
prolein and ammonia are obtained. 
The following prolamines have been described by Osborne: 
Zein from maize 
Hordein from barley 
Gliadin from wheat and rye 
Bynine from malt. 
84 If the residue remaining after the alcohol extraction of pro- 
lamines be treated with dilute acid or dilute alkali, another pro- 
tein is recovered. Members of this group are called glutelins. 
Glutenin, from the wheat is an example of this class. 
1. Extraction of Gliadin. 
Mix some flour into a thick paste with warm water, 
and tie up in a piece of cloth. Work this paste under 
tepid water, changing it frequently until the washings 
are free from starch. What remains? Carefully grind 
up the residue in a mortar, containing a little sand, 
with 5 portions of 70% alcohol, using about 25 cc. 
each time. Save the alcohol washings and the residue 
insoluble in alcohol. The alcoholic solution will contain 
gliadin. Filter. 
2. Protein Color Reactions with Gliadin. 
Dilute the filtrate with water. What happens? 
Try all protein color reactions. Caution: Alcohol is 
oxidized by concentrated nitric acid. Add small amounts 
of acid at a time—cool continuously under the water 
tap. 
3. Glutenin. 
Test the solubility of the residue which consists 
mainly of glutenin. With a solution of glutenin, per- 
form the protein color reactions. 
(d.) Albuminoids. 
The members of this group are important constituents of 
the animal skeleton or cutaneous structure. As a rule, they all 
occur in an insoluble state in the organism, and uniformly re- 
sist solution by ordinary protein solvents as well as by all except 
the most vigorous chemical reagents. They are therefore char- 
acterized by their inactivity. 
Among the members of this class may be mentioned the 
keratins, elastin, collagen, reticulin and others. 
1. Keratin. 
The albuminoid, keratin, makes up the major part 
of hair, wool, horn, hoof, feathers, nails and the epi- 
85 dermal layer of the skin. Keratins, as a group, pos- 
sess very similar properties. They are insoluble in the 
usual protein solvents, and are not acted upon by the 
gastric or pancreatic juices. They respond to Millon's 
reaction and Xanthoproteic reaction, and are character- 
ized by containing large amounts of loosely held sul- 
phur. Pure keratin may be prepared by successive 
treatment with artificial gastric juice, artificial pan- 
creatic juice, boiling alcohol and boiling ether. Each 
process should last for at least 24 hours. 
Using scoured wool, test the solubility of keratin 
in the ordinary protein solvents. Perform the protein 
color reactions upon small amounts of wool. (Do not 
try to put the wool into solution before performing the 
tests.) 
2. Collagen and Geletin. 
Collagen, or gelatin-forming substance, occurs ex- 
tensively in vertebrates. It is the chief constituent of 
the fibrils of the connective tissue (as ossein) and of 
the organic substances of the bony tissue. By contin- 
ued boiling with water, especially if a little acid is 
present, collagen is converted into gelatin. Gelatin 
is of somewhat variable composition, depending upon 
the collagen, from which it was made. 
Gelatin solutions are not precipitated on boiling 
or by mineral acids, acetic acid, alum, basic lead ace- 
tate, or metallic salts. They are precipitated when acidi- 
fied with acetic acid by potassium ferro-cyanide on care- 
ful addition of the reagent. Gelatin is precipitated by 
tannic acid in the presence of salts, by acetic acid and 
sodium chloride; by mercuric chloride in the presence 
of hydrochloric acid and sodium chloride; by meta- 
phosphoric acid; phosp'homolybdic acid in the presence 
of hydrochloric acid; also by alcohol, especially in the 
presence of neutral salts. The biuret reaction is posi- 
tive, but the Hopkins-Cole reaction is negative. Mil 
Ion’s reaction and the xanthroproteic reaction, when 
given, are probably due to impurities. 
86 (a.) Preparation of Gelatin. 
Carefully clean the shaft of a long hone. Break 
or cut to small bits, then weigh out a sample of about 
10 grams. Place the material in a flask and cover with 
about 60 cc. of dilute hydrochloric acid (1-20). Al- 
low the material to remain for three or four days. Di- 
lute hydrochloric acid dissolves the inorganic portions 
of the bone, leaving the collagen. Note the efferves- 
cence due to the presence of carbonates. The acid solu- 
tion is poured off. Save for Exp. 101, page 134. The 
remains of the bone are allowed to stand over night in 
a dilute solution (1-10) of sodium carbonate, then 
boiled in 100 cc. of water for one or two hours. The 
collagen undergoes hydration, and is converted into the 
soluble substance gelatin. The core of bone untouched 
by the acid usually remains. Evaporate the solution to 
25 cc. volume, and allow to cool. A firm jelly should 
form if the solution is sufficiently concentrated. If the 
solution gelatinizes, add an equal volume of water and 
heat again. This solution may be used for the follow- 
ing tests. If the solution is not pure, it is advisable 
to prepare one from commercial gelatin. 
(b.) Precipitation Tests and Color Reactions. 
With a gelatin solution perform protein precipitation and 
color tests. Tabulate your results, and explain them to the in- 
structor. 
Conjugated Proteins 
(Compounds of simple proteins and some other non-protein 
group. The non-protein group is usually acid in nature.) 
(a.) Chromoproteins. 
j Haemoglobin is an example of this group and is composed 
of a nonprotein pigment substance which contains iron (haem- 
atin) and the protein globin, which belongs to the class of his- 
tones. 
Hemoglobin is soluble in water and dilute salt solutions. It 
is decomposed by acids and alkalies into its constituents. Heat 
87 causes it to coagulate. It is generally insoluble in organic sol- 
vents. 
Work upon the compound will be given later under blood. 
(b.) Glycoproteins. 
Glycoproteins are compound proteins, which yield upon de- 
composition a protein substance, on the one hand, and carbohy- 
drates or derivatives of these on the other, but no purine bodies. 
Certain glycoproteins contain no phosphorus. These are mucin 
substances, chondroproteins and hyalogens. Others contain 
phosphorus (phosphoglyco-proteins). The glycoproteins yield- 
ing-no phosphorus may be divided into two groups respective to 
the carbohydrate radical split off. These are mucin substances 
and chondroproteins. Mucins yield upon cleavage an amino- 
sugar, which in most cases is glucosamine. Chondroproteins, on 
the other hand, yield upon cleavage a protein and chondroitin- 
sulphuric acid. 
1. Preparation of a Mucin. 
Extract an umbilical cord for twenty-four hours 
with 10% barium hydrate solution in a stoppered flask. 
Filter. To the filtrate add acetic acid, drop by drop, 
which precipitates the mucin. Allow to settle, filter, and 
wash with water. Mucins may also be prepared from 
saliva by precipitation with acetic acid. 
a. Try the solubility of mucin in water, potassium hy- 
drate, calcium hydrate solution, and in a 5% 
hydrochloric acid. 
b. Perform the protein color tests with mucin. 
c. Boil mucin for some time with dilute hydrochloric 
acid (1-3), allow to cool, neutralize with sodic 
hydrate solution and test for a reducing sugar 
with Fehling's solution. Where did the reduc- 
ing carbohydrate come from? 
(c.) Phosphoproteins. 
This group of substances contains the protein molecule and 
some, as vet undefined, phosphorus-containing substances other 
88 than a nucleic acid or lecithin. They occur extensively in the 
animal and plant kingdoms. Casein of milk is the best stud- 
ied phosphoprotein. Lecithalbumins occur in ovovitellin. In 
this body they are supposed to occur as a combination of pro- 
tein and lecithin. Similar substances also occur in fish eggs. 
These substances containing lecithin can be removed only with 
difficulty or incompletely from proteins by a mixture of alcohol 
and ether. 
1. Using Casein as an example of this class, perform the 
following experiments: 
a. Solubility in water, acids and alkalies. 
b. Protein color reactions. 
c. Test for phosphorus. (Note: To do this, fuse 
a little in a crucible with sodium carbonate 
and sodium nitrate. Dissolve the fused mass, 
when cold, in water, and test for phosphoric 
acid by means of ammonium molybdate.) 
d. Digest a portion for some time at 40°C. with 
pepsin-hydrochloric acid. Filter and test the 
filtrate for proteins. What is the insoluble 
residue ? 
(d.) Nucleoproteins—Nuclein—Nucleic Acid. 
Note: The extended discussion of nucleoproteins contained 
in this section is admittedly out of place in a laboratory manual 
of this type. It is inserted with the hope that the facts pre- 
sented will clarify the student’s mind after reading the section 
upon this subject in the text-Took. 
The nucleoproteins are compounds of nucleic acid with a 
protein. Typical nucleoproteins are characteristic constituents 
of the cell nuclei. 
Nucleoproteins are insoluble in water, soluble in dilute al- 
kali, from which they may be precipitated by carbon-dioxide or 
dilute acetic acid. 
From the researches of Jdammarsten (1894) two nucleo- 
proteins are recognized as extractable from glandular tissue. 
89 Hammarsten gave to these the names of a and /?—nucleoprotein. 
The oc -nucleoproteins can be prepared from the cloudy aqueous 
gland extracts, by precipitation with acetic acid. The oc -nucleo- 
protein consists of various salts of protein with nucleic acid 
in which the protein is in excess. Upon digestion with pepsin- 
hydrochloric acid part of the protein is digested away, leaving 
a mixture of protein and acid salts. This indefinite mixture 
is called “nuclein.” It is resistant to the further action of pep- 
sin-hydrochloric acid. 
The is obtained by rapidly heating a sus- 
pension of ground tissue in water to boiling, filtering off the 
coagulum, and then upon the addition of acetic acid to the fil- 
trate, a flocculent precipitate is obtained. The oc -nucleoprotein 
remains in the heat coagulum in this case. Jones states that there 
“is no evidence to show that is a constituent of 
the cell nucleus.” 
Nucleoproteins are of special interest on account of the nu- 
cleic acids which they contain. Nucleic acids are split down 
by acid hydrolysis into three essential constituents. These are: 
1st, phosphoric acid; 2nd, a carbohydrate radical (hexose 
or pentose), and 3rd, a nitrogenous base or bases belonging to 
either the group of purine bases or pyrimidine bases. 
The nucleic acids derived from oc -nucleoproteins, will yield 
upon complete hydrolysis two purine bases, and also two pyri- 
midine bases in addition to the phosphoric acid and the carbo- 
hydrate. 
The formula given on following page, shows the present 
state of our knowledge as to the composition of a nucleic acid. 
90 Phosphoric Acid 
Carbohydrate 
Base 
Guanine Group 
(Purine Base) 
Thymine Group 
(Pyrimidine Base) 
Cytosine Group 
(Pyrimidine Base) 
Adenine Group 
(Purine Base) 
Thymus Nucleic Acid. 
From the structure of the thymus nucleic acid, it is seen 
that two types of linkages between nucleotids occurs. Jones be- 
lieves all four parts of yeast nucleic acid to be linked by ties 
similar to that connecting the thymine group to the cytosine 
group in the above formula. Nucleic acids containing four parts 
as indicated, occur in oc-nucleoproteins. Therefore, oc-nucleo- 
proteins are compounds of protein with a tetranucleotid. 
The /Tnucleoproteins, on the contrary, are compounds of pro- 
tein with an acid “guanylic acid,” which is a mono-nucleotid. One 
of the possible formulae for this nucleic acid is given below: 
Pentose 
( d-ribose) 
Puri ne 
IGuajii ne) 
Phosphoric 
Acid 
Guanine mono-nucleotid. 
91 The following table from Jones, gives a general idea of the 
composition of nucleic acids from nucleoproteins. 
Hydrolytic Products of Nucleic Acids 
Of Plant Origin 
1. Phosphoric Acid 
Of Animal Origin 
1. Phosphoric Acid 
2. Guanine 
3. Adenine 
4. Cytosine 
5. Thymine 
6. (Hexose) Laevulinic 
Acid 
Purine 
Bases 
2. Guanine 
3. Adenine 
4. Cytosine 
'5. Uracil 
Pyrimidine 
Bases 
6. Pentose 
Jones furthermore suggests that the animal nucleic acids 
of various origin are identical with one another. 
1. Preparation of Thymus Nucleic Acid (Jones) 
To a boiling mixture of 2 liters of water, 100 grams 
of sodium acetate and 33 grams of caustic soda in a 
flask, add in small successive portions, 1000 grams of 
trimmed and finely ground thymus gland. The tissue 
should dissolve quickly, if not, heat for a short time 
over a free flame. Immerse the flask in a briskly boil- 
ing water bath for 2 hours, stirring occasionally. Now 
dilute with 1/3 volume of hot water, and faintly but 
distinctly acidify with 50% acetic acid. Usually 100 
cc. acid is required, the final addition should, however, 
be carefully controlled by testing with litmus paper. 
If too much acid is added, it should be neutralized by 
addition of dilute sodium hydroxide. Small portions 
should from time to time during neutralization be test- 
ed for filterability. When the acidity favorable for 
rapid filtration has been obtained, heat the material 
to hard boiling, then filter through paper using a hot 
water funnel. The filtration should be fairly rapid, a 
green slime remaining on the paper. The filtrate will 
gelatinize upon cooling. Evaporate the filtrate to about 
750 cc. on the steam bath. Pour the hot concentrated 
solution slowly into 1000 cc. of 95% alcohol. Allow 
to stand over night. Sodium nucleate settles out as a 
92 white spongy mass, from which the brown fluid should 
be decanted and the remaining portion pressed out with 
a spatula, leaving the material in one cohesive mass. 
Wash the residue of sodium nucleate by decanta- 
tion respectively with 80% and 95% alcohol. Press 
out as thoroughly as possible after the last treatment 
with alcohol. Now add 300 cc. of boiling water, trans- 
fer to a suitable flask, and heat on the water bath. 
In half an hour or less, insoluble phosphates will col- 
lect, leaving a perfectly transparent interstitial fluid. 
To lower the viscosity so that the fluid will filter read- 
ily, 10 cc. of 20% caustic soda are added, and after 
thoroughly mixing, the solution is filtered, using a hot 
water funnel. The clear filtrate is acidified with acetic 
acid, then poured into 700 cc. of 95% alcohol. Sodium 
nucleate is precipitated as before, then washed with 80, 
90 and 95% alcohols, finally transferring to a mortar, 
where it is ground to a fine white powder under abso- 
lute alcohol. The ground mass is now washed onto a 
filter with absolute alcohol, drained thoroughly and 
transferred to a watch crystal and placed in a des- 
sicator over sulphuric acid until thoroughly dry. Yield 
about 33 grams. 
This is the sodium salt of thymus nucleic acid, 
it should he fine and white, and not hygroscopic. This 
method is applicable to the preparation of similar prod- 
ucts from the pancreas and spleen. 
Properties: A 4 per cent, solution of sodium nu- 
cleate in warm water becomes gelatinous when cooled 
to the room temperature. With this solution or one 
slightly more concentrated, the following properties 
may be demonstrated. 
a. The viscosity of the fluid is decreased by the ad- 
dition of either acetic acid or sodium hydrox- 
ide. The solution may be changed back and 
forth from gelatinous to fluid by the alternate 
addition of acid and alkali. 
93 b. The positive optical rotation is greatest in a neu- 
tral fluid, and like the viscosity, is markedly low- 
ered by the addition of either acid or alkali. 
c. The specific rotation falls by dilution with water, 
but more rapidly by dilution with acid or alkali. 
d. The rotation is lowered by heating a solution from 
20° to 40°C., but again rises when the solution 
cools. 
Derived Proteins 
Products resulting- from chemical or physical changes, or 
the action of enzymes upon native proteins. 
(a.) Primary Derived Proteins. 
1. Proteans. 
If edestin (globulin, see page 84) be prepared 
by extracting crushed hemp seed with 5% warm 
(60°C.) sodium chloride for one-half hour, then fil- 
tered, and filtrate allowed to cool very slowly (this 
may be obtained by placing the filtrate contained in a 
flask into an earthen ware jar or crock, filled to the 
proper height with water at 60° C., then allowing this 
system to cool) beautiful crystals of edestin separate 
out. Filter off the edestin. 
Dissolve some of the edestin (globulin) in a 0.2% 
hydrochloric acid, allowing it to stand for one-half 
hour. The solution must now be neutralized with 0.5% 
sodium carbonate, and the precipitate which is edestcin 
(protean) filtered off. 
With the precipitate try the solubility in water, 
sodium chloride, and dilute acid and alkali. 
Apply the protein color reactions and do the pro- 
tein precipitation tests. 
How does this protein dififer from a globulin or 
albumin ? 
2. Metaproteins. 
There are two principal metaproteins, the acid 
metaprotein (so called acid albuminate) and the alkali 
94 metaprotein (so called alkali albuminate). Aside from 
solubilities, these two types differ in their nitrogen and 
sulphur content when made from the same protein. 
The acid metaprotein contains more of these elements, 
as loss occurs due to the action of the alkali used in 
forming alkali metaproteins. 
To prepare an alkali metaprotein from albumin, 
proceed as follows: 
Separate the white from the yolk of an egg, and 
place it in an evaporating dish. To it add 40% potas- 
sium hydroxide solution, DRO'P BY DROP, stirring 
continuously until the albumin has attained the con- 
sistency of a jelly. This is known as Lieberkiihn’s jelly, 
and is the desired alkali metaprotein. 
The jelly may now be cut into thin strips, and 
the alkali leached out by covering and decanting the 
material with distilled water several times. 
Place some of the material in a beaker, add suf- 
ficient water to cover it and then heat gently. If the 
material fails to dissolve note the fact, and then add 
a drop or two of alkali. When in solution, cool and 
divide into two parts. With the first part do the pro- 
tein precipitation tests and the biuret reaction. To 
the second part, add dilute hydrochloric acid until neu- 
tral, noting the odor of hydrogen sulphide emitted. 
Filter off the precipitate, wash with water, and using 
the solid material perform the protein color reactions, 
including the test for lead blackening sulphur. 
3. Coagulated Proteins. 
With pieces of the white of a hard boiled egg, 
perform solubility and color tests. 
(b.) Secondary Derived Proteins. 
1. Proteoses and Peptones. 
There are several proteoses (protoproteose, hetero- 
proteose and deuteroproteose) resulting from proteo- 
lysis. They are differentiated mainly by their solubili- 
95 ties in salt solutions, especially in ammonium sulphate 
solutions of varying concentrations. 
The “peptones” sold for bacteriological purposes 
contain a large amount of proteose. 
It is impossible to take up the proteoses withoul 
defining their properties in relation to the peptones. 
There are at least two peptones recognized, the ampho- 
peptone, and the antipeptone. 
The proteoses as a class represent protein cleavage 
products less completely digested than the peptones. Nei- 
ther proteoses or peptones are coagulable by heat. They 
both are extremely soluble in water, and diffuse rap- 
idly, the peptone having even greater diffusion than 
the proteoses. Peptones differ from proteoses chiefly 
by the fact that they are non-precipitable by ammon- 
ium sulphate, give no precipitate with potassium ferro- 
cyanide and acetic acid, potassium mercuric iodide and 
hydrochloric acid, picric acid, and trichloracetic acid. 
Peptones are precipitated by phosphotungstic acid, 
phosphomolybdic acid, absolute alcohol, and tannic acid. 
Many of the precipitants when added in excess dis- 
solve the precipitate. 
(a.) General Properties of Proteoses and Peptones. 
Using Witte’s or some other commercial “peptone” try the 
solubility in various solvents (water, alcohol, dilute acid and 
dilute alkali) and with a solution perform the protein color re- 
actions. 
(b.) Separation of Proteoses from Peptones by Means of 
Ammonium Sulphate. 
Weigh out 5 grams of Witte’s or another commercial pep- 
tone, and make a solution with a small amount of hot water (50 
cc. if possible). To this add an equal volume of saturated am- 
monium sulphate (thereby giving you a half saturated solution). 
A precipitate of primary proteoses (protoproteose and hetero- 
proteose) should be observed. Place the solution in a pyrex 
beaker, and heat to boiling. Add solid ammonium suplhate until 
96 the solution becomes saturated. The secondary proteose (deu- 
teroproteose) now separates out and the solution contains the 
peptone. If a rubber tipped stirring rod be worked about in 
the beaker, the sticky proteose may be collected and removed. 
Dissolve the proteose in water in a beaker, precipitate the am- 
monium sulphate by adding barium carbonate, boil, filter, then 
concentrate the filtrate upon the steam bath. With the concen- 
trated filtrate, perform the protein precipitation tests. 
The saturated ammonium sulphate solution containing the 
peptone should be cooled, and filtered through cloth. A por- 
tion of the filtrate is diluted with water, barium carbonate added 
as above, boiled, filtered, and the filtrate concentrated upon the 
steam bath. With the concentrated solution so obtained re- 
peat the protein precipitation reactions, and compare with those 
given by the proteose solution above. 
82.—Problem. Obtain two unknown proteins from the in- 
structor. Run through the color reactions and precipitation 
tests and report to the instructor the class and name of the pro- 
tein found. In some cases the student may receive a sample 
containing more than one protein. Identify and report each. 
These are some of the ultimate cleavage products of the 
protein molecule. 
AMINO ACIDS 
83.—A. Synthesis of Glycocoll 
Dissolve 20 grams of monochloracetic acid (OH2C1-COOH) 
in a flask by means of 50 cc. of cold concentrated ammonia. Cool 
with ice and saturate the solution with ammonia gas, shaking 
constantly in ice water to avoid any rise of temperature. Cover 
the flask with a watch crystal and allow to stand over night in 
a basin of cold water. This prevents any appreciable rise in 
temperature, due to the completion of the chemical reaction. 
Take the flask out of doors and pour the contents into a 
large evaporating dish and leave for twenty-four hours to al- 
low the escape of ammonia. At the end of this time, gently 
boil the contents of the dish until all the free ammonia is gone. 
Large amounts of cupric oxide will be dissolved, if all of the 
97 ammonia is not expelled. Add freshly precipitated cupric oxide 
until some remains undissolved after five minutes boiling. (Cu- 
pric oxide is prepared by adding a slight excess of sodium hy- 
drate to a dilute copper sulphate solution, and washing several 
times by decantation until the precipitate is free from alkali.) 
Filter the hot solution to remove the excess of cupric oxide. 
Evaporate the filtrate to a small volume (75-100 cc.) and allow 
it to cool. Do not carry to dryness. Upon standing overnight, 
an abundant precipitate of the copper salt of glycocoll should 
be obtained. Filter off these crystals on a Buchner funnel, and 
wash the precipitate successively with 60, 80, and 90 per cent, 
alcohol until the wash alcohol no longer shows a reaction for 
chlorine. (Note: Acidify the test portion with a few drops 
of nitric acid before adding the silver nitrate solution.) 
To prepare free glycocoll from this salt, proceed as fol- 
lows: Dissolve the copper salt in 150 cc. of hot water, then 
add a little freshly precipitated aluminum hydrate, and pass 
hydrogen sulphide gas into the liquid until saturated. 
(Note: Aluminum hydrate is prepared by precipitation 
from aluminum sulphate by ammonia and washing by decanta- 
tion until neutral. The hydroxide takes no part in the above 
reaction, but acts only to assist in the precipitation of the col- 
loidal copper sulphide.) 
Heat the liquid to boiling and filter. Concentrate the fil- 
trate to about 20 cc. and precipitate the glycocoll by the grad- 
ual addition of alcohol. Filter the glycocoll on to a Buchner 
funnel, and wash once or twice with alcohol. Transfer the 
crystals to a watch glass, and place in a protected place to dry. 
Weigh the dry precipitate and calculate the yield in per cent 
of the theoretical. Determine the purity of the product by a 
nitrogen determination. 
B.—Amino Acids by Acid Hydrolysis. 
The preparation of cystine from wool: (Fblin) 
Put 50 grams of scoured wool into a 500 cc. Florence flask. 
Add 100 cc. of concentrated hydrochloric acid, and connect the 
flask with a condenser. (If a good hood is available, a two and 
one-half foot glass tube inserted through the cork serves as a 
98 suitable air condenser.) Heat the flask in a water bath until 
the wool has been dissolved. Remove from the water bath, dry 
the flask thoroughly, boil gently over a very small flame until 
the biuret reaction is negative. Boil for one hour after the 
biuret reaction is entirely gone. This digestion requires from 
three to five hours. After completely digesting the mixture al- 
low to cool, and add solid sodium acetate until no free mineral 
acid can be detected in solution by means of Congo red paper. 
Explain the disappearance of mineral acid. Allow the mixture 
to stand 1 week. Filter the contents of the flask on a Buchner 
funnel, and wash once or twice in cold water. Dissolve the 
precipitate in 150 cc. of water, to which has been added 5 cc. 
(more if necessary) of concentrated hydrochloric acid and about 
20 grams of purified bone black. Boil the mixture for 5 to 10 
minutes. Filter again with a suction pump and collect the fil- 
trate. The filtrate is heated to boiling and JUST neutralized by 
the addition of hot concentrated sodic acetate solution. Use 
congo red paper as an indicator. (AN EXCESS OF SODIC 
ACETATE MUST BE AVOIDED.) The precipitate formed 
consists of cystin and should be very white and pure. Examine 
a few crystals under the microscope and sketch their appear- 
ance. Filter them off after five minutes standing. Allow the 
liquid to cool. Tyrosin and leucin will crystallize out in the 
filtrate. Identify microscopically. Test the cystin crystals for 
sulphur bv means of lead acetate and sodic hydrate. 
(Note: To prepare pure bone black, the commercial sam- 
ple is allowed to stand over night in an excess of dilute HC1. 
Then filter, and digest over a flame with a second portion of 
dilute hydrochloric acid. This process is repeated until the acid 
digestion mixture fails to give a test for phosphate. The char- 
coal is then thrown on to a large filter paper in a funnel, and 
washed free from chlorides.) 
99 DIGESTION 
The following experiments are intended to give the student 
an idea of the action of digestive ferments upon the foods taken 
into the body. 
Salivary Digestion 
84.—Collect about 50 cc. of saliva by chewing a piece of 
paraffine. Filter, and test the clear filtrate as follows:— 
(a.) Reaction.—Test with litmus. 
(b.) Mucin.—To 5 cc. add a few drops of dilute acetic 
acid and record the result. 
(c.) Proteins.—Filter (b) to remove the mucin. Test the 
filtrate by the xantho-proteic test. 
(d.) A sulphocyanide.—Add to some saliva a drop or two 
of very dilute and acidulated (FTCl) solution of Fe2Clfi. Report 
the result. Then add a few drops of a solution of HgCF. What 
is the result ? What other substances give a similar reaction with 
ferric chloride, and how may they be distinguished from each 
other and from sulphocyanic acid? 
(e.) Action of saliva upon starch.—Take some filtered 
saliva in a test tube, and place in a water-bath at 40° C. for five 
or ten minutes. Put some starch paste into a second test tube, 
and place this also in the water-bath for a while, then mix the 
two (10 cc. of starch paste to 3 cc. of undiluted saliva) and 
return to the water-bath. The starch is changed first to soluble 
starch (if originally a thick paste, it becomes fluid and loses its 
opalescence), then to erythro-dextrin, which gives a red color 
with iodine, and finally to achroo-dextrin, which gives no reac- 
tion with iodine, and then to maltose. Prove these changes 
as follows:—Every minute or two take out a drop of the mix- 
ture, place it on a porcelain plate and add a drop of iodine solu- 
tion. This gives first a blue color, showing the presence of 
starch; later a violet color, due to the mixture of the blue of 
the starch reaction with the red caused by the dextrin: next a 
reddish-brown, due to erythro-dextrin alone (starch being ab- 
sent) and finally no reaction at all with iodine, proving the ab- 
sence of starch and erythro-dextrin. The fluid now contains 
100 achroo-dextrin and maltose. Test for the latter with Fehling’s 
solution and with Barfoed’s reagent. 
At what stage in the process does maltose appear? Explain. 
(f.) Influence of conditions on ptyalin and its amylolytic 
action—Report and explain the results of the following experi- 
ments :— 
1. Boil a few cc. of the saliva, then add some starch paste, 
and place in the water-bath at 40° C. After five minutes test for 
sugar. 
2. Take two test tubes; put some starch paste in one, and 
saliva in the other, and cool them to 0° C. in a freezing mixture. 
Mix the two solutions, and keep the mixture surrounded by ice 
for several minutes, then test a portion for sugar. Now place 
the remainder in the water-bath at 40° C. and after a time test 
for sugar. 
3. Carefully neutralize 20 cc. of saliva with very dilute 
HC1 (about 0.05%) and dilute the whole to 100 cc. Test the 
action of this neutralized saliva on starch. 
4. To 5 cc. of starch paste add 10 cc. of 0.2% HO, and 
5 cc. of neutral saliva, and expose the mixture for awhile at 
40° C., and test for sugar. 
5. To 5 cc. of starch paste add 10 cc. of 0.5% solution of 
Na2COs and 5 cc. of neutral saliva, and expose the mixture for 
awhile at 40°C., and test for sugar. 
6. Carefully neutralize (4) and (5) and again test the 
action of the two on starch. 
(Note: By repetitions of (4) and (5) using reagents of 
regularly increasing degrees of dilution, the percentage of each 
which does not interfere with the action of ptyalin on starch can 
be determined. In order to obtain uniform results, a diluted 
saliva should be used.) 
7. Mix a little uncooked starch with saliva, expose to a 
temperature of 40° C. for awhile, and test for sugar. 
Gastric Digestion 
85.—The following solutions will be found in the labora- 
tory :— 
101 A. —A 0.2% solution of HC1.—This is prepared by diluting 
6.5 cc. of concentrated H'Cl (Sp. Gr., 1.19) with distilled water 
to one liter. 
B. —A solution of pepsin.—Prepared by dissolving two 
grams of pepsin in 1000 cc. of water. 
C. —A pepsin-hydrochloric acid solution.—Prepared by dis- 
solving two grams of pepsin in 1000 cc. of solution A. 
Or, add to 150 cc. of solution A about 10 cc. of the glycer- 
ine extract of the mucous membrane of the stomach. 
(a.) Take fire test tubes and label 1, 2, 3, 4, 5. Fill as in- 
dicated below. Place in a water-bath at 40° C. and examine 
an hour later and again the next day. 
1. —3 cc. pepsin solution -f- 10 cc. water -f- a few shreds 
of fibrin. 
2. —10 cc. 0.2% HC1 -f- a few shreds of fibrin. 
3. —3 cc. pepsin -f- 10 cc. 0.2% HC1 and a few shreds of 
fibrin. 
4. —3 cc. pepsin -f- 10 cc. 0.2% HC1, boil, cool, and then 
add a few shreds of fibrin. 
5. —3 cc. pepsin solution -f- 10 cc. 0.2% HC1 and a few 
shreds of fibrin which have been tied firmly together into a hall 
with a thread. 
Make a note of all changes. 
(b.) Filter (3). Neutralize with dilute Na2C03. Filter 
again. Why? Test the filtrate for the Biuret reaction. 
(c.) To 5 grams of fibrin add 30 cc. of the pepsin solu- 
tion and 100 cc. 0.2% HQ. Set in the water-bath at 40° C., 
stirring frequently and leave in the water-bath over night. Ob- 
serve the undigested residue on the following day and also a 
slight flocculent precipitate. What is this precipitate? 
Filter and carefully neutralize the filtrate. A precipitate 
varying with the progress of the digestion will form. What 
is it? 
Remove this by filtration and saturate this filtrate with 
(NH4)2S04. Filter. Save precipitate and filtrate. Of what 
does each consist? 
102 (d.) Dissolve the precipitate of Ex. (c) in water and try 
the following tests:— 
1. —Biuret reaction. 
2. —Effect of boiling. 
3. —Test with HNOa, as in performing test for albumin 
in the urine. 
(e.) To the last filtrate of Ex. (c) add an equal volume 
of 95% alcohol and stir thoroughly. The peptones will collect 
in a gummy mass about the stirring rod. 
1. —Determine the solubility of peptones in water. 
2. —What is the effect of heat when so dissolved? 
3. —Try the Biuret reaction. 
(f.) Demonstration of the Rennet Enzyme.—Place 10 cc. 
of milk in each of three test tubes. Label the test tubes 1, 2, 3. 
To 1 add a drop of a neutralized glycerine extract of the 
mucous membrane of the stomach (made from the stomach of 
a calf). 
To 2 add a drop of a neutralized glycerin extract and boil 
at once. 
To 3 add a few cc. of (NH4)2C204 solution and then a 
drop of the glycerine extract. 
Place these tubes in the water-bath at 40° C. and examine 
after five or ten minutes. Explain result in each case. 
Continue heating tube 3 for half an hour; then add 2 to 3 
drops CaCl2 solution. The liquid instantly solidifies. Why? 
(g). Digestion of Casein.—Determine the products of the 
digestion of the curd from the last experiment. 
(h.) Tests for free hydrochloric acid.—Try each of the 
following tests with (1) HIC1 (0.2%, 0.05%, and 0.01% suc- 
cessively) ; (2) lactic acid (1%) ; (3) mixtures containing equal 
volumes of (1) and (2). Tabulate the results. 
1.—Topfer's Reagent.1—Use one or two drops of a 0.5% 
1 Organic acids and acid salts produce a somewhat similar reaction with Conge 
red, and with Topfer’s solution, but in solutions more concentrated than those ordi- 
narily found in the stomach contents. 
103 alcoholic solution. In the presence of free mineral acids a car- 
mine-red color is obtained. 
2:—Gunzburg’s Reagent.—Phloroglucin, 2 gms.; vanillin, 
1 gm.; alcohol, 100 cc. Place two or three drops of the solution 
to be tested in a porcelain dish, add one or two drops of the 
reagent, and evaporate on a water-bath. In the presence of free 
hydrochloric acid a rose-red color develops. 
3. —Tropaeolin OO.—Use one or two drops of a saturated 
alcoholic solution. 
4. —Congo-Red.—Use filter paper which has been dipped 
into a solution of the reagent and then dried. 
(i.) To 5 cc. egg albumin in solution add 1 cc. of 0.2% 
HO. Mix thoroughly and test for the presence of free HC1, 
What is the result? How do you explain it? Repeat the test 
using a solution of peptone in place of the egg albumin. 
(j.) Tests for Lactic Acid.—Uffelmann's reagent. Mix 
10 cc. of a 4% solution of carbolic acid with 20 cc. of water, 
and add a drop or two of ferric chloride. 
To 5 cc. of the reagent add a few drops of the lactic acid 
solution. Note the canary yellow color. 
Does the presence of free HC1 interfere with this reaction? 
A more delicate reagent is obtained by adding three or four 
drops of a 10% ferric chloride solution to 50 cc. of water. Such 
a solution has a very faint yellow color, which is distinctly in- 
tensified by lactic acid. 
Using 5 cc. of this nearly colorless solution for each ex- 
periment, note the effect of (1) 0.2% HC1, (2) di-hvdrogen 
sodium phosphate, (3) alcohol, (4) glucose, (5) cane sugar. 
What conclusions do you reach concerning the value of this 
test, when applied directly to the gastric contents? 
The test is best applied to an aqueous solution of the ether- 
eal extract of the gastric contents:—Add to the contents two 
drops of H'Cl, boil to a syrup, and extract with ether. Dis- 
solve the residue obtained upon evaporation of the ether in a 
little water, and test for lactic acid. 
104 (k.) Quantitative Estimation of Free Hydrochloric Acid, 
and of Total Acidity. 
1. Free Hydrochloric Acid.—To 10 cc. of unfiltered gas- 
tric juice add three to four drops of a solution of Topfer’s rea- 
gent. Titrate the acidity with N/10 NaOH, taking the dis- 
appearance of the carmine red color as an end point. This point 
represents fairly sharply the neutralization of the free hydro- 
chloric acid in the contents used. Acidity is generally (clini- 
cally) recorded as the number of cc. of N/10 NaOH required to 
neutralize 100 cc. of stomach contents. Report your analysis 
m this way, and also calculate the per cent of free hydrochloric 
acid present. Gunzburg’s Reagent may also be used to deter- 
mine the end point. 
2. —Total Acidity.—Add two or three drops of phenolph- 
thalein to the porcelain dish containing the stomach contents 
which has just been titrated for free hydrochloric acid. Continue 
the titration until a permanent pink color is obtained. This end 
point indicates the neutralization of all acid or acid salts in the 
stomach contents. Report the total acidity as required under 
free hydrochloric acid (remembering to include the free hydro- 
chloric acid in your calculation). 
Pancreatic Digestion 
The experiments illustrating the action of the pancreatic 
juice will be performed with an extract prepared as follows:— 
Digest one part of finely divided pig’s pancreas with four times 
its weight of 25% alcohol, for two or three days, and filter. 
86.— (a) Proteolytic action.—To 25 cc. of a 1% solution 
of Na2C03 add a few drops of the pancreatic extract. Place 
some pieces of fibrin in this liquid and keep in the water-bath 
at 40° C. till the fibrin has disappeared (one to two hours prob- 
ably). Observe the digestion from time to time. Note that the 
fibrin does not swell and dissolve as in gastric digestion, but 
that it is eaten away from the edges. 
Filter. What is the precipitate? Carefully neutralize the 
filtrate with 0.2% HC1. Another precipitate may appear. What 
is this? 
105 Again filter, if necessary, and test the filtrate for proteoses 
and peptones as directed under gastric digestion. 
(b.) Formation of leucin and tyrosin.—Perform a simi- 
lar experiment, using boiled fibrin and adding a few drops of a 
20% alcoholic solution of thymol, or a few drops of chloroform 
water. Why use boiled fibrin, and why add thymol or chloro- 
form? Digest for forty-eight hours, and then examine as fol- 
lows :—Filter, neutralize, and concentrate by evaporation on the 
water-bath. Crystals of tyrosin (and possibly leucin) usually 
separate. Examine microscopically. 
If there is sufficient tyrosin, try Millon’s test. In a portion 
of the residue left after separation of the tyrosin, test for tryp- 
tophan by adding, drop by drop, a very dilute solution of bro- 
mine water. The liquid becomes pale-red, then violet. To the 
remainder of the residue add twice its volume of 95% alcohol, 
which precipitates proteoses and peptones. Filter, concentrate 
by slow evaporation, and set aside to crystallize. Sketch the 
crystals of leucin and tyrosin. 
(c.) Amylolytic action.—To some starch paste in a test- 
tube, add a drop or two of the pancreatic extract and place in 
the water-bath at 40° C. After a few minutes, test for sugar 
and report the result. 
(d.) The lypolvtic (fat-splitting) action.—For the demon- 
stration of this action, refer to experiment No. 24, page 36. 
106 URINE ANALYSIS 
All urines for quantitative analysis should be collected over 
a definite period, usually 24 hours. Folin gives the following 
specific directions for this collection. He says, “The only cor- 
rect way to collect 24 hour urines is to begin the metabolism 
period immediately after passing the night urine in the morning 
(i.e., immediately upon rising). Note the time and then collect 
all the urine passed up to the same hour the following morning. 
Urines so collected must be placed in a cool and preferably 
dark place. Unless the entire analysis can be completed within 
12 hours a preservative should be added. As these urines are 
not to be analyzed for glucose, the most convenient method of 
preserving the specimen is to add 5 cc. of a 5% solution of 
thymol in chloroform. Since most samples require a preserva- 
tive, it is best, as a matter of routine, to place the thymol-chloro- 
form solution in the container before any urine is added. In 
this way complete preservation of all material is assured. 
When all urine for the period has been collected, it should 
be carefully measured, the specific gravity taken, the color and 
the reaction to litmus paper noted. If time is not available for 
the complete analysis of the urine at once, the estimation of 
uric acid, creatinine, ammonia, acidity and phosphates should be 
made the first day. If the specimen has been properly pre- 
served, the remaining urinary constituents may be estimated at 
a later laboratory period. 
In order that the student may have an idea as to the val- 
ues he may reasonably expect from his analyses, the following 
table, taken from Folin (Am. Jour, of Physiol. XIII.) is ap- 
pended. 
Diet. 
High Protein 
Low Protein 
Volume of urine. .. 1170 
cc 
. .385 
cc. 
Total Nitrogen .. . 
16.08 
gms. 
.. 3.60 
gms. 
Urea-Nitrogen .... 
14.07 
87.5%.. 
... 2.20 
61.7% 
Ammonia-Nitrogen. 
0.49 . 
3.0%... 
... 0.42. 
... .11.3% 
Uric Acid-Nitrogen. 
0.18 
1.1%.. 
.. . 0.49 
2.5% 
Creatinin-Nitrogen. 
0.53 
3.6%.. 
... 0.60. 
17.2% 
Undeterm. Nitrogen 
0.85 
4.9%.., 
... 0.27. 
7.3% 
107 Total Sulph. as SOa 
3.64. . 
... 0.76.. 
« 
Inorganic Sulphates 
(SO.) 
3.27 • - 
. .90.0%.. 
.. 0.46.. 
. .60.5% 
Etherial Sulphates 
(SO.) 
0.19.. 
.. 5.2%.. 
.. 0.10.. 
. .13.2% 
Neutral Sulphur 
(SO,) 
0.18 . . 
.. 4.8%.. 
. . 0.20. . 
. .26.3% 
Titrable acidity cc. 
N/10 
805 0 .. 
. .224.0 .. 
Mineral Acidity .. . 
389. • - 
..123. .. 
Organic acidity ... 
407. .. 
..201. .. 
Total Phosphates, 
gms. P205 
4.1 .. 
.. 1.0 .. 
Chlorine gms. Cl,.* 
6.1 .. 
.. 1.6 .. 
87. Total Nitrogen. 
(a.) Kjeldahl Method: 
Into an 800 cc. Kjeldahl flask 
Pipette 5 cc. of Urine. 
Add 15 cc. concentrated sulphuric acid 
Add 2 cc. of 5% copper sulphate 
Add a few glass beads or bits of glass to prevent bump- 
ing. Place on the digestion rack and digest until clear, or at 
most, a light straw color. Cool. Add about 400 cc. of dis- 
tilled water and 2 drops of alizarine red; then prepare a receiv- 
ing flask containing 25 or 30 cc. of N/10 HC1, which is placed 
in position below the still. Place the Kjeldahl flask on the rack. 
Add the calculated quantity of strong sodium hydroxide, stopper 
the flask, rotate to mix the caustic soda, then light the burner 
and distil as directed under b, page 10. 
Note: To determine the proper amount of alkali required 
for the Kjeldahl distillation, pipette 1 cc. of concentrated sul- 
phuric acid into a beaker containing about 25 cc. of distilled 
water. Add a drop of alizarin red, then add the strong caustic 
soda from a Mohr pipette, (noting the yolume), drop by drop, 
stirring the beaker after the addition of each drop, until the acid 
is neutralized. From the amount required to neutralize 1 cc. 
of concentrated sulphuric acid, calculate the quantity needed for 
108 15 cc. To this figure add 3 cc. to insure a slight excess of alkali. 
Calculate the total nitrogen in the total 24 hour specimen of 
urine, and record this value in the table. 
(b.) Folin-Farmer Method: 
Preparing a urea solution so that 1 cc. contains approxi- 
mately 1 mg. of nitrogen. Pipette out by means of the Ostwald 
pipette, 1 cc. of diluted urea solution into a large Pyrex test tube 
(200 x 25 mm.). Add 1 cc. of concentrated H2S04, 1 gram 
of K,S04, 1 drop of 10% CuS04 solution, and a glass bead. 
Boil over a micro-burner for ten minutes (in the hood). Al- 
low the digestion mixture to cool until it just becomes viscous. 
(Tt must not solidify.) Then add about 6 cc. of water, at first 
adding a few drops at a time and shaking the tube gently, and 
then adding it more rapidly. If the tube is allowed to cool too 
much, the water is liable to cause the contents to solidify. Tn 
this case it is best to add the water rapidly and with vigorous 
shaking. In adding the water, care should be taken to wash 
down the sides of the test tube. Place a piece of rubber tubing 
about one and one-half inches long, provided with a pinch-cock, 
over the end of the long glass tube, which is inserted through 
one hole of the rubber stopper belonging to the aeration appar- 
atus. Draw up 3 cc. of sodic hvdrate into this tube, and retain 
by means of a pinch cock. (The tube should be marked at this 
point bv a scratch.) Stopper the tube securely with this appar- 
atus, taking care that the long glass tube does not project into 
the test tube so far as to be shoved through the bottom. Con- 
nect the apparatus to the absorption tube placed in a receiving 
flask. The receiving flask should contain 10 cc. of N/50 HO, 
to which has been added 100 cc. of water and 3 drops of alizarine 
red. Care should be taken in connecting the apparatus to have 
all joints air tight and the absorption tube extend to the bottom 
of the receiving flask. Release the pinch-cock, thus allowing 
the alkali to flow into the test tube. Connect with the com- 
pressed air and turn on carefully, so that one or two bubbles 
shall mix the solutions. Run the air slowly for two minutes, 
then rapidly for fifteen minutes. At the end of this time all of 
the ammonia has gone over. Remove the delivery tube and 
109 rinse it with water into the receiving flask. Titrate the excess 
of acid with N/50 sodic hydrate solution. Calculate from the 
results of analysis the nitrogen content of the urea solution, 
and compare with the theoretical figure. Repeat the determina- 
tion upon this solution until consistent and accurate results are 
obtained. 
Apply the above method to urine as follows: 
Take the specific gravity of the urine and dilute accord- 
ing to the following table: 
Sp. Gr. 
Dilution 
Take cc. 
Dilute to 
1.018 or less 
10 cc. 
50 cc. 
1.018 to 1.030 
.... 10 times 
10 cc. 
100 cc. 
1.030 or over . . . . 
20 times 
10 cc. 
200 cc. 
The dilution should be made by means of a pipette and volu- 
metric flask. The diluted urine contains approximately 0.7 to 
1.5 mg. Nitrogen per cc. 
Pipette 1 cc. of this diluted urine into the large Pyrex test 
tube by means of the Ostwald pipette. Proceed as indicated 
above for the urea solution. Check your results against those 
obtained by the Kjeldahl method. 
(c.) Total Nitrogen by Folin’s Direct Nesslerization 
Method: 
The urine is diluted as indicated in the previous method. 
Pipette 1 cc. of diluted urine, using an Ostwald pipette into a 
large Pyrex test tube (200 x 25 mm.). 
Add (ordinary pipette) 2 cc. Phosphoric-Sulphuric acid di- 
gestion mixture. Add a glass bead or pebble. 
Heat over a micro-burner, having the test tube only a few 
cm. above the top of the burner and using a low flame; until 
nearly all the water has been driven off. When foaming stops 
and dense sulphuric acid fumes appear within the test tube cover 
it with a small watch .glass (2 to 5 min.) Regulate the flame 
so that only a trace of fume escapes from the tube and continue 
digestion until the material is clear, with only a blue, green, or 
light straw tint (one-half to three min. more). After this color 
is noted (due to the copper) continue the heating about 1 min- 
110 ute. (The total heating time after the watch crystal has been 
placed in the tube should never be less than 2 minutes.) 
Remove the flame and allow the test tube to cool for 2 
mins. Add water and transfer quantitatively to a 100 cc. volu- 
metric flask, using about 65 cc. of water. 
Standard Solution: Pipette into a second 100 cc. volumet- 
ric flask exactly 20 cc. of Standard Ammonium Sulphate solu- 
tion. This contains 1 mg. of Nitrogen. Add 40 cc. distilled 
water. Add 2 cc. acid digestion mixture. 
Nesslerization: To each of the above flasks add 30 cc. 
Folin's modified Nessler Reagent. Fill to the mark with dis- 
tilled water; stopper with a clean rubber stopper and mix. The 
Nesslerization of the Standard and Unknown should be done as 
nearly together as possible. A sediment may develop. This can 
be removed by centrifuging a portion of the sample. If the 
sediment is reddish in color, the determination is of no value 
and should be repeated. 
Set the standard for 20 mm. in the colorimeter and after 
checking against itself (by placing some of the Standard in the 
cup) make the color comparison of the unknown. 
Calculate the total nitrogen of the 24 hour sample of urine 
bv the following equation : 
20 
reading x dilution x cc- *n 24 hr. specimen = grams of nitro- 
gen. 
Solutions Required 
L Standard Ammonium Sulphate Solution. 20 cc. = 1 
milligram of nitrogen. 
Weigh out exactly 0.2358 gram of special ammon- 
ium sulphate (see Folin and Farmer, Jour. Bio. Chern. 
XI., 496, 1912). Dissolve in water and make to 1 
liter. Preserve with chloroform. 
2. Phosphoric-Sulphuric Acid Digestion Mixture. 
Measure 300 cc. HsP04 (85%) into a tall cyl- 
inder. Add 100 cc. cone. H2S04—set aside for pre- 
cipitation of CaS04. (Settles slowly.) 
111 Pipette off 100 cc. of clear acid mixture. 
Add 10 cc. of 6% CuS04, then add 100 cc. dis- 
tilled H20. 
3. Nessler's Solution: Folin’s Modification of the Ness- 
ler-Winkler Reagent. 
v(a.) Prepare a mercuric potassium iodide solu- 
tion ' as follows: 
Take a 500 cc. Pyrex Florence Flask; 
Add 150 gms. potassium iodide, 110 gms. iodine, 
100 cc. distilled water, 140-150 gms. mercury. 
Shake vigorously for 7 to 15 minutes, or until dis- 
solved iodine has nearly disappeared. Solution becomes 
hot. Watch the color. When red color has faded to 
pale red, cool in running water, shaking continuously 
until reddish iodine color has been replaced by green- 
ish color of double iodide. Time of complete operation 
about 15 mins. Decant liquid from the mercury. Wash 
mercury thoroughly, with a number of changes of dis- 
tilled water, adding to decanted part of liquid. Finally 
make decanted liquid up to 2 liters with distilled water. 
Final solutions should be nearly clear. 
(b.) Prepare a Saturated Sodium Hydroxide So- 
lution. 
Prepare an adequate amount of completely satur- 
ated sodium hydroxide solution, which requires about 
55 gms. NaOH per 100 cc. Allow to cool, then decant 
clear supernatant liquid. Determine strength of NaOIi 
by titration, then calculate quantity necessary to make 
a 10% solution. Allowable variation 9.5% to 10.5%. 
Prepare the Nessler's Solution 
Select a large bottle, preferably of brown glass, having a 
capacity of over 5 liters. 
| Into the bottle pour 3500 cc. of 10% sodium hydroxide 
solution. Add 750 cc. of the double iodide solution; 
Add 750 cc. distilled water. 
Mix thoroughly by shaking. 
112 15 cc. of the above Nessler's solution contains enough alkali 
for the neutralization of 1 cc. of phosphoric-sulphuric acid mix- 
ture. It has also the proper alkalinity for the development of 
color with ammonia matching the standard solution at a volume 
of 50 cc. 
Note: It is sometimes difficult to obtain a clear solution 
when Nessler’s reagent is added to so concentrated a solution as 
that contained in the 100 cc. flask. Muddy solutions are of no 
value. If it is necessary a 200 cc. volumetric flask may be 
used, the solutions diluted to about 150 cc. before the addition 
of 30 cc. of Nessler’s reagent. 
It is imperative that the Nessler's reagent be free from 
suspended matter. If necessary such material may be removed 
by filtering the solution through the asbestos mat of a freshly 
prepared Gooch crucible, using suction. 
88.—Determination of Ammonia Nitrogen 
(a.) Folin-Macallum Areation Method: 
The student should learn the technic of this method 
using the solution prepared as directed in No. 8, page 
9. (1 cc. contains between 1-1.25 mgs. nitrogen). Con- 
sistent results checking with the theoretical should be 
obtained before proceeding with urine. 
Procedure: Using the Ostwald pipette, measure 
out 2 cc. of the ammonium sulphate solution into a 
large Pyrex test tube. Add one-half cc. of the am- 
monia reagent and 2 drops of kerosene to the tube, 
and stopper immediately. Connect the delivery tube 
with the absorption apparatus and with the flask con- 
taining 10 cc. N/50 hydrochloric acid as in the total 
nitrogen method above. The air current should run 
for fifteen minutes. Remove the delivery tube, rins- 
ing it with water and titrate as before. Compare the 
nitrogen content of the solution with the theoretical 
and with that previously obtained. Repeat your de- 
terminations until consistent and accurate results are 
obtained. 
113 When the method has been learned, substitute 2 
cc. of urine for the 2 cc. of ammonium sulphate solu- 
tion and proceed as directed. Calculate the grams of 
ammonia nitrogen in the 24 hour specimen, and also its 
percentage of the total nitrogen. 
Note: The ammonia reagent is prepared by dis- 
solving 150 grams of potassium carbonate in water, 
adding to this 150 grams of potassium oxalate dis- 
solved in water. Finally add water until the total vol- 
ume is 1 liter. 
The potassium oxalate is added to prevent the pos- 
sibility of loss of ammonia through formation of an 
insoluble ammonium magnesium phosphate compound. 
(b.) Permutit Method'—Direct Nesslerization—Folin. 
The student should check this method with 1 cc. 
of the ammonium sulphate solution as in the previous 
method before proceeding with urine. 
Procedure: Weigh out 2 grams of purified Per- 
mutit on the laboratory trip scales. Place it in a 200 
cc. volumetric flask. Add 5 cc. distilled water. 
Add 2 cc. of urine by means of an Ostwald pi- 
pette. 
Rinse urine down with a little water, 1-5 cc. 
Shake gently and continuously for 5 minutes. 
Rinse the permutit to the bottom of the flask with 
25 to 40 cc. of water. 
Decant off the water. 
Add water again (25 to 40 cc.) and decant. 
Repeat the above washing again if the urine con- 
tained much bile. 
To the Permutit, after decanting the last water, 
add 2 cc. of 10% NaOH. Mix by rotating the flask, 
then add water until the flask is about two-thirds full. 
Set this flask aside until you have prepared the Stand- 
ard Ammonium Sulphate solution and have diluted it 
to a similar volume (see below). 
114 Shake the flask for 10 to 20 seconds, then add 20 
cc. of Folin’s modified Nessler reagent, gently shak- 
ing the flask as the Nessler reagent is added. Add 
distilled water until the flask is filled to the mark, then 
stopper with a clean rubber stopper, and mix thoroughly 
by inverting the flask several times. 
Preparation of the Standard Ammonium Sulphate Solution 
Pipette 20 cc. of the Standard Ammonium Sulphate solution 
containing 1 milligram of nitrogen into a second 200 cc. volu- 
metric flask. A weaker standard may be prepared if necessary 
by taking only 15 or 10 cc. of the above solution. 
Add 2 cc. of 10% NaOH. 
Add distilled water until the flask is two-thirds full. 
Add 20 cc. of modified Nessler’s reagent, as directed above. 
Dilute with water to the mark, stopper flask and mix con- 
tents. 
Note: It is important that the standard and unknown 
solutions be nesslerized as nearly together as possible. 
Compare the solutions in the colorimeter, setting the stand- 
ard at 20 mm., as directed under total nitrogen. 
Calculate the amount of ammonia nitrogen in grams in the 
24 hour specimen, and also the percentage of total nitrogen. 
Preparation of a Purified Permutit: Permutit is the trade 
name for an artificial zeolite of complex chemical nature. It may 
be looked upon as a sodium aluminum silicate, in which the 
sodium is easily exchanged for ammonia or vice versa, depending 
upon their respective concentrations. 
In ordering Permutit indicate it as follows, “Permutit pre- 
pared according to Folin” to secure the proper grade. Before 
being used it is washed once with 2% acetic acid and twice with 
distilled water. If desired, this may be done in the volumetric 
flask before the addition of the urine. 
Permutit may be used repeatedly if washed free from al- 
kali and treated with 2% acetic acid as above. The purified ma- 
terial should be air dried, as oven temperature decreases its 
efficiency. 
115 89.—Urea Nitrogen. Folin’s Application of the 
Urease Method. Areation and Direct Nesslerization. 
The student should learn the technic of this method using 
a urea solution of known concentration. If 2 to 3 grams of 
urea be accurately weighed out, dissolved in water and made 
to a volume of 1 liter; 1 cc. of that solution will contain a 
little more than 1 milligram of urea nitrogen. To facilitate the 
class work, a solution so prepared may be obtained from the 
stock room. Caution: This solution requires no further dilu- 
tion as in urine. 
Procedure: Dilute the urine according to its specific grav- 
ity as directed under section b, total nitrogen above. 
Pipette 1 cc. of urea solution or diluted urine into a large 
Pyrex test tube. (This test tube should previously be rinsed 
with nitric acid, to remove any trace of mercury; then well 
washed with distilled water.) 
Add 1 to 2 drops (not more) of phosphate buffer. 
Add 1 cc. of urease solution. 
Place in a beaker of warm water (40° C. to 55° C. as shown 
hy the thermometer) for 10 to 15 minutes. 
The procedure to this point is the same irrespective of the 
mean by which the ammonia formed by urease digestion is to be 
evaluated. Complete the determination by either of the follow- 
ing methods. 
a.—Areation and Titration 
Pipette 10 cc. N/50 hydrochloric acid into a 250 cc. Florence 
flask. Add 2 drops of alizarine red. Use this to receive the 
ammonia, as indicated in the areation method under total nitro- 
gen. 
To the large test tube containing the dilute urine and urease, 
Add 2 drops kerosene. 
Draw 2 cc. 10% NaOlH up into the air tube of the areation 
apparatus, holding it there by the short piece of gum tubing and 
pinch cock fitted over the end (see total nitrogen method). 
Stopper the test tube. 
Connect to the perforated tube in the receiving flask. 
116 Complete the procedure and back titrate with N/50 NaOH 
as directed under the Folin-Farmer method for total nitrogen. 
From your results calculate the number of grams of urea 
nitrogen in the 24-hour specimen. Remembering that this 
method gives not only the urea nitrogen, but also the ammonia 
nitrogen; subtract the ammonia nitrogen as determined under 
No. 88 above, the remainder then equals the grams of urea nitro- 
gen per 24 hours. Calculate also in percentage of the total nitro- 
gen. 
b.—Direct Nesslerization 
At the end of the 15-minutes digestion period, transfer the 
contents of the large Pyrex tube to a 200 cc. volumetric flask, 
rinsing the tube with distilled water and then adding water until 
the flask contains about 150 cc. of fluid. Prepare the standard 
solution as follows: 
Pipette 20 cc. standard ammonium sulphate solution (con- 
taining 1 milligram of nitrogen) into a second 200 cc. volu- 
metric flask. 
Add 1 cc. of urease solution. 
Add 1 to 2 drops of phosphate buffer. 
Add water to a volume of 150 cc. 
To each flask (standard and unknown) add 20 cc. of Ness- 
ler’s reagent. (It is sufficiently accurate to measure out Nessler’s 
reagent with a cylinder). The flasks should be gently rotated 
while the Nessler’s reagent is being added. To each, add dis- 
tilled water to the 200 cc. mark. Stopper, and invert to thor- 
oughly mix the solution. 
Compare the solutions in the colorimeter, setting the stand- 
ard at 20 mm. From this calculate the combined urea and 
ammonia nitrogen in the 24-hour specimen. Subtract the 
ammonia nitrogen, the difference being the urea nitrogen. 
Also calculate the percentage of urea nitrogen to the total 
nitrogen. 
Two solutions require preparation: 
1. Jack Bean Urease Solution: 
Weigh out 3 grams of Permutit on the trip scale. 
Place it in a 250 cc. Florence flask. Wash once with 
117 2% acetic acid, then twice with distilled water. Add 
5 grams of Jack Bean meal, then 100 cc. of 15% alco- 
hol. Shake gently and continuously for 15 minutes. 
Now place a fluted filter paper in a large funnel. Pour 
the material from the flask into the funnel, then cover 
with a watch crystal, collecting the filtrate in a number 
of small bottles. These bottles should be stoppered, 
then placed in the ice box. When required, one bottle 
may be opened and depended upon to retain its strength 
for 1 week. The unopened bottles retain their activity 
for 6 weeks, when kept in the refrigerator. 
2. Phosphate Buffer Solution : 
Folin recommends two phosphate buffer solutions, 
one a mixture of the mono- and di-sodium phosphates 
(1:2) and the second, a solution of 14 grams of sodium 
pyrophosphate (Na4P2O7-10 H2OJ in sufficient half nor- 
mal ortho-phosphoric acid to make 100 cc. 
For work with classes in this laboratory, we prefer the first 
mentioned buffer mixture. It is easily prepared, keeps unchanged 
and gives excellent results. To prepare it, dissolve 69 grams of 
monosodium phosphate (NaH2P04-H20) in warm distilled 
water, add 179 grams of crystallized disodium phosphate 
(Na2HP04- 121HoO) and sufficient water to get it into solution, 
finally making the volume to 1000 cc. when cold. 
90.—Uric Acid 
a. Preparation from Urine, and Qualitative Tests. 
1. Preparation of Uric Acid from Urine. 
Place 500 cc. of urine in a large beaker, and add 
25 grams of ammonium sulphate. Stir until the 
ammonium sulphate is completely dissolved. Add 10 
cc. of strong ammonia and allow to stand over night. 
Crystals of ammonium urate should form. Filter 
these off, and wash two or three times with a little 
water, containing some ammonia. Place the crystals 
in a small beaker containing a few cc. of hydrochloric 
acid, and allow it to stand until the next day. Ex- 
amine the crystals under the microscope. 
118 2. Reactions of Uric Acid. Murexide Test. 
Place a few crystals of uric acid in a small por- 
celain evaporating dish. Add 2 drops of concentrated 
nitric acid, and evaporate cautiously to dryness on 
the water bath, or over a very low flame. A red, or 
yellowish-red color appears. After the residue is 
cooled, add a drop of very dilute ammonium hydrox- 
ide solution. A purplish-red color should appear. The 
development of color is due to the formation of a 
substances called murexide. A purplish-violet color is 
obtained if potassium hydrate is used instead of 
ammonium hydrate. This is due to the formation of 
the potassium salt of murexide. Repeat the test, using 
caffein instead of uric acid. (Note: The color 
developed by means of potassium hydrate disappears 
quickly on warming with uric acid, thus distinguish- 
ing it from purines.) 
3. Folin-Dennis Reaction. 
Dissolve a few crystals of uric acid in 2 cc. of 
very dilute sodic hydrate solution. Add 1 cc. of the 
uric acid reagent (to be described later), and then 
10 cc. of saturated sodium carbonate solution. A 
pronounced blue color should be obtained. 
b. Quantitative Determination of Uric Acid. Titration. 
Folin-Shaffer Method. 
1. Solutions Required: 
One liter of N/20 potassium permanganate solu- 
tion (1.5780 gms. of KMn04 per liter). The Folin- 
Shaflfer reagent, consisting of 500 grams of ammo- 
nium sulphate, 5 grams of uranium acetate, and 60 
cc. of 10% acetic acid in 560 cc. of distilled water. 
A uric acid solution prepared by dissolving 0.5 to 
0.6 grams of uric acid in 600 cc. of water, to which 
has been added 1 gram of lithium carbonate. The 
solution is then diluted to one liter. 
2. Checking of Solutions: 
The above solutions should be checked against 
each other in the following manner: 100 cc. of the 
119 uric acid solution is pipetted out into a beaker. 
Twenty cubic centimeters of concentrated sulphuric 
acid are added to the solution, which is titrated imme- 
diately with N/20 potassium permanganate solution. 
The first permanent pink coloration should be taken 
as an end point. Each cubic centimeter of potassium 
permanganate equals 3.76 mgs. of uric acid. 
3. Preliminary Experiment: 
Measure out two samples of 100 cc. of the uric acid 
solution into a beaker. Add 10 grams of solid 
ammonium sulphate and stir until dissolved. Then 
add 5 cc. of concentrated ammonia. Cover the 
beaker with a watch crystal, and allow it to stand for 
forty-eight hours. Filter off the precipitated ammo- 
nium urate. Open the filter paper and wash the pre- 
cipitate back into the beaker by means of a very fine 
jet of water. Not more or less than 100 cc. of water 
should be used in this operation. Add 20 cc. of con- 
centrated sulphuric acid, and titrate immediately as 
described under 2. The results of the two determina- 
tions should agree. 
4. Determination of Uric Acid in Urine: 
Pipette out 150 cc. of fresh urine into each of 
two beakers. Add exactly 37.50 cc. of the Folin- 
Shaffer reagent.* Stir vigorously. Allow the solu- 
tion to stand for a few minutes until it settles, and 
then decant off the clear liquid. If the solution does 
not settle readily, it should be filtered. Measure out 
125 cc. of the filtrate (which corresponds to 100 cc. 
of urine) into a beaker. Add exactly 5 cc. of con- 
centrated ammonia, and after covering the beaker 
with a watch crytsal, allow it to stand for forty-eight 
hours. At the end of this time, filter off the ammo- 
nium urate onto a small filter paper. Wash the pre- 
cipitate five or six times with small portions of 10% 
ammonium sulphate solution. Wash the precipitate 
back into the beaker, and finish the determination, as 
described above. Calculate the 24-hour quantity of 
120 uric acid and of uric acid nitrogen in the specimen 
of urine. 
♦This reagent precipitates the phosphates in the 
urine. Substances of a mucoid nature, which are 
present, are removed during this precipitation. The 
object of this precipitation is to remove those sub- 
stances other than uric acid, which may be oxidized by 
potassium permanganate. The ammonium sulphate 
serves to decrease the solubility of the ammonium 
urate, which is formed later in the procedure by the 
addition of ammonia. 
c. Colorimetric—Determination of Uric Acid in Urine. 
Benedict-Hitchcock Modification of the Folin-Macal- 
lum-Denis Procedure. 
This method gives trustworthy results with an amount of 
uric acid ranging between 0.7 and 1.3 milligrams. Ordinarily 
2 cc. of urine is sufficient to obtain the required quantity of uric 
acid, but with very dilute urines, 3 cc. or even 4 cc. may be 
required. 
Procedure: Pipette 2-4 cc. urine (Ostwald pipette) into a 
15 cc. centrifuge tube. 
Add water to a volume of 5 cc. 
Add 15-20 drops ammoniacal silver mangesium mixture. 
Mix thoroughly by stirring with small glass rod. 
Centrifuge 1 to 2 minutes. 
Decant all possible fluid. 
Drain standing on filter paper for two minutes. 
Add 2 drops of 5% potassium cyanide. 
Stir with small glass rod previously used for j/2 minute, or 
until the precipitated silver urate is dissolved. 
Add 10 to 15 drops water. 
Stir thoroughly. Any precipitate remaining is probably 
magnesium ammonium phosphate. It will do no harm. 
Add 2 cc. uric acid reagent. 
Stir thoroughly. 
Add 10 cc. of 20% sodium carbonate (anhydrous). 
Transfer to a 50 cc. volumetric flask. 
Allow to stand minute. Dilute to 50 cc. with distilled 
water. 
121 Compare in the colorimeter with a standard, prepared sim- 
ultaneously, in the following manner: 
Uric Acid Standard: 
Pipette 5 cc. standard uric acid solution (1 milligram uric 
acid) into a 50 cc. volumetric flask. 
Add 2 drops of 5% potassium cyanide. 
Add 2 cc. uric acid reagent. 
Add 10 cc. of 20% sodium carbonate. 
Allow to stand y2 minute. Dilute to 50 cc. volume with 
distilled water. 
Set the standard at 15 mm. 
Calculation: = mgs. uric acid in quantity of urine 
unknown ' 
taken. From this calculate the grams of uric acid, and uric acid 
nitrogen per 24 hours. Also calculate the percentage of total 
nitrogen. 
Solutions Required: 
1. Ammoniacal Silver Magnesium Mixture: 
3% silver lactate solution 70 cc. 
Magnesia mixture 30 cc. 
Concentrated ammonium hydroxide 100 cc. 
2. Magnesia Mixture (used above) : 
Dissolve— 
175 gms. magnesium sulphate 
350 gms. ammonium chloride 
in 1400 cc. distilled water. 
Add 700 grams concentrated ammonium hydroxide. 
3. 5% Potassium Cyanide. Label bottle “Poison”—Handle 
with Care. 
4. Modified Uric Acid Reagent. 
Place the following material in a l)4-liter Pyrex flask: 
100 grams of sodium tungstate. 
30 cc. of orthophosphoric acid (85%). 
20 cc. of concentrated hydrochloric acid. 
750 cc. of distilled water. 
Boil for \]/2 hours, using a reflux condenser. Cool, 
make up to 1 liter with distilled water. 
122 5. A 20% solution of sodium carbonate. The anhydrous 
salt must be used. 
6. Standard Uric Acid Solution. Dissolve together: 
9 grams pure dibasic sodium phosphate (Na2HP04- 
12HX>) 
1 gram pure monobasic sodium phosphate (NaH2P04- 
H.O) 
in 300 cc. hot distilled water. 
Filter if not perfectly clear. 
Add hot distilled water to 500 cc. volume. 
When filtered, pour this hot solution upon exactly 
200 milligrams of pure uric acid, suspended in 5 to 
10 cc. of distilled water, in a 1-liter flask. Shake 
gently until the uric acid is completely dissolved. 
Cool, add exactly 1.4 cc. of glacial acetic acid, dilute 
to 1 liter with distilled water, stopper and mix. Add 
5 cc. of chloroform to prevent formation of moulds. 
Five cubic centimeters of this solution contains 
exactly 1 milligram of uric acid. When properly pre- 
served, in a well-stoppered bottle, this solution shows 
no change upon keeping. 
d.—Benedict-Franke Direct Colorimetric Method for Uric Acid. 
This method is the latest and simplest reliable colorimetric 
method for uric acid in urine. If the urine contains albumin, it 
must be removed by slightly acidifying with acetic acid, coagu- 
lated by boiling, and removed by filtration. 
Procedure: Dilute the urine so that 10 cc. will contain 
between 0.15-0.30 mg. uric acid. (Usually 5 cc. of urine diluted 
to 100 cc. is satisfactory.) 
Pipette 10 cc. diluted urine into a 50 cc. volumetric flask. 
Add 5 cc. of 5% sodium cyanide solution from a burette. 
Add 1 cc. of arseno-phosphotungstic acid reagent. 
Mix, by shaking; allow to stand 5 minutes and then dilute 
to 50 cc. with distilled water. 
Prepare at the same time the following standard so that the 
color in the two solutions will be developing simultaneously. 
Standard Solution: Pipette 10 cc. standard uric acid solu- 
tion into a 50 cc. volumetric flask. 
123 Add 5 cc. of 5% sodium cyanide solution from the burette. 
Add 1 cc. of arseno-phosphotungstic acid reagent. 
Mix by shaking; allow to stand 5 minutes, then dilute to 50 
cc. volume as indicated above. 
Compare the solutions in the colorimeter, setting the stand- 
ard at 15 mm. 
15 
Calculation: —milligrams of uric acid in 10 cc. of 
R 
the diluted urine used. 
Calculate the grams of uric acid and of uric acid nitrogen 
in the 24-hour sample. Also calculate the percentage of uric 
acid nitrogen to the total nitrogen. 
Solutions: 
1. Arseno-phosphotungstic Acid Reagent. Put 100 grams 
of sodium tungstate (C. P.) into a 1000 cc. Pyrex flask. Add 
600 cc. of water and when dissolved add 50 grams of pure arsenic 
acid (P205). Add 25 cc. of 85% ortho phosphoric acid and 20 
cc. of concentrated hydrochloric acid. Boil for 20 minutes, cool, 
and dilute to 1000 cc. 
2. A 5% solution of sodium cyanide. This should be pre- 
pared fresh each week. 
3. Standard Uric Acid Solution . Ten cubic centimeters 
of this solution contains 0.2 mg. of uric acid. To prepare it, 
pipette 50 cc. of the phosphate standard solution given on page 
123, No. 6, into a 500 cc. volumetric flask. Add distilled water 
to a volume of 400 cc., then add 25 cc. of dilute hydrochloric 
acid (1 part HC1 plus 9 parts of distilled water), and dilute to 
500 cc. with distilled water. Mix. Prepare a fresh standard each 
week. 
91.—Creatinin 
a. Jaffe Test. Place 100 cc. of urine in a beaker and add 
to it 20 cc. of a 5% alcoholic solution of picric acid. The pre- 
cipitate is a picrate of creatinin. Allow the solution to stand for 
one-half hour, then filter off the crystals and wash with a very 
little cold water. Dissolve a part of the precipitate in dilute 
sodium hydrate solution. Slightly acidify the mixture, and notice 
the change in color. Repeat the experiment with 10 cc. of urine, 
124 adding from 3 to 5 cc. of a saturated aqueous solution of picric 
acid and 1 cc. of a 10% sodium hydroxide solution. The inten- 
sity of the color is dependent upon the amount of creatinin pres- 
ent. Folin makes use of this test for his quantitative estimation 
of creatinin in urine.1 
b. Determination of Creatinin in Urine. 
1. Folin’s Original Method. Place 10 cc. of urine in a 500 
cc. volumetric flask. Add 15 cc. of a saturated aqueous solution 
of picric acid and 5 cc. of 10% sodium hydroxide solution. Mix 
the liquids thoroughly, and allow them to stand for exactly 
five minutes. During this interval pour a little N/2 potassium 
bichromate solution into each of the two cylinders of the Dubosq 
colorimeter. Adjust the depth of the liquid in the left-hand 
cylinder to 8 millimeters. Make a few preliminary readings by 
comparing the color in the two halves of the field. The two 
readings should be identical. At the end of five minutes, fill the 
flask up to the 500 cc. mark with tap water. Shake the solution 
so that it is thoroughly mixed. Read immediately by replacing 
the potassium bichromate solution in the right-hand cell with this 
creatinin solution. If the creatinin content of the urine is such 
that a reading of over 15 or under 5 millimeters is obtained, 
repeat the determination, varying the amount of the urine taken, 
so that the color of the solution may approximate the standard 
more closely. 
When 10 milligrams of pure creatinin are put into solution, 
and the color developed as stated for urine, exactly 8.1 milli- 
meters of this solution balance 8 millimeters of an N/2 potas- 
sium bichromate solution. The potassium bichromate solution 
is permanent in color, whereas the color of the creatinin potas- 
sium picrate fades rapidly. Determine the amount of creatinin 
in the volume of urine taken by the following equation: 
a: 8.1:: 10: x 
where a is the observed reading in millimeters, x is the milli- 
grams of creatinin in the volume of urine taken. The propor- 
tion is inverse on account of the fact that the instrument is so 
constructed that the greater the color, the lower the numerical 
1 A very interesting paper dealing with the nature of the reaction between 
picric acid and creatinin in a cold alkaline solution was published by Chapman. See 
“On Jaffe’s Colorimetric Method for the Estimation of Creatinine.” A. C. Chapman, 
The Analist, XXIV., 475, 1909. 
125 value of the reading. The creatinin equivalent of the potassium 
bichromate is substituted, i. e., 8.1 millimeters. Calculate the 
24-hour quantity of creatinin and also of creatinin per kilogram 
and per pound of your body weight. 
2. Folin’s New Method. Pipette (Ostwald pipette) 1 cc. 
to 2 cc. urine into a 100 cc. volumetric flask. Mark this urine. 
Pipette (Ostwald) 1 cc. of standard creatinin solution (con- 
taining 1 mg. creatinin) into a second 100 cc. volumetric flask. 
Mark this standard. 
To each flask: 
Add 20 cc. of saturated picric acid solution. 
Add (Mohr pipette) 1.5 cc. 10% sodium hydroxide solu- 
tion. 
Allow both flasks to stand for 10 minutes. 
Dilute at the end of 10 minutes to 100 cc. volume. 
Stopper and mix and compare in the colorimeter, setting 
the standard at 20 mm. 
20 
Calculation: — = milligrams of creatinin in the 
unknown 
quantity of urine taken. 
Calculate the total amount of creatinin in the 24-hour sample 
of urine. Also calculate the percentage of creatinin nitrogen. 
Determine the number of milligrams of creatinin per kilogram of 
body weight. 
Solutions Required: 1. The picric acid used for the above 
creatinin estimations must be carefully purified according to 
the method of Folin and Doisy (Jour. Biol. Chem. 28-349, 1916- 
17). From the purified picric acid, as a supply a saturated 
aqueous picric acid should be prepared. 
2. Standard creatinin solution may be most conveniently 
made by weighing out 1.61 grams of creatinin zinc chloride, 
and dissolving it in 1 liter of N/10 hydrochloric acid. One cubic 
centimeter of this solution contains 1 milligram of creatinin. 
92.—Creatin 
Folin has worked out a method whereby the creatin occur- 
ring in the urines of children and adults suffering from severe 
tissue destruction, as in fevers, may be transformed to creatinin, 
126 and estimated along with the pre-formed creatinin. It should 
be remembered that creatin gives no color with an alkaline solu- 
tion of picric acid. Normal adult individuals have no creatin in 
their urines, unless eating large amounts of fleshy foods, in which 
it exists preformed. 
Folin’s Procedure (Creatinin plus Creatin). 
Pippette (Ostwald pipette) 1 cc. urine into a 300 cc. Florence 
flask. 
Add 20 cc. saturated picric acid solution. 
Weigh the flask so prepared on the laboratory trip scale. 
Add 150 cc. water. 
Boil gently for 45 minutes. 
Weigh again, and unless this weight is within 3 or 4 grams 
of the original, return to the flame and boil rapidly to its original 
weight. 
Cool to room temperature. 
Add 1.5 cc. 10% sodium hydroxide. 
Allow to stand 10 minutes. 
Transfer at the end of 10 minutes to a 100 cc. volumetric 
flask. 
Add distilled water to the mark. 
Stopper—shake, etc. 
Compare in the colorimeter with the standard described 
above under Creatinin. The standard should, of course, be 
freshly made up, and the color developed simultaneously in both 
standard and unknown. 
From the value obtained, subtract the value for the pre- 
formed creatinin, the difference is the creatin. Calculate the 
total amount of creatin in the 24-hour specimen of urine. 
Folin gives the following shorter method which may be used 
if an autoclave is available. 
Pipette (Ostwald) 1 cc. of urine into a 100 cc. volumetric 
flask. 
Add 20 cc. saturated picric acid solution. 
Cover mouth of flask with tin foil. 
Heat in autoclave at 115°-120° C. for 20 minutes. 
Cool to room temperature. 
Add 1.5 cc. sodium hydroxide. Proceed as above. 
127 93.—Inorganic Sulphates—Folin Method 
a. The student must now learn to use the Gooch crucible. 
This crucible, when fitted with a proper mat of asbestos, is used 
in place of filter paper to collect the precipitated barium sulphate. 
Furthermore, the subsequent process of ignition and weighing of 
precipitates are most easily accomplished. 
In order to be of most service, the Gooch crucible must be 
provided with an asbestos mat which will permit rapid filtration 
with moderate vacuum, and yet retain the finest particles of 
precipitate. 
To form the asbestos mat, proceed as follows: Shake thor- 
oughly the stock bottle of “asbestos soup,” then pour about 50 
cc. into a small beaker. Place the Gooch crucible in the rubber 
holder, disconnecting the rubber tubing from the suction flask. 
Agitate the asbestos soup in the beaker, then pour about 15 to 20 
cc. into the crucible, allowing it to drain completely. Now apply 
very gentle suction, causing the fibres to “felt” together without 
plugging the holes in the crucible. This should give a firm and 
uniform felt of 1/32 inch thickness. Wash the mat thoroughly 
with distilled water while the suction is turned on, using about 
500 cc. This should remove all loose fibres. Stop suction, dis- 
connect rubber tubing, remove crucible, empty flask, rinse flask 
with distilled water, then return crucible to holder. Open suc- 
tion cock for medium suction and connect tubing to flask. Now 
pour 100 to 200 cc. more distilled water through crucible, exam- 
ining the filtrate for loose asbestos fibres. If none are visible in 
filtrate when poured into beaker for examination, the mat is in 
condition for drying. 
Place the crucible in a small, clean beaker, labeled with your 
name, and put into the drying oven (or steam oven) for 20 
minutes. At the end of this time, remove the crucible and ignite 
over medium Bunsen flame, while resting on a porcelain crucible 
cover, supported by a clay triangle, on an iron tripod. 
After the crucible has been ignited for ten minutes, transfer 
with clean crucible tongs to the dessicator. Allow to cool thor- 
oughly (45 min. at least), weigh, and record weight. Re-ignite, 
cool in dessicator, and re-weigh. Repeat until weights are con- 
stant within 0.0003 gm. 
128 Always handle prepared crucible with clean tongs. When 
crucible is to be used for filtration, always turn on gentle suction 
before fitting into rubber adapter on funnel. Never flood felt in 
bottom of crucible with strong current of water or solution, but 
pour down side of crucible, using stirring rod as guide, with suc- 
tion turned on. Always test crucible before using, for leaks in 
mat by running 100 cc. distilled water through it, examining fil- 
trate for asbestos fibre. If leaks are discovered, discard, and re- 
make mat. Two crucibles should be prepared in the above man- 
ner. 
b. The student should run a sulphate determination upon 
an ammonium sulphate solution of known strength. 
Procedure: Pipette into a small beaker 25 cc. of the ammo- 
nium sulphate solution prepared as directed on page 8, No. 8. 
Dilute with water to about 100 cc. Add 10 cc. of a 20% solu- 
tion of sodium chloride (free from sulphates). Acidify by the 
addition of 5 cc. of concentrated hydrochloric acid. Add care- 
fully, drop by drop and without shaking, 15 cc. of 5% barium 
chloride solution. Allow to stand quietly for V2 hour, then stir to 
completely precipitate all the sulphate. After 1 hour, filter the 
precipitated barium sulphate into the Gooch crucible, and wash 
with distilled water until the filtrate no longer gives a test for 
chlorides. Dry the crucible and precipitate, ignite, cool in the 
dessicator and weigh. From the increase in weight, due to the 
barium sulphate, calculate the amount of sulphur (as S03) in 
the 25 cc. of ammonium sulphate solution. Compare with the 
theoretical figure. 
Repeat the above, using duplicate determinations on 25 cc. of 
urine. From the determinations calculate the amount of inor- 
ganic sulphate (in grams of S03) in the total 24-hour sample. 
Also later, calculate the per cent of inorganic sulphates to the 
total sulphur (as S03). 
Note: Folin claims that the addition of 10 cc. of sodium 
chloride to the urine is unnecessary. He also states that no 
hydrolysis of ethereal sulphates occurs even if the precipitated 
barium sulphate is not filtered off at the end of 1 hour. 
94.—Total Sulphates—Folin Method 
Pipette out 25 cc. of urine into a beaker, and add 5 cc. of 
129 concentrated hydrochloric acid. Cover the beaker with a watch 
crystal and boil very gently for fifteen minutes. Cool; add suffi- 
cient water to make the volume 100 cc., then add barium chloride 
and proceed as in the above determination, No. 93. 
Note: The ethereal sulphates are decomposed when boiled 
with the hydrochloric acid. 
Calculate the total sulphates as S03 in the 24-hour urine. 
Later calculate the percentage to total sulphur in terms of SOs. 
95.—Ethereal Sulphates 
Procedure No. 93 gives the inorganic sulphate content, 
while procedure No. 94 gives the total sulphates occurring in 
urine. The latter contains both the inorganic and the ethereal 
sulphates. It is evident, therefore, that by subtracting the value 
obtained for the inorganic sulphates from that obtained for total 
sulphates, the remainder will represent the ethereal sulphates 
present in urine. Figure this quantity in terms of SOa in the 
24-hour amount of urine. Later calculate its percentage of total 
sulphur (as SOs). 
96.—Total Sulphur — Denis’ Modification of Benedict’s 
Method 
Pipette 25 cc. of urine into a 300 cc. Pyrex beaker. Add 
exactly 5 cc. of the oxidizing reagent. Evaporate the mixture 
to dryness on the water bath. Place in the hot-air oven, and dry 
at 110° C. for one-half hour. Gently heat the beaker over the 
flame of a Fletcher burner until the organic matter is burned off, 
then gradually increase the temperature until the beaker is at a 
dull red heat, and continue at this temperature for ten to fifteen 
minutes. Allow to cool. 
Add 20 cc. of dilute hydrochloric acid (1-4), and warm 
gently until the residue goes completely into solution. Add 100 
cc. of hot water. Heat the solution to boiling and add, drop 
by drop, 25 cc. of 10% barium chloride solution. Filter, wash 
free from chlorides, dry in the oven, and ignite as described 
above. Calculate the total sulphur in terms of sulphur and S03 
in the 24-hour amount of urine. 
(Note: Vigorous oxidation is necessary to decompose cer- 
tain organic sulphur-containing substances in urine. These bodies 
are decomposed, and their sulphur oxidized to sulphate by the 
130 reagent. The value of the so-called “neutral sulphur” is obtained 
by subtracting the value obtained in the determination of total 
sulphates from the value obtained in this determination (both 
being figured as S or SOa). 
All sulphur determinations must be run in duplicate. Solu- 
tion : The oxidizing reagent is made by dissolving 25 gms. of 
copper nitrate, 25 grams of sodium chloride (free from sulphate) 
and 10 grams of ammonium nitrate in water, making the final 
volume to 100 cc. 
97.—Total Phosphates. Quantitative Estimation by the 
Uranium Acetate Method 
Place 50 cc. of urine in a small beaker or a small Erlen- 
meyer flask, and add 5 cc. of a special sodium acetate solution. 
Heat the mixture to boiling. Run into the hot mixture from the 
burette a standardized solution of uranium acetate. The mixture 
should be added in small portions, boiling after each addition, 
and stirred with a glass rod. The faintest permanent brownish- 
red color, which is produced when a drop of the mixture is 
touched against a few crystals of powdered potassium ferrocyan- 
ide on a tile, should be taken as the end point. Caution: Rinse 
rod off before returning to beaker. 
Repeat the determination with another specimen of urine, 
and calculate the total phosphate content of the 24-hour speci- 
men. 
The values should be recorded both in grams of phosphorus 
pentoxide (P205), and as cubic centimeters of tenth-normal acid- 
ity (7.1 milligrams of P205= 1 cc. N/10 acidity). 
Solutions: The sodium acetate solution is prepared by dis- 
solving 100 grams of sodium acetate in 800 cc. of distilled water. 
To this, 100 cc. of 30% acetic acid is added, and the mixture 
made up to 1 liter. 
The uranium acetate solution is prepared by dissolving about 
34 grams of uranium acetate in water. Fifty cubic centimeters 
of strong acetic acid is added. After the salt is in solution, the 
volume is made up to 1 liter with distilled water. The solution 
should be filtered if not perfectly clear. 
The solution is standardized against a monopotassium phos- 
131 phate solution, prepared by weighing out exactly 1.3102 grams 
of recrystallized potassium dihydrogen phosphate (KH2P04), 
dissolving it in water, and diluting to a volume of 250 cc. Fifty 
cubic centimeters of this solution contain 0.1 gram of phosphorus 
pentoxide. Fifty cubic centimeters of the phosphate solution are 
pipetted out into a beaker and 5 cc. of a special sodium acetate 
solution are added. This is heated to boiling, and the uranium 
solution carefully run in until no further precipitation of phos- 
phate is noticed. Continue adding the uranium solution until a 
drop of the mixture, when removed and touched against a few 
crystals of powdered potassium ferrocyanide, produces a faint 
brownish-red color. The quantity of uranium acetate solution 
required to titrate the phosphate mixture is equivalent to 0.1 gm. 
of phosphorus pentoxide (P2Os). The value of 1 cc. of the 
uranium solution is obtained by dividing 0.1 gm. (the value of 
the phosphate solution) by the number of cubic centimeters of 
uranium acetate solution used. This value, when determined, 
should be recorded on the label of the bottle. 
98.—Acidity of Urine—Quantitative Estimation of the Acidity 
of Urine—Folin Method. 
Titrate the acidity of 25 cc. of urine, to which has been 
added 5 cc. of a saturated solution of potassium oxalate. Phenol- 
phthalein should be used as an indicator. Titrate the acidity of 
25 cc. of urine without the addition of potassium oxalate and 
compare with the value obtained in the above titration. The dis- 
turbing influence of calcium salts forming with the phosphates 
present a tri-basic phosphate as the alkali is added, is prevented 
by the addition of the potassium oxalate. The effect of the 
ammonia upon phenolphthalein is also somewhat decreased by 
the addition of this salt. Calculate the total acidity in a 24-hour 
specimen of urine in terms of cubic centimeters of N/10 acid. 
From these figures, what do you conclude as to the condition of 
phosphates in urine? 
99.—Chlorides—Volhard Method 
Pipette out 10 cc. of urine into a 100 cc. volumetric flask. 
Add 50 cc. distilled water, 5 cc. of a 5% ammonium iron alum 
solution, and 5 cc. of concentrated nitric acid. Add 20 cc. of 
standard silver nitrate solution by means of a pipette. Fill up 
to the mark with distilled water, and shake. Filter the mixture, 
132 and pipette our 50 cc. of the filtrate. Titrate this with a standard 
ammonium sulphocyanate solution. The amount of sulphocy- 
anate solution used, when multiplied by 2, gives at once the 
excess of silver nitrate. The difference between this and 20 
represents the quantity required to precipitate all of the chlorides 
in 10 cc. of urine. Calculate the number of milligrams of sodium 
chloride in the 24-hour specimen of urine. 
Solutions: 
Silver nitrate solution contains 7.2650 grams of purest 
obtainable silver nitrate dissolved in water and made up to 250 
cc. volume. One cubic centimeter of this solution equals 10 
milligrams of sodium chloride. 
The ammonium sulphocyanate solution is made by dissolv- 
ing 3.25 gms. of the salt in water and making up to 250 cc. vol- 
ume. One cubic centimeter of this solution should be exactly 
equivalent to 1 cc. of the silver nitrate solution. The solutions 
should be titrated against each other to determine if this ratio is 
correct. 
100.—Jndican—Folin Method 
Put 10 cc. of urine in a long test tube, and add 2 cc. of a 
5% copper sulphate solution and 5 cc. of chloroform. Add to 
this mixture 17 cc. of concentrated hydrochloric acid. Place the 
thumb across the mouth of the test tube, and shake vigorously 
for one-half minute. A blue color, sometimes tinged with red, 
appears in the chloroform. This is due to the presence of indican 
in the urine, and the development of color is proportional to its 
amount. This qualitative test is frequently made roughly quan- 
titative by using the color of Fehling’s solution as a standard, 
calling it 100. 
133 BONE1 
101.—A. Gelatin.—Gelatin is prepared as described on page 87. 
B. Mineral Constituents: 
1. Test for phosphoric acid as follows: To a portion of 
the hydrochloric acid solution from Experiment 81-D-2-A (p. 
87), add nitric acid, then twice its volume of a solution of 
ammonium-molybdate, and warm gently. A yellow precipitate of 
ammonium-phosphomolybdate should form. 
2. Treat some bits of bone with dilute nitric acid, and test 
a small portion for chlorides by the addition of silver nitrate. 
3. Test for calcium and magnesium as follows: Add to 
a portion of the hydrochloric acid solution an excess of ammonia. 
Calcium phosphate and magnesium phosphate are thrown down 
as a white precipitate. Filter off the precipitate, and to the filtrate 
add ammonium oxalate. A white precipitate shows the presence 
of calcium, not in the form of a phosphate. Wash the precipitate 
upon the filter paper and dissolve in dilute hydrochloric acid. 
To this solution add ferric chloride, drop by drop, until a drop 
of the solution gives a yellowish precipitate when mixed with a 
drop of ammonium hydrate. Add sodium carbonate until nearly 
neutralized, and then barium carbonate to precipitate ferric phos- 
phate. Filter, heat the filtrate, and precipitate the barium by 
adding just the right amount of dilute sulphuric acid. Filter 
off the barium sulphate. The filtrate contains calcium and mag- 
nesium phosphates. The calcium phosphate is precipitated after 
making the solution alkaline with ammonia by the addition of 
ammonium oxalate. This salt should be added slowly and as 
long as a precipitate of calcium oxalate forms. Filter off the 
precipitate, and add to the filtrate sodium phosphate, which, in 
the ammoniacal solution, precipitates magnesium as a white am- 
monium magnesium phosphate. 
1 Consult Mathew’s Chapter XV. 
134 MILK1 
102.—Determine the specific gravity of milk. 
Proteins of Milk 
103.—Pipette out 5 cc. of milk into a Kjeldahl flask, and 
add 20 cc. of sulphuric acid, 10 grams of potassium sulphate, 
and 1 cc. of 5% copper sulphate. Heat gently until the charred 
mass ceases to froth. Then increase the heat and continue the 
digestion for about two hours, or until all of the carbonaceous 
matter has been oxidized. From this point, continue the deter- 
mination as described under total nitrogen, Experiment No. 9. 
Knowing the specific gravity and the volume of the milk used, 
determine its nitrogen content. Also figure this result in terms 
of protein by multiplying the nitrogen content by the factor 6.25. 
Caseinogen 
104—Mix about 20 grams of milk with 40 cc. of a saturated 
solution of magnesium sulphate, then add the salt in substance 
until no more will dissolve. The precipitate consists of casein- 
ogen admixed with a little fat and lacto-globulin. Filter off the 
precipitate, wash it thoroughly with a saturated solution of mag- 
nesium sulphate (preserving the filtrate and washings for the 
determination of lactalbumin). Transfer the filter and precipi- 
tate to a Kjeldahl flask, and determine the nitrogen content 
according to the directions given in the previous experiment. 
Calculation: Multiply the total nitrogen by the factor 6.25 to 
obtain the amount of casein. Determine also the percentage of 
casein nitrogen to the total nitrogen. 
Lactalbumin 
105.—To the filtrate and washings from the determination 
of caseinogen add Almen’s2 reagent until no more precipitate 
forms. Filter off the precipitate and determine the nitrogen 
content according to the method given under protein. Multiply 
the total nitrogen by the factor 6.25 to obtain the amount of 
lactalbumin. 
Lactose 
106.—Lactose is prepared in Experiment 50, page 56. 
1 See Mathew’s p. 306. 
2 Almen’s reagent may he prepared by dissolving 5 grams of tannin with 
240 cc. of 50% alcohol and adding 10 cc. of 25% acetic acid. 
135 Determination of Milk Sugar 
107.—Pipette out 50 cc. of milk into a small flask and add 
25 cc. of neutral lead acetate solution. Connect the flask to a 
condenser, and heat the mixture with shaking, over a small flame 
until it begins to boil. Cool quickly under the tap. Filter the 
precipitate onto a small filter, collecting the filtrate in a 100 cc. 
volumetric flask. Repeat the filtration with the same filter until 
the filtrate is perfectly clear. Wash the precipitate on the filter 
with distilled water until the filtrate is made up to a volume of 
100 cc. Determine the sugar in the solution so obtained by 
means of the polariscope. The 200-millimeter tube should be 
used, as the rotation will then give the sugar content of the 50 
cc. of milk taken. 
Determination of Fat 
108.—Babcock Method.—See Exp. 22, page 33. 
136 BLOOD1 
109.—Centrifugalize blood and note the separation into 
three layers? What are these? 
Blood Corpuscles 
110.— (a) Examine a drop of blood under the microscope. 
Sketch the red and white corpuscles. 
(b) Note the difference between the corpuscles of mam- 
mals, and those of birds and reptiles. 
(c) Note the effect upon the red corpuscles produced by 
the addition of (1) water; (2) a concentrated solution of salt. 
(d) Isotonic Solutions.—Starting with a 2.5% salt solu- 
tion, determine by successive dilutions with an equal volume of 
water, the minimum percentage strength of a salt solution from 
which the corpuscles do not take up water. 
Haemoglobin Crystals 
111.—Place a drop of defibrinated rat’s blood on a slide, add 
a drop or two of water, mix, and cover with a cover-glass. 
Sketch the crystals which separate after a few minutes. Or, add 
a few drops of ether to some blood in a test tube, shake thor- 
oughly until the blood becomes “laky,” and then place the tube 
on ice till crystals appear. 
Absorption Spectra 
112.—The principle of the spectroscope will be explained, 
and the absorption bands given by solutions of haemoglobin and 
its derivatives demonstrated. Prepare solutions for examination 
as follows: 
1. Oxy-haemoglobin.—Use dilute blood (1 cc. defibrinated 
blood to 50 cc. of water). 
2. Haemoglobin (reduced haemoglobin).—Add to blood a 
few drops of strong ammonium sulphide, or 1 or 2 drops of 
freshly prepared Stokes’ reagent.2 
Shake the solution of haemoglobin with air, and note the 
rapid change to oxy-haemoglobin. Change the same solution 
1 Mathew’s Chapter XII. 
2 Stokes’s reagent is prepared as follows:—Dissolve 2 parts of ferrous sul- 
phate, and 3 parts of tartaric acid in water, and add ammonia to distinct alkaline re- 
action. There should be no permanent precipitate. 
137 of oxy-haemoglobin to haemoglobin and the reverse two or three 
times and note the facility with which haemoglobin takes up and 
loses oxygen. 
3. Carbon-monoxide haemoglobin. — Pass a current of 
illuminating gas through a dilute oxy-haemoglobin solution for 
a few minutes and filter. Note the change of color. Try the 
efifect on the solution of (1) ammonium sulphide; (2) Stokes’ 
reagent; (3) potassium ferricyanide; (4) shaking with air. Note 
the stability of the compound. 
4. Methaemoglobin. — Add to dilute defibrinated blood 
(1 :15) two drops of a freshly prepared concentrated solution of 
potassium ferricyanide. Note the change. Note the efifect pro- 
duced by the addition of reducing agents. 
5. Acid haematin.—Add a few drops of glacial acetic acid 
to dilute defibrinated blood and warm gently. The color becomes 
brownish owing to the formation of acid haematin. 
6. Alkali haematin.—Add two or three drops of a strong 
NaOH solution to diluted blood, and warm gently till the color 
changes to a brownish green. 
7. Reduced haematin, or haemochromogen.—Add to a 
solution of alkali-haematin a few drops of ammonium sulphide 
and warm gently; or, add to a solution of reduced haemoglobin 
a few drops of concentrated NaOH solution. 
8. Haematoporphyrin (iron-free haematin).—To 10 cc. of 
concentrated sulphuric acid in a test tube add 5 drops of blood, 
and shake thoroughly. Note the wine-red or violet-red color 
and the two absorption bands. Dilute with a large excess of 
water, neutralize the acid with an alkali, dissolve the precipi- 
tated haematoporphyrin in ammonia, and note the four absorp- 
tion bands. 
Make drawings of all the bands seen and compare with a 
table of spectra.1 
Haemin Crystals (Teichmann’s Test) 
113.—Place a bit of powdered dried blood on a glass side, 
1 Hawk’s Practical Physiological Chemistry. 
138 add a minute crystal of NaCl (fresh blood contains sufficient 
NaCl) and two drops of glacial acetic acid. Cover with a cover- 
glass and warm gently over a flame until bubbles appear. On 
cooling, dark-brown rhombic crystals, often crossed, separate 
(chloride of haematin). Similar crystals can be obtained by 
using an alkaline iodide or bromide in place of NaCl. 
To apply this test for the detection of blood pigment in urine, 
coagulate the albumin present by heat, and filter. The blood 
pigment adheres to the precipitate and may be detected as above 
described, using the washed and dried precipitate. The haematin 
may also be precipitated by means of a solution of sodium 
tungstate acidified with acetic acid. 
Coagulation of Blood 
114.— (a) Observe the phenomena of coagulation as it takes 
place (a) in a test tube; (b) in a drop of blood, examined under 
the microscope. 
(b) Add a few cc. of blood to 5 cc. of a 0.75% salt solu- 
tion (normal saline) and filter or place in a test tube surrounded 
by ice till the corpuscles settle. Transfer the plasma to a watch- 
glass, and note the result. Conclusions? 
Conditions Influencing Coagulation 
115.— (a) Cold.—Receive blood into a test tube surrounded 
by ice. Note the effect on coagulation. 
(b) Neutral salts.—Mix one volume of a 27% solution 
of MgS04 with four volumes of blood (salted plasma). Note 
the result. Dilute some of this salted plasma with 10 volumes 
of water. What takes place? 
(c) Oxalate solutions.—Allow 100 cc. of blood to run into 
5 cc. of a 6% solution of potassium oxalate. Mix. The blood 
remains fluid (oxalate plasma). Dilute some of this with water 
and note the result. 
(d) Relation of calcium salts.—To oxalate plasma add a 
few drops of a 2% calcium chloride solution. What is the result? 
Explain. 
139 Proteins of Blood-plasma 
116.— 
(a) Serum-albumin. 
(b) Serium-globulin. 
Using blood serum, separate and identify these two proteins. 
(c) Fibrinogen.—Fibrinogen is a globulin found in blood, 
lymph, etc., along with paraglobulin. Like paraglobulin it re- 
sponds to all the general precipitants and tests, and in addition 
gives the reactions with C02, dialysis, and MgS04. It is easily 
distinguished from paraglobulin by two reactions, viz., its power 
to coagulate, i. e., to form fibrin when acted on by fibrin ferment, 
and its temperature of heat coagulation. Demonstrate these 
two properties in this way: 
1. Take two specimens, one of salted plasma and one of 
serum. Dilute each five or ten times with water. The salted 
plasma will colt after a certain time, owing to the presence of 
the fibrinogen; the serum will not clot. 
2. Take two other specimens, one of salted plasma and one 
of serum, and making use of the apparatus for the determination 
of the temperature of coagulation, heat them both slowly to 
50°-60° C. The salted plasma will show a scanty precipitate of 
coagulated fibrinogen. The serum will not be affected. Dilute 
the heated specimen of salted plasma five to ten times; no clotting 
will occur, as all the fibrinogen has been removed. 
Fibrin 
117. 
(a) Note its physical properties. 
(b) Note action of 0.2% hydrochloric acid. 
(c) Apply the protein color tests. 
118. —What are the proteins of plasma; serum; clot? What 
is the difference between serum and defibrinated blood? 
140 BILE1 
Color 
119.—What is the difference between the color of human 
bile and ox bile? Explain. 
Reaction 
120.—Dilute some bile with four parts of water. Immerse 
a strip of red litmus paper, then remove and wash with water. 
Note the reaction. 
NUCLEO-ALBUMIN 
121.—Dilute bile with twice its volume of water, filter if 
necessary, and add acetic acid. What is the precipitate? How 
distinguished from mucin? 
122.—Filter (above) and test the filtrate for proteins. Report 
the result. 
Separation of Bile Salts 
123.—Mix 20 cc. of bile with animal charcoal to form a 
thick paste, and evaporate on the water-bath to complete dryness. 
Pulverize the residue in a mortar, transfer to a flask, add 25 c.c 
of absolute alcohol, and heat on the water-bath for half an hour 
Filter. To the filtrate add ether till a permanent precipitate 
forms. Let the mixture stand for a day or two and then filter 
off the crystalline deposit of bile salts. Save the filtrate which 
contains cholesterin. 
Pettenkofer’s Test for Bile Acids 
124.—Put some bile diluted with an equal volume of water 
in a wine glass, add a few drops of a 10% solution of cane sugar, 
then incline the glass and pour concentrated H2S04 carefully 
down the side of the glass, so as to form a layer beneath the 
bile. Mix the two liquids gradually by stirring with a glass rod. 
A cherry red, changing to a reddish purple color, soon develops. 
The success of this test depends upon keeping the temperature 
below 70° C. Explain the reaction. 
125.—Repeat above, using a dilute solution of bile salts. 
2 Mathews. Paere 40fi. 
141 Bile Pigments 
126.— (a) Gmelin’s Test.—Take some bile in a wine glass 
and add yellow HN03 in the manner described for H2S04 in 
No. 124. Notice the play of colors—beginning with green and 
passing through blue, violet, and red, to yellow—at the junction 
of the two liquids. Explain. 
(b) Iodine Test.—Place 10 cc. of dilute bile in a test tube, 
and add slowly two or three cc. of dilute tincture of iodine, so 
that it forms an upper layer. A bright green ring forms at the 
line of contact. (Note: There are several modifications of 
Pettenkofer’s test and of Gmelin’s test.) 
Cholesterin 
127.—Examine under the microscope the crystals obtained 
by the cautious evaporation of the alcohol-ether filtrate of No. 
123 or that obtained under No. 128. 
For color reactions, refer to demonstrations. 
Analysis of Biliary Calculi 
128.—Pulverize, and extract several times with water to 
remove bile. Dry and extract with a mixture containing equal 
parts of alcohol and ether, which dissolves cholesterin. If there 
is a residue insoluble in the alcohol and ether mixture, treat it 
with dilute HC1. An effervescence indicates a carbonate 
(CaCOs). Copper, if present, will be found in the HC1 solution. 
Any insoluble residue still remaining is washed with water and 
treated with chloroform which dissolves bilirubin. For purifica- 
tion remove chloroform by evaporation, treat with alcohol and 
ether, re-dissolve in chloroform, filter if necessary, and evaporate 
till bilirubin begins to separate. Upon the addition of alcohol, 
bilirubin separates as an orange precipitate. 
Action of Bile in Digestion 
129.— (a) Take three test tubes. In one mix 10 cc. of 
bile and 2 cc. of neutral olive oil: in the second, 10 cc. of bile 
and 2 cc. of rancid olive oil; in the third, 10 cc. of water and 
2 cc. of neutral oil. Shake and place in a water bath at 40° C. 
for some time. Note the extent and the permanency of the 
emulsion in each case. 
142 (b) Into each of two funnels fit a filter-paper. Moisten 
one with water, and the other with bile, and into each pour an 
equal volume of olive oil. Set aside for twelve hours (with a 
beaker under each funnel). Do you notice any difference in the 
rate of filtration? 
143 THE VITTU PRINT SHOP 
CHICAGO