QUANTITATIVE DROP ANALYSIS

XIII. THE FORMOL TITRATION OF AMINO NITROGEN*

BY ROBERT C. SISCO, BURRIS CUNNINGHAM, AND PAUL L. KIRK

(From the Division of Biochemistry, University of California Medical School,
Berkeley)

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(Received for publication, January 8, 1941)
In the course of certain studies of the metabolism and nitrogen
utilization of animal tissues growing in in vitro culture, it became
necessary to determine changes in amino nitrogen content of the
culture medium. This material, composed of blood plasma and
embryo extract or fractions of these constituents, was available
only in small quantity and had a very low content of amino nitro-
gen, making necessary the application of drop scale methods for
its analysis.
Although numerous procedures have been employed for the
determination of amino nitrogen in various types of materials,
relatively few have been found applicable for ultramicrodetermina-
tion of this constituent. Linderstrom-Lang and Holter (1)
first adapted the alcohol titration method to the ultramicro scale
for determination of hydrolysis of peptides of low molecular weight
by thin sections of plant and animal tissues. In this type of
substrate, the method was shown to be both adequate and simple.
For application to biological materials of more complex composi-
tion, the form01 titration originated by Sorensen (2) has proved
more satisfactory on the macro scale than most other methods.
In line with this, Borsook and Dubnoff (3) recently developed an
ultramicroprocedure in which the form01 titration was carried out
electrometrically with the glass electrode, along the lines in-
vestigated earlier by Dunn and Loshakoff (4). No data were
quoted for evaluation of their procedure except a claim of better
than f2 per cent accuracy. It is well recognized (5, 2) that the
* Aided by grants from the Rockefeller Foundation and the Research
Board of the University of California.
1
2 Quantitative Drop Analysis. XIII

form01 titration is an empirical method which yields quite different

recoveries with different amino acids and with different titration
conditions. With the unknown mixtures found in biological
materials, the recovery will not usually be quantitative or known,
but may have considerable comparative value. Only a-limited
number of .amino acids yield recoveries of approximately 100 per
cent, a few dropping to values as low as 50 to 80 per cent. In view
of these facts, it is possible that the claim of Borsook and Dubnoff
refers to precision rather than accuracy, or that compensating
errors in their particular material balanced the known deficiencies

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of recovery.
It seemed desirable to ascertain whether indicators could be
used for drop scale determinations of amino nitrogen by the drop
scale form01 titration, and to compare the data so obtained with
those obtained by the procedure of Borsook and Dubnoff. The
method here described, while definitely simpler than the electro-
metric titration, is shown to have a comparable accuracy and
precision when tested with pure amino acids, and to agree quite
well with the published data obtained by use of the macro form01
titration. Its application to the analysis of embryo extract was
also incompletely studied in connection with recovery of added
amino acids when various deproteinizing agents were used. This
work will be presented in detail in a later publication.

EXPERIMENTAL

Reagents-
1. Stock amino acid solutions were prepared by weighing the
proper quantity of each to yield a 0.01 M solution, with purified
and analyzed samples of amino acid and redistilled water. The
amino acids used were glycine, glutamic acid, serine, lysine,
histidine, and phenylalanine, all of which were highly purified
samples available in the laboratory.
2. Formaldehyde solutions were prepared from Merck reagent
grade formaldehyde, 36 to 38 per cent, diluted with redistilled
water to various strengths, usually 1 volume of formaldehyde
with 2 volumes of water.
3. Indicator solution, usually phenolphthalein in 0.03 per cent
solution in 50 per cent alcohol. This was diluted with an equal
volume of water before use.
 Sisco, Cunningham, and Kirk 3

Apparatus-The apparatus used was, for the most part, the

standard drop analysis equipment,l a preliminary description of
which has been published by Kirk (6). The burette was an im-
proved form of the universal capillary type shown, respectively,

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FIG. 1. Front assembly of the universal type capillary burette

FIG. 2. Rear assembly of the universal type capillary burette

in front and rear views in Figs. 1 and 2. It had a total capacity of

about 0.1 ml., and was capableof a precision in reading of ho.03
1 All apparatus, except electrical measuring instruments, was obtained
from the Microchemical Specialties Company, Berkeley.
4 Quantitative Drop Analysis. XIII

L2 Samples and reagents were measured with the same pre-

cision by use of capillary pipettes calibrated with mercury for
content, and washed out with a little water to avoid drainage
errors. Their form and that of the pipette control are shown in

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FIG. 3. Capillary pipette and syringe control

7---- ..--

I.
f /iI -

I\\\\\\\\\\W
FIG. 4. Cross-section of the glass electrode titration vessel. B repre-
sents an inverted glass electrode; C, a burette; D, a reference calomel cell;
E, a glass tube; A, cup assembly, consisting of a, a central block; b, a lower
cup; c, an upper inverted cup.

cross-section in Fig. 3. The pipettes were constructed with a

taper on the upper end so as to slip through a rubber gasket
encasedin a metal fitting on the end of a 0.5 ml. syringe barrel.
* 1 X = 1 microliter = 0.001 ml. (6).
 Sisco, Cunningham, and Kirk 5

Electrometric titrations were performed by use of a glass

electrode used in conjunction with a vacuum tube galvanometer
and a Leeds and Northrup hydrogen ion potentiometer. The
details of the glass electrode vessel may be seen in cross-section in
Fig. 4. B is an inverted glass electrode constructed from Corning
No. 015 glass by blowing a bulb and sucking in a depression, the
latter serving as a titration vessel. All of the bulb except the
depression was coated with paraffin on the outside. The bulb
was filled with 0.1 N hydrochloric acid solution saturated with
quinhydrone, inverted, and inserted into the chamber, the lower

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part of which had been filled with the same reference solution.
The outside chamber A was constructed of the synthetic plastic,
lucite, by use of a lathe, being made in three parts, b a lower cup,
a a central block drilled as shown, and an upper inverted cup (c)
also drilled for insertion of a reference calomel cell D, a burette C,
and a glass tube E. Through the last, nitrogen was blown on
the surface of the sample to produce a whirling action, which
effectively stirred it and prevented entrance of atmospheric
carbon dioxide during titration.

Procedure
Indicator Method-A sample (about 50 X of 0.01 M amino acid
solution) was carefully measured with rinsing into a porcelain
titrating dish. To this a known quantity (usually about 10 X)
of diluted phenolphthalein solution was added, and the sample
titrated to a faint pink color by use of standard (about 0.02 N)
sodium hydroxide. This preliminary end-point was chosen be-
cause it obviated the necessity of using a second indicator. A
volume of formaldehyde, diluted as stated above and equal to the
volume of the sample, was then added. The mixture was again
titrated to the phenolphthalein end-point while being stirred.
A blank value was determined with distilled water instead of the
sample to correct for indicator and formaldehyde titers. These
values normally ran from 0.5 to 1.0 X. The volume used in the
second titration of the sample was then corrected by subtraction
of the corresponding blank and calculated in terms of amino
nitrogen. In case of ammonia or ammonium salts being present
in the sample, it is necessary to make proper analysis for their
presence. Theoretically, the ammonium ion is nearly quantita-
6 Quantitative Drop Analysis. XIII

tively determinable by use of the form01 titration, but the re-

covery is, to a considerable extent, dependent on the particular
shade of pink chosen as the end-point before addition of formalde-
hyde. Complete recovery of ammonia may not be obtained by
the method given, and it is always advisable to run a controlled
determination on known amounts of ammonia in order to make
proper allowance for this factor. The independent estimation of
ammonia and the application of the recovery factor properly
corrected the amino nitrogen values for the ammonia present.
Electrometric Method-With the electrometric set-up as de-

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scribed, the titration was run by essentially the same method as
outlined by Borsook and Dubnoff (3). In this case, the sample
was measured as before and placed in the depressed cup of the
glass electrode. It was adjusted to pH 7, the electrode being
used to determine this point. Formaldehyde previously adjusted
to pH 5 was added in equal volume, and the mixture titrated to a
final pH of 8. A blank value was determined as before and
subtracted from the titer of the unknown. In order to assure the
reliability of the electrode, frequent checks against standard
buffers were made, and a constancy to ZIZ~millivolts throughout
the period of titration was maintained.

Results
Indicator Method-In developing a proper procedure for drop
scaleform01 titration, it was necessary to study the various factors
concerned. There are only two important variables which may
cause difficulty; namely, the effect of formaldehyde concentration
and the effect of the indicator chosen. The first factor was studied
by making a series of determinations on 0.01 M solution of serine,
with variable concentrations of formaldehyde. The data are
shown in Table I. It is readily apparent that the optimum con-
centration was 1 volume of the formaldehyde diluted with 2
volumes of redistilled water to give a final formaldehyde con-
centration after titration of about 2.7 per cent. This figure is
definitely lower than that used by Sorensen (2), and was somewhat
lower than that of Borsook and Dubnoff (3). That this factor
may vary on the drop scale from the optimum concentration
when larger volumes are used is entirely possible and not contrary
to experience with other reagents. In order to check the effect
of the end-point chosen, thymolphthalein, methyl red, phenol red,
 Sisco, Cunningham, and Kirk 7
cY-naphtholphthalein, phenolphthalein, and alizarin yellow were
used. Of this group of indicators, phenolphthalein was found to
be definitely the most favorable. Thymolphthalein has been
widely used, but this indicator did not allow a clear end-point on
the drop scale. The other indicators, except phenol red, gave
poor end-points in the presence of formaldehyde. Phenolphthalein
gave better recoveries than the latter indicator owing to its higher
pH of color change. Some fading of end-point is occasionally
encountered with phenolphthalein owing to absorption of carbon
dioxide from the atmosphere. Appreciable error from this source

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was avoided by titrating reasonably rapidly to the first perceptible
pink, and disregarding fading after this point. Table II shows
TABLE I
Effect of Formaldehyde Concentration
Sample, 50.87 X of 0.01 M serine solution = 7.12 y of N.
-
Final
Formaldehyde dilution formaldehyde Recovery
concentration

per cent Y per cent

36-38% undiluted 8 4.70 66
4.76 67
1 volume HCHO to 1 volume re- 4 5.84 82
distilled water 5.48 77
1 volume HCHO to 2 volumes re- 2.7 6.62 93
distilled water 6.83 96
1 volume HCHO to 3 volumes re- 2 6.05 85
distilled water 6.19 87

the results of the application of this simple titration method to the

determination of amino nitrogen in six amino acids. These were
chosen to represent all of the general types of amino acids without
any effort to include all of the available ones. Recovery data
indicate that, with the exception of certain neutral and dicar-
boxylic acids, definitely low results are to be expected. This
finding is again in accordance with that of S@rensenand subsequent
investigators. In general, the data agree rather closely with those
of S@rensen.
In view of the rather wide use of glass electrodes as indicator
instruments in the form01 titration, the electrode technique de-
scribed was applied to the same series of amino acids. The
details of formaldehyde concentration and end-points chosen were
8 Quantitative Drop Analysis. XIII

taken from Borsook and Dubnoff’s description (3). An initial

pH of 7 was used, and the formaldehyde added had been pre-
viously adjusted to pH 5. The mixture was finally titrated to
pH 8, all of these values being those used by the above authors.
The results of these titrations are shown in Table II.
TABLE II
Indicator and Electrometric Form01 Titrations of 0.01 Y Amino Acid Solutions

Indicator method Electrometric method

Amino acid
“i?? Amino
Am?

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Re- vole Ami Ro-
c( m?rJ ume Ni way
sample fo:d a=1 found
--
x 7 Pm x 7 Y Per
Y cent cent
Glycine 50.87 7.12 7.08 99 93.90 13. 11.62 88
7.01 98 11.78 90
7.09 99 11.49 89
7.09 99 11.62 88
Glutamic acid 50.87 7.12 6.85 96 56.36 7. 7.88 100
7.15 101 8.08 102
32.38 4.53 4.74 104 7.88 100
4.74 104 7.90 101
Serine 50.87 7.12 6.56 92 95.85 13. 13.17 98
6.56 92 12.05 90
6.48 91 12.73 95
6.37 90 11.54 86
Phenylalanine 50.87 7.12 4.97 70 50.87 7. 4.52 64
5.12 72 4.55 64
4.11 58 4.47 63
5.08 71 4.57 64
Histidine 50.87 14.23 9.46 67 50.87 14. 11.61 82
9.56 67 11.52 81
9.41 66 11.57 81
9.55 67 11.64 82
Lysine 50.87 14.23 10.16 72 50.87 14. 6.84 48
10.60 74 7.04 49
9.92 70 7.24 51
10.12 71 7.32 51
-

In three cases, the recovery figures are equal to, or greater than,
those of the indicator method. In the remainder, they are even
lower. The variability between results with the same amino
acid is at least as great in most instances as with the indicator
procedure.
 Sisco, Cunningham, and Kirk 9

The general inferiority of the electrometric results to those of

the indicator method is unquestionably not due to the use of the
glass electrode, but rather to the particular pH values chosen in
the titration. It is, for example, not clear why formaldehyde
solutions should be adjusted to pH 5, which allows a large blank.
As a general rule, a final pH of 9 is theoretically better than one of
8 as used in this study. The choice of these values by the authors
quoted was undoubtedly made from empirical considerations
which were correct for their particular system, but not useful for
pure amino acids nor for systems of other types. In any case, it

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is preferable from a theoretical standpoint to employ a glass
electrode because it is free of most of the criticisms which may be
made of the indicator method. At the same time, the greater

TABLE III
Indicator Form01 Titration of Ammonia
Sample, 73.04 X of 0.01 M ammonium chloride solution = 10.22 y of N.

AmmoniaNfound Recovery
I
pm cent
7.756 74
7.55 74
7.62 74
7.67 75
7.90 77
7.53 74

simplicity and speed of the latter method justify its use for all but
the most critical determinations.
Interference of Ammonia-It is well known that ammonia is
titrated in the form01 titration and, if present along with the
amino acid, will appear as an error in the determination. The
interference from this material may be eliminated by its complete
removal (aeration from the basic solution), or more commonly by
determining the ammonia separately and subtracting an appro-
priate correction. Since the ammonia may not be determined
quantitatively by the form01 titration, the determination of this
correction factor must be made by determining the percentage
recovery of ammonia under the conditions used. Ammonia
has a dissociation constant very close to that of phenolphthalein,
10 Quantitative Drop Analysis. XIII

a fact which makes the recovery quite dependent on the shade of

color chosen in the adjustment of the first end-point. For this
reason, different analysts will obtain different recovery figures
which will, however, usually be quite consistent with the particular
analyst. Table III shows typical data obtained by one analyst
who was running the determination for the first time. It is seen
that a reasonable precision is obtained, yielding a negligible
variation in the amino acid figures when the ammonia recovery is
used merely as a correction.

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SUMMARY

A drop scale form01 titration for determination of amino nitrogen

with simple indicators is described.
The results of the indicator method are compared with results
obtained by the method of Borsook and Dubnoff in which electro-
metric titration is used.
The method is shown to be comparable with the macro form01
titration in recovery and accuracy, but applicable to very much
smaller quantities of material.

BIBLIOGRAPHY

1. LinderstrZm-Lang, K., and Holter, H., Z. physiol. Chem., 201, 9 (1931).

2. SGrensen, S. P. L., B&hem. Z., 7, 45 (1907).
3. Borsook, H.: and Dubnoff, J. W., J. Biol. Chem., 131, 163 (1939).
4. Dunn, M. S., and Loshakoff, A., J. Biol. Chem., 113, 359 (1936).
5. Levy, M., J. Biol. Chem., 99, 767 (1932-33).
6. Kirk, P. L., Mikrochemie, 14, 1 (1933).
QUANTITATIVE DROP ANALYSIS: XIII.
THE FORMOL TITRATION OF AMINO
NITROGEN
Robert C. Sisco, Burris Cunningham and Paul
L. Kirk
J. Biol. Chem. 1941, 139:1-10.

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