QUANTITATIVE INVESTIGATIONS OF AMINO ACIDS AND PEPTIDES I. QUANTITATIVE FORMOL TITRATION BY MEANS OF THE GLASS ELECTRODE * BY MAX S. DUNN AND ABE LOSHAKOFF (From the Chemical Laboratory, University of California at Los Angeles, Los Angeles) (Received for publication, November 18, 1935) A considerable number of the analytical procedures which have been proposed for the quantitative determination of amino acids have been found to be generally applicable to the analysis of amino acids in plant extracts, protein hydrolysates, and clinical fluids, where extreme accuracy is unimportant or unattainable. How- ever, the methods in common use are notably unsatisfactory for the precise analysis of amino acids. Often the results are only semiquantitative because of interfering side reactions, uncertain color changes of indicators at the end-point of titrations, or other uncontrollable factors. The present work of the authors has been predicated on the assumption that none of the older methods’ which has been de- * A preliminary report was given before the Protein Conference at Berkeley, December, 1934. Financial assistance in this work has been received from the research funds of the University of California. The authors are indebted to Professor G. Ross Robertson for valuable suggestions during the course of this work and for the loan of his glass electrode apparatus. 1 The methods referred to here are the Van Slyke nitrous acid (l), the Sorensen form01 titration (2), the Foreman alcohol titration (3), the Linderstrom-Lang acetone titration (4), the Folin fi-naphthoquinone sul- fonate (5), and the Harding-MacLean ninhydrin (6) procedures. The Ashmarin modified form01 titration (7), the Halford molecular boiling point rise (8), and the specific rotation methods were not considered because of their uncertain reliability or limited applicability. The determination of the purity of amino acids by recrystallization to constant solubilities (9), conductivities (lo), or heats of combustion (11) should be dependable, although laborious, procedures. 359 by guest on June 13, 2018 http://www.jbc.org/ Downloaded from
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QUANTITATIVE INVESTIGATIONS OF AMINO ACIDS AND PEPTIDES
I. QUANTITATIVE FORMOL TITRATION BY MEANS OF THE GLASS ELECTRODE *
BY MAX S. DUNN AND ABE LOSHAKOFF
(From the Chemical Laboratory, University of California at Los Angeles, Los Angeles)
(Received for publication, November 18, 1935)
A considerable number of the analytical procedures which have been proposed for the quantitative determination of amino acids have been found to be generally applicable to the analysis of amino acids in plant extracts, protein hydrolysates, and clinical fluids, where extreme accuracy is unimportant or unattainable. How- ever, the methods in common use are notably unsatisfactory for the precise analysis of amino acids. Often the results are only semiquantitative because of interfering side reactions, uncertain color changes of indicators at the end-point of titrations, or other uncontrollable factors.
The present work of the authors has been predicated on the assumption that none of the older methods’ which has been de-
* A preliminary report was given before the Protein Conference at Berkeley, December, 1934.
Financial assistance in this work has been received from the research funds of the University of California.
The authors are indebted to Professor G. Ross Robertson for valuable suggestions during the course of this work and for the loan of his glass electrode apparatus.
1 The methods referred to here are the Van Slyke nitrous acid (l), the Sorensen form01 titration (2), the Foreman alcohol titration (3), the Linderstrom-Lang acetone titration (4), the Folin fi-naphthoquinone sul- fonate (5), and the Harding-MacLean ninhydrin (6) procedures. The Ashmarin modified form01 titration (7), the Halford molecular boiling point rise (8), and the specific rotation methods were not considered because of their uncertain reliability or limited applicability. The determination of the purity of amino acids by recrystallization to constant solubilities (9), conductivities (lo), or heats of combustion (11) should be dependable, although laborious, procedures.
vised for the analysis of amino acids can be relied upon for the attainment of truly quantitative results. It seems probable, as pointed out by Levy (12) for the form01 titration method, that about f0.5 per cent is the limit of accuracy and precision for these procedures.
It has been a common practise to determine the purity of amino acids and peptides by a Kjeldahl or Dumas analysis. While it has been shown that total nitrogen may be estimated by these methods with an error of only 0.1 to 0.2 per cent (13), analyses of this precision indicate less accurately the purity of the material when nitrogenous impurities are present.2
In a recent paper, Nadeau and Branchen (14) described a new method by which amino acids in glacial acetic acid are titrated with perchloric acid by means of crystal violet indicator or the chloranil electrode. Results of high precision were obtained in the direct calorimetric titration of glycine with crystal violet in- dicator and weight burettes. However, deviations of considerably greater magnitude between duplicate determinations as well as between the calorimetric values and those found by micro-Dumas analyses, were reported for ten additional amino acids.
The present method was elaborated because of the need for a procedure by which the purity of amino acids, required for the physicochemical investigations under way in this Laboratory, could be determined with quantitative accuracy. As the result of the authors’ investigations it has been found that amino acids in aqueous-formaldehyde solution may be titrated with standard alkali by means of the glass electrode with both a precision and accuracy of f0.1 per cent.
EXPERIMENTAL
Materials-The amino acids and some of the peptides were highly purified samples prepared in this Laboratory. The pep- tides, obtained from Hoffmann-La Roche, Inc., were used with- out further purification.
The solutions of carbonate-free sodium hydroxide were stand- ardized against acid potassium phthalate and Bureau of Standards
2 If the analytical error from the determination of the nitrogen content of glycylglycine by the Kjeldahl or Dumas method were 0.1 per cent, 1.26 per cent of glycine could be present and escape detection.
benzoic acid with the use of carefully calibrated instruments throughout.
A 37.5 per cent formaldehyde solution of purest commercial grade, treated with basic magnesium carbonate to remove formic acid, was used in all of the experiments. Dearing (15) has stated that formaldehyde may be maintained permanently neutral by treatment with basic magnesium carbonate. In the present ex- periments, there was no visible pink color when 12 ml. of the treated 37.5 per cent formaldehyde solution were filtered, diluted to 50 ml., and tested with 1 ml. of phenolphthalein solution. However, a glass electrode determination showed that this solu- tion was slightly alkaline (pH 7.69) owing, presumably, to dis- solved basic magnesium carbonate. The special purification pro- cedures employed by Euler and Euler (16) and Levy (17) werenot used because the formaldehyde solutions prepared by these authors were slightly acid owing, probably, to formic acid.
Apparatus-The glass electrode apparatus, described by Robert- son (18), was used with a few modifications. A silver-silver chloride electrode, prepared essentially by the method of Mac- Innes and Beattie (19), replaced Robertson’s calomel electrode (CJ. The silver-silver chloride electrode was connected by a 1 N potassium chloride bridge, with liquid junction, to a reference solution of 0.1 N hydrochloric acid in the glass electrode bulb.
The electrodes, made from No. 015 Corning glass, consisted of extremely thin glass bulbs with an average resistance of 0.15 megohms. Resistances were determined by comparison of the glass electrode potentials required to produce a given deflection on the galvanometer scale with the potential needed to give the same deflection with a standard, approximately 2 megohm resist- ance. A maximum change of f2 millivolts was observed during the life of the electrodes (2 or 3 months). Before each titration in which pH values were determined, the glass electrode was stand- ardized against buffers of known pH determined with the quinhy- drone electrode to an accuracy of 0.01 unit. It was shown that the glass electrode acted theoretically as a hydrogen electrode, since the apparent potential interval per pH unit between 2 and 8 was 0.0587 at 23’.
Potentiometric Titration-To carry out a titration, approxi- mately 0.2 to 0.4 gm. of the amino acid, dried for 3 hours at 80”
and 10 mm. in a modified Abderhalden drier, was dissolved in 38 ml. of distilled water. 12 ml. of 37.5 per cent formaldehyde, which had been treated with basic magnesium carbonate and filtered, were added. In the case of alanine, norleucine, phenyl- alanine, and a-aminobutyric acid 45 ml. of distilled water and 20 ml. of formaldehyde solution were used. The motor stirrer was started and stirring continued throughout the titration. About 1 ml. less than the calculated volume of an approximately 0.3 N standard solution of sodium hydroxide was added slowly over a period of about 10 minutes. The potential of the system was measured after about 1 minute. Approximately 0.2 ml. incre- ments of standard base were added, and after 4 minute intervals to permit depolarization of the glass electrode, the voltages were read. Titrations were carried from 0.1 to 1.0 ml. beyond the equivalence point, depending on the magnitude of the voltage change per ml. of base. The end-points of the titrations were determined from a plot of AE/AV against V (AE/AV signifies the change in voltage per unit change in volume of base).
For the attainment of smooth curves it was found necessary to avoid adding any water during the titration, to add the base directly to the solution away from the stirrer, and to measure the volumes of base with an error 5 0.05 per cent.
DISCUSSION
Complete titration data and AE/AV values from the analysis of three samples of one amino acid, glycine, are given in Table I. In order to conserve space detailed analytical information on six additional amino acids and nine peptides has been omitted. In the latter cases summaries of the experimental results are listed in Tables II and III.
The precision obtained in the titration of amino acids is indicated by the differential plots, shown in Fig. 1, of the experimental data from the analysis of three glycine samples. In these determina- tions 30.73, 30.57, and 33.50 ml. were the corrected volumes of 0.2963 N sodium hydroxide solution required for the titration of glycine Samples I, II, and III respectively. The percentages of the theoretical equivalent weights calculated from these corrected end-point volumes of standard base were 99.59, 99.74, and 99.70. The mean deviation for these experimental values was found to
be ~0.06 per cent. Mean deviations, ranging from ~1~0.03 to ~0.10 per cent, and an average mean deviation of f0.05 per cent, were calculated from the analytical data for all of the amino acid determinations given in Table II.
The deductions of Eastman (20) and Roller (21) seem to indi- cate the degree of precision which can be attained in the form01 titration of amino acids. These authors have shown that the condition of an inflection in the titration of a weak acid by a strong base is that cKA > 27Kw, where KA is the apparent ioniza-
TABLE I
Titration Data and AE/AV Values from Analysis of Glycine
The weights of samples taken were: Sample I, 0.6858 gm., Sample II, 0.6816 gm., and Sample III, 0.7471 gm. Time intervals of 4 minutes, 0.2963 N sodium hydroxide, and a temperature of 22” were used in all of the titrations. The expression, AE/AV, signifies the change in voltage per unit change in volume of base.
tion constant of the acid, Kw the ion product constant of water, and c defined by the equation l/c = l/a + l/b. In the latter equation a and b are the initial concentrations of acid and base respectively. It was shown further that an inflection point should appear, and that a titer deviation of 0.03 per cent between the stoichiometric end-point and the inflection point values should result, when c = 0.1 and KA is lo-lo. Since, in the present in- vestigations, c was approximately 0.1 and KA (in aqueous formal- dehyde solution) of the amino acid with the smallest apparent
in this study were known to be purified products, it appears that they were not of high purity or homogeneity. However, only a limited number of peptide analyses could be made, owing to the small amount of available material.
The glass electrode analysis of amino acids and peptides appears to be an accurate measure of purity when only inert contaminants
TABLE III
Summary of Experimental Data from Analysis of Peptides
* Prepared by Sidney Fox from diketopiperazine. t Hoffmann-La Roche product. $ Prepared by Thorpe Deakers from chloroacetyl chloride and glycine
by the method of Dunn, Butler, and Deakers (27). $ Prepared by Thorpe Deakers from chloroacetyl chloride and diglycyl-
glycine. 11 Hoffmann-La Roche product. This material was very hygroscopic. 7 Prepared by Lee Read from a-bromocaproyl chloride and glycine.
are present. While strongly acidic or basic impurities would de- stroy the accuracy of the determinations, fortunately such sub- stances may be readily removed by simple purification procedures. When an amino acid is the impurity, quantitative results can be expected from titrations with the glass electrode in those cases in which there is a lO,OOO-fold (22) difference between the apparent acid dissociation constants of the component substances. The
authors’ data on the titration in aqueous solution of d-glutamic acid in the presence of glycine illustrate t’he validity of this principle.
On the other hand, peptide titrations would be subject to a different interpretation. Even in aqueous formaldehyde solution there is not more than a loo-fold difference between the apparent
SAIIPLE II SAVPLE III
b
IrlR. b.ZY63’ N N&O H
FIG. 1. Curves showing the volumes of standard base required to titrate glycine Samples I, II, and III respectively. The volume of standard base at the equivalence point is indicated in each cast by an arrow extending upward from ihe base-line.
acid dissociation constants of a peptide and its amino acid con- stituents. While it could be assumed that the end-point would be sharp in titrating a peptide which contained a small amount of an amino acid impurity, the equivalents of base at the inflection point should be the sum of the equivalents of peptide and amino acid in the sample. Furthermore, the number of equivalents of base at the inflection point would be greater than that taken for
analysis, when the latter is calculated on the assumption that the sample was pure peptide.
An experiment was performed to test the validity of these con- clusions.4 In the titration of a mixture containing approximately 96 per cent of glycylglycine and 4 per cent of glycine, it was found that the AE/AV values rose sharply to a maximum during the addition of three 0.03 ml. portions of standard base at the inflec- tion point. Hence, the total volume of base, 19.66 ml. at the equivalence point, could be estimated precisely. The equivalents of base used in the titration exceeded by 3.0 per cent those calcu- lated on the assumption that the sample was wholly glycylgly- tine. However, the number of base equivalents was only 0.63 per cent higher than the sum of the equivalents of the glycyl- glycine and glycine taken for analysis. This value is considered to be within the limits of the probable experimental error since the latter is the summation of three titration errors.
A minimum error of approximately 1.0 per cent appears to be inherent in many of the titration procedures which have been proposed for the analysis of amino acids and peptides. A com- mon difficulty in Sorensen’s classical form01 titration procedure (a), published in 1908, and in many later modifications has been the inability to provide end-points which are sharply defined. The precision and accuracy of Sorensen’s original method have not been greatly improved by the use of end-points more alkaline than Sorensen’s third color stage with phenolphthalein; by the substitution of thymolphthalein (23) and other indicators for phenolphthalein; by the employment of media containing ethyl alcohol and other solvents in addition to water and formaldehyde; by the use of varying concentrations of formaldehyde; or by the determination of equivalence points with the hydrogen electrode (17) or conductive apparatus (24).
The Linderstrom-Lang (4) and the Nadeau and Branchen (14) titrimetric methods seem to be more satisfactory than other pro- cedures. The latter method was discussed earlier in the present paper. Linderstrom-Lang titrated amino acids in concentrated
4 A mixture, containing 0.005361 equivalent of glycylglycine (0.7169 gm. of 98.9 per cent purity) and 0.000420 equivalent of glycine (0.0361 gm. of 99.8 per cent purity), was titrated in aqueous formaldehyde solution with 0.2963 N sodium hydroxide solution with the use of the glass electrode.
aqueous acetone with 0.1 N 90 per cent alcoholic hydrochloric acid with the use of naphthyl red as indicator. Under optimum conditions of indicator color and acetone concentration the aver- age experimental error was approximately 0.5 per cent.
The conditions which Levy (12) deduced from theoretical con- siderations to be of fundamental importance in the form01 titra- tion of amino acids were adopted in the present work. Within practical limits, the amino acid solutions and the diluting fluids were of the highest possible concentration. The amino acid solu- tions were at approximately pH 6 prior to the addition of formalde- hyde, the end-point of the titration was at about pH 9, no correc- tion was made for the volume of standard base required to bring the aqueous formaldehyde solution to the end-point pH of the amino acid titration, and the concentration of the formaldehyde solution at the end of the titration was between 6 and 9 per cent.
According to Levy’s calculations (12), the intrinsic error is 0.5 per cent, when monoaminomonocarboxylic acids are titrated under the most favorable conditions. In the present studies it, has been demonstrated that the inherent error is f0.1 per cent. A plau- sible explanation for this disagreement would seem to be the prob- able uncertainties in the evaluation of the equation which Levy set up as an expression for the error in the form01 titration at the stoichiometric point.
SUMMARY
A method for the quantitative determination of monoamino- monocarboxylic acids, consisting essentially of a form01 titration by means of the glass electrode, has been described. The pre- cision attainable in the analysis of amino acids and peptides was found to be 3~0.1 per cent. The method was found to have an inherent, accuracy of fO.l per cent. The probable effects of different types of impurities on the accuracy of the method were discussed.
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