ANALYTICAL CHEMISTRY
1300 Table V. Effect of Silver Sulfate on Moore Method for Determining Oxygen Consumed Regular . With AgzSOc % of % of Compounda Mean Range theory Mean Range theory 26 Acetic acid 2.4 1014 62 95.1 38 362 17 A 1anin e 33.5 870 80.6 25 1790 97.7 1804 26 a-Amino-n-caproic acid 98.5 40 240 260 28 Benzene 7.8 8.1 18 1306 1748 18 34 n-Butyric acid 71.8 96.1 252 410 95 Chlorobenzene 26.4 41.4 176 2095 2413 43 o-Cresol 83.2 95.8 54 619 1672 33 E t h y l alcoholb 29.7 80.1 67 1074 1264 14 Furoic acid 83.6 97.6 57 626 17 78 Glutamic acid 63.9 100 980 Lactic acid0 882 13 45.4 484 82.7 28 Oleic acid 2248 83 62.4 1805 77.7 150 fi 17 34 20 Pyridine 0.8 1.3 2018 7 4 . 2 2494 Sodium stearate 36 91.7 124 671 22.5 704 21.4 Toluene 89 40 1528 1206 46.8 36.9 839 Turpentine 706 1295 1573 94 Valene 95.9 78.9 77 5 Ten replicates of each compound run with and without silver catalyst. b 87% CxHnOH. 0 Impure grade of lactic acid.
compounds resulted in varying degrees of improvement on oxygen-consumed values, but substantial improvement was obtained in 14 of the 17 compounds used. Particularly good results are obtained when using the catalyst on short-chain carbon acids. Certain compounds tend to precipitate the silver and either nullify its effects or actually result in a lower oxygen-consumed value than the method without catalyst. This is generally true when the sample has a high concentration of chlorides. The precipitated silver also causes some difficulty in determining the end point, olving to turbidity effects. Sample 3 in Table I was one of those which decreased the oxygen-consumed value upon addition of silver sulfate. Silver sulfate as a catalyst is recommended as a complement to the regular procedure rather than a substitute, as apparently many wastes show incompatibility. If work is to be done on a given type of waste, it would be beneficial to use both the regular and the catalyzed methods and select the one giving the better results. Neither the Ingols nor the iodate method is applicable
with the silver sulfate because of the reaction with the added iodide before titration. CONCLUSIONS
Application of statistical procedures to the data leads to the conclusion that the Moore method is preferable to the other methods tested for the determination of oxygen consumed in organic wastes. This method also provides greater ease of manipulation and applicability to a large variety of samples and requires less time. The use of silver sulfate as a catalyst for dichromate reactions in determination of oxygen consumed appreciably extends the useful range of the procedure. On most compounds or mixtures and especially with straight-chain acids an increase in the theoretical oxidation is noted. With a few compounds, or in the presence of high chloride concentration, the use of the catalyst is precluded, making i t necessary to check both modifications. Certain materials such as benzene, toluene, and pyridine are not oxidized by either procedure. LITERATURE CITED
(1) Am. Public Health Assoc., New York, “Standard Methods for the Examination of Water and Sewage,” 9th ed., p. 122, 1946. (2) Dzyadzio, A. &Vodosnabzhenie I., i Sanit. Tekh., No. 8-9, 117-25 (1938). (3) Ettinger, M. B., U. S. Public Health Service, Environmental Health Center, Cincinnati, Ohio, unpublished memoranda, 1950. (4) Ingols, R. S., and Murray, P. E., Water & Sewage Works, 95, 113-17 (1948). (5) Johnson, P. W., Halvorson, H. O., and Tsuchiya, H. M., Abstracts of 109th Meeting, AM.CHEM.SOC., p. 2S, Atlantic City, 1946. (6) Madison, K. M., Division of Water, Sewage, and Sanitation Chemistrv. 113th Meeting AM. CHEM.SOC.. Chicago. 1948. (7) Moore, W. k:,Kroner, R. C, and Ruchhoft, C: C., A N ~ LCHEM., . 21, 953-7 (1949). (8) Muers, M. M., J. SOC.Chem. Id., 55, 71T (1936). RECEIVEDSeptember 13, 1950. Presented before the Division of Water, Sewage, a n d Sanitation Chemistry at the 118th Meeting of the AMERICAN CHEMICAL SOCIETY,Chicago, 111.
Determination of Glutamine and Asparagine in Plant Tissue Extracts G . W. BUTLER‘ Plant Chemistry Laboratory, Department of Scient@ Industrial Research, Palmerston North, New Zealand
V
ARIOUS methods for the estimation of glutamine are based
on its anomalous decomposition to form pyrrolidone carboxylic acid and ammonia. The most widely used is the modification of Vickery, Chibnall, and coworkers (16) of the original method of Chibnall and W-estall (3). This consists of measuring the ammonia formed when a plant extract is heated a t 100” C. and pH 6.5 for 2 hours. This method is not specific, as other constituents of plant extracts such as urea, allantoin, and asparagine may also liberate ammonia under these conditions. To overcome this lack of specificity, Pucher and Vickery ( I S ) developed a method for the quantitative extraction of pyrrolidone carboxylic acid, with its subsequent estimation through an amino nitrogen determination of the glutamic acid formed by acid hydrolysis, Measurement of the carbon dioxide liberated by ninhydrin before and after hydrolysis a t 1 Present address, Botanical Laboratory, University of Lund, Lund, Sweden.
100” C. and pH 6.5 has been made the basis of a method ( 8 , 1 8 ) . Archibald (1) and Krebs (10) used hydrolysis with glutaminase preparations from dog kidney and Clostridium welchii, respectively, Krebs claiming a high degree of specificity for his method. The position with regard to asparagine is much less satisfactory, in that the only method available is through hydrolysis with 1N sulfuric acid at 100’ C. for 2 hours; the asparagine concentration is calculated by subtracting the values for glutamine amide nitrogen and ammonia nitrogen from the total amide nitrogen value. Vickery et al. (16), in their original description of the method, emphasized its lack of specificity and pointed out that errors in the determination of glutamine must be reflected in asparagine values. That this caution is justified is amply demonstrated by results with rhubarb (19) and tobacco (18), which were inexplicable on current hypotheses of amide metabolism. Vickery and coworkers stress that results using indirect
V O L U M E 23, NO. 9, S E P T E M B E R 1 9 5 1
1301
The specificity of the methods available for the estimation of asparagine and glutamine appeared open to question. Independent check methods were therefore devised. The amides were separated from each other and from other interfering substances by means of paper chromatography. Hydrolysis and estimation of the amides were carried out in one operation in Conway units. In a comparison with other methods, good agreement was obtained with a method for the estimation of glutamine using a glutaminase preparation. The agreement with a method based on differential acid hydrolysis was unsatisfactory for both amides, especially asparagine. Although the glutaminase method is suitable for the estimation of glutamine, the technique described is the most specific at present available for the determination of asparagine.
methods of analysis must be interpreted with reservations, and that in doubtful cases substantiation of the results either by independent analytical methods or by direct isolation is necessary. Further evidence of the danger of applying the standard indirect methods of analysis to the amide fraction has accumulated in this laboratory during the past 4 years ( 2 , 14). Some hundreds of determinations of glutamine, asparagine, and urea have been performed on plant tissues grown under varying conditions of nitrogen nutrition and prepared for analysis by widely different procedures. Although in most cases serious discrepancies have not been apparent, a relatively large number of tissues have yielded results that could not be explained by the known behavior of these compounds. It has become increasingly apparent that progress towards a better understanding of the processes involved requires more specific methods of analysis than those so m-idely used in investigations on plant nitrogen metabolism. All the methods in current use are applied to the complex mixture resulting from an aqueous or alcoholic extraction of the fresh or dried tissue. The method described is based on the paper chromatography of plant extracts ( 6 ) ,so that a considerable purification is achieved before the final analysis is made. REAGENTS
As a solvent for chromatography the phenol-water system was used. The phenol was purified by distillation in vacuo from zinc in an all-glass apparatus and was renewed for each determination. The hydrolyzing agent was saturated potassium hydroxide solution. Standard 0.001 N hydrochloric acid was prepared by the dilution of standard 0.1 N acid, incorporating ethyl alcohol and Taschiro's reagent (methyl red-methylene blue) according to Conway (6). The alkali was barium hydroxide solution, 0.002 N . Glutamine was prepared by the method of Vickery, Pucher, and Clark (16). Purity was 95% as determined by amide analysis. Asparagine was prepared by the method of Vickery, Pucher, and Deuber (17). Its purity was 97% as determined by amide analysis. EQUIPMENT
Freeze-drying apparatus, a micrometer pipet (accuracy = 0.05 PI.), apparatus for chromatography, standard Conway units (No. l),and a 0.26m3. Conway microburet. EXTRACTION OF T I S S U E
Perennial rye grass was used as the plant tissue. Ten grams of fresh grass or the equivalent amount of dried grass was blended for 3 minutes in a Waring Blendor with 100 ml. of ethyl alcohol a t room temperature (8). Eighty-seven per cent alcohol was used for fresh tissue and 80% for dry, so that in each case the final alcohol concentration was 80%. The fibrous material was centrifuged off and the solution concentrated by vacuum distillation a t 40" C. until all alcohol was removed. The residual aqueous solution was transferred to a measuring cylinder together with washings from the vacuum distillation flask, made up to 25 ml., and centrifuged. Twenty milliliters of this extract were pipetted into a large test tube and lyophilized for approximately 20 hours. -4t the conclusion of drying, air was admitted gradu-
ally through a bubbler, in order not to disturb the residue in the test tube. A known volume of water (0.5 to 5.0 ml., according t o the probable amide content of the leaf), was added, the residue was carefully dissolved and the solution was filtered if necessary. PROCEDURE
Strips of Whatman KO,1 filter paper (11 X 46 cm.) were taken and a t 8 to 10 cm. from one end of each strip a line 5.5 cm. long was drawn parallel to the end and starting 1 cm. from the edge. A point was marked 1.5 em. from the other edge, lying on an extension of the line. Ttventy microliters of solution were applied in the form of a narrow band along the line and 3 PI. were placed on the spot, using the micrometer pipet. After the solution had been allowed to dry, the strips were placed in the chromatography vessel, e uilibrated with the vapor phase of the phenol-water mixture, a n 1 irrigated with solvent in the normal way, using the descending technique (4). The system phenol-water constituted a satisfactory solvent, as ammonia and the amides known to be present were well separated. Approximate Rp values for relevant substances are ammonium ion 0 to 0.20, asparagine 0.40, glutamine 0.55, arginine 0.55, alanine 0.55, and urea 0.73. It is obvious that the glutamine band will always contain such free alanine and arginine as are present. Alanine presents no difficulty, while preliminary experiments showed that arginine does not yield ammonia under the conditions of hydrolysis employed (see Table 11). T o obtain maximum resolution, the chromatograms were irrigated with solvent for 40 hours, in which time the solvent front had usually passed off the end of the paper. The papers were dried in a current of air a t room temperature (20" C.). A strip along the side of the paper containing the control spot was cut out and developed in the usual way with ninhydrin. With the aid of this developed chromatogram, rectangular strips of paper enclosing the glutamine and asparagine bands were cut out, with strips of comparable width to act as blanks. These sections were usually 4 cm. wide, though a 5-cm. cut was necessary if the amide concentration was high or if the band was somewhat diffuse owing to imperfect conditions during the chromatographic stage. It was advisable a t this stage to label the papers in pencil. No difficulties arose from the application of a band rather than a spot to the paper. Hydrolysis of the amides and quantitative estimation of the ammonia produced were done in one operation, using essentially the procedure described by Conway ( 5 ) for the determination of ammonia nitrogen in the range 0 to 14 micrograms. Preliminary trials showed that various mixtures of saturated potassium carbonate solution with potassium hydroxide did not give quantitative recoveries of both amides when these were adsorbed on filter paper. Saturated potassium hydroxide solution was found satisfactory and was used throughout the investigation. Each paper strip was cut into small pieces, which were distributed about the outer chamber of a Conway unit. The paper was moistened slightly with about 5 drops of water, 1 ml. of standard acid was delivered into the central chambe:, and the cover was sealed on, using as fixative white petroleum jelly hardened with p a r a h wax. Approximately 1 ml. of cold saturated potassium hydroxide solution was introduced from a quick delivery pipet and the unit was incubated for 3 hours a t 37" C. Adding cold alkali to moistened paper was necessary to avoid excessive mercerization, which tended to cause leakage of alkali into the middle compartment. After 3 hours, the units were
ANALYTICAL CHEMISTRY
1302 Table I. Treatment L-nwashed
Paper Blank NHI N ,
y/32 Sq. Crn. Paper
Alkali-naphed
1.87 i 0.26 3 . 5 0 i 0.30 2.69 iO,l5 0.72 jz 0.13 0.78 i 0.05 1.10 i 0.48 0.28 i 0.27
Table 11. Recoveries of Glutamine and Asparagine in Presence of Possible Interfering Substances (Values are mean of duplicate determination) A ccompanyinp Mean Recovery, % Substances Asparagine Glutamine Urea 98.8 96.5 Arginine 95.2 98.3 Ammonia 91.8 93.5 Urea 91.6 87.1 Ammonia ITrea I 93.7 92.2
’I
*
:,mp%I21
withdrawn one at a time and the excess acid was titrated with 0.002 h’ barium hydroxide solution, using a Conway microburet.
It was found desirable to perform analyses in triplicate. Strict precautions were necessary to prevent contamination of the paper with ammonia both from the laboratory air [cf. Martin and hlittelmann (11)], and from the atmosphere of the chromatography vessels. The latter should be cleaned out monthly and 2 days allowed for the atmosphere of the vessels to become saturated with the vapor of the phenol-water mixture. The papers rested on a clean glass surface during the various manipulative stages of the operation and were handled n.ith forceps, evcept when the strips were being placed in the chromatography vessel. BLAYK CORRECTIOY
Other investigators ( 7 , 11) a-ho have attempted to use paper chromatography as the basis for quantitative estimation of nitrogenous compounds have found that the presence of interfering substances in the paper constitutes a serious difficulty. This is also true of the proposed method, where the chief source of error lies in the presence in the paper of substances which give tise to ammonia on treatment with strong alkali. Moreover, there is considerable variation from sheet t o sheet and within Pheets, as can be seen in Table I. The data in the table are delived both from untreated sheets and from sheets washed with 0.002 S potassium hydroxide solution and distilled water prior . Each value is the result of analyzing six strips of 32 sq. em. area, the standard size for the analytical determinations. Although the blanh value is reduced by the washing procedure, the standard deviation, which is a measure of the variability within a single sheet, is not markedly improved. The method finally adopted is based on the distribution along unwashed sheets of ammonia precursors (and of ammonium) from RF 0 to 0.9 when plant extracts are chromatographed. The result of a series of such determinations is evpressed as the curve shown in Figure 1. The ammonia-producing substances are concentrated a t RF 0 to 0.20 (ammonium ion and possibly unknown substances), RF 0.35 to 0.45 (asparagine), RF 0.50 t o 0.60 (glutamine), and RP 0 85 to 1.00 (contaminants from the paper and possibly unknown substances). Strips for blank determinations were then cut from intervening portions of the paper and gave an absolute value of 0.5 to 1.5 micrograms per 32 sq. cni. strip with a standard deviation of 0.10 to 0.26 microgram.
The form of the curve did not vary significantly, regardless of the previous history of the tissue, while the presence of urea in the band a t RF 0.70 to 0.80 caused no increase when the blank strip was cut from that area. The peaks due to glutamine and asparagine are shown as being symmetrical, but the experimental method was actually incapable of revealing any symmetry. The peaks would be asymmetrical if the adsorption isotherms were nonlinear. I n addition to the variable paper blank, there is a substantially constant blank from the diffusion apparatus of 0.3 microgram of nitrogen, This is presumably due to traces of amnionin in the fixative. RESULTS
A series of eyperiments was performed to arrive a t a figure for the average recover>- of asparagine and glutamine. Forty individual determinations of both amides gave the following mean recoveries with standard deviations: glutamine 95.5 f 5.7y0,asparagine 96.5 + 7.8%. The range over which amide nitrogen can be applied to the paper with the retention of good resolution of the compounds is 0 to 12 micrograms of amide nitrogen (for each amide), with the optimum a t 10 micrograms. The error is greatest a t low concentrations of amide, somewhat smaller a t higher concentrations; the deviations given above really correspond to those observed a t the 6 microgram of nitrogen level. A comparison with the deviations given in Table I for “blank nitrogen” indicates the importance of this error.
1
I
so Z
a CL
v
0 - 2 0.4
0.6
0.8
1.0
RF V A L U E Figure 1. Distribution of Ammonia-Producing Substances in Chromatograms of Plant Extracts
That the recoveries were unaffected by the presence of comparable amounts, on an “amide” nitrogen basis, of ammonium salts, urea, arginine, or unknown constituents is shown in Tables 11, 111,and IT.’. The results and standard errors for asparagine and glutamine in grass extracts, obtained using the proposed method, are compared in Tables I11 and IV with the results and standard errors obtained using tn-o other methods: A slight modification of the hydrolytic procedure of Vickerh , Chibnall, and coworkers (16). Interference by ure‘a was obviated by a pretreatment of the extract n-ith urease. The glutaminase method of Krebs (10)using the cell-free extract of Hughes and Williamson (9).
V O L U M E 2 3 , NO. 9, S E P T E M B E R 1 9 5 1
1303 DISCUSSIOS
In T:ible 111, a comparison of methods is made, using extracts of tissues which had undergone a variety of treatments dwigned t o achieve large differences in amide levels. In Table I\- the
The agreement between the paper niethod and the glutaminase method is generally good. When allowance is made for the fact that 95y0 recoveries were obtained by the paper method for pure solutions, there appear to be no systematic differences between the results of the two methods but only random errors. Two satisfactory and entirely independent methods for glutamine eatimation are now available. It is impossible to claim absolute specificity for t,he paper method, as other metabolites, possessing the sanie RF value as asparagine or glutamine and capable of giving ammonia under the conditions of hydrolysis employed, may be found to occur in other plant tissues or under other conditions of nitrogen nutrition. .$part froin extract 5, the hydrolytic method for glutnniine agrees with the other two methods. This extract, honcver, is typical of a considerable number of tissues analyzed in this lahoratory over the past 4 years. Separation of the aniide constituents by paper chromatography may be concluded to bc more specific than differentiation by hydrolytic procedures. Generally, there is agreement bet,ween the two "asparagine" values only when the asparagine is present in small quantity. Here the large error generally involved in the determination of asparagine by the hydrolytic procedure (especially in thc presence of much glutamine) could well mnsk differences in the analyses. I n extracts 5 and 10, n-here nap:iragine is present in higher concentrations, the paper mcthoti givci much lower rewlts. It
recovery of added asparagine and glutamine by all three methods The concentrations are espressed in both tables as milligrams of amide ammonia or urea nitrogen per 100 grams of fresh weight of grass ("mg. yoJ').Foi. Tahle I11 all the analyses were done in quadruplicate. For Table IV, the glutaminase and hydrolytic analyses were done in duplicate and the paper analyses in triplicate. I n calculating the standard errors of the glutaminase and hydrolytic determinations, the variability of the particular titer concerned was alone conqi icred. As these estimations are made by difference, there is : i i i a,L.iitional incomputable error, equal to the deviation of the ptirticular blank titer being considered (glutamine titer in the case of asparagine, etc.) from the true figure. The error should normally be small, but nevertheless the standard errors given are conservative. No correction factor has been applied to the results froin the paper met,hod to allow for recoveries froin pure solutions of l r w than 100%. is shown for two estracts.
*
For the "culture" experiment recorded in Table I11 (extracts
A, 7, and 8 ) , two samples of leaves each of 100 grams fresh weight
\\ere rut from a single rye grass plant and were cultured for 20 hours in dilute solutions of ammonium sulfate and urea, respectively. The grass for extract 8 was dried in an electrically heated forced draft oven at 80" C. and was complete in 30 minutes.
Tahle 111. Comparison of Methods so. I 2
:3 .I
5
6
,
Description of Extract From plant "fertilized" with ammonium sulfate From plot, fertileed with ammonium milfate. lime notash, and phosphate I-rea added t o ext r a c t of rye grass plant From plant fertilized with urea From plot fertilized with urea Leaves cultured in urea solution Leaves cultured in urea solution and d n d
8
Leaves cultured in a i n n i ~ n i u msulfate soliltion
Aininonia S .\Ig. 7c
Crea S ,
wz
7c
Paper
R
10
Description of Extract From plot fertilized with-urea Extract i 8 . 7 mg.,% @utamine a m i d e N Theoretical Extract +17.8 mg. % , glutamine amide N +16.2 mg. yo asparagine amide N Theoretical From plot fertiljaed with ammonium sulfate Extract f6.0 mg.,% glutemin8 amide N +6.1 mg. Yo asparagine amide N Theoretical E x t r a c t +12.0 mg. % glutamine amide N +12.1 mg. % aaparsgine amide N
0 12
134iOO3
1 9 i O - L
1 75
0 30
G 19 i 0 36
...
5 40
0.64
4.i2
4 i 4 i 0 11
5 9 3 i. 0 . 0 7
5.54
0 65
0
io
7.30 & 0.68
8 4 i z 0 J-l 25.0 & 0 8
31.6
f
41.8 & 1 . 5
0 7
12.0
=t0
6 70
20.6
19.8 r 0 . 8
5 78
17 8
13.4
9.08
13 0
633&031
14 0
-0.5
Single determination.
+0
2
19.2 i 0 . 6
4ininonia s ,Mg. o/o
Urea N , .\Ig. %
0 95
-0.13
1 73
-0
1 23
-005
25
5 9 3 ~ 1 3 19.0 & 1 G
-0.4
3.89
=r0
46
3 38 i 1 . 1 8
23
1 39
= 0.16
0 73 i 0 . 9 0
8.19 f 0.12
2.33
+0
1.90 I 1 . 2 0
I
0 52
=0
0.6
.7
2.1
*
5 7 x 0 8
0 . 8 7 10 18
192zkOi
1 78 r 0 . 1 3
~~
39.1
*
2.4
1.29 i 0.1s 0 49 & 1 . 6
1.7
=
1 4
A~paragine.Mg. % Amide S Paper Hydrolytic
6 8,5 & 0 . 1 0
13.6 & 0 3 13 .j
13 2 i 0 5 5 14 3
16 2 13.6
2 1 8 r 0 2 22 6
2 1 2 * 0 2 23 6
2 3 4 i 0 3 24 i
310-06 30 4
4 52
0 33
5 80 f 0 60
352,040
10 6 & 0 30 in 6
10 6 & 0 . 2 0 11 8
9.90 9.6
16.1 & 0.30 16.5
113.5~ 17.8
15.6
f
0 28
4 6 3 i 0 0.5
2 85
0 18
10 0
+0
05
10 6
16.8
80
1.8
2 . 8 0 i 0 03
4 80
0 13
0.90
I
= 0.5
0 5 1 I O 02
Glutairline. -~ XIg. To Amide 4 Glutaininane Hydrolytic
2 68
2 38
14 6
1 3 1 0 2
Recovery- Experiment
Puper
16.6 a
Asparagine, Xlg. % Amide N Paper Hydrolytic
0 88
Table IV. SO.
Glutamine, blg. % Amide N Glutaminase Hydrolytic
f
0 2
I
I
1 3 1 ~ 0 2
14 2"
...
0.4
. .
* 0.20
15.2 f 0.2
,..
...
708+040
1 3 . 7 i. 5 13.2
17.1 19.2
* 15
1304
ANALYTICAL CHEMISTRY
would be highly desirable to have an independent analytical method for asparagine of comparable specificity to the glutaminase method for glutamine. The standard errors for the paper and glutaminase methods are of the same order for the determination of glutamine. For asparagine, the standard errors for the paper method are considerably smaller than those for the hydrolytic method. The error would be diminished by the use of paper with a lower blank correction. No significant error appeared to result from the extra manipulative steps required in the paper method between the extraction of the tissue and application of the concentrate to the paper. If the rigorous standards of cleanliness necessary for a microchemical technique are observed, errors from contamination can be avoided. An important advantage of the paper method is the ancillary information it yields regarding the distribution of amino acids and fluorescing substances in the extract. Its major disadvantage is that the analysis is spread over 4 days. The actual manipulative time amounts to 8 hours per extract, done in triplicate by one operator and including the tissue extraction step. On the other hand, a number of analyses can be made a t the same time, dependingonly on the capacity of the freeze-dryer and chromatography apparatus. The rectangular strips of paper cut from the chromatograms can be stored without deterioration in a desiccator containing sulfuric acid until it is convenient to commence the final step. The “cold alcohol” extraction procedure of Bathurst and ,411ison (8)was found excellent for quantitative paper chromatographic purposes. The use of alcohol has the advantage that the extract is free of peptides and proteins and has a low concentration of inorganic salts. On removal of the alcohol, waxes separate out, leaving a solution very suitable for paper chromatography. I n the experimental method outlined, an extraction macroprocedure has been followed by a microchemical estimation. Where the supply of biological material is limited, the extraction and concentration steps could be adapted to a micro scale. The recoveries of glutamine and asparagine from paper chromatograms are interesting in the light of the observations of Woiwod (!No),who found, using Whatman No. 4 paper, that the recoveries obtained for glycine, valine, and leucine decreases with increasing RFvalue and with increasing distance of migration down the paper. This effect was not apparent in the present study, although it may be a partial explanation of the consistently low recoveries from pure solutions. The drying of the chromatograms at low temperatures is important because of the interaction of phenol with amino acids above 50’ C., as pointed
out by Fowden and Penney ( 7 ) , and also because of the instability of glutamine. One of the factors possibly contributing to the success of the proposed method is that the hydrolysis of the amides is performed on the paper, using a reagent which mercerizes the cellulose and is likely to reach all the amide molecules inside the cellulose fibers. The necessity for an elution step is avoided, with a saving of manipulative time. ACKNOWLEDGRIENT
The author is deeply indebted t o his colleague, J. L. Mangan, who, ITith the technical assistance of J. F. Fisher, conducted analyses for the comparative lvork by two standard methods. Thanks are also due to J. G. Fraser for the preparation of the glutamine and asparagine samples used. Finally, he wishes to acknowledge the invaluable assistance of James Melville, both in the preparation of the manuscript and from stimulating discussions held during the course of the investigation. LITERATURE CITED
(1) Archibald, R. hl., J . Bid. Chem., 154, 643 (1944). (2) Bathurst, N. O., and Allison, R. M., N . 2. J. Sci. Tech., in press. (3) Chibnall, 8. C., and Westall, R. G., Biochem. J., 26, 122 (1932). (4) Consden, R., Gordon, 8 . H., and Martin, A. J. P., Ibid., 38, 224 (1944). (5) Conway, E. J., “Microdiffusion Analysis and Volumetric Error,” rev. ed., London, Crosby Lockwood & Sons, 1947. (6) Dent, C. E., Stepka, W., and Steward, F. C., Nature, 160, 682 (1947). (7) Fowden, L., and Penney, J. R., Ibid., 165, 846 (1950). (8) Hamilton, P., J . Bid. Chem., 158, 375 (1945). (9) Hughes, D. E., and Williamson, D. H., Biochem. J . , 43, xlv (1948). (10) Krebs, H. A,, Ibid., 43, 51 (1948). (11) Martin, A. J. P., and Mittelmann, R., Ibid., 43, 353 (1948). (12) Seuberger, A., and Sanger, F., Ibid., 36, 662 (1942). (13) Pucher, G. W., and Vickery, H. B., IND. ENG.CHEJI.,SNAL. ED., 12, 27 (1940). (14) Reifer, I., and Melville, J., Trans. XIth Intern. Conf. Pure and Applied Chem. (Supplement to Chemistry and Industry, 1948). (15) Vickery, H. B., Pucher, G. W., and Clark, H. E., J . Biol. Chem., 109, 39 (1935). (16) Vickery, H. B., Pucher, G. W.,Clark, H. E., Chibnall, A. C., and Westall, R. G., Biochem. J., 29, 2710 (1935). (17) Vickery, H. B., Pucher, G. W.,and Deuber, C. G., J . Bid. Chem., 145, 45 (1942). (18) Vickery, H. B., Pucher, G. W., Leavenworth, C. S.,and Wakeman, A. J., Conn. Agr. Expt. Sta., Bull. 374, (1935). (19) Vickery, H. B., Pucher, G. W.,Wakeman, A. J., and Leavenworth, C. s.,Ibid., 424 (1939). (20) Woiwod, A. J., Biochem. J.,45, 412 (1949). RECEIVED October 13, 1950.
Determination of Traces of Chloride Potentiometric Titration to the Apparent Equivalence Potential I. M. KOLTHOFF AND P. K. KURODA School of Chemistry, University of Minnesota, Minneapolis, .Minn.
I
N T H E classical method of potentiometric titrations the end point is taken at the location of the maximum in the A E / A c curve. When the equilibrium constant of the reaction is unfavorable and the dilution is high, either no maximum occurs or A E / A c changes so little at the end point that it cannot be determined with a n y degree of accuracy. Under such conditions a potentiometric titration still can yield rapid and accurate results when reagent is added until the a p parent equivalence potential is attained. From the practical point of view a serious limitation of this method is that the
equivalence potential, in general, changes with the ionic strength and the kind of electrolyte in the solution titrated. Moreover, the exact determination of the equivalence potential is impossible, owing to a n unknown liquid junction potential. From an analytical viewpoint these difficulties can be almost completely eliminated by carrying out the titration in a “supporting electrolyte” of high ionic strength and suitable composition. The “apparent” equivalence potential in such a medium can be found b y classical methods. For practical purposes it may be desirable to use various supporting electrolytes.
