1252
ANALYTICAL CHEMISTRY
Research Foundation, for valuable suggestions for the preparation of this paper and the statistical analysis. LITERATURE CITED
(1) Bliss, C. I., Ann. Math. Statistics, 17, 232-7 (1946). (2) Bliss, C . I., and Cattell, McK., Ann. Rev. Physiol., 5 , 479-539 (1943). (3) Bliss, C. I., and Marks, H. P., Quart. J . Pharm. and Pharmacol., 12,82-110,182-205 (1939). (4) Crandall, W. A,, and Burr, Muriel &I., Federation Proc., 6, 246-7 (1947). (5) Finney, D. J., Quart. J . Pharm. and Pharmacol., 18, 77-82 (1945). (6) Greene, R. D., and Black, A , J . Am. P h a r m . Assoc., Sci. Ed., 32,217-20 (1943). (7) Kent-Jones, D. W., and Meiklejohn, M., Analyst, 69, 330-6 (1944). (8) Kerly, Margaret, Biochem. J., 38, 423-5 (1944). (9) Light, A. E., and Clarke, &I. F., J. Biol. C h a . , 147, 739-47 (1943).
(10) Oser, B. L., Proc. I n s t . Food Technol., [ 6 ] ,169-84 (1946). (11) Pennington, D., Snell, E. E., and Williams, R. J., J . Biol. Chem., 135,213-22 (1940). (12) Pharmacopoeia of the C.S,.4., XII, First bound supplement, pp. 79-81,1943. (13) Price, S. A,, ’Irature, 156, 171-2 (1945). (14) Price, S. A , . and Graves. H. C. H.. Ibid.. 153. 461 (1944) (15) Roberts, E. C., and Snell, E. E., J . Biol. Chem., 163, 499-509 (1946). (16) S h h , hi. E., Horowitz, 31., Gelman, h.C., and Sass, hI., Science, 115,517-19 (1952). (17) Snell, E. E., and Strong, F. AI., IND.EXG.CHEM.,AXAL.ED., 11,346-60 (1939). (18) Society of Public Analysts, Analytical Methods Committee, Sub-committee on Vitamin Estimations, A n a l y s t , 71, 397406 (1946). (19) Toennies, G., and Frank, H. G., Growth, 14, 341-51 (1950). (20) Wood, E. C., Analyst, 71,l-14 (1946). (21) Ibid., 72,84-90 (1947). (22) Wood, E. C., and Finney, D. J., Quart. J . Pharm. and PhamnaCOZ., 19, 112-27 (1946). RECEIVEDfor review January 5, 1953.
Accepted April 14, 1953.
Accuracy of Quantitative Paper Chromatography in Amino Acid Analysis-Addendum R. C. SALANDERI, MARCUS PIANO, AND A. R. PATTON Chemistry Department, Colorado A & M College, Fort Collins, Colo. and Chism recently published a “quantitative” P method for paper chromatography of amino acids. In response t o inquiries regarding the accuracy of the method as well as ATTON
(6)
certain unpublished details of procedure, the following is presented. METHOD
One-microliter spots of 5 to 100 m M acid solutions are applied to Whatman No. 1 paper with a self-filling transfer-type pipet capillary, which is wiped dry before each application. Ascending chromatography is conducted for 18 to 24 hours in a suitable solvent. The solvents most often used are 80% phenol (Merck reagent grade) and water-saturated 2,4-lutidine (Matheson Co.). After development the solvent is allowed to evaporate a t room temperature for 18 to 24 hours. The papers are then sprayed with 0.3% ninhydrin in 95% ethyl alcohol and color development is allowed to take place in the dark a t room temperature for 18 hours. Maximal absorbances are read through the vertical axis of the colored spots in the Welch Densichron. Concentrations are derived from comparison with standard series run on the same chromatogram. The standard series curves are made by plotting peak absorbances against molar concentrations in a geometric progression. Increment curves which represent the distribution of color density in a spot are obtained by recording the absorbance every 3 mm. along the vertical axis of the spot.
Table I. Added, m M 6.25 5.00 2.50 1.25 5.00 5.00 5.00 5.00
densest area of the color spot as the concentration increases. Figure 1, B, is an example of a standard series curve for alanine. Alanine increment curves a t the higher concentrations are shown in Figure I, C. Using this technique, with water-saturated lutidine as solvent, 35 replicate spots of a casein hydrolyzate showed the sample to contain 8.3% lysine with a standard deviation of 1.5%. Block ( 3 ) gives 8.5% as an approximate value for lysine in casein. To test recoveries, various concentrations of lysine and alanine were added to the casein hydrolyzate as s h o m in Table I. The student method described by Patton ( 5 ) was tested by giving an “unknown” alanine solution to each of 11 students, none of whom had attempted quantitative paper chromatography before. Using the Welch Densichron and uncalibrated capillary .OS M
Recovery of Alanine a n d Lysine Recovered, mA4 Recovery of Alanine 5.60 5.37 2.50 1.22 Recovery of Lysine 5 00 5.00 4.75 5.12
Recovered, % ,0125
89.5 107.4 100.0 97.5
Rf
(mm)
,025
9 5
MOLAR C O N C E N T R A T I O N
1,
100.0 100.0 95.0 102.4
DATA AND R E S U L T S
D
Figure 1, A , shows increment curves of 1-pl. spots of alanine in the molar concentrations listed. Spot diameter increases with concentration and there is a slight increase in the R, of the 1 Present address, Department of Agricultural Biochemistry, Rutgers University, New Brunswick, N. J.
\\ \I Rf
(mmh
I
.os
0.1
0.E
MOLAR CONCENTRATION
Figure 1
1253
V O L U M E 25, N O . 8, A U G U S T 1 9 5 3 pipets, they reported values of 25, 22, 28, 24, 34, 26, 26, 36, 26, 23, and 25 millimoles of alanine, respectively; the correct value was 25. DISCUSSION
R-hen maximal densities are used, the standard curves break down a t the higher concentrations, as can be seen in Figure 1, D. Therefore, the standard curves cannot be extrapolated. Sample concentrations must be adjusted, usually by dilution, to fall within the limits of the standard series. One- or 2-11]. spots of solution give the best results. Application of larger volumes often results in hazy color spots that are large and difficult to read satisfactorily. I n such cases the maximal color density may be found a t the periphery rather than in the interior. Such a spot is useless for a quantitative measurement by optical scanning. Block ( I ) obtained good iesults with a 5 4 . pipet; however, in subsequent work ( 2 ) 2.5-pl. pipets have been employed and the use of 1-11]. pipets is being considered. If spots larger than 1 or 2 111. are to be used, the concentration of the solution and the standard series must be reduced accordingly. Figure 1, C and D.shows that the top concentration that can be used in a standard series when a 1 4 . pipet is employed is 0.05 M or a total concentration in the spot of 0.05 micromole. Very often the 5-pL spot exhibits excess diffusion in the paper: however, if this size of pipet is employed, the highest value in the standard series would be 0.01 h ' or 0.01 micromole. The lower concentrations of the standard series would then be serial dilutions of this 0.01 11.1 amino acid and would be extremely dilute and susceptible to dilution errors. This standard series would probably be much too dilute for satisfactory results. Block ( I ) recommends drying the paper at 30" C.; while this is not excessively hot or much above room temperature, the
authors' chromatograms have been dried a t room temperature (20" to 22" C.). The present work confirms that of Brush and coworkers (4). They showed amino acid destruction on chromatograms which were heated wet with solvent. If chromatograms wet with phenol or lutidine are sprayed with ninhydrin, the amino acid color spots are red and pink rather than the normal purple-blue. I t is therefore important to remove as much of the solvent as possible before spraying. A period of 18 to 24 hours of solvent evaporation has proved sufficient to permit proper color development. Several grades of paper have been tested and Whatman No. 1 filter paper appears to be a very satisfactory stratum for the color spots. By setting the Densichron to read 1.43 and recording several hundred readings in areas chosen a t random, the standard deviation in absorbance was found to be 0.013. Quantitative paper chromatography in its present state of development compares favorably with older methods of amino acid analysis (microbiological and chemical) as to both accuracy and reproducibility. Furthermore, it can be used to determine certain amino acids for which there was no previous suitable method. LITERATURE CITED
(1) Block, R. J., ASAL. CHEM.,22, 137 (1950). (2) Block, R. J., personal communication, 1952. (3) Block, R. J., and Balling, D., "Amino Acid Composition of Proteins and Foods," 2nd ed., Springfield, Ill., Charles C Thomas, 1951. (4) Brush, M.K., Boutwell, R. K., Bartin, B . P., and Heidelberger, C., Science, 113, 4 (1951). ( 5 ) Patton, A. R., J. Chem. Educ., 28, 629 (1951). ( 6 ) Patton, A. R., and Chism, P., - 4 ~ 4 CHEM., ~ . 23, 1683 (1951). RECEIVED November 3, 1952. Accepted M a y 21, 1953. Published with the approval of the director, Colorado Agricultural Experiment Station, as Scientific Series Paper 396. Supported in part b y a grant from The Nutrition Foundation.
Microdetermination of DDT in River Water and Suspended Solids BEN BERCK Dirision of Entomology, Department of Agriculture, Winnipeg, Man., Canada
of D D T are effective in controlling mosquitoes and black flies ( 2 ) . In studies in which D D T was used as a black fly larvicide ( 6 ) , information was sought on the D D T content of the river water a t various distances downstream and on the role of suspended solids in affecting the amount and effectiveness of the DDT. This report deals with methods found useful in that regard. For determining small amounts of D D T in the South Saskatchewan River, where amounts as low as 1 microgram per liter were anticipated, three colorimetric methods ( 7 , 12, I S ) were considered. On the basis of satisfactory accuracy, precision, and specificity in exploratory tests, the Schechter-Haller method (12) \+asselected as the foundation for the methods described below. The following auxiliary aspects were given attention. In a limited side study, with recovery data as criteria of suitability, extraction technique Jyas developed empirically. Prospects of meeting the anticipated sensitivity limits through increasing the size of sample were investigated. It was found that samples between 1.50 and 1.75 liters could be handled in 2-liter, Squibb separatory funnels. The problem posed by suboptimal amounts of D D T \vas met by analyzing only the suspended solids for DDT. This was based on the finding that D D T is adsorbed to an appreciable extent by the suspended solids fraction (6). Thus, larger samples of n-ater-e.g., 30 liters-may be used, resulting in a corresponding increase in sensitivity. Since the amount of D D T adsorbed appears to vary x i t h kind, particle size, and the total amount of suspended solids, such analyses were ICRO amounts
used mainly to provide an approximation of the D D T present in suboptimal samples. Interferences present in the water and suspended solids produced a yellow to amber color a t the colorimetric stage. Davidow's ( 5 ) modification of the method of Schechter, Pogorelskin, and Haller (11) was effectivein eliminating the interferences, but the recovery of D D T was lowered. From recovery data so obtained, a correction curve was constructed. To increase the absorbancy of the final solution, the tetranitroD D T obtained from the sample was dissolved in a decreased volume of benzene and sodium methoside. Lowry-Bessey microcells (@, which have a capacity of less than 1 ml., were used, and the final solution was prepared with 0.7 ml. of benzene and 1.4 ml. of 2.40 A; sodium methoxide. Further lowering of percentage transmittance by dissolving the tetranitro-DDT in less than 0.7 ml. of benzene and 1.4 ml. of sodium methoxide was impractical owing to increased absorption of the blank. Figure 1 shows that these modifications result in a lowering of the slope of the curve, with resulting greater photometric accuracy. The data for Figure 1 were obtained 15-ith acetone solutions of D D T of a grade similar to that used in the field tests (6). Applying Ringbom's method ( I O ) for determining the inaccurate region of the standard curve (C, Figure l), it was found that 4 to 30 micrograms of D D T was the most accurate range under conditions described herein. Residues containing less than 4 micrograms were considered suboptimal, and where these were encountered, 10 micrograms of D D T were added (stand-
