Table IV. Analysis of Miscellaneous Triple Carbonate Samples
% Nitrate Ion exchange, Ultraviolet brucine Method Sample SO. method 220mp 210mp 1 0.40 0.39 0.38 0.39 2 0.29 0.28 0.28 0.29 3 0.60 0.59 0.60 4 0.96 0.98 0.98 0.97 5 0.81 0.79 0.79 0.79 6 0.21 0.23 0.21 0.22 7 0.15 0.16 0.15 0.15 8 0.47 0.47 0.48 0.48 9 0.55 0.57 0.57 0.56
The brucine values are averages of from three to six determinations. Ultraviolet values are averages of three determinations, except for the second set a t 220 mp, which were run in routine fashion by a second analyst and represent one or two determinations. At 210 mp the maximum average deviation on a single sample was i 0.01170 nitrate, and the average of all such deviations mas =t0.005% nitrate. At 220 mp (considering only values based on three determinations) the corresponding results were ~t0.017 and =t0.0077,. For the brucinemethod the values were 10.019 and 1 0 . 0 1 0 ~ .
Table V.
Elimination of Cation Interferences on Sample Containing 0.5070 Nitrate
Nitrate Found, yo Direct Triple Carbonate Method Sample Containing 220mp 210mp 0.73 0.227GFe+++,0.225& Cu++, 0.84 0.73 0,22% Pb+0.85 0.74 0.86 -4v. 0.85 0.73 Corrected for Fe+++,Cu++, Pb + + a 0.51 0.51 Using factors in Table 11.
Difference hlethod 220mp 210mr 0.50 0.51 0.50 0.50 0.52 0.51 0.51 0.51
Ion Exchange Separation, 210mp 0.50
0.50
0.50
0.50
0
ELIMINATION
OF CATION INTERFERENCES
T o make the procedure more generally applicable, several variations can be used to eliminate or correct for interfering cations. The general method is to pass a weakly acid perchloric acid solution of the material through a cation exchange resin t o remove cations, adjust the acidity, and read the solution directly. The resin should be thoroughly mashed with dilute perchloric acid immediately before use and the washings tested to make sure the ultraviolet absorption is negligible. -4second method is to take t x o equal aliquots of the sample solution, fume one thoroughly with perchloric acid to expel nitrates, and use it as a background for the other. This “difference method” mag fail if the oxidation state of the metal ion is changed in the process as, for example, with chromium(II1). If iron(lI1) is the main interference, the solution may be read a t 260 mp (1) and a correction applied.
The first two procedures are illustrated in Table V on a synthetic sample Tvith a triple carbonate base composition and iron, lead, and copper impurities. Methods for eliminating the interference of anions are beyond the scope of this paper. However, certain anions could be precipitated with silver which, in turn, could be precipitated with chloride. LITERATURE CITED
\
,
Bastian, R., Sf7eberling, R., Palilla, F., ANAL.CHEX. 28, 459 (1956). Buck, R. P., Singhadeja, S., Rogers, L. B., Zbid., 26, 1240 (1954). Dolance, A.. Healv. P. W., IND.ENG. C H E ~ANAL. . “ED.17. 718 (1945). Komarmy, J. M., Brokch, SV. J., Testerman, RI. K., Anal. Chim. Acta 7, 349 (1952). LIacDonald, A. M. G., Znd. Chemist 31, 515 (1955). S o l l . C. A.. IND. ENG.CHEM..ANAL. ED. 17, 426 (1945).
RECEIVED for review May 27, 1957. Accepted August 23, 1957.
Determination of Carbon-14 Steroids on Paper Chromatograms DAVID L. BERLINER, OSCAR V. DOMINGUEZ, and GARTH WESTENSKOW Departments of Anatomy, Biological Chemistry and Radiobiology, University o f Utah College o f Medicine, Salt take City 7 2, Utah
b The major points of loss in the chromatographic determination of radioactive steroids are the steps involving elution and plating. Steroids may b e quantitated directly on paper chromatograms by using @ray selfabsorption factors and measuring radioactive areas recorded on a strip counter. The method makes possible the study of physiological quantities of steroids from biological tissue and fluids.
P
Chromatography is one of the most widely employed analytical techniques in tracer studies of steroid metabolism. The removal and quantitation of steroids from paper chromatograms require several steps (elution, plating, and counting), which are often accompanied by loss of material. Therefore quantitation of steroids directly from the chromatogram would be advantageous. The purpose of this study was to deAPER
velop a more sensitive method for determination of steroids following paper chromatography. A technique for determining steroids directly on a paper chromatogram, a more sensitive counting apparatus for evaluating the radioactivity, and a method for determining correction factors for self-absorption of low energy beta rays have been developed. The method has been successfully applied to the determination of steroids VOL. 29, NO. 12, DECEMBER 1957
1797
isolated from tissues and fluids (1, 49. APPARATUS AND METHODS
The principal paper chromatographic techniques employed ere those described by Zaffaroni (6). I n a few studies the Bush system was used ( 5 ) . The steroids nere cortisol 4-C" (specific activity 1.467 me. permmole) andprogester0ne-4-C'~ (L4.0.5 mc. per mmole). Chromatograms 3 em, nide and 45 cm. long were analyzed. Known quantities of mixtures of nonradioactive and radioactivc steroids (specific activities ranging from 2.4 to 365 counts per minute per y) were prepared. To maintain a constant amount of material 100 y of steroid was applied on each of 30 chromatographic strips. The chloroform-formamide system (6) was used for cortisol-4-C14, and the hexane-formamide system (6) for progesterone-4-CI4. After chromatography, the papers ryere dried and the radioactivity was counted in a strip scanner system (designed and constructed a t the Radiobiology Department. Vniversity of Utah, by Westenskow) (Figure 1). The scanning system uses a SuclearChicago Model D-47 gas flow counter and Micromil windon operated as a Geiger-Muller counter. A thin brass disk with a slit 1 cni. wide is placed between the Micromil \\-indow and the transport strip. The detector is surrounded by 2 inches of lcad shielding in its housing. The chromatograms are taped to an aluminum transport strip 2 inches wide and 48 inches long, which is gear-driven by a synchronous electric motor and gear train identical to that used in the Brown recording potmtiometer chart drive system. The scanner is designed so that the strip is driven under the detector slit a t the rate a t n hich the recorder chart is moved past the recording pen. Scanning rates of 6, 10, and 12 inches per hour have been uscd. The rate may be changed by making identical gear changes in the strip drive and the recorder chart drive gcxar trains. The detector is operated from a precision count rate meter which has a maximum sensitivity of 100 c.p.m. full scale. Additional rangcs of 300, 1000, 3000, 10,000, 30,000, 100,000, and 300,000 c.p.m. full scale are available. It was necessary to use a long time constant (about 150 seconds) in the rate meter circuit for the lorn counting range. -4 representative pattern obtained from the strips analyzed in the counters is depicted in Figure 2. The areas, A , and altitudes, H , of the radioactive zones were measured and corrected to the scale of 100, taking this as a unity area x scale . Subsequent to this 100 analysis the 3-em. wide papers were cut into 1-em. lengths and each section was counted with a Nuclear-Chicago C-110 automatic sample changer and D-47 gas flow counter, both with, C W . and without the Micromil window ( S W ) . The sum of the counts on paper, P , corresponding to the area from each chromatogram was recorded. The zone where
1798
ANALYTICAL CHEMISTRY
the radioactivity was present was eluted, E, with methanol. Because of background activity in the paper, the same area of paper was eluted, irrespective of the quantity of radioactivity present. An aliquot of the eluent (or the total sample) was plated on aluminum plates and the radioactivity measured in both counters (Figure 2). All samples were counted long enough to give an error of 5% or less. The following factors were calculated to determine the quantity of steroid (both counters) : P / A , P / H , E / P , E / d , and E/H
in which P = c.p.m. from paper, E = c.p.m. from the total zone after elution, 4 = area under the curve, and H = height of peak ( A and H from strip counter register. The ratios between ~ i n d o wand windowless counters were determined for each piece of paper and each aliquot present in the aluminum plates. The standard deviations and coefficients of variation were also determined (Table 1). RESULTS
The results are summarized in Tables
I and 11. When the CW counter is used, the coefficients of variation and the standard deviations are smaller than when the S W counter is used. This means that, on the basis of reproducibility, the C W counter is the more reliable instrument. The sensitivity is higher in the STV counter because the low energy beta rays are not screened; however, the variation is greater, probably because of the presence of small quantities of solvent in the sample, as in preparing the paper chromatograms formamide is applied to them ( 6 ) ,and this solvent is difficult to evaporate complc tely after chromatography. The presence of the Alicromil windon. in the CJV counter screens the lon energy beta rajs. The difference between the ratios of the counts in the
( PSW
papers p ~ W = 1.29) and those on the eluates (FW ' E S W = 1.58) is 18.47,, which corresponds to the screening of the low energy beta rays by the hlicromil window. The absorption of beta rays by the T h a t m a n S o . 1 filter paper used for chromatography in these studies was in the range of 61.27,. The smallest coefficients of variation from all the factors determined are the factors E / P and E,IA using the CTY counter. This means that the correlations between area under the curve and radioactivity on the paper in respect to radioactivity in the eluate are more significant than the other factors. If the log area measurements obtained from the recording strip counter are plotted against the log of counts per minute originally applied on the
Table II.
Factor
Averages, Standard Deviations, and Coefficients of Variation
Radioactive Steroid Directly Applied, C.P.M. 2000 4000 8000, 16,000, 20,000 a b a h a a a h
P
Averagc and Variations =tC *CV
'/tw sw
3.26 4.45
2.85 3.72
2.82 3.30
2.98 3.87
2.88 4.07
2.84 3.45
2.57 3.45
2.67 3.22
2.86 3.69
0.193 0.394
6.75% 10.70%
sw
1.73 2.38
1.40 1.77
1.29
1.51
1.42 1.85
1.65 2.34
1.29 1.57
1.17 1.57
1.26 1.46
1.40 1.81
0.183 n ,342
13.10% 18.90%
sw
2.12 2.44
2.05 2.46
2.18 3.10
2.00 2.75
2.12 2.41
2.08 2.30
2.00 2.32
2.12 2.08 2.84'2.58
0.059 0.268
2.87% 10.40%
"/&SW
10:83
6.00 9.11
6.15 10.24
5.95 10.65
6.09 9.85
5.91 7.91
5.13 8.00
5.67 9.12
5.84 9.46
0.324 1.05
5.55% 11.10%
3.66 5.74 1.36 1.57
2.86 4.35 1.26 1.52
2.82 4.69 1.17 1.66
2.85 5.10 1.30 1.79
3.50 5.65
2.69 3.59 1.24 1.34
2.33 3.63
2.50 4.15 1.20 1.61
2.90 4.61 1.29
0.430 0.781 0.080 0.120
14 .80% 16.90% 6.2% 7.6%
"I&
sw
P.SW/P.CW E . S W / E .CW
1.42
1.62
Figure 1.
1.35
1.56
1.58
Strip Counter
1. Brown recording potentiometer
2. Count rate meter 3. Nuclear Chicago D-47 gas flow counter 4. Strip drive mechanism ~
5. Lead shielding 6. Gas supply, helium 98.7%-hutane 1.3% 7 . Chromatogram strip transport
Figure 2. factors
Procedure for determination of
VOL. 29, NO. 12, DECEMBER 1957
1799
paper chromatogram, a linear curve is obtained (Figure 3). DISCUSSION
The use of a factor based on the height, H , of the radioactive zone would be restricted to a single steroid in a rigidly controlled system, because steroids near the origin are in narrow and compact bands, whereas those migrating near the front are more broad and diffuse. This holds true for all the radioactive steroids employed in biological experiments (4) (~ortisone-4-C~~, 11P-hydroxyandro~tenedione-4-C~~, and 4-pregnene17,20,21 - triol - 3,11 - dione - 4 - CY4)). The results have all fallen within i8% of the factor E / A , which is 6.12 (Table I). Therefore, if the area recorded by the strip counter (Figure 2) is multiplied by this factor, the amount of radioactivity present in the paper can be determined with this accuracy. This eliminates the necessity of elution steps @), FT-hich often result in loss of appreciable quantities of steroid.
E / P is a useful factor, which has been employed in previous studies (9). When a known quantity of steroid is chromatographed, the recovery from the zone of migration of the steroid in the Zaffaroni chromatographic systems varies from 80 t o 92%. The percentage of radioactivity not accounted for in the chromatographed zone can be demonstrated a t the origin and front and in the streaking zones. The distribution of radioactivity outside the chromatograph zone is usually greater in the Bush system (5) of chromatography, because of the low capacity of this system in the presence of fats in biological extracts. When the area method is applied to the determination of radioactive steroids extracted from biological fluids and tissues, the percentage loss can be determined by adding a known quantity of nonradioactive steroid to the extract before chromatography (1). The loss of nonradioactive steroid in the chromatographed zone is proportional to the loss of the radioactive steroids, when they are identical and in mixture.
A quantitative ultraviolet strip recording spectrophotometer in combination with the strip counter employed in the work should be of great value in the simultaneous determination of radioactive and nonradioactive steroids directly on paper chromatograms. LITERATURE CITED
(1) Berliner, D. L., Proc. SOC.Esptl. Biol. & M e d . 94, 126 (1957). (2) Berliner, D. L., Salhanick, H. A,, ANAL.CHEX 28, 1608 (1956). (3) Berliner, D. L., Wiest, TV. G., J . Biol. Chem. 221, 449 (1956). (4) Berliner, M. L., Berliner, D. L., Dougherty, T. F., Proc. Am. Assoc. Cancer Research 2, 94 (1956).
(5) Bush, I. E., (‘Recent Progress in Hormone Research,” 1‘01. IX, p. 321, Bcademic Press, Xew York, 1954. (6) Zaffaroni, A., Ibid., Vol. VIII, p. 51, 1953. RECEIVED for review Sovember 15, 1956. Accepted July 27, 1957. Work supported in part by research grants from the S a tional Cancer Institute, National Institutes of Health, U. S.Public Health Service, and the American Cancer Society upon recommendation of the Committee on Growth, National Research Council.
Accuracy of Quantitative Paper Chromatography in Amino Acid Determination Using Direct Photometry HENRY R. ROBERTS and MICHAEL G. KOLOR Research laboratories Division, National Dairy Products Corp., Oakdale, 1. I., N. Y.
A study has been made of the accuracy and precision of the quantitative paper chromatographic procedure developed by McFarren and Mills for determination of amino acids in protein hydrolyzates. Known solutions of 20 common amino acids were prepared and analyzed. Each amino acid was assayed nine times, the average value obtained, and the per cent deviation of this average value from the known value calculated. Eleven acids gave average values which differed from the theoretical value by less than 2%, 13 less than 370, 17 less than 5%. Only lysine, norleucine, and tyrosine had values more than 5% in excess of the true values. Statistical evaluation of precision showed that the assays can b e repeated with good reproducibility. ARIOUS quantitative methods have been developed for the determination of amino acids by paper chromatography (3, 4, 18). A number of investigators (9-5, 7, 10, 11, 13-15, l Y ,
1800
ANALYTICAL CHEMISTRY
13, 20) have shown that the direct photometric or maximum color density procedure yields results which agree with other more involved methods and is convenient, less tedious, and more rapid. This paper presents data on the accuracy and precision of McFarren and Mills’ ( I S ) maximum color density procedure for amino acid determination. I n determination of the amino acids content of p-lactoglobulin, McFarren and hlills’ results were comparable to those obtained by other methods. Their appraisal of the procedure was based only on this comparison. The present study is based on analysis of a known standard solution of amino acids, for without a comparison iTith a standard, accuracy has no meaning. I n addition to ascertaining the accuracy and precision of McFarren and Mills’ technique, this study also ivas attempted:
To point out that the procedure gives excellent separation of the amino acids as round or elliptical spots, the lack of which is often offered as an argument
against the use of the maximum density procedure. To offer additional proof that the maximum density technique, as a quantitative procedure, can be used with confidence. EXPERIMENTAL
hIcFarren and Mills’ one-dimensional descending paper chromatographic procedure ( I S ) uses seven solvent systems a t a selected p H to separate each amino acid from all others in a mixture containing 20 common amino acids. The paper is buffered at the same p H as the solvent. Strips of Khatman No. 1filter paper are dipped into the appropriate buffer solution ( I d ) , suspended by one end, and dried in air. Each solvent system is a two-phase system. The waterrich layer is placed in the bottom of the chamber. To achieve the desired separations, the bottoms of the chambers must be kept wet with the water-rich phase. The developing solvents, equilibrated a t 22’ C. in a temperaturecontrolled room m-hich houses the chromatographic chambers, are prepared as follows: SOLVENT 1. Equal volumes of phenol
