molar response seems unwarranted, since the results were dependent on the applied potential chosen for study. As a further complication, the results obtained with two different detectors a t the same potential were similar but not identical (compare values at 800 volts in Tables I1 and 111), suggesting that the observed molar responses are also dependent to a certain extent on the design of the detector. I n this regard, the molar areas with the 2.5cm. detector were considerably greater than those obtained with the I-cm. detector. This observation may be due to differences in the amount of radioactive material in the two detectors. As a result of this study, several points may be made regarding quantitative microanalysis of steroids by gas chromatography employing the argon ionization process for detection. Since no set of operating conditions has been found by which the argon detector may be made to give uniform molar responses independent of structure, standards must be used for each steroid to be measured, using graphs such as those in Figure 1 for the conversion of observed areas into concentration. This method, though somewhat cumbersome, provides precise analyses, especially when the standards are determined daily. For example, it has been possible, in this laboratory, to measure accurately the cholesterol content of lipides isolated from human serum and from mixed animal-vegetable diets. By a similar procedure involving standards, Wotiz and Martin have determined urinary estrogens on a routine basis by gas chromatography (16).
Since the relative molar response for each steroid is also a function of the applied potential on the detector, it is necessary, after analyses have been made of the standards, to maintain a constant voltage throughout a given series of analyses. The use of a stepping switch is preferred for this purpose, since it is difficult to regulate the voltage in a reproducible manner with a continuously variable control. Aside from the problems of quantitation introduced by variations in molar response, interesting questions may be raised concerning the mechanism of the process. The most pronounced effect, that of decreasing sensitivity with increasing oxygen content, as well as secondary effects such as those observed with changes in the nature of the functional groups, may be attributable to recombination phenomena similar to those observed by Lovelock with an electron capture ionization detector (9). While the argon ionization and electron capture detectors are different in design and mode of operation, the effects with steroids may be related to differences in response shown in the electron capture detector with various halogen-containing materials. Although these studies have not been extended to other types of biological mixtures such as amino acids, urinary aromatic acids, and similar mixtures in which functional groups vary, it may well be that in all such cases, molar response will be dependent to a degree on the nature and number of functional groups. Hopefully, studies of such mixtures will provide further data from which may be obtained a more
complete understanding of this complexity of the argon ionization process and its relation to electron capture. LITERATURE CITED
(1) Beerthuis, R. K., Recourt, J. H., hrature 186,372 (1960).
(2) Bottcher, C. J. F., Clemens, 3. F.G.,
van Gent, C. M., J. C h r m a t o7.~ 3, 582
(1960). (3) Eglinton, G.,Hamilton, R. J., Hodges, R., Raphael, R. H., Chem. & Ind. (London) 1959,955. (4) Farquhar, J. W., Insull, William, Jr.,
Rosen, Paul, Stoffel, Wilhelm, Ahrena E. H., Jr., Nutrition Reus. 17, 8 (SUPP1.l (1960). (5) Haahti, E. 0. A., VandenHeuvel, W. J. A., Horning, E. C., J. Am. Chm. SOC.83,1516 (1961). (6) Haahti, E. 0. A,, VandenHeuvel, W. J. A., Horning, E. C., J. Org. Chem. 26,626 (1961). (7) Horning, E. C., Moacatelli, E. A., Sweeley, C. C., Chem. & Ind. (London) 1959, )sky, 751.S.R., Landowne, R.A,, ANAL. ( 8 ) Lil: _ . CHEM.33 818 (1961). (9) Lovelock J. E.,Ibid., 33, 162 (1961). (10) Nicolaides,. N.,. J. Chromatog. - 4,. 496 . (isso). (11) Sweeley, C. C., Horning, E. C., Nature 187, 144 (1960). (12) VandenHeuvel, W. J. A,, Horning, E. C., Bwchem. Biop&s. Research Communs. 3,356 (1960). (13) VandenHeuvel, W. J. A., Sweeley, C. C.. Horninn. E. C.. Ibid.,. 3.. 33 (1960)'. (14)VandenHeuvel, W. J. A., Sweeley, C. C.. Horninn. E. C., J . Am. Chem. Soc. 82,3481(ig66j. (15) Wotiz, H. H., Martin, H. F., J . Bi0Z. Chem. 236, 1312 (1961). I ,
RECEIVED for review November 14, 1960. Accepted September 13, 1961. Work supported by grants from the National Institute of Arthritis and Metabolic Diseases (A-4307) and the American Cancer Society.
uantitative P per Chro atogra phy f Plasma Amino Acids odification of: the Binitrophenylation Procedure of Levy CARL PERAlNO and ALFRED E. HARPER Department of Biochemistry, University of Wisconsin, Madison 6, Wis.
b A paper chromatographic procedure has been devised for the quantitative determination of 15 of the free amino acids present in blood plasma. The procedure involves the preparation of the dinitrophenyl derivatives of the amino acids, separation e f these derivatives by two-dimensional paper chromatography, elution of the derivatives from the paper, and mPasurement of the absorbances of the resulting solutions, using a spectro-
photometer. This method is sufficiently sensitive to permit accurate determination of the amino acids in 1 ml. of plasma, and it can readily b e adapted to the analysis of large numbers of samples. The samples need not b e desalted prior to analysis. Leucine and isoleucine appear as one spot on the chromatogram; methionine, tryptophan, histidine, arginine, and ornithine are not satisfactorily determined b y this procedure.
I
1954 Levy (8) described a method for the quantitative paper chromatography of free amino acids. The method involves the reaction of the amino acids with l-fluoro-2,4-dinitrobenzene (FDNB); separation of the yellow dinitrophenylated amino acids by two-dimensional paper chromatography; elution of the derivatives from the paper; and measurement of the absorbance of each derivative, using a spectrophotometer. Although the N
VOL. 33, NO. 13, DECEMBER 1961
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adaptability of this technique for blood m i n o acid analysis has been suggested by Levy (2), and in the comprehensive review by Biserte et al. (I), it has seldom been used for this purpose. This report describes a procedure, based on the original method of Levy (2), which has been used with success in our laboratory for the quantitative estimation of 15 of the free amino acids present in blood plasma. PROCEDURE
All reagents used are of the highest purity obtainable commercially. A given volume of plasma is deproteinized by adding an equal volume of 10% (v./v.) perchloric acid and removing the precipitated protein by centrifugation. Trichloroacetic acid is not used as a deproteinizing agent, because its solubility in ether would cause it to interfere in subsequent stages of the analysis. Two milliliters of deproteinized plasma (equivalent to 1 ml. of the original plasma) are transferred to a 50-ml. glass-stoppered Erlenmeyer flask. The pK of the solution is adjusted to 9 (pH paper) with 5N sodium hydroxide and 5 ml. of 5% (w./v.) sodium bicarbonate are added. Wherever possible, subsequent operations are carried out in total darknessLe., either in a darkened room or in equipment covered with a black clothowing to the light sensitivity of the derivatives (5). Subdued light (15-watt light source) is used in steps requiring visual manipulations. Two-tenths milliliter of a 2% (v./v.) solution of FDNB in absolute ethanol (ca. 35 pmoles of FDNB) are added to the reaction mixture, which is then shaken for 80 minutes a t 40' C. ( 2 ) . (The number of micromoles of FDNB added is approximately five times as great as the total number of micromoles of amino acids in the sample.) After the reaction mixture has cooled to room temperature, it is acidified by the addition of 0.75 ml. of 5N hydrochloric acid. The mixture is then transferred to a 60-ml. separatory funnel equipped with a Teflon stopcock and extracted with several small volumes of peroxide-free ether, until the ether layer no longer becomes yellow. The ether extracts (containing the ether-soluble dinitrophenylated amino acids) are collected in a 250-ml. Erlenmeyer flask; sufficient anhydrous sodium sulfate (ca. 30 grams) is added to remove any contaminating aqueous phase; and the ether solution is filtered quantitatively through glass wool into another 250-ml. Erlenmeyer flask. The sodium sulfate is washed thoroughly with fresh ether, the washings being added to the main sample. The ether is then removed from the sample by evaporation in vacuo. The samples are kept in vacuo for approximately 5 hours after the ether has been entirely evaporated, in order to remove, by sublimation, a significant fraction of the dinitrophenol which is formed in a side 864
e
ANALYTICAL CHEMISTRY
reaction during the dinitrophenylation step (1, 9)The derivatives are then dissolved in a small volume of acetone and spotted quantitatively on a sheet of Whatman No. 1 filter paper (18 X 221/2 inches) at a point 5 inches from each of two adjacent edges. Sufficient acetone washings are employed to transfer all the yellow color from the flask to the paper. The spotting is performed by allowing the acetone solution to fall dropwise onto the paper while a stream of air (room temperature) is directed against the underside of the spotting area. The paper should be permitted to dry completely after each drop, in order to keep the spot as small as possible. The drop rate can be satisfactorily controlled through the use of a Teflon stopcock equipped with a metering valve (Kimble, Vari-Flo, No. 4100P, 2-mm. size). One end of the tubing attached to the stopcock is drawn out to a fine tip and the other end is widened slightly in the shape of a funnel. The unit is then suspended vertically over the spotting area, with the fine tip approximately 2 cm. from the swface of the paper. The tubing above the stopcock is filled with the acetone solution and the drop rate is adjusted by means of the metering valve. Once the proper rate has been obtained, the unit can be left unattended until the tube is empty. When the spotting is completed, t,he chromatogram is placed in a chro2-METHYL-1-BUTASOL, I-PROPANOL, "3,
H20
Y
OH-PRO
Figure 1 , Diagrammatic representation of chromatographic map of dini-
trophenyleted amino acids (Solid spots represent amino acids which can be analyzed by procedure descrlbed in text) cys. Cystine glu. Glutamic acid asp. Aspartic acid gluNHz. Glutamine rer. Serine thr. Threonine OH-pro. Hydroxyproline pro. Proline ala. Alanine Val. Valine leu. Leucine Ileu. Isoleucine phe. Phenylalanine try. Tryptophan lyr. Lysine tyr. Tyrosine DNP-OH. Dinitrophenol DNP-"2. Dinitroaniline
matography cabinet, arranged for descending chromatography, so that development will proceed along the long axis of the paper. The solvent system for the first dimension is then prepared by mixing together 100 volumes of 2-methyl-2butanol, 15 volumes of 5N ammonium hydroxide, and 15 volumes of 1-propanol. When the mixture is shaken, a clear, monophasic, unsaturated solution results. This system, which was devised in our laboratory, is equally as efficient, easier to prepare, and far less hazardous than the "toluene" system used by Levy (8). A portion of this solvent is placed in the bottom of the chromatography cabinet (inside dimensions of cabinet: 16 X 24 X 25 inches) and complete equilibration of the tank atmosphere with the solvent vapors is allowed to occur (4 to 6 hours). The chromatogram is then developed until the leading amino acid spot approaches the bottom edge of the paper (ca. 48 hours). At this point the paper i s removed from the cabinet and suspended in a hood until it has thoroughly dried (4 to 6 hours). Development in the second dimension is also carried out by the descending technique,using a 1.5Mphosphate buffer (pH 6) which is X.OM in NaHaPBr and 0.5M in Na2HPQ (2). No prior equilibration period is necessary for development in the second dimension. It is sufficient to place water in the bottom of the tank at the time development is begun. As before, the development is terminated when the leading spot approaches the bottom edge of the paper (ca. 16 hours), and the chromatogram is hung in a hood until dry. The individual amino acid derivatives are precisely located on the chromatogram by viewing it under ultraviolet light, and are identified by position (Figure 1). The position of the dinitrophenol can be verified by placing a drop of dilute hydrochloric acid on the spot in question. The acidified area will become colorless if the spot is dinitrophenol. Each derivative spot is cut out and placed in a test tube, sodium bicarbonate solution (1% w,/v,) is added until the paper is completely submerged, and the tubes are either heated for 20 minutes a t 55' to 60' C. (9) or allowed to stand overnight a t room temperature. The absorbmces of the resultant extracts are determined in a Beckman DU spectrophotometer against a 1% sodium bicarbonate blank, The absorption maxima are 385 mp for the proline and hydroxyproline derivatives, and 360 mp for the remaining derivatives (I, 9). The concentration of each amino acid in micromoles per 100 ml. of plasma is oaIcuIated by multiplying together the observed absorbance, the eluted sample volume in milliliters, and the appropriate conversion factor (Table I). The factors were derived from experimental data obtained by the procedure described above (using standard solutions of the amino acids in place of tbe plasma s a =
ple); and each incorporates a ratio of amino acid concentration to absorbance, and a correction for dilution.
Table I.
RESULTS
Column 2 in Table I shows the results of recovery experiments in which 0.2 to 1.0 pmole of each amino acid was added to 1 ml. of plasma. The average recoveries of 12 of the amino acids fall between 87 and 105'%, The recoveries for lysine, alanine, and cystine, although low, are sufficiently consistent at various amino acid levels to permit reliable comparisons. When the same plasma sample was analyzed by means of the Beckman amino acid analyzer and the paper chromatographic procedure, the results (columns 3 and 4, Table I) were in fairly close agreement. The discrepancies noted in the lysine and alanine values would be expected on the basis of the recovery results. DISCUSSION
The procedure described above has been successfully employed in our laboratory for the analysis of a large number of small blood samples. The chief advantages of this method have been found to be: SENBITIVITY. One milliliter of plasma is sufficient for analysis.
Amino Acid Threonine Serine Leucine Goleucine} Glycine
Analytical Results
%
Conversion Factors
Recoveriea
7.2 7.2 7.6 9.0 6.2 7.4 7.6 6.2 10.0 7.6 8.3 10.0 6.0 7.6 5.8
105 i 6" 102 7 102 i a 99 & 6 99 f 1 4 99 97 f 6 96 i 4 96 f 1 94 f 6 5 91 87 f 2 80 f 7 74 i 8 ~ 6 3f 2
Proline Valine Glutamic acid EITdroxproline ubmme Aspartic acid Phenylalanine Tyrosine Cystine a Standard deviation.
*
* *
EFFICIENCY.An average of 10 Dlasma samples per week are anilyzed (in our laboratory) by one person using two chromatography cabinets and one spotting device. TECHNICAL SIMPLICITY. No desalting is required; and the derivatives are visible during separation.
Comparative Analysis of Single Plasma Sample, pmoles/100 MI. FDNB Amino acid analyzer method 60 42 15 33 14
63 36
16
...
10
. a .
32
... .,.
10 20 30
...
34 14 11 18 36
... *..
8 30 42
...
LITERATURE CITED
(1) Biserte, G., Holleman, J. W. H o b
man-Dehove, J., Sautiere, P.,
matog. 2, 225 (1959).
5. Chro-
(2) Levy, A. L., Nature 174,126 (1954). (3) Pollara, B., von Korff, R. W., Biochem. et Biophys. Acta 39,364 (1960). RECEIVED for review June 8, 1961. Accepted September 25, 1961. Contribution from the Department of Biochemistry, University of Wisconsin, Madison 6, Wis. Published with the approval of the Director of the Wisconsin Agricultural
The chief disadvantages are: Leucine and isoleucine coincide on the chromatogram. Methionine, tryptophan, histidine, arginine, and ornithine are not satisfactorily determined by this procedure.
Experiment Station. Supported in part by a grant from the National Live Stock and Meat Board, Chicago, Ill.
Sampling and Recor ing Systems in Gas graphy-Time-of-Flight Mass Spectrometry A. A. EBERT, Jr. Research and Development Division, Organic Chemicals Deparfment, Jackson laboratory, E. 1. du Pont de Nemours & Co., Inc., Wilmington, Del. The combination of a gas chromatograph with a time-of-flight mass spectrometer has proved to be extremely useful for the rapid and positive identification of unknowns in complex mixtures. The mass spectra of the components can be scanned and recorded simultaneously with the running of the chromatogroph. A getterion pump has been used successfully to maintain the high vacuum in the spectrometer. This provides a clean system with little background. The input system to the spectrometer is designed to trap the sample from the column and allow it bo leak into the spectrometer long enough t6 allow the analog output system to record the spectrum. This normally requires less
than 1 minute. Usable spectra can b e obtained of components in very low concentrations (0.1 p.p.m.) and even when they are only partly resolved b y the chromatograph.
T
HE combination of a gas chromatograph (GC) with the Bendix timeof-flight (TOF) mass spectrometer has proved to be an extremely useful tool for the rapid and positive identification of chromatograph cuts. Chromatographic data alone are often ambiguous, and the identification of sample components by this means can be obtained with assurance only by the expenditure of considerable time and eEort. The GC-TOB combination was first
described by Gohlke (1) in 1959. Since that time many improvements have become available, which simplify the operation of the equipment and make the spectral recording more precise and readable. A new input system has been devised which provides the option of introducing the chromatograph cut directly into the spectrometer or employing a cold-trapping technique whereby the sensitivity may be increased a thousand times. Our experience with trapping samples from a gas chromatograph and transferring them to a mass Spectrometer has shown this to be a tricky procedure, much too slow for routine work and often showing gross contamination, usually with water. VOL. 33, NO. 13, DECEMBER 1961
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