followed in the range of 1 to 20 pg of Fe(AA)3 per ml, which is equivalent to 0.16-3.2 pg of iron per ml. The Fe(AA)r contents of several samples of uncured terpolymer were determined. Results are listed in Table 11. These results are within the precision attainable with photometric methods. The method is rapid and suitable for row tine determinations.
Nitrosoresorcinol forms complexes with several cations (3). It was not considered necessary to study the effects of these ions because they are not generally found with the terpolymer. RECEIVED for review Oct. 11, 1967. Accepted Nov. 20, 1967.
Infrared Spectra of Microgram Amounts of Amino Acid Phenylthiohydantoins Eluted from Thin Layers of Silica Gel Marvin Murray Department of Pathology, University of Louisville School of Medicine, 101 W . Chestnut St., Louisville, Ky. 40202 Gerald F. Smith’ Department of Chemistry, University of Louisville, Louisville, Ky.
THINLAYER CHROMATOGRAPHY, in itself, is an invaluable tool for the separation and purification of compounds in both simple and complex mixtures. Special techniques have been developed recently which render feasible the quantitation of compounds from thin layers of silica gel using ultraviolet spectrometry ( I , 2). Combination of the above with infrared analysis of microgram amounts of compounds separated on thin layers would greatly enhance the analytical capabilities of those researchers working with small amounts of organic compounds. The attainment of infrared spectra in the microgram range has been the goal of several investigations. Various approaches and techniques have been studied. A bibliography of these is included in a recent report by Price, Sunas, and Williams (3), who have been able to obtain spectra in the 5-pg range by use of a special, positioning liquid sample cell. The method described below involves relatively simple and inexpensive instrumentation and materials. In our laboratory, analyses are performed upon fibrin samples, separated from both autopsy and surgical thrombi, and other protein specimens for their N-terminal amino acid content. The method used involves the formation of phenylthiohydantoins by the reaction of phenylisothiocyanate with the terminal amino groups and subsequent liberation of the terminal amino acid derivative from the protein molecule. The procedure used, in essence, is that described by Blomback and Yamashina (4). The phenylthiohydantoins are separated on thin layers of silica gel and quantitated as described previously (2). In many cases the array of products and by-products observed on the resulting chromatograms make positive qualitative identification virtually impossible. Infrared spectral analysis of the compounds on the chromatographic spots in question has enabled us to identify, with certainty, the products from these reactions. 1
Graduate trainee in Molecular Pathology.
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(1) R. D. Spencer and B. H. Beggs, J . Chromatog., 21, 52 (1966). (2) G. F. Smith and M. Murray, Anal. Biochem. (in press). (3) G . D. Price, E. C. Sunas, and J. F. Williams, ANAL.CHEM., 39, 138 (1967). (4) B. Blomback and I. Yamashina, Arkil: Kemi, 12, 299 (1958).
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ANALYTICAL CHEMISTRY
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used EXPERIMENTAL
Reagents and Materials. Spectral grade 1,2-dichloroethane containing 0.0003% residue is used. The solvent is kept in a dark bottle which is flushed with nitrogen before recapping. Chromatographic solvents and solvent systems have been described previously (2, 5). Phenylthiohydantoins were purchased from the Sigma Co., St. Louis. Precoated silica gel (250 p ) thin layer plates were purchased from Brinkmann Instruments, Inc., Westbury, N. Y. The layer is Silica Gel FZs4,containing a special organic binder and a manganese-activated zinc silicate fluorescent indicator (fluorescing at about 250 mp). Silver chloride plates (1 mm X 10 mm X 30 mm) are purchased from Harshaw Chemical Co., Cleveland, Ohio. Potassium bromide windows are prepared by pressing about 0.5 gram of the powder with a special die (1-cm diameter) having on its center surface a micro-die (1-mm diameter X 0.5-mm height). The resulting KBr disks are 1 mm thick containing a cylindrical cell whose dimensions are that of the micro-die. Such disks remain clear for months stored over PZOS. ( 5 ) K. Randerath, “Thin Layer Chromatography,” Academic Press, New York, 1963, p 104.
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Infrared spectra are recorded on a Perkin-Elmer Model 237 B double beam grating spectrophotometer. The beam condenser used is Perkin-Elmer No. 127-1271, which reduces the sample beam slit image to 0.5 mm X 4 mm at the sample position, Ordinate scale expanded spectra are recorded with Perkin-Elmer NO. 220-0058 accessory attached to a IO-mV strip chart recorder (10-inch width). Methods. Thin layer chromatography is performed upon reaction product mixtures and phenylthiohydantoin standards. Chromatography and removal of the desired spots from the layers are performed exactly as previously described to ensure the extraction of pure compounds (2). Elution of a compound from a spot area is performed by allowing the removed spot area to stand overnight under 1 ml of spectral dichloroethane in a 1-cm-diameter test tube. The tube is centrifuged at about 300 rpm and about 80% of the eluate is transferred to a clean tube by a Pasteur pipet, taking care not to disturb the packed silica gel particles. The transferred eluate is evaporated with a gentle stream of nitrogen to a volume of 5-25 pl, which is then spotted onto an AgCl plate or KBr welled disk with a micropipet. Evaporation of the solvent deposits the sample as a thin film. Spectra are recorded after horizontal and vertical adjustment of the sample holder in the beam condenser to attain maximum absorbance. The base line is set by attenuating the reference beam, and water vapor bands are eliminated by adjustment of the balance control. All spectra are recorded with the slit program designated as "normal."
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RESULTS AND DISCUSSION
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Ramachandran, Epp, and McConnell have shown that the infrdred spectra of amino acid phenylthiohydantoins are sufficiently different to warrant their use for identification purposes. The spectra presented below were chosen to be a survey of the practicality of the procedure described here. Figures 1 and 2 show the spectra obtained from the elution of 10 pg of tyrosine and alanine phenylthiohydantoin standards after thin layer chromatography. The actual spectral samples must be less than 10 pg as indicated by the above sampling technique. Also there is probably incomplete elution of these compounds into dichloroethane (2). Figure 3 demonstrates the resolution obtained with a 5-pg sample of phenylalanine phenylthiohydantoin (after chromatography) compared with that obtained from a 20-pg sample applied from solution without chromatography. (6) L. K. Rarnachandran, A. Epp, and W. B. McConnell, ANAL. CHEM., 27,1734 (1955).
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Curve B. Spectrum of aspartic acid PTH 15 r g N-terminal analysis product recovered in 1 ml of dichloro ethane plus 1 drop of acidic ethanol; AgCl window used
Curve C. Spectrum of blank silica gel area eluted with 1 ml of dichloroethane; AgCl window used VOL 40, NO. 2, FEBRUARY 1968
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Figure 6. Curve A . Spectrum of valine PTH 5 pg of standard was chromatogrammed, then recovered; AgCl window used. Spectrum ordinate expanded 2 X onto 10 inch wide chart
Curve B. Spectrum of valine PTH 1.5 pg N-terminal analysis product; AgCl window used. Spectrum ordinate expanded 10 X onto 10 inch wide chart
The glycine derivative (27 pg), Figure 4, and the aspartic acid derivative (15 pg), Figure 5 , curve B, are from N-terminal analyses. The amounts of compounds eluted were calculated by the ultraviolet spectrometric quantitation of the products from a duplicate analysis on the same thin-layer plate. Elution of the aspartic acid derivative is performed by adding one drop of acidic ethanol (made to pH 1 with concd HCl) to the 1 ml of dichloroethane elutant. Elution of the dicarboxylic acid derivative, with only dichloroethane and subsequent sampling, deposits the compound in a form in which the carboxyl group is in the anionic state, as shown in Figure 5 , curve A (1580 cm-l and 1370 cm-l). Exposure of such a sample to HC1 vapor for a few seconds changes the spectrum to that of curve B, Figure 5 . The same behavior is observed when handling glutamic acid phenylthiohydantoin. However, by making the eluting solution acidic as described above, the compound is obtained in its acidic form. Figure 6, curve B, shows a portion of the spectrum of 1.5 pg valine phenylthiohydantoin, also separated from an Nterminal analysis, The spectrum had to be expanded by 1OX for comparison purposes. Curve A , Figure 6 is the spectrum of a 5-pg valine phenylthiohydantoin standard (after chromatography) expanded 2 X. The absorbance scale indicated is the true absorbance of the standard spectrum and about twice the actual absorbance of the unknown spectrum. Figure 7 is included to demonstrate the spectral limitations with the technique presented in this report. The sample, glutamine phenylthiohydantoin, was spotted directly onto a
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ANALYTICAL CHEMISTRY
welled KBr disk without prior chromatography. The loss in resolution with a 1-pg sample (curve B, Figure 7) compared with a 2-pg sample (curve A ) is consistently observed. Occasionally, a 1-pg sample or less will yield a discernible spectrum, but only by use of ordinate scale expansion. However, 2-pg samples consistently yield spectra resolved well enough for identification purposes. The infrared spectra of all of the common amino acid phenylthiohydantoins sampled as films from dichloroethane are identical to the crystalline state spectra which have been reported (6) except those of the aspartic and glutamic acid derivatives, which yield liquid state spectra (6). Figure 5 , curve C , shows the base line obtained from elution of a blank area with 1 ml of dichloroethane and subsequent sampling of any residue. The curve is identical to that obtained with a clean AgCl plate in the sample beam. The solvent contains 0.0003% residue. However, no spectral interference is observed in the eluted samples. If this solvent is exposed for lengthy periods to air and light, apparent oxidation and/or polymerization reactions occur, rendering the solvent unsuitable for this sampling technique. Spectral acetone and methanol contain a ketonic residue (0.0003-O.0005%) which yields an intense spectrum upon evaporation of 1-ml amounts. However, these solvents are available commercially as "residue-free." Other solvents have not been examined. In conclusion, a method has been presented with which one can successfully obtain infrared spectra of organic compounds in the microgram range after separation on thin layers of silica gel. The use of expensive instrumentation or special instrumental modification has not been necessary. RECEIVED for review September 27, 1967. Accepted November 7, 1967. Work supported by National Institutes of Health Training Grant in Molecular Pathology-HE-5664, and American Heart Association Grant No. 67-642.
