Sample Preparation for Infrared Spectra of Agar Supported Aqueous Solutions of Amino Acids Margaret
L. Tarver and Lawrence M. Marshall,
Department of Biochemistry, College of Medicine, Howard University,
Washington, D. C.
T
report presents a convenient procedure for the preparation of 100-micron slices of agar mounts of amino acids in aquecus solutions for infrared absorption studies in the rock salt region. .Ilthough I'einberg, Rapson, and Taylor ( I ) first observed the applicability of agar for this purpose, their films, calculated from their esperimental conditions, were more than 1.5 mm. thick. In addition, the dried films of these investigators v;ere designed less for a support for an aqueous sample than they were for a mechanical support for the absorber. Crystallization of the latter in the film did not hinder the reported infrared absorption. HIS
EXPERIMENTAL
Five hundred milligrams of each amino acid were adcled to a freshly prepared 1% agar solut'ion that had
FFiEOUENCY ( C M - ' )
cooled to 40" C. The solvent for the agar was water when the amino acids were examined in the hydrochloride form, but 0.05JI phosphate buffer. pH 7 . 2 was used for the examination of the amino acids in a medium basic to their isoelectric points. The agar solution was poured into the barrel of a 50-1111. syringe that contained a plunger but that had its needle end rut off. The barrel end of the syringe with the plunger a t the bottom and the cut end a t the top vias supported over a microtome (.\nierican Optical Co., Model 880) in such a manner that the plunger reyted on the microtome mounting disk. By this arrangement, a 100-micron mechanical ad\,aiicenient of the mounting disk supporting the plunger extruded the agar gel precisely 100 microns. This syringe arrangement with microtome \vas similar t o that miployed by Lauffer ( 3 ) for his diffusion studies in agar. The slices, obtained by cutting them
with a wire 0.003 inch in diameter, were mounted over a rectangular a i m ture 2 X 0.8 cni. ivhirh had kern rut out of a cardboard 8 cm. long and 5 cm. wide. The size of the cardboard permitted it to .slide iiito the sample holder of the Perkin-Elmer ;\lode1 21 infrared sl)ectrolihotonieter. Ikf'ore the mounted sample waq placed in the sample beam of the ~l)e'trol)hotonietrr, the fre3hly mounted sliw \vas allo\ved to dry in air on the cardboard as a film for the time periods indicated. The sample was then scanned b e t n e m 2 and 13 micron*. The referrncr beam contained a 100-micron elicr of agar that had been dried over phosphorus pentoxide for 2 1 hour-. Samples were scanned repeatedly until the \vatcr loss from the dice pertnittcd the denionstration of the characteristic ab.;orption bands of the amino acid Films were examined a t 10x magnification with a Cycloptic stereomicroscope (-1inerican
4 Figure 1. Infrared spectra of glycine and D-alanine in aqueous solution in agar Spectrum above that for glycine hydrochloride i s for a g a r dried for 107 and 6 5 2 4 hours. For each amino acid the indicated times-i.e., minutes-are the drying periods after mounting the wet 1 00-micron d g a r slices
" 100-1
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WAVELEWGTH
Figure 2.
8 9 NCRCUS
IC
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Infrared spectra of t-threonine
Conditions a r e the same as those for Figure 1 except that threonine in the crystalline state was dried for 2 4 hours
VOL. 36, NO. 7, JUNE 1 9 6 4
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Instrument Co.) to decide whether crystallization occurred within the film. Thirty-seven minutes were required for a single scan. The resolution setting was 980 and that for the pen speed was 7. The water content of the agar slice was determined by weighing after drying in air, in the infrared beam or under vacuum, or over phosphorus pentoxide as indicated. RESULTS AND DISCUSSION
Spectra in agar of glycine and alanine in buffer p H 7.2 and of these amino acids as their hydrochlorides appear in Figure 1. Similar spectra for threonine appear in Figure 2. When the water loss from the agar slices was examined, slices weighing 546.8 and 650.48 mg. lost 482.1 and 520.0 mg. of water 107 minutes after 65 minutes after slicing slicing-Le., plus 42 minutes in the infrared beam. This water loss was approximately 83% of the original slice weight. When these same slices remained in the sample beam
for 42 minutes more, the total water loss was 90% of the original sample weight. When slices with a mean weight of 625.9 mg. ( u = 0.097)' were dried over phosphorus pentoxide the water loss was 585.6 mg. ( u = 0.0245). This water loss corresponded to approximately 93% of the original slice weight. The presence of the 1720 cm.-' absorption band for the hydrochloride but the absence of this absorption in buffer solution relate our spectra to those of Gore ( 2 ) in aqueous solution. In his studies deuterium oxide was used to avoid interference at 1640 em.-' Absorption in this region does not interfere with our demonstration of the 1720 ern.-' carbonyl absorption noted throughout for the amino acid hydrochlorides. Indeed, slices can be dried until very little absorption shows (Figure 1) at 3350, 640, or 800 cm.-', the well known regions of the spectrum where water absorbs. Of course, the reference beam contains an agar film but even then, in all cases, 65 minutes after mounting the sample the amount
of water present finds the film opaque to infrared light. Thirty-seven minutes later, however, the characteristic absorption bands are seen; a t this time the film contains approximately 80 pl. of water. Although all of this water might not be free as solvent, adequate water was present for solvent for the amino acid. The difference between threonine hydrochloride in the crystalline and dissolved states indicates that solution under these conditions of sample preparation changes the absorption of the hydrochloride form of a t least this acid just as it changed the absorption of the dipolar form observed by Gore ( 2 ) . LITERATURE CITED
( 1 ) Feinberg, J. G., Rapson, H. D., Tavlor, M. P., Nuture 181, 763 (1958).
(2) Gore, R. C., Barnes, R. B., Peterson, E., ANAL.CHEM.21,382 (1949). (3) Lauffer, M.A,, Schantz, E. J., Bibchemistry 1 , 658 (1962).
SUPPORTED in part by a research grant (AI-04819) and a training grant from the United States Public Health Service.
System for Vaporizing Samples Directly in Ion Source of a Mass Spectrometer Gerard 1. Kearns,' Research Division, W. R. Grace & Co., Clarksville, Md.
to be analyzed mass must be in the vapor state. Normally, the material under study is vaporized in an inlet system and slowly diffuses into the ionization region. Many inlet systems can be heated to obtain sufficient sample pressure (about lop2 torr) from materials with very low pressures at room temperature (1, 2 ) . The insertion and heating of samples directly in the ionization region reduces the sample pressure requirements several orders of magnitude. The temperature to which the sample must be heated to obtain the necessary pressure, therefore is reduced proportionately. This permits the analysis of many thermally unstable materials. Heating the sample directly in the ion source has many other useful applications. I t makes it possible to obtain maw spectra of materials of very low volatility (3,5 ) . The products from deliberate thermal degradation (4) can be analyzed with a minimum number of collisions. Gaseous products evolved when solid materials-e.g., cataly-tq-are heated can also be detected and identified in this manner (6). This technique is especially valuable when used with a rapid scanninge.g., time-of-flight-mass spectrometer. The sample systems described in the literature ( 2 , 5 ) are not easily adaptable bimERIAL
A spectrometrically
1 Present address, Picker Nuclear Division, Picker X-Ray Corp., Khite Plains, N. Y.
1402
ANALYTICAL CHEMISTRY
to many spectrometers. A new system has been developed which can be used not only with all time-of-flight mass spectrometers, but with any instrument with a n accessible ion source-e.g., sector magnetic field instruments. EXPERIMENTAL
Apparatus. The basic requirement for a n y sample system which enters the ion source region is t h a t the sample be inserted and/or withdrawn without appreciably affecting the low pressure (10-6 torr) of the spectrometer. The system which has been developed utilizes: (a) a linear-motion high vacuum feed-through; (b) a high conductance isolation valve; (c) a vacuum lock pumped by an external vacuum
A:
Normal
Swag*lok
I connlslor
system; and (d) a vacuum-tight sample holder (probe). A linear-motion high vacuum feedthrough is usually the most difficult component to obtain because it must maintain a vacuum-tight connection while the sample probe is manipulated. A simple way has been devised to accomplish this with a standard '/,-inch Swagelok (Crawford Fitting Co., Cleveland, Ohio) connector (Fig. 1.4). These connectors are constructed with a restriction which allows '/(-inch tubing to be inserted only about 3/8 inch. The vacuum seal, however, is made by the compression of ferrules and not by the end of the tubing. The opening in the body of the connector is enlarged (Figure 1B) so the tubing can pass through the entire unit without affecting the vacuum seal (Figure IC). Teflon ferrules are used and they not only make the seal, but also provide a surface for sliding the tubing easily through the apparatus. Figure 2 is a schematic drawing of the apparatus as attached to a timeof-flight mass spectrometer. A '/*-inch ball valve is used as the isolation valve. The vacuum lock [the volume between the connector (a) and the ball valve] is evacuated through the valved vacuum line. The sample probe is inserted through the linear-motion feed-through ( a ) . All connections except the Swagelok nut are silver-soldered to reduce the possibility of leaks. The sample system is installed so the sample can be positioned with 2 cm. of the electron beam. The magnet assembly of the spectrometer used to collimate the ionizing electron beam was modified
