Infrared Microspectrophotometry Using Reflecting-Type 6X Beam Condensing Optics in Reference and Sample Beams MARIO SPARAGANA’ and W. 6. MASONZ Departments o f Medicine and Biochemistry, School o f Medicine and Dentistry, The University o f Rochester, Rochester, N. Y,
b Microspectrophotometry carried out with reflecting-type 6X beam condensing optics i n both the reference and sample paths of a Perkin-Elmer Model 2 1 infrared spectrophotometer i s described. Microsamples may b e used to cancel absorptions due to solvents or other interfering substonces. Compensation of atmospheric bands i s excellent and I, i s constant within 3=5% over the range 2 to 15 microns. Absorbonce values are about 5% greater than those obtained in the normal operating mode. Full-scale spectra ore obtained with samples weighing 1 to 5 pg. . Use of electronic ordinate expansion in combination with the dual beam condensing unit has permitted spectra to be recorded with samples as small os 0.05 Peg.
1
NFRARED MICROSPECTROPHOTOMETRY
has not been used to full advantage in analybis of trace constituents occurring in biological materials because of difficulties in preparing microsamples having sufficient purity for profitable infrared study. Especially troublesome are contaminants introduced during final steps of purification. Significant contamination is also introduced during incorporation of microgram samples into KBr for preparation of micropellets. Poor success in eliminating these residual impurities prompted the authors to investigate techniques wherein differential infrared spectrophotometric measurements could be made using micropellets in both the reference and sample beams. It is impractical to attempt cancellation of interfering absorptions by using a macro “blank” in a compensating, but nonequivalent, reference path because of difficulties in preparing a macro “blank” which is chemically representative of the microsample. 1 Present address, PostdoctoralResearch Fellow (AF-8627-C1), National Institute of Arthritis and Metabolic Diseases, University of Rochester. 2 Present address, National Institutes of Health Clinical Center, Bethesda, Md. (on sabbatical leave).
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Figure 1. Dual beam condensing unit employing reflecting-typ( optics in both sample and reference beams. Source housing has been removed to permit better ,^. view of unit in position on Perkin-Elmer Model 2 1 IR spectrophotomei,,
Ford et ul. (9) have described a double-beam infrared microspectrophotometer employing a double-beamin-time system and reflecting optics having 8X magnification, with which they obtained good spectra using samples apparently weighing about 10 rrg. It would seem that much smaller samples could bc accommodated. Some modification of their sample mounting arrangement would most likely he required to permit inclusion of a micro “blank” in the reference path. Hilger and Watts Ltd. have advertised (3) an attachment for their H800 double-beam recording infrared spectrophotometer that has a 1OX reflecting microscope in each beam, but they give no indication of performance. To our knowledge, there have been no comparable developments with other instruments. Mohl (5) has reported briefly on unsatisfactory twin microscopes for a double beam infrared spectrophotometer. Commercially available lens-type beam
condensers similar to the unit described by White et ul. (7), although fitting directly into both reference and sample beams, require samples in excess of 10 pg., which are too large to satisfy aims of the present work. The reflecting-type 6X beam condensing system recently made available hy Perkin-Elmer (6) for their Models 21, 221, and 421 infrared spectrophotometers provides full-scale spectra with samples weighing 1 to 5 pg. and seemed ideal for use in a dual unit. There was concern, however, that nonuniform illumination of the optical wedge might introduce appreciable photometric error. Such a dual system should provide better equalization of sample and reference paths than can he obtained by inserting a 1meter path-length gas cell into the reference beam, as recommended by Rlout and Abbate (I). With precise compensation of atmospheric hands electronic and a relatively constant IO,
M ONOC H RO M AT0 R
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IMAGE
lyi I REFERENCE BEAM MSR-MICROSAMPLING REGION
M3,M4-6 I OFF-AXIS ELLIPSOID
Figure 2.
Optical schematic for dual beam condensing unit
ordinate expansion can be utilized to rcduce sample requirements below the limits achievable through optical micro~pcctrophutoinetrralone. APPARATUS
The dual unit consists of two standard 6X beam condensers mounted on a single base plate (Figure 1). The base plate is kinematically mounted to a
sub-base (not shon-n), attached by screws to the spectrophotometer. Potassium bromide pellets (in metal disks), or liquid filled cells, are positioned in the sample and reference paths by holders supplied with the individual beam condensing units. Figure 2 shows the optical schematic. The sample beam is diverted by mvrors M1 and M2 and falls upon ellipsoidal mirror -Ma which produces a sixfold reduced image of the source in the rnicrosampling region. This image is magnified by the matching ellipsoid (?iiq),and subsequently reflected to the normal entrance slit position of the monochromator. An analogous path is followed by the reference beam, with the image falling upon the optical medge. When properly aligned, the reflecting system transmits 30 to 35% of the energy normally available in each beam. iMetal disks having central holes measuring 1.5 nim. in diameter may be so positioned in the microsampling regions that essentially all this energy passes through the central holes. Disks having 0.5-mm. central holes, however, produce appreciable attenuation of the beams, and transmit only about 10% of the normally available energy. Careful optical alignment is required for mork with 0.5-mm. pellets; otherwise, energy transmission is reduced, and Zo becomes unsatisfactory. Optical alignment is not disturbed when the dual system is removed for storage, and repositioning is easily accomplished by means of the kinematic mounting. Potassium bromide pellets (1.5- and 0.5-mm. diameter) were pressed using a Perkin-Elmer ultramicro die. The 1.5-mm. pellets weighed about 5 mg.; 0.5 mm. pellets weighed about 0.5 mg. The metal disks (13 mm. 0.d.) in R hich the pellets were formed provided support for mounting both sizes. Samples were ground with KBr in a motor-
driven tool steel mortar and pestle (Fisher Scientific Co.). Sodium chloride ultramicro cavity cells having a path length of 0.05 mm. and exposed cross section of 1 x 4 mm. (Type D, Connecticut Instrument Corp.) were used for recording solution spectra. In all instances, spectra were recorded using the maximum slit program (1000) and source current of 0.5 amp. A scan rate of 1 to 2 minutes per micron was used with the I X ordinate scale. With 5X ordinate expansion, the scan rate was 3 to 5 minutes per micron. PHOTOMETRIC ACCURACY
Two 1.5-mm. KBr pellets were used to evaluate photometric accuracy. Each pellet weighed about 5 mg. One
contained about 10 gg. of hydrocortisone and was pressed from a ponder prepared by grinding 4.0 mg. of hydroreference compound) cortisone (U.S.P. with 2.0 grams of KBr (IR quality, Harshaw Chemical Co.). The other pellet contained about 60 pg. o f hydrocortisone and was pressed from a sixtimes more concentrated powler. The reference spectrum (Figure 3C) for the ppllet containing 10 pg. of hydrocortisone was recorded with the pellet in the normal sampling position-Le., in the sample beam at the slit image position just inside the monochromator cover. This required a special disk positioning device in the sample path (4, but did not entail auxiliary optics. An adjustable grid was used to attenuate the reference beam. Spectra 3.4 and 3B were obtained with the beam condensing unit in place. In spectrum 3A, the 1.5-mm. pellet was recorded against an empty 1.5-mm. hole on the reference side. In spectrum 3B, a 0.5-mm. pellet was simulated as follows: Disks having empty 0.5-mm. holes nere inserted on both reference and sample sides. The 1.5-mm. pellet used for the previous spectrum was then superimposed (monochromator side) upon the sample disk. Similarly, a disk having an empty 1.5mm. hole was superimposed upon the reference disk. Spectrum 3B was then recorded. Three similar spectra were recorded for the second pellet. Absorbance measurements were made by the base line technique. Figure 4
W A V U f f i l H (MICRCM)
Figure 3. A.
E. C.
Spectrophotometer performance with dual beam condensing optics
1.5-mm. pellet Simulated 0.5-mm. pellet 1.5-mm. pellet in normal sampling position
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Figure 4. Photometric accuracy (1.5mm. pellet)
shows a plot of absorbance with beam condensing optics and 1.5-mm. pellets against absorbance for the corresponding band when the pellet was in the normal sampling position. Similar data for simulated 0.5-mm. pellets are shown in Figure 5. Dotted lines indicate 1 to 1 relationships. The regression equation for the 1.5-mm. pellet data 1.08 2; for the 0.5-mm. is y = 0.00 pellet, y = 0.01 1.01 t.
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DIFFERENCE SPECTRA
Total Compensation. Figure 6 illustrates the degree of compensation that can be achieved with microgram amounts of material in the form of pellets. Two similar 0.5-mm. pellets, each containing about 1 pg. of hydrocortisone, were prepared (Spectra A and B ) . The difference spectrum ( A us. B ) compares favorably with the 10 (air) line obtained with empty 0.5-mm. holes in both the reference and sample paths. Compensation for One Component of a Mixture. Reference spectra of pure estrone and @-estradiolare shown in Figure 7. These two compounds differ only in the hydroxyl and keto
ABSOREANCE N NORMAL SAMPLING "9SITION
Figure 5. Photometric accuracy (simulated 0.5-mm. pellet)
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ANALYTICAL CHEMISTRY
2
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Figure A and B.
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8 9 1 WAVELENGTH (MICRONS)
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Difference spectrum illustrating total compensation
Matched 0.5-mm. pellets each contoining about 1 pg. of hydrocortisone
groups a t the 17-position. Spectrum 8 4 obtained lvith a micropellet containing about 4 pg. of sample, resembles that of estrone, but obvious differences exist. In 8B a micropellet containing about 2 pg. of estrone (sufficient to provide compensation of the 5.8-micron carbonyl band) m s included in the reference path. Comparison of this difference spectrum n ith the reference spectrum (SC) clearly indicates the
Figure 7.
presence of @-estradiol in the original mixture. Compensation Using Microcells. I n some instances, it is adrant,ageous to be able to record difference spectra using liquid filled cells of small volume. Figure 9 A illustrates solvent compensation using tv-o ultramicro cavity cells filled with chloroform. Only 0.2 p l . of solvent is required to fill the exposed cross section of these crlls,
Spectra of chemically similar compounds
Estrone. 2 pg. in a 0.5-mm. pellet @:Estradiol. 4 pg. in a 0.5-mm. pellet air. Empty 0.5-mm. disks
Spectrum 11B Eas recorded with the same pellet used for 11A, but with 5X ordinate expansion. Differences between spectra 1lB and 11C in the 2.9and 6.1-micron regions are thought to be due to the larger amount of water, relative to phenobarbital, in the more dilute powder. DISCUSSION
W A Y H f f i T H (MICRONS1
Figure 8. A. 8. C.
Compensation for one component of a mixture using 0.5-mrn. pellets
Estrone plus fi-estradiol against air Estrone plus &estradiol against estrone &Estradiol against air
although the total volume is 0.8 p l . when dead-space is included. Compensation is satisfactory, except in regions of intense absorption. In 9B a solution of phenobarbital in chloroform (20 pg. per pl.) was used in the sample cell. This spectrum agrees well with the reference spectrum (9C) recorded with 0.5 pg. of phenobarbital in a 0.5-mm. KBr pellet.
A 0.5-mm. pellet containing about 0.05 wg. of phenobarbital was pressed from the more dilute powder, and it gave spectrum 11A. A similar pellet, pressed from the more concentrated powder, gave the reference spectrum (1lC).
Although there was concern that appreciable photometric inaccuracy might result from introduction of beam condensing optics into the reference path, present data indicate this error t o be small (+8’7,, for 1.5-mm. pellets; +lyGfor 0.5-mm. pellets). The difference between results with the two sizes of pellets probably relates to different portions of the optical wedge being illuminated in the two cases. Conceivably, realignment of the beam condensing optics might improve performance with the 1 . 5 - m . pellets. The most intense bands had transmittances of about 9.5’7,0/0.This corresponds to the region where discontinuities occur in the optical wedge and may account for the scatter of points observed a t larger absorbances (Figures 4 and 5 ) . The linear relationship betn-een absorbances measured with the dual unit and values obtained with pellets in the normal sampling position indicates that the dual unit is quite satisfactory for qualitative work. With care, quantitative data of useful nature can also be obtained.
ORDINATE EXPANSION
Compensation of atmospheric absorptions and constancy of lo (AIR, Figures 6 and 7) are adequate to permit electronic ordinate expansion with microsamples. An example of the resulting increase in sensitivity is given in Figure 10. Two powders (grinding technique), containing respectively 4.1 and 0.82 pg. of hydrocortisone per mg. of KBr, were used to prepare 0.5-mm. pellets. The spectra relative to air are shown in Figure 10. It is difficult to identify the more dilute sample (IOA) until 5X ordinate expansion is carried out (10B). The reference pellet (lOC) contained about 2 pg. of hydrocortisone, in contrast with 0.4 pg. present in the pellet used for spectra 1OA and 1OB. Greater sensitivity can be achieved with compounds having more intense absorptions, Figure 11. In tkis case, two powders containing respectively 1 pg. and 0.1 pg. of phenobarbital per mg. of KBr were prepared by serial dilution, using a grinding technique.
Figure A.
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Solvent compensation using ultramicro cavity cells
Chloroform against chloroform Phenobarbital in chloroform against chloroform Reference phenobarbital spectrum (KBr pellet against air)
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Figure 10.
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8 9 WAVELENGTH (MICROMI
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0.5-mm. KBr pellet containing 0.4 pg. of hydrocortisone a t 1 X 5X expansion
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Reference hydrocortisone spectrum
Careful optical alignment is required
t o obtain a relatively flat l o line with empty 0.5-mm. holes in both beams (Figure 7 ) . When adjustment is less optimum, as in Figure 6, I o decreases gradually toward either longer or shorter wavelengths. Even when IO is quite flat, there appears to be incomplete cancellation of atmospheric absorptions. This may be due to a slight wavelength difference between the two beams, however, rather than inequalities in path length, since the bands characteristically show symmetrical displacement above and below the 10 line (derivative shape) and are often larger than would be expected on the basis of possible differences in path length. In any case, the effect is small with good optical alignment. A technique wherein difference spectra can be recorded with microgram amounts of materials offers considerable potential usefulness beyond cancellation of absorptions due to solvents or to residual impurities arising in separation procedures. Close chemical and physical similarities between components of a mixture frequently prevent their being completely separated. The usefulness of difference spectra in such a situation is illustrated with estrone and 8-estradiol (Figure 7 ) . In applying difference spectra, reference compounds must be treated in a manner comparable t o that used for the unknown, Otherwise, as is well known, polymorphism or reactions with reagents or solvents, or between constituents, 246
ANALYTICAL CHEMISTRY
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may result in spurious spectral differences. The advantage of being able t o use a micropellet in the reference beam lies in being able t o subject the reference compound to a procedure that closely approximates that used for the unknown.
It is instructive to compare sensitivities achieved with micropellets and with liquid-filled microcells. The microcell used to record the spectra shown in Figure 9 had a volume of 0.2 pl., plus 0.6 pl. of dead space. This is the smallest commercial cell (Type D, Connecticut Instrument Co.) known to the authors. It will be noted (Figure 9) that a 0.5-mm. diameter micropellet gave about eightfold greater sensitivity, neglecting dead-space of the cell. If dead-space is included, an allowance must be made for losses incurred in the micropellet technique. In our experience, this amounts to about 50y0 when samples in the 1-pg. range are incorporated into 1.0-mg. of KBr using a lyophilization procedure. The over-all advantage realized by using micropellets, therefore, amounts to about 15-fold greater sensitivity. Because of the good optical compensation obtained with the dual beam condensing unit, it is possible to carry out 5X ordinate expansion throughout the entire 2- to 15-micron region without adjustments of base line. This considerably facilitates collection of data. The reduction of sample requirement achievable through combination of ordinate expansion and optical microspectrophotometry should permit practical application of infrared microspectrophotometry to much smaller samples than has hitherto been possible. In principle, the front-surface reflecting optics used in the dual unit should permit microspectrophotometric measurements to be made over a much wider
z Y
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Figure 1 1. A.
0.5.mm.
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Ordinate scale expansion
KBr pellet containing 0.05 pg. of phenobarbital at 1 X
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Reference Phenobarbital spectrum
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range of tvavelengths than have been included in the present work. In this respect, the unit is much superior to a dual system that could be constructed using lens-type condensing optics.
Leon Schwartz for photographing the illustrations; to Barbara Moyer for recording Some of the spectra; and to E. H. Keutmann for his continued interest in this work.
ACKNOWLEDGMENT
LITERATURE CITED
The authors gratefully the University of Rochester Atomic Energy Project (USAEC) for the use of its recording infrared spectrophotometer, and the Connecticut Instrument CO. for donating the ultramicro caVitY cells. The authors are also indebted to
(1) Blout, E. R., Abbate, M. J., J. Opt. ,yoe. A ~45,1028 . (1955). (2) Ford, M. A., Price, W. C., Seeds, W. E., Wilkinson, G. R., Ibzd., 48, 249 (1958). (3) Handbook of Scientific Instruments and Apparatus, p. 249, The Physical
Society, London, 1957.
(4) Mason, W. B., Van Slyke, H. N., Abstracts, Pittsburgh Conference on
halytical Chemistry and Applied Spec-
troscopy, Pittsburgh, Pa., hfarch 1957.
( 5 ) Mohl, F., Acta Chem. Scand. 12, 1356
(1958). (6) The Perkin-Elmer Corp., Instrztrnent News 12, (3), 3 (1961): (7) White, J. U., Weiner, S., Alpert, N. L., Ward, W. M., ANAL. CHEY.30, 1694 (19%). RECEIVED for review July 12, 1961. Accepted November 9, 1961. Twelfth Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Pittsburgh, Pa., March 1961. Work was supported by Grant C-1003 from the National Cancer Institute.
Direct Titration of Phenols by Bromination CALVIN 0. HUBER' and JOANNE M. GILBERT2 Rockford College, Rockford, 111.
b Phenols are commonly determined by bromination titrations in which all open ortho and para positions are substituted. The titration ordinarily involves the addition of excess brominating agent and iodometric back titration of excess bromine. A direct titration in glacial acetic acid is proposed. The presence of an appropriate amount of pyridine provides a reasonable reaction rate. The end point is determined by constant current potentiometry using platinum foil electrodes. The titration curve is interpreted from voltammetric data. Quantitative and reproducible results were obtained for phenol and several substituted phenol samples. Errors and deviations were ordinarily within 1 %. The method is relatively simple, rapid, and convenient.
T
HE DETERMINATION of phenols by bromine substitution was first proposed by Koppeschaar (6). The development and application of this classical method are reviewed by Kolthoff and Belcher (4). Sources of difficulty in the method are bromination of alkyl side groups, oxidations, replacement reactions, and precipitation of partially brominated products. The classical method involves addition of excess bromate to an aqueous bromide solution containing the sample, addition of iodide, and titration of iodine
Present address, Department of Chemistry, University of Wisconsin-Milwaukee, Milwaukee 11, !Vis. * Present address, Department of Cheniistry, University of Wisconsin, Madison 6 , Wis.
with thiosulfate using starch as indicator. Ingberman (3) has reviewed some of the difficulties of the classical method and has developed a modified titration procedure which provides stoichiometric titration for many phenols. Elemental bromine dissolved in glacial acetic acid is used as titrant, glacial acetic acid as titration solvent, and pyridine as catalyst. The end point is determined iodometrically using aqueous potassium iodide and standard thiosulfate solutions, as in the classical method. Using these modifications, Ingberman titrated a large number of phenols within a 1 to 2% error level. The development of a direct titration method with resulting improvement in speed and convenience seemed desirable. Direct titration requires a stoichiometric, rapid reaction and a suitable end point detection method. Such a direct titration was developed. Constant current potentiometry (6) was used for end point indication. Good results were obtained for several phenols tested. METHOD
In the Ingberman procedure, 25.00 ml. of 0.15M bromine in glacial acetic acid is added t o about 3 meq. of sample. About 0.25 ml. of pyridine is added, and the mixture is permitted to react for 2 to 20 minutes. The required reaction time must be ascertained for each unknown material analyzed. Investigation in this laboratory showed that titration with 0.15M bromine in glacial acetic acid in the presence of similar amounts of pyridine gave stoichiometric results, but the time required for titration was 1 to 2 hours. The replacement of pyridine with other similar compounds, to increase the rate
of the reaction, as attempted. Compounds tested were: quinoline, aniline, P-picoline, ypicoline, o-toluidine, quinolinic acid, 4-(2-hydrosyethyl)pyridine, triethylamine, piperidine, and nicotinic acid. No signiiicant increase in reaction rate was observed for any of the compounds tested. Variation of the amount of pyridine used, however, resulted in a sharp change in reaction time. The use of 3 ml. of pyridine resulted in a titration time of less than 20 minutes. Further increase in pyridine concentration did not increase the rate significantly, but only added to the blank. A preliminary study of the relationship between rate of reaction and pyridine concentration was made by observing the time required for reaction when 25.00 ml. of 0.15Af bromine in glacial acetic acid was added to 40 ml. of glacial acetic acid solutions containing 0.400 grams of p-phenylphenol and various amounts of pyridine. A plot of log time vs. log concentration (Figure 1) indicates that the reaction order with respect to pyridine changes by more than a factor of two over the concentration range studied. The existence of pyridinium bromide perbromide and of pyridine perbromide complexes in acetic acid has been reported (1, 2 ) . I t seems probable that the attacking agent is a pyridine-bromine complex. Possibly a t higher pyridine concentrations, the electrophylic attack by the pyridine-bromine complex is the rate determining step. The kinetics and mechanism of this reaction deserve further study. End point indication was by constant current potentiometry, which has the advantages of being readily interpretable, applicable to nonaqueous solutions, and yielding end point signals which can be read directly. Two identical platinum foil electrodes, 1.0 X 0.5 cm., were spaced 1.0 cm. apart. The conVOL 34, NO. 2, FEBRUARY 1962
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