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Infrared Analysis of Suspended Particulates with Millipore Filters and Attenuated Total Reflection ROBERT W. HANNAH The Perkin-Elmer Corp., Nonvalk, Conn;
JAMES 1. DWYER Millipore Filter Corp., Bedford, Mass.
b Attenuated total reflection has been characterized as being essentially a surface effect. One implication of this is that for a given onalyris only a fraction of a milligram of material properly presented to the totally rdecting face is required ta produce a good spectrum. Membrane filters, because of their surface retention ability, not only accomplish a separation of particulate material from fluid media but alsa permit a nearly ideal presentation of the sample to the ATR crystal. In addition, substrate interferences which complicate transmission results, are reduced considerably. This method is especially voluable where the material to b e examined is present in very low concentrations normally difficult to isalate from the fluid media or of a microscopic particle size. Several diverse examples are described which illustrate the utility of the technique.
HE
of infrared mettrometry to the analysis of part& lates suspended in a fluid media must, in general, he preceded by a separation and concentration of the particles. Millipore filters, which are sheets of polymeric material perforated with millions of precisely controlled small pores, have been used for some years for separations of this general kind, The separation may then be followed by a wide variety of analytical procedures depending on the system to be investigated. It is the purpose of this paper to describe the unique combination of Millipore filters and a& tenuated total reflection to yield a very flexible and sensitive analytical procedure. Attenuated total reflection is essentially a surface effect. The distance over which interaction between the sample and the radiation occurs, the so-called depth of penetration, is generally less than a few microns. Thus, when ATR is applied to a given sample, the examination reduces to a measurement of the surface properties and not of the hulk properties. This implies that a few micrograms of sample,
1
properly presented to the ATR crystal, are sufficient to yield a good infrared spectrum. The application of Millipore filters to the separation of suspended particulates accomplishes, in a single step, both the separation and proper sample preparation for ATR analysis. The particulates are distributed on the planar surface of the filter in the form of an extremely thin film such that nearly all of the sample can interact with the radiation. Although substrate interferences are reduced or, in some cases, eliminated, compensated techniques may be necessary for some samples. Except for cutting portions of the filter to fit the ATR accessory, no additional sample preparation is required. A number of spectra have been prepared to illustrate the flexibility and potential uses of such a technique. The applications presented are divided into four general categories: powders dispersed in liquid vehicles; colloidal trace contaminants present in liquids; aerosol mists, fumes, ete.; and biochemical.
Figure 1.
Three-reflection ATR occes-
APPLICATION
EXPERIMENTAL
The infrared spectra were obtained with a Perkin-Elmer Model 521 spectrophotometer equipped with the single and triple-reflection ATR accessofies, The prism material was KRS-5 and the angle of incidence was between 41" and 50" in all cases. All sample preparations were performed with standard Millipore and holders. parameters specific to a given sample are presented in the discussion associated with that sample. RESULTS AND DISCUSSION
Powders Dispersed in Liquid VePaint analysis can be difficult and time consuming. Nearly all of the components will have absorption bands in the infrared spectrum. I n the past it was desirable, but tedious, t o separate liquid vehicles from the dispersed pigments. The application of Millipore filters eliminates the tedium and satisfies the separation requirement in one easy step. Vehicle, free of pigment, passes through the filter while the dispersed pigment and toners are collected on the surface
hicles.
of the filter. The residual vehicle is removed with an inert volatile thinner and portions of the filter are presented to the three-reflection ATR accessory (Figure 1). The pigment spectra shown were obtained by a modification of this procedure. An aliquot of paint was diluted with petroleum ether and filtered through a 0.22-micron pore size filter. This w&s followed by several rinses with petroleum ether. The entire filtering process required about 5 minutes. Figure 2 shows the spectra of 45 pg. and 5 pg. of pigment from a red paint obtained with the three-reflection ATR at an angle of incidence of 45'. These well defined spectra permitted identification of the pigment as toluidine red. A very weak spectrum of about 1 pg. was obtained by the same technique and this probably represents the lower limit of sensitivity. The sample weights shown were determined from the weight gain and area of the filter and the illuminated area of the three-reflection ATR accessory. The upper curve shows no interferences while the lower VOL 36. NO. 12. NOVEMBER 1964
2341
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Figure 10. A. ATR spectrum of solids from exhaust of a two-cycle engine 6. Exhaust from a four-cycle engine VOL. 36, NO. 12, NOVEMBER 1964
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of the exhaust solids and aerosols 'is essentially that of a paraffinic hydrocarbon. There is evidence for some oxidation in the presence of a weak carbonyl band a t 1750 em.-' but,, for the most part, the spectrum is very clean. The cleanliness of spectrum 10A is even more apparent in comparison with the spectrum 10B. The sample was collected from the exhaust of a fourcycle engine. The aut,omobile accelerator was pumped while sampling, producing a considerably enriched spectrum. Hydrocarbons are obviously present. Oxidation has been more severe and there is evidence for the presence of additives in t,he gasoline, which are possibly sulfonates, as well as some raw gasoline. Biochemical. The final application was chosen purely as a n example of a technique. Biomedical and biochemical investigations have not normally included infrared spectrometry. Perhaps one of the reasons for this is that most of the systems studied contain water and most infrared spectroscopists are reluctant t,o work with water solutions. One interesting area of biochemist,ry is the metabolic mechanisnis used by microorgankms. Bacteria are marvelously adaptive. Give them certain nutrients and they thrive. Give them a completely different nutrient and they change their entire metabolic process.
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Figure 1 1 . Compensated ATR spectrum of Serratia marcescens cultured on Wilson's media on 0.22-micron pore size filter
There has been some work done on infrared analysis of microorganism metabolism. Needless to say, it is a complex subject. There are a number of metabolic pathways and it may be said that the present metabolism is conditioned by the past. An innoculum of Serratia marcesens in physiological saline was filtered through a 0.22-micron filter. The bacteria were retained on the filter surface. Wilson's media was introduced to the back of the filter and the filter was incubated for 24 hours a t 32" C. The filter was then dried and a portion was placed in contact with the ATR crystal with the angle set a t 45". A second ATR unit containing a blank piece of filter was used in the reference beam a t a slightly higher angle and 5X ordinate expansion was employed (Figure 11). Amide I, 11, and I11 bands a t 1630, 1525, and 1230 cm.-l are indicative of the extended form or pleated sheet form of proteins or polypeptides ( 1 ) . The assignment of
the band near 1025 cm.-' is a little uncertain but it is probably due to the dextran capsule making up the shell of the bacteria. Another culture grown on a second, but similar media, yielded the same spectrum. ACKNOWLEDGMENT
We acknowledge the contributions of Seymour Goldwasser of Lever Brothers Research a t whose suggestion we explored this technique, and Robert Gore who aided in the interpretation of the spectra. LITERATURE CITED
(1) Nlay, Leopold, A p p l . S p e c t ~ y . 18, 5
(1964).
(2) Sloane, Howard, ANAL. CHEY. 35,
1556 (1963).
RECEIVEDfor review June 1, 1964.
Accepted August 14, 1964. Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Pittsburgh, Pa., 1964.
Estimation of a Few Nanograms of Fluoride in Presence of Phosphate by Use of Liver Esterase CHARLES McGAUGHEY and ELLERY C. STOWELL Denfal Research Laborafory, Medical Research Programs, Veterans Administration Hospital, Long Beach, Calif, The method of Linde, based on the inhibition of liver esterase by fluoride, has been modified to permit the estimation of a few nanograms of fluoride, even in the presence of large amounts of phosphate. Enzyme activity was measured by titration of the butyric acid formed from ethyl butyrate with NaOH. An acetone powder containing the stable esterase was prepared, and the optimal conditions for inhibition of the enzyme by fluoride were determined. The best conditions in terms of both efficiency and results: use of nitric acid-washed glass weighing bottles as incubation vessels; incubation volume, 1.25-2.5 ml.; pH, 3.0-3.3; 25 PI. of acetone powder suspension ( 2 mg. per ml,); 0.5 mi. of ethyl butyrate solution ( 1 :200 in
2344
ANALYTICAL CHEMISTRY
water); room temperature (inhibition improving somewhat with decreased temperature); incubation time, 1-1.5 hours.
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of a method for quantitatively determining nanogram (ng., 10-9g.) levels of fluoride in biological tissues has stimulated investigations in this laboratory of the potentials of several of the currently available procedures for adaptation to a submicro scale. Of the methods examined ( 2 , 3, 5 , 6 , 8, 9, 1.2, 1 4 ) , that of Linde (6) appeared to offer the best possibility of attaining the required degree of sensitivity. This method is based on the extraordinary SUSceptibility of the enzyme lever esterase ESIRABILITY
to inhibition by fluoride, and it is capable of est,imating a minimum of approximately 0.1 pg. of fluoride in a volume of 100 ml. -4 disadvantage associated with the other methods is that separation of fluoride from phosphate is required for successful colorimetry, and we found this difficult to accomplish on a submicro scale with an acceptable degree of precision. In cont'rast, our preliminary findings with the method of Linde suggested that although the sensitivity is lowered by the presence of phosphate, this interference is moderate and can be controlled. Early attempts to reduce the scale of the method resulted in intolerable losses of sensitivity and precision, and the method suffered from the additional disadvantage that the
