library. It is evident that the compressed format search has retained enough information to yield results as in- formative as those returned by traditional search meth- ods.

IV. CONCLUSION

The Karhunen-Loeve transformation has been utilized to compress an infrared spectral library. It was found that a 185 dimensional spectrum could be linearly trans- formed into a 37 dimensional representation with little or no loss of information. This represents a fivefold reduction in the size of the reference library, and a proportional reduction in the time required to perform a search. Within the limited number of cases tested, the Karhunen-Loeve compressed search compares favorably in accuracy to other search systems.

1. L. E. Kuentzel, Anal. Chem. 23, 1413 (1951). 2. D. H. Anderson and G. L. Covert, Anal. Chem. 39, 1288 (1967). 3. F. E. Lytle and T. L. Brazie, Anal. Chem. 42, 1432 (1970). 4. S. R. Lowry and T. L. Isenhour, J. Chem. Inf. Comp. Sci. 15, 212 {1975). 5. H. B. Woodruff, S. R. Lowry, and T. L. Isenhour, J. Chem. Inf. Comp. Sci. 15,

2071 {1975}.

6. J. Pupan, D. Hadzi, and M. Penca, Comp. Chem. i, 77 (1976}. 7. P. F. Dupvis, A. Djikstra, and J. H. van der Mass, Fresenius Z. Anal. Chem.

290, 357 (1978). 8. P. F. Dupvis, A. Djikstra, and J. H. van der Mass, Fresenius Z. Anal. Chem.

291, 27 {1978}. 9. C. S. Rann, Anal. Chem. 44, 1669 (1972).

10. K. Schaarschmidt, R. Reimer, and E. Steger, Z. Chem. 14, 374 (1974}. 11. Yu. P. Druhyshev, R. S. Nigmatullin, V. I. Lobanov, I. K. Korobeincheva, V.

S. Boshkarev, and V. A. Koptyug, Vestn. Akad. Nauk. SSSR 40, 75 {1970}. 12. E. C. Penski, D. A. Padowski, and J. B. Bouck, Anal. Chem. 46, 955 (1974). 13. R. C. Fox, Anal. Chem. 48, 717 (1976). 14. K. Tanabe and S. Saeki, Anal. Chem. 47, 118 (1975). 15. L. M. Powell and G. M. Hieftje, Anal. Chem. 47, 118 (1975). 16. G. T. Rasmussen and T. L. Isenhour, Appl. Spectrosc. 33, 371 (1979). 17. P. T. Funke, E. R. Malinowski, D. E. Martire, and L. Z. Pollara, Sep. Sci. 1,

661 (1966). 18. D. G. Howery, Am. Lab. 8, 14 (1976}. 19. R. J. Rummel, "Applied Factor Analysis" (Northwestern Univ. Press, Evans-

ton, Ill., 1970}. 20. E. R. Malinowski and D. G. Howery, Factor Analysis in Chemistry. {Wiley-

Interscience, New York, 1980}. 21. R. W. Rozett and E. McL. Petersen, Anal. Chem. 47, 1301 {1975). 22. Y. T. Chen and K. S. Fu, Inform. Control 375 (1968). 23. K. Fukunaga and W. Koontz, IEEE Transaction on Computers C-19, 311

(1970). 24. M. de Bruin, P. J. Korthoven, R. P. W. Duin, F. C. A. Groon, and C. C.

Bakels, J. Radioanal. Chem. 15, 18kl (1973). 25. L. R. Malinowski, Anal. Chem. 49, 612 (1977}. 26. H. F. Kaiser, Educ. Psych. Meas. 20, 141 (1960). 27. S. L. Grotch, Anal. Chem. 43, 1362 (1971).

Breaking the One Second Barrier: Fast Kinetics of Protein Adsorption by FT-IR

R. MICHAEL GENDREAU Biological Spectroscopy Facility, Battelle's Columbus Laboratories, Columbus, Ohio 43201

T h e B a t t e l l e Biological Spectroscopy Facility is currently study- ing the adsorption of blood proteins onto various surfaces using the combination of Fourier transform infrared spectroscopy (FT-IR) and attenuated total reflectance (ATR) optics. We have advanced our capabilities to study this phenomenon to the point w h e r e useful infrared spectra are obtained at 0.8-s intervals. This high-time resolution is helping to probe more accurately these important blood-surface interactions. Index Headings: F o u r i e r transform infrared spectroscopy; Bio- medical spectroscopy; Protein adsorption kinetics.

INTRODUCTION

These biomedical experiments being carried out at Battelle depend upon our ability to sort the numerous protein interactions occurring as whole blood contacts various surfaces. The dominant feature of these interac- tions is the extreme speed with which they occur. Be- cause FT-IR is the technique being used to study these interactions, the ability to obtain spectra at short enough intervals to follow this adsorption accurately was desired.

When these studies were initiated, 40 scans of the interferometer at I s/scan were needed. This 40-scan (40-

Received 1 October 1981.

Volume 36, Number 1, 1982

s) interval provided an infrared spectrum with the high signal-to-noise ratio needed to allow the necessary sub- tractions and scale expansions to study protein adsorp- tion events. Extremely high signal-to-noise is needed because the complex aqueous solution with which we are working (blood} requires numerous spectral subtractions before the desired information is obtained.

With modifications in hardware and software in 1980, we were able to use 25 scans instead of 40 to obtain the required signal-to-noise ratio, and the scans could be obtained at 0.2 s/scan, giving an infrared spectrum every 5 s. Even spectra every 5 s appeared not to be fast enough. Adsorption and desorption kinetics are ex- tremely rapid when whole blood is used, and higher time resolution was sought. In 1981, detector and software changes were made which allow useful spectra to be obtained using only four interferometer scans. This pro- vides a time resolution of 0.8 s which has given us the opportunity to follow the adsorption kinetics much more accurately. Also, having infrared spectra spaced at such close intervals allows us to confirm transient features. A spectral feature that previously was only present in one spectrum will now (if it is not an artifact} be seen in three or four of the spectra, greatly increasing our confidence in the interpretation of transient features. This has

APPLIED SPECTROSCOPY 47

proven to be important to us because very fast transient events apear to be occurring during the initial phases of blood-surface contact, and several spectra showing a feature greatly increases our confidence in the interpre- tation of such features. This is discussed briefly later.

I. RESULTS

Fig. 1 illustrates adsorption kinetics obtained using this high-time resolution. In the 1 min shown in this figure, 75 spectra were measured for base line corrected band intensities to obtain this plot. Because half of the total amount of protein adsorbed in this experiment was adsorbed within 20 s, time resolution of this magnitude is necessary.

Fig. 2 compares the desorption kinetics (loss of protein when saline is circulated) for two runs of whole blood adsorbing onto germanium. More than 200 infrared spec- tra from each experiment are represented by this plot. From this plot, it can be seen that the slope of the desorption curve is reproducible in both experiments. The starting amounts actually were slightly different, so the experiment with less adsorbed protein was normal- ized so that the desorption rate could be compared. Again, having the high-time resolution allows us to follow

. [ ~ L E ~ O 0 ~ ..... [ ................ 7 . 7 . - ~ ~ o ~ ~O....eE,.AN ZU. Jo ................................ ~ ..................................... i ..................................... i ................

. . . . . . . . . . . . ~, ............. ~ ..................................... i ..................................... i ........

~O~ol/ ........................ i i i l i i i i i ..................................... ~ ..................................... ii .... • . .2G .$ .7S * !, •

T IHE IN HINUT[$

FIG. 1. Adsorption kinetics for whole blood adsorbing onto germanium. Spectra were obtained at 0.8-s intervals, base line corrected band intensities measured, and these intensities plotted for the bands listed. Seventy-five spectra are represented by this plot.

, [ ........ N.i., i DE$ORPTIOH KIHETICS i

: t ........ ~ ......... t ......... ~.- . .-~-i ......... i ......... i ......... i .................. i ......... i ......... i .................. i . . . . . . . . . . I - ~ ~ . . . . ~ ,,.,D~ ,,,~ .........

o . o . z . 4 . 6 . I l .O I . I t . 4 t . 6 t .O l . l ~ / . ! 1 . 4 ! . 6 I . I 3 . •

TIME IN MINUTES

FIG. 2. The desorption rates of two whole blood adsorbed onto ger- manium experiments are compared in this figure. The Amide II band from each experiment was measured for more than 200 spectra and the resulting plot obtained.

4 8 V o l u m e 3 6 , N u m b e r 1 , 1 9 8 2

l l l l l g l l l l L l l I 1 I / hd~ l I l l d ' l ~ l t f l ,

i . ~ .............. ..l HUHAH UB/GE (8S) ] ....... "T ..... I i I I .............. I ............. • / S O L I D LAYER RDSORB£DI

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WRVEHUMBERS

FIG. 3. Adsorption of human whole blood onto germanium. Dashed line, this spectrum represents the proteins adsorbed during the first 24 s of blood contact; solid line, this spectrum represents the proteins adsorbed from 8 to 24 s of blood contact {spectral subtraction of 24-s spectrum minus 8-s spectrum). Absence of 1361 cm ~ band in the solid spectrum illustrates that this component is only adsorbing in the first few seconds.

I I /11 I L S - l e SECOND SPECTRA

*4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. |

1S00 1400 1300 1200 I l e o t l UAVENUH|ER$

Fro. 4. Adsorption of human whole blood onto germanium. This spec- trum represents those proteins adsorbed between 10 and 18 s after initial blood contact.

I

. 27 .........................

,20 ..............

, 0 6 ............

1 | 0 0

~='=~:-:° '¢=i: / i i | HUHAH UHOLE |LOOD ......

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................... F T W \

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Fro. 5. Adsorption of human whole blood onto germanium. This spec- trum represents those proteins adsorbed between 20 and 28 s after initial blood contact.

subtle changes in the adsorbed film as it undergoes desorption.

One of the major advantages of this high-time resolu- tion is the confirmation of transient features. Fig. 3 illustrates spectra generated by subtracting successive spectra to obtain the spectrum of protein adsorbed at

time intervals from 0 to 24 s. The dotted spectrum represents the proteins adsorbs from 0 to 24 s. The solid line represents proteins adsorbed between 8 to 24 s. As indicated by the arrow, a feature at 1361 cm -~ is noted in the total layer which is absent in the 8- to 24-s layer, indicating that this component adsorbed in the first 5 to 10 s only. Because a number of spectra were collected in this time interval, we could both confirm that this is a real feature and accurately follow its adsorption kinetics during the first 10 s of blood-surface contact.

Figs. 4 and 5 further illustrate the rapidity with which the protein adsorption profile changes. Fig. 4 illustrates a spectrum of those proteins that adsorbed from 10 to 18 s; Fig. 5 shows those proteins adsorbing for 20 to 28 s. It is obvious that these two spectra are quite different, and thus we need a capability to study these rapid events in real time.

II. SUMMARY

Current state-of-the-art FT-IR hardware and software is allowing us to perform complex biomedical experi- ments with a time resolution of 0.8 s. This time resolution provides a signal-to-noise ratio that allows us to study small differences between successive layers and to profile changes in the composition of the adsorbed layer as a function of time.

Obviously, the faster spectra are obtained, the lower the signal-to-noise ratio and thus sensitivity. Even though great strides have been made in sensitivity, biomedical FT-IR awaits further improvements in the near future. ACKNOWLEDGMENTS

This work was supported by National Institutes of Health Grant 1-RO1 HL- 24015. The author would like to thank Robert J. Jakobsen for his review and comments about this manuscript.

Effect of Spectrometer Resolution on Absorption Spectra

A. K. ARORA and R. KESAVAMOORTHY Reactor Research Centre, Kalpakkam 603 102, India

The effect o f spectrometer resolution on the parameters of an absorption spectrum is studied by numerical convolution. The dependencies of observed full width a t half maximum (FWHM) and peak absorption on spectrometer resolution, true FWHM and peak absorption of the absorption band is obtained. T h e observed dependencies are explained qualitatively. O v e r the u se fu l r a n g e o f parameters, coupled empirical relations are obtained which estimate the parameters of the true spectrum f r o m the parameters of the observed spectrum within 1.5%. The usefulness of the coupled empirical relations is demonstrated by applying them to C--H stretching mode of CHC13.

Index Headings: Absorption spectroscopy; Resolution; Slit width effects; Estimation of true parameters.

INTRODUCTION

It is well known in all branches of spectroscopy that a spectrum gets distorted due to finite resolution of the spectrometer. 1-3 The distortion of the spectrum is signif- icantly large if the full width at half maximum (FWHM) of the spectrometer resolution function (SRF) is more than one tenth of the FWHM of the true spectrum. There have been many attempts 3-s to estimate the extent of distortion caused by SRF and to recover the entire spectrum from the experimentally observed spectrum and the known SRF. Some work has been reported 9-13 to extract only the relevant parameters of the true spectrum like FWHM, peak intensity, or the integrated intensity. Wertheim e t al . 9 and Torkington 1° have considered the possibility of fitting a sum of Lorentzian and Gaussian

Received 3 June 1981.

Volume 36, Number 1, 1982

line profiles to convoluted line shapes of Lorentzian with Gaussian, Lorentzian with triangular and Gaussian with triangular profiles. Empirical relations have been sug- gested 11-13 to estimate the true FWHM of a Raman line from the observed spectrum. The situation with absorp- tion spectrophotometers is more complex as the observed FWHM (2Fe) depends not only on true FWHM of the band (2F) and spectrometer resolution but also on the peak absorption. Similarly the observed peak absorption depends also on the ratio of true FWHM of the band to the FWHM of the SRF (2Fs). Ramsay 1 has considered a triangular SRF and a Lorentzian absorption spectrum and has given methods to estimate the true integrated intensity of the band from the observed integrated inten- sity. The methods are either based on certain assump- tions about the observed band shape or need the correc- tion for the residual area under the wings of the absorp- tion band. Roseler 2 has considered a Gaussian SRF and a Lorentzian spectrum and has shown that the results are not significantly different from those for triangular SRF. No attempt has been made to estimate accurately the true FWHM and peak absorption by means of em- pirical relations. In the present investigation the effect of a triangular SRF on the FWHM and peak absorption of a Lorentzian absorption spectrum has been studied by numerical convolution over the most useful range of the ratio of true FWHM of the band to FWHM of the SRF and the peak absorption. The observed dependencies of the observed FWHM and peak absorption on F, Fs and true peak absorption are explained qualitatively. Coupled empirical relations between the parameters are obtained to estimate true FWHM and peak absorption. The use-

APPLIED SPECTROSCOPY 49