Anal. Chem. 1988, 60, 2609-2612
2609
Spectral Interference Refractometry by Diode Array Spectrometry Giinter Gauglitz* and Jiirgen Krause-Bonte Institut fur Physikalische Chemie, Auf der Morgenstelle 8, D-7400 Tubingen, Federal Republic of Germany
Harry Schlemmer and Axel Matthes Carl Zeiss, Zentralbereich Forschung, Postfach 136911380,0-7082 Oberkochen, Federal Republic of Germany
A new method of detection Is glven for flowing liquid streams by introducing Interferometric reflectance measurements and new apparatus. The caiculatlon of the spectral dlsperslon rather than the determination of the classic absorption spectrum is used by applying mathematical transformation of the Interferencespectrum. This allows measurements outside of absorption bands for up to now spectroscopically undetectable samples and extremely small detectlon volumes of less than 80 nL. The constructed apparatus, the experimental iimitatlons, the algorithms used, and future developments are dlscussed. The quality of the method is proven by experimental results. A flrst potential application is glven for hlghperformance ilquid chromatography.
Electronic spectroscopy in ultraviolet (UV)and visible spectral range has become a general detection method in transmission spectroscopy, even in in-line process control of fluid liquid streams, and in high-performance liquid chromatography (HPLC). The development and commercial availability of diode array spectrometers have brought further progress. Spectral information increases the chance to identify components during production steps of pharmaceutics via in-line remote sensing (1)and allows the control of peak purity as well as supports the quantification of components in chromatographically nonresolved peaks (2,3).Furthermore spectral detection helps to realize artifacts by changes in refractive index caused by eluent gradients. Quite a few substances, which are of some interest in biological processes and in separation techniques of liquid chromatography, as amino acids, nucleotides, and saccharides, only absorb in the short wavelength range close to 200 nm (4,5).In this case the detection is quite problematic, because of the absorption of the eluent itself in this wavelength range, and the decreasing sensitivity of UV detectors-especially of diode arrays (6). Whereas absorption takes place at a specific wavelength, which will be characteristic for the substance, interaction between molecules and electromagnetic radiation is not restricted to such wavelength bands. In contrary the dielectric constant as well as the refractive index will be influenced by polarizing effects of the inducing electromagnetic field vector of the radiation in the whole visible and ultraviolet range (7, 8). This effect is demonstrated by the curve of dispersion, given in the upper part of Figure 1. The refractive index becomes a complex variable (7) as soon as the interaction between radiation and molecules causes an energy transfer to the molecules (absorption). Then, besides the real part of the curve of dispersion (upper part of Figure l),an imaginary part will be found (lower part of Figure 1)at such wavelengths. This principle of absorption has been used for convenience in flowing liquid streams to control in-line chemical reactions as well as to detect chromatographicallyresolved peaks. But, because of the physical limitation by the product of concen0003-2700/88/0360-2609$01.50/0
tration times absorption coefficient and optical path length, in general the latter is chosen to 5-10 mm in practice to get as low as possible limit of detection. For this reason most commercial equipment will be fixed to 4-8 pL cell volume. A reduction of this volume-as is necessary, e.g., in the techniques of microbore separations in HPLC-is possible but will increase the limit of detection. As mentioned above and discussed for the curve of dispersion, such principle of transmissive detection will be restricted to wavelengths of absorption. These may be for some classes of substances in a wavelength region of unfavorable detector sensitivity. Therefore refractive index detectors have been used at these applications for quite a long time (9-11). The disadvantage of the method of refractivity is its poor limit of detection being somewhat a factor of lo3 worse than absorbance. Besides, measurement of refractive index at one wavelength can be neither specifk with respect to the sample’s identity nor very quantitative because of problems with the reference, temperature, and pressure. Furthermore the linear range is smaller than that obtained by using absorption detectors. Refractive index detectors, which work by difference measurement between sample and reference cell (Fresnel, deflection refractometer, interferometer), use cell volumes of 1.5-15 pL, whereby reduction of volume will increase the limit of detection. Recently unconventional methods of detection were introduced (12-15)to the single wavelength detection in HPLC with microcell volume. The deflection of a highly focused laser beam by the sample can be correlated with the absorption of the detectable compounds. The limit of detection is very low (12)and can be decreased up to 2.8 X lo4 refractive index units by using an excimer laser and a second laser beam for the deflection measurement. This thermal crossed beam arrangement (12-15)heats the sample in dependence on its absorbance and increases the deflection of the laser beam. Even though this approach appears promising and sensitive, the sophisticated setup, the excimer laser, and the single wavelength detection has to be overcome in practice. Nowadays interferometric methods are discussed in literature as promising detection systems (16,17).For this reason our aim was to develop a new general method of detection in flowing liquid streams, especially applicable to HPLC, which offers a very small volume of the cell, uses the advantage of refractive index measurements, increases the information by spectral detection, uses commercially available equipment (diode array), and is fully computerized. The idea was (a) to combine the independence of selected small wavelength areas by measurement of refractive index with the possibility of increasing the signal to noise (S/N) ratio at regions of anomalous dispersion (area of absorption, see Figure l),(b) to obtain in principle spectral information, which allows peak identification, gives the chance to handle peak overlaps in HPLC, and avoids artifacts, and (c) to favor a cell volume as small as possible. 0 1988 American Chemical Society
2610
ANALYTICAL CHEMISTRY. VOL. 60, NO. 23, DECEMBER 1, 1986
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THEORY A light beam, penetrating a thin fhof medium with lower refractive index (n < nJ, will be reflected in part at the surface of the thin film and a t its back (see Figure 2, beam a and h). Both reflected beams a and b will show interference depending on the angle of incidence and the product of refractive index times the thickness d of the thin film.By use of Fresnel's law and geometric considerations, the measured interference pattern ImIA)is created by the superposition of the beams a and h. The measured result can he written as
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Similar results will be obtained for the minima. This method of measurement requires small optical path lengths. Therefore a cell was constructed (18)that contained two polished sapphire half spheres (Figure 3) with a path length of 7.3 pm. The cell volume is approximately 80 nL. The path length of the microcell and typical refractive indices ( n = 1.3) cause a distance hetween the extrema of the interference pattern of 5 nm (at 400 nm) to 15 nm (at 720 nm), as can be seen in Figure 4a. According to the Rayleigh miterion 2.5 nm (spedral distance of diodes, 0.83 nm) will be resolved by the array (512 diodes) with a spectral range of 30W720 nm. The interference spectrum has to he corrected by the reference signal of the light source (wavelength dependent) 1 : " and the dark signal of the diodes Id1h) by
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This interference spectrum (Figure 4a) has to he smoothed by using either Savitzky-Golay (20) or SPLINE (21) algorithms. A correct transformation into the spectral dispersion will only be successful if the wavelength scale of the spectrometer is calibrated with extreme expenditure. The quality can be controlled by the measurement of the wavelength-independent dispersion spectrum of an air gap.
ANALYTICAL CHEMISTRY, VOL. 60, NO. 23,DECEMBER 1, 1988
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The known path length d allows the calculation of many points of the curve of spectral dispersion, given in Figure 4b, for water as an example.
EXPERIMENTAL SECTION Figure 5 gives a block diagram of the used apparatus, containing a fast scanning diode array spectrometer (C. Zeiss, Oberkochen) with a holographic grating for high optical throughput and distortion-free imaging, large numerical aperture (NA = 0.2), a 512-element diode array (EG&G,Reticon, Munich type G/10), and a fast integrated 1Zbit analog to digital converter. The optical components are adjusted in a new ceramic housing. The light source is a continuous wave halogen lamp. A special interface SSK (C. Zeiss) and a 8088/8087 (Intel) microprocessor unit, running C (DigitalResearch) under CP/M 86, take charge of the data acquisition and the evaluation. The time for the calculation of one spectral dispersion was approximately 1.5 8. Recently the SSK and the 8088/8087 were replaced by a 68020 microprocessor system with arithmetic coprocessor unit 68881 (Motorola). Data acquisition is done via three P I 0 ports (68230, Motorola) all modular on a VME-bus system (Klein-Elektronik, Heilbronn), using graphics (GRAZ4,Eltec). It is run by a real-time multiuser, multitasking operating system (PDOS). Thereby the readout of
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the diode array (minimal integration time 5.6 ms) could be reduced to 13.5 ms and evaluation time could be reduced drastically. The cell is considered to be used in an analytic device for the kinetic examination of reaction procedures (22) by HPLC containing a high-pressure pump (Gynkotek, Model 600/200) and RP-18 columns.
RESULTS AND DISCUSSION The presented method for the determination of spectral dispersion allows one to measure changes in the refractive index better than 8 X refractive index unit (RIU). The systent noise itself amounts to (2-3) X refractive index unit. This limit of detection is determined by the spectral resolution of the spectrometer and the S/Nratio of the diode array. Both parameters have become better in the new generation of equipment and will be certainly optimized in the next future. Then, the quality would get closer to the RI values, published in literature (1.5 X RIU) (23). It is an advantage of the presented method to determine the spectral dispersion absolutely, that means no reference cell is necessary. Slow fluctuations of temperature, caused by changes of enthalpy of reaction on the column during chromatography, result a drift in the base line (4 X lo4 RIU/"C), which are demonstrated in Figure 6a. They will not affect the signal. In contrast, the pulsations of the HPLC pump will cause noise
ANALYTICAL CHEMISTRY, VOL. 60, NO.
2812
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methods of refractivity as crossed beam photothermal refraction, thermal prism, or photothermal deflection. We understand the presented method as a new approach of detection, which certainly can be optimized in the future by the announced improvement of the diode arrays and use of higher resolving grating. The present state of the art for the presented method is given by figure 7. It demonstrates the result of injecting 0.5-fiL of glucose (5.0 pg) in the HPLC setup. Finally, the first experiments with measurement of the anomalous part of the spectral dispersion gave another success-promising tool. As can be shown by Figure 8 for a solution of crystal violet, the changes of refractive indices with wavelength are high, compared to measurementsof the normal part. Furthermore the maximum of the absorption band can be identified. The aim will be to use both real and imaginary parts of the spectral dispersion at any wavelength to yield more information and increase sensitivity. For this reason this detection method promises to be a new successful approach to solve problems with on-line measurements in biotechnology, in difficult biological samples, and in using extremely small detection volumes. The success depends on the idea of interferometric transform, the high standard detection cell, second generation of diode array spectrometry, modern microprocessor development, and advanced programming tools.
LITERATURE CITED 1.3.80
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(24 X RIU/bar), which has to be reduced by flow damping. The large effects, given in Figure 6b, are caused by influence of pressure changes on the refractive index as well as changes in the optical path length in the cell. The latter can be reduced by improvement of the cell fixing but will be a problem in any sensitive method, which measures refractive indices in flow systems. Optimization of the adjustment of the detection cell to the outlet of the HPLC column has minimized the dead volume such that the apparatus offers a new approach in HPLC application. Even though the normal part of the spectral dispersion is not very characteristic and will not allow the identification of different samples, spectral curves will overcome the information of common refractometry at one wavelength. Interferometric refractometry has the following advantages compared to normal HPLC/UV-vis detectors: (a) the detection volume can be extremely small, (b) no reference cell is necessary, (c) the spectral detection also allows the measurement of the anomalous dispersion, which gives comparable informationto absorption, and (d) the possibility of measuring outside of absorption bands, an advantage in chromatography of material, which only absorb in the far-ultraviolet region. Presently there is a disadvantage of a detection limit some orders worse than absorption spectroscopy or sophisticated
(1) Tschulena, G. Presented at the APV-Seminar of Pharceutlcal Industry, Darmstadt, Feb 1988. (2) Fell, A.; Scott, H. P. J. Chromatogr. 1983, 273, 3-17. (3) Schieffer, G. W. J. Chromafogr. 1985, 319, 387-391. (4) Verhaar, L. A. Th.; Kuster, B. F. M. J. Chromatogr. 1981, 220, 313-328. (5) Simpson, R. C.; Brown, P. R. J. Chromatogr. 1987, 400, 297-305. (6) EG&G RETICON, product information 08175, 1980. (7) Bbttcher, C. J. F. Theory of Electric Polarisation, Elsevier: Amsterdam, 1952. (8) Attkins, P. W. Physical Chemistry, 3rd ed.;Oxford University Press: Oxford, 1986. (9) Syndsr, L. R.; Kirkiand, J. J. Inhoducfion to Modern LlquM Chromatography, 2nd ed.; Wiiey: New York, 1979. (10) Colin, H.; Jauimes, A.; Gulochon, G.; Corno, J. J . Chromatogr. Sci. 1979, 17, 485-491. (11) Engelhardt, H. Practice of High Performance Liqukl Chromatography, 1st ed.; Springer: Heidelberg, 1986. (12) Bornhop, D. J.; Nolan, T. G.; Dovichi, N. J. J . Chromatogr. 1987, 384, 181-187. (13) Bornhop, D. J.; Dovichi, N. J. Anal. Chem. 1987, 59, 1632. (14) Dovichi, N. J.; Nolan, T. G. Anal. Chem. 1987, 5 9 , 2803. (15) Bornhop, D. J.; Dovichi, N. J. LC-OC 4987, 5 , 427. (16) Wolfbeis, 0. S., Tubingen, personal communication, 1988. (17) Gaugli, G. Procsedings des Workshops Computer in der Chemie; Springer: Berlin, 1986; pp 165-200. (18) Machler, M.; Sachse, R.; Schlemmer, H. (Carl Zeiss) Ger. Offen. DE 3414260. (19) Schiemmer, H.; Miichler, M. J. Phys. E . 1985, 18. (20) Woodruff, S. D.; Yeung. E. S. Anal. Chem. 1982, 54. 1174-1178. (21) Savitzky. A.; Golay, M. Anal. Chem. 1984, 36, 1627. (22) Spith, H. A@r/fhmen fiir elemenfare Ausgleichsmodelle; R. Oldenburg Verlag: Munchen Wien, 1973. (23) Gauglitz, G.; Schmid, W. Chromatographia 1987, 23,395-400.
RECEIVED for review November 18, 1987. Resubmitted July 12,1988. Accepted August 29,1988. We thank the Fond der Chemischen Industrie for financial support.
