CHROMATOGRAPHIC SYSTEM BASED ON AMORPHOUS SILICON PHOTODIODES

D. Caputo, G. de Cesare, C. Manetti*, A. Nascetti, R. Scipinotti Dept. of Electronic Engineering, University La Sapienza, via Eudossiana, 18 00184 Rome (Italy) *Dept. of Chemistry, University of Rome La Sapienza, Piazzale Aldo Moro 5, Rome (Italy) Abstract In this work, we present a novel thin layer chromatography system based on fluorescence detection by means of a linear array of amorphous silicon photodiodes. The photodiodes are optically coupled to a thin layer chromatography plate to monitor, in real-time, the separation of the components of a mixture during the chromatographic run. We designed a horizontal development chamber with UV transparent window and integrated eluent tank. The resulting system is extremely compact and ensures fast analysis using only small amounts of eluent. With our system, we analyzed different mixtures of fluorescent inks and other molecules such as fluorescein. Real-time data of sensors located in different position are used to detect the composition of the mixture. The various components can be determined from the time of the transit of the different species in front of the sensors. Furthermore, from the measured data it is possible to extract additional information as the transport properties of the stationary phase or the velocity profile along the run for each component of the mixture. Introduction Chromatography is a method of separating mixtures of two or more compounds [1]. The separation is accomplished by the distribution of the mixture between two phases: one that is stationary and one that is moving. Different compounds will have different solubility and adsorption to the two phases between which they are partitioned. Among the different chromatography methods the Thin Layer Chromatography (TLC) is a solid-liquid technique in which the stationary phase is silica gel, while the moving phase is constituted by solvents with different polarity [2, 3]. TLC involves spotting the sample to be analyzed near one end of a glass that is coated with a thin layer of silica gel. The glass substrate, which can be the size of a microscope slide, is placed in a covered jar containing a shallow layer of solvent. As the solvent rises by capillary action up through the adsorbent, differential partitioning occurs between the components of the mixture dissolved in the solvent along the stationary adsorbent phase. When the solvent front reaches the other edge of the stationary phase, the plate is removed from the solvent reservoir. The separated spots are typically visualized with a scanner by exciting their fluorescence with an ultraviolet light or by a suitable reaction procedure. In this work, we introduce a novel system for in-situ realtime analysis of the chromatographic run. The basic idea presented here is the integration of a set of photosensors on the TLC plate. This is possible by using the hydrogenated amorphous silicon (a-Si:H) technology that permits to deposit high-quality photodiodes on glass substrates [4]. The structure of the proposed TLC plate with integrated photosensors is reported in Figure 1. One side of a glass substrate is covered with a layer of Indium Tin Oxide (ITO) that represents the transparent bottom-electrode of the photodiode. The photodiode is an amorphous silicon ntype/intrinsic/p-type stacked structure deposited by Plasma Enhanced Chemical Vapor Deposition (PECVD). The photodiode top-contact is a three metal Cr/Al/Cr stack. The structure is passivated with a 8 m thick Cyclotene layer [5]. The other side of the glass substrate is coated with a 200 to 800 m thick layer of a solid adsorbent (usually silica or alumina) that constitutes the stationary phase for the thin layer chromatography. By irradiating the TLC plate with a UV lamp during the chromatographic run it is possible to monitor, in real-time, the transit of the components of the mixture in front of the photodiode. Different components can be then distinguished from their arrival time. By using a linear array of photodiodes it is possible to observe the chromatographic run at different grade of separation of the mixture components. This gives the ability to optimize the development time in order to achieve the desired resolution of a given separation. This helps to shorten the analysis time by terminating the run when the components of interest have reached the desired degree of separation, eliminating the differences in homogeneity and surface activity from plate to plate. Furthermore, the width of each photocurrent peak, associated with the transit of a fluorescent component in front of a photodiode is related to the local velocity of that component leading to additional information as the transport properties of the stationary phase or the velocity profile along the run of each component of the mixture. As an additional advantage, the proposed system can be handled in the same way as the conventional plates from the spotting of the mixture to the possibility of scan with conventional systems. In particular, thanks to the low cost technology of the amorphous silicon, the TLC plates

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Figure 1: Schematic view of the TLC plate with integrated amorphous silicon photodiode

1-4244-1245-5/07/$25.00 2007 IEEE

Figure 2: Prototype of the 16-pixel linear array of amorphous silicon photodiodes to be optically coupled to conventional TLC plates

presented here are disposable, making them attractive for practical applications. Experiment As a first step toward the development of the system described above, we deposited a linear array of amorphous silicon photodiodes on a glass substrate covered with a transparent conductive oxide. Then we optically coupled the sensor glass to a standard TLC plate. This modular approach allowed us to setup the entire system by tuning its single components individually. In Figure 2, a picture of the glass substrate with a linear

array of sixteen amorphous silicon photodiodes is reported. The pixel size (0.5x1mm2) and pitch (1 mm) were chosen according to the width of the chromatographic zone and the thickness of the glass substrate and of the TLC plate. The sensors are n-type/intrinsic/p-type a-Si:H stacked structures deposited by PECVD in our three chamber reactor. The deposition parameters have been chosen for minimizing the dark current and achieving a spectral response centered around 500 nm, to match the fluorescence emission of a wide range of substances including the fluorescein used in the following experiments. The current-voltage characteristic measured in dark condition is plotted in Figure 3. The dark current density is about 10-10 A/cm2 at -0.5 V.The quantum efficiency of the same sensor is reported in Figure 4. To test our system we designed a development chamber with a UV transparent quartz window to be able to illuminate the TLC plate during the chromatographic run. The chamber is made of PTFE (Teflon) to prevent chemical interaction with the moving phase (eluent). The development chamber includes the housing for the TLC plate and the sensor substrate ensuring an optimal optical coupling between them. The volume of the chamber is very small to guarantee fast saturation with the eluent vapors. Consequently, very small amounts (less than 5 ml) of eluent are needed to perform a chromatography run, reducing the costs and the risk associated with harmful eluents. A 10 ml eluent tank is included in the chamber. A drawing of the development chamber is reported in Figure 5. The experimental setup has been mounted on a optical bench. A mercury lamp filtered by a Jobin-Yvon H10 monochromator supplies the UV radiation at 254.3 nm. The incident power is 2.3 W. The photocurrent of selected photodiodes is measured by a Keithley 236 Source-Measure unit. The development chamber is placed horizontally in the optical path and the incident radiation is focused with UV optics on the selected sensor. Results Experiments have been performed with several mixtures

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Figure 4: Quantum efficiency of the a-Si.H photodiode

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Figure 5: Prototype of the horizontal development chamber with UV transparent window and integrated eluent tank

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of fluorescent molecules that have been spotted on conventional TLC plates. The same linear array has been used for all the experiments. In Figure 6 the experimental results achieved with a solution of a single fluorescent component (cumarine) is shown. The cumarine has been dissolved in water and ethanol has been used as eluent for the chromatography. The graph shows the real-time signal of a sensor located in the center of the array. The photocurrent peak detected after 6 minutes has to be ascribed to the transit of the cumarine in front of the selected photodiode. From system characterization we have estimated an average noise current around 0.1 pA [6], and then from figure 6 a very good signal-to-noise ratio can be

appreciated. The tail observed after the transit of the cumarine is due to impurities contained in the solution. A second experiment has been performed on undiluted ink of a fluorescent marker using ethanol as mobile phase. In Figure 7a, the signal of a photodiode located in correspondence of the initial part of the chromatography run is reported. Although at this point the complete separation of the mixture is still not achieved, the system clearly identifies three fluorescent components present in the analyzed sample. The real-time signal is in agreement with the image of the developed TLC plate observed under UV illumination and reported in Figure 7b. The direction of the chromatographic run is from left to right in the picture. The fourth component observed in Figure 7b is not detected by the sensor because the corresponding band has still not reached the photodiode, whose position is indicated by the arrow in the figure. The rightmost peak in the picture corresponds to the photocurrent peak observed after 400 s. The higher velocity of this component with respect to the others is also evident from the narrower width of the corresponding photocurrent peak due to the lower transit time in front of the photodiode.

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b) Figure 7: a) Real time signal of a chromatography run performed on a solution of a highlighter ink. b) picture of the developed TLC plate under UV illumination. The position of the sensor, whose signal is reported in a) is indicated by the arrow

Figure 6 Real-time signal of a photodiode during a chromatography run performed on a solution of cumarine.

Conclusions An innovative TLC system based on the optical coupling between a thin layer chromatography plate and a linear array of a-Si:H photodiodes has been presented. The system results very compact thanks to a small, ad hoc designed horizontal development chamber, where a UV-transparent quartz window and the eluent tank are integrated. During the chromatographic run an UV radiation is impinging on the TLC plate and the photocurrent induced in the a-Si:H sensor by the fluorescence of the analytes can be monitored in real-time. Experimental results achieved by analyzing different mixtures demonstrate the suitability of our system for realtime chromatography. Acknowledgments Authors wish to aknowledge Ing. Domenico Pellegrino for helpful collaboration. References 1. Scott, R. P. W., Techniques and practice of chromatography, Marcel Dekker (New York, 1995). 2. Sherma, J., Fried, B. Handbook of Thin-Layer Chromatography, 2nd ed. Marcel Dekker (New York, 1996). 3. Hahn-Deinstrop, E.: Applied Thin-Layer Chromatography. Wiley-VCH Verlag, (Weinheim, 2000) 4. Street, R. A., Technology and Applications of Amorphous silicon, Springer (Berlin, 2000), pp. 147-156. 5. http://www.dow.com/cyclotene/index.htm 6. Caputo D., de Cesare G., Nascetti, A. Negri R.Scipinotti R., Amorphous Silicon Sensors for Single and Multicolor Detection of Biomolecules, in press on IEEE Sensor