Microcomputer-controlled interface between a high performance liquid

containing powdered KCI, which Is held In a carousel. When most of the peak has been deposited, the carousel Is rotated and the remaining solvent is ...
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Anal. Chem. 1980, 52, 1394-1399

Microcomputer-Controlled Interface between a High Performance Liquid Chromatograph and a Diffuse Reflectance Infrared Fourier Transform Spectrometer Donald 1.Kuehl and Peter

R. Griffiths”

Department of Chemistry, Ohio University, Athens, Ohio 4570 I

A microcomputer-controlled interface between a high performance liquid chromatograph (HPLC) and a diffuse reflectance infrared Fourier transform (DRIFT) spectrometer is descrlbed. The solution elutlng from the UV detector of the HPLC Is concentrated by about a factor of ten in a short heated tube. When any peak is present in the concentrator tube, the solution emerging from the tube is passed dropwise onto a cup, Containing powdered KCI, whlch Is heid In a carousel. When most of the peak has been deposfted, the carousel is rotated and the remaining solvent is eliminated during the time H takes for the foilowlng HPLC peak to be deposited. The DRIFT spectrum of each peak is measured after the solvent eliminatron step. Submicrogram detection llmits are observed for all samples of low volatility, and chromatographlc resoiutlon Is maintained even for closely separated peaks.

In the past few years, gas chromatographs (GC) have been interfaced with several different types of Fourier transform infrared (FT-IR) spectrometers for the on-line measurement of t h e infrared spectra of peaks as they elute from the GC column (1-8). T h e sensitivity of GC/FT-IR systems is now high enough that FT-IR spectrometry may be used as a viable alternative or complement to mass spectrometry (MS) for the qualitative analysis of mixtures whose components are sufficiently volatile and thermally stable to be separated by GC. For mixtures containing nonvolatile or thermally labile components, the separation is usually effected by high performance liquid chromatography (HPLC). The on-line spectrometric characterization of HPLC peaks, whether by MS or FT-IR, is considerably more difficult than the corresponding measurement made on GC peaks. Several HPLC/MS (see Refs. 9 and 10 for reviews), and HPLC/FT-IR systems (11-13) have been described and a t least one of each is commercially available but with detection limits which are well in excess of the corresponding GC/MS and GC/FT-IR systems. T h e difference in sensitivity between GC/FT-IR and H P L C / F T - I R systems is due to the interference caused by t h e strong infrared absorption of the mobile phase. In a conventional HPLC/FT-IR system (11-13), the effluent from the column is passed through a flow-cell, and interferograms are continually measured and stored during the entire chromatographic run. At the end of the chromatogram, the solution spectra are computed and the absorption bands due to the solvent system are subtracted out. I n order to keep solvent bands from “blacking out” excessively wide regions of the spectrum, the pathlength of the flow-cell must be kept quite small-typically about 100 pm for most solvents used for normal phase HPLC. For 3-mm diameter cells of this pathlength, the volume is less than 1 pL, and only a small fraction (usually less than 1%)of each eluting sample is present in the cell a t any moment during the measurement of the spectrum of the solute, thereby drastically reducing the efficiency of the measurement (14). The disadvantages of this type of HPLC/FT-IR measure0003-2700/80/03521394$01 .OO/O

ment have been summarized by us in a previous article ( 1 5 ) , and in the same paper several different approaches for eliminating the solvent prior to the measurement of the infrared spectrum of the solute were discussed. By far the most successful of these approaches involved a device in which the effluent from the HPLC column is concentrated and then dropped onto KC1 powder. T h e remaining solvent is rapidly evaporated leaving only the solute on the KCl, and the diffuse reflectance infrared Fourier transform (DRIFT) spectrum of the solute is then measured. Only very scant details of this device were given, since the paper in which it was described was of a review nature. In the following paper, we will describe the construction and performance of the system in more detail.

EXPERIMENTAL Spectrometry. A model 296 interferometer (Digilab, Inc., Cambridge, Mass.) was modified by the manufacturer so that its mirror travels at a rate of 6.4 mm/s, four times faster than the usual scan rate of this interferometer, thereby increasing the modulation frequencies in the interferogram. A wide-range mercury cadmium telluride (MCT) photoconductive detector (Infrared Associates, New Brunswick, N.J.) was used, and across most of the spectrum the D* of this detector was almost doubled at the higher mirror velocity in comparison to the performance before the conversion. With this mirror velocity, 100 scans at 8 cm-’ resolution could be signal-averaged in 21 s. Data collection and any subsequent spectral manipulations were performed on the data system of a Digilab FTS-20 spectrometer using its interactive multitasking executive programs. Diffuse reflectance spectra were measured using the optical system developed in this laboratory and described previously (16, 17) with one change. The ellipsoidal collecting mirror of this earlier design was replaced by a 12.5-mmfocal length paraboloidal mirror (Special Optics, Little Falls, N.J.), increasing the efficiency of the system by about a factor of two. The reference material and substrate for the deposition of HPLC peaks was KCl, purchased as “random cuttings“ from Harshaw Chemical Co. (Solon, Ohio), which was crushed in a Wig-L-Bug (Crescent Dental Manufacturing Co., Chicago, Ill.) and dried in a vacuum oven prior to use. This KCl was considerably more pure than powdered ACS grade KC1 purchased from several other sources. Chromatography. The high performance liquid chromatograph used in this work was purchased from Tracor (Austin, Texas) and consisted of a Model 995 pump, Model 980A low pressure solvent programmer, and Model 960 254-nm ultraviolet detector. Most of the separations were performed by normal-phase HPLC using a 24-cm long, 4-mm i.d. column of Partisil 10 (Whatman. Inc., Clifton, N.J.), and a few separations were performed using 5-pm microparticulate silica columns purchased from Rheodyne (Berkeley, Calif.). A Rheodyne Model 7920 20-pL closed-loop injector was used on the Tracor Chromatograph. All solvents were purchased from Fisher Scientific (Parkersburg, W.Va.). Microcomputer. The HPLC/FT-IR system which will be described was controlled by a KIM-1 microcomputer board (MOS Technology, Norristown, Pa.). The board contains a 6502 microprocessor, 1 Kbytes of RAM, 2 Kbytes of ROM on which the monitor programs reside, a hexadecimal key pad and a 6-digit LED display. In addition, a “Memory Plus” expansion board was purchased from The Computerist (Chelmsford,Mass.). This board has 8K of additional RAM memory, space for 8K of EPROM, 1980 American Chemical Society

ANALYTICAL CHEMISTRY, VOL. 52, NO. 9, AUGUST 1980

and an 1/0chip called a versatile interface adapter (VIA). This device provides most of the functions which were used to interface the microprocessor to the HPLC, the HPLC/FT-IR interface, and the data system of the FTS-20 spectrometer. HPLC/DRIFT Interface. Mechanical Construction and Basic Operation. The automated system for collection of HPLC peaks is based around a sample carousel which supports 31 equally spaced sample cups. The sample wheel was precision machined from 0.5-inch aluminum stock to assure high accuracy of sample positioning. The carousel is keyed to a 0.5-inch stainless steel shaft supported by two heavy duty ball bearings mounted in an aluminum bearing cage which was mounted on a 0.5-inch aluminum base. The wheel is rotated by an ac chart drive motor via a belt drive reduction pulley. The carousel is locked into each position through the use of a solenoid with a stainless steel locking pin. Precision spaced holes corresponding to the sample positions are used to retain the locking pin. The sample cups are 5 mm in diameter and 19 mm high, and the sample well was 4.5 mm by 2.5 mm deep. A fine mesh wire screen was used to support the KCl powder in the sample cups as the sample cups were hollow to assist in solvent evaporation. In order to effectively purge the beam of atmospheric water vapor and yet still allow free rotation of the carousel into the outside atmosphere, a special purge box was constructed. The purge box was constructed of aluminum sheet metal of the minimum volume required to enclose the DRIFT optics, the source, interferometer, and detector. A slot was cut into the side of the purge box adjacent to the sample position and part of the carousel wheel fitted into the slot so that part of the sampling wheel was inside the purge box and part outside. Slots were also cut for the sample cups to pass through the side of the purge box upon rotation of the wheel. Thus the slot in the purge box is effectively blocked off by the carousel wheel with the only open holes being where the sample cups pass through the purge box. However, in view of the low volume of the purge box (about 11 L) and the relatively good flow rate of dry air (better than 5 L min-'), an excellent purge of the system was achieved. The effluent from the HPLC is passed through the UV detector and sprayed into a concentrator tube. The effluent sprayer was constructed from melting point capillary tubes which were drawn out to a fine tip using a Bunsen burner. The capillaries were held in place by a Teflon holder. The concentrator was constructed from 2-mm i.d. Pyrex tubing which was about 15 cm long. A small bend was put near the end of the tube which allows for the drops to collect and drip onto the sample cups. The glass tube was heated by asbestos-covered nichrome wire connected to a Variac. Near the end of the concentrator tube, a small capillary tube was placed which was connected to an aspirator through a solenoid valve. This allows the deposition of the effluent onto the sample cups to be controlled. When the valve is open, the concentrated effluent is drawn off to waste and when the valve is closed the effluent will be deposited onto a sample cup. There are four active positions on the carousel (Positions 1, 2,3, and 5) used during the HPLC/DRIFT measurements, and one other (Position 4) at which the sample cups reside immediately before the measurement of the DRIFT spectrum, as shown in Figure 1: position 1,where the sample is deposited on the KCl powder; positions 2 and 3, where a slow stream of air is drawn through the KCl for the final solvent elimination step; and position 5,where the DRIFT spectrum of the deposited sample is measured. The signal from the UV detector of the chromatograph is monitored by the microcomputer and, when the signal exceeds a certain threshold level, a series of coordinated events, separated in time by several seconds, is initiated by the microcomputer. The effluent from the HPLC is continuously sprayed into the concentrator tube using nitrogen, and the gas flow rate and the temperature of the tube are set so that about 90% of the solvent evaporates and the solution emerging from the bottom of the tube is concentrated by about a factor of ten. The concentrated solution emerging from this tube may then either be deposited on the KCl or drawn to waste through a glass capillary tube, depending on the position of a solenoid valve. When the signal from the first HPLC peak (peak A) exceeds a preset threshold level, a time delay, t D , set by the operator, allows the leading edge of the peak to travel from the detector to the bottom of the concentrator tube. At this

POSITION , I

1395

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POSITION 2

POSITIONING

POSITION 3-

"i: POSITION 4-

POSITION 5

c)

: ?

e-

- - _ _PURGE -

e

COVER

8-

Figure 1. Schematic diagram of the carousel used in the HPLCIDRIFT interface. A small light bulb is held above one of the positioning holes and a photodiode is held below it, to allow the position of the carousel to be monitored. The sample is deposited at position 1, the solvent is eliminated at positions 2 and 3, and the spectrum is measured at position 5

point the solenoid valve closes and the solution drips onto the KCl sample cup at position 1. t~ seconds after the HPLC signal returns below the threshold level, the solenoid valve is reopened so that the solution emerging from the concentrator tube is drawn off to waste. A t this point the carousel is rotated so the cup containing the solute from peak A is moved into position 2. Here a slow stream of air is drawn through the cup to eliminate the remaining solvent. Air is drawn through the hollow sample cup through a hole in the bottom of the sample wheel where the sample cups screw in. An aluminum manifold connected to a piece of rubber tubing butts up flush to the bottom of the sample wheel to draw air through the sample cup, effectively evaporating the residual solvent. When the second peak in the chromatogram, peak B, is sensed, the above procedure is repeated, and after the carousel has been rotated, peak C is deposited at position 1, while the solvent is eliminated from peak B at position 2 and peak A is held at position 3. At this position we have the option of either drawing air through the sample (as at position 2) or not. Most solvents u e d for normal phase HPLC are sufficiently volatile that only one solvent elimination step is needed for all solvent bands to be eliminated from the spectrum, and so we rarely used this position. After peak C has eluted, the carousel is rotated for the deposition of peak D, with peak A being held a t position 4. This procedure is repeated for the deposition of peak E, but a t this point peak A has reached position 5 and interferograms are signal averaged over the complete time the sample cup stays in this position. This process continues until each peak (or the first 31 for a mixture containing many resolved components) is collected and characterized. The final four rotations are not triggered from the HPLC detector but rather data are collected over a predetermined period of time for each of the final four peaks. Microcomputer Interface and Electronics. All operations are controlled through the KIM- 1 microcomputer using the VIA on the Memory Plus expansion board to provide most of the functions needed to interface the microprocessor to the HPLC, the HPLC/DRIFT interface, and the spectrometer. All software was written in 6502 machine code through the aid of a cross assembler and 6502 simulator on an IBM 370/ 158 computer. The software generated on this system could be loaded directly to the microcomputer through an RS-232 port to a 20-mA current loop converter. At the beginning of a chromatographic run, a number of parameters are entered into the program through the microcomputer keypad. It is assumed that the chromatogram has been previously measured and the number of peaks is known. This assumption is reasonable since for any sample the nature of the solvent, and often the solvent gradient, must be optimized to ensure good resolution and a reasonable elution time. The time delay, tD, required for a peak to travel from the HPLC detector to the end of the sample concentrator was determined empirically using a colored component. If the flow rate of the

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ANALYTICAL CHEMISTRY, VOL. 52, NO. 9, AUGUST 1980

mobile phase was not changed, t~ remained constant. The value of t D in seconds is entered in the appropriate memory location. The number of peaks in the chromatogram is also entered, together with the number of any peaks which are to be skipped, e.g., solvent peaks or a peak which has already been characterized. Data acquisition, rotation of the carousel, control of the solenoid valves, etc., are all actuated (after appropriate delays) when the signal from the HPLC detector passes a preset threshold voltage. The level detector was made by coupling a precision potentiometer in tandem with the chart recorder potentiometer. The potentiometer is used as a voltage divider, and a comparator circuit with a small amount of hysteresis is adjusted to trigger at the desired voltage level. The comparator output is then rectified and converted to a TTL level by a 5-V switching transistor, and input to the microcomputer. The FT-IR spectrometer is set up using Digilab's GC/FT-IR software. This software can be used in several different ways, one of which allows interferograms to be signal-averaged when the signal from the GC detector exceeds a certain threshold level (3). Data acquisition is controlled by a simple 'ITL signal whose level depends on whether or not the GC signal exceeds the threshold. Each time the logic level goes high, interferograms are signal-averaged in a different file until the level returns low. On our HPLC/DRIFT interface, the W L level signal is replaced by the TTL signal derived from the KIM-1 microcomputer which is applied to the appropriate minicomputer input pin in the FTS-20 data system. The microprocessor program is started by depressing the GO key on the keypad. The microcomputer then sits in a loop until the sample is injected. At this point an external pin on the VIA is brought to logic level low. The microprocessor then initializes the programmable 1/0 devices and starts the system clock. The system clock utilizes a programmable counter on the VIA which generates an interrupt every 0.05 s at which time the microcomputer will update a counter. When the level detector detects a peak, the signal generates an interrupt to the microcomputer at which time the clock is read and the delay, t ~is ,added to this time. This value is stored in a table along with a byte which indicates whether the peak was going high or low at the time. The VIA pin for peak detection can be programmed to generate an interrupt on a zero-to-one transition or a one-to-zero transition so that the chromatographic peak transitions are known by the computer and the proper byte can be loaded in the table with the time. In the meantime, between interrupts the microcomputer is scanning the clock and comparing the time values with the peak table values. When the two values are equal, the microcomputer jumps to the appropriate subroutine and, as for the case when a peak is going high, will switch off the solenoid valve by applying the appropriate signal to the appropriate output pin. When the microcomputer determines the time to stop depositing the peak, a series of events occurs. First the solenoid valve reopens (stopping the deposition) and the motor is turned on along with the solenoid to unlock the wheel. As the wheel turns, a photodiode senses each passing sample position. An input pin on the VIA decrements a preloaded counter for every pulse generated. When the counter counts down to zero (when the desired number of positions have passed), an interrupt is generated and the microcomputer turns off the motor and locking solenoid, locking the wheel into the proper position. This would bring the deposited peak into the drying stage. If this were the first peak of the chromatogram, no peak would yet be under the DRIFT optics for spectral measurement, and so the software would not initiate data collection. When the first peak is brought into position under the DRIFT optics, the microprocessor initiates the first data collection after a delay time of about 1 s t o allow all mechanical vibrations to die out. In this case, the data collection will continue until the next peak is deposited and the wheel begins its next rotation. Since the carousel motor, locking solenoid, and solenoid valve were powered by 110 VAC, they were switched upon receiving a TTL level signal from the microprocessor using a 5-A triac through an optically isolated coupler. A t the end of a chromatographic run, the microcomputer ignores any more signals generated by the HPLC detector trigger. The remaining samples which have not been measured are rotated through the remaining positions for drying and spectral mea-

surement. The wheel remains at each position for a predetermined amount of time. After the last peak is measured, the wheel rotates one more position and data are collected at this position until the run is aborted from the spectrometer. This allows for a reference spectrum to be obtained which is close to the sample spectrum in water content due to atmospheric exposure of the KCl powder.

RESULTS AND DISCUSSION Optimization of Variables. Concentrator Tube. The rate a t which the sample passes through the concentrator tube and the concentration factor are controlled both by t h e temperature of the concentrator tube and the sprayer gas flow rate. T h e gas flow rate should be the minimum consistent with effective nebulization of the effluent from the HPLC (about 10 L min-' with a n HPLC flow rate of about 2 mL/min). Increasing the gas flow rate does change the concentration factor. At too great a flow, t h e drops formed a t t h e end of the concentrator tube are disturbed and loss of sample occurs. Also the upper flow limit is greatly restricted by the small capillary tube used. In practice the most effective control of the sample concentrator is found by adjusting the temperature of the tube. In this way the concentration factor can be varied from about 2 or 3 to 1 all t h e way to infinity for most nonaqueous H P L C solvents. The sample cups have a finite capacity for the concentrated eluent, with a maximum capacity of about 3 drops (approximately 0.2 mL). At any greater volume deposited across the peak width, overflow of the sample cup occurs with a corresponding loss of sample and sensitivity. An optimum concentration factor would be where only one drop of concentrated effluent were deposited per peak. This allows only a narrow range of concentrator flow rate which must be carefully determined and so we prefer depositing two drops which leaves a small margin for error. One problem with setting t h e concentrator flow rate for a particular peak width (1-3 drops per peak) is that for a n isocratic chromatogram which is sufficiently complex (i.e., a large difference in the retention time between t h e first and last peaks), the peaks a t t h e end of the chromatogram are significantly wider than those at the beginning. In this case if the concentrator flow rate is optimized for peaks a t the beginning of the chromatogram, i t will not be optimized for those at the end. In this case the volume of effluent will be too great for the sample cups with the later peaks and a corresponding decrease in sensitivity will occur. This problem also occurs in the GC/FT-IR experiment, where the light-pipe is optimized for a certain peak width and for all others is less than optimum. However in the H P L C / D R I F T experiment it should be possible to change the concentrator flow rate and optimize it for any particular peak width quite easily, while in the GC/FT-IR experiment changing the light-pipe volume is much more difficult. I n GC/FT-IR, this problem can be minimized by using temperature programming and similarly, in HPLC/FT-IR, gradient elution helps to overcome this problem. Even though these procedures help to minimize peak broadening, the problems are not completely eliminated. However we believe that the HPLC/DRIFT method is much more easily adaptable for maximum sensitivity of a given chromatogram than GC/FT-IR. Aspirator Drying. T h e next variable considered which would possibly affect the sensitivity of the system was t h a t of the drying aspirator flow rate. After deposition of the concentrated effluent onto the KCl, the wheel rotates to bring this sample cup to a drying position (position 2). Air is drawn through the sample cup a t this position to evaporate the volatile solvent. T h e rate of aspiration through t h e sample cup was studied as a function of sensitivity. Intuitively, one may see that, if the aspirator flow rate were very high, t h e concentrated solution could be drawn too rapidly through the

ANALYTICAL CHEMISTRY, VOL. 52, NO. 9, AUGUST 1980 ~~

Table I. Sample Retention in HPLC/DRIFT System waveconcen- sample length, column, trator, cup, % % 9b solute nm Butter Yellow azobenzene naphthalene

396 315 311

100 100 100

60 67 20

65 45 8

KC1 powder with minimal deposition of the solute on the surface of the KCl. To test for this possibility, 100 pL of a 0.1% solution of caffeine in chloroform was deposited using a micropipet onto sample cups filled with KC1, and the sample cup was immediately aspirated a t different flow rates. A micropipet was used t o ensure that an exactly known amount of sample was deposited on the KC1. T h e flow rate through the aspirator was varied from 2 scfm, the minimum flow rate required to ensure drying over a 30-s single stage drying period, up to 10 scfm, the maximum aspirator flow rate. After drying, the spectrum of the caffeine was measured and the intensity of the bands from different aspirator flow rates compared. I t was found t h a t , within the range of these flow rates, no significant change in band intensities could be measured. Therefore, to ensure thorough drying of the sample, the higher aspirator flow rates were preferable. Since the sensitivity of the measurement does not change with the aspirator flow rate, it was suspected that the solute may have some affinity for the KCl. If this were the case, the top surface of the KCl would be expected to contain the majority of the solute. T o test this hypothesis, a solution of a relatively nonpolar solute, azobenzene, was deposited onto a KC1 filled sample cup as described above. After the infrared spectrum was measured and converted to Kubelka-Munk units, the top fraction of the KCl layer was scraped off to a depth much less than a millimeter and the spectrum measured. It was found that greater than 75% of the total solute resides in this top layer of the 2.5-mm deep KC1 bed. This confirms that some adsorption of the solute by the KC1 does occur and probably helps contribute somewhat to the excellent sensitivity of the technique. The same results were noted with several other solutes so that this effect is not a characteristic of azobenzene alone. Particle Size of KC1. The particle size of the KC1 powder in the sample cup does not appear to affect the sensitivity of the measurement to a very great extent. We have found that if large crystals of KCl are ground in a Wig-L-Bug for about 2 min (giving an average particle size of about 30 pm), measurements may be made with close to the greatest sensitivity and with a reasonably short sample preparation time. Sample Volatility. T h e device described in this paper requires the samples to be considerably less volatile than the solvent. Some solutes which are separable by HPLC have a fairly high volatility, so that loss of sample could occur through the sample concentration and solvent elimination stages of the system. To check on the importance of this problem, two relatively volatile solids, azobenzene ( m p 71 “C) and naphthalene ( m p 80 “C), were run on the H P L C / D R I F T system a n d the loss of sample a t each stage were determined. T h e results were compared to those obtained from a relatively nonvolatile substance, Butter Yellow from Stahl’s test dye. T h e samples were collected, eluted if necessary, and diluted to 5 mL in a volumetric flask. This was done a t each of three positions, directly from the column, from the end of the concentrator tube, and from the KCl sample cup after aspiration. The amounts a t each stage were monitored by UV/VIS spectrophotometry. T h e results are shown in Table I. Due to the difficulty in reproducing the depositions and manual collections, the range of the individual results was

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quite high (on the order of i=20%) and the results reflect these inconsistencies. However, they yield a good estimate of the sample loss at each stage for both the volatile and nonvolatile samples. I t can be seen that the majority of solute is lost a t the concentration stage. The reason for this is twofold. One reason is t h a t a certain amount of solute is lost in the form of an aerosol which escapes from the concentrator tube. Also some loss of sample occurs through the capillary which controls the deposition. Even when the solenoid valve controlling the aspirator is turned off, a certain amount of effluent is lost by capillary action up the capillary tube. This could be remedied by applying a slight positive pressure to this capillary when the solenoid valve closes. For volatile samples, it seems t h a t the majority of solute is lost in the concentrator tube. But even with the “worst case” of naphthalene, the sensitivity of the technique should be great enough to detect low microgram amounts of volatile solids. I t may also be noted that the more volatile compounds would usually be separated by GC rather than by HF’LC. Sample Stability. Since some complex molecules are thermally labile, they can never be separated by GC because they decompose a t the temperatures required for separation. Therefore an important factor to consider in an HPLC/FT-IR interface is the maximum temperature to which the sample is exposed. T o determine this parameter, thermocouples were used to monitor the temperature of the concentrator tube. For a typical mobile phase (82% hexane, 18% ethyl acetate) and a concentration factor of about 7 to 1,the highest temperature to which the solute is exposed was 39.5 “C. This is generally far too low to have any harmful effects on most compounds. Also the spray gas used is nitrogen to minimize possible oxidation of the compounds being separated. Although air is currently drawn through the cup in the final solvent elimination stage, it should not be hard to design a cover for performing the drying and measurement steps in a nitrogen atmosphere. (Many spectroscopists use liquid nitrogen boil-off for purging their spectrometer.) Under these conditions only the most sensitive compounds could not be run on the H P L C / D R I F T system. System Performance. Good spectra of samples separated by normal-phase HPLC have been obtained and the performance of the system, both in terms of detection limits and maintaining the chromatographic resolution, has exceeded our initial expectations. Identifiable spectra from submicrogram quantities of all nonvolatile samples injected into the HPLC can be obtained as shown in our previous report ( 1 5 ) . T h e detection limits appear to be determined by chemical interferences rather than the intrinsic noise level of the spectra. T h e source of the interference may be any or all of the following: residual solvent remaining after the drying stage; contaminants in the solvent eluting from the chromatograph; contaminants sorbed onto the KCl from the atmosphere; and contaminants present in small quantities in the KC1. T h e first interference is dependent on the nature of the solvent-the more volatile and less polar is the solvent, the smaller is the problem. We have used hexane/2-propanol gradients quite frequently and rarely see any trace of bands of 2-propanol in the spectrum, even though it has a fairly low volatility. Methanol bands, on the other hand, will often appear in the spectrum even though methanol is considerably more volatile than 2-propanol. T h e explanation appears t o be related to the fact that KCl is slightly soluble in methanol and much less soluble in 2-propanol. T h e principal contaminant sorbed onto the KC1 from the atmosphere is water. This problem can usually be eliminated by treating the KC1 reference in the same way that the sample is treated, but on very humid days or when very large ordinate expansions are required, water bands are often observed in

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ANALYTICAL CHEMISTRY, VOL. 52, NO. 9,

AUGUST 1980 TRANS

TANS

‘RANS

CIS

t

z

0

F 0

z

A

8

3 LL

:I I

a Y

A

W

m CIS

1

3 Y

L

H I MIN.

Figure 2. The chromatogram of cis- and trans-azobenzene, with the natural isomer ratio, separated on Partisil 10 with a mobile phase of (A) 40% ethyl acetate, 60% hexane and (6)100% ethyl acetate. The resolutions for (A) and (6)are 4.0 and 0.8, respectively

the spectrum. DRIFT spectrometry yields much more intense absorption bands than conventional transmission measurements (18). Therefore it is essential to keep the concentration of contaminants from all sources to an absolute minimum in order to maintain low detection limits in the HPLC/DRIFT interface. T h e chromatographic resolution is maintained surprizingly well through the concentration and deposition steps. This may be illustrated by the separation and measurement of the infrared spectra of the cis and trans isomers of azobenzene. In commercial azobenzene, the trans isomer is the major component and the cis isomer represents about 1.7% of the mixture. T h e two isomers were separated by HPLC using isocratic elution using a mixture of hexane and ethyl acetate as t h e solvent a t a flow rate of 2 mL/min. T h e separation was varied by increasing the proportion of ethyl acetate in t h e mixture, a n d with 100% ethyl acetate the resolution of the peaks due to the cis and trans isomers was 0.8, see Figure 2. T h e data collection trigger was set to be about 80% of the full height of the peak due to the cis isomer. There are four bands of azobenzene in the frequency region between 800 and 600 cm-’ which vary in relative intensity depending on the particular isomer. No contamination of the minor component from the major components in the infrared spectrum was observed, until the chromatographic resolution was 0.8, when a small amount of cross contamination could be seen (see Figure 3). It may be mentioned that the quantity of the minor component was only 350 ng. This experiment is very demanding, since the minor component is in the tail of the peak due to the major component and is present in a quantity approximately 60 times smaller than the major component. Until this point, HPLC/FT-IR systems have not possessed the sensitivity to identify minor components of mixtures separated by adsorption chromatography, largely because of the problems afforded by the presence of the mobile phase. T h e most impressive work t o date in which the solvent has not been eliminated prior to the measurement of the infrared spectra of the H P L C peaks has involved size exclusion chromatography where the choice of solvent does not seriously affect the separation (13). For size exclusion chromatography, such good infrared transmitting solvents as CCll and CS2 may be used as the mobile phase, but even with these solvents, some of the spectrum is still “blacked out”. Spectral subtraction techniques are not yet powerful enough to enable all solvent bands to be removed from the spectrum when gradient

7-J e50

cm-I C m-’ Figure 3. Spectra measured from the chromatograms shown in Figure 2. From the relative band intensities, the trans isomer (98.3% abundance) is present at less than 10% of the concentration of the cis isomer (1.7 % abundance) when the peaks are just resolved (R = 0.8)

elution is needed for the chromatography. With the HPLC/DRIFT technique, all of the above problems have been largely solved. An additional advantage is that each component is collected in the sample cups. If an unacceptable spectrum is obtained by the on-line method, the cup may be returned to the infrared beam when the chromatogram is complete and signal-averaged for a longer period of time to improve the signal-to-noise ratio. T h e sample may also be removed from the KCl using a nonaqueous solvent and characterized by other instrumental techniques off-line. There are, of course, a few limitations of this method. The first is that solutes must be significantly less volatile than the solvent; this is certainly true for most samples separated by HPLC since volatile solutes are usually separated by gas chromatography. The second limitation is that, as yet, water has not been successfullyeliminated because of its high surface tension and latent heat of vaporization, thereby limiting separations to normal-phase adsorption or size exclusion HPLC. However it may be noted that HPLC/FT-IR methods using flow-cells also cannot be used with aqueous phases because of the strength of the absorption bands of water. Finally the solute must be detected by the HPLC detector for the solvent eliminator t o function successfully. Although this limitation is fairly serious, we believe t h a t the HPLC/ DRIFT interface described in this paper represents only the first phase in the development of a totally automated HPLC/FT-IR system. In the final system, the effluent from the HPLC will almost certainly be continuously deposited, thereby obviating the need for a trigger actuated by the signal from the HPLC detector. In summary, the HPLC/DRIFT interface enables complete infrared spectra of all detected peaks to be routinely measured with a sensitivity which is considerably greater than is available by other infrared methods.

LITERATURE CITED (1) Low, M. J. D.; Freeman, S. K. Anal. Chem. 1987, 3 9 , 194. (2) Low, M. J. D. Anal. Lett. 1988. 1 , 819. (3) Kizer. K. L. Am. Lab. 1973, 5(8), 40. (4) Azarraga, L. V. “GCIIR with submicrogram sensitivity”, presented at 5th Annual Symp. on Recent Adv. Anal. Chem. of Pollutants, Jekyll Island, Ga., 1975. (5) Wall, D.; Mantz. A. W. Appl. Specfrosc. 1977, 3 1 , 5 2 5 . (6) Coffey, P.: Mattson, D. R.; Wright, J. C. Am. Lab. 1978, 1 0 ( 5 ) , 126.

1399

Anal. Chem. 1980, 52, 1399-1402 (7) Krishnan, K.; Curbelo. R.; Chiha, P.; Noonan. R. C. J . Chromatogr. Sci. 1979, 77, 413. (8) Mattson, D. R.; Julian, R . L. J . Chromatogr. Sci. 1979, 77, 416. (9) McFadden, W . H. J . Chromatogr. Sci. 1979, 77, 2. (IO) Arpino. P. J.; Guiochon, G. Anal. Chem. 1979, 57, 682A. (11) Kizer, K. L.; Mantz, A. w.; Bonar, L. c. Am. Lab. 1975, 7(5), 85. (12) Vidrine. D. W.; Mattson, D. R. Appl. Spectrosc. 1978, 32, 502. (13) Vidrine. D. W. J . Chromatogr. Sci. 1979, 77, 477. (14) Grlffiths, P. R. Appl. Spectrosc. 1977, 31, 284. (15) Kuehl, D.;Griffiths. P. R. J . Chromatogr. Sci. 1979, 17, 471. (16) Fuller, M. P.; Griffiths, P . R. Anal. Chem. 1978, 50, 1906. (17) Fuller, M. P.; Griffiths, P. R . Am. Lab. 1978, 10(10), 69.

(18) Fuller, M. P.; Griffiths, P. R.. Appl. Spectrosc., in press, 1980.

RECEIVED for review December 10, 1979. Accepted April 7, 1980. This work was in s u p p o r ~ dby Grant N ~m , o4333 from the U.S. Environmental Protection Agency. Additional financial support from the Alcoa Foundation, the Standard Oil Company of Ohio, the Baker Fund of Ohio University, and Merck, Sharp and Dohme is also gratefully acknowledged.

Detector Based on Optical Activity for High Performance Liquid Chromatographic Detection of Trace Organics Edward S. Yeung," Larry E. Steenhoek, Steven D. Woodruff, and J. C. Kuo Ames Laboratory and Department of Chemistry, Iowa State University, Ames, Iowa 5001 1

A mlcropolarlmeter based on laser optics is interfaced to HPLC for application In trace organic analysis. With the use of selected Glan prisms, selected ceil-wlndow material, and air-based Faraday rotators, it is possible to obtain extinction ratlos four orders of magnitude better than that in standard instruments. A detection limit of 0.5 pg is demonstrated in the separatlon of sugars In a 200-pL detection volume. Applications to the HPLC of untreated human urine are also presented.

T h e increased concern over the environment and the increased use of chemical information in clinical diagnosis have led t o a surge of interest in trace organic analysis. With the availability of capillary column gas chromatography (GC), particularly when interfaced to mass spectrometers (MS), the volatile organics no longer present major problems to the analytical chemist. On the other hand, high performance liquid chromatography (HPLC) still suffers from lack of resolution, and the corresponding LC-MS interface is still far from being ideal. The need for sensitive and selective HPLC detectors is therefore obvious, especially those that are based on entirely different physical or chemical properties than are normally associated with standard instruments. T h e limitations of available optical detectors for HPLC are many. Refractive index detectors suffer from a lack of sensitivity and selectivity. In cases where gradient elution is needed for proper separation, the problems are even more severe. Absorption detectors have been the most successful, b u t generally require the presence of convenient absorption bands, which means only unsaturated compounds are detectable. The use of far-UV detectors is limited by the possible choice of chromatographic eluents. T h e choice of eluents is also a problem with Fourier transform infrared detectors ( I ) , where many other technical problems still exist. Many of these optical techniques can be improved with the laser as a light source, and these have been discussed by Yeung (2). Naturally one can use chemical derivatization to overcome some of these difficulties, either before or after separation, but convenience a n d reliability are degraded. T h e rotation of polarized light by optically active molecules has resulted in relatively few applications in chemical analysis. Accurate determination of the optical rotary power of a sample 0003-2700/80/0352-1399$01 .OO/O

can provide information regarding its isomeric purity, and can be used for quality control in pharmacological and food-related industries (3). Instrumental limitations, however, do not allow the extension t o micro and trace analysis. There have been some attempts in the use of optical activity to monitor column chromatography ( 4 , 5). Again, the extension to HPLC has not been demonstrated owing to technical difficulties. An HPLC detector based on optical activity seems to possess many advantages in the problematic areas of organic analysis. Since most chromatographic eluents are not optically active, one will not be limited in the choice of eluents or gradients. Such a detector is extremely selective, so that complex samples can be handled. At the same time, since conformation is so specific a property for biological processes, there exist a large number of environmental and clinical systems where optically active species are the most important ones. T h e availability of a sensitive micropolarimeter will therefore benefit organic analysis when coupled to HPLC, and will broaden the applicability of spectropolarimetry in general. We present in the following a working detector for H P L C based on optical activity. A detailed description of the experimental system is given since it is the key to the success. Applications to the separation of sugars and human urine are also presented.

EXPERIMENTAL A schematic of the HPLC detector is shown in Figure 1. About 0.5 W of 514.5-nm radiation was used from a Control Laser (Orlando, Fla.) Model 554 argon ion laser. A l-m fl crown glass lens was used to collimate the light through the flow cell. A pair of selected (see below) 8-mm aperture Glan prisms, Karl Lambrecht Corp. (Chicago, Ill,),Model MCT-25E8-45, served as the polarizer and the analyzer respectively. The prisms were mounted in rotational stages with a resolution of degrees, obtained from Aerotech, Inc. (Pittsburgh, Pa.), Model ATS-301R. The rotational stages were in turn mounted rigidly on a vibrationisolated optical table, Newport Research Corp. (Fountain Valley, Calif.), Model RS-410-8. The separation between the prisms was kept at about 2 m to reduce stray light. Appropriate apertures were also used at various points in the optical path for the same reason. The flow cell was machined from an aluminum cylinder 10 cm long and 5 cm in diameter. A 1.58-mm diameter hole was drilled along its axis as the active region, which was intercepted a t both ends at 60° by 0.89-mm bores to interface with the chromatographic plumbing. The cell was held rigidly with spring-loaded positioners at each end for optical alignment. Cell C 1980 American Chemical Society