Anal. Chem. 1991, 63,782-787
702
Reversed-Phase Liquid Chromatography/Fourier Transform Infrared Spectrometry Using Concentric Flow Nebulization Andrew J. Lange and Peter R. Griffiths* Department of Chemistry, University of Idaho, Moscow, Idaho 83843
David J. J. Fraser' Department of Chemistry, University of California, Riverside, Riverside, California 92521
A novel technique for interfacing a microbore reversed-phase hlgh-perfmance llquld chromatograph and Fourier transform infrared (FT-IR) spectrometer has been developed. Warm helium gas Is passed through the outer of two concentric fused-silica tubes while the effluent from a liquid chromatograph Is passed through the inner tube. The tubes are housed in a vacuum chamber equipped with a rotatable stage used for solute deposition. With this approach, solute spots less than 150 pm In diameter can be obtained from aqueous mobile phases. I n this feasibility study, a two-step procedure was developed in whkh transmlsslon spectra of the deposJted solutes are measured by using an FT-IR microscope afler the solutes have been deposited onto a ZnSe plate. Complete elimination of the solvent from a mobile phase containing up to 100% water at a flow rate of at least 50 pL/min is possible. The minimum identtfiabie quantity in a measurement tlme of 45 s is less than 500 pg. On-line measurements uslng the same technique are shown to be feasible.
The interfacing of high-performance liquid chromatography (HPLC) and Fourier transform infrared (FT-IR) spectrometry has been of interest since the first demonstration of its feasibility 15 years ago (1). Two types of interfaces have been developed, one involving the use of simple flow cells, and the other using various solvent elimination techniques. The use of flow cells is generally hindered by absorption by the solvent but has been effective in certain cases (2, 3). Solvent elimination techniques have proven to be fairly straightforward with nonaqueous mobile phases (4-12), but reversed-phase separations have proven to be far less easily managed because of the low volatility of aqueous mobile phases. The first solvent elimination HPLC/FT-IR interface, which employed the "buffer-memory" technique, was designed by Jinno and co-workers (4-8). The eluites from a microbore H P liquid chromatograph were continuously deposited on a moving KBr window while the mobile phase was gently evaporated under a slow stream of nitrogen. The eluites were deposited in a continuous narrow band about 1.5 mm wide, and the stage was translated a t 1.25 mm/min. After separation, the window was moved into the beam condenser of an FT-IR spectrometer. The transmission spectrum of each eluite was measured, and the chromatogram could be constructed from the infrared data. Spectra were obtained a t 0.5-mm intervals on the window, giving a temporal resolution of 24 s. Both because of the nature of the substrate and because of the high surface tension and heat of vaporization of water, this interface could only be used with nonaqueous mobile phases of the type used for normal-phase HPLC.
* Author to whom correspondence should be addressed.
Current address: Xerox Corp., 800 Philips Rd, Bldg 139-064A, Webster, NY 14580.
An alternative interface, in which the eluites from normal-phase HPLC columns were deposited on powdered KCl, after which the diffuse reflectance infrared spectrum was measured, was first described by Kuehl and Griffiths (9,lO). Although this approach allowed detection limits to be reduced below 100 ng when microbore HPLC columns were used for the separation ( I I ) , it suffered the disadvantage that fractions had to be collected discretely. Although Kalasinsky et al. indicated that continuous deposition could be achieved by using this technique (12),the use of powdered substrates for HPLC/FT-IR measurements has never proved to be popular because of the experimental difficulties involved. In view of the contemporary inportance of reversed-phase HPLC, the complete removal of aqueous solvents from HPLC column effluents is necessary if HPLC/FT-IR is to be generally useful. Several methods for extracting water from aqueous mobile phases to facilitate solvent removal for diffuse reflectance have been described. Duff et al. (13) and Conroy et al. (14) described an interface in which the solutes emerging from the HPLC column were continuously extracted into an immiscible solvent (usually dichloromethane), while Kalasinsky et al. (12, 15) developed a method for water removal through a chemical reaction with 2,2-dimethoxypropane: CH,C(OCHJ,CH, + HzO 2CH3OH + (CHJ&O (1) The products of this reaction (methanol and acetone) are more easily evaporated than water. With this method, complete water removal was achieved for mobile phases containing up to 80% H20. For both of these techniques (13-15), analytes were deposited on a powdered substrate, and spectra were measured by diffuse reflectance. An interface based on a concept similar to the buffermemory technique but that is suitable for reversed-phase separations with aqueous mobile phases was described by Gage1 and Biemann (16,17). The effluent from the column was nebulized with nitrogen gas, and deposits were made in a 1-2 mm-wide helical track on a reflective metal substrate. Spectra of the deposited eluents were then taken by reflection-absorption (RA) spectrometry. Identification limits for the buffer-memory and the HPLC/RA-FT-IR interfaces are about the same (approximately 100 ng) (18). The main limitations to obtaining lower detection limits with this technique are the need for a relatively thick film for RA spectrometry a t -45O incidence (19) and for a reduced spot diameter to increase the film thickness. de Haseth and co-workers have modified the MAGICLC/MS (Monodisperse Aerosol Generation Interface Combining Liquid Chromatography and Mass Spectrometry) to work for HPLC/FT-IR at flow rates of up to 0.5 mL/min (20). The major modification is that an infrared transparent plate on which eluites are deposited is substituted for the ionization chamber of the mass spectrometer. This plate is removed from the vacuum chamber after deposition and placed in the beam of an FT-IR spectrometer equipped with beam-condensing optics. A deposition diameter of 0.44 mm has been achieved
0003-2700/91/0363-0782$02.50/00 1991 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 63, NO. 8, APRIL 15, 1991
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Figure 1. Design elements incorporated into the concentric flow nebulizer. (A) Thermospraying, (6) hydrodynamic focusing, (C) concentric flow nebulization.
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with this device, and depositions from injections of 100 ng have been demonstrated (22). Detection limits of MAGIC-LC/ FT-IR of 400 pg were also reported (22). In any direct deposition chromatography/FT-IR interface, it is advantageous to deposit the sample in a spot with the smallest possible diameter. For any reduction in the diameter of the sample, the path length, and hence the absorbance, increases by the square of that amount. T o achieve subnanogram detection limits for any real-time chromatography/ FT-IR interface, the diameter of the deposited eluite must be less than 200 pm (23). T o this end, several techniques for eliminating the solvent from aqueous HPLC effluents were investigated in our laboratory. The first design applied the principle of the thermospray HPLC/MS interface (24); see Figure 1A. Unfortunately, in most cases, the analyte sprayed out in a diffuse plume instead of a narrow jet, and deposits with a sufficiently small diameter could not be attained (25). An alternative means of reducing the diameter of a liquid stream is by hydrodynamic focusing, in which a concentric gas flow reduces the diameter of the liquid stream through the Bernoulli effect. When the diameter of the liquid becomes too small, however, Rayleigh instability causes the stream to break up into monodisperse droplets that spread out rapidly; see Figure 1B (25). Concentric flow nebulization has been used for several types of flame spectroscopy (26). The flowing outer sheath gas imparts momentum to the inner core, and as the inner liquid gains momentum, the end of the liquid stream is broken up into droplets as the surface tension of the liquid is overcome. The difference in velocity between the sheath gas and the liquid stream is usually so great that turbulence occurs near the end of the sprayer, which is useful for atomic spectroscopy nebulizers but not for creating a compact HPLC/FT-IR deposition. By heating the sheath gas in the concentric flow nebulizer, evaporation of the liquid decreases the amount of turbulence and yields a narrow droplet stream; see Figure 1C. Although the droplets are probably not monodisperse, even aqueous solvents can be evaporated rapidly so that the solute can be deposited in a small area on a substrate held 1 mm or less from the end of the tube. An interface between a normal-phase liquid chromatograph and an FT-IR spectrometer based on this principle was described earlier (27). The substrate for this work was a layer
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Figure 2. Exploded view of HRC-FTIR interface. (A) Electrical feed-throughs, (B) inlet for chromatographic effluent, (C) inlet for He gas, (D) chromel heating wire, (E)concentric fused-silica tubes, (F) rotatable stage for solute deposition, (G)horseshoe magnet and bar magnet for stage rotation, (H) micrometer for vertical stage positioning.
of powdered KCl on a ZnSe or metallic substrate, and spectra were measured by diffuse reflectance or diffuse transmittance spectrometry. However, use of the KCl powder as the substrate meant that this technique was not applicable to reversed-phase HPLC. In light of the conclusions made by Pentoney e t al., who showed that transmission spectrometry was the optimum sampling technique for SFC/FT-IR (19), and the direct deposition GC/FT-lR data obtained by Haefner e t al. (28),we wanted to use a simple ZnSe window as the substrate for reversed-phase HPLC/FT-IR measurements using the concentric flow nebulizer. The deposition described in this paper is based on the above principle. It shares many aspects of the gas chromatography and supercritical fluid chromatography (SFC) direct deposition (DD) interfaces with FT-IR spectrometers that have been developed in our laboratory (28-32).
EXPERIMENTAL SECTION Apparatus. An exploded view of the interface is presented in Figure 2. I t consists of two concentric fused-silica tubes, the inner diameter (i.d.) and outer diameter (0.d.) of which are selected to be consistent with the flow rate of the mobile phase. The outer tube is wrapped with chromel thermocouple wire used for rediameter, 2.5 R/cm, Omega Engineering, sistance heating (0.003-in. Inc., Stamford,CT) connected to a variable-power supply (Variac). for 20 mm. Helium gas is The wire is wrapped at 1 turn/" passed down this tube and is warmed conductively through the walls of the outer tube by the wire. Helium is used as the sheath gas in preference to, say, nitrogen because the higher thermal conductivity of helium allows better heat transfer to the mobile
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F w e 4. Flow-injectbn depositions using methyl violet 2B. The mobile phase for these depositions was 97:3 H,O/MeOH, flowing at a rate of 2 pL/min. The inner tube i.d. was 20 pm. (A) 60 ng injected, 75 X 100 pm spot; (B) 15 ng injected, 60 X 85 pm spot; (C) 840 pg, 78 X 81 pm spot after subtraction of the Impurity peaks; (D) same as C but without impurity peak subtracted.
Flgure 3. Close-up of stage shown in Figure 1. (A) Chrome1 heating wire, (B) outer tube carrying He gas, (C)inner tube for carrying chromatographic effluent, (D) zinc selenide plate used as substrate for solute deposition, (E) rotatable stage.
phase. The tubes are connected to a stainless steel male tee ('/I6 in. X in.X '/I6 in.) (Swagelok) and supported by custom-drilled no-hole ferrules (VG-2, Alltech Associates, Deerfield, IL). Gas flow is directed through one arm of the tee and liquid flow through another. The gas flow rate required is determined by the flow rate of the mobile phase; the highest gas flow rate used in this work was approximately 80 mL/min. The chromatographic effluent passes through the inner tube, which protrudes 1 mm from the end of the larger tube. These tubes are housed in an aluminum vacuum chamber equipped with a rotatable stage for solute deposition. The vacuum chamber is connected to a solvent trap (cooled by a mixture of methanol and dry ice), which is in turn connected to a rotary pump. A ZnSe plate is used as the solute substrate (see Figure 3), with the end of the inner tube being between 500 and 125 pm above the plate. A micrometer attached to the floor of the vacuum chamber (with two O-rings) permits vertical positioning of the stage to within 10 pm, and a small horseshoe magnet allows the stage to be turned manually without breaking the vacuum seal. Although all but the last of the experiments described in this paper were made more in the manner of flow injection analysis than HPLC, the flow rates investigated are designed to show the feasibility of a reversed-phase HPLC/FT-IR interface for chromatographs equipped with either packed capillary or microbore columns. For the measurements reported in this paper, the analytes were deposited from aqueous solutions of a single solute with the stage stationary. The stage was rotated manually after each injection. For the initial flow-injection studies, the sample compounds were dissolved in 97:3 H,O/MeOH (unless otherwise noted), injected into a 60-nL injection loop (Valco, Houston, TX), and passed down the inner tube into the nebulizer. Later, solutions in 100% water were injected. Studies were made at two solvent flow rates, 2 and 50 pL/min, that were selected to simulate the flow rates of packed 0.25-mm-i.d. and I-mm4.d. microbore columns, respectively. At 2 pL/min, a 100-pm-o.d., 20-pm-i.d.
fused-silica inner tube and a i"-pm-o.d., 500-pm4.d. fused-silica outer tube (PolymicroTechnologies, Phoenix, AZ) were used. For a solvent flow rate of 50 pL/min, a 150-pm-o.d., 50-pm-i.d. fused-silica inner tube was installed, and the outer tube was constructed from a 2.0-mm-o.d., 1.70-mm4.d. aluminosilicate capillary tube (A-M Systems, Inc., Everett, WA). For the lower flow rate, a Microfeeder HPLC syringe pump (Azumadenkikogyo Co. Ltd., Tokyo, Japan) was used. A reciprocating HPLC pump (Gilson Co., Middleton, WI) was employed for the investigations made at the higher flow rates. Separations were done with a 1-mm x 25cm odadecylsilane (C,& column (S3 ODS2, Phase Separations Inc., Norwalk, CT) with a mobile phase flow rate of 50 pL/min. Chromatograms were measured by using a ultraviolet detector (Spectroflow 757, Kratos Analytical Instruments, Ramsey, NJ) equipped with a capillary UV flow cell (LC Packings, San Francisco, CA). Solute spectra were measured off-line with a Perkin-Elmer Model 1800 FT-IR spectrometer equipped with a Spectra-Tech IR-Plan microscope. The microscope apertures were set to encompass the image of the spot (typically about 100 pm), and 100 scans at 4-cm-' resolution were taken in the transmission mode for each spectrum (for a 45-s data acquisition time).
RESULTS AND DISCUSSION The results of a 60-nL injection of the dye, methyl violet 2B (MVZB), dissolved a t concentrations of 1 mg/mL, 250 pg/mL, and 14 pg/mL in 97:3 H,O/MeOH are shown in Figure 4. These data show that the minimum identifiable quantity (MIQ) of an off-line measurement performed in the flow-injection mode at 2 fiL/min is well below 1ng. A siliceous impurity, with a large abosrption at 1110 cm-', is codeposited with the solute. When the injected quantity is small, this feature must be removed by spectral subtraction if an authentic spectrum is to be measured (as illustrated in Figure 4, parts C and D). A deposition from a blank injection allowed to accumulate for 5 min was collected; its spectrum is shown in Figure 5. Because this deposition was allowed to build up over several minutes, the absorbance of the resulting spot is higher than would be seen in a typical HPCL/FT-IR measurement. This spectrum can be used for spectral subtraction but is rarely necessary for injected quantities greater than 10 ng. Experiments to determine (and eliminate) the cause of this band are under way.
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Table I. Computerized Spectral Search Results from the Caffeine Spectrum Shown in Figure 6 hit
name
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1 2 3 4 5 6 7 8
caffeine in KBr disk (UI) fenethylline in KBr disk (GSCL) 0.4% caffeine in KBr disk, air reference (UI) 0.4% caffeine in KBr disk, KBr as reference (UI) pentifylline in KBr disk (GSCL) pentoxofylline in KBr disk (GSCL) 7,2-(chloroethyl)theophylline in KBr disk (GSCL) proxyphylline in KBr disk (GSCL) caffeine in KBr disk (GSCL) dyphylline in KBr disk (GSCL) 1,3-dimethylbarbituricacid in KBr disk (GSCL) 0.2% caffeine in KBr by DR (UI) 0.04% caffeine in KBr by DR (UI) fenethylline in KBr disk (GSCL) methylprilon in KBr disk (GSCL)
0.288 0.299 0.321 0.327 0.354 0.377 0.443 0.493 0.499 0.504 0.549 0.560 0.565 0.621 0.671
9
10 11
12 13
14 15
1000
Flgure 5. Blank flow-injection deposition using a mobile phase of 100% H,O at a flow rate of 2 pLlmin. I
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Figure 6. (A) Injection/deposition of 62 ng of saccharin (120 pm spot), under the same conditions as Figure 4. (B) KBr reference spectrum.
An injection/deposition of caffeine under the same conditions is shown in Figure 6A. Even though caffeine sublimes rapidly when heated under a vacuum, a useful spectrum was still obtained. Thus, the heating required to eliminate the solvent a t a flow rate of 2 pL/min is insufficient to cause extensive loss of sample. When this spectrum was searched against the Georgia State Crime Laboratory (GSCL) library of infrared spectra of commonly abused substances, the reference spectrum of caffeine only gave the sixth best hit. An identical result had previously been found in our laboratory when caffeine was separated by supercritical fluid chromatography and identified by a direct deposition SFC/FT-IR interface (33). Apparently the optical thickness of caffeine for the GSCL reference spectrum was too great, causing a reduction in the relative peak heights of the intense C=O stretching modes and the weaker bands at lower wavenumber. Several other reference spectra measured either by transmission of a KBr disk of caffeine or by diffuse reflectance (DR) of a dispersion of caffeine in KCl powder were added to the
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Figure 7. (A) Injection/depositionof 62 ng of saccharin (120 pm spot), under the same conditions as Figure 4. (B) Sodium saccharin KBr reference spectrum from GSCL library, (C) 0.8% saccharin in KBr by transmission, (D) 2.3% saccharin in KBr by diffuse reflectance.
library. These spectra yielded hits nos. 1,3,4,12, and 13 with the best match being the spectrum of a KBr disk of caffeine (Figure 6B). The search results with the new spectra of caffeine added to the GSCL library are summarized in Table I. The GSCL reference spectrum of caffeine is the 9th best match, with all the top 10 hits being theophyllines. It should be noted that the reference spectrum of caffeine has been the only spectrum in the GSCL library that we have felt it necessary to replace. The HPLC/FT-IR spectrum of saccharin measured in the same way (see Figure 7A) was also searched against the GSCL library, and the results are listed in Table 11. Although saccharin was injected in its free acid form, the closest matching spectrum in the library was that of the sodium salt; see Figure 7B. Thus, in the process of nebulization/deposition, the saccharin was converted to its salt, even though no salts
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Table 11. Computerized Spectral Search Results for the Saccharin Spectrum Shown in Figure 7 hit 1
2 3 4 5 6 7 8 9 10 11 12
13 14 15
name sodium saccharin in KBr magnesium sulfate in KBr calcium sulfate in KBr rescinnamine in KBr
1,8-dihydroxyanthraquinonein KBr polythiazide in KBr procaine (free base) in KBr dioxybenzone in KBr methylparaben in KBr n-butyl p-aminobenzoate in KBr oxybenzone in KBr doxylamine succinate in KBr flufenamic acid in KBr hydroflumethiazide in KBr calcium hvdroeen Dhosuhate in KBr
B S 0 R
HQI 0.488 0.521 0.549 0.560 0.565 0.576 0.587 0.587 0.593 0.593 0.604 0.610 0.615 0.621 0.621
were present in the solvent. The spectra of free saccharin measured as a KBr disk and by diffuse reflectance are shown for reference in Figure 7, parts C and D, respectively. The cause of salt formation is currently being investigated. The initial data from our lab suggested that it was useful to include approximately 3% methanol in the aqueous mobile phase to reduce solvent wetting of the interface tube and hence to reduce solute adsorption on the walls of the inner tube of the interface. All of our early work was therefore performed with a mobile-phase composition of 97:3 H,O/MeOH. After further optimization of the flow parameters, however, the addition of methanol was found to be unnecessary, and all current depositions are being made from 100% water. The equipment needed to perform packed capillary HPLC is not commonly available in many laboratories in the U S . On the other hand, the pumps, injectors, and detectors required for 1-mm-i.d. microbore columns are commercially available from several sources. The feasibility of applying the interfaces shown schematically in Figures 2 and 3 with a flow rate directly compatible with microbore HPLC (50 pL/min) was therefore investigated. After increasing the diameters of both the inner and outer tubes (as described in the Experimental Section), complete elimination of 100% water was effected. As would be expected, complete elimination of water/methanol or water/acetonitrile mixtures requires a somewhat smaller potential across the heater wire than the elimination of 100% water. The spectrum of a deposition of 60 ng of MV2B from 100% H 2 0 at a mobile phase flow rate of 50 pL/min is shown in Figure 8A; a reference spectrum of pure MV2B dispersed in KCl measured by diffuse reflectance is included in Figure 8B to illustrate the complete elimination of water. One critical factor for achieving the smallest possible spot size with this interface is that the inner tube must be concentric with the outer tube. Centering of the inner tube is paricularly important for higher flow rates. It is also important to maintain a laminar flow of the heated sheath gas to achieve a compact deposition. For studies using a mobile phase flow rate of 50 pL/min, the inner tube was centered by tying a short length of chrome1 wire around the inner tube a t two locations along its length and securing the ends to opposite walls of the outer tube. This arrangement permitted adequate centering of the two tubes while allowing a laminar flow of sheath gas. Flow-injection depositions for two chemically similar analytes, namely, theophylline and caffeine, using a mobile phase flow rate of 50 pL/min, are shown in Figure 9. The mobile phase for this deposition was H20/CH3CN (8515) to allow comparison with the HPLC/FT-IR data devised in the next paragraph. It was anticipated that the higher heat input required to eliminate the solvent a t 50 pL/min might be
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Flgure 8. (A) Flow-injection deposition of 60 ng of MVPB (injected) using a mobile phase of 100% H,O at 50 pL/min. (B) 0.1 % MVPB in KBr by diffuse reflectance.
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Flgure 9. Injectionldepositions of (A) 60 ng of theophylline (120 X 90 pm spot) and (B) 60 ng of caffeine (114 X 123 p m spot) using a mobile phase of 85% H,0/15% CH,CN at 50 pL/min. enough to cause the more volatile caffeine to sublime from the ZnSe plate after deposition before a spectrum could be measured. If the ZnSe plate was held under the warm helium gas for a prolonged period, this is exactly what happened. By rotating the plate after each deposition and removing it from the vacuum chamber after only a few depositions had been made, it was possible to prevent the plate from heating up, and a good deposition of caffeine was obtained. Cooling of the stage was also tried, but no better results were attained. The concentration of solutes entering the nebulizer after flow injection should only be slightly less than their concentration in the injected solution. On the other hand, in light of the peak-broadening processes that occur in a chromatographic column, the analyte concentrations in the effluent from an HPLC will be significantly reduced. Thus,if an anlyte is a t all volatile, one might expect to observe greater sample
ANALYTICAL CHEMISTRY, VOL. 63, NO. 8, APRIL 15, 1991
a pressure pulse causes the droplet stream emerging from the nebulizer to diverge instantaneously, presumably leading to a slight spreading of the deposit, and hence a decrease in absorbance. The delay introduced by the retention of the analytes and the dampening effect of the column minimize this effect for a true HPLC/FT-IR measurement. The fact that both the spectra of caffeine, which is relatively volatile, and theophylline, which is far less volatile, are more intense in Figure 11 than in Figure 9 indicate that sample loss by vaporization is not a problem for these analytes. In summary, these results indicate the feasibility of reversed-phase HPLC/FT-IR measurements by concentric flow nebulization at flow rates up to at least 50 pL/min. Higher flow rates have not yet been investigated. The spectra are directly searchable against KBr-disk reference spectra. Maintaining the deposition stage near room temperature seems to be necessary to preseve some solutes at sufficiently low vapor pressures to prevent their sublimation. An on-line version of this interface that incorporates a computer-controlled moving stage capable of being heated or cooled (28) is currently being developed.
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Flgure 10. Separation of 60 ng of theophylline (peak C) and 60 ng of caffelne (peak F). The injection was made at point A, and the ZnSe plate was moved at points B, D, E, and G.
LITERATURE CITED (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11)
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Flgure 11. Spectra of deposited peaks from Figure 10. (A) Deposition of theophylline (peak C, Figure 10) (120 X 120 pm spot). (B) Deposition of caffeine (peak F, Figure 10) (82 X 114 pm spot).
losses during deposition of analytes emerging from an HPLC column than for flow injection. To check on this premise, caffeine and theophylline were separated on a 1-mm-i.d. CI8column with a mobile phase of 85:15 H20/CH3CN. The chromatogram (measured with an ultraviolet detector at 274 nm) is shown in Figure 10. Caffeine (denoted as peak C) was deposited between points B and D with the substrate stationary. Similarly theophylline (peak F) was deposited between points E and G. The measured spectra are shown in Figure 11. By comparison of the ordinate scales of Figures 9 and 11,it can be seen that band intensities are actually greater when the sample has been passed through an HPLC column than if the flow-injection mode is used. The delay between injection and deposition in the flow-injection experiment was only 0.5 s. When the injector is activated,
(23) (24) (25) (26) (27) (28) (29) (30) (31) (32) (33)
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RECEIVED for review October 16,1990. Accepted January 18, 1991. Financial support for this work through Grant No. R-814441-0from the US.Environmental Protection Agency is gratefully acknowledged.
