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Anal.’ Chem. W04, 56,2632-2636
A New Transport Detector for High-Performance Liquid Chromatography Based on Thermospray Vaporization Lily Yang, G. J. Fergusson, and M. L. Vestal* Department of Chemistry, University of Houston, Houston, Texas 77004
A new detector for HPLC has been developed which employs thermospray deposition of the sample onto a movlng stalnless steel belt wlth subsequent vaporization or pyrolysis into a flowing gas stream for detectlon in a conventional GC detector. Most of the present work has employed a photolonization detector (PID), but some work has also been done with an electron capture detector (ECD). The new detector is superficially slmiiar to earlier transport devices in that a movlng surface Is required and the sample Is detected after the solvent is removed. The major unique feature of the present approach Is that the sample Is vaporized and removed without belng deposlted as liquid on the movlng surface. Performance wlth the P I D was evaluated using amlno aclds and peptides, and performance wlth ECD was checked by using chlorinated pesticides. Detection limits in these initial studies were typically In the 10-400 pM range. The system performs satisfactorily with both aqueous and nonaqueous solvents at flow rates up to at least 2 mL/min.
It is generally recognized that the greatest need for future development in high-performance liquid chromatography (HPLC) is detectors (1,2). Many attempts have been made toward this goal (3-5), but a universal and sensitive detector is not yet available. The most widely used detectors in modern LC are photometers based on ultraviolet (UV)and visible absorption. The shortcomings of UV detectors are well-known. In recent years, fluorescence and electrochemical detecotrs have gained increasing popularity owing to their selectivity and high sensitivity for certain classes of compounds, but these do not provide a general solution. An approach to monitoring effluent from an HPLC column is to transform the liquid effluent to gas and use GC detecotrs, since several superior gas chromatographic (GC) detecotrs exist including flame ionization (FID),electron capture (ECD), and photoionization (PID). Direct liquid introduction interfaces for these HPLC/GC detectors have been suggested (6-8), which vaporize the total HPLC effluent from the analytical column prior to detection. Either the entire vaporized effluent or a known fraction of this is then transferred to a GC detector. This requires that the vaporized mobile phase not interfere with detection of vaporized samples. Attempts to eliminate the mobile phase prior to detection have led to transport detectors, in which a conveyor receives the column effluent and transports it into an evaporator, where the solvent is evaporated from the conveyor. A separate stream of carrier gas feeds sample components to a GC detector (9-14). The detection limits achieved by these earlier transport detectors was somewhat disappointing. Transport devices have been used successfully as LCJMS interfaces (15,16), and some of the problems associated with the earlier systems have been addressed in this and later work (17-19). One of the major problems with the transport devices, particularly for reversed-phase LC, is that i t is difficult to rapdily vaporize polar solvents from a surface without losing a significant and uncontrolled fraction of many samples. 0003-2700/84/0356-2632$01.50/0
Various approaches to solving this problem have been studied in connection with LC/MS interfacing including a segmented flow reactor (ZO), use of microbore LC (21), and spray deposition either by gas flow nebulization (17-19) or by thermospray (22). Spray deposition allows samples somewhat less volatile than the solvent to be efficiently transferred to the surface of a moving belt while most of the solvent is vaporized and removed without deposition as liquid on the surface. This paper describes a new “thermospray” transport detector for HPLC which greatly improves the detection limit and chromatographic performance over earlier transport detectors. The thermospray transport detector is superficially similar to the earlier transport detectors in that a moving surface is required and the sample is detected after the solvent has been removed. The major difference with the thermospray approach is that the sample is sprayed onto the surface with most of the solvent being vaporized and removed without being deposited as liquid on the moving surface. In the thermospray vaporizer, sufficient heat is transferred to effect nearly complete vaporization of the liquid at the rate with which it is supplied. The effluent from the vaporizer is a superheated mist carried in a supersonic jet of vapor. Nonvolatile molecules are preferentially retained in the droplets of the mist (23). A moving surface is placed perpendicular to the vapor jet. When the distance between the vaporizer nozzle and the moving surface and the temperature of the vaporizer are chosen correctly for a given effluent flow rate, most of the droplets containing samples and a very small amount of solvent will deposit onto the moving surface. The thermospray deposition efficiency can be high and stable if both a stable effluent flow rate and a stabilized heat source are available.
EXPERIMENTAL SECTION A schematic diagram of the LC detector employing thermospray deposition of the sample onto a moving belt is shown in Figure 1. The effluent from the LC enters the vaporizer through a stainless steel capillary tube (typically 0.015 cm i.d. by 0.15 cm 0.d.) the end of which is brazed into a copper block (3.0 cm X 2.5 cm X 1.2 cm). The block is heated by two commercial 100-W cartridge heaters (Wattlow, St. Louis, MO.) normally operated at substantially below their rated power. A thermocouple is embedded in the copper block to monitor the temperature of the vaporizer. The distance between the vaporizer nozzle tip and the belt is 1.2 cm and a vent under the belt beneath the vaporizer nozzle allows solvent vapor to be aspirated away. The belt used in this system is stainless steel (EBTEC), 54.5 cm long and 0.3 cm wide. It is driven by a synchronous motor (I rpm) at a linear velocity of 0.2 cm/s. The belt carries the sample into a pyrolyzer chamber which is surrounded by two stainless steel blocks. These blocks are heated by two commercial 100-W cartridge heaters and a thermocouple embedded in the bottom block indicates the temperature of the pyrolyzer. Carrier gas passes through the pyrolyzer chamber and carries the vaporized or pyrolyzed sample into a GC detector. Results presented in this paper were obtained by using the thermospray transport device described above coupled to either a photoionization detector (PID) or an electron capture detector (ECD) designed for use as GC detectors. The ECD was a Valco Model 140-N (Valco Instruments, Houston, TX), and the PID detector was constructed in our shops and is superficially similar 0 1984 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 56, NO. 14, DECEMBER 1984 c;pr
Carrier gas
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LC effluent
i 60K
vapor out
2 50-
L
\moving
bail
Flgure 1. Schematic diagram of LC detector employing thermospray deposition of the sample onto a moving belt and sample detection by vaporization or pyrolysis of the sample into a standard GC detector.
to those available commercially (HNU, Inc., Newton, MA). The volume of the active portion of the cell was 0.2 cm3 and it was connected to the pyrolyzer chamber by a stainless steel tube (0.15 cm i.d. by 0.3 cm 0.d. by 10 cm long) insulated with asbestos. The cell was heated by a 100-W cartridge heater and a thermocouple mounted on the cell surface indicated the cell temperature. A krypton resonance lamp with MgF window (Model 108-10.0 eV, Scientific Services, Rocky Hill, NJ) was used as the light source. The LC mobile phase was pumped to the vaporizer by a Constametric I HPLC pump (Laboratory Data Control, Division of Milton Roy Co., Riviera Beach, FL) through a 10'mL buffer reservoir located upstream from the sample injection valve. Sample solutions were injected with a syringe loading sample injection valve (Mdoel7125, Rheodyne, Inc., Berkeley, CA) with a 20-pL sample loop. Except for those results which involve separation of mixtures, the samples were carried by the mobiIe phase directly to the vaporizer without passing through an LC column. Chemicals used were obtained from the following sources: amino acid, Sigma Chemical Co., St. Louis, MO; aldrin, Applied Science Laboratories, Waltham, MA, peptides, United States Biochemical Corp., Cleveland, OH. Methanol was HPLC grade purchased from Fisher Scientific, Medford, MA, and acetic acid (glacial) from Matheson, Coleman and Bell, Norwood, OH. Water used was deionized and purified with a Milli-Q water purification system from Millipore Corp., Bedford, MA, and vacuum degassed with ultrasonic agitation. All mobile phases were filtered through a 0.5-pm filter.
RESULTS AND DISCUSSION The thermospray transport system is compatible with a variety of gas-phase detectors. In the present work, the characteristics of thermospray deposition for use in a transport system for LC detection were evaluated by coupling to the PID detecotr described above, using phenylalanine as a test solute and deionized water as the solvent. Most of these initial experiments were done without an LC column in place in order to quickly and efficiently evaluate the performance of thermospray sample deposition with particular mobile phases (e.g., pure water) without the additional complications imposed by retention on an LC column. The effect of vaporizer temperature on the relative deposition efficiency is shown in Figure 2 , where responses to 1nmol of phenylalanine are plotted as a function of vaporizer temperature. There is a maximum on the curve a t a temperature of 247-248 "C where the deposition efficiency is about 69% as determined below. As can be seen, the deposition efficiency drops rapidly with either an increase or decrease in the termperature of the vaporizer. The peak was significantly broadened when the vaporizer temperature was lower than the optimum. At the optimum temperature for a certain flow rate nearly complete vaporization occurs before the jet strikes the surface and a visible 1-2 mm spot is observed on the belt, as shown in Figure 3A. At higher temperatures, complete vaporization is achieved as indicated by the lack of an observable spot on the belt and deposition efficiency is low.
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Flgure 2. A plot of the response of 1 nmol of phenylalanine injected as a function of the temperature of the vaporizer. Chromatograms show the actual peak at different temperatures on the vaporizer. Conditions were as follows: solvent (water) flow, 0.6 mL/min; carrier gas (He) flow, 400 mL/min; pyrolyzer temperature, 420 OC.
A
B
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-
belt surtace visible spot
completle vaporitat ion
flooding Figure 3. Appearance of the surface of the moving belt beneath the thermospray vaporizer at different operating temperatures: (A) optimum temperature on the vaporizer: (B) higher temperature than the optimum; (C) lower temperature than the optimum.
At low temperature the jet is very wet which results in flooding of the belt surface (Figure 3C) leading to problems similar to those associated with the earlier transport detectors. These observations correlate with the observed peak shapes as indicated in Figure 2, where chart records of responses observed at various vaporizer temperatures are superimposed on the plot of peak area vs. vaporizer temperature. When the temperature is too low, belt flooding causes the peaks to be broad and low, and when the temperature is too high, the intensity falls off due to premature sample vaporization. At higher temperature significant modulation is observed due to more pronounced effects from flow variation introduced by imperfections in the HPLC pumping system. The sensitivity and detection limits were determined at the optimum vaporizer temperature by injections of 20-pL samples of known concentrations. Peak current was determined as peak height minus the signal from a blank run. Data were plotted as peak current vs. sample injected and were fitted to a linear equation. The MDQ was determined by extrapA). olating to a peak current twice the noise level (5 x Responses of the PID cell to triplicate injections of phenyl-
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ANALYTICAL CHEMISTRY, VOL. 56, NO. 14, DECEMBER 1984 A-
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Table I. Characteristics of the Thermospray Transport Photoionization Detector with Phenylalanine as Test Solution in H 2 0
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minimum detectable quantity linear dynamic range background current noise level sensitivity peak width at half-height (20-pL injection)
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Table 11. Minimum Detectable Quantities (pmol) for Various Amino Acids glycine iso1eucine alanine proline methionine asparagine
Figure 4. Analysis of 20-pL aliquots of serial dilution of ph nylalanine in deionized water with the thermospray transport PID. Triplicate injections of (A) blank, (6)20 pmol, (C) 40 pmol, (D) 0.1 nmol, (E) 0.2 nmol, and (F) 0.4 nmol in 20 pL of water were used. Full scale is 1 X lo-’’ A. Conditions were as follows: solvent (water) flow, 0.8 mL/min; vaporizor temperature, 247 O C , pryolyzer temperature, = 430 OC;
carrier gas (He) flow, 220 mllmin.
/
o,o,o~
/ PHENYLALANINE (nmolr)
Figure 5. log-log plot of the response as a function of nmol of phenylalanine injected under conditions given in Figure 4. The dashed line corresponds to sample placed directly on the beit and the solld line to samples transferred by thermospray deposition from water flowing at 0.8 mL/min.
alanine into deionized water flowing at 0.8 mL/min are shown in Figure 4 for serial dilutions from 0.4 nmol per injection (F) down to 20 pmol (B). Trace A shows the signal spanning three blank injections. The data on response of the thermospray transport PID detector as a function of sample size are summarized in Figure 5 , where the mean peak areas are plotted vs. nanomoles of phenylalanine injected. At least three replicate determinations were made for each sample size, the standard deviations are indicated by the error bars. The line shown in Figure 5 corresponds to the least-squares fit to the
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37
glutamic acid arginine histidine serine phenylalanine
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200 12
200
data given by log Y = 1.14 log X + 2.22 with correlation coefficient 1.00 (0.9996). Y is the average response (peak area) and X is sample size (nanomoles). As can be seen in Figure 5, the response is essentially linear over the range from ca. 0.01 to 100 nmol of phenylalanine injected, Le., over a range of lo4. At higher samples sizes, some deviation from linearity is observed. The absolute deposition efficiency for the present thermospray transport device was determined by comparison of the response for phenylalanine injected with the response to the same size sample placed directly on the belt. One-microliter aliquots of serial dilutions of phenylalanine in water were directly placed on the stopped belt. After the sample solution dried, the belt transport was started and the sample detected by the PID cell in the usual way. The dashed line on Figure 5 , shows the response for samples placed directly on the belt. The ratio of thermospray response vs. direct deposition indicates an average deposition efficiency of 69%. Performance characteristics of the thermospray transport PID detector determined for phenylalanine in water are summarized in Table I. The major contributor to the peak width appears to be the PID cell, and it appears that minor design changes may allow the peak broadening to be reduced. Detection limits were determined for a number of other amino acids; the results are summarized in Table 11. Since the peak shapes and background levels are essentially the same in all cases, the variations in detection limits also reflect variations in sensitivity for these compounds. There appears to be no simple correlation with sample volatility, and it appears that the major factor determining sensitivity is the relative ionization efficiency in the PID cell. One of the problems encountered with earlier transport systems is that “ghost” peaks are frequently observed due to recycling of residual components on the belt from previous injections. The extent of this problem with the present detection system is illustrated in Figure 6 where the responses to triplicate injections of 4 nmol of phenylalanine are shown together with the peaks corresponding to recycling. In this case the first recycle peak is only about 2% of the original, and the second recycling is not detected. From these and similar results it appears that the sample is being quite efficiently vaporized in the pyrolyzer but that a small fraction may leak out through the belt exit slit and recondense on the belt in this cooler region. Modest redisgn of the pyrolyzer cell may remove this effect. For less volatile samples it is expected that the recycling problem may be more severe, and some kind of belt cleanup device, such as those used in recent LC/MS transport systems (15),may be required. The transport detector is applicable to samples covering a wide range of volatility and may be used with almost any
ANALYTICAL CHEMISTRY, VOL. 56, NO. 14, DECEMBER 1984 IO,
I
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Figure 6. Detector response to 4.0 nmol of phenylalanine: 1, 2, and 3 were three replicate injections; l', 2', and 3' were their first recycles, l", 2", and 3" were second recycles. Full scale was 1 X lo-'' A and belt cycle time was 4.5 min.
A B Flgure 8. Sample of performance of the thermospray transport system with electron capture detection. Response corresponds to duplicate 20-pL injections. A contains 1 ng each and B 0.5 ng each. Conditions were as follows: solvent (MeOH) flow, 0.6 mL/min; carrier gas (N2) ftow, 90 mL/min; ECD temperature, 300 O C ; pyrolyzer temperature, 290 O C ; attenuation, 1024.
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Figure 7. Response of the thermospray transport PID detection to trlplicate injectbns of some dlpeptides: full scale 1 X lo-'' A; pyrolyzer temperature, 425 O C ; solvent (water)flow, 0.8 mL/mln; carrier gas (He) flow, 400 mLlmin; sample size, 2 nmol each: (A) Gly-Phe, (B) Ile-Glu, (C) Gly-Asp, (D) Gly-Ser.
type of gas chromatographic detector. A sample of results obtained with the PID system for dipeptides is shown in Figure 7. In these results there is some apparent peak tailing which was not observed with the amino acids. This may indicate that the temperature of the pyrolyzer and/or that of the PID cell and transfer line was too low for these less volatile samples, but further work will be required to establish operating conditions for different classes of compounds and to more clearly delineate the range of samples for which useful results can be obtained. Some preliminary experiments have also been conducted using the thermospray transport device with the electron capture detector. Some samples of results obtained for the pesticide aldrin are shown in Figure 8. These results were obtained by using methanol as the mobile phase, but comparable results have been obtained by using chlorinated solvents. The fact that essentially no increase in background level is observed when methanol is replaced by dichloromethane, for example, indicates that solvent removal in the thermospray transport system is quite effective. Several similar pesticides (e.g., aldrin, lindane, etc.) were detectable at subnanogram levels even though the transport efficiencies for these more volatile compounds was substantially less (ca.
Flgure 9. Chromatograms of a mixture of six amino acids: (A) thermospray transport PID detection at vaporizer temperature 430 O C and carrier gas (He) flow 130 mL/min; (B) on-line UV detection at 254 nm and 0.005 AUFS. 5-10%) than that obtained with the amino acids.
Chromatographic performance of the thermospray transport detector with photoionization has been evaluated by comparison with UV detection at 254 nm using a mixture of amino acid standards separated on a reversed-phase column. An example is shown in Figure 9. In this example a mixture of 5 nmol of each of six free amino acids was injected into a IBM CI8 column (4.5 X 15 cm, 5 pm) using a flow rate of 0.8 mL/min of 20% methanol in dilute acetic acid (pH 4.4). A UV detector (Waters Model 440) was on line before the PID transport detector. As can be seen from Figure 9, only phenylalanine is detected in the UV while all six amino acids give comparable response with the PID transport detector. The small peaks in the UV trace at low retention times do not correspond exactly to the amino acid retentions and probably correspond to low-level UV absorbing impurities in the sample. The offset of the two traces in Figure 9 corresponds to the transit time of the sample on the belt from the deposition region to the vaporizer/pyrolyzer. A small but significant amount of band broadening can be detected in the signal from the transport device. For example, the width at half height of the phenylalanine peak in the PID signal is about 5 s more than that from the UV detector. The major source of this loss in resolution appears to be dispersion introduced in the removal of sample from the belt or in the PID cell rather than in the deposition step. This was determined by comparing the response for direct deposition of a sample onto the belt
2636
Anal. Chem. iga4, 56, 2636-2642
with that obtained by thermospray deposition.
(3) Locke, D. C.; Dhlngra, B. S.;Baker, A. D. Anal. Chem. 1982, 5 4 , 447. (4) Stoiyhwo, A.; Colin, H.; Guiochon, G. J . Chromafogr. 1983, 265, 1. (5) Hayes, M. J.; Lankmayer, E. P.; Vouros, P.; Karger, B. L.; McGuire, J. M. Anal. Chem. 1983, 55, 1745. (6) Nota, G.; Palombarl, R. J . Chromatogr. 1971, 62, 153. (7) Willmott, F. W.; Dolphin, R. J. J . Chromatogr. Sci. 1974, 12, 695. (8) Xle, K. H.; Krull, I . S.;Drlscoll, J. N. Paper delivered at the Plttsburg Conference on Analytical Chemistry, 1983, March, Paper No. 076. (9) James, A. T.; Ravenhill, J. R.; Scott, R. P. W. Chem. Ind. 1984, May (2), 746. (10) Stouffer, J. E.; Kersten, T. E.; Krueger, P. M. Biochim. Biophys. Acta 1984, 93, 191. (11) Karmen, A. Anal. Chem. 1968, 38, 286. (12) Maggs, R. J. Chromatographia 1988, 1 , 43. (13) Coil, H.; Johnson, H. W., Jr.; Polger, A. G.; Selbert, E. E.; Stross, F. H. J . Chromatogr. Sci. 1989, 7, 30. (14) Scott, R. P. W.; Lawrence, J. G. J . Chromatogr. Sci. 1970, 8 , 65. (15) Scott, R. P. W.; Lawrence, J. G. J . Chromafogr. 1974, 99,395. (16) McFadden, W. H.; Schwartz, H. L.; Evans, S. J . Chromatogr. 1978, 122, 389. (17) Smith, R. D.; Johnson, A. L. Anal. Chem. 1981, 53,739. (18) Smith, R. D.; Burgen, J. E.; Johnson, A. L. Anal. Chem. 1981, 53, 1603. (19) Hayes, M. J.; Lankmayer, E. P.; Vouros, P.;Karger, B. L.; McGuire, J. M. Anal. Chem. 1983, 55, 1745-1752. (20) Kirby, D. P.; Vouros, P.; Karger, B. L.; Hldy, B.; Peterson, B. J . Chromatogr. 1981, 203, 139-152. (21) Games, D. E.; Hewlins, M. J.; Westwocd, S.A.; Morgan, P. J. J . Chromatogr. 1982, 250, 62-67. (22) Hardln,’E. D.; Fan, T.-P.; Vestal, M. L. Anal. Chem. 1984, 56, 2-7. (23) Vestal, M. L. I n t . J . Mass. Spectrom. Ion Phys. 1983, 4 6 , 193-197.
CONCLUSION The present work demonstrates that thermospray deposition overcomes some of the problems associated with earlier transport detectors for LC. In particular it provides sensitive detection for a wide variety of samples and can efficiently deal with an solvent (including water) at essentially any flow rate. Some loss in chromatographic fidelity is detectable, but it appears likely that this loss can be significantly reduced by redesigning the system involved in removing the sample from the belt and transporting it to the gas-phase detector. A more serious problem which has not yet been addressed involves the cleanliness of the moving belt itself. At present our detection limits are primarily set by background noise due to unwanted materials being transported on the belt along with the samples. These appear to be due primarily to relatively nonvolatile impurities in the solvents and tend to buildup with running time. Thus it appears that a practical device for general use will require the addition of an efficient device for cleaning the belt on the return or the use of an inexpensive single pass surface.
LITERATURE CITED (1) Johnson, E. L.; Stevenson, R. “Basic Liquid Chromatograph”; Varian Associates: Palo Alto, CA, 1978; p, 270. (2) Snyder, L. R.; Kirkland, J. J. Introduction to Modern Liquid Chromatography”, 2nd ed.; Wliey-Intersclence: New York, 1979; p 863.
RECEIVED for review April 16,1984. Accepted August 6,1984. This work was supported by National Institute of General Medical Sciences, NIH, under Grant GM 24031.
Interface of a Reverse-Phase High-Performance Liquid Chromatograph with a Diffuse Reflectance Fourier Transform Infrared Spectrometer Christine M. Conroy and Peter R. Griffiths*
Department of Chemistry, University of California, Riverside, California 92521 Pamela J. Duff’
Department of Chemistry, Ohio University, Athens, Ohio 45701 Leo V. Azarraga
Environmental Research Laboratory, U.S. Environmental Protection Agency, Athens, Georgia 30613
An approach to the Interface of a reverse-phase hlgh-performance llquid chromatograph and a Fourler transform lnfrared spectrometer has been developed In whlch the solutes eluting from the column are contlnuously extracted Into dlchloromethane. The applicatlon of both flow cell and solvent ellmlnatlon techniques was explored. Coextracted water and methanol Into the CH,CI, precluded the use of flow cells. I n the solvent ellmlnatlon Interface, the solutes are first extracted into dichloromethane and subsequently concentrated by dlfferentlal evaporatlon. The solutes are then deposited on gently heated KCI powder, and thelr dlffuse reflectance spectra are measured. The interface operates In a contlnuous fashion without the need for an auxlllary detector. Spectra can be measured at submlcrograrn sensitlvlty using thls Interface.
Present address: DRDAR BLI, Ballistics Research Laboratory, Aberdeen Proving Ground, D 21005.
hI/
Several techniques have been reported by which the Fourier transform infrared (FT-IR) spectra of species eluting from a high-performance liquid chromatograph (HPLC) could be measured. To date, the most popular HPLC/FT-IR interface has involved a simple flow cell through which the column effluent is continuously passed. Although this approach has been relatively successful for separations performed by sizeexclusion chromatography (SEC) (1-4), it has been notably unsuccessful for high-resolution separations by normal-phase (NP) or reverse-phase (RP) chromatography. Several reasons account for the poor performance of NP-HPLC/FT-IR and RP-HPLC/FT-IR interfaces in which a simple flow cell is used, the most important of which is related to the infrared absorption of the mobile phase. Many of the SEC/FT-IR results which have been reported have involved the use of simple chlorinated molecules as the mobile phase. These solvents are almost ideal for infrared spectrometry since their spectra exhibit appreciable “windows” at path lengths of 0.5 mm and even 1mm. Nevertheless, even
0003-2700/84/0356-2636$01.50/0 1984 American Chemical Society
