Anal. Chem. 1984, 56, 2681-2684 (13) Deming, S. N.; Turoff, M. L. Anal. Chem. 1078, 50, 546-548. (14) Price, W. P., Jr.; Edens, R.; Hendrlx, D.C.; Deming, S. N. Anal. Biochem 1979, 93,233-237. (15) Price, W. P., Jr.; Deming, S. N. Anal. Chim. Acta 1970, 708, 227-231. (18) Sachok, B.; Stranahan, T. J.; Deming, S. N. Anal. Chem. 1981, 53,
.
268 I
70-74. (17) Jenke, D. R.;Pagenkopf, G. K. Anal. Chem. 1984, 56,85-88.
RECEIVED for review July 5, 1984. Accepted September 4, 1984.
New System for Delivery of the Mobile Phase in Supercritical Fluid Chromatography Tyge Greibrokk,* Ann Lisbeth Blilie, Einar J. Johansen, and Elsa Lundanes
Department of Chemistry, University of Oslo, P.O.Box 1033, Blindern, 0315 Oslo 3, Norway
The delivery of pressurized mobile phases for supercritical fluid chromatography (SFC) has usually been achieved by pumping the fluids in the liquid phase either by syringe pumps or by membrane pumps. The only commercial SFC instrument available so far makes use of a diaphragm pump (1). For the formation of pressure gradients, pressure control rather than flow control has been recommended in SFC (2, 3). However, in systems with added modifiers and partiylarly with modifier gradients, flow control rather than pressure control could well be preferable for the same reasons as in HPLC. Since the retention in supercritical chromatography systems is 46t a linear function of the pressure, but directly related to the density and to the dielectric properties of the fluids ( 4 ) , the pumping system should contain both flow control and pressure control, in our opinion. With the present development toward microbore columns, the possibility of forming modifier gradients at low flow rates should also be considered. This would require a second pump with reproducible delivery of 10 pL/min and less. Today the majority of the HPLC pumps are of the standard reciprocating type (piston pumps), due to easy and rapid solvent shift, unlimited solvent delivery and good properties for gradient elution. Most modern piston pumps include both flow and pressure control, as well as microflow ability. Previous reports have contained claims that piston pumps are not suitable for SFC, either due to a lack of pressure control or due to a lack of pulse-free operation (2). The purpose of this investigation was to examine whether piston pumps can deliver supercritical fluids at reproducible flow rates and whether the pulses created by such a system are acceptable or not. Carbon dioxide was chosen as the supercritical fluid, since this seems to be the most commonly used fluid in SFC today.
turning the cylinder upside down) and a thermally insulated f in. stainless steel transfer line. A 2-pm filter was inserted in the line to prevent particles in the cylinder from entering the system. A Waters Associates Model 6000 A pump with square pump heads was modified in the following way: (1)Two additional check valves (SSI-02-0129Scientific Systems Inc., State College, PA) were connected both to the inlet and to the outlet. The valves were placed in a homemade block of stainless steel containing cooling channels (Figure 1). (2) Cooling channels were drilled in the solid outer part of both pump heads (Figure 2). (3) The pump heads and check valves were connected with thermally insulated tubing to a Julabo F 10 V refrigeration unit pumping methanol at -8 O C through the whole system (Figure 3). (4) The electronics module was removed from the pump housing in order not to risk any problems from condensed humidity. A purifier column (500 X 7 mm) filled with 28 mesh activated carbon (Alfa Products, Danvers, MA) was inserted prior to the injector (Figure 3) in order to improve the C 0 2 quality. The purifier column acted as an efficient pulse damper as well, in addition to a Orlita PDM 3.350 pulse damper (Orlita Dosiertechnik, 63 Giessen, FRG). A stop valve was inserted prior to the injector. Thus, the pump and the check valves could be held at the equilibrium pressure of the fluid during stops, column replacement, etc. Modifiers were added to C02 by using a microflow pump (Waters Model 590) and a T-piece of tubing or, better, by using a homemade high-pressure low-volume solvent mixer. Injectors. Samples were injected in standard micro HPLC injectors (Rheodyne 7410 or Valco C1 4W). The Rheodyne injector was used at room temperature, while the Valco injector was heated to temperatures above the critical temperature by a block heater. With a flame ionization detector, the samples were dissolved in dichloromethane or in carbon disulfide. Columns. The columns (CP-Spher C18,250 X 1.3 mm) were kept at 40-60 "Cin a gas chromatograph oven or with a separate column heater. All connections were made from thermally insulated 1/16 in. stainless steel tubing with 0.1 mm inner diameter. Detectors and Restrictors. Three detectors were utilized, two UV detectors (Perkin-ElmerLC-55 and Shimadzu-SPD-2AM) and one flame ionization detector (Hewlett-Packard5790 A). The UV detectors could be used at pressures up to approximately 200 bar without leaks or other problems. The restrictor following the UV detector was made by crimping the end of the 1/16 in. stainless steel tubing and keeping it at 80-100 "C in order to avoid blocking by solid COz. The restrictor in the FID was made by crimping the end of a piece of platinum tubing 0.4 mm 0.d. and 0.1 mm i.d. (Goodfellow Metals, Cambridge, UK). The restrictor was located 4-6 mm below the jet tip of the FID.
EXPERIMENTAL SECTION Delivery of Fluid. Liquid COz (standard grade) was transferred from the cylinder to the pump by an eductor tube (or by
RESULTS AND DISCUSSION COz Delivery. The actual delivery of COSwas measured as gas at atmospheric pressure by connecting a tube from the
A study has been undertaken to examine the propertles of reciprocal HPLC pumps In supercrltlcal fluld chromatography. After small modifications, Including cooled-down pump heads and extra check valves, a stable and reproduclble flow of supercritical COz was obtained. With an efflclent pulse damper and a UV detector the pumping contribution to the baseline noise was measured to be less than absorbance unlts at most wavelengths. The short-term reproduclblllty of retentton tlmes and peak helghts was measured to be 1-2% (coefficient of variation).
0003-2700/84/0356-2661$01.50/0
0 1984 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 56, NO. 14, DECEMBER 1984 COOLING BLOCK FOR T W O SSI CHECK-VALVES
coolant 200-
/
MATERIAL COOLING CHANNELS I N / O U T TUBING
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The check valve cooling system for the pumping of liquid LEFT PUMPHEAD [MIRROR IMAGE
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front view
COOLING CHANNELS
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The pump head cooling system for a piston pump delivering
Flgure 2.
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Components and connections of the supercriticai fluid chromatography system in use: (1) particle filter, 2 pm; (2) T-coupling and shut-off valve; (3) soft seat check valve; (4) pump head; (5) T coupling; (6) pulse dampener [Waters 6000 A]; (7)pressure transducer; ( 8 ) column, activated carbon; (9) shut-off valve; (10) solvent mixer; (11) pulse dampener [Orlita]; (12) injector; (13) analytical column: (14) UV detector; (15) restrictor: (16) gas flowmeter. Flgure 3.
outlet of the restrictor to a calibrated gas flowmeter. The nominal flow rate of liquid COz was set between 0.1 and 0.5
02
03
04 05 nominal flow (rnl/minl tiquld C02
Flgure 4. Relationship between the nominally set flow rates of liquid COP and the experimentally determined flow rates of gaseous CO,.
mL/min, with 0.1-mL intervals, using a restrictor which created a pressure of approximately 150 bar at 0.2 mL/min. The actual gas flow is shown in Figure 4, together with the reproducibility measured as the coefficient of variation based on 10 measurements. A linear relationship between the nominal and the actual flow was obtained in the area 0.2-0.5 mL/min. No leaks were ever detected at the piston seals, a fact which probably can be attributed to the efficient cooling of the pump heads. The extra set of check valves was needed in order to obtain a stable flow of COz. After the initial round of experiments it was observed that the soft seat check valves released small pieces of material from the O-ring within the valve, which eventually plugged the tubing. The damage to the O-rings seems to be caused by large pressure variations. At high pressure the rings absorb COz, which causes the rings to swell when the absorbed COz is released. Combined with mechanical stress this will eventually break up the ring. The absorption and release of COz were demonstrated by placing the rings in a small autoclave, applying the equilibrium pressure of compressed COz, and then rapidly releasing the pressure. By immediate measurement of the size of the swollen rings, the absorption of C 0 2 in O-rings made from different polymers could be studied. In the check valve the O-ring is subjected to pressure variations between the equilibrium pressure of the fluid and the total pressure of the system. The major damage to the rings, however, was obtained with sudden pressure pulses between the system pressure and atmospheric pressure. Of the polymers examined none was well suited for the purpose, mainly due to high absorption of COz, but the original Kalrez (Du Pont) rings were by far found to give the best results. By gentle handling one complete set of Kalrez rings lasted almost 1000 h of run time, while others lasted less. Pulsing. Without the additional pulse dampening unit the pressure varied by approximately 7 bar in each pulse (measured directly on the pressure transducer of the pump), which resulted in an excessive noise level. With the Orlita pulse dampener and the carbon column, the pumping noise was
ANALYTICAL CHEMISTRY, VOL. 56, NO. 14, DECEMBER 1984
BASELINE NOISE ( m A U )
*251
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I1
0.15
2683
10.11
i
0 0
lo
0.10
2 00 250 WAVELENGTH (nm1
300
Figure 5. Base-line noise of the Shimadzu SPD-PAM detector, measured with N, in the flow cell (0)and then connected to the SFC system (0). The coefficient of variation of the noise at each wavelength was determined to 10-20%.
BA !LINE NO E ( m A U )
0
IO
20 tR ( m i n l
Figure 7. Separation of toluene (I), naphthalene (2), biphenyl (3), fluorene (4), phenanthrene (5), anthracene (6), fluoranthene (7), benchrysene (10,ll). zo[a]fluorene (8), pyrene (9), and triphenylene The flow of liquid CO, was increased from 0.2 to 0.4 mL/min over a 5-min period. The pressure on the pump was measured to 125-180 bar. Detector was a Shimadzu SPD-PAM at 254 nm. Cblumn temperature was 37 OC. Injector temperature was 32 OC.
+
0.8
0
0.6 0
0.4 0 O
0.i
.
0
0
0
8
0
O e 8 e 8
200 250 WAVELENGTH (nm 1
e 8
300
Flgure 6. Base-line noise of the LC-55 detector, measured with N, In the flow cell (0)and then connected to the SFC system (0).
reduced to approximately 7 x lod absorbance units, measured on the Shimadzu UV detector (Figure 5 ) . With the LC-55 detector the noise contribution from the pump did not make a measurable addition to the base line noise level, due to the higher inherent detector noise (Figure 6). A demonstration of the use of a UV detector is shown in Figure 7 . Another flow/pressure gradient, with the flame ionization detector, is shown in Figure 8. With the present FID a higher flow
5
0 1 8 12 tRi(rn1I-J Flgure 8. Separation of the Cz0,C, C, C,, CZB,C30rC3, and CJ4 alkanes (approximately 0.1 pg each), by a flow program of 0.2-0.4 mL/min of liquid CO,. Detector was a Hewlett-Packard FID modified with a platinum restrictor. Column temperature was 60 OC. Injector temperature was 50 OC.
than 0.4 mL/min of liquid COz, corresponding to about 140 mL/min of C 0 2 gas, extinguished the flame. Older flame ionization detectors, constructed for packed GC columns only, may allow higher flow rates of C 0 2 (5).
2884
Anal. Chem. 1984, 56, 2684-2686
Reproducibility. The short-term (10 injections) reproducibility of peak heights was measured (as the coefficient of variation) to 1.8% for 11 different polycyclic aromatic hydrocarbons. The same short-term reproducibility of retention times was measured to 1.3%. The short-term pressure variations with the pulse dampener were measured to f 5 psi. Since the long-term pressure variations are of the order of f100 psi, the long-term variation of retention times could be significantly higher than the short-term variation. Accordingly, the use of internal standards may become more important in SFC than in HPLC.
LITERATURE CITED (1) Gere, D. R., Application Note AN 800-2 (Hewlett-Packard Co., Avondale, PA), 1983. (2) Peaden, P. A,; Lee, M. L. J . Liq. Chomatogr. 1982, 5 , 179. (3) Van Lenten, F. J.; Rothman, L. D.Anal. Chem. 1976, 4 8 , 1430. (4) Schneider, G. M. I n "Extraction with Supercritical Gases"; Schneider, G. M., Stahl, E., Wilke, G., Eds.; Veriag Chemie: Basel, 1980. (5) Rawdon, M. G. Anal. Chem. 1984, 56, 831.
RECEIVED for review July 23, 1984. Accepted September 11, 1984.
Quantitative Gas Chromatography without Analyte Identification by Ultrasonic Detection Kristen J. Skogerboe and Edward S. Yeung*
Department of Chemistry and Ames Laboratory, Iowa State University, Ames, Iowa 50011
The response of the ultrasonlc gas chromatographlc detector is used to determine the absolute welght of an unknown analyte without a callbratlon curve. The method does not require that any physical propertles of the analyte be known. The procedure for determlnlng the welght concentratlon of an unknown requlres that Its response be measured in two carrier gases wlth dlfferent molecular weights but equal molar heat capacltles, such as He and Kr. A detectablllty of 1 pg of hexane was found using He as the carrier gas.
The velocity of sound in a gas has for 70 years been a basis for gas analysis (1). The idea has been applied to gas chromatographic (GC) detection ( 2 , 3 )leading to the commercial availability of an ultrasonic detector (4). The ultrasonic detector is a universal detector which measures changes in the speed of a sound wave traversing the effluent of a GC column by comparing the phase shift of the wave to a reference signal (5). So far, the ultrasonic detector has seen limited laboratory use, despite a demonstrated dynamic range of 6 orders of magnitude (6). This is probably due to a reported detectability in the nanogram range (7), which is substantially inferior to those provided by most other GC detectors. It is generally assumed that quantitative analysis must be preceded by identification of the unknown and subsequent preparation of a calibration curve from standards. We have recently shown that this approach is unnecessary (8). The new quantitative concept has been demonstrated in liquid chromatography (LC) using refractive index (9),absorbance (IO),conductivity ( I I ) , and indirect polarimetry (12) for detection. The extension of this idea to the gas phase is important, since roughly 50% of all current separations are performed using GC. In GC, even though retention times may change, the elution order of analytes does not change when different carrier gases are used, as can happen in LC upon changing the mobile phase. Therefore, correlation, or assignment of peaks, in a sample which contains two or more unidentified peaks when the carrier gas is changed is not a problem.
Quantitative GC without analyte identification has been reported with a mass detector (13), which is essentially a microbalance that monitors the gain in weight due to adsorption of the analyte onto activated charcoal. Detectability is poor and is in the l-Mg range. The gas density detector for GC (14) has the potential of providing absolute quantitation (15). Normally, when quantitation is performed, a response factor is obtained from the known molecular weights of the analyte and the carrier gas (16-18). In separate studies, such a detector has been adapted for determining molecular weights of analytes (19-21). Thus, absolute quantitation should be possible by combining these two approaches. However, no experimental verification of this capability for absolute quantitation has been reported. Furthermore, the gas density balance has poor detectability, slow response times, and occasionally nonlinear response (22),all of which limit its usefulness. In this paper, we shall examine the properties and response functions of an ultrasonic detector for GC. We shall show that good detectability is achievable and that quantitation can be performed without analyte identification.
THEORY The response of an ultrasonic detector is well-known (2). For a GC peak containing the carrier gas, c, and the analyte, x, the measured phase change, A+, is predicted by
s is the distance between the transducers, f is the frequency of the sound wave, n is the mole fraction, M is the molecular
weight, C, is the molar heat capacity at constant pressure, and y is the heat capacity ratio (constant pressure/constant volume). To arrive at eq 1,one assumes that both gases are ideal, the gas mixture is homogeneous, and the mole fraction n is small. These conditions are always satisifed in GC. If one integrates the response in eq 1over the entire GC peak, one obtains a peak areea, Sc, for the analyte in this carrier gas, such that
0003-2700/84/0356-2684$01.50/00 1984 American Chemical Society
