Instrumentation
J o h n C. F j e l d s t e c Milton L. L e e Department of Chemistry Brigham Young Universtty Provo. Utah 84602
The ever-increasing demands on chromatographic separations stimulate the continual investigation of new techniques and new approaches to old techniques. One area that is receiving increased attention today is the use of supercritical mobile phases. These fluids offer intermediate viscosities and solute diffusivities between those of liquids and gases. Hence, higher mobile phase linear velocities (faster analysis times) and higher separation efficiencies per unit time are possible when compared to high-performance liquid chromatography (HPLC). In addition, the greater densities of supercritical fluids compared to gases lead to solute solubility and mobile phase selectivity as important parameters in the separation process. These parameters are also characteristic of HPLC, but they are largely nonexistent in gas chromatography (GC). With presently available technology, both packed and capillary column supercritical fluid chromatography (SFC) techniques are possible and of interest. Although greater sample capacities are obtained with packed columns in all forms of chromatography, SFC with packed columns suffers from the same limitation fundamental to the use of packed columns in either GC or HPLC-the pressure drop across the column limits the length and hence the total chromatographic 0003-2700/84/0351-619A$O1.50/0
0 1984 American Chemical Society
efficiency (total number of theoretical plates) obtainable. Furthermore, since retention is a function of the pressure gradient along the'column length, pressure drops are expected to influence the reproducibility of retention measurements. Although packed-column SFC can be used to chromatograph thermally labile compounds and compounds beyond the volatility range of GC, its main advantage over HPLC is reduced analysis time (increased resolution per unit time). In comparison, the permeability of a capillary column is much higher than a packed bed, and long column lengths can be used. This leads t o significantly higher total separation efficiencies. Therefore, the use of capillary columns in SFC gives the added advantage of high efficiency, and the technique is particularly useful for the analysis of complex mixtures or for the separation of related compounds that cannot he separated on the basis of mobile phase selectivity alone. Mobile phase composition and temperature are the main parameters controlling solute retention in HPLC and GC, respectively. These characterktics give rise to gradient elution in HPLC and temperature programming in GC. Although both of these programming modes can be applied in SFC, a third powerful programming alternative is density (pressure) pro-
gramming. Density programming is most effective at pressures near the iritical point where the change in density with pressure is greatest. Unfortunately, density programming through this region leads to resolution losses if there is a pressure drop across the column ( I ) , the significance of which depends on the magnitude of the pressure drop and the degree of resolution ioss that can be tolerated. This is usually not a limiting factor in capillary SFC, and rhe full density range can be used. Since the first report of the use of capillary SFC only three years ago (Z), tremendous progress has been made in instrumentation and column wchnology. Easy-to-operate systems can he readily constructed from commercially available components. Conventional GC and HPLC detectors, as well as mass spectrometry, have been successfully used. In this article, both the instrument design requirements and the contemporary instrumentation for capillary SFC will he described. instrument Design Requirements Recent theoretical studies ( I ) have indicated that small-diameter capillary columns of less than 100-pm i.d. are necessary for use in SFC to obtain high efficiencies in reasonable analysis times. For instance, using supercritical n-pentane as mobile phase, a
ANALYTICAL CHEMISTRY. VOL. 56. NO. 4, APRIL 1984
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Condlllons. Flame o n zation detecta (F DI. GC chromalogram. 2 M X 300-pmi d lusedaidca colmn:SE-54 stationa,y phase ( 0 . 2 5 . p ~lilm hickness). temperawe program hom 40 "C to 265 OC at 4 OC mm-' anar an inilial Cmin isothermal period: hydrogen carrler gas SFC chromatogram. 3Cm X 5 0 - p m ~ db . & s i ica Column: SE-54 stat onary pnase IO 25-prn 11 m th.Cknes: C02m0b.k pnasa at 40 'C; wnsiw program lrom 0 225 to 0.70 m--' at 0.005 g mL-' m n-' ansr an an lial 15-mi" isocon lertic (constant density) p e r i d
lished stationary phase coating procedures have been reported ( 4 ) ;elevated temperatures (5) and mixed solvents (6)greatly facilitate the coating process. Since the solvating power of supercritical fluids is often sufficient to cause migration of conventional stationary phases (7),cross-linking is essential to render these phases insoluble. The free-radical initiator, azo-tbutane, has been successfully used to cross-link these stationary phases inside the small-diameter capillaries ( 4 ) . The required use of small-diameter capillary columns places stringent demands on the mobile phase pumping system. Flow rates on the order of several WLmin-1 are required. In contrast to HPLC pumping systems, pressure control instead of flow control is needed, and pulseless operation is more critical. In addition, provision must he made for density (or pressure) programming of the mobile phase during the chromatographic run. The method of sample introduction into the chromatographic column is important in preserving the chromatographic efficiency. For 5Ogm-i.d. columns, injection volumes of less than 100 nL are typical (I).Studies have shown that injection at the existing mobile phase pressure is the most desirable ( 8 ) . The only requirement for the chromatographic oven is that the column temperature be accurately and precisely controlled. Even slight fluctuations in temperature can significantly alter the density of the supercritical fluid and hence the retention of solutes. The column temperature is normally held constant during the chromatographic run, although temperature programming may become a desirahle alternative in the future. A distinct advantage of capillary SFC is its compatibility with both conventional GC and HPLC detectors. However, optical detector cell volumes must he extremely small and able to withstand high pressures, and flamebased detectors must be modified for fluid decompression and expansion into the flame jet. Similar requirements apply to coupled systems such as SFC-Fourier transform infrared spectrometry (SFC-FT-IR) and SFC-mass spectrometry (SFC-MS).
34-m X 50-pm-i.d. column should produce 160 000 theoretical plates ( k = 5) in 2.3 h a t 10 times the optimum mobile phase linear velocity. In practice, a 27-m X 50-pm4.d. column coated with a cross-linked methylphenylpolysiloxane stationary phase demonstrated over 90 000 theoretical plates for coronene a t k = 7 (3).Similarly, using supercritical carbon diox-
Contemporary Instrumentation SFC has instrumental requirements in common with both GC and HPLC. For this reason it has been possible to take advantage of technological developments from both. The major requirements of the capillary column were borrowed from GC. HPLC tuhing and injectors were used for sample introduction. Methods of detection came from both GC and HPLC. One
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ide as mobile phase, 69 000 theoretical plates (k = 3.9) were generated using a 23-m X 50-pm-id. column (4). These efficiencies compare well with the performance of a typical 20-mm X 300pm-i.d. column in GC (Figure 1). The preparation of small-diameter columns for capillary SFC is not as straightforward as for regular-bore columns. Modifications of the estab-
ANALYTICAL CHEMISTRY, VOL. 56, NO. 4. APRIL 1984
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Injectors.Valves.Filters.Columns.Good advice. When you use HPLC, make the important connection, Rheodyne. Our components both sirnplity your work and extend the capabilities of your system. Just check our connections. 1.Sample injectors. The Model 7125 injects either a fully-loaded or partially-loaded sample loop. Other injectors include the pneumatically-actuated 7126, the 7410 for microsamples and the 7010 for full-loop loading only. 2. Column selection valve. Model 7066 selects one of up to five columns. 3. Switching valves. Type 70 Valves switch flow for backflushing. sample enrichment, sample cleanup, etc. 4. Solvent switches. Low-cost Teflon valves switch low-Dressure
streams. 5. Pressure relief valve. Model 7037 protects equipment from overpressure. 6. Column inlet filter. The 7302 keeps particles from damaging columns. 7. Columns. Guard. presaturator and analytical columns. 8. Fittings. As for good advice, you'll do better HPLC if you ask us for free copies of Technical Notes 1, 2, 3 and 4.They contain practical how-to-do-it information for both the beginner and the experienced chromatographer. For Tech Notes and product literature, please address Rheodyne, Inc., PO.Box 996, Cotati, California 94928, U.S.A. Phone (707) 664-9050
Spliner ITjector
Figure 2. Sch-
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:diagram of instrumentation for capillary SFC
development unique to SFC was modification of the HPLC pump to obtain mobile phase pressure control. A schematic diagram representing capillary SFC instrumentation is shown in Figure 2. Because of the low tlow rates (several p L min-') required for capillary SFC, pulseleas syringe pumps have been used. Typical HPLC syringe pumps are designed for flow control and require additional control electronics for pressure programming (9). The required modification involves comparing the pump pressure a$ sensed from an electronic pressure transducer and a reference voltage, supplied by a digital-to-analogconverter (DAC).A positive voltage signal results from this comparison when the signal from the DAC is greater than the signal from the transducer; this indicates that the pump is below the required pressure. This voltage is converted to a frequency which drives the pump, compressing the syringe reservoir and bringing the system up in pressure. Because the difference hetween the desired and measured pressure is translated into a frequency proportional to the difference, stable pressure control is achieved. The DAC plays an important role in pressure and density programming. By interfacing the DAC to a computer, it becomes possible to generate pressures under software control (10). Many different density programs are useful in capillary SFC. Among these are linear pressure, linear density, and asymptotic density ( 1 0 ) . Incorporation of an algorithm which converk a desired supercritical tluid density into a pressure makes density programming possible. One nlgorithm in. volves approximating the densitypressure relationship of a supercritical fluid with an nth-order polynomial. 622A
Densities can be determined from compressibility data for fluids near their critical points. Nonlinear density programming can also be generated by computer software and appears to be useful for the separation of bomologons series. In SFC, late-eluting homologues tend to converge in elution a t a certain density. Equal spacing between adjacent homologues is accomplished by programming asymptotically to this convergence density (10).
Various injector arrangements have been investigated. A high-pressure internal sample loop valve injector, such as those used in microbore HPLC, used in conjunction with a splitting arrangement, has been found to be most acceptable for introducing a small band of sample onto the bead of the chromatographic column (8).The inlet splitter was constructed from readily available chromatographic hardware and allows for a variety of chromatographic conditions. For programmed elution, split ratios from zero to over 100 can be used, depending on sample concentration and method of detection. When a universal detector is used (such m a n FID), a high split ratio is preferred to prevent excessive tailing of the solvent peak into the Chromatographicrun. In fluorescence detection, the solvent is most often undetected. allowine for low split ratios. At the outlet of the column, both flow restriction and detection are required. In the case of UV and fluorescence detection (2,8,11),restriction takes place after the capillary flow cell. When GC-type detectors are used (121, the restriction takes place such that the carrier expands to a gas and passes into the detector in the normal fashion. Two successful flow restrictors have been used small-bore fused-
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silica (5-10gm id.) and platinumiridium tubing. Fused-silica tubing, ranging from 2 to 20 cm in length, is useful for flow restriction in GC-type detectors (12). Mass spectrometry interfacing has primarily made use of platinum-iridium tubing with the end pinched to establish proper flow rates and ion source pressures ( 1 3 , 1 4 ) . Both UV-absorption and fluorescence detectors have been used in capillary SFC ( 2 , 8 , 1 1 ) .Due to the low dead-volume requirements imposed by the use of small-diameter (
