Milos Novotny Stephen R. Springston Chemistry Department Indiana University Bloomington, Ind. 47405
Paul A. Peaden John C. Fjeldsted Milton L. Lee
Report
Chemistry Department Brigham Young University Provo, Utah 84602
Capillary Supercritical Fluid Chromatography Rapid separations of respectable efficiency for nonvolatile solutes can be achieved by use of capillary columns in conjunction with supercritical fluids
Capillary columns were first de scribed for gas chromatography (GC) in 1957 by M.J.E. Golay, who utilized long, open tubular columns uniformly coated with a thin layer of stationary phase (1). Although many technologi cal improvements were necessary in the years following Golay's invention, the method has recently revolution ized many areas of scientific research. This is primarily due to its high re solving power, allowing the separation, detection, and quantitation of hun dreds of components from a sample in a single chromatogram. The practical separating capability of capillary gas chromatography is presently unparalleled. However, the method is somewhat restricted by the limited volatility and thermal stability of many organic compounds. Mixtures of less volatile compounds can be ana lyzed by high performance liquid chromatography (HPLC), but large numbers of theoretical plates can only be obtained at the expense of a long analysis time. Achievement of the maximum number of theoretical plates in capillary HPLC is presently complicated by the extremely small column diameters required for low plate heights. This requirement is a consequence of solute diffusivities and mobile phase viscosities. These are the two most important properties gov erning chromatographic efficiency (2, 3) and are primarily a function of the mobile phase and its physical proper ties. While encouraging results were recently obtained (4) with open microtubular columns as small as 30 μπι i.d., theoretical analysis by Knox and 0003-2700/81 /0351-407AS01.00/0 © 1981 American Chemical Society
Gilbert (5) indicates the need to work with columns of about 10 μπι and de tectors with volumes of a few nL to maximize efficiency, a formidable challenge at present. Clearly, other alternatives are needed for highly efficient separations of large, less volatile molecules. A system is needed that provides somewhat faster solute diffusion than in liquids (permitting larger, more "reasonable" column diameters) and mobile phase viscosities sufficiently low so as to per mit long columns to be exploited. Si multaneously, such a system should be suitable for the separation of large and thermally labile molecules. Qualita tively, these conditions are met by use of open capillary columns in conjunc tion with a supercritical fluid as the mobile phase. It is reasoned in this ar ticle and supported by preliminary data that, under certain experimental conditions, this combination can pro vide a nearly ideal chromatographic system for the above-mentioned mo lecular species. The pioneering work on supercriti cal fluid chromatography in packed columns by Klesper et al. (6) clearly demonstrated the potential of this ap proach in separating thermally labile substances. Later work of Giddings et al. (7-9) and Sie and Rijnders (10-13) with both dense gases and supercriti cal fluids extended this interesting di rection in separation science both the oretically and experimentally. How ever, these developments of the late 1960s were largely overshadowed by the advent of HPLC. Furthermore, technical difficulties with supercritical
fluids discouraged additional research. The most recent advances have been reviewed by Klesper (14). A very serious limitation of super critical fluid chromatography with packed columns is the pressure gradi ent generated by the column packing, as discussed by Novotny et al. (15). Ironically, while a decrease in particle size leads to much greater efficiency in liquid chromatography (where the mobile phase is practically noncompressible), the reverse is observed with supercritical fluids (15). This point was reemphasized by Gouw and Jentoft (16), who compared the effects of an increasing pressure gradient along the column in supercritical fluid chro matography to a decreasing tempera ture program in gas chromatography. Both have disastrous consequences as far as the column efficiency is con cerned. The advantages of capillary column "openness" are well known in GC and will become even more evi dent with supercritical fluid chroma tography, if the whole system (from the point of sampling to the point of detection) can be maintained under proper conditions of high pressure and low pressure drop. Supercritical fluid chromatography should provide superior migration of labile and less volatile substances through a capillary column when com pared to GC. Fluids compressed to their critical points and above exhibit an "extraction effect" on chromatographed samples similar to solvation in HPLC. The effect of "extraction" may be predicted based on the second virial coefficient of the system (70) or
ANALYTICAL CHEMISTRY, VOL. 53, NO. 3, MARCH 1981 · 407 A
Table 1.
Potentially Useful Mobile Phases
Mobile phase
Critical pressure
Critical temperature
Corresponding density
n-Pentane Dlchlorotetrafluoroethane Isopropanol Carbon dioxide Sulfur hexafluoride
33.3 atm 35.5 47.0 72.9 37.1
196.6 °C 146.7 253.3 31.3 45.6
0.232 g/mL 0.582 0.273 0.448 0.752
the Hildebrand solubility theory (9), although such predictions are first ap proximations at best. The point of practical interest is that while ordi nary gases can be compressed to pro duce these effects (9), similar condi tions can be met with some other fluids at pressures and temperatures well within the limits of ordinary HPLC technology. Some examples are shown in Table I. Attention will be fo cused on fluids with low critical tem peratures, since they will be useful for separating thermally labile com pounds. Optimum conditions for capillary supercritical fluid chromatography will now be briefly considered. If we introduce the initial restriction of a maximum allowable pressure drop, P m a x , between the inlet and outlet of a capillary column of radius r, the maxi mum column length, L m a x , is given by the integrated Poiseuille equation: (1) 8ημ where η is the mobile phase viscosity and μ is the average linear velocity of the fluid. The number of theoretical plates, N, for such a column is: N--
(2)
Η Since both L and Η (plate height) are functions of μ, the maximum efficien cy, iV max , can easily be found (3). However, the maximum number of plates applies here to conditions of in finite length and zero flow. If, instead, the column is operated under condi tions of optimum efficiency per unit length, more realistic efficiencies may be calculated. The minimum plate height, i i m ; n , can be derived from the Golay equa tion, assuming that resistance to mass transfer in the stationary phase is neg ligible: Hmin = 0.577 Τ
(3)
This condition occurs at:
- _M
—
_
Mopt —
D
48
(4)
where Dm is the diffusion coefficient of a solute in the mobile phase. Sub-
stituting equations (1), (3), and (4) into equation (2) yields the maximum number of plates that can be generated from a column of fixed length: Nn
32TJD„
(5)
This equation is similar to that de rived earlier by Giddings (3) for the limiting number of plates used to compare the relative merits of gas and liquid chromatography. The differ ence is that equation (5) assumes μ = μ ορί , whereas Giddings's expression holds only for L =
