Focus
Advances in Chromatography 1982 The 17th International Symposium on Advances in Chromatography was held in Las Vegas, Nev., April 5-8, the week after this spring's ACS national meeting in the same town. The symposium featured a series of well-attended and lively sessions on topics such as capillary column gas chromatography, gas chroma tography for environmental analysis, high-performance thin-layer chromatog raphy, and new developments in liquid chromatography. The feature of this year's symposium was a series of sessions on gas chromatography-mass spectrometry in honor of E. C. Horning, who was in attendance. The symposium chairman was A. Zlatkis. Field flow fractionation and high-performance thin-layer chromatography, two of the topics under consideration at this year's symposium, are described in the following pages.
Field Flow Fractionation: Steric Transition Field flow fractionation (FFF) "will eventually prove applicable to almost any complex mixture of soluble or suspendable molecules, macromolecules, or particles," wrote J. Calvin Giddings in 1981 (Anal. Chem. 1981,53, 1170-78 A). Though FFF is not strict ly speaking a chromatographic tech nique (there is no stationary phase in FFF), interest in the technique has grown in recent years as chromatographers and others who use chromatog raphy in their research have become more aware of its applicability to an exceptionally broad range of samples. FFF is not just a separation tech nique. In addition to separating com ponents, FFF can be used to deter mine effective molecular weights, par ticle densities, and particle diameters. In addition, FFF is a nondestructive technique, so collected samples can be analyzed by other methods, such as spectroscopy, after elution. Recently the technique has been applied to samples as diverse as river water, fly ash, subcellular particles, and even to viruses and live cells. Because it can handle samples over an unprecedent 0003-2700/82/0351-787 A$01.00/0 © 1982 American Chemical Society
ed range of effective molecular weights (approximately 103 to 10 18 ), FFF is attracting particular attention among those interested in biological applications. Giddings, who originally developed the FFF technique in the mid-1960s, reviewed FFF theory in a presentation at the symposium coauthored by M. N. Myers, F.-S. Yang, J.-P. Chang, and K. D. Caldwell. There are two separation modes in FFF, normal and steric. In both modes, the separation is carried out in a rectangular channel
with a narrow dimension (50-500 μηι deep). A force field of some type (it might be an electrical field, centrifugal force, a thermal gradient, or some other field) is applied to the channel as the sample solution or suspension flows through. The field will tend to push all the sample particles toward one wall of the channel. In normal FFF, the particles form a Brownian motion cloud that extends a short distance into the channel (Fig ure 1). Separation in FFF is possible because the solvent flows at different
Figure 1 . Band of sample particles forms a Brownian motion cloud close to the wall of the channel when the field is imposed ANALYTICAL CHEMISTRY, VOL. 54, NO. 7, JUNE 1982 · 787 A
Focus
For most field strengths, steric FFF begins to take «ffect when particle di ameters exceed 1 μτη. Beyond this point, particles are generally large enough and heavy enough that normal gravitation alone is sufficient to push all the particles down to the wall. In addition, Brownian displacement de creases as the particles increase in size. However, because these particles are so large, they tend to protrude toward the center of the channel be cause of their very size, and despite the fact that they are in contact with the wall. So in the steric FFF region (all particles larger than about 1 μπι), there is an inversion of elution order. Unlike normal FFF, the large particles tend to move more quickly than the smaller particles in steric FFF, and the separation mechanism in the steric mode is thus based on inherent parti cle size rather than on the ability of a particle to undergo Brownian diffu sion (Figure 3). "The concept of steric FFF looks simple," explained Giddings, "but is complicated by a number of phenome na that we're still investigating. With steric FFF we can separate red blood cells out of whole blood, and we have analyzed ground coal for use in coal liquefaction, where the particle size
region, you can decrease the field and get it to elute in the normal region, within limits of course," explained Marcus Myers, Giddings's colleague. According to Karin Caldwell, anoth er colleague, "What we'd like to do is set our experimental conditions so that we work in a clean domain, either totally outside the influence of steric effects or totally in the steric domain." If you suspect that the particle size range of your sample spans the steric transition, a quick run at two different field strengths will reveal whether or not foldover is taking place. "Very sel dom do you have particles that range from, say, 0.1 μτη up to 15 Aim," said Caldwell. "Your sizes are by nature just not that broad." But in addition to providing prob lems, the steric transition provides op portunities to extract more informa tion out of the FFF experiment. Parti cle density and shape information for polydisperse samples are available in the steric transition region. According to Caldwell, "We are trying to refine our understanding of the steric effect to the point where we can interpret what's happening in the foldover re gion, and because we feel there is so much information potentially avail able there." Although nobody seems to be nam ing names, the word is that commer cial instrumentation for FFF may be appearing soon. These instruments should make it possible for a wide spectrum of scientists to have access to this versatile technique.
Normal FFF
Steric FFF
• · • · · Flow
•
Flow
· Signal
velocities at different points within the channel. The velocity profile of the solution in the direction of flow is shaped like a parabola, with short ve locity vectors near the walls and the longest vectors in the center of the channel (Figure 2). The flow rate, in fact, approaches zero near the walls, including the wall to which the parti cles have been forced. Thus, explained Giddings, if the particles in a sample are relatively small, the Brownian cloud they form will be more diffuse, and they will be carried along more rapidly, since the solvent velocity is higher away from the wall. Larger par ticles, which tend to interact more strongly with the field, will hug the wall more tightly, will not elute as quickly, and are thus separated from smaller particles (Figure 3).
Signal
Figure 2. Parabolic flow profile of chan nel velocity vectors in FFF
distribution is an important parame ter." Giddings's group has worked with particles as large as 100 μια in di ameter, corresponding to an effective molecular weight of 10 18 . As mentioned above, FFF can han dle particles differing in effective mo lecular weight by 15 orders of magni tude, with norrnal FFF most useful for the smaller particles and steric FFF handling the particles larger than about 1 μτη. The only potential prob lem is that there is a kink in that range, involving the transition from normal to steric mode. Whereas the small particles elute more quickly in normal FFF, it is the large particles that elute first in steric FFF. Thus, there is an inversion of elution order between the two modes. If you have a complex sample that spans the steric and normal regions, then the fractogram may contain an overlap region where the elution curve folds back and particles of different size elute at the same time. Theoreti cally, one should never see particles, at least of a certain density, beyond a certain elution volume when foldover occurs. This maximum elution volume is the point at which the elution curve folds back upon itself. The foldover phenomenon is a dis advantage when working with a broad-range sample that happens to span the foldover point. However, a fractogram with an overlap region can often be unfolded by changing the field strength. "If a particle is so big that it's starting to elute in the steric
Time
JX
—
Time
Figure 3. Reversal of elution order in the transition between normal and steric FFF
788 A · ANALYTICAL CHEMISTRY, VOL. 54, NO. 7, JUNE 1982
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HPTLC: Taking Off Thin-layer chromatography (TLC) has never been one of the most glam orous chromatographic techniques. But in the past few years improve ments have been realized in TLC sep aration efficiency, detection limits, and sample throughput. These im provements have led to a wider accep tance of TLC as a powerful separating tool and to a new name for the tech nique: high-performance thin-layer chromatography (HPTLC). According to Colin F. Poole of Wayne State Uni versity, "The performance break through in TLC was not a result of any specific advance in instrumenta tion or materials, but was rather a cul mination of improvements in practi cally all of the operations of which TLC is comprised." A good general reference on HPTLC is the article by David C. Fenimore and Chester M. Davis that appeared in ANALYTICAL C H E M I S T R Y last year (1981,53, 252-66 A). In conventional TLC, a thin layer of stationary phase (commonly silica gel) is coated onto a rigid backing plate. The sample is spotted onto the edge of the plate. This same edge of the plate is then placed in contact with the mo bile phase, which moves up the plate by capillary action, developing the chromatogram as it proceeds. Each sample component migrates differen tially along the plate according to its relative attraction for the mobile and stationary phases. High-Performance vs. Conven tional TLC. HPTLC differs from con
Table 1. TLC 1
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1
ventional TLC primarily as a result of improvements in spotting techniques, plate technology, and detection. Poole compared the two modes of TLC in the paper he presented at the sympo sium, coauthored by Sheila A. Schuette (see Table I). The sample volume in HPTLC is reduced from the conventional case, the starting spot diameter is smaller, and the developed spots are therefore also smaller in size. The decreased sample volumes and spot diameters in HPTLC have'been made possible by improvements in spotting methods. With some of the ingenious spotting devices now commercially available, it is possible to spot many more samples per plate than was formerly possible. HPTLC, according to Poole, should be capable of nearly an order of mag nitude improvement in number of the oretical plates compared to the con ventional technique. This increased efficiency results in some measure from the smaller particle size (and narrower particle size distribution) sil ica gel used in the manufacture of HPTLC plates. Detection limits are about an order of magnitude better, and sample throughput is higher. HPTLC vs. HPLC. In his sympo sium address on therapeutic drug monitoring, Arthur Karmen described one reason why scientists are becom ing increasingly interested in HPTLC. (Karmen was also one of this year's Tswett Chromatography Medal awardees; see Anal. Chem. 1982,54, 584 A). Karmen, who is chairman of
Comparison of Conventional and High- Performance Parameter
Conventional TLC
Sample volume Starting spot diameter Diameter of separated spots Solvent migration distance Separation time Plate height Effective theoretical plates Separation number Particle size, average Particle size, distribution Adsorbent layer thickness Detection limits, absorption Detection limits, fluorescence Sample tracks per plate
1-5 μ\3 - 6 mm 6-15 mm 10-15 c m 3 0 - 2 0 0 min 30 μνη
