Milos Novotny Department of Chemistry Indiana University Bloomington, Ind. 47405
In 1981 an article in ANALYTICAL CHEMISTRYdescribed a relatively new direction in modern liquid chromatography (LC) (I). Since that time, microcolumn LC, which was initiated during the late 1970s, has undergone extensive development. Whereas the main purpose of the 1981 article was to acquaint the analytical chemistry community with some unique features of miniaturized LC systems and their potential for various separations and measurements, this article provides a summary of the recent progress in microcolumn LC and, once again, points out new directions. Owing to the small number of laboratories engaged in microcolumn LC prior to 1981, the earlier article was comprehensive of the various developments of that time. The present article necessarily excludes this luxury, hecause the number of papers and presentations in this area has mown suhstantially. Microcolumn LC is now about a decade old. The initial developments toward LC miniaturization in the United States (2-5) and Japan (6-8) were quickly followed by a number of lahoratories worldwide. Although the varI
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ious groups pioneering this research might have had distinctly different objectives in mind, the most important advantages of using miniaturized columns became generally appreciated in a relatively short period of time. Most of these advantages remain attractive today: higher column efficiencies, improved detection performance, various benefits of drastically reduced flow rates, and the ability to work with smaller samples. Priorities have changed over the years as different applications have varied the emphasis of these unique capabilities of the miniaturized systems. Miniaturization is a general trend common to science and technology. Indeed, many developments in modern analytical chemistry have required miniaturization of instrumental components (in electrochemistry, spectroscopy, and separation science alike). Consequently, our ability to work with small dimensions (micrometers) and volumes (nanoliters and below) has improved dramatically in the past several years. Besides the obvious benefits in the area of column fabrication, miniaturization has made it feasible to exploit certain laser and microelectrode technologies for the sake of improved detection. A scientific dialogue among the analytical disciplines has been mutually rewarding. Microcolumn LC recently has had a growing number of enthusiastic follow-
ANALYTICAL CHEMISTRY, VOL. 60, NO. 8, APRIL 15. 1988
ers in spite of slow progress in the necessary instrumentation and commercialization. During 1984-85, three monographs on the subject appeared ( S l l ) in , addition to numerous review articles. The correct role of microcolumn LC is not to replace conventional (4.6-mm id.) columns in modern LC, a fact that analytical chemists increasingly appreciate. However, microcolumn LC has certain strengths in a growing number of applications in which conventional LC cannot effectively compete. Microcolumn LC is viewed as one of the several miniaturized separation techniques that have recently been under intensive development. The others are capillary gas chromatography (GC), capillary supercritical fluid chromatography (SFC), and capillary electrophoresis. It can be argued that the invention of the capillary column for GC some 30 years ago was the first dramatic miniaturization attempt in the field of separation science (12,13).It is now emphasized that miniaturized separation columns, whether used in various forms of chromatography or in electrophoresis, share similar technologies and instrumental requirements. This emphasis has been of benefit to advances in all of these microcolumn seDaration techniques. The cloud of controversy that is perhaps typical for a new development no longer surrounds microcolumn 1.C as it 0003-270018810360-50OAl5Ot 5010
@ 1988 Amer can Chemical Society
did in 1981, when the first feature article was published ( 1 ) . Although still primarily a research tool, microcolumn LC is more widely accepted in various laboratories and for various sample types. It is one of the most active areas in analytical separation science, as evidenced by the steady increase in publications and presentations a t both major chromatographic symposia and specialized meetings. This REPORT begins with a review of the recent advances in microcolumn technology, followed by a description of the state-of-the-art miniaturized instrumentation. New detection opportunities are particularly emphasized. Finally, some unique applications of microcolumn LC that are caused primarily by column advances and new detector types are demonstrated.
Cdurnn developments For clarification, we will often refer to a classification of microcolumns of three different types: open tubular columns, partially packed capillary columns, and tightly packed capillary columns (Figure 1).Although this general division was also used in the earlier article
( 1 ) and in other reviews (14-16), the column dimensions have changed radically since then. Some confusion in terminology still exists; this was addressed in a recent editorial (17). Throughout this article, the terms “microcolumn” he., a separation column with a typical i.d. of a small fraction of a millimeter) and “capillary” may he interchanged. The latter term is not necessarily restricted to the open tubular geometry. The term “microbore,” an obvious misnomer, unfortunately has been associated by a number of workers with the 1-mm i.d. dimension. Columns of this diameter and larger will not be discussed here. In assessing the separation potential of various microcolumns,one must apply appropriate criteria for column performance. Whether the characteristic dimension of a microcolumn is the particle size (packed columns) or the column radius (open tubular columns), under optimum linear velocity of the mobile phase, the plate height, H,is roughly equal to twice that characteristic dimension. To evaluate the column’s separation potential based on ita H-value is misleading if the column
I length, L, and the analysis time, t , are not considered. A large number of theoretical plates, N = LIH, is often achieved in LC at the expense of long analysis time. Comparison of different microcolumns under dissimilar conditions of chromatographic analysis is quite adequately obtained using the socalled separation impedance, E, introduced by Bristow and Knox (18).
where Ap is the pressure gradient required for the separation, 7 is the solvent viscosity, and k is the capacity factor. It is shown that the separation impedance is proportional to the square of the plate height (H)divided by the column permeahility (KO), or, alternatively, is equal to the square of the reduced plate height (h = H / d ) multiplied by a column resistance factor, 6 = d2/K0,where d is the particle diameter
Open tubul capillary i.d. 3-50
p
I‘ Stationary phase, a liquid or finel!’ dispersed solic
Partiles of adsorbent may be chemically
Particles of adsorbent or a support with 2onded phase (3-20 urn)
modified 15-30 pm)
~~~~~~
I
~~
~
Figure 1. Types of LC microcolumns (dimensions reported are those currently used). ANALYTICAL CHEMISTRY, VOL. 60,
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or the i.d. of an open tubular column. Table I gives estimates of such values for different types of microcolumns (19). The obvious mass transfer considerations for the diffusion-controlled sorption-desorption kinetic phenomena in chromatographic columns have long centered around particle size as the characteristic dimension of a packed column in LC. The remarkable success of modern LC is, in fact, directly attributable to “miniaturization” of packing particles. Considerably less attention was paid initially to the potential benefits of decreasing the column diameter. In fact, using widely accepted packing procedures, small column diameters (and greater column lengths) generally yield inferior results; hence the extensive use of 0.25 m X 4.6 mm i.d. columns in the practice of modern LC. This is a t least a partial explanation of why the pioneering studies by Scott and Kucera (2,3),employing 1-m segment columns with 1-mm i.d., initially were met with considerable indifference. Because a decrease of column i.d. is continuously emphasized for a variety of reasons, tightly packed capillary columns based on the fused-silica tuhing material and various LC packings are increasingly popular. For reasons that are not yet fully understood, the column tubing material appears to play a considerable role in the efficiency of a slurry-packing process, and the fusedsilica columns originally developed for capillary GC currently provide the most desirable results (20).These slurry-packed capillary columns, introduced by research groups in the United States and Japan (21-24), were not covered in the 1981 article (I),yet they may well be a key development of the microcolumn LC field. The technology of tightly packed capillaries has improved substantially over the past several years. Typical column diameters are between 200 and 300 pm, although columns as small as 44-pm i.d. were recently packed and resulted in excellent performance (25). Such columns can now be slurrypacked to a length of one meter or even longer, yielding total numbers of theo502A
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retical plates well over 100,000. An example of such high performance is showninFigure 2, which represent sthe separation of sulfur-containingpolycyclics isolated from a fossil fuel material (26);the plate number measured on a column two meters long was more than 200,000 theoretical plates. The plate height values of such columns, packed with 5-pm particles and used in the reversed-ohase mode..aDDroach _. the theoretical. Extremelv. high - reDroducihilitv of the column parameteis for reveisedphase columns has been demonstrated (22,25,27).A surprising recent finding is that the reduced plate height of slurry-packed capillaries decreases as a function of the column radius (25).Hvalues below 2 have been achieved (25, 28) with 50-pm columns packed with 5pm particles. The highest plate number thus far measured (1.95 m X 44 pm i.d., 5-um C,a Darticles) has been 226,000 in
33 min (25)! Reproducible preparation of slurrypacked capillary columns is demonstrated in Figure 3, where five successively prepared columns have been evaluated at different mobile-phase velocities (28). Although most successful studies have thus far been conducted with reversed-phase columns, a recent communication from this laboratory has also demonstrated respectable efficiencies for 5-pm silica adsorbent and other polar materials (diol-, cyano-, and amino-bonded phases) (29). The study demonstrated that considerably different packing procedures may be needed for packing materials of diverse chemical natures. To date, attempts to prepare efficient columns with particles smaller than 5 pm have not been very successful. Partially packed (semipermeable) capillary columns (Figure 1) are prepared by using a special column technology. A narrow-bore, thick-walled glass tube is first tightly packed with a suitable sorption material. Subsequently, a glass-drawing machine is, used to draw a length of the packed tube into a capillary of suitable diameter (4). Individual particles are drawn inside this capillary, and a number of them actually become imbedded in the column wall a t the melting point of the glass. In the original work on these columns by Tsuda and Novotny (4), a high ratio of column diameter to particle size was employed. Kinetic evaluation of such columns indicated that the column performance was a sensitive func-
Flgure 2. High-resolutii omtograph , large cllc aromatic compounds emacted from solvent-refined CWI. (Adapted with prmigsim horn Reterence 26.)
ANALYTICAL CHEMISTRY, VOL. 60, NO. 8, APRIL 15, 1988
containing polycy-
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