Analytical chemists are constantly seeking new tools which permit them to s a v e the increasingly complex problems with which they are confronted. Sometimes a revitalized form of an old tool appears which offers new opportunities. Liquid chromatography (IC) now fits this category. New forms of IC are now emerging which promise that this technique will be one of the most powerful and widely used analytical approaches. 1 1
Columns for Modern Analytical liquid Chroma-
Modern Lc Vs. Traditional Lc
The phrase “modern liquid chromatography” may need some explanation. I n the newer forms of IC, the basic interactions within the column (mostly chemical) are still those which have been used for many decades. However, the speed of the separation processes, mostly physical in nature, have been greatly increased. Separations by IC now can be carried out 100-1000 times faster than by traditional column IC approaches, and 10-100 times faster than by thin-layer chromatography. This increase in speed is accomplished with columns that may be reused many times before they must be discarded. With no attention from the operator, detection and measurement of separated components is continuously accomplished by a device which monitors the column effluent and produces a chromatogram directly. While liquid flow in traditional IC is achieved by gravity or low-inlet pressures, modern IC makes use of high-pressure pumps, with operating pressures in the 5005000-psi range. I n fact, the im36A
0
ANALYTICAL CHEMISTRY, VOL. 43, NO. 12, OCTOBER 1971
proved performance of modern IC is the result of being able to use these higher column inlet pressures with long, narrow-bore columns containing small particles of packing. These columns are normally operated a t relatively high carrier velocities; thus, the elution of complex samples in relatively short times is possible. This approach is in sharp contrast to traditional IC, in which columns of relatively large diameters are packed with larger particles and operated with gravity flow or low pressure and a t very low linear carrier velocities. The significant advantage of modern IC is improved column efficiency, resulting in the separation and analysis of more complex and difficultly separable mixtures. I n addition, many more analyses can be performed in a given time period. Our understanding of column dynamics in high-speed IC now provides a basis for optimizing the technique for its practical use in a wide variety of applications. Lc Vs. Gc
Only about 20% of the known compounds lend themselves to analysis by gas chromatography (gc) owing to insufficient volatility or thermal instability. However, IC does not have this limitation and is ideally suited for the separation of nonvolatile or unstable materials. Ionic compounds (amino acids, nucleotides), labile, naturally-occurring compounds, polymers, and high molecular weight, polyfunctional compounds are conveniently analyzed by IC but cannot be handled by gc. But most importantly, the
REPORT FOR ANALYTICAL CHEMISTS
J. J. KIRKLAND Experimenta I Station Industrial and Biochemicals Dept. E. I. du Pont de Nemours & Co., Inc. Wilrnington, Del. 19898
Modern liquid chromatography makes use larger variety of moving and stationary phases available in IC permits a much wider range in selectivity. While gc is, a t present, a faster, more convenient technique which should be used for the analysis of appropriate samples, IC is an important alternative. Often a mixture can be more easily separated by IC, even though it can be gas chromatographed satisfactorily. Another important advantage of IC is the virtual lack of effects originating from the support material. I n gc, such effects sometimes lead to incomplete recoveries and poorer separations. Since the column is the heart of the separating system, much effort has recently been expended in optimizing column design and operation. This paper will summarize the present state-of-the-art of columns designed for high-speed, highperformance IC. Particular attention will be given to the specialized commercial packing materials which have recently been developed for this technique. Only columns for liquid-partition, liquid-solid (adsorption), and ion-exchange chromatography will be discussed, since exclusion chromatography generally involves column packings requiring a somewhat different technology. Column Design
The tubing used to construct high-performance IC columns may be heavy wall glass or the more commonly used precision-bore stainless steel tubing. It appears that the smooth internal walls of
of high-pressure pumps with operating pressures usually in the 500-5000-psi range. These higher column inlet pressures combined with long, narrow-bore columns containing small particles have produced high-speed, high-performance liquid Chromatography these materials are important in producing columns of high efficiency ( I ) . Straight columns are generally preferred, and lengths of 50 or 100 cm are convenient. Columns filled with packings and then coiled have shown a significant decrease in efficiency (1, 2 ) . T o obtain a large number of theoretical plates, one may connect individual straight columns in series using low deadvolume fittings to produce longer columns with little loss in total theoretical efficiency ( 1 ) . Columns are usually operated in a vertical position with the flow being directed either up or down to the packing. However, for convenience, columns can be operated in a horizontal position, provided the column has been packed in an optimum manner to prevent channeling due to the settling of the packing while in this position. Columns should also be connected to the detector with low dead-volume fittings designed to eliminate dead pockets of carrier.
Circulating air baths or water jackets are used to control the temperature of the column, preferably to less than t 0 . 2 " C . Temperature control is especially important in liquid-liquid partition and ion-exchange chromatography, which are more influenced by this parameter than adsorption or exclusion chromatography. I n addition to other variables, column efficiency is dependent on the particle size of the packing material and the internal diameter of the column ( 3 ) . For analytical studies, columns with a 2-3 mm i.d. provide a good compromise between sample capacity, the amount of packing used, amount of solvent required, and column efficiency. While columns of < 2 mm i.d. are generally found to be less efficient ( 4 ) , highly efficient columns up to about 11 mm i.d. have been demonstrated in special circumstances (5, 6 ) . Highest column efficiency results when solute molecules never reach the disturbed wall area of the packing within the column, a condition called the infinite-diameter phenomenon ( 3 , 6 ) . The shape and particle size of the packing in a column has a major effect on column efficiency. Less dense, irregularly shaped supports, such as the diatomaceous earths and silica gel, are difficult to pack homogeneously in a column with particle sizes of
