A nalytical chemists are constantly seeking new tools which permit them to solve the increasingly complex problems with which they are confronted. Sometimes a revitalized form of an old tool appears which offers new opportunities. Liquid chromatography (lc) now fits this category. New forms of lc are now emerging which promise that this technique will be one of the most powerful and widely used analytical approaches. Modern Lc Vs. Traditional Lc
The phrase "modern liquid chromatography" may need some explanation. In the newer forms of lc, 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 lc now can be carried out 100-1000 times faster than by traditional column lc 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.
Columns for Modern Analytical Liquid Chromatography
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 lc is achieved by gravity or low-inlet pressures, modern lc makes use of high-pressure pumps, with operating pressures in the 500— 5000-psi range. In fact, the im36 A .
ANALYTICAL CHEMISTRY, VOL. 43, NO. 12, OCTOBER 1971
proved performance of modern lc 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 at relatively high carrier velocities; thus, the elution of complex samples in relatively short times is possible. This approach is in sharp contrast to traditional lc, in which columns of relatively large diameters are packed with larger particles and operated with gravity flow or low pressure and at very low linear carrier velocities. The significant advantage of modern lc is improved column efficiency, resulting in the separation and analysis of more complex and difficultly separable mixtures. In addition, many more analyses can be performed in a given time period. Our understanding of column dynamics in high-speed lc 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, lc 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 lc but cannot be handled by gc. But most importantly, the
REPORT FOR ANALYTICAL CHEMISTS
J. J. KIRKLAND Experimental Station Industrial and Biochemicals Dept. E. I. du Pont de Nemours & Co., Inc Wilmington, Del. 19898
Modern liquid chromatography makes use larger variety of moving and sta tionary phases available in lc per mits a much wider range in selec tivity. While gc is, at present, a faster, more convenient technique which should be used for the analysis of appropriate samples, lc is an impor tant alternative. Often a mixture can be more easily separated by lc, even though it can be gas chromatographed satisfactorily. Another important advantage of lc is the virtual lack of effects originating from the support material. In gc, such effects sometimes lead to in complete recoveries and poorer sep arations. Since the column is the heart of the separating system, much effort has recently been expended in opti mizing column design and opera tion. This paper will summarize the present state-of-the-art of col umns designed for high-speed, highperformance lc. Particular atten tion will be given to the specialized commercial packing materials which have recently been developed for this technique. Only columns for liquid-partition, liquid-solid (ad sorption), and ion-exchange chro matography will be discussed, since exclusion chromatography gener ally involves column packings re quiring a somewhat different tech nology. Column Design
The tubing used to construct high-performance lc 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 (1). Straight columns are gener ally 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). To 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 the oretical efficiency (J). 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 po sition, provided the column has been packed in an optimum manner to prevent channeling due to the set tling 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 jack ets are used to control the tempera ture of the column, preferably to less than ±0.2°C. Temperature control is especially important in liquid-liquid partition and ion-ex change chromatography, which are more influenced by this parameter than adsorption or exclusion chro matography. In addition to other variables, column efficiency is dependent on the particle size of the packing ma terial and the internal diameter of the column (S). 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 demon strated 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 con dition 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 ho mogeneously in a column with par ticle sizes of
