Over the past 40 years the practice of chromatography has witnessed a continuing growth in almost every respect: the number of chromatographers, the amount of published work, the variety and complexity of samples being separated, separation speed and convenience, and so on. However, this growth curve has not moved smoothly upward from year to year. Rather the history of chromatography is, one of periodic upward spurts that have followed some major innovation: partition and paper chromatography in the 1940s, gas and thin layer chromatography in the 1950s, and the various gel or size-exclusion methods in the early 1960s. A few years later it was possible to foresee still another of those major developments that would revolutionize the practice of chromatography: a technique that we call modern liquid chromatography.
What do we mean by “modern liquid chromatography”? Liquid chromatography (LC) refers to any chromatographic procedure in which the moving phase is a liquid, in contrast to the moving gas phase of gas chromatography. Traditional column chromatography (whether adsorption, partition, or ion-exchange), thin-layer and paper chromatography, and modern LC are each examples of liquid chromatography. The difference between modern LC and these older procedures involves improvements in equipment, materials, technique, and the application of theory. Samples are usually stored in special HPLC vials. In terms of results, modern LC offers major advantages in convenience, accuracy, speed, and the ability to carry out difficult separations.
To appreciate the unique value of modern LC it will help to draw two comparisons:
• Liquid versus gas chromatography.
• Modern versus traditional LC procedures.
The tremendous ability of gas chromatography (GC) to separate and analyze complex mixtures is now widely appreciated. Compared to previous chromatographic methods, GC provided separations that were both faster and better. Moreover, automatic equipment for GC was soon available for convenient, unattended operation. However, many samples simply cannot be handled by GC. Either they are insufficiently volatile and cannot pass through the column, or they are thermally unstable and decompose under the conditions of separation. It has been estimated that only 20% of known organic compounds can be satisfactorily separated by GC, without prior chemical modification of the sample. LC, on the other hand, is not limited by sample volatility or thermal stability. Thus, LC is ideally suited for the separation of macromolecules and ionic species of biomedical interest, labile natural products, and a wide variety of other high molecular-weight and/or less stable compounds, such as the following:
Liquid chromatography enjoys certain other advantages with respect to GC. Very difficult separations are often more readily achieved by liquid than by gas chromatography, because of:
Chromatographic separation is the result of specific interactions between sample molecules and the stationary and moving phases. These interactions are essentially absent in the moving gas phase of GC, but they are present in the liquid phase of LC – thus providing an additional variable for controlling and improving separation. A greater variety of fundamentally different stationary phases have been found useful in LC, which again allows a wider variation of these selective interactions and greater possibilities for separation. Finally, chromatographic separation is generally enhanced as the temperature is lowered, because intermolecular interactions then become more effective. This favors procedures such as LC that are usually carried out at ambient temperature. Liquid chromatography also offers a number of unique detectors that have so far found limited application in GC:
Although GC detectors are generally more sensitive and also provide unique selectivity for many sample types, in many applications the available LC detectors show to advantage. That is, LC detectors are favored for some samples, whereas GC detectors are better for others. A final advantage of liquid versus gas chromatography is the relative ease of sample recovery. Separated fractions are easily collected in LC, simply by placing an open vessel at the end of the column. Recovery is quantitative and separated sample components are readily isolated, for identification by supplementary techniques or other use. The recovery of separated sample bands in GC is also possible but is generally less convenient and quantitative.
Consider now the differences between modern LC and classical column or openbed chromatography. These three general procedures are illustrated in figure below In classical LC a column is often used only once, then discarded. Therefore, packing a column (step 1, “bed preparation”) has to be repeated for each separation, and this represents a significant expense of both manpower and material. Sample application in classical LC (step 2), if done correctly, requires some skill and time on the part of the operator. Solvent flow in classical LC (step 3) is achieved by gravity feeding of the column, and individual sample fractions are collected manually. Since typical separations require several hours in classical LC, this is a tedious, time-consuming operation. Detection and quantitation (step 4) are achieved by the manual analysis of individual fractions. Normally, many fractions are collected, and their processing requires much time and effort. Finally, the results of the separation are recorded in the form of a chromatogram: a bar graph of sample concentration versus fraction number.
The advent of paper chromatography in the 1940s and thin-layer chromatography (TLC) in the 1950s greatly simplified the practice of analytical liquid chromatography. This is also illustrated in Figure 1.1. Bed preparation in TLC or paper chromatography (step 1) is much cheaper and simpler than in classical LC. The paper or adsorbent-covered plates can be purchased in ready-to-use form at nominal expense. Sample application is achieved rather easily, and solvent flow is accomplished by placing the spotted paper or plate into a closed vessel with a small amount of solvent. Solvent flow up the paper or plate proceeds by capillary action, without the need for operator intervention. Finally, detection and quantitation can be achieved by spraying the dried paper or plate with some chromogenic reactant to provide a visible spot for each separated sample component.
The techniques of paper and thin-layer chromatography greatly simplified liquid chromatography and made it much more convenient. A further advantage, particularly for TLC, was that the resulting separations were much better than in classical LC and required much less time-typically 30-60 min rather than several hours. However, certain limitations were still apparent in these open-bed methods:
Despite these limitations, TLC and paper chromatography became the techniques of choice for carrying out most LC separations. Let us look now at modern LC. Closed, reusable columns are employed (step 1, image above), so that hundreds of individual separations can be carried out on a given column. Since the cost of an individual column can be prorated over a large number of samples, it is possible to use more expensive column packings for high performance and to spend more time on the careful packing of a column for best results. Precise sample injection (step 2) is achieved easily and rapidly in modern LC, using either syringe injection or a sample valve. Solvent flow (step 3) is provided by high-pressure pumps. This has a decided advantage: controlled, rapid flow of solvent through relatively impermeable columns. Controlled flow results in more reproducible operation, which means greater accuracy and precision in LC analysis. High-pressure operation leads to better, faster separation. Detection and quantitation in modern LC are achieved with continuous detectors of various types. These yield a final chromatogram without intervention by the operator. The result is an accurate record of the separation with minimum effort.
Repetitive separation by modern LC can be reduced to a simple sample injection and final data reduction, although the column and/or solvent may require change for each new application. From this it should be obvious that modern LC is considerably more convenient and less operator dependent than either classical LC or TLC. The greater reproducibility and continuous, quantitative detection in modern LC also lead to higher accuracy and precision in both qualitative and quantitative analysis. As discussed in Chapter 13, quantitative analysis by modern LC can achieve a precision of better than ±0.5% (1 standard deviation or S.D.). Finally, preparative LC separations of multigram quantities of sample are now proving relatively straightforward.
Modern LC also provides a major advance over the older LC methods in speed and separation power. In fact, LC now rivals GC in this respect. Modern LC also features a number of new column packings that provide separations that were previously impossible. Similar determinations by classical, physical methods required literally months of work, as compared to the 10 min today. Most important, all these advantages of modern LC are now routinely available with commercial LC equipment and supplies. What we have called modern LC has been referred to by other names: high-performance or high-pressure LC (HPLC), high-speed LC, and simply liquid chromatography (LC). In the present book we refer to modern liquid chromatography in columns as LC. Where high-pressure operation is to be contrasted with low-pressure LC, we use the term HPLC to define the usual technique that employs high pressure. Recently, an improved version of TLC has been introduced, and referred to as high-performance TLC (HP-TLC). It has been implied that this new technique will displace modern LC from many of its present applications. This seems to us an overoptimistic assessment of the potential of HP-TLC (e.g., la, Ib). However, TLC itself has proved to be a complementary technique that can be used effectively in conjunction with LC, and any improvement in HP-TLC will only increase the value of TLC in these applications.