Concepts and Principles of High Performance Liquid Chromatography

OVERVIEW

This chapter introduces some of the basic concepts and principles of liquid chromatography, providing background on the development of high performance liquid chromatography (HPLC) and briefly describing the basic system components.

The chromatogram is introduced as the record of the separation, and we identify the information it does and does not contain. Examples of different chromatographic profiles are presented and interpreted.

A strategy for the selection of the stationary phase is developed based on a discussion of the mechanism underlying the separation involved in gel filtration, reverse-phased, and ion-exchange chromatography.

The mobile phase will be considered, including its composition, preparation, and use.

The problem of column maintenance, particularly when the column is used for enzyme assays, is discussed, cleaning solutions are recommended, and a method for monitoring column performance described.

2.1 INTRODUCTION

Chromatography, the separation of classes or groups of molecules, in principle requires two phases. In liquid chromatography one of the phases is liquid and the other a stationary phase, often bonded to a solid. In days gone by, a stationary phase widely used in biochemistry laboratories was potato starch, and graduate students often found themselves up to their elbows in white "gooey stuff" making large batches of starch. After mixing, the starch would be poured into rectangular forms, where it hardened for later use as the solid phase for chromatographic analyses.

The sample was applied to one end of the block (or column if the stationary phase was poured into a vertical cylinder) and eluted from the other end (the bottom of the column) by allowing a mobile phase or solvent to flow through the stationary phase. Since the molecules of the sample are carried along by the mobile phase, the time required for a group of molecules to emerge from the stationary phase, other things being equal, was a property of the packing material. Those that emerged in the shortest time were considered not to have been affected by or to have interacted with the stationary phase, whereas those that emerged later did interact. Emergent time—or, as it is more often called, elution time—could be affected by two parameters: the distance (i.e., the path) that the molecules traverse as they pass through the packing, and the rate (velocity) at which they travel through the packing. These two parameters may be expressed in the relationship

rate of travel

2.2 THE INTRODUCTION OF HPLC

It has been known since the early days of liquid chromatography that the size of the particle used for the stationary phase affects the separation, or resolution, in a rather direct way: the smaller the particle, the better the separation.

However, with columns that used gravity to pull the mobile phase around the particles, a lower limit to particle size was reached, since the smaller the particles, the tighter they packed, eventually cutting off the flow of solvent. Thus, a pressurized solvent delivery system able to pump the mobile phase through the packing became necessary. Of course, as such pumps were developed, the old packings were found to collapse with the increased pressure, and new packing materials were required that could withstand these pressures. Together, pumps and new packing materials provided resolution and separation not achieved with earlier methods. As a fringe benefit, the new technique considerably shortened the time required to carry out separations. Separations that took hours or even days are now accomplished in minutes.

Another fringe benefit is the increased sensitivity provided by the detectors. Thus, it is no longer necessary to resort to the old ploy I was shown rather jokingly during my graduate school days. I was examining the results of an experiment in which radiolabeled compounds had been used, and I remarked to a fellow student on the incredibly low number of counts obtained during the experiments, expressing the wish for more counts. This prompted my fellow student to turn to the counter and change the counting-time dial from 1 minute to 10 minutes. Needless to say, a recount of the same samples produced more counts. In HPLC it is not necessary to resort to such tactics to obtain increased sensitivity. Rather, the geometry and low volume of the flow cells used in the detectors work in our favor.

2.3 BASIC COMPONENTS AND OPERATION

An HPLC system, shown schematically in Figure 2.1, consists of a solvent reservoir, which contains the eluent or mobile phse; a pump, often called a solvent delivery system; an injector through which the sample is introduced into the system without a drop in pressure or change in flow rate; the analytical column, which is usually stainless steel and contains the solid packing or stationary phase; and a suitable detector to monitor the eluent.

SOLVENT RESERVOIR

SOLVENT RESERVOIR

Figure 2.1 The components of an HPLC system: Solvent flow is from top to bottom. This diagram is representative only; the boxes are not drawn to the scale of actual system components.

Also shown in Figure 2.1 are two other components: a precolumn and a guard column. The precolumn, located in the system between the solvent reservoir and the pump, acts to filter out any impurities in the mobile phase before they reach the pump heads and the analytical column. In the case of analytical columns made of silica, impurities in the solvents can result in leaching of the silica. If solvents are made daily and are filtered (see below), a precolumn may not be needed.

The guard column, by virtue of its location between the injector and the analytical column, functions to remove any insoluble material and other debris that might have been injected and would otherwise clog the analytical column. For example, when used with enzyme assays, the guard column will remove precipitated proteins or other insoluble material carried over from the incubation mixtures. Since guard columns themselves can get clogged, if the pump is to maintain a constant flow rate it must generate greater pressures to drive the solvent through the clogged filter. This increase in pressure, referred to as "back pressure," can be eliminated by cleaning and repacking the guard column.

To operate an HPLC system, the sample is introduced through the injector into the system and is then pushed through the analytical column by the constant pumping of solvent (or mobile phase) from the reservoir through the system (Fig. 2.1). The mobile phase can be delivered in two ways: isocrati-cally, that is, at constant composition, or in the form of a gradient, when the composition is varied. Chapter 4 explains how to decide which form to use and also describes injectors in more detail. Additional information about the operation of pumps can be obtained by consulting the references.

Following its emergence from the other end of the column, the eluent flows through the detector. Detectors operate on various principles. For example, some monitor the ultraviolet, visible, or fluorometric properties of molecules: others monitor radioactivity; and still others monitor differences in oxidation-reduction potential and refractive index. These detectors are listed in Table 2.1 together with some examples of the specific reactions with which they have been used.

2.4 COUPLING THE COMPONENTS: ON THE PERILS OF FERRULES

While one of the most confusing steps for the new user of HPLC is deciding what equipment to order, an even more difficult and frustrating step occurs after the equipment has arrived and connections must be made between the solvent delivery system (the pump), the various columns, and the detector. For the new user quickly discovers that connections are not made with the more familiar, easy-to-use flexible plastic, but with stainless steel tubing, which cannot be cut with scissors or easily coupled with plastic connectors. Thus special tools must be used for cutting, and connections must be made with

TABLE 2.1 Detectors and Their Applications

Detector

Reaction analyzed"

1. UV spectrometer

ATP -» ADP + Pi IMP -* Ino + P¡ FoTP -> cFoMP cFoMP -» FoMP

2. Fluorometer

3. Radiochemical

4. Electrochemical

5. Refractive index

L-Dopa -* dopamine L-5'-Hydroxytryptophan -» serotonin Maltoheptose -* oligosaccharides

" FoTP; formycin 5-triphosphate.

nuts and bolts and fittings called ferrules. Unfortunately, ferrules are not all the same, and once in place they are not easily removed.

The ferrule shown schematically in Figure 2.2 is swaged, that is, attached, when the end of one piece of stainless steel tubing is coupled to another. The coupling itself involves a bolt (B) and a nut (N), which are assembled as follows. The bolt is placed on the tubing, followed by the ferrule, and the nut is threaded to the male bolt, trapping the ferrule between and thus swaging the ferrule to the tubing. The appearance of the ferrule on the tubing before compression is shown in the upper panel of Fig. 2.2, and its appearance within the fitting after compression in shown in the lower panel. To avoid damage to the ferrules, these fittings should not be overtightened. Keep in mind, as

Figure 2.2 Cross-sectional diagram of the components used to couple two pieces of stainless steel tubing. Top: Units before swaging. T, tubing to be joined; B, "male" bolt; F, female; N, "female" nut. Bottom: After swaging. The pressure of the bolt on the nut has forced the ferrule to seal the joint between the two ends of the tubing.

Figure 2.2 Cross-sectional diagram of the components used to couple two pieces of stainless steel tubing. Top: Units before swaging. T, tubing to be joined; B, "male" bolt; F, female; N, "female" nut. Bottom: After swaging. The pressure of the bolt on the nut has forced the ferrule to seal the joint between the two ends of the tubing.

well, that the connecting tubing should be short and the connectors few, to avoid excess space where mixing can occur.

2.5 THE CHROMATOGRAM

Information about the separation is displayed on a chromatogram, which is obtained by converting the detector output to an electrical signal and following this signal on a recorder as a function of the time after the loading of the sample. Figure 2.3 shows a representative HPLC chromatogram of a sample containing two species of compounds, A and B. In this example, while both enter the column at the same time (with the injection of the sample), compound B traverses the column at a faster rate than compound A. As shown in Figure 2.3, compound B will emerge and be detected first, followed by A. The time of injection of the sample, marked on the chromatogram by the arrow, is taken as zero time, and the time after injection is determined from the speed of the recorder. Of course, the rate of fluid flow is held constant and is controlled by the pump setting. Under these conditions, the chromatogram will show the elution of A and B as a function of time after loading the sample (injection time). Many investigators change the variable elution time to the more useful parameter elution volume by multiplying the flow rate, expressed in milliliters per minute, by the reciprocal of the chart speed, expressed in minutes per centimeter, to give the new unit of milliliters per centimeter. This maneuver allows the length unit (cm) on the chart to be converted to volume. The elution volume is especially useful if it becomes necessary to change the flow rate from run to run or if chromatograms obtained in different laboratories under different flow rates are to be compared.

INJECT

INJECT

TIME

Figure 2.3 A representative HPLC chromatogram showing the separation of compounds A and B. The time of injection is taken as zero time, and the elution position is shown as a function of time after injection. The amount of each compound in the original sample is given by the peak height or area, as represented on the tracing by the letters B and A.

Detector Intensity

TIME

Figure 2.3 A representative HPLC chromatogram showing the separation of compounds A and B. The time of injection is taken as zero time, and the elution position is shown as a function of time after injection. The amount of each compound in the original sample is given by the peak height or area, as represented on the tracing by the letters B and A.

Figure 2.4, which shows chromatograms of the same sample obtained at different flow rates, illustrates the usefulness of the volume unit. Whereas in Figure 2.4A the sample was eluted at a flow rate of 2 mL/min, in Figure 2.4B the flow rate was 1 mL/min. A superficial analysis of these data would suggest that the two peaks in A and the two peaks in B represented four different compounds. However, if the same data are expressed as a function of elution volume, as shown in Figure 2.5, the two sets of peaks are easily seen to have similar retention volumes. Thus, based on this criterion, they are the same.

2.6 INTERPRETATION OF THE CHROMATOGRAM

In addition to showing that species such as A and B have been separated, chromatograms provide other information. For example, the shape of a curve provides information about the efficiency of the separation. With a system operating at high efficiency, peaks will be narrow and spikelike (Fig. 2.6/4), while broad-based peaks suggest low efficiency (Fig. 2.6B). These results may be due in part to such factors as a luck of uniformity in either the size or homogeneity of the particles used in the stationary phase. Alternatively, broad peaks may indicate heterogeneity in the sample, as is often observed when the pH of the mobile phase is too near the pK of the molecules being separated.

The appearance of the peaks on chromatograms can also provide information about the quality of the resolution. Thus, if the compounds are well separated, the second peak will emerge only after the detector has completely

TIME (min)

Figure 2.4 A comparison of the chromatography of the same two compounds carried out at flow rates of 1 mL/min (A) and 2 mL/min (B).

CO ac

Figure 2.5 Data from Figure 2.4 replotted as a function of elution volume. The volume was determined by the multiplication of the flow rate (mL/min) by the reciprocal of the chart speed (cm/min). Expressed in this manner, each unit of distance is converted to a unit of volume.

returned to the baseline (Fig. 2.1A). Failure to achieve baseline separation (Fig. 2.7B) indicates poor resolution and suggests that something must be done to allow the second component to be retained longer—either slow its rate or increase the distance it must travel.

The resolution of any two components, therefore, is a ratio relating the distance between the apex of the peaks and the distance between their bases. With baseline separation, the bases of the peaks do not overlap. In the absence of baseline separation, however, the apex of each peak may be separate while the bases overlap. A mathematical expression can be written to describe this

Figure 2.6 HPLC profiles of two components separated on two columns operating (/t) at high efficiency and (B) at low efficiency.

Figure 2.6 HPLC profiles of two components separated on two columns operating (/t) at high efficiency and (B) at low efficiency.

Figure 2.7 HPLC profiles of two compounds separated on column showing (A) good separation (resolution) and (B) poor resolution.

relationship be dividing the distance between the peaks, shown by the symbol delta (A) in Figure 2.8, by half the sum of the width of the bases, giving a numerical value for resolution (Fig. 2.8).

The symmetry of each peak can provide information about the sample. Tailing (Fig. 2.9A) suggests some heterogeneity in the sample—either real or introduced by the chromatographic conditions. Flat-topped peaks (Fig. 2.9B) suggest that the capacity of the column has been exceeded.

Of course, the magnitude of the signal from the detector can be used as a measure of the relative amount of each sample. While arbitrary units of area

Figure 2.8 Representative HPLC chromatogram to illustrate a method for calculation of resolution R. The separation of two components labeled A and B) is shown. The width of each peak is shown by arrows and the symbol W, while the distance between peaks is shown by the symbol A. Resolution may be defined as h-A-H

Figure 2.8 Representative HPLC chromatogram to illustrate a method for calculation of resolution R. The separation of two components labeled A and B) is shown. The width of each peak is shown by arrows and the symbol W, while the distance between peaks is shown by the symbol A. Resolution may be defined as

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