Instrumentation
'hromatography I
Laverne Berry Institute of Applied Physical Chemistry University of Saarlandes. Saarbrucken. Germany Barry L. Karger' Department of Chemistry Northeastern University. Boston. Mass. 021 15
Proper selection of componentparts can spell t h e difference between success and failure in high-pressure liquid-chromatographic analysis. W i t h a n appreciation of t h e performance characteristics of currently available p u m p s and injectors, a worker should be able to improve his chances for achieving the goals of his separation problems
High-pressure liquid rhromiitography tllPl.Ci husemerredoverrhe last few yearsas a majur sepuration and analytical tool. Speed5 and eiticiencies equivalent tu gar-rhromntographir pertmnance are now being achiwed, and nen developments arr rontinually Iieing hrought t ~ t hA. numhrr oireierences detail HP1.C' t /Mi,; torpurl,"'eioithiinrtirle. we shall assumr some rudimentary knowl edgeofthis method. A n important aipert of HP1.C is
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ter (B) (-2-5,~m pore size) to prevent particles from damaging the pump. Sometimes, the pump and reservoir are part of the same unit (e.g., gas displacement pump). If a pulsating pump is used, then a pulse dampener (D) is required in the flow stream when using the refractometric or ultraviolet detector. If a dampener is not inserted, the noise level of the chromatographic signal from the detectors may he excessive, significantly limiting the detection level of the system. The next component in the flow line is a precolumn (E),This is used in liquid-liquid chromatography (LLC) to assure saturation of the stationary phase in the eluent. In other modes of chromatography, including columns of bonded phases, the precolumn is not used. Typically, the precolumn consists of a short length of
large particle diameter (-100-150 pm) diatomaceous earth support (to minimize pressure drop) which is heavily loaded with stationary phase. A pressure gauge (F), typically a Bourdon tube, is inserted close to the column to memure column inlet pressure. The Bourdon tube can also he employed as a pulse dampener. Owing to the requirement of rapid solvent changeover, a flow-through Bourdon gauge is desirable. The sample is inserted through the injection system ( G )onto the column (H) where separation takes place. It then travels into the detector (I) with readout on a recorder or other data handling device (K). The flow from the detector can either he collected a t (J)or recycled into the solvent reservoir, a s shown by the dotted line. Eluent recycling is useful when only small volumes of expensive solvent
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of component parts can o,ften spell the difference between SLiccess and failure. Figure 1 presents a block diagram of the general HPI>C system for isocratic operation. T he eluent flows from a solvent resei.voir (A) to the pump ( C )with a n int,erveningfil-
vhom correspondence should be d.
Figure 1, Diagram of liquid-chromatographic apparatus ANALYTICAL CHEMISTR
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are available hut is not recommended for careful work over extended time periods. The dotted line from the point immediately before the injection system to the detector indicates the possibility of passing eluent through the reference cell of a UV or refractive index detector. In HPLC a vast number of samples of widely different properties can he separated, analyzed, and/or prepared in pure form. A variety of chromatographic retention mechanisms can he used; moreover, the mobile phase can strongly influence results (e.g., gradient elution). The conditions for chromatographic operation will thus vary greatly from one problem to the next. Consequently, it is not possible to have a single apparatus that will he optimum or even acceptable in all cases. The worker should he aware of the limitations of a system in relation to his particular goals and, if possible, should judiciously select equipment components. Two areas of HPLC equipment have not received sufficient attention-pumps and injectors. Moreover, the sparse discussion of these topics (especially pumps) has been somewhat conflicting (7-9). In this paper we critically evaluate currently available components and suggest their relative merits in relation to the various separation problems that are faced. It is not our purpose to compare commercial equipment; however, to assist the reader, the Appendix lists those companies that have pumps and injectors available explicitly for HPLC.
Pumps Depending on the goals of separation, the pump can often he the limiting factor in determining the performance of the liquid chromatograph. The main features of the pump that need to he considered are flow constancy, maximum pressure, noise level in the detector resulting from pulsations, minimum and maximum flow, and convenience of operation. HPLC pumps often have many additional features, such as gradient elution capabilities, pressure readout, direct calibration in flow, and dampening systems, which further distinguish their performance. A few examples illustrate the types of considerations in selection of a pump. First, most detectors in current use in HPLC are concentration sensitive, in which case the area of the bandA can he expressed as
where R = response factor, rn = mass of solute, and F ~ =volumetric flow rate of the mobile phase. The preci82OA
sion in quantitation is dependent on the constancy of flow from one run to the next (quantitation on an absolute basis) or within a run (quantitation on an internal standard basis). Secondly, if detection oftrace components is necessary, the minimum detectability may he limited by the base line noise generated by pump pulsations. Thirdly, if the goal is fast separation, the speed may he limited by the maximum operating pressure of the pump. Fourthly, under preparative scale conditions involving wide diameter columns, the maximum flow rate of the pump may he the limiting factor on throughpd of sample. Finally, in scouting for the optimum solvent(s) for a particular separation. the ease with which solvents
may he changed may he an important consideration. Commercially available systems can he classified generally into three groups (Figure 2): syringe-type, reciprocating, a n d constant-pressure pumps. The general properties of each of these types will now he described, followed by a critical comparison of the pumps from the point of view of the type of chromatography required to achieve a specific goal. Syringe-Type Pumps. In general, these pumps work on the principle of solvent displacement by a piston advancing a t a constant rate in a piston chamber with the generation of pulseless flow. Typically, the pumps employ large-volume chambers (250500 ml) with slow moving pistons and
- -- - - - - PULSELESS
F~
Back Pressure 6'
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Out
Check Valves 8
In
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P
PULSATING
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Fl
I Back Pressure
Figure 2. Types of pumping systems used in HPLC. Linear change in flow with pressure is shown in each case; however, change is likely to be nonlinear, especially for reciprocating and syringe-type p u m p s A, syringe type: B. reciprocaling; C , ConStan1 pressure
* ANALYTICAL CHEMISTRY, VOL. 45. NO. 9, AUGUST 1973
have high-pressure capabilities (3000-7000 psi). They have the further capability, when operated in tandem, of allowing the use of multishaped forward and reverse gradients and simple flow programming. Syringe-type pumps are often considered to generate a constant flow independent of the operating pressure up to the pump rating. Over a moderate pressure range this assumption is valid; however, as pressure is increased, liquids become compressible to a significant extent (e.g., -4% changes for most liquids from 15 to 6000 psi). The compression of a large volume of eluent in the piston chamber can lead to some flow change, as indicated on the diagram (the change need not be linear); however, the flow change is not excessive. Yet, in gradient elution where the solvent viscosity (and hence pressure) can vary by a factor of 3 or more, the compressibility of the liquid should be kept in mind. Because of the limited pumping volume, one often finds two syringe pumps operated synchronously. One other characteristic worth noting is that these pumps tend to be relatively expensive. Reciprocating Pumps. These pumps employ small-volume chambers with reciprocating pistons or diaphragms to drive the flow against a back pressure. Two check valves are synchronized with the piston (or diaphragm) drive to allow alternate filling and emptying of eluent from the chamber. The flow is characterized as pulsating, and some dampening or other appropriate action is necessary, when using flow-sensitive detectors. In their simplest configuration, reciprocating pumps are relatively inexpensive and thus find frequent use in HPLC. They have the further advantage, relative to the syringe-type pumps, that a continuous supply of mobile phase is available. Many workers mistakenly believe that simple reciprocating pumps produce constant flow; however, a measurable deviation (likely nonlinear) from constancy is experimentally observed with solvent or back-pressure changes (Figure 2, B). This deviation results from the compressibility of the solvent a t high pressures, as well as leakage a t the check valves and seals. Recently, more expensive reciprocating pumps which can partially compensate for these flow changes have appeared on the market (dual-head, special drive pumps). Three types of reciprocating pumps are in common use: dual-head, special drive pumps, single-head, sinusoidal drive pumps, and multiplehead, sinusoidal drive pumps. These pumps can be distinguished according to the methods used to minimize the pulsating pumping noise.
The first type of reciprocating pump employs two small-volume piston chambers (0.1-0.4 ml), with piston delivery a t a steady, nonsinusoida1 rate. One chamber fills while the other provides flow to the system. Minimal pulsation is introduced by this procedure (especially a t high pumping frequency), and flow-sensitive detectors can often be used a t high sensitivities. An added feature is the correction for flow changes caused by variations in solvent composition or back pressure. In one case, electronic “compressibility” correction of the piston drive rate as the pressure increases is used, and in the other, air operated (pneumatic) check valves are employed. Careful start-up and conditioning procedures must be followed to prevent leaks from the pump and to insure long pump life. For the single-head, sinusoidal drive pump, a significant pulsation is produced, resulting in detector noise. A pulsation dampener must be employed to minimize this noise, the extent of dampening being a function of the detector in the system (e.g., refractometer > UV detector). This device behaves in a fashion similar to an electrical capacitor by storing energy during the pressurizing stroke and releasing it during the refilling stroke. Pulsation dampening has been achieved by using as capacitors gas, springs, and coiled metal tubes such as found in pressure gauges (Bourdon tubes). With gradient elution a dampener with a flow-through design is necessary. The third type of commercially available reciprocating pump uses multiple heads, each with a sinusoidally driven piston operating in an oilfilled piston chamber. Each chamber is isolated from the solvent that is being pumped by means of a thin steel membrane (diaphragm).As the piston is well lubricated, it can be driven at high speed without significant wear, thus producing less detector base line noise for a given flow rate (and piston size) than the single-head pump operating without the oil-filled chamber. Pulse dampening, however, still proves useful in many circumstances with multiple-head pumps. Constant-PressurePumps. Constant-pressure pumps typically employ gas pressure either to drive or to regulate the pressure of the eluent. There are three types of constant pressure pumps in use: direct gas displacement, gas amplifier, and pressure-regulated pumps. As shown in Figure 2, the flow generated from these pumps is strongly dependent on the back pressure. The gas displacement pump employs direct gas pressure to drive the eluent and is thus generally limited by the available gas cylinder pressure
( - 2500 psi). One potential problem with this type of pump is gas dissolving a t the high-pressure end and bubbling out a t the low-pressure end, i.e., in the detector cell. One can minimize this effect by keeping the gas-liquid interfacial area small with a long narrow tube to contain the eluent. This configuration has appeared in the pumping systems of some of the least expensive liquid chromatographs. Alternatively, one can prevent gas-liquid contact by use of baffles or a fluid such as mercury or oil. Collapsible plastic bottles (Figure 2), plastic bellows, metal bellows, and sliding pistons have also been selected. Direct gas displacement pumps are limited not only to moderate pressures but also to fixed volumes of eluent. Thus, as with syringe-type pumps, they can only operate for a limited time before refilling is necessary, leading to inconvenience in column conditioning and in preparative scale chromatography. Moreover, gradient elution cannot normally be used. Finally, care must be taken that the back pressure is maintained constant for constant flow and good quantitation. Yet, this pumping system is inexpensive and low detection limits can be achieved. since the flow produced is nonpulsating. The gas amplifier pump is similar to a gas displacement pump that employs a piston to drive the eluent; however, the area of the gas piston is larger than the area of the eluent piston. Since the pressure on the eluent is proportional to the ratio of the areas of the two pistons, a low-pressure gas source can be used to generate high liquid pressures. In this commonly used constant-pressure pump, a valving arrangement is included that rapidly refills the empty eluent chamber (-70 ml). Thus, this system can pump continuously and can achieve relatively high volumetric flow rates with no pulsation. As with reciprocating pumps, rapid solvent changes are also possible. The pressure-regulated pump typically uses a reciprocating piston pump to generate a pulsing flow of eluent and a pressure regulator to maintain the pressure constant. Three types of pressure regulators have found use in HPLC: the gas pressure regulator, the spring pressure regulator, and the continuous gas displacement pump. The gas pressure regulator has the advantage over the spring regulator in that it can be operated over a wider pressure range and a t higher pressures. The continuous gas displacement pump (CDP) incorporates a controlled leak section to pulse dampen the flow from a reciprocating pump and to control the pressure ( 1 0 ) .These
ANALYTICAL CHEMISTRY, VOL. 45, NO. 9, AUGUST 1973
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pumping systems can be set up with relatively little expense beyond the reciprocating pump.
Choice of Pump A wide variety of pumps is available with quite different characteristics. The choice of the proper pump for a particular laboratory is complicated, involving many factors. Such considerations as price, serviceability, and ease of operation are obviously important. Moreover, if the laboratory is to be faced with the separation of a wide variety of mixtures in which chromatographic conditions are unknown, then the speed a t which the solvent can be changed in the system may need to be taken into account. These matters are quite well covered by the instrument manufacturers and need not be discussed in detail here. What is not brought out, however, is the relationship of the pump type to HPLC mode of operation. Yet, this relationship is quite important, if one wishes to use his system fully. In this context, mode of operation means type of HPLC (liquid-solid, liquidliquid), elution mode (isocratic or gradient), and sample capacity of the column (analytical, preparative). (These three categories are not meant to be totally independent or inclusive.) In Table I we present a comparison of the different pumps on the basis of these modes of operation. We will now discuss this table in detail. Chromatographic Type. Consideration of the nature of the sample will determine the type of chromatography needed to perform the separation. The chromatographic type and column dimensions will, in turn, es822A
tablish the approximate flows, pressures, detection limits, and volume of eluent (operating time) required. These four operating conditions (bottom of Table I) will define the suitability of a pump for the given problem (++, + , o r -). In Table I, LSC and LLC are considered in terms of small particle diameter (
