Equipment design

Most commercial systems can be purchased in a form which includes gradient elution capabilitie, a variable vavelength detecter capable of wavelengths down to 200 nm, and/or a refractive index detector and a low dead-volume injector.

There are a large number of liquid chromatographs available and the choice can be rather bewildering. Almost all current manufacturers produce a reliable instrument and an individual's choice is often determined by factors such as good local service from a particular firm and the amount of money available to purchase the instrument. The researcher should also carefully consider teh experimental requirements of the separations that are to be achieved; for example, a 254nm UV detector is of little use for monitoring the separation of pepetides and proteins which are best detected at 206-226 nm, or a saturated lipid, which does not exhibit significant absorption above 195 nm, will require another form of detection, e.g. refractive index.

Most modern systems are based on a constant volume reciprocating pump which can generate pressures of up to 6000 psi, which is a practical upper limit as many columns do not have a satisfactory life-time much above 5000 psi. On the other hand, the use of the viscous eluants such as propanol can require pressures of 3000 psi. Pulse free pumps are neccessary as pulsations within the system can result in a noisy detector baseline and thus raise the detection limits in sensitive assays. Multi-head reciprocating pumps have been designed to operate in an asymmetric manner and thus compensate for variations in flow rates during the strokes of piston. Such a system when used with small volume pistons allows the rapid generation of almost pulse free gradients which is an important feature for the analysis of biological samples. It is essential that the pump(s) maintain constant operating conditions so that retention times in consecutive replicate assays should agree by 0.5%.

A major difference between many commercial systems is the use of one or two pumps for the generation of a solvent gradient. The gradient can be formed on either the low or high pressure side of the pump system. Either system can be used to generate reliable gradients provided small-volume piston pumps are used and teh mixing vessel has a minimal total volume (0.6 mL or less) which allows rapid and complete mixing of the solvents. A new advance in pump design has occurred with teh development of microprocessor controls which allow the use of a single multi-headed pump for the generation of complex gradient shapes.

An alternative to teh continuous gradient is the stepwise change of the mobile phase by means of a solvent select value. If there is a sufficiently large number of solvent changes, the " stepwise gradient" approaches the true gradient. Since this system requires only the pump it is less expensive and with an optimal set-up the performance of this system can approach that of a true gradient apparatus.
Other options which individual separations can require are temperature control, flow programming, complex gradient generation, variable wavelength UV detection, UV scanning of peaks during a run, fraction collecting, recycle mode, higher flow rates for preparative columns, fluorescent detection, automatic injecion and sampling, and intergration facilities. A major advantage of microprocessor controlled facilities is that the instrument can be programmed to execute a multi-step gradient analysis and then re-equilibrate the column for the next analysis. On the more expensive instruments, the control system can allow unattended methods development with repeated injections under different chromatographic conditions, thus allowing rapid development of the optimum separation conditions. Since the analysis of a biological sample depends on teh complex interaction of a number of variables, a microprocessor control system is not an expensive luxury but rather an important part of the modern liquid chromatograph.