Instrumentation pumps, injectors and column design

In the first part of this chapter, the different designs of LC pumps are dealt with, from the simple mechanical instruments which were used to pioneer the field, to the microprocessor-controlled pumps available today. Later, some other related topics ate considered, such as the problems associated with the introduction of samples, and the means available for connecting up the highly pressurised HPLC system. 

In attempting to design an ideal pump for HPLC, the following features should be built into it. 

The pressure experienced by the pump as it forces mobile phase through the column is known as the column back pressure. This will vary of course, from column to column (according to dimensions and particle size) and with the flow rate and viscosity of the mobile phase used. The relationship between these parameters is expressed in the following equation  

In the above relationship, the back pressure is measured in bars. This is just one of a number of systems of units used in HPLC to measure pressure. The SI unit of pressure is the Pascal (Pa), although this is not used very often in this context, probably because of the large and unwieldy numbers needed to describe typical HPLC pressures. Other units that may be encountered include pounds per square inch (psi), which tends to be the favoured unit in the United States, and its metric equivalent kg/cm.  

For reference, 1 bar is equal to 1 atmosphere of pressure, which is approximately equal to 14psi, 1 kgcm or 10 Pa. 

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Because of the interest in narrow-bore and microbore columns, instrument manufacturers now have developed solvent delivery systems that are capable of accurately pumping at the low flow rates typically required for microbore applications (>10 / L/min). In addition, injectors have been designed that are capable of introducing the smaller sample volumes, and detector cells are available that are small enough to monitor the reduced sample volumes passing through the detector. Thus narrow-bore and microbore applications are possible with readily available instrumentation, and reports may be found in the literature. 

Commercially available HPLC instrumentation was originally designed for use with standard-bore columns (4.6 mm I.D.). Detector flow cells were optimized for maximum sensitivity with these analytical columns, injectors were designed to introduce microliter quantities of sample, and pumps were designed to be accurate and reproducible in the milliliter flow-rate ranges commonly employed with standard-bore columns. However, these instruments are not well suited for use with small-bore columns, as the dispersion introduced by the large volumes is detrimental to the separation. In addition, the reproducibility and accuracy of the pumping system at the low flow rates required are questionable. 

Liquid chromatograph. The instrument should be one that is designed for use with stainless steel columns and is equipped with a pumping system able to maintain a pressure of 15 MPa a UV spectrophotometer detector able to measure UV absorbance at 254nm and a loop-type injector. 

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