HPLC equipment column designs

Most of the time, the pressure is measured after the eluent pump. On well-designed HPLC equipment, the operating pressure is mainly due to the pressure drop in the column. 

Applied Biosystems Ltd. recently made an addition to the list of HPLC equipment devoted to proteins. The range consists of a protein sequencer (470 A), a protein purification system (130 A), a peptide synthesiser (430 A) and an amino acid analysis system (120 A). The chromatographic module is a microbore HPLC system specificaUy designed for use with very small quantities of material. The columns consist of Brownlee cartridges. Flow rates in the order of 10 to 100... 

Over the last decade, the most significant developments in liquid chromatography, specifically in HPLC, have been in the areas of instrumentation and equipment [49-52], advances in stationary phase chemistry and their design [51, 52], improvements in column design and dimensions [53, 54], use of different additives in mobile phases [55] and sample preparation techniques [38,39]. All of these factors have helped to significantly improve the way separation-scientists develop their methods. 

Column size is another important consideration. For equipment designed for most routine laboratory HPLC situations the relative sensitivity of APTelectrospray instruments is better at low flow rates (0.2-0.8 mL/min) whereas the relative sensitivity of APCI instruments is enhanced at high flow rates (0.5-2 mL/min). As a result, small columns are appropriate for API-electrospray/MS and, if only one or two compounds of interest are found in a particular sample, high-resolution separations are not necessary. For APTelectrospray analysis of complex samples, 150 mm x 4.1 mml.D., 3 pm columns (flow 0.5-1.0 mL/min) are usually sufficient. For drug quantification involving analysis of single or low numbers of compounds, small columns such as 30 mm x 2.1 mm I.D., 3.5 pm columns (flow rate 0.2-0.4 mL/min) provide sufficient separation and a saving in both column cost and solvent utilization. The reduced injection volume required for the small columns often results in better resolution and increased sensitivity. 

With many laboratory instruments, equipment specifications alone control the decision of which instrument you should buy. However, HPLC systems are so flexible, can run so many types of columns, and have enough control variables, that hardware decisions alone are insufficient in helping you decide which system you need to solve your application problems. I finally designed a diagram to aid in explaining how to buy an HPLC system (Fig. 2.1). 

Installation qualification establishes that the instrument is received as designed and specified, that it is properly installed in the selected environment, and that this environment is suitable for the operation and use of the instrument. During installation, one should 1) Compare the equipment, as received, with the purchase order 2) check documentation for completeness or for any damage 3) install hardware (computer, equipment, fittings and tubings for fluid connections, columns in HPLC,... 

There Is no doubt that narrow-bore packed columns pose very stringent requirements on the design of the liquid chromatograph. Modifications of existing commercial equipment for the practice of narrow-bore packed column HPLC are given below ... 

Safety devices such as air sensors and pressure transducers are built into preparative HPLC units, together with a series of valves. These devices create dead volume and contribute to the extra column volume. A large-scale chromatography unit is composed of valves for selection of buffers and feed solutions, at least two pumps, the separation column, and, in most cases, at least one detector. Instead of a fraction collector, a combination of valves is often used. These sources of dead volume create typical washout kinetics, which contribute exponentially to the band-broadening processes. For the industrial scale, the equipment is mainly customer designed. For medium scale, modular units are available [51]. Attention should be paid to extra column volume when systems are compared. Extra column effects are an important parameter of the quality of a system and should be considered when a system is purchased. 

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