Column efficiency in HPLC

Fields reported that continuous silica xerogels prepared from potassium silicate solutions could be used as highly permeable support media, and exhibit reasonable chromatographic efficiency in HPLC [23]. Minakuchi et al. reported the preparation and evaluation of continuous porous-silica columns that provide a much higher column efficiency in HPLC than do conventional columns packed with particles [13-16,18], The monolithic columns prepared in a capillary can also be used in CEC. 

Guillaume, Y. Guinchard, C. Study and optimization of column efficiency in HPLC Comparison of two methods for separating ten benzodiazepines. J.Liq.Chromatogr., 1994, 17, 1443-1459... 

SOURCE C. S. Young and R. J. Weigand, An Efficient Approach to Column Selection in HPLC Method Development," LCGC 2002, 20, 464. 

Figure 25-29 Separation of six compounds on (a) phenyl- and (b) Cle-silica columns with 3-p.m particle size using 35 65 (vol/vol) acelonitrile/0.2% aqueous trifluoroacetic acid. Column size 7 x 53 mm flow rate = 2.5 mL/min. [From C. S. Young and R. J. Weigand, "An Efficient Approach to Column Selection In HPLC Method Development." LCGC 2002,20.464. Courtesy Alltech Associates.]...

On the other hand, the lack of internal pore structure with micropellicular sorbents is of distinct advantage in the analytical HPLC of biological macromolecules because undesirable steric effects can significantly reduce the efficiency of columns packed with porous sorbents and also result in poor recovery. Furthermore, the micropellicular stationary phases which have a solid, fluid-impervious core, are generally more stable at elevated temperature than conventional porous supports. At elevated column temperature the viscosity of the mobile phase decreases with concomitant increase in solute diffusivity and improvement of sorption kinetics. From these considerations, it follows that columns packed with micropellicular stationary phases offer the possibility of significant improvements in the speed and column efficiency in the analysis of proteins, peptides and other biopolymers over those obtained with conventional porous stationary phases. In this paper, we describe selected examples for the use of micropellicular reversed phase... 

HPLC theory could be subdivided in two distinct aspects kinetic and thermodynamic. Kinetic aspect of chromatographic zone migration is responsible for the band broadening, and the thermodynamic aspect is responsible for the analyte retention in the column. From the analytical point of view, kinetic factors determine the width of chromatographic peak whereas the thermodynamic factors determine peak position on the chromatogram. Both aspects are equally important, and successful separation could be achieved either by optimization of band broadening (efficiency) or by variation of the peak positions on the chromatogram (selectivity). From the practical point of view, separation efficiency in HPLC is more related to instrument optimization, column... 

Capillary electrochromatography (CEC) is a high-efficiency microseparation technique in which mobile-phase transport through a capillary (usually 50- to lOO-pm I.D., packed with stationary-phase particles) is achieved by electroos-motic flow instead of a pressure gradient as in HPLC (342-349). The absence of backpressure in electroosmotic flow allows the use of smaller particles and longer columns than in HPLC. In the reversed-phase mode, CEC has the potential to yield efficiencies 5 to 10 times greater than reversed-phase HPLC. 

A major requirement for column tubes in HPLC is constancy of the column diameter when changing the pressure drop along them. If this is not so, the stability of the packing is affected on changing the pressure drop, which destroys the column efficiency. All available polymeric tubes, such as PTFE, nylon or plastic tubes, show a definite pressure dependence of the tube diameter. Apart from this, polymeric materials, except PTFE, are easily attacked by organic solvents. For these reasons polymeric materials are unsuitable as column tube materials in HPLC. Recently, however, the application of a radial compressed PTFE column for preparative LC was reported (Waters Assoc.). 

Modern high-performance liquid chromatography (HPLC) has been developed to a very high level of performance by the introduction of selective stationary phases of small particle sizes, resulting in efficient columns with large plate numbers per metre. There are several types of chromatographic columns used in HPLC. 

Below the dotted line in Table I we list less fundamental differences between the two methods. Column lengths tend to be somewhat shorter in HPLC using small particle PB as a consequence of the high efficiencies that can be generated with the smaller particle sizes. For analytical scale HPLC, tube diameters of 3-4 mm are selected however, for preparative scale, tube diameters of 1 cm or above are not uncommon. 

At this point it is worth considering the demands made on the instrumentation for operation with wide bore columns and, in particular, the adaptation of analytical Instruments for this purpose [596,597]. The pump requirements for preparative separations differ from those in analytical HPLC as the ability to generate high flow rates at moderate backpressures is crucial to the efficient operation of wide bore columns. A flow rate maximum of 100 ml/min with a pressure limit of 3000 p.s.i. is considered... 

The efficiency, or plate count of a column N is often calculated as 5.54 (tr/a)2, where tr is the retention time of a standard and a is the peak width in time units at half-height.1 2 5 This approach assumes that peaks are Gaussian a number of other methods of plate calculation are in common use. Values measured for column efficiency depend on the standard used for measurement, the method of calculation, and the sources of extra-column band broadening in the test instrument. Therefore, efficiency measurements are used principally to compare the performance of a column over time or to compare the performance of different columns mounted on the same HPLC system. 

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