Efficiency Band Broadening

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... 

The electroosmotic flow profile is very different from that for a phase moving under forced pressure. Figure 12.40 compares the flow profile for electroosmosis with that for hydrodynamic pressure. The uniform, flat profile for electroosmosis helps to minimize band broadening in capillary electrophoresis, thus improving separation efficiency. 

In order to achieve the best efficiency the SEC column should be operated at optimized operating parameters. The most important ones are flow rate [cf. van Deemter equation for band-broadening effects (21)], sample viscosity (depends on molar mass and concentration of the sample), and injection volume (7). 

Traditionally, column efficiency or plate counts in column chromatography were used to quantify how well a column was performing. This does not tell the entire story for GPC, however, because the ability of a column set to separate peaks is dependent on the molecular weight of the molecules one is trying to separate. We, therefore, chose both column efficiency and a parameter that we simply refer to as D a, where Di is the slope of the relationship between the log of the molecular weight of the narrow molecular weight polystyrene standards and the elution volume, and tris simply the band-broadening parameter (4), i.e., the square root of the peak variance. 

The flow profiles of electrodriven and pressure driven separations are illustrated in Figure 9.2. Electroosmotic flow, since it originates near the capillary walls, is characterized by a flat flow profile. A laminar profile is observed in pressure-driven systems. In pressure-driven flow systems, the highest velocities are reached in the center of the flow channels, while the lowest velocities are attained near the column walls. Since a zone of analyte-distributing events across the flow conduit has different velocities across a laminar profile, band broadening results as the analyte zone is transferred through the conduit. The flat electroosmotic flow profile created in electrodriven separations is a principal advantage of capillary electrophoretic techniques and results in extremely efficient separations. 

Efficiency Degree of band broadening for a given retention time. It is expressed as the number of theoretical plates, N, or as the height equivalent to a theoretical plate, HEPT. 

The major advance in the way in which column eluate is deposited on the belt was the introduction of spray deposition devices to replace the original method which was simply to drop liquid onto the belt via a capillary tube connected directly to the outlet of the HPLC column. These devices, based on the gas-assisted nebulizer [5], have high deposition efficiencies, transfer of sample can approach 100% with mobile phases containing up to 90% water, and give constant sample deposition with little band broadening. 

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. 

Injection systems of a capillary gas chromatography should fulfil two essential requirements (i) the injected amount should not overload the column (ii) the width of the injected sample plug should be small compared with band broadening due to the chromatographic separation. Good injection techniques are those which achieve optimum separation efficiency of the column, allow accurate... 

The efficiency of a chromatographic separation can be described by the height equivalent of a theoretical plate (H), where lower values of H correspond to more efficient separations. The Van Deemter equation describes the relationship between H and mobile phase flow velocity (u) as the sum of three major terms, A, B, and C, each of which represents a different contribution to band broadening in a chromatographic column. 

Obviously, the main purpose for the introduction of CL detection coupled to CE separations is inherent to the development and improvement of sensitive and uncomplicated devices to achieve a decrease of the band broadening caused by turbulence at the column end, together with the attractive separation efficiency of CE setups. With this purpose in mind, Zhao et al. [83] designed a postcolumn reactor for CL detection in the capillary electrophoretic separation of isoluminol thiocarbamyl derivatives of amino acids, because, like other isothiocyanates, isoluminol isothiocyanate has potential applications in the protein-sequencing area. 

Monoliths Low backpressure, suited for conventional HPLC Higher separation efficiency by column coupling Rugged against delay volume and extra-column band broadening fast column re-equilibration Reduced maintenance on pumps and injector seals Reduced need for sample pre-treatment... 

The choice of carrier gas and gas flow control are critical for successful GC. The carrier gas does no more in the separation process than its name implies it carries the vapor phase analyte molecules along the column. As such, it must be inert, non-toxic, inexpensive, highly pure and must provide efficient transport with minimal band broadening. For packed column GC, nitrogen is the most commonly used carrier gas, followed by helium. For capillary column GC, the most common carrier gas is helium, followed by hydrogen and nitrogen. 

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