Sample overload

The major cause of peak asymmetry in GC is sample overload and this occurs mostly in preparative and semi-preparative separations. There are two forms of sample overload, volume overload and mass overload. 

The low numbers of routine applications of IMS have been attributed to the ease of sample overload and to the... 

Stalberg, O., Westerlund, D., Rodby, U. B., and Schmidt, S. (1995). Determination of impurities in remoxipride by capillary electrophoresis using UV-detection and LIF-detection — principles to handle sample overloading effects. Chromatographia 41, 287—294. 

The major cause of peak asymmetry in LC is sample overload and this occurs mostly in preparative and semi preparative LC. There are two forms of sample overload, volume overload and mass overload. Volume overload results from too large a volume of sample being placed on the column and this effect will be discussed later. It will be seen that volume over load does not, in itself, produce asymmetric peaks unless accompanied by mass overload. Mass overload which, as discussed above, is accompanied by a distortion of the normally linear isotherm, can cause very significant peak asymmetry and, in fact, seriously impair the resolution obtained from the column. 

Sensitive to alkali metal contamination and sample overloading... 

Baseline drift Normal effect of gradient delivery Sample overload Buildup of retained compounds on column... 

Often TLC is used for fast screening of mobile phases, since the stationary phases used in prep LC are also available for TLC. The TLC R value should be equal to or less than 0.3. In prep work, the separation is usually optimized so that the column can then be run with a sample overload and still produce adequate separations. Overloading will cause the plate number and partition ratio to decrease, and chromatograms produced this way are not pretty to look at. 

The numerous reasons which can account for various deviations from the ideal FFF retention theory were discussed in the corresponding sections. Here, additional problems are treated which can complicate FFF measurements and significantly distort the results obtained. General requirements for a successful FFF measurement include precise flow control and flow rate precise temperature measurement precise determination of t0 and tr correct relaxation procedure control of sample overloading and integrity and control of mixed normal and steric retention effects as well as wall adsorption control. Some of these complications cannot be avoided so one must correct for these effects, usually in a sem-iempirical and partially very complicated fashion. 

Litzen and Wahlund systematically studied error sources like temperature effects, sample overloading, sample adsorption to the accumulation wall membrane and influences of the carrier liquid composition, that occur with Fl-FFF [455] the latter has already been discussed above. It was shown that preservation of constant channel temperature is very important as repeated measurements of an identical sample resulted in gradually decreasing retention times due to increasing channel temperature caused by frictional heat, especially when using high flow rates. As constant channel temparature is usually not fulfilled with the standard Fl-FFF channels, which simply operate at room temperature without any temperature control, this is an important point to consider. 

Peak tailing is the most commonly observed effect of sample overloading. In essence, in most cases this effect is associated with nonlinear adsorption isotherms. In Chapter 2 the relationship of the retention volume and the derivative of the excess adsorption isotherm of the analyte on given stationary phase surface was derived. If the isotherm is linear within the injected concentration region, all components of the chromatographic zone are moving... 

For templates interacting only weakly with the functional monomer, recognition is often only seen when using the same solvent used as diluent. Often, however, no selectivity is observed even then. The most common reasons for the lack of selectivity in these cases are sample overloading and/or slow mass transfer [59]. Decreasing the sample load leads to an increase in both retention and selectivity. Furthermore,... 

Band tailing causes inferior resolution and reduced precision. Thus conditions resulting in tailing or asymmetric peaks should be avoided. Peak asymmetry or band tailing can arise from several sources partially plugged column frits, void(s) in the column, buildup of sample components and impurities on the column inlet following multiple sample injections, sample overload, solvent mismatch with reference to the sample, chemical or nonspecific interactions (e.g., silanol effects), contamination by heavy metals, and excess void volume in the HPLC system. 

An alternative way to minimize sample overloading is to use higher concentrations of buffer salts. This approach has its limits, as increased salt concentrations lead to increased electrical currents, power dissipation, and thus thermal overloading. 

The second mode is referred to as the sample overload technique and sacrifices column efficiency and resolution in favour of sample throughput. The use of wide diameter columns (>10mm) packed with large microporous particles (>50pm) allows isolation of gram quantities of material in relatively short elution times as the column can be operated at high flow rates without seriously degrading resolution. By its nature this technique... 

Vacuum chromatography is simple, rapid, and convenient. Optimum sample loads are similar to flash chromatography. However, it is not unusual to use sample overload conditions to separate simple mixtures by stepwise gradient elution, or to simplify mixtures for further separation. Under these conditions the sample loads may reach 10 % (w/w), or even higher, of the bed mass. 

The peaks eluted from OTC columns are of very low volume and this can create technical difficulties as the volumes of the flow cells in conventional detectors (5-10 jal) are much greater than the eluted peak volumes. If such detectors were used with OTC columns band dispersion could result, thereby negating the inherent advantages of these columns. Therefore, the flow cell volume should be in the range 0.01-1 /il. Similarly, there is a requirement in capillary systems for both a minimal dead volume and a reduction in the injection volume necessitating the development of speciahsed pre-concentration and sampling techniques. One further restriction of OTC columns is their small internal diameter which can result in sample overloading only quantities of less than 10 ng should be used. 

---