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that is quoted for most organic solvents in liquid chromatography is about 110 x 10-6


. If the compressibility of the liquid is not set correctly, then the pump will potentially deliver the wrong flow rates and cause an increase in the level of noise originating from the pump. Compressibility settings are unlikely to change during an assay but might create retention shifts when instrument systems are changed.


Atm-1 Pump seals and pistons


Pump seals and pistons are often thought to be a cause of incorrect flow delivery, however in reality it is rarely the seals or piston. If the seals are suspected, then the installation of new ones should be done. Great care should be taken, particularly if very high pressures are being employed, as the seals have to be bedded in properly. With the pistons, the use of buffers can result in small scratches which can cause leaks and make accurate solvent delivery impossible.


Solvents


Mobile phase selection can play an important role in the reproducibility of the retention time for an analyte. Particular attention should be paid to the equilibration time and also the sensitivity of the analytes to effects such as pH and injection solvent. The following sections will discuss the implications of incorrect selection of solvent, either in mobile phase or injection solvent.


Equilibration issues


Another consideration is the equilibration time that is given at the end of the chromatographic run. This has been discussed in a previous Chromatography Today helpdesk articles, and so it will not be discussed in great detail here. However, this tends to become significant when the method is being transferred between different systems or columns. Often, if there is an issue this will be highlighted by poor peak shapes, with the affected peaks typically fronting. Figure 2 shows the effect of not having the correct equilibration time with the first injection looking fine, but with shifts in the retention time being observed with subsequent injections.


Figure 3. Effect of changing the pH on acidic, basic and neutral compounds, printed with kind permission M. Euerby.


Injection solvent


The final issue to consider is the injection solvent. The choice of the injection solvent may be affected by analyte solubility and also by any sample pre-treatment that may have occurred prior to the separation. It is important to understand that chromatography is fundamentally based on a partitioning effect between a mobile phase and a stationary phase, and that when the compound is injected the mobile phase is formed partially by the injection solvent. For very strong injection solvents, the compounds of interest will remain predominantly within the mobile phase and not interact with the stationary phase, with the retention times shifting towards the solvent front as a consequence. As with the incorrect use of equilibration times this may also result in poor peak shapes. As with all of the previous causes of retention time shift, it would be anticipated that this would affect both enantiomeric forms equally, however if only one enantiomer is being seen, which may reflect the synthetic pathway, it can result in a large uncertainty with the assay. Other injection considerations such as pH, buffer strength and matrix components can affect the potential partitioning between of the enantiomers and the stationary phase.


Conclusion


With chiral separations, where resolution is often marginal between enantiomers, there are a variety of potential pitfalls that separation scientists should be careful of. Being aware of the parameters that can cause a shift in the retention time will ensure the development of robust assays to ensure that good decisions are made based upon accurate results.


Figure 2. The effect of not equilibrating the column effectively before an injection. pH


Stability of the retention time for any compound, whether it is chiral or achiral, can be very dependent on the variation of the log D as a function of the pH. In particular, it is important to keep the pH away from any pKa points for that the compound, since this will result in inherently unstable retention times. Although this may not affect the elution order, this will have a detrimental effect on the accurate determination of the any compound. Figure 3 illustrates the effect that pH has on log k. It can be seen for neutral compounds that there is no appreciable effect.


It should be noted that modern pumps have built-in diagnoses or there are sophisticated check-out and performance verification procedures provided by manufacturers, so many of the suggestions in this article may not be so pertinent to newer pumps. It should also be noted that these guidelines are relevant for all HPLC separations and not specifically for chiral separations, where mostly isocratic, premixed solvent are used.


References 1 J. Dolan, Jun 01, 2008 LCGC North America 26 (6) 532–538,


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