Column ageing

Van Nederkassel, A. M., Aerts, A., Dierick, A., Massart, D. L, Vander Heyden, Y. Fast separations on monolithic silica columns method transfer, robusmess and column ageing for some case smdies. /. Pharm. Biomed. Anal. 2003, 32, 233-249. 

The use of CSPs provides several advantages such as the speed of analysis, the possibility of analyzing or purifying enantiomers in complex mixtures, the reproducibility of the analysis and its flexibility. However, a disadvantage of these CSPs is their relative low stability, i.e., they can be subject to relatively fast column aging. 

Temperature, reagent source, instrument, column age, column type, pH, mobile phase, detector type... 

The main objective of system suitability is to recognize whether or not system operation is adequate given such variability as chromatographic columns, column aging, mobile-phase variations, and variations in instrumentation. 

It is important, first, to realize that efficiency is not a function solely of the column. Bad extracolumn parameters, such as detector cell volume or tubing diameters, can make the best column in the world look terrible. Second, efficiency measurements are very poor ways of comparing or purchasing columns unless all other parameters are constant. Many columns are bought and sold because they have a higher plate count than someone else s column. The efficiency calculations could have been made with different equations, on different compounds, on different machines, at different flow rates, all of which will have a profound effect on efficiency. The only valid use of plate counts that I have found is in column comparisons where all other variables are equal, or in following column aging over a period of days or months. 

Column problem. (Not a common cause of erratic retention. As a column ages, retention times gradually decrease.)... 

Klausen J, Ranke J, Schwarzenbach RP. Influence of solution composition and column aging on the reduction of nitroaromatic compounds by zerovalent iron. Chemosphere 2001 44 511-517. 

As the column ages, it becomes necessary to increase the molarity of solvent A to continue to separate arginine and threonine. The addition of 3 M sodium acetate at pH 5.5 in 5 ml increments, as needed, will raise the molarity a sufficient amount. 

A neat reference mixture was prepared that contained 17 pure NHCs plus pyridine and quinoline as retention index markers (Table 1 Fig. 2). Relative amounts were adjusted to give approximately equal peak sizes. Triplicate analyses were performed on the 65 °C headspace above this mixture and above several wastewaters. To minimize any effect of column aging on peak retention times, the reference and wastewater replicates in each test series were alternated. Reproducibilities were excellent with standard deviations averaging 0.12 retention units overall and 0.05 retention units for the reference mixture. Reference-peak to sample-peak correlations were performed by a variety of statistical procedures including z tests. Student-t tests and, later, the procedure described in Section 3.2. 

The effects of sample preparation variability on assay variability are well known and should be considered when acceptable variations within the analytical method are set in place. Pipetting errors, sample collection errors, time, and temperature of sample preparation may all contribute to slight differences in the amount of analyte extracted or prepared within a given sample. Additionally, HPLC instrumentation may also exhibit injector or flow rate variability leading to differences in retention times and peak responses. Column aging and buildup of lipids and proteins within the HPLC components may ultimately cause pressure fluctuations and mechanical problems if the instrument is not properly maintained. 

Column aging = 4 days at 240°C under normal flow rate. Column temperature = 185 °C... 

Other Cobarm Aging Mechanisms Her we will discuss a few miscellaneous column aging mechanisms that have not been covered in other sections. These mechanisms are specific to certain columns, but the principles are generally instructive. 

Peak asymmetry is a common practical measure of the quality of a column. As columns age, the peak asymmetry usually deteriorates thus, one observes tailing peaks. Peak tailing is undesirable, since it can affect the quality of peak integration, especially when the ignal-to-noise ratio is low or when peaks are only partially resolved. This tailing can have multiple causes, as will be discu ed in later sections. 

Hydrolysis of the stationary phase or other kinds of column aging may even occur during methods development. A finished method that needs to be used for a long time or by other workers should therefore always be verified on a new column, preferentially prepared from the same batch of packing. Column kits for this purpose are available fi om some manufacturers. 

In reversed-phase chromatography, tailing may show up or increase as the column ages. Tliis is due to a partial and slow hydrolysis of the stationary phase. This process exposes additional silanols, which cause or increase peak tailing. This is a natural part of the column aging process. If the column lifetime is too short as a result of this effect, you may want to incorporate amine modifiers into the mobile phase, even if they are not needed with a brand-new column. 

In this section, we will discuss various causes of retention-time problems and examine their causes. We will discuss the influence of temperature, mobile-phase composition, column contamination, column aging, and various other topics. We will also briefly discuss again column-to-column reproducibility. 

The chapter is divided into two sections. In the first section we deal with retention time problems that are random in nature. The second section then covers the cases where the retention times are drifting in one direction only. If we can differentiate between these cases, we can immediately exclude some causes of the observed problem. For example, random fluctuations of retention times are not a symptom of column aging. 

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