Column lifetime

Protecting the columns with guard columns increases the lifetime. 

1 Issaq, H.J. (ed.) (2002) A Century of Separation Science, Marcel Dekker, Inc. 

2 Cassiano, N.M., Lima, V.V., Oliveira, R.V., de Pietra, A.C., and Cass, Q.B. (2006) Development of restricted-access media supports and their application to the direct analysis of biological fluid samples via high-performance liquid chromatography. Analytical and Bioanalyticcd Chemistry, 384. 1462. 

3 Svec, F. and Tennikova, T. (Eds) (2012) Monoliths, Journal of Separation Science, 

De Beeck, J.O., Eeltink, S., Detobel, F., Gardeniers, H., and Desmet, G. (2012) Separations using a porous-shell pillar array column on a capillary LC instrument Journal of Separation Science, 35, 

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Other parameters that have been used to characterize column performance include the column pressure drop and the column flow resistance.1617 The column pressure drop is simply the difference in pressure observed when the column is or is not in-line. The column flow resistance normalizes for particle diameter, solvent viscosity, and column length. One may also wish to compare issues of cost per analysis and column lifetime in evaluating a column.18... 

The pH used in the first C18 separation dimension was rather high (pH 10), however, no peptide loses or carryover, due to on-column precipitation, were observed. Peak shape was comparable to peptide analysis at low pH. Modern stationary phases, based on hybrid silica and stable alkyl bonding chemistry, are well suited for chromatography at extreme pH without compromising column lifetime or analysis-to-analysis reproducibility (Wyndham et al., 2003). 

The PO mode is a specific elution condition in HPLC enantiomer separation, which has received remarkable popularity especially for macrocyclic antibiotics CSPs and cyclodextrin-based CSPs. It is also applicable and often preferred over RP and NP modes for the separation of chiral acids on the cinchonan carbamate-type CSPs. The beneficial characteristics of the PO mode may arise from (i) the offset of nonspecific hydrophobic interactions, (ii) the faster elution speed, (iii) sometimes enhanced enan-tioselectivities, (iv) favorable peak shapes due to improved diffusive mass transfer in the intraparticulate pores, and last but not least, (v) less stress to the column, which may extend the column lifetime. Hence, it is rational to start separation attempts with such elution conditions. Typical eluents are composed of methanol, acetonitrile (ACN), or methanol-acetonitrile mixtures and to account for the ion-exchange retention mechanism the addition of a competitor acid that acts also as counterion (e.g., 0.5-2% glacial acetic acid or 0.1% formic acid) is required. A good choice for initial tests turned out to be a mobile phase being composed of methanol-glacial acetic acid-ammonium acetate (98 2 0.5 v/v/w). 

In reversed-phase HPLC, column temperature is a strong determinant of retention time and also affects column selectivity. A column oven is therefore required for most automated pharmaceutical assays to improve retention time precision, typically at temperatures of 30-50°C. Temperatures >60°C are atypical due to concerns about thermal degradation of the analytes and column lifetimes. Exceptions are found in high-throughput screening where higher temperatures are used to increase flow and efficiency. Ambient or snb-ambient operation is sometimes found in chiral separations to enhance selectivity. Column ovens... 

The pore diameter of the silica support should lie between 100 and 500 A. With pore diameters below 100 A the values-of retarded peaks are much higher than for pore diameters of 100 A or larger. At pore diameters above 500 A, even at moderate eluent velocities, mechanical erosion of the stationary liquid phase dimi.nishes column lifetime. 

For purification, scale-up considerations are important even in the earliest phases of development. It is important to avoid the use of purification techniques of limited scale-up potential even for early clinical production because thorough justification of process changes and demonstration of biochemical comparability are necessary prior to product licensure. For successful scale-up, it is important to understand the critical parameters affecting the performance of each purification step at each scale. Conversely, it is important to verify that the scaled-down process is an accurate representation of the scaled-up process, so that process validation studies, such as viral clearance and column lifetime studies, can be performed at the laboratory scale. 

To increase the lifetime of the column, a precolumn or guard column is often used. The guard column is usually filled with the same stationary phase as the analytical column is between 0.4 to 1 cm long. The guard column is inexpensive and can be changed periodically. It is also recommended to pass samples through a filter with a porosity of less than 0.5pm (Fig. 3.6) before analysis to increase the column lifetime. 

Column. Although the advantage of the normal-phase column is the high resolution in the separation of all tocopherols and tocotrienols, the silica packing material in a normal-phase column is very reactive to strong polar chemicals. Any high-polarity compounds in the sample extract and mobile phase will diminish the column performance and shorten the column lifetime. It is very important to ensure that the sample extract is free of water and metal ions, and it is also advisable to regenerate the column routinely. 

Chromatographic resins contain a great deal of surface area, and a clear understanding of what is occurring on the surface in terms of carryover of risk factors is not always possible. Cleaning and sanitization, as well as column lifetime, are issues that must be addressed in development and validated at pilot or full scale. 

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