Reversed-phase columns temperature effects

The effect of temperature on the acid base chemistry of the stationary phase can also play a role in separation. Free silanol groups on the stationary phase may exhibit changes in acid base chemistry with temperature [28]. Also, reverse phase columns with amine, amide, or acidic functional groups will be affected by the interaction of the temperature, the ionization state of the stationary phase, the mobile phase acidity, and the ionization state of the solute. Most non-linear van t Hoff plots can be rationalized in these terms, but it is difficult to predict a priori what the effects will be on a given system. Thus, it is important to characterize the system under study if a simple change in temperature produces unexpected effects. 

An isocratic HPLC method for screening plasma samples for sixteen different non-steroidal anti-inflammatory drugs (including etodolac) has been developed [29]. The extraction efficiency from plasma was 98%. Plasma samples (100-500 pL) were spiked with internal standard (benzoyl-4-phenyl)-2-butyric acid and 1 M HC1 and were extracted with diethyl ether. The organic phase was separated, evaporated, the dry residue reconstituted in mobile phase (acetonitrile-0.3% acetic acid-tetrahydrofuran, in a 36 63.1 0,9 v/v ratio), and injected on a reverse-phase ODS 300 x 3.9 mm i.d. column heated to 40°C. A flow rate of 1 mL/min was used, and UV detection at 254 nm was used for quantitation. The retention time of etodolac was 30.0 minutes. The assay was found to be linear over the range of 0.2 to 100 pg/mL, with a limit of detection of 0.1 pg/mL. The coefficients of variation for precision and reproducibility were 2.9% and 6.0%, respectively. Less than 1% variability for intra-day, and less than 5% for inter-day, in retention times was obtained. The effect of various factors, such as, different organic solvents for extraction, pH of mobile phase, proportion of acetonitrile and THF in mobile phase, column temperature, and different detection wavelengths on the extraction and separation of analytes was studied. 

The Effects of Eluent Temperature. m agreement with previous studies (67), both solute retention and separation selectivity were found to increase significantly as the eluent temperature was reduced from 30 to 0 C, as shown in Fig. 6 for the enantiomers of tryptophan. The change is much larger than what is observed on an octadecylsilica reversed-phase column and the improved separation selectivity is attributed to more pronounced inclusion complex formation. Solute retention decreases precipitously when the temperature of the eluent is raised to or above 60 C, an important fact that can be utilized advantageously for the rapid removed, of strongly adsorbed components (such as displacers) from the cyclodextrin-silica column. 

D. V. McCalley, Effect of temperature and flow-rate on analysis of basic compounds in high-performance liquid chromatography using a reversed-phase column,/. Chromatogr. A 902 (2000), 311-321. 

Vasanits, A. and Molnar-Perl, I., Temperature, eluent flow-rate and column effects on the retention and quantitation properties of phenylthiocarbamyl derivatives of amino acids in reversed-phase high-performance liquid chromatography, J. Chromatogr., A, 832,109, 1999. 

In HPLC, a sample is separated into its components based on the interaction and partitioning of the different components of the sample between the liquid mobile phase and the stationary phase. In reversed phase HPLC, water is the primary solvent and a variety of organic solvents and modifiers are employed to change the selectivity of the separation. For ionizable components pH can play an important role in the separation. In addition, column temperature can effect the separation of some compounds. Quantitation of the interested components is achieved via comparison with an internal or external reference standard. Other standardization methods (normalization or 100% standardization) are of less importance in pharmaceutical quality control. External standards are analyzed on separate chromatograms from that of the sample while internal standards are added to the sample and thus appear on the same chromatogram. 

Reversed-phase HPLC can separate polyphenolics of extracts on the basis of polarity. HPLC easily produces better resolution among chemically similar compounds in extracts than conventional chromatographic methods. The operating temperature of the column during reversed-phase HPLC analysis should be controlled for data reproducibility. A change in temperature produces only a minor effect, however, on band spacing in reversed-phase HPLC and produces essentially no effect in normal-phase HPLC (Lee and Widmer, 1996). A range of ambient temperatures is widely used, and elevated temperatures are often applied. The retention times of the peaks are dependent upon the type of column and the combination of various solvents used in the method. 

Most HPLC applications used for phenolic analysis simply allow the room temperature to determine the operating temperature of the column, but elevated temperatures of between 30°C and 40°C are often applied for phenolics and derivatives in apples (14), carrots (15), apple juice (6,13), bilberry juice (16), and for cis-trans isomers of caffeic and p-coumaric acids in wines (17). Generally, a change in temperature has only a minor effect on band spacing in reversed-phase HPLC and has essentially no effect in normal-phase separations. Thermostatic control of the column temperature is generally recommended to provide reproducible retention. 

The pronounced discrepancy between the measured dynamic 15 °C-elution curve and its extrapolated reversible-thermodynamic part, shown in Fig. 7, represents a direct proof of the inadequacy of the reversible Eq. (3) in the dynamic region of the column (PDC-effect). Moreover, the experiment shows immediately that the polymer of the mobile phase has to dissolve in the gel layer within the transport zone to a considerably higher extent than is allowed by the partition function (4) in a reversible-thermodynamic equilibrium between the gel and the sol at the same column temperature. As a consequence, a steady state, i.e. a flow-equilibrium, must be assumed in the system sol/gel within the considered transport zone, governing the polymer trans-... 

Figure 20 shows why Casper s reversible PDC-concept1,2) cannot explain the observed high resolution of the PDC-column at low column temperatures the corresponding reversible contributions to the activation enthalpy of a higher P-mer in the gel phase are too low for such an effect (dashed lines in Fig. 20). 

On the other hand, the lack of internal pore structure with micropellicular sorbents is of distinct advantage in the analytical HPLC of biological macromolecules because undesirable steric effects can significantly reduce the efficiency of columns packed with porous sorbents and also result in poor recovery. Furthermore, the micropellicular stationary phases which have a solid, fluid-impervious core, are generally more stable at elevated temperature than conventional porous supports. At elevated column temperature the viscosity of the mobile phase decreases with concomitant increase in solute diffusivity and improvement of sorption kinetics. From these considerations, it follows that columns packed with micropellicular stationary phases offer the possibility of significant improvements in the speed and column efficiency in the analysis of proteins, peptides and other biopolymers over those obtained with conventional porous stationary phases. In this paper, we describe selected examples for the use of micropellicular reversed phase... 

Fig. 10. Chromatograms illustrating the effect of pairing ions on the separation of the dipeptides Ala-Tyr (1), Pro-Tyr (2), and Leu-Tyr (3) by reversed-phase chromatography. Column /x-Bondapak C flow rate 2 ml/min temperature 20 C eluents A, 5% methanol-95% water-50 mJVf KHjP04-5 mM HjPOi, pH 3.0 B, same as A plus 5 laM n-hexyl sulfonate C, 50% methanol-50% water-50 mW KH1PO4-5 mW HjP04-5 mA/ do-decyl sulfate, pH 3.0. Reprinted with permission from Hearn el al. (29). Copyright by Elsevier Scientific Publishing Co., Amsterdam.

The separation of chiral compounds will be discussed in Chapter 22. However, the separation of diastereomers can be accomplished using achiral stationary phases. Another alternative is the use of chiral columns for the separation of diastereomers in either the reversed-phase or normal-phase mode. The use of achiral bonded phases without chiral additives, such as phenyl and alkyl bonded phases for the separation of diastereomeric pharmaceutical compounds, is acceptable. Different selectivities can be obtained by employing stationary phases containing varying functionalities (phenyl, polar embedded moieties). The effect of aqueous mobile-phase pH, temperature, and type of organic eluent (acetonitrile versus methanol) can also play a dramatic role on the separation selectivity of diastereomeric compounds. 

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