Temperature effects chromatography

So far the plate theory has been used to examine first-order effects in chromatography. However, it can also be used in a number of other interesting ways to investigate second-order effects in both the chromatographic system itself and in ancillary apparatus such as the detector. The plate theory will now be used to examine the temperature effects that result from solute distribution between two phases. This theoretical treatment not only provides information on the thermal effects that occur in a column per se, but also gives further examples of the use of the plate theory to examine dynamic distribution systems and the different ways that it can be employed. 

Lochmiiller, C. H., Moebus, M. A., Liu, Q., and Jiang, C., Temperature effect on retention and separation of poly(ethylene glycoljs in reversed-phase liquid chromatography, /. Chromatogr. Sci., 34, 69, 1996. 

S. J. Marin, B. A. Jones, W. D. Felix, and J. Clark, Effect of high-temperature on high-temperature liquid chromatography column stability and performance under temperature-programmed conditions,/. Chromatogr. A 1030 (2004), 255-262. 

C. Panagiotopoulos, R. Sempere, R. Lafont, and R Kerherve, Sub-ambient temperature effects on the separation of monosaccharides by high-performance anion-exchange chromatography with pulse amperometric detection-application to marine chemistry, J. Chromatogr. A 920 (2001), 13-22. 

In gas chromatography the most successful solution to the resolution of complex mixtures has been that of programmed temperature gas chromatography. In this technique all partition ratios are decreased over the entire length of the column with time. When a separation is started at a relatively low temperature, the partition ratios of most components are so large that they are almost completely immobilized, or frozen, at the inlet of the column. The components with small partition ratios can move normally along the column. As the temperature is raised, partition ratios decrease, and the other solutes successively reach temperatures at which they have vapor pressures that enable them to be eluted at a reasonable rate. In effect, each compound tends to be eluted at its optimum temperatme for the heating and flow rates chosen. 

If the dioxetane is a solid, recrystallization is obviously the method of choice. It is critical to use metal-free solvents since traces of metal ions can lead to extensive decomposition. In the case of volatile crystalline dioxetanes, prepurification via sublimation can be advantageous. In some systems low temperature column chromatography is effective. In the case of liquid 1,2-dioxetanes, unless they are sufficiently volatile for low-temperature distillation such as 3,3-dimethyl-1,2-dioxetane, repeated low-temperature column chromatography is the only means of purification. 

For reproducible separations by IPC, it is important to thermostat the column. Temperature effects in IPC are more important than in some other liquid chromatography methods. 

The programming procedure usually involves three stages. An initial isocratic period is introduced to efficiently separate the early eluting peaks with adequate resolution. The isocratic period is followed by a linear increase in column temperature with time, which accelerates the well-retained peaks so that they also elute in a reasonable time and are adequately resolved. The effect of linear programming can be calculated employing appropriate equations and the retention times of each solute predicted for different flow rates (see the entry Programmed Temperature Gas Chromatography). To do this, some basic retention data must be measured at two temperatures and the results are then employed in the retention calculations. The tempera-... 

Since this review was completed, a number of interesting developments have been obtained in this laboratory and elsewhere in various protein fractionation techniques at subzero temperatures. Let us mention a work on isoelectric focusing and electrophoresis by M. Per-rella, A. Heyda, A. Mosca and L. Rossi-Bemardi (Anal. Biochem., in press) a work by C. Le Peuch and C. Balny on the sequential elution of proteins bound by hydrophobic interaction chromatography [FEBS Lett. 87,232(1978)] a work by K. Andersson, Y. Benyamin, P. Douzou, and C. Balny about organic solvents and temperature effects on desorption from immunoadsorbents (J. Immunol. Methods, in press, 1978). 

Figure 31-7 Effect of temperature on gas chromatograms, (a) Isothermal at 45°C (b) isothermal at I45°C (c) programmed at 30°C to 180°C. (From W. E. Harris and H. W. Habgood, Programmed Temperature Gas Chromatography, p. 10. New York Wiley. 1966. Reprinted with permission of the author.)...

Here we must distinguish between the effect of temperature both before and after the development (drying of the plate) and the effect of heat during chromatography. In the first case, the choice of a more suitable solvent system can prevent loss or decomposition of volatile or temperature-sensitive test substances. In the second case, development should be performed at lower temperatures, e.g. in a TLC Thermo-Box (see Section 4.2.3 Effect of Temperature in Chromatography ). 

The high salt concentrations used can present a challenge for the HPLC equipment. Pumps with seal-wash are prrferred. If chloride buffers are used, HPLC systems with nonmetallic fluid paths are recommended. It is also advisable to flush the salt solution out of the system when it is not in use. Temperature and pH can affect the separation and should therdbre be controlled. But both the temperature effect and pH effect are smaller than in other forms of chromatography (with the exception of size-exclusion chromatography). 

Minimizing the temperature effects discussed above could be obtained with the use of polymer micelles or polymer surfactants [81-83], whose CMC is zero, and even in nonaqueous solvent, the micelle is stable. Although several polymer surfactants are commercially available, no such surfactant is widely accepted, probably because SDS, CTAB, or CTAC, and bile salts are superior to polymer surfactants as the pseudostationary phase in MEKC. Although microemulsion electrokinetic chromatography (MEEKC) is not discussed in this chapter but covered in Chapter 4 by Altria and colleagues, a similar optimization strategy to that in MEKC applies to MEEKC [84-86]. Since... 

C. D. Wick, J. I. Siepmann, W. L. Klotz and M. R. Schure, Temperature effects on the retention of -alkanes and arenas in helium-squalane gas-liquid chromatography experiment and molecular simulation, /. Chromatogr., A, 2002, 954, 181-190. 

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