Temperature effects, HPLC

This chapter will describe both the theoretical understanding of temperature effects and the practical considerations needed in the laboratory. It will also provide a range of examples of how temperature has been applied to improve separation with emphasis on applications since the previous edition of the Handbook of HPLC [12], It is not the intent to provide a complete review of all applications. 

There are a number of limitations on the use of extremes of temperature in HPLC. Clicq et al. [91] note that instrumental issues become increasingly limiting as one goes to very high temperatures and flow rates. They suggest that most separations will occur below 90°C where there are less instrumental constraints. As detailed below, column bleed can limit the selection of columns. Highspeed separations require a faster detector response than many systems allow and constrain extra column volume. This is especially true for narrow bore columns and sub-2 jam particles. In many cases, the additional speed gained above the temperature limits of commercial HPLC ovens will not be worth the additional expense and complexity required. For macromolecules, the effect of extreme pressure can also impact retention time as noted by Szabelski et al. [92]. 

The high-pressure cells and temperature control units are similar to the ones described by Betts and Bright (29). Samples for analysis were prepared by directly pipetting the appropriate amount of stock solution into the cell. To remove residual alcohol solvent, the optical cell was placed in a heated oven (60 °C) for several hrs. The cell was then removed from the oven, connected to the high-pressure pumping system (29), and a vacuum (50 pm Hg) maintained on the entire system for 10-15 minutes. The system was then charged with CF3H and pressurized to the desired value with the pump (Isco, model SFC-500). Typically, we performed experiments at 10 /xM PRODAN and there was no evidence for primary or secondary interfilter effects. HPLC analysis of PRODAN subjected to supercritical solvents showed no evidence of decomposition or additional components. 

Fig. 3 Influence of temperature in HPLC and CE. In HPLC, this effect was studied using a mobile phase consisting of 25.0 mM citrate buffer, 45.0% methanol, 4.00 mM sodium octane sulphonate, pH=3.25, and T=24.0°C, 30.0°C, and 39.0°C. In CE, the electrolyte used was 25.0 mM citrate buffer, with pH=3.70, 14.0 kV voltage, and T=25.0°C, 30.0°C, and 40.0°C. (View this art in color at www.dekker.com.)...

Martin, J. Mendez, R. Negro, A. Effect of temperature on HPLC separations of penicillins. J.Liq.Chromatogr, 1988,11, 1707—1716 [simultaneous ampicilUn, cloxadUin, penicillin G, penicillin V, piperacillin colvunn temperature 15-55°]... 

The so-formed tetracyclic products 117a-g are of particular note due to their unusual rotameric properties the bridgehead aromatic group encounters significant steric barrier to rotation about the sp -sp carbon-carbon bond, despite its lack of orffto-substituents. Indeed in some instances, the rotamers can be separated by low-temperature preparative HPLC These are the first compounds in which such an effect has been noted. 

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). 

So we have seen how complex the influence of temperature in HPLC can be, but we have learned so many helpful rules that the user should really apply temperature effectively in method optimization. Especially the combination of temperature and mobile phase composition maybe an excellent wayto optimize methods. Temperature should always be considered as an important method variable. If a method uses temperatures below 40 °C, the reason for this should always be challenged. There are three important conclusions for this chapter that should take away all myths and misconceptions about the use of temperature in method speed-up. 

The temperature, in HPLC usually between 10 and 60 °C, is an effective additional optimization parameter. The corresponding Excel table (Table 3.4 shows the table header) was developed by U.D. Neue. By entering the desired temperature in cell B3, A oo and S are adjusted according to the above-mentioned equations. 

Many factors, such as solvent viscosity, diffusion rates, and column type, contribute to temperature effects in protein RP-HPLC. Temperature may also directly affect protein conformation or aggregation. For example, whereas fresh extracts of maize zein resolved well upon RP-HPLC [42], even at low temperature, purified zeins, especially after lyophilization and storage, separated poorly [53] (Fig. 5). Resolution was restored, however, at 70 C. These results suggest that zeins may aggregate and polymerize via hydrogen bonds between glutamine... 

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