The Effect of Temperature on Retention

As with any equilibrium constant, the effect of temperature on K (distribution coefficient) is described by the van t Hoff equation  

We have already assumed that Vstat mob constant and thus have seen that K is proportional to k. Extending this assumption, if and V ,ob 

Making the approximation that, over the fairly narrow range of temperatures at which HPLC is used AH will be independent of temperature, we can take the integral 

SOME PRACTICAL WARNINGS REGARDING VARIATIONS IN TEMPERATURE (INCLUDING USE OF COLUMN HEATERS) 

Manymethods now specify the use of a column heater to run separations at higher temperatures, thus decreasing retention times and obtaining more rapid results. This can be very effective, but it is important to remember the following  

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The effect of temperature on retention time was investigated by Scott and Reese (3), who measured the retention volume of the solutes o-dinitro-benzene, 2-ethoxy naphthalene and p-chlorophenatole over a range of temperatures. The chromatographic conditions used are as follows,... 

FIGURE 4.11 The effect of temperature on retention and selectivity in packed column SFC and open tubular GC. Conditions 15 cmx250 xm ID capillary packed with 5 xm porous (300 A) silica particles encapsulated with P-CD polymethylsiloxane (10% w/w) and end capped with HMDS, 160 atm, CO2, FID, 10 cmx 12 xm ID restrictor. (B) 25 mx250 xm ID cyano-deactivated capillary cross-linked with P-CD polymethylsiloxane (0.25 xm d ) He FID. (Adapted from Wu, N. et al. 2000. J. Microcol. Sep. 12 454-461. With permission.)... 

If the HPLC mobile phase is operated close to the pA of any solute or if an acidic or basic buffer is used in the mobile phase, the effects of temperature on retention can be dramatic and unpredicted. This can often be exploited to achieve dramatic changes in the separation factor for specific solutes. Likewise, the most predictable behavior with temperature occurs when one operates with mobile phase pH values far from the pA s of the analytes [10], Retention of bases sometimes increase as temperature is increased, presumable due to a shift from the protonated to the unprotonated form as the temperature increases. As noted by Tran et al. [26], temperature had the greatest effect on the separation of acidic compounds in low-pH mobile phases and on basic compounds in high-pH mobile phases. McCalley [27] noted anomalous changes in retention for bases due to variations in their pA s with temperature and also noted that lower flow rates were needed for optimal efficiency. 

The effect of temperature on retention has been described experimentally,(4-8) but the functional dependence of k with temperature has only recently been described.W A thermodynamic model was outlined relating retention as a function of temperature at constant pressure to the volume expansivity of the fluid, the enthalpy of solute transfer between the mobile phase and the stationary phase and the change in the heat capacity of the fluid as a function of temperature.(9) The solubility of a solid solute in a supercritical fluid has been discussed by Gitterman and Procaccia (10) over a large range of pressures. The combination of solute solubility in a fluid with the equation for retention as a function of pressure derived by Van Wasen and Schneider allows one to examine the effect of solubility on solute retention. 

The effect of temperature on retention was studies using n-hexadecane on a 20 m, 50ji I.D. fused silica capillary column coated with an OV-17 phase using FID detection. The OV-17 was cross-linked in-situ to decrease its solubility in the supercritical fluid. The stationary phase film thickness was calculated to be 0.25pm. The... 

A similar linear logarithmic relationship, known as a van t Hoff plot, usually exists between adjusted retention data and the reciprocal of column temperature in gas, liquid (constant composition) and supercritical fluid (constant density) chromatography. The effect of temperature on retention is based on the Gibbs-Helmholtz equation and has a sound thermodynamic basis, Eq. (1.9)... 

The effects of temperature on carotenoid content can be considered from three perspectives (1) evaluation of stability or retention of carotenoids, (2) study of the chemical changes (isomerization, oxidation, epoxy-furanoid rearrangement), and (3) their effects on the nutritional value and other carotenoid actions in humans. The first two topics are discussed in the following sections. The third is presented in Section 3.2.4.1 of Section 3.2. 

The effect of temperature on the RP-HPLC behaviour of /(-carotene isomers has been extensively investigated and the results were employed for the separation of carotenoids of tomato juice extract. Carotenoids were extracted from food samples of 2g by adding magnesium carbonate to the sample and then extracted with methanol-THF (1 1, v/v) in a homogenizer for 5min. The extraction step was repeated twice. The collected supernatants were evaporated to dryness (30°C) and redissolved in methanol-THF (1 1, v/v). Separations were performed on a polymeric ODS column (250 X 4.6 mm i.d. particle size 5/.an). The isocratic mobile phase consisted of methanol-ACN-isopropanol (54 44 2, m/m). The flow-rate was 0.8 or 2.0 ml/min. The effect of temperature on the retention times of lycopene and four /(-carotene isomers is shown in Table 2.11. The data indicated that the temperature exerts a considerable influence on the retention time and separation of /(-carotene isomers. Low temperature enhances the efficacy of separation. 

The effect of temperature on the retention of a series of -methyl amino acids was investigated on a TE CSP, by using either subambient or elevated temperatures (1.5-50°C) [93]. Linear van t Hoff plots were observed in the studied temperature range, and the apparent changes in enthalpy entropy (AS°), and Gibbs free... 

Rojkovieova, T. et al.. Study of the mechanism of enantioseparation. Vll. Effect of temperature on retention of some enantiomers of phenylcarbamic acid derivates on a teicoplanin aglycone chiral stationary phase, J. Liq. Chrom. Rel. TechnoL, 27, 1653, 2004. 

Column pressure usually has little effect on enantioselectivity in SFC. However, pressure affects the density of the mobile phase and thus retention factor [44]. Therefore, similar to a modifier gradient, pressure or density programming can be used in fast separation of complex samples [106]. Later et al. [51] used density/temperature programming in capillary SFC. Berger and Deye [107] demonstrated that, in packed column SFC, the effect of modifier on retention was more significant than that of pressure. They also showed that the enhanced solvent strength of polar solvent-modified fluid was nof due fo an increase in densify, caused by fhe addition of fhe liquid phase modifier, buf mainly due fo fhe change in composition. 

Gant et al. (175) examined the effect of temperature on resolution and on selectivity, retention factors, and plate number, which determine the magnitude of resolution. They found that these data can be used together with the lempeniliire dependence of solvent viscosity to optimize iinaivsis rate with required resolution. This is of particular interest when RFC is used for automated repetitive analyses of lar e numbers of samples. 

In addition to the mobile phase composition, the effect of other parameters such as temperature, flow rate, pH, and structure of the analytes were also studied, but only a few reports were available in the literature. In 1995, Lin and Maddox [66] studied the effect of temperature on the chiral resolution of amino acids and esters. The temperature was varied from 5°C to 25°C and it was reported that the resolution improved at low temperature. Hyun et al. [48-50,67] carried out the effect of temperature on the chiral resolution of amino alcohols, amines, fluoroquinolones, and other drugs. Again, lowering of temperature resulted in better resolution. The effect of temperature on the chiral resolution of phenylalanine, phenylglycine, and 2-hydroxy-2-(4-hydroxy-phenyl)-ethyl amine is shown in Table 5 [50], which indicates an increase in retention factors at lower temperature, but the best separation occurred at 20°C. These experiments indicated the exothermic nature of chiral resolution on CCE-based CSPs. Lin and Maddox [66] also studied the effect of flow rate on the chiral resolution of... 

The effect of temperature on solute retention in SFC has been investigated experimentally (5,8) and described theoretically.(9)... 

Figure 3 shows the effect of temperature on the capacity factor of p-nltroanillne, from 0°C to 77°C. A mobile phase consisting of 10 methanol/water was employed. The retention at 0°C was 23.62 min. while at 77°C was 2.28, a ten fold decrease. This decrease in retention may be attributed to many factors such as increased solubility of the p-nitroaniline with increase in temperature, which results in less solute-stationary phase, and an increase in solute-mobile phase Interactions increase in mass transfer, and decrease in the pressure. Also the binding constant of any solute with cyclodextrin goes to zero at 80°C (11). 

The next variable investigated was the effect of temperature on the analyte retention. The effect of temperature on the retention and selectivity of the para and ortho isomers at wpHs 2, 8, and 8.6 was studied (figures 8-31, 8-32, and 8-33). The effect of temperature could be used to optimize the run time and the apparent efficiency of the separation. At a buffer pH of w2.0, the effect... 

This difference is also evidenced by the temperature dependence of the number of theoretical plates for the two enantiomers. The template molecule shows hardly any temperature dependence because of increased two-point binding with slow binding rates at higher temperatures, whereas the number of theoretical plates for the second enantiomer increases rapidly, as expected (see Fig. 4.3a) [21]. The effect of concentration on retention is also very different for two enantiomers (see Fig. 4.3b). The wrong enantiomer does not show much effect, whereas retention of the template molecule is greatly increased at lower concentrations, at which only the most selective cavities are occupied by the template molecule. The selectivity therefore increases sharply [49]. It is interesting to notice that this chromatographic... 

Fig. 4,3. Differences in the effect of temperature on the number of theoretical plates and of the amount of chromatographed substance on the retention of template molecules and their enantiomers, (a) Temperature dependence of the number of theoretical plate Wh) in the resolution of D-la and L-la on a polymer imprinted with 1 [21]. (b) Dependence of the capacity factor k on the amount of chromatographed substance in the resolution of l- and of D-phenylalanine anilide on a polymer imprinted with L-phenylalanine anilide [49],...

In addition to molecular weight, thermal FFF is used to measure transport coefficients. For example, the measurement of thermodiffusion coefficients is important for obtaining compositional information on polymer blends and copolymers (see the entry Thermal FFF of Polymers and Particles). Thermal FFF is also used in fundamental studies of thermodiffusion because it is a relatively fast and accurate method for obtaining the Soret coefficient, which is used to quantify the concentration of material in a temperature gradient. However, the accuracy of Soret and thermodiffusion coefficients obtained from thermal FFF experiments depends on properly accounting for several factors that involve temperature. In order to understand the effect of temperature on transport coefficients, as well as the effect on thermal FFF calibration equations, a brief outline of retention theory is given next. 

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