Size Exclusion Chromatography

In size exclusion chromatography (SEC), the selectivity of the separation is not affected significantly by the

Figure 6 Chromatograms of 16 polynuclear aromatic hydrocarbons at 204 atm for different mobile-phase conditions: (A) 0.70 : 30 mol ratio methanol-H2O at 26°C; (B) 0.70 : 0.30 mol ratio methanol-H2O at 60°C; (C) 0.49 : 0.21 : 0.30 mol ratio meth-anol-H2O-CO2 at 26°C; (D) 0.49 : 0.21 : 0.30 mol ratio methanol-H2O-CO2 at 60°C. 1, Benzene; 2, naphthalene; 3, acenphthalene; 4, fluorene; 5, phenanthrene; 6, anthracene; 7, fluoranthene; 8, pyrene; 9, benzo[a]anthracene; 10, chrysene; 11, benzo[b]fluoranthene; 12, benzo[k]fluoranthene; 13, benzo[a]pyrene; 14, dibenzo[a,h]anthracene; 15, benzo[ghi]perylene; 16, indeno[1,2,3-cd]pyrene. (Reproduced with permission from Lee and Olesik (1994).)

Figure 6 Chromatograms of 16 polynuclear aromatic hydrocarbons at 204 atm for different mobile-phase conditions: (A) 0.70 : 30 mol ratio methanol-H2O at 26°C; (B) 0.70 : 0.30 mol ratio methanol-H2O at 60°C; (C) 0.49 : 0.21 : 0.30 mol ratio meth-anol-H2O-CO2 at 26°C; (D) 0.49 : 0.21 : 0.30 mol ratio methanol-H2O-CO2 at 60°C. 1, Benzene; 2, naphthalene; 3, acenphthalene; 4, fluorene; 5, phenanthrene; 6, anthracene; 7, fluoranthene; 8, pyrene; 9, benzo[a]anthracene; 10, chrysene; 11, benzo[b]fluoranthene; 12, benzo[k]fluoranthene; 13, benzo[a]pyrene; 14, dibenzo[a,h]anthracene; 15, benzo[ghi]perylene; 16, indeno[1,2,3-cd]pyrene. (Reproduced with permission from Lee and Olesik (1994).)

choice of mobile phase because the separation is based solely on the entropic partitioning of the solute between the bulk mobile phase and the stagnant solvent in the pores. Therefore, efficient separations are highly desirable in SEC. Often, to increase the total efficiency of a separation, analytical columns are connected in series until the maximum pressure of the chromatographic pumping system is approached. SEC is frequently used at elevated temperatures to improve the solubility of high MIC samples and this also lowers the mobilephase viscosity and allows more columns to be linked together to increase the total efficiency of the separation.

Enhanced-fluidity liquid mixtures can improve the chromatographic performance of SEC without need ing to increase the temperature. When CO2 was added to tetrahydrofuran (THF) for the separation of polystyrene standards, improved efficiency and decreased separation time resulted. For a mobile-phase velocity of approximately 0.8 cms"1, the reduced plate height for polystyrene (Mw 12 600) decreased by a factor of 2 by adding 0.40 mol% CO2 to the THF. In addition, the 40 mol% CO2: 60 mol% THF mixture had a viscosity that was approximately 50% that of pure THF, which resulted in an approximate 50% decrease in the separation time for the separation. However, when greater proportions of CO2 were added to the THF, the solvent strength diminished significantly, which caused ad-sorptive interactions to compete with the exclusion mechanism.

Figure 7 Chromatogramsofthe NISTSRM 1597 coal tar standard using 0.49 : 0.21 : 0.30 mol ratio methanol-H2O-CO2at 26°Cand 204 atm for (A) one and (B) four columns (A) u = 0.143 cm s_1; AP=22.0atm; (B) u= 0.145 cm s_1; AP= 127.5 atm. (Hypersil C18150 x 1 mm packed with 5 ^m diameter particles.) 1, Naphthalene; 2, acenpthalene; 3, fluorene; 4, phenanthrene; 5, anthracene; 6, fluoranthene; 7, pyrene; 8, benz[a]anthracene; 9, chrysene; 10, benzo[b]fluoranthene; 11, benzo[k]fluoranthene; 12, perylene; 13, benzo[a]pyrene. (Reproduced with permission from Lee etal. (1995).)

Figure 7 Chromatogramsofthe NISTSRM 1597 coal tar standard using 0.49 : 0.21 : 0.30 mol ratio methanol-H2O-CO2at 26°Cand 204 atm for (A) one and (B) four columns (A) u = 0.143 cm s_1; AP=22.0atm; (B) u= 0.145 cm s_1; AP= 127.5 atm. (Hypersil C18150 x 1 mm packed with 5 ^m diameter particles.) 1, Naphthalene; 2, acenpthalene; 3, fluorene; 4, phenanthrene; 5, anthracene; 6, fluoranthene; 7, pyrene; 8, benz[a]anthracene; 9, chrysene; 10, benzo[b]fluoranthene; 11, benzo[k]fluoranthene; 12, perylene; 13, benzo[a]pyrene. (Reproduced with permission from Lee etal. (1995).)

Both enhanced-fluidity SEC and high temperature SEC have limits to the scope of their application. Enhanced-fluidity SEC is limited by the solvent strength of the high fluidity mixtures and high temperatures can cause decomposition of the polymer. Therefore the combination of increased temperature and use of enhanced-fluidity SEC might often be the best choice to improve the chromatographic performance in exclusion separations.

By adding 30 mol% CO2 to THF and increasing the temperature from 24 to 80°C, the slope of a plot of reduced plate height versus linear velocity decreases substantially. For example, with styrene as the solute, the C coefficient in the van Deemter equation decreases to a value that is only 17% of the C value when THF at 24°C is the mobile phase (Figure 8). With such a flat slope, SEC separations can be accomplished at high velocities with minimal loss in efficiency.

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