Programmed elution —

Figure 6.8 Separation of Triton X-114 by SFC using prograMmed elution on a 10 cm x 2 mm I.D. Nucleosil column, 3 micrometer packing, at 170 C with UV detection at 278 nm. The separation on the left was performed under isobaric conditions at 210 bar with a mobile phase of carbon dioxide -t- methanol (2 + 0. 5) ml/min. The separation in the center was obtained using a ccmt. sition gradient from 0.025 to 0.4 ml/mln over 8 min with other conditions as above. The separation on the right was obtained using a pressure program from 130 to 375 bar over 8 min with the same mobile phase used for the isobaric sepeuration. (Reproduced with permission from ref. 57. Copyright Preston Publications, Inc.)...

Figure 2.5 Flow rate programming elution of aromatic compounds. Experimental conditions as Figure 2.2 except the flow rate. Flow rate increased from 1 to 2 ml min-1.

The important aspect of programmed elution techniques with respect to optimization criteria is that the peak width does not increase with the retention time in a manner corresponding to eqn.(1.16). In programmed analysis a constant peak width is wanted throughout the chromatogram (see section 6.1). 

Another aspect of programmed elution that will affect the quality of the chromatogram is the variation ( drift ) of the baseline during the program. Methods to reduce the baseline drift (or blank signal) and other aspects of programmed analysis will be discussed further in chapter 6. 

The effects of changes in a parameter during a programmed elution will generally be the product of two independent factors ... 

Certainly different select vities and efficiencies are observed with the two chromatograms. The resolution is favor2Jt>le in the temperature programmed elution for the volatile compounds, the contrary is true for the least volatile substances. 

Figure 9.14 Pressure-programmed elution of a mixture of polystyrenes of M 580,2100,4250 and 9000. Column 58 cm x 0.2 mm I.D. Develosil 100 (10 /zm) mobile phase, 5% ethanol in n-hexane...

An alternative to programmed elution can be the coupling of two (or even more) columns with different stationary phases. This technique is known as multidimensional chromatography. The first column, for example, will separate the sample according to polarity groups. Then selected fractions are switched online to the second column, where the fine separation into chemically pure compounds takes place. 

Even excellent chromatographic columns have rather low peak capacities (e.g., = 86 for N = 20 000 and max = 10). The number of adequately resolved peaks in a real chromatogram is in fact markedly lower because their k values are distributed in an unpredictable manner and because the peaks differ in size (see Statistical Peak Overlap below). A way to overcome this limitation is programmed elution, i.e., running temperature programs or solvent gradients. 

Pavel, J. Cesla, P. Hajek, T. Vohralik, G. Vynuchalova, K. Fischer, J. Optimization of separation in two-dimensional high-performance liquid chromatography by adjusting phase system selectivity and using programmed elution techniques. J. Chromatogr. A, 2008, 1189, 207-220. 

Gacia-Lavandeira, J. Martmez-Pontevedra, J.A. Lores, M. Cela, R. Computer-assisted transfer of programmed elutions in reversed-phase high-performance hquid chromatography. J. Chromatogr. A, 2006,1128, 17-26. 

Packed column (SE-30, 12-ft) temp, programmed (eluted complexes)... 

Gregory and Feldstein (94) developed an ion-paired, reversed-phase HPLC method for individual Be vitamers extracted with sulphosalicylic acid from different foods. Using a ternary solvent program, elution of nutritionally active Be vitamers from the analytical column was complete within 30 min. PLP was determined as its hydroxysulfonate derivative, following postcolumn introduction of a buffered solution of sodium bisulfite. This method was found suitable for vitamin Be analysis in foods of both plant and animal origin. Recoveries for PLP and PL from pork loin were <90% it was suggested that these vitamers were not completely released from muscle proteins, even in the presence of 5% sulfo-salicylic acid. 

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