Dynamic thermal focusing of chromatographic separations
There is provided a method for thermally focusing the elution times of compounds of a liquid chromatography system comprising a pre-column and a separation column as well as to provide a liquid chromatography system, wherein the chromatographic separation of a mixture of substances is optimized.
1. A method for thermally focusing an analyte passing through a pre-column and a separation column for liquid chromatography, which method comprises the steps:
operation of the pre-column, during an enrichment phase for enriching a sample in the pre-column, at a first temperature that is lower than ambient temperature and the operating temperature of the separation column and lower than 15° C.;
heating the pre-column, after completion of the enrichment phase, to a second temperature that is higher than the operating temperature of the separation column and higher than 20° C.;
eluting the analyte contained in the pre-column to the separation column;
cooling of the sample to the operating temperature of the separation column;
eluting the analyte contained in the separation column; and
thermally desorbing non-eluted sample from the separation column by increasing the temperature to a temperature that is higher than the second temperature of the pre-column;
wherein the ID of the pre-column is from 150% to 750% of the ID of the separating column.
2. The method according to claim 1 , wherein said second temperature that is at least 10° C. higher than the operating temperature of the separation column.
3. The method according to claim 1 , wherein the first operating temperature of the separation column is gradually raised to a level that is at least as high as the second temperature of the pre-column during elution in order to also facilitate focused separation of analytes that have a strong interaction with the stationary phase.
4. The method according to claim 1 , wherein the first temperature of the pre-column is lower than 5° C., and said second temperature of the pre-column is higher than 45° C.
5. The method according to claim 1 , wherein the first temperature of the pre-column is lower than 2° C., and said second temperature of the pre-column is higher than 55° C.
6. The method according to claim 1 , wherein said second temperature is at least 20° C. higher than the operating temperature of the separation column.
7. The method according to claim 1 , wherein said second temperature is at least 30° C. higher than the operating temperature of the separation column.
8. The method according to claim 1 , wherein said second temperature is at least 40° C. higher than the operating temperature of the separation column.
9. The method according to claim 1 , wherein the first temperature of the pre column is lower than 10° C., and said second temperature of the pre-column is higher than 35° C.
10. The method according to claim 9 , wherein the ID of the pre-column is from 200% to 500% of the ID of the separating column.