( bsequently becomes more tedious with th...)Separation of Peptides by Hydrophilic Interaction Chromatography Using TSKgel Amide-80

The effect of ACN concentration on peptide retention was also examined. Opposite of RPLC, increasing the percent organic increases retention of polar compounds with HILIC stationary phases. As evident in Figure 2 , the higher the initial concentration of ACN, the better the resolution. Resolution of all peptides is possible above an 85% initial ACN concentration. Furthermore, recovery of the various peptides averaged 92% as shown in Table 1 . Recovery with the Amide-80 column was higher when compared to a column packed with an 80 pore size, end capped ODS material(3).

Reproducibility and repeatability were determined by examining both the variability between injections and the variability between columns. Ten replicates of a peptide mixture were injected on a new TSKgel Amide-80 column and a TSKgel Amide-80 column that had seen over 500 injections. As expected, the coefficient of variation for the retention time of the various peptides between injections averaged below 0.4% for both columns. The coefficient of variation for peak heights, which relates directly to efficiency, averaged 1.2% and 2.4% for the new and used columns respectively. Figure 3 provides a visual comparison of traces from a new and used column indicating that good reproducibility and long lifetimes can be achieved with this method and column type.

Conclusions:
The separation of polar compounds has long been problematic for reasons related to retention, peak shape and reproducibility. The data shared within in
'"/>

Source: