Gradient peptide separation

Stadalius, M. A., Gold, H. S., and Snyder, L. R., Optimization model for the gradient elution separation of peptide mixtures by reversed-phase high-performance liquid chromatography. Verification of retention relationships, /. Chromatogr., 296, 31, 1984. 

For analytical separations it is important to run a full gradient based separation since this will recognize and allow subsequent identification of the majority of impurities present in the crude peptide. This impurity identification may eventually... 

In cases where mass transfer is rapid, as is the case with most small molecule separations, then isocratic elution can offer advantages such as automatic fraction reprocessing and solvent recycle. However, with larger synthetic objectives the rate of mass transfer is comparatively low so isocratic elution leads to band broadening and subsequently to recovery of the peptide at high dilution. Most preparative HPLC based peptide separations are carried out under gradient and overload conditions that allow for maximum throughput in terms of time and quantity. 

Fig. 4. An onflow gradient LC-NMR spectrum of a mixture of peptides separated using a CH3CN/D2O solvent gradient. (Reproduced from Keifer6 with permission.)...

Figure 9.22 Separation of hydrolysis products of the action of tadpole collagenase on DNP-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Argby reversed-phase HPLC. Upper tracing shows the gradient employed. The reaction was initiated at zero time by the addition of 4 /xg of enzyme. At the indicated times, a 4.0 /xL aliquot erf the reaction mixture was injected onto the HPLC column and the peptides separated. (From Gray and Saneii, 1982.)...

The coupling of ESI and MS with a pressurized CEC system (PEC) has been shown to separate peptides [4]. This particular study of Schmeer et al. utilized a commercial reversed phase silica gel, Gromsil ODS-2,1.5-pm packing material, already utilized in capillary HPLC for peptide separations. It was never made perfectly clear why this particular packing material was selected or why PEC was selected for MS interfacing over conventional, isocratic CEC conditions. It is possible that the EOF alone with this packing material was insufficient to elute all peptides in a reasonable time frame and, thus, pressurized flow was introduced. No gradient elution PEC was demonstrated in this particu-... 

Figure 5.4-1. Schematic overview of typical reversed phase-HPLC with precolumn and column loading for peptide separation. The central switching valve has two positions In position one, the sample is loaded onto the precolumn with the loading pump. After trapping and desalting of the sample, the valve is switched and the precolumn is integrated into the flow of the gradient pump. By increasing the amount of solvent B, the single peptides are separated on the separation column and afterward detected with a UV-detector and/or by mass spectrometry.

The common gradient for peptide separation consists of a binary solvent system containing in solvent A 0.1% formic acid and in solvent B 0.1% formic acid and 84% acetonitrile. If no high-sensitive MS detection is required, formic acid can be replaced by TFA. For solvent B, the amount of TFA has to be reduced to 0.08% to prevent baseline shift during the gradient run time. 

When analyzing the molecular weight of peptides from larger proteins (>30 kDa) a gradient of up to 100 min improves peptide separation. 

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