Sample self displacement for purification of a peptide

The purification of synthetic peptide objectives is open to many modes of separation due to the hydrophilic, hydrophobic and 

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. 

Once the stationary phase and gradient conditions have been selected there are two options for practical applications. The first option is to maximize the column loading such that a reasonable level of resolution is maintained allowing peak skimming to achieve the required purity. The second option is to overload the column to such an extent that the main component displaces earlier running components of a mixture. 

Self-displacement will operate with almost any mode of chromatography including reversed phase as demonstrated above, normal phase, chiral and ion-exchange. The following procedures provide a generic approach to development of a 

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