What effect do surfactants have on RP-HPLC separations?          

The separation on a C₁₈ column of the protein sample containing SDS was much worse (Fig. 1B) than the separation of the protein sample without SDS (Fig. 1A). Subsequent chromatography of the sample without SDS, however, showed no deterioration
(Fig. 1C), confirming that the SDS was removed in the gradient and did not harm the column or affect subsequent separations.

Results on a C₄ column showed parallel but slightly better results than those obtained on the C₁₈ column (Figure 2). The presence of SDS in the protein sample affected the chromatography (Fig. 2B), however the effect was less than on the C₁₈ column (compare with Fig. 2A). The SDS was removed in the gradient and did not affect the column or subsequent separations (Fig. 2C).

Figure 1 Effect of surfactants on C18 RP-HPLC of polypeptides                  Surfactants affect chromatography (B) but do not harm column or subsequent separations (C). Conditions: Column: Vydac 218TP54 Eluent: 24-95% ACN in 0.1% TFA over 30 min at 1.5 ml/min Sample: ribonuclease, insulin, lysozyme, myoglobin and ovalbumin

Figure 2 Effect of surfactants on C4 RP-HPLC  of polypeptides Surfactants affects chromatography (B) but does not harm column or subsequent separations (C). Conditions: Column: Vydac 214TP54 Eluent: 24-95% ACN in 0.1% TFA over 30 min at 1.5 ml/min Sample: ribonuclease, insulin, lysozyme, myoglobin and ovalbumin

Peptide separations are seriously affected by the presence of surfactant. Even trace amounts of SDS in a peptide sample or protein digest can reduce separation efficiency significantly. Peptide maps of a protein digest containing small amounts of SDS showed that even small amounts of SDS affected the digest separation and higher amounts virtually destroyed resolution (Figure 3).

Figure 3 Effect of surfactants on peptide map The presence of even trace amounts of SDS causes a loss in resolution in a peptide map. Conditions: Column: Vydac 218TP52 (Narrowbore) Eluent: 2-80% ACN with 0.06% TFA over 120 min at 0.25 ml/min Sample: tryptic digest of carboxymethylated transferrin

Although surfactants usually degrade RP-HPLC peptide separations, the use of octylglucoside, urea and guanidine in the eluent have produced beneficial results in some cases.

Surfactants usually degrade RP-HPLC polypeptide separations, however they do not harm the column. If surfactants are present in the sample, we recommend using a C4 reversed-phase column or removing the surfactant prior to chromatography.