Sample Abstract

A RADIOCHEMICAL STUDY OF IRREVERSIBLE LOSS OF PROTEIN IN REVERSED PHASE HIGH PERFORMANCE LIQUID CHROMATOGRAPHY.

PAUL C. SADEK, PETER W. CARR (Univ. of Minn., Chem. Dept., Minneapolis, MN, 55455), LARRY D. BOWERS (Univ. of Minn., Dept. Lab. Med. & Pathol., Minneapolis, MN, 55455), & LOUIS C. HADDAD, (3M Co., Anal. & Properties Res. Lab., St. Paul, MN, 55144).

The loss of protein during RP-HPLC separations has long been recognized as a major disadvantage in small scale preparative and analytical work. We present here the results of a study on protein loss to various components of the chromatography system using I-125 labeled ovalbumin, lysozyme, and cytochrome c.

Small amounts (10 - 600 ng) of labeled protein were injected onto 10 X 4.6 mm columns. The effect of contact time, sample load, and type of packing material were studied. Small elution fractions were collected over the range 0<k’<250 after which the system was dismantled and all components counted.

Different packing materials exhibited substantially different levels of bound protein (6-25X). Standard stainless steel HPLC frits were found to absorb up to 33X of the protein sample and were replaced by stainless steel meshes which performed much better (<<1 X absorption).

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