In RPLC mobile phase is commonly water or aqueous buffers, methanol, acetonitrile or tetrahydrofuran, or mixtures of them.

TLS Online TPP Program

#Id: 4711

#Unit 13. Methods in Biology

Common colorimetric and UV absorption assays

TLS Online TPP Program

#Id: 4712

#Unit 13. Methods in Biology

The electronic transitions of the peptide bond occur in the far UV. The intense peak at 190 nm, and the weaker one at 210–220 nm is due to the p!p* and n!p* transitions.

TLS Online TPP Program

#Id: 4713

#Unit 13. Methods in Biology

A number of amino acids (Asp, Glu, Asn, Gln, Arg and His) have weak electronic transitions at around 210 nm. Usually, these cannot be observed in proteins because

they are masked by the more intense peptide bond absorption.

TLS Online TPP Program

#Id: 4714

#Unit 13. Methods in Biology

The most useful range for proteins is above 230 nm, where there are absorptions from aromatic side chains While a very weak absorption maximum of phenylalanine occurs at 257 nm, tyrosineand tryptophan dominate the typical protein spectrum with their absorption maxima at 274 nm and 280 nm, respectively

TLS Online TPP Program

#Id: 4715

#Unit 13. Methods in Biology

Cystine (Cys2) possesses a weak absorption maximum of similar strength as phenylalanine at 250 nm. This band can play a role in rare cases in protein optical activity or protein fluorescence.

TLS Online TPP Program

#Id: 4716

#Unit 13. Methods in Biology

Proteins that contain prosthetic groups (e.g. haem, flavin, carotenoid) and some metal–protein complexes, may have strong absorption bands in the UV/Vis range.