Detection Techniques

The detection of peptides suffers from the same difficulties as described for amino acids. Additionally only a few amino acids (phe, try, tyr and to a lesser extent his, arg, gln, asn) provide residues with strong chromophores.

Measuring UV absorbance at low wavelengths ( < 220 nm) is the commonest mode of detection to give limits of detection of about 1 ^gmL-1 ( & 10~5-10~6 mol L_1) which are sufficient for most applications. Spectra obtained by a photodiode array detection support identification of impurities in pept-ide synthesis due mainly to the absence of the characteristic absorbance of aromatic residues at 220 nm (Figure 7).

Indirect techniques can be applied as for amino acids.

Detection of trace amounts of peptides requires more sensitive methods and sensitivity can be improved by fluorescence methods.

This approach faces the same difficulties as UV absorbance detection in that only try and, to a lesser extent, tyr and phe exhibit native fluorescence when excited at 280 nm (Xe-lamp). However, this 'natural specificity' facilitates selective identification of try-containing peptides. In addition, indirect fluorescence detection using salicylic acid for anionic charge pep-tides (basic buffers) or quinine for the positive mode (acidic buffer) have been applied.

To accomplish lower detection limits for a broader range of species derivatization techniques have to be applied and all the agents described for amino acids can be used for the derivatization of peptides.

Increased interest is being paid to mass spectromet-ric techniques for the characterization of peptides, especially soft ionization techniques like electron spray ionization (ESI). A promising approach towards nonfragmented peptides is the matrix-assisted laser desorption ionization with time-of-flight mass spectrometers (MALDI-TOF).

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