I would like to quantify bradykinin potentiating peptides in a standart solution using HPLC (c18 column) Does anyone have a protocol for quantifying peptides using HPLC vis?
Dear @Samyr Querobino
HPLC is extremely versatile for the isolation of peptides from a wide variety of synthetic or biological sources. The number of applications of HPLC in peptide and protein purification continue to expand at an extremely rapid rate. Solid-phase peptide synthesis and recombinant DNA techniques have allowed the production of large quantities of peptides which need to be highly purified. The design of multidimensional purification schemes to achieve high levels of product purity further highlight the power of HPLC techniques in the analysis and isolation of peptide samples. The complexity of the mixture to be chromatographed depends on the nature of the
source and the degree of preliminary clean-up that can be performed. In the case of synthetic peptides, RPC is generally employed both for the initial analysis and the final large scale purification. HPLC techniques are then introduced at the later stages following initial precipitation, clarification and preliminary separations using soft gel. Purification protocols therefore need to be tailored to the specific target molecule. The key factor that underpins the development of a successful separation protocol is the ability to manipulate the retention of the target molecule so that it can be resolved from other contaminating components.
This chapter thus provides an outline of the general theory of chromatography and the factors that control both the retention time and peakwidth of solutes undergoing separation in terms of the parameters that control resolution. This information can then be used to understand the approaches used to perform separations with specific modes of chromatography as outlined in the remaining chapters in this book
HPLC of Peptides and Proteins
Volume 251
Methods and Protocols
Edited by
Marie-Isabel Aguilar
Article Micropreparative HPLC of peptides and proteins
Because HPLC is a 'comparative' technology you will have to find a pure primary reference standard 1st rather than asking for a protocol. Try the USP or EDQM.
You can always use a 99% pure Sigma standard if it is identified and qualified (MS, UV, FTIR, NMR...). These are usually on their 'Certificate of Analysis'.
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