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The theoretical molecular weight of your peptide can be confirmed by mass spectroscopy. In general, is is considered that your target peptide is found in the sample if a peak fits with the molecular weight of the full length and fully deprotected peptide. Also, if the corresponding peak as far more intense than the others, it suggests that the target compound represents the major part of the analyzed material.
Some specificities, such as disulfide bon formation, can shift the main constituent peak (due to the loss of 2 hydrogen atoms) and should be taken into account when analyzing the mass spectra.
Peptide purity refers to the amount of correct peptide in a peptide product. This is determined by analytical reversed phase HPLC and corresponds to the percentage of correct peptide compared to impurities absorbing at 220nm. Impurities include deletion and truncated sequences, partly protected sequence and side products but do not include water and salts as these molecules do not absorb at this wavelength.
The net peptide content corresponds to the percentage of peptides relative to non-peptide impurities. In other words, the net peptide content and the peptide purity are both necessary to determine the amount of target peptide in a sample.
The net peptide content is generally determined by amino acid analysis. This technique allows for quantification of free amino acids, as well as amino acids released by amino acid containing compounds such as peptides and proteins. Basically, for net peptide content analysis, the sample is hydrolyzed and derivatized with a reagent high resolution separation using HPLC columns. This allows detecting pmol levels of amino-acids using a single wavelength UV detection.
TFA is a strong acid commonly used in peptide synthesis to remove the target compound from the solid-phase resin and for HPLC purification. When it comes to peptides intended for preclinical and clinical studies, TFA is highly undesirable. Thus, it should be quantified and removed. While most of the unbound TFA is removed during the lyophilization step, ion exchange chromatography is necessary to remove TFA counterions bounded to the free amino termini and to positively charge amino acids. This technique is commonly used for TFA quantification as it can be automated and is highly sensitive. TFA quantification is based on anion exchange chromatography meaning that the stationary phase is positively charged. A stationary phase functionalized with quaternary ammonium group is commonly used. To increase the signal-to-noise ratio, the effluent is passed throught a suppressor decreasing the background conductance of the mobile phase while increasing the conductance of the analyte ions.
Please note that the TFA can be removed from the sample by HPLC, anion exchange chromatography or using a specific lyophilization protocol. Of course, ProteoGenix can remove TFA from your peptide upon request.
The water content analysis is performed by Karl Fischer titration. Two types of Karl Fischer titration exist:
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