Amino Acid Analysis (AAA)
Description: Amino acid analysis is the gold standard to determine peptide quantities. During amino acid analysis the peptide is hydrolyzed under acidic conditions to release free amino acids. After pre-column derivatization, labeled amino acids are separated by RP-HPLC and detected in a fluorescence detector. This method is to determine the peptide content.
When is it needed: Determination of the amino acid composition in a peptide or protein product.
Precise determination of the net peptide content to ensure experimental accuracy and consistency.
Description: Counter ions such as TFA or HCL present during peptide synthesis can also be present in the final product. Ion exchange chr omatography is often the method of choice used for the detection and quantification of counter ions in a product.
When is it needed: Removal of TFA in peptides is needed for cellular assays, active pharmaceutical ingredient (API) formulations, as well as for some manufactured peptide products.
Description: Endotoxin tests allow testing for bacterial pyrogens such as liposaccharides (LPS) and other pyrogens. The Limulus amoebocytes lysate (LAL) from horseshoe crabs is used for this. Gel-clot, turbidimetric and chromogenic tests allow detection of endotoxins at levels as low as 0.001 EU/ml.
When is it needed: Endotoxins are components of gram-negative bacterial cell walls. It is advised to test for their presence in peptide products if they are used for cellular assays since they can decrease cell viability or cause an immune response.
Moisture Content Analysis
Description: For many products moisture analysis is often a critical part during quality assurance and control. In synthetic peptides, even after a extensive lyophylization step small amounts of water can be present the amount of which depends on the hygroscopic nature of the peptide. In general, the Karl Fisher method is used for moisture analysis of peptides.
When is it needed: The water content in peptides can vary from batch to batch in peptide products. Moisture analysis will help determining the proteins content in peptide preparations needed to ensure accuracy in peptide stock solutions.
Peptide Content Analysis
Description: The peptide content in a peptide based product can be determined using amino acid analysis as well as the analysis of elemental nitrogen.
When is it needed: This test is needed when the accurate peptide content is needed, for example, to prepare exact peptide stock solutions.
Description: For this test peptides are usually dissolved in water or appropriate buffer systems and a pH meter is used to determine the pH value.
When is it needed: Salts as well as the nature of peptide sequences can can influence the pH of peptide solutions. This test is needed to ensure that a peptide solution has the desired pH value.
Description: For this test peptides are systematically dissolved in different solvents. The resulting peptide concentration in each solution is measured and the solvent that allows the highest amount of peptide to be dissolved is determined. Specifically, hydrophobic and longer peptides may have the need for the selection of special solvents or buffer.
When is it needed: Hydrophobic and longer peptides can have different specific solubilities. This is particularly true for peptides containing tryptophan, isoleucine, leucine, phenylalanine, methionine, valine and tyrosine when part of a peptide sequence.
TFA Counter-Ion Removal
Description: Peptide products purified with standard methods contain TFA salts. Because of this and depending on the peptide sequence various amounts of TFA can be present in product. Ion exchange chromatography among other methods can be used to remove or exchange TFA counter ions.
When is it needed: This removal step is needed when peptides need to be used for cellular assays, cell culture development, active pharmaceutical ingredient (API) formulations, as well as for some manufactured peptide products.