Research On Amino Acid Composition In Biologicals

一、Analysis of amino acids in animal cell culture medium by HPLC with OPA/FMOCA pre-column derivatization high performance liguid chromatography(HPLC) method for detemination of 20 amino acids in animal cell culture medium was performed by using o-phthaldialdehyde(OPA) and 9-fluorenylmethyl chloroformate(FMOC) as the derivatization reagents and the detection was carried out with fluorometric detection combined a C-18 column using gradient elution. The separation of 20 amino acids was achieved within 40 minutes on a reversed-phase column(Waters XBridge? Peptide BEH C18 column,250mm×4.6mm×3.5μm) with fluorometric detection(wavelength at 338 nm and 262nm) by elution buffers based on sodium dihydrogen phosphate solution and acetonitrile/methyl alcohol/water(45/45/10).The method linearity,high accuracy, good repeatability,has a correlation coefficient between 0.9988 and 0.9994. The presented method is suitable for the quantitation of animo acids in animal cell culture medium.二、The detection of amino acids in protein and peptidesThe sample of this test is consists of 20 amino acids.After the degration of sample,we detect it by HPLC.The relative molar ratio is control,we get that arginine, tyrosine, isoleucine, leucine is between 0.80 and 1.20,aspartic acid, phenylalanine(Dphenylalanine) is between 1.60 and 2.40.proline is between 2.40 and 3.60,glutamic is between 3.40 and 4.60,glycine is between 4.20 and 5.80.Considering the deviation in the experiments,the deviation range of limit value and theoretical value is ±20%。三、Amino acid sequencingThis experiment combines classical biochemical methods and detection methods.We improve it on the basis of the classical methods, combining with the MALDI-TOF,and optimaize the Edman degration,so the purpose of protein sequcing quickly comes true.The main idea of this experiment is speculating protein sequence by detecting peptid...