Amino acid analysis optimization

The study of cell culture supernatants and media was chosen to exemplify the utility of the optimized quaternary HPLC method. The rate at which amino acids are consumed by protein synthesis or other metabolic pathways can be quantified by performing amino acid analysis on supernatants of the protein producing cell cultures. Optimization of target protein expression can then be achieved by feeding the culture concentrated supplements rich in those amino acids that are rapidly consumed. 

All the various methods for hydrolysis, chromatography, and derivatization, combined with the need to optimize these methods for the different matrices make amino acid analysis somewhat daunting. It is hoped that this chapter has served as an introduction and will aid in either the selection of a laboratory to conduct the analysis or as an aid to those who are beginning or conducting the analysis. 

Carboxylic acids with a halide, hydroxy or amino group in the a-position form no dimers (Table 2, No. 11), except when two or three fluorine atoms are pr ent there (Table 2, No. 12). A large amount of work has been devoted to the coupling of fluoro-carboxylic acids (Table 2, Nos. 13-16) due to interesting properties of the produced fluorohydrocarbons. By statistical analysis optimal conditions for Kolbe ela trolysis of perfluorinated acids have been calculated [135]. 

Anions of weak acids can be problematic for detection in suppressed IEC because weak ionization results in low conductivity and poor sensitivity. Converting such acids back to the sodium salt form may overcome this limitation. Caliamanis et al. have described the use of a second micromembrane suppressor to do this, and have applied the approach to the boric acid/sodium borate system, using sodium salt solutions of EDTA.88 Varying the pH and EDTA concentration allowed optimal detection. Another approach for analysis of weak acids is indirect suppressed conductivity IEC, which chemically separates high- and low-conductance analytes. This technique has potential for detection of weak mono- and dianions as well as amino acids.89 As an alternative to conductivity detection, ultraviolet and fluorescence derivatization reagents have been explored 90 this approach offers a means of enhancing sensitivity (typically into the low femtomoles range) as well as selectivity. 

A universal eluent system was proposed for analysis of amino acids in biological fluids by IEC on the cation exchange resin Ostion LG ANB134. A study was carried out on the effect of the carrier pH on RP-HPLC of amines, using a Cs column and octyl sulfate as ion pairing reagent. Optimal results were obtained for the analysis of catecholamines... 

A cluster analysis of the amino acid structures by PCA of the A -matrix is shown in Figure 6.5a note that PCA optimally represents the Euclidean distances. The score plot for the first two principal components (preserving 27.1% and 20.5% of the total variance) shows some clustering of similar structures. Four structure pairs have identical variables 1 (Ala) and 8 (Gly), 5 (Cys) and 13 (Met), 10 (He) and 11 (Leu), and 16 (Ser) and 17 (Thr). Objects with identical variables of course have identical scores, but for a better visibility the pairs have been artificially... 

An experimental design approach was also used in Reference 26 for the chiral analysis of amino acid derivatives. The screening and optimization schedule followed... 

Analysis using a CMPA is usually resolved on a nonchiral column. A transient diastereomeric complex is formed between the enantiomer and the chiral component in the mobile phase, similar to the complexes formed with chiral stationary phases. A review by Liu and Liu (2002) cites several papers where addition of CPMAs has been used in analyzing amphetamine-related compounds. Some CPMAs include amino acid enantiomers, metal ions, proteins, and cyclodextrins. Advantages of this method of analysis include the use of less expensive columns and more flexibility in the optimization of chiral separation (Misl anova and Hutta, 2003). 

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