HPLC chromatography amino acids

V-1 from acid and alkaline hydrolyzates, SCX-HPLC of amino acids, a mixture of purified crosslinks and hydroxylysine b purified cross-link V-2 c amino acids from an acid hydrolyzate (6 M HCl) of reduced bovine dentin retained on a phenylboronate agarose column after purification as high molecular weight fractions by repeated size exclusion chromatography d as c, alkaline hydrolyzate (2 M KOH). Injections (c, d) resulted from 18 and 52 mg collagen originally hydrolyzed, respectively. 1 = 111 (HP) 2 = V-2 3 = IV 4 = V-1-1 (DHLNL) 5 = HLNL (bovine tendon) 6 = VI (histidinoalanine ) 7 = hydroxylysine 8 = VI (lysinoalanine). 

Figure 13.23 HPLC of amino acid derivatives detected by 254 nm UV absorption (a) 200 pmol of PTC-amino acid standard, including PH-S PH-T, hydroxyproline (OH-P), galactosamine (Gal), norleucine (NLE, 1 nmol IS), excess reagent (Re), and other amino acids designated by one-letter codes listed in Figure 13.22 and (b) analysis of a human fingerprint, taken up from a watch glass using a mixture of water and ethanol. (Courtesy of National Gallery of Art and the Andrew W. Mellon Foundation From Cazes, J., Encyclopedia of Chromatography, Marcel Dekker, Inc., New York, 2001. With permission.)...

Gas Chromatography (gc). A principal advantage of gas chromatography has been the faciUty with which it can be combined with mass spectrometry for amino acid identification and confirmation of purity. The gc-mass spectrometry combination offers the advantage of obtaining stmctural information rather than the identification by retention time in hplc. 

FIGURE 4.22 HPLC chromatogram of amino acids employing precolumn derivatiza-tion with OPA. Chromatography was carried out on an Ultrasphere ODS column using a complex tetrahydrofuran methanol 0.05 M sodium acetate (pH 5.9) 1 19 80 to methanol 0.05 M sodium acetate (pH 5.9) 4 1 gradient at a flow rate of 1.7 mL/min. 

Although these Boc derivatives underwent methylation with poor selectivity (compared to 3-amino-N-benzoyl butanoates [106] and Z-protected methyl 4-phen-yl-3-aminobutanoate [107]), epimers were succesfully separated by preparative HPLC or by flash chromatography. However, saponification of the methyl ester caused partial epimerization of the a-stereocenter and a two-step (epimerization free) procedure involving titanate-mediated transesterification to the corresponding benzyl esters and hydrogenation was used instead to recover the required Boc-y9 -amino acids in enantiomerically pure form [104, 105]. N-Boc-protected amino acids 19 and 20 for incorporation into water-soluble /9-peptides were pre-... 

There are many proteins in the human body. A few hundreds of these compounds can be identified in urine. The qualitative determination of one or a series of proteins is performed by one of the electrophoresis techniques. Capillary electrophoresis can be automated and thus more quantified (Oda et al. 1997). Newer techniques also enable quantitative determination of proteins by gel electrophoresis (Wiedeman and Umbreit 1999). For quantitative determinations, the former method of decomposition into the constituent amino acids was followed by an automated spectropho-tometric measurement of the ninhydrin-amino add complex. Currently, a number of methods are available, induding spectrophotometry (Doumas and Peters 1997) and, most frequently, ELISAs. Small proteins can be detected by techniques such as electrophoresis, isoelectric focusing, and chromatography (Waller et al. 1989). These methods have the advantage of low detection limits. Sometimes, these methods have a lack of specifidty (cross-over reactions) and HPLC techniques are increasingly used to assess different proteins. The state-of-the-art of protein determination was mentioned by Walker (1996). 

Consider one small molecule, phenylalanine. It is an essential amino acid in our diet and is important in protein synthesis (a component of protein), as well as a precursor to tyrosine and neurotransmitters. Phenylalanine is one of several amino acids that are measured in a variety of clinical methods, which include immunoassay, fluorometry, high performance liquid chromatography (HPLC see Section 4.1.2) and most recently MS/MS (see Chapter 3). Historically, screening labs utilized immunoassays or fluorimetric analysis. Diagnostic metabolic labs used the amino acid analyzer, which was a form of HPLC. Most recently, the tandem mass spectrometer has been used extensively in screening labs to analyze amino acids or in diagnostic labs as a universal detector for GC and LC techniques. Why did MS/MS replace older technological systems The answer to this question lies in the power of mass spectrometer. 

The most popular current techniques for amino acid analysis rely on liquid chromatography and there are two basic analytical methods. The first is based on ion-exchange chromatography with post-column derivatization. The second uses pre-column derivatization followed by reversed-phase HPLC. Derivatization is necessary because amino acids, with very few exceptions, do not absorb in the UV-visible region, nor do they possess natural fluorescence. 

Isolation of individual amino acids started about 1820 by 1904 all of the naturally occurring amino acids in proteins had been isolated except methionine (Mueller, 1922) and threonine (Rose, 1937). One of the earliest methods for the separation of amino acids was through the differential volatility of their methyl or ethyl esters (Emil Fischer, 1901). This approach led to the discovery of valine, proline, and hydroxyproline. [In the 1970s Fischer s method was modified for microanalysis of proteins, separating the amino acid esters by gas phase chromatography. Separation is now usually performed by hplc (high pressure liquid chromatography).]... 

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