Amino acid analyzers

The entire operation is carried out automatically using an amino acid analyzer and is so sensitive that as little as 10 -10 g of the peptide is required. 

Complete hydrolysis of a peptide gives a mixture of amino acids. An amino acid analyzer identifies the individual ffluino acids and detennines their molar ratios. 

To determine the structure of a protein or peptide, we need to answer three questions What amino acids are present How much of each is present in what sequence do the amino acids occur in the peptide chain The answers to the first two questions are provided by an automated instrument called an amino acid analyzer. 

Post-column reaction is a common feature of many special types of analyses, the most well-known being the amino acid analyzer that uses ninhydrin with a post-column reactor to detect the separated amino acids. In general, derivatization and post-column reactor systems are techniques of last resort. In some applications they are unavoidable, but if possible, every effort should made to find a suitable detector for the actual sample materials before resorting to derivatization procedures. 

The soluble tryptic peptides of 130 mg a chain of Hb-St. Claude were separated on 0.9 x 60 cm columns of Chromobead resin type P (Technlcon Instruments, Dowex 50-X4) at 37°C using the procedure described earlier (16). The method uses a gradient of volatile pyrldlne-acetlc acid buffers of differing molarities and pH as follows first gradient, 666 ml 0.1 M, pH 3.1, and 333 ml 1.0 M, pH 5.0 and second gradient, 166 wl 1.0 M, pH 5.0, and 332 ml 2.0 M, pH 5.0. The amino acid composition of Isolated fragments was determined with a Splnco model 121 automated amino acid analyzer (Beckman Instruments)... 

The polyamines putrescine, cadaverine, spermidine, and spermine, which are seen at elevated levels in some victims of cancer, were separated on a Technicon (The Technicon Company Chauncey, NY) TSM Amino Acid Analyzer packed with an 8% divinylbenzene-co-polystyrene sulfonated resin with post-column ninhydrin detection.111 Amines such as ethanolamine, noradrenaline, hexamethylene diamine, methoxytryptamine, spermine, and spermidine were separated from amino acids on a DC-4A cation exchange resin.112 A similar approach, using a Beckman Model 121M amino acid analyzer equipped with an AA-20 column, was also successful.113 A Polyamin-pak strong cation exchange column (JASCO) was eluted with a citrate buffer for the detection of putrescene, spermine, cadaverine, and 1,5-diaminohex-ane from rat thymus.114 A post-column o-phthaldehyde detection system was used. 

Figure 6 Separation of amino acids on conventional ion exchange Beckman 120B Amino Acid Analyzer. Column 15 cm. Eluent 0.35 M sodium citrate buffer, pH 5.28. Flow rate 30 ml/hr. Temperature 50°C. Note that the separation requires approximately 6 hours. Compare to a modern separation shown in Figure 7. (Reproduced with permission from Beckman Instruments Fullerton, CA.)...

Beecher, G. R., Design and assembly of an inexpensive, automated, microbore amino acid analyzer separation and quantitation of amino acids in physiological fluids, Adv. Exp. Med. Biol., 105, 827, 1978. 

Hadzija, O. and Keglevic, D., Simple method for the separation of amino acids, amino sugars, and amino alcohols related to the peptidoglycan components on a standard amino acid analyzer, /. Chromatogr., 138, 458, 1977. 

Lange, H.W. and Hempel, K., Automated chromatography of aromatic acids, aldehydes, and alcohols with an amino acid analyzer, /. Chromatogr., 59, 53,1971. 

Atassi, M. Z., Perlstein, M. T., Rosenblatt, M. C., and Rocek, R, Fully automated simple analytical peptide chromatography on the amino acid analyzer at the 10 8 mole level, Anal. Biochem., 49, 164, 1972. 

Hartley, R. W., Peptide analysis and preparation with an amino acid analyzer, Anal. Biochem., 46, 676, 1972. 

The use of amino acid analysis to characterize protein hydrolyzates is well known, and IEC is an integral part of the commercial amino acid analyzer.61 Determinabon of individual amino acids in physiological fluids such as plasma and cerebrospinal fluid can be important in the etiology of various diseases.62 63 Of particular current interest is the measurement of total... 

Marnela et al. [57] used an amino acid analyzer using fluorescence detection to determine penicillamine in urine. Urine is analyzed on a Kontron Chromakon 500 amino acid analyzer containing a column (20 cm x 3.2 mm) of AS70 resin in the Li (I) form. Buffers containing LiOH, citric acid, methanol, HC1, and Brij 35 at pH 2.60, 3.20, and 3.60 are used as mobile phases (0.4 mL/h). The fluorescence reagent is prepared by the method of Benson and Hare. Detection is at 450 nm (excitation at 350 nm). The analyte response is linear from 0.025 to 10 mM, with a limit of detection of 25 pM. 

The use of the relative proportions and racemization properties of amino acids as a means of identifying contaminated samples would have several advantages. First of all, modern amino acid analyzers are very sensitive analytical instruments. Systems are... 

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. 

FIGURE 4.8 Racemization tests employed for acquiring information on stereomutation. Couplings are carried out, and the isomeric content of the products is determined by a variety of techniques. AAA = amino acid analyzer GLC = gas-liquid chromatography. 

J Gerhardt, K Nokihara, R Yamamoto. Design and applications of a novel amino acid analyzer for D/L and quantitative analysis with gas chromatography, in JA Smith, JE Rivier, eds. Peptides Chemistry and Biology. Proceedings of the 12th American Peptide Symposium, Escom, Leiden, 1992, pp 457-458. 

Problems such as diffusional limitations and the analysis of catalyst composition occur with solid-phase catalysts. Much work has been done on diffusion in bound enzymes (for reviews, see 24 and 88). In our work we used ninhydrin, which is a reagent ideal for surface analysis amino acid analysis is used wherever possible. Amine depletion as followed by ninhydrin is not exact, but some quantitative guides are obtained. Certainly synthetic catalysts must be made with bonds other than amide bonds and components other than those compounds that are detectable on the amino acid analyzer. 

Protein Sequencing. The xylanase protein sequence was obtained by stepwise automated E an degradation ter an initial double-coupling protocol, using an Applied Biosystems model 470A gas-phase sequenator equipped with an on-line Applied Biosystems model 120A PTH-amino acid analyzer 13). 

Amino Acid Analyses. Samples of native and ozonized lysozymes were hydrolyzed in evacuated, sealed tubes with 6N hydrochloric acid at 110 C for 24 hr. After being cooled to room temperature, the solution was adjusted to pH 2 with NaOH solution and brought to mark in a volumetric flask with sodium citrate buffer pH 2.3. A portion containing a suitable amount of the amino acids was applied to a Beckman 12OB amino acid analyzer. 

For many years, automated amino acid analyzer using lEC and postcolumn derivatization with nin-hydrin (or less frequently with fluorescamine) has been the most popular technique for amino acid determination. Amino acids are separated in their free form by employing stepwise elution with sodium- or lithium-based buffers. 

In recent years, the wide diffusion of precolumn derivation agents able to increase analyte hydro-phobicity and hence its retention on an apolar phase allowed a gradual replacement of dedicated amino acid analyzer with more versatile and less expensive RP-HPLC systems. 

Naulet et al. [223] compared three chromatographic techniques lEC on an automatic amino acid analyzer, HPLC (with phenylisothiocyanate derivatization) and GC for the determination of the free amino acid content in 64 orange juices from different countries. The consistency of the different methods was estimated by considering the mean standard deviation for the set of amino acids observed. The best consistency was observed between lEC and HPLC. 

Peptides (and proteins) are hydrolyzed with acid in amino acid analyzers to give the kind and number of each amino acid constituent. 

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