Automated amino acid analyzer

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)... 

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. 

Automated amino acid analyzers are available from a number of manufacturers (e.g., Beckman, Biotronik, Dionex, LKB, Pickering). These are integrated systems in which the column resin, buffer system, and various instrumentation parameters have been co-optimized for that particular system. All are reported in the literature to perform very well. The advantage to purchas-... 

In 1944, about the time Sanger determined the primary structure of insulin, two-dimensional paper chromatography became available for analyzing amino acids of protein samples.32 This method allowed Sanger to analyze 20 amino acids in a single run with considerably less sample and time compared to the previous methods. The development of an automated amino acid analyzer in 1958 by Spackman, Stein and Moore had made further progress.33 This first amino acid analyzer performed an analysis with 1 pmol of sample in 20 hours. Due to the continuous improvements made on amino acid analyzers,... 

Alarcon (1976) developed a method for quantifying 3-hydroxypropylmercapturic acid (MCA), a known metabolite of acrolein, in urine. This method involves acidification of the urine to convert MCA to S - (3 - hydroxypropyl) - L - cysteine. The amount of S - (3 - hydroxypropyl) - L - cysteine can then be quantitated using an automated amino acid analyzer. 

Sample Derivatization. For HPLC analyses, many analytes are chemically derivatized before or after chromatographic separation to increase their ability to be detected. For example, in automated amino acid analyzers, eluted amino acids are reacted with ninhydrin in a postcolumn reactor (see Chapter 20). The resulting chromogenic species are then detected with a photometer. Other examples include labeling amino acids or other primary amines with dansyl or fluorescamine tags either before or after the chromatographic step. 

A typical chromatogram obtained from the separation of a mixture of amino acids using an automated amino acid analyzer. 

The automated amino acid analyzer depends on ion-exchange chromatography (117) and is now a routine tool for the analysis of amino acid mixtures (118). This most advanced machine can detect as little as 10 pmol in ninhydrin reaction analysis. One-half to two hours are required for each analysis. An analysis chart is shown in Figure 2. 

The modem automated amino acid analyzer based on HPLC (Hancock, 1984) can perform complete analysis in less than 60 m with a sensitivity as low as Ipmol of each amino acid. Various derivatization protocols for high performance (high-pressure) chromatography (HPLC) use fluorescent and UV-absorbing tags, such as 1-dimethylamino... 

In the 1950s, W. H. Stein and S. Moore conducted pioneering work at Rockefeller Institute to understand the structure of the enzyme ribonuclease, for which they were awarded the Nobel Prize in 1972. Continual need to measure amino acids led them to explore amino acid separations by ion exchange. By 1958, their design led to the first commercial automated amino acid analyzer— a revolutionary advance for biochemistry. 

Place the microvials into the autosampler of the amino acid analyzer system. Put 100-/rl aliquots of OPA and FMOC reagents as well as 1 ml of 0.4 N borate buffer into the autosampler and start the fully automated amino acid analyzer system as described in the manufacturer s manual. 

Studies by Heathcote (1979) indicate that quantitative densitometric TLC analyses of amino acids will provide results equivalent to that obtained from automated amino acid analysis procedures. In a study by Mack et al. (1979) on amino acids in mosquitoes, qualitative TLC procedures were first used to determine amino acid profiles in the tissue and hemolymph, and, subsequently, the automated amino acid analyzer was applied for quantification. The study of Mack et al. (1979) should be useful to those interested in qualitative and quantitative analysis of amino acids from biological samples. 

Figure 11 Ultra-trace level analysis of an amino acid standard using the automated, capillary-scale amino acid analyzer. A 2 pL, low nanomolar amino acid standard (top trace) and blank (bottom trace) is derivatized and preconcentrated using the automated amino acid analyzer. The samples were contained in a polypropylene, 384-weU microtiter plate. The reagents were added, mixed, and siphoned for injection using the fused silica needle of the autosampler (Figure 14). The time axis is truncated to highlight the elution window of the amino acid derivatives. All separation and detection parameters are as described in Figure 9.

Sequence analysis can only be conducted on a pure protein. First, the amino acid composition is determined after acidic hydrolysis. The procedure (separation on a single cation-exchange resin column and color development with ninhydrin reagent or fluorescamine) has been standardized and automated (amino acid analyzers). Figure 1.10 shows a typical amino acid chromatogram. 

When the polypeptide strand has been purified, the next step in structural analysis is to establish its composition. To determine which amino acids and how much of each is present in the polypeptide, the entire chain is degraded by amide hydrolysis (6 N HCl, 110°C, 24 h) to give a mixture of the free amino acids. The mixture is then separated and its composition recorded by an automated amino acid analyzer. 

AAA analyzed using automated amino-acid analyzer according to Spackman, D. H., Stein, W. H., Moore, S. Anal. Chem. 30,1190—1206 (1958). lEC ion exchange chromatography according to the procedure of Moore, S., Stein, W. H. J. biol. Chem. 192, 663—681 (1951). 

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