Amino acids analyser

The separation takes place in a column of sulphonated cross-linked polystyrene resin, which is a strong cation exchanger. The matrix of the resin is strongly anionic in nature (S03 ) and at the low pH used initially, the amino acids will be positively charged and will be attracted to the negatively charged sulphonate groups. 

Although the amino acids have a considerable affinity for the resin, the sodium ions are constantly present in a much higher concentration and, as a result, the equilibrium of the above equation is shifted to the right and the amino acids are displaced from the resin. Thus the molarity of the eluting buffer affects elution and when the ionic concentration of the buffer is increased, the amino acids are eluted more rapidly from the column. 

In the prototype analysers, two columns were often needed to achieve complete separation of all the amino acids. A 50-100 cm column was used to separate the acidic and neutral amino acids and a 5-10 cm column for the basic amino acids, each with a diameter of 1 or 2 cm, but today s instruments use single columns with narrower diameters. As peak width is proportional to the square root of the column length, these glass or stainless steel columns give narrow peaks and improved separation of closely related amino acids. 

The composition and pH of the buffer should be accurate to 0.001 mol l-1 and 0.01 pH units. Most methods rely on the sequential application of a series of buffer solutions of increasing pH and molarity, with the initial pH around 3.2. Sodium citrate or, preferably, lithium citrate buffers are used, which incorporate a detergent (BRH 35), an antioxidant (thiodiglycol) and a preservative 

The temperature of the resin column must be carefully maintained to avoid changes in both the pH of the buffers and ionization of the amino acids. Although increasing temperature usually results in faster elution, the effect may be variable for different amino acids and the relative elution positions can be altered, making interpretation of results difficult. The temperature often chosen is 60 °C although lower temperatures are sometimes required to resolve two similar amino acids. Temperature programming, which entails an alteration in temperature at a specified time in the separation procedure, is widely used. 

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The product quaUty considerations for nonphotosynthetic microorganisms are similar to those for algae. Tables 6 and 7 present composition and amino acid analyses, respectively, for selected bacteria, yeasts, molds, and higher fungi produced on a large pilot-plant or commercial scale. Table 8 summarizes results of proteia quaUty and digestibiUty studies. 

LPC Product Quality. Table 10 gives approximate analyses of several LPC products. Amino acid analyses of LPC products have been pubhshed including those from alfalfa, wheat leaf, barley, and lupin (101) soybean, sugar beet, and tobacco (102) Pro-Xan LPC products (100,103) and for a variety of other crop plants (104,105). The composition of LPCs varies widely depending on the raw materials and processes used. Amino acid profiles are generally satisfactory except for low sulfur amino acid contents, ie, cystine and methionine. 

Acknowledgment. The author is indebted to A. Baldesten for performing amino acid analyses on Ustilago cytochrome c. 

The question was whether impurities were present in the samples analysed (Bada et al., 1983). In a more recent publication, Cronin and Pizzarello (1997) reported amino acid analyses using Murchison material in which an excess of L-enantiomers was present. Contamination with terrestrial biological material can be ruled out, as the amino acids in question are not proteinogenic a-methylamino acids, which occur either extremely seldom or not at all in terrestrial life forms, were detected. GLPC/mass spectrometry (MS) analysis gave the following enantiomeric excess (ee) values ... 

Amino acid analysers based on ion exchange resins are available commercially. These achieve good separations of amino acid mixtures. Fluorescent derivatives of separated amino acids constitute a very sensitive means of detecting these compounds in seawater [256,258]. Fluorescent derivatives that have been studied include o-phthalaldehyde [259], dansyl [260], fluo-rescamine [261], and ninhydrin [261]. 

The amino acid analyser using fluorescamine as the detecting reagent has been used to measure 250 picomoles of individual amino acids routinely [262], and dansyl derivatives have been detected fluorometrically at the 10 15 M level [260]. Where the amounts of amino acid are high enough, the fluorescamine method, with no concentration step, can be recommended for its simplicity. At lower concentrations, the dansyl method, with an extraction of the fluorescent derivatives into a non-polar solvent, should be more sensitive and less subject to interferences. For proteins and peptides, the fluorescamine method seems to be the most sensitive available method. 

In this connection, it is interesting to note that Gardner [263] isolated free amino acids at the 20 nmol/1 level from as little as 5 ml of sample by cation exchange, and measured concentrations on a sensitive amino acid analyser equipped with a fluorometric detector. 

The classic work of Dawson and Pritchard [264] on the determination of a-amino acids in seawater uses a standard amino acid analyser modified to incorporate a fluorometric detection system. In this method the seawater samples are desalinated on cation exchange resins and concentrated prior to analysis. The output of the fluorometer is fed through a potential divider and low-pass filter to a comparison recorder. 

Although this technique is relatively straightforward and automated amino acid analysers are commercially available, it is subject to a number of disadvantages that limits its usefulness in bi-opharmaceutical analysis. These include ... 

Standard analytical procedures were used to evaluate the composition of ingredients. Of the proximate analyses, nitrogen, lipids, and crude fiber were measured by American Oil Chemists Society (AOCS) methods (12) and moisture and ash by Association of Official Analytical Chemists (AOAC) methods (13). Amino acid analyses were performed by gas-liquid chromatography (14) except for tryptophan, which was analyzed colormetrically Tl5). In addition to these assays, certain tests of ingredient safety or spoilage were also performed, which space does not permit to be reported in this paper, to assure that ingredients met accepted standards for food safety (16). 

Chemical,Biological and Physical Tests on Blends. Reported chemical evaluations on the freshly prepared blends include proximate analyses and amino acid analyses. The same analytical procedures described above for ingredients were also used on the blends. 

In recent years, a number of workers have published amino acid analyses of the sweet potato (38, 43, 35, 22, 18). The overall picture is that the sweet potato amino acid pattern is of good nutritional quality but that the variability of individual amino acids both within the same cultivar and across cultivars is very high. For example, Walter et al. (44) reported that with the exception of aromatic amino acids, every essential amino acid has a score of less than 100 in one or more cultivars. The amino acid score is defined as the g of amino acid in 100 g of test protein divided by the number of g of that amino acid in the FAO/WHO reference pattern times 100. Bradbury et al. (22) showed that, for the same cultivar, environmental effects on the amino acid patterns is significant. For three cultivars, they found a mean percent standard deviation for all amino acids of 24.2,... 

Certainly, a vast amount of experience has been gained by the widespread use of conventional amino acid analysers. They offer high reliability, accuracy, reproducibility and can separate complex samples. Because conventional analysers can be fully automated, they are widely used in routine analysis. However, the method is limited by the sensitivity which can be achieved using ninhydrin as the derivatizing agent. Sensitivity can be increased by using ortho-phthaldialdehyde (OPA) instead, but where extremely high sensitivity is required, HPLC is the method of choice. 

This latter method is mainly used when a single substance in a sample is being determined but where the analysis involves the quantitation of many or all of the components of the sample, e.g. in an amino acid analyser, the former method is the more suitable. 

The aqueous reagent is stable at room temperature and the reaction proceeds quickly without requiring heat. The method is approximately ten times more sensitive than the ninhydrin method and is particularly useful when the quantitation of many amino acids is being carried out using amino acid analysers or HPLC. However, the fluorescent yield of individual amino acids varies and fluorescence values must be determined for quantitative work in the same manner as the colour values for ninhydrin. 

The identification and quantitation of the individual amino acids in a mixture is often required in metabolic studies and investigations of protein structure. The use of thin-layer chromatography or electrophoresis may be adequate to indicate the relative amounts and number of different amino acids in a sample but the use of gas-liquid chromatography or an amino acid analyser is essential for quantitative analysis. 

The use of reverse-phase columns with pre-column derivatization of the amino acids offers an acceptable alternative to the dedicated instrumentation of an amino acid analyser or separation by HPLC followed by post-column derivatization. 

Figure 10.18 Schematic diagram of an amino acid analyser using the ninhydrin reagent for quantitation.

Successful and reproducible separations require a steady buffer flow rate and this is achieved with either a constant pressure or a constant displacement pump. These pumps are designed to deliver a constant rate of fluid independent of the resistance to the flow and recent developments in pump design permit the production of a precise and pulseless flow this has contributed towards the increased analytical precision and sensitivity that can now be achieved with amino acid analysers. The choice of flow rate is dependent upon the type of resin, the dimensions of the column and overall design of the instrument and this varies between models. 

Figure 10.19 Amino acid analyser trace. Separation of a complex physiological standard mixture of amino acids in 3.5 hours using lithium citrate buffers and ninhydrin detection 10 nmol of each amino acid, including the internal standard, nor-leucine, were applied to the column (0.3 X 35 cm) in a total volume of 50 ml. 

Elution from the ion-exchange column in an amino acid analyser is affected by which of the following ... 

A strong cation-exchange resin is used in the amino acid analyser BECAUSE... 

From 1951, Moore and Stein at the Rockefeller Institute refined the quantitative separation of amino acids on Dowex-50 which led to fully automated amino acid analyses. In early models two columns were needed one of 100 cm to separate most of the acidic and monobasic monocarboxylic acids between pH 3-11 and a short, 15 cm column for the basic amino acids which were eluted at pH <7. The columns operated above room temperature to give more rapid results, and the elution was monitored automatically by quantitative ninhydrin reactions. By the late 1950s a protein hydrolysate could be analyzed overnight. 

Surface-modified silica gels are used for a variety of separations, but organic polymer-based stationary phase materials are more useful for long-term operations, such as for an amino acid analyser and size-exclusion liquid chromatography. 

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. 

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