Ion-exchange chromatography analysis

Ion exchange chromatography analysis or preparation of amino acids and peptides... 

An amylase, previously detected in barley and described as a new barley amylase, has been further purified by immunoadsorption and ion-exchange chromatography. Analysis by isoelectric focusing and immunochemical techniques showed that the enzyme preparation did not contain the normal a-and 8-amylases usually found in barley and malt. The enzyme had a very low isoelectric point ca. pH 3.0) and was identified as an a-amylase on the basis of its action pattern on amylose. 

Ion exchange chromatography analysis is typically based on use of a 25-cm Whatman Partisil-10 SCX strong cation exchange column and a mobile phase of methanol or acetonitrile/water. The mobile phase contains a salt, such as ammonium formate, and the pH may be adjusted by addition of acetic acid. Elution is in order of decreasing alkyl character, and retention time is increased by decreasing the concentration of the salt in the eluent. Most often, conductivity detection is not used, but rather direct or indirect UV absorbance or refractive index detection. 

Examples of the application of ion-exchange chromatography to the analysis of (a) inorganic anions, (b) inorganic cations, (c) antifreeze, and (d) vitamins. (Chromatograms courtesy of Alltech Associates, Inc. Deerfield, IL). 

Ohta and Tanaka reported a method for the simultaneous analysis of several inorganic anions and the cations Mg + and Ca + in water by ion-exchange chromatography. The mobile phase includes 1,2,4-benzenetricarboxylate, which absorbs strongly at 270 nm. Indirect detection of the analytes is possible because their presence in the detector leads to a decrease in absorbance. Unfortunately, Ca + and Mg +, which are present at high concentrations in many environmental waters, form stable complexes with 1,2,4-benzenetricarboxylate that interfere with the analysis. 

The automated amino acid analy2er 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 Htde 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. 

Fig. 2. Amino acid analysis by automated ion-exchange chromatography. Standard column, 4.6 mm ID x 60 mm Ninhydrin developer. Computer print out indicates retention time (RT), height and area of peaks, and the ratio of the height of an amino acid in the sample to the height of a standard amino acid.

An ion chromatographic system that included column switching and gradient analysis was used for the deterrnination of cations such as Na", Ca ", Mg ", K", and NH" 4 and anions such as Cf, NO, NO , and in fog water samples (72). Ion-exchange chromatography compares very well with... 

Adsorption and ion exchange chromatography are well-known methods of LC. In adsorption, one frequently selects either silica or alumina as stationary phase for separation of nonionic, moderately polar substances (e.g. alcohols, aromatic heterocycles, etc.). This mode works best in the fractionation of classes of compounds and the resolution of isomeric substances (J). Ion exchange, on the other hand, is applicable to the separation of ionic substances. As to be discussed later, this mode has been well developed as a tool for analysis of urine constituents (8). 

Ion exchange chromatography has also been extensively applied to the analysis of nucleic acid components (3,14,35),... 

As an example, consider the separation of the creatine kinase isoenzymes, MM, MB, and BB. Mercer has used classical ion-exchange chromatography (DEAE - Sephadex - A50) for the resolution of these three isoenzymes (44) To speed up the separation and ultimately to allow an automated analysis,... 

Hence, once a B ion is freed from the resin, it is immediately complexed and there is much less tendency for it to be resorbed lower down the column as would happen with a stable cationic species. This is an illustration of separation by elution analysis. Its most important application is in the separation of rare earths. When used on a laboratory scale in chemical analysis, this separation technique is known as ion-exchange chromatography. 

It would be easier to describe those classes of compounds not normally separated by RPLC than to catalogue the applications to which RPLC has been turned. Applications for reversed phase can be found in virtually every area of analysis and are reviewed regularly in the journal Analytical Chemistry. RPLC has not been in general use for the analysis of inorganic ions, which are readily separated by ion exchange chromatography polysaccharides, which tend to be too hydrophilic to separate by RPLC polynucleotides, which tend to adsorb irreversibly to the reversed phase packing and compounds which are so hydrophobic that reversed phase offers little selectivity. 

The analysis of amino acids involves chromatographic issues similar to those encountered in analysis of simple amines. Underivatized amino acids have, with a few exceptions, weak UV absorbance and a strong tendency to interact with stationary phases in undesirable ways. Underivatized amino acids are normally separated with ion exchange chromatography, then visualized post-column by reaction with ninhydrin, o-phthaladehyde (OPA), or other agents. Underivatized tryptophan and the metabolites kynurenine, 3-hydroxykynurenine, kynurenic acid, and 3-hydroxyanthranilic acid, were separated on a Partisphere 5-p ODS column with fluorescent detection.121... 

Ishimitsu, S., Fujimoto, S., and Ohara, A., Quantitative analysis of the isomers of hydroxyphenylalanine by ion-exchange chromatography, Chem. Pharm Bull., 24, 2556, 1976. 

Houpert, Y., Tarallo, P., and Siest, G., Amino acid analysis by ion-exchange chromatography using a lithium elution gradient. Influence of methanol concentration and sample pH, /. Chromatogr., 115, 33, 1975. 

Elution with salt pulses A multiple step elution is performed by the introduction of, for example, 5%, 10%, 25%, 50%, and 100% of 1.5 M sodium chloride in 19 mM phosphate buffer (pH 2.5) containing 5% methanol. Each step is for 10 min and run at 0.5 mL/min. This elution method compromises analytical system dimensionality, as the peak capacity of the ion-exchange chromatography (IEX) step is equal at most to the number of salt steps. However, in the second dimension only one or two columns are needed and there is no particular limitation in the second dimension separation time as peptides are eluted in portions in a controlled manner. However, the number of salt steps is limited by the total analysis time. In this case the multidimensional system is relatively simple. 

Vanrensen, I. and Veit, M. (1995). Simultaneous determination of phenolics and alkaloids using ion-exchange chromatography for sample preparation. Phytochemical Analysis 6 121-124. 

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