Amino acids reversed-phase

Reversed-phase LC is becoming increasingly popular for the separation of amino acids. Reversed-phase columns are readily available commercially and exhibit higher efficiencies than most commercially available ion-exchange columns. They are also compatible with aqueous samples, since water is generally a major component of the mobile phase. Therefore, it is not necessary to employ additional sample preparation steps in order to produce a sample in a nonaqueous environment. 

Analytical Properties (i-Cyclodextrin (cycloheptamylose) normal phase separation of positional isomers of substituted benzoic acids reverse phase separation of dansyl and napthyl amino acids, several aromatic drugs, steroids, alkaloids, metallocenes, binapthyl crown ethers, aromatics acids, aromatic amines, and aromatic sulfoxides this substrate has seven glucose units and has a relative molecular mass of 1135 the inside cavity has a diameter of 0.78 nm, and the substrate has a water solubility of 1.85 g/ml, although this can be increased by derivatization Reference 13-28... 

Ethylene diamine tetraacetic acid and nitroacetic acid Reverse phase column Agtrichloro acetic acid (pH<2) Amino acids, citric acid and fiilvic acid do not interfere Carbon paste electrode [28]... 

Amino acids, nucleosides, carboxylic and sulphonic acids Reversed phase [116]... 

For most free amino acids and small peptides, a mixture of alcohol with water is a typical mobile phase composition in the reversed-phase mode for glycopeptide CSPs. For some bifunctional amino acids and most other compounds, however, aqueous buffer is usually necessary to enhance resolution. The types of buffers dictate the retention, efficiency and - to a lesser effect - selectivity of analytes. Tri-ethylammonium acetate and ammonium nitrate are the most effective buffer systems, while sodium citrate is also effective for the separation of profens on vancomycin CSP, and ammonium acetate is the most appropriate for LC/MS applications. 

The mixture of free amino acids is reacted with OPA (Fig. 7-8) and a thiol compound. When an achiral thiol compound is used, a racemic isoindole derivative results. These derivatives from different amino acids can be used to enhance the sensitivity of fluorescence detection. Figure 7-9 shows the separation of 15 amino acids after derivatization with OPA and mercaptothiol the racemic amino acids may be separated on a reversed-phase column. If the thiol compound is unichiral, the amino acid enantiomers may be separated as the resultant diastereomeric isoindole compound in the same system. Figure 7-10 shows the separation of the same set of amino acids after derivatization with the unichiral thiol compound Wisobutyryl-L-cysteine (IBLC). 

The stability of the CBI derivative is sufficient for its isolation and complete characterization (11), an accomplishment that is not realized with most OPA adducts. Thus, the CBI derivatives of a number of representative amino acids and amines have been isolated and their fluorescent properties determined as a function of the media and other relevant parameters encountered in reverse-phase HPLC (RP-HPLC). 

Heinrikson, R.L. and Meredith, S.C. 1984 Amino acid analysis by reverse-phase high-performance liquid chromotography Precolumn derivatization with phenylisothiocyanate. Analytical Biochemistry 136 65-74. 

Amino acid analysis, by reverse-phase HPLC, of acid-hydrolyzed uncross-linked recombinant resilin and cross-linked recombinant resilin clearly shows the presence of dityrosine in the cross-linked sample (Figure 9.3c). Further evidence of the presence of dityrosine was obtained by UV irradiation (Xmax,ex 315 nm Xmax,em 409 nm). Dityrosine endows natural resilin with pH-dependent blue fluorescence [38] on UV irradiation. The cross-linked recombinant resilin material was similarly fluorescent, strongly suggesting dityrosine cross-links. 

A simple and rapid method of separating optical isomers of amino acids on a reversed-phase plate, without using impregnated plates or a chiral mobile phase, was described by Nagata et al. [27]. Amino acids were derivatized with /-fluoro-2,4-dinitrophenyl-5-L-alanine amide (FDAA or Marfey s reagent). Each FDAA amino acid can be separated from the others by two-dimensional elution. Separation of L- and D-serine was achieved with 30% of acetonitrile solvent. The enantiomers of threonine, proline, and alanine were separated with 35% of acetonitrile solvent and those of methionine, valine, phenylalanine, and leucine with 40% of acetonitrile solvent. The spots were scraped off the plate after the... 

Water-in-oil microemulsions (w/o-MEs), also known as reverse micelles, provide what appears to be a very unique and well-suited medium for solubilizing proteins, amino acids, and other biological molecules in a nonpolar medium. The medium consists of small aqueous-polar nanodroplets dispersed in an apolar bulk phase by surfactants (Fig. 1). Moreover, the droplet size is on the same order of magnitude as the encapsulated enzyme molecules. Typically, the medium is quite dynamic, with droplets spontaneously coalescing, exchanging materials, and reforming on the order of microseconds. Such small droplets yield a large amount of interfacial area. For many surfactants, the size of the dispersed aqueous nanodroplets is directly proportional to the water-surfactant mole ratio, also known as w. Several reviews have been written which provide more detailed discussion of the physical properties of microemulsions [1-3]. 

Figure 8.43 Separation of enantiomers using complexation chromatography. A, Separation of alkyloxiranes on a 42 m x 0.2S mm I.O. open tubular column coated with 0.06 M Mn(II) bis-3-(pentafluoro-propionyl)-IR-camphorate in OV-ioi at 40 C. B, Separation of D,L-amino acids by reversed-phase liquid chromatography using a mobile phase containing 0.005 M L-histidine methyl ester and 0.0025 M copper sulfate in an ammonium acetate buffer at pH 5.5. A stepwise gradient using increasing amounts of acetonitrile was used for this separation.

Aguilar, M. I., Hodder, A. N., and Hearn, M. T. W., High-performance liquid chromatography of amino acids, peptides, and proteins. LXV. Studies on the optimisation of the reversed-phase gradient elution of polypeptides. Evaluation of retention relationships with (3-endorphin-related polypeptides, /. Chromatogr., 327, 115, 1985. 

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