OPA-ALS

A number of more sophisticated curve resolution procedures for multi-component mixtures have been developed over the years. The most well-known of these procedures are IPCA [77], SIMPLISMA [78] and OPA-ALS [79]. All rely on an a priori estimate of the number of observable species present, most if not all rely on the concept of a pure wavelength i. e. a spectral window where only one component is present, and all have difficulties with trace components i. e. components whose spectral features are embedded in the signal noise. In practice, both the prior estimate of the number of observable species present and the need for pure wavelengths are very serious restrictions, especially for catalytic systems where so many species are simultaneously present. ... 

In Section 4.5.3, discussion was restricted to the application of BTEM to organome-tallic and homogeneous catalytic systems using MIR spectroscopy. However, it should also be noted that BTEM has been successfully applied to other problems in the chemical sciences using FTIR [104], RAMAN, FTIR and RAMAN [105, 106], as well as MS [107, 108]. A one-to-one comparison between BTEM and SIMPLISMA, OPA-ALS, has appeared [109]. 

MEKC-DAD data (54). Studies from Kaniansky and coworkers have focused on using factor analysis, including ITTFA, WFA, and orthogonal projection approach (OPA), for the feasible identification of orotic acid at low concentration level in urine matrices (55, 56). The mathematical resolution of anionic surfactants that cannot be separated electrophoretically has been accomplished by OPA-ALS (57). 

Recent scientific investigations of natural polyphenols have demonstrated their powerful antioxidant property (Niki et al, 1995). Several classes of polyphenols have been chemically identified. Some of these are grape polyphenols, tea polyphenols, soy polyphenols, oligomeric proanthocyanidines (OPA) and other natural polyphenols of the flavone class. Rice bran polyphenols are different from the above in that they are p-hydroxy cinnamic acid derivatives such as p-coumaric acid, ferulic acid and p-sinapic acid. Tricin, a flavone derivative, has also been isolated from rice bran. 

All these observations underscore the potential for application of appropriate OPAs to the destruction of organophosphorus compounds with anticholinesterase activity (Cheng and Calomiris 1996). However, since, hydrolysis results in release of fluoride, the possibility of its subsequent incorporation into organic substrates to produce fluoroacetate and 4-fluorothreonine (Reid et al. 1995) may be worth consideration. 

For 2PA or ESA spectral measurements, it is necessary to use tunable laser sources where optical parametric oscillators/amplifiers (OPOs/OPAs) are extensively used for nonlinear optical measurements. An alternative approach, which overcomes the need of expensive and misalignment prone OPO/OPA sources, is the use of an intense femtosecond white-light continuum (WLC) for Z-scan measurements [71,72]. Balu et al. have developed the WLC Z-scan technique by generating a strong WLC in krypton gas, allowing for a rapid characterization of the nonlinear absorption and refraction spectra in the range of 400-800 nm [72]. 

Merino-Merino et al. [32] used the OPA reagent (o-phthaldehyde condensed with 2-mercaptoethanol) to separate penicillamine enantiomers after their derivatization. Racemic and (/q-penicillamine were dissolved in aqueous 0.5 M NaOH, and treated with the derivatizing solution (methanolic o-phthaldehyde and 2-mercaptoethanol in 0.4 M potassium borate buffer solution of pH 10). The reaction mixture was set aside for 2 min at room temperture, whereupon a portion of solution was analyzed by HPLC. The method used a Cyclobond column (25 cm x 4.6 mm) maintained at 5 °C, a mobile phase of ethanol/1% triethylammonium acetate (1 1 pH 4.5) eluted at... 

OPA in combination with chiral thiols is one method used to determine amino acid enantiomers. A highly fluorescent diastereomeric isoindole is formed and can be separated on a reverse-phase column. Some of these chiral thiols include N-acetyl-L-cysteine (NAC), N-tert-butyloxy-carbonyl- L-cysteine (Boc-L-Cys), N-isobutyryl- L-cysteine (IBLC), and N-isobutyryl- D -cysteine (IBDC). Replacing OPA-IBLC with OPA-IBDC causes a reversal in the elution order of the derivatives of D- and L-amino acids on an ODS column (Hamase et al., 2002). Nimura and colleagues (2003) developed a novel, optically active thiol compound, N-(tert-butylthiocarbamoyl)- L-cysteine ethyl ester (BTCC). This reagent was applied to the measurement of D-Asp with a detection limit of approximately 1 pmol, even in the presence of large quantities of L-ASP. 

There are two major approaches to achieve enantiomeric separation of d- and L-amino acids. The first involves precolumn derivatization with a chiral reagent, followed by RP-HPLC [226], while the second involves direct separation of underivatized enantiomers on a chiral bonded phase [227], Weiss et al. [209] determined d- and L-form of amino acids by applying derivatization with OPA and chiral /V-isobutyryl-L-cysteine. 

As an alternative to RP columns some authors used cation-exchange columns. Salazar et al. [301] used an Alkion column (4x 150mm) heated at 40°C to separate OPA derivate of biogenic amines extracted from complete feeds and animal tissues, while Saccani et al. [302] used an lonPac CS17 column and a gradient elution of methanesulfonic acid to separate biogenic amines in meat products. 

Moret et al. [280] applied precolumn derivatization with Dns-Cl and OPA to the same sample (vegetables) extracts and compared the results. Figure 19.6 reports HPLC traces obtained for a spinach sample (Kromasil C18 column, 250x4.6mm i.d., 5[tm particle size). 

FIGURE 19.6 HPLC trace of a spinach sample derivatized with (A) OPA and (B) Dns-Cl. (From Moret, S. et al., Food Chem., 89, 355, 2005. Copyright 2005. With permission from Elsevier.)... 

Since then, there have been a number of reversed-phase separations employing precolumn derivatization. Interestingly, fluorescamine (not frequently employed for RP-HPLC of amino acids with precolumn reaction) has been reported for taurine analysis in milk (197) and human plasma (198). Precolumn derivatization with OPA/2-mercaptoethanol has been reported for the analysis of infant formula and human breast milk (199). Although not the principal focus of the study, Carratu et al. (200) report taurine values in parenteral solutions as determined by FMOC. In an excellent article, Woollard and Indyk (201) report the dansylation of taurine for its determination in a wide variety of dairy-related products. Subsequently, the same authors report the results of a large collaborative study (202) for the determination of taurine (again, by dansylation) in milk and infant formula. This study afforded an overall interlaboratory RSD of 7.0% and established a lower limit for determination at 5 mg taurine per 100 g of product. 

The specific peptide composition can be used to characterize foods. Abe (124) separated the peptides carnosine, anserine, and balenine from the white and red muscle of nine species of marine fishes. Carnegie et al. (37,38) developed an HPLC method using a Partisil-lOSCX column with 0.2 M lithium formate at a pH of 2.9 and a temperature of 40°C under isocratic conditions with postcolumn derivatization using OPA to separate the dipeptides of histidine, anserine, carnosine, and balenine from the muscles of various species (pork, chicken, beef, lamb, and mutton) in order to identify the origin of the meat used in meat products. The concentration of balenine and the balenineranserine ratio were higher in pork than in the other meats, and these relationships were useful in determining the presence of pork in mixtures with other meats. 

O-Phthaldialdehyde (OPA) is an amine detection reagent that reacts in the presence of 2-mercaptoethanol to generate a fluorescent product (for preparation, see Section 4.1, 2-mercaptoethanol) (Fig. 91). The resultant fluorophore has an excitation wavelength of 360 nm and an emission point at 455 nm. OPA can be used as a sensitive detection reagent for the HPLC separation of amino acids, peptides, and proteins (Fried et al., 1985). It is also possible to measure the amine content in proteins and other molecules using a test tube or microplate format assay with OPA. Detection limits are typically in the microgram per milliliter range for proteins. 

Fluorimetric methods for the determination of amino acids are generally more sensitive than colorimetric methods. Fluorescamine (4-phenyl-spiro[furan-2(3H),l -phthalan]-3,3 -dione) and o-phthaldialdehyde (OPA) substances are used for protein analysis. Fluorescamine reacts with amino groups to form fluorophores that excite at 390 nm and emit at 475 nm (Weigele et al., 1972). Applications of fluorescamine include monitoring the hydrolysis of K-casein (Beeby, 1980 Pearce, 1979) and quantification of proteins, peptides, amino acids in extracts (Creamer et al., 1985). OPA produces fluorescence on reaction with 2-mercaptoethanol and primary amines, with strong absorption at 340 nm. Lemieux et al. (1990) claimed that this method was more accurate, convenient, and simple for estimating free amino acids than the TNBS, ninhydrin, or fluorescamine methods. 

Ultrafiltration 1 Low pressure exclusion chromatography Cut-off membrane 10000 Da 1 Sephadex LH-20 RP-HPLC Ultrasphere ODS column Absorbance at 214 nm and fluorescence of the OPA derivatives Acedo et al. (1994)... 

Proteins precipitation 4. Low pressure exclusion chromatography Ethanol 95% 4 Sephadex G-10 RP-HPLC Nova-Pak CIS column Scanning from 190 to 2S0 nm and fluorescence of the OPA derivatives Bartolome et al. (1997)... 

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