O-Phthalaldehyde reagent

A fluorescence detection system for sialic acid has also been reported [39]. For the detection of sialic acid, o-phthalaldehyde reagent was used for the fluorescence measurement of amino residues in the polymer matrix. According to the authors, when sialic acid was bound to the polymer, the fluorescence intensity was increased, because the binding of the template increased the permeability of the o-phthalaldehyde reagent because of the swelling change. The increase in fluorescence was proportional to the amount of sialic acid bound. 

Detector F ex 335 em 455 following post-column reaction with o-phthalaldehyde reagent solution (Pierce) pumped at 1 mL/min. The mixture flowed through a 250 X 4.6 column packed with 40 pm glass beads (Whatman) to the detector. 

Detector F ex 340 em 455 following post-column derivatization. The column effluent mixed with an o-phthalaldehyde reagent (Pierce) and flowed through a reaction coil (PCR 520, Applied Biosystems) at 33° to the detector. 

Figure 1. Protocol 1 standard amino acid analysis. A calibration mixture containing 1.0 nmol of each amino acid was injected onto a 0.40 x 13 cm bed of Dionex DC-5 A cation-exchange resin. Four discrete buffer solutions were pumped through the column at 18 ml/hr to achieve the indicated separation. Column temperature 45°C changed to 65°C at 12 min. Eluent pump pressure was 55 atm at 45°C. o-Phthalaldehyde reagent was added to the column effluent to produce fluorescent derivatives, which were continuously monitored using a fluoro-meter and strip chart recorder.

Note If netilmicin is to be chromatographed alone it is recommended that the methanol content of the mobile phase be increased (e.g. to 23 -I- 7), in order to increase the value of the hRf. The detection limit for the substances in the application tested was more sensitive using DOOB reagent on RP layers than when NBD chloride, fluorescamine or o-phthalaldehyde were employed. The derivatives so formed were stable and still fluoresced after several weeks if they were stored in the dark. 

Another reagent that readily forms fluorescent derivatives with primary amines is o-phthalaldehyde (trade name "Fluoropa"). The reaction proceeds in aqueous solution in the presence of a mercaptan at a pH of 9-11 producing an isoindole. 

The derivatives have an optimum fluorescence at an excitation wavelength of 340 nm and an emission wavelength of 455 nm. The adduct is relatively stable at a pH of 9-11 but it rapidly degrades to a non-fluorescent residue at low pH values. Consequently, when used as a pre-column derivatizing reagent the pH of the mobile phase should be kept fairly high, o-phthalaldehyde has been employed for derivatization in the analysis of dopamine (29), catecholamines (30) and histamines (31). 

Note o-Phthaldehyde in the presence of mercaptoethanol or cysteine has already been discussed as a reagent [4]. The present monograph describes the use of o-phthal-aldehyde in the presence of sulfuric add. There are, in addition, a number of applications, which have been described, employing o-phthalaldehyde without any additives e. g. for the detection of primary arylamines, histamine, histidine and histidylpeptides [5-71. 

For fast reactions (i.e., < 1 min.), open tubular reactors are commonly used. They simply consist of a mixing device and a coiled stainless steel or Teflon capillary tube of narrow bore enclosed in a thermostat. The length of the capillary tube and the flow rate through it control the reaction time. Reagents such as fluorescamine and o-phthalaldehyde are frequently used in this type of system to determine primary amines, amino acids, indoles, hydrazines, etc., in biological and environmental samples. 

Derivatization of primary amino acids with o-phthalaldehyde (OPA) is simple and the poor reproducibility due to the instability of the reaction product can be improved by automation and the use of alternative thiols, e.g. ethanthiol in place of the 2-mercaptoethanol originally used. An alternative fluorimetric method using 9-fluoroenylmethylchloroformate (FMOC-CL) requires the removal of excess unreacted reagent prior to column chromatography. This procedure is more difficult to automate fully and results are less reproducible. However, sensitivity is comparable with the OPA method with detection at the low picomole or femtomole level, and it has the added advantage that both primary and secondary amino acids can be determined. 

A comparative study was made of the RP-HPLC analysis of free amino acids in physiological concentrations in biological fluids, with pre-column derivatization by one of the four major reagents o-phthalaldehyde (73) in the presence of 2-mercaptoethanol, 9-fluorenylmethyl chloroformate (90), dansyl chloride (92) and phenyl isothiocyanate (97, R = Ph) (these reagents are discussed separately below). Duration of the analysis was 13-40 min. Sensitivity with the latter reagent was inferior to the other three however, its use is convenient in clinical analysis, where sample availability is rarely a problem. The derivatives of 73 were unstable and required automatized derivatization lines. Only 92 allowed reliable quantation of cystine. All four HPLC methods compared favorably with the conventional ion-exchange amino acid analysis188. 

Latent fingerprints on paper have been revealed by combining the amino acids present with reagents such as ninhydrin (see 37), dansyl chloride (92), fluorescamine (154), 4-chloro-7-nitrobenzofurazan (127a) and o-phthalaldehyde (see reaction 7). To avoid some problems encountered with these reagents it was proposed to use 1,8-diazafluorenone (155), leading to the formation of highly fluorescent ylides (156)349. 

Fluorescence is not widely used as a general detection technique for polypeptides because only tyrosine and tryptophan residues possess native fluorescence. However, fluorescence can be used to detect the presence of these residues in peptides and to obtain information on their location in proteins. Fluorescence detectors are occasionally used in combination with postcolumn reaction systems to increase detection sensitivity for polypeptides. Fluorescamine, o-phthalaldehyde, and napthalenedialdehyde all react with primary amine groups to produce highly fluorescent derivatives.33,34 These reagents can be delivered by a secondary HPLC pump and mixed with the column effluent using a low-volume tee. The derivatization reaction is carried out in a packed bed or open-tube reactor. 

Fluorometric detection has also been employed for the determination of sulfonamides in edible animal products, because it confers the advantages of selectivity and sensitivity. Although sulfonamides possess weak native fluorescence, their sensitive lluorometric detection necessitates use of precolumn or postcolumn derivatization producing the corresponding fluorescent derivatives. The most commonly used derivatizing reagent for precolumn derivatization is fluorescamine (217, 228, 230, 238, 239), while for postcolumn derivatization fluorescamine (231), and o-phthalaldehyde (OPA), and -mercaptoethanol (219) are most often used. 

N Nimura, T. Kinoshita. o-Phthalaldehyde-A -acetyl-t.-cystcine as a chiral derivatization reagent for liquid chromatographic optical resolution of amino acid enantiomers and its application to conventional amino acid analysis. J Chromatogr 352 169-177, 1986. 

Method (manual). An aliquot portion (0.1 ml) of effluent containing the amino acid is mixed with 3 ml of a solution containing 1.5 ml of o-phthalaldehyde (10 mg/ml in ethanol), 90 ml of sodium tetraborate buffer (0.05 M, pH 9.5) and 1.5 ml of a solution of 2-mercaptoethanol (5 mg/ml) in ethanol. The latter reagent should be freshly prepared each day. The reaction mixture is permitted to stand for 5 min and the intensity of fluorescence is measured in a fluorimeter (340-nm excitation, 455-nm emission). The buffered reagent may be also useful as a spray reagent for amino acids separated by TLC, although such an investigation has not been reported. 

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