Phthalaldehyde Reagent

Dipping solution Make 0.1 g o-phthalaldehyde (phthaldialdehyde, OPA) and 0.1 ml 2-mercaptoethanol (2-hydroxy-l-ethanethiol) up to 100 ml with acetone. 

Storage The reagent solution is stable for several days when stored in 

In the presence of 2-mercaptoethanol o-phthalaldehyde reacts with primary amines to yield fluorescent isoindole derivatives [20]  

Immerse the dried chromatogram for 1 s in the reagent solution and then heat to 40 - 50 °C in the drying cupboard for 10 min. 

Substance zones are produced that mainly yield blue fluorescence under long-wavelength UV light (A = 365nm) (indoles occasionally fluoresce yellow [15]), colored zones are also produced occasionally. The fluorescence is stabilized by immersing in 20% methanohc polyethylene glycol solution [5]. 

---

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. 

LLE liquid-liquid extraction SPE solid-phase extraction SPM solid-phase microextraction MAE microwave-assisted extraction SEE supercritical fluid extraction FMASE focused microwave-assisted Soxhlet extraction PSDB polystyrene-divinylbenzene NVPDB A-vinylpyrrolidane-divinylbenzene SPE solid-phase extraction LLE liquid-liquid extraction GPC gel permeation chromatography OPA f -phthalaldehyde reagent TAD thermally assisted desorption FI flow injection... 

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.

The most widely appHed colorimetric assay for amino acids rehes upon ninhydrin-mediated color formation (129). Fluorescamine [38183-12-9] and (9-phthalaldehyde [643-79-8] are popular as fluorescence reagents. The latter reagent, ia conjunction with 2-mercaptoethanol, is most often used ia post-column detection of amino acids separated by conventional automated amino acid analysis. More recently, determiaation by capillary 2one electrophoresis has been developed and it is possible to determine attomole quantities of amino acids (130). 

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). 

The initial discoveries of the extension of the aromatic ring of the ortho-phthalaldehyde (OPA) to a naphthalene-2,3-dicarboxaldehyde (NBA) and the substitution of cyanide (CN ) for 2-ME as the nucleophile have provided the Center with a much more versatile reagent system (5,11), which maintains the sensitivity for primary aliphatic amines and amino acids, and now is known to form fluorescent products with oligopeptides, proteins, and other related analytes that possess a primary amine function (Equation 1). 

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. 

The c-phthalaldehyde reaction compares favourably with the ninhydrin reaction in several respects. The reagent is stable and is in an aqueous form, which eliminates the use of potentially toxic chemicals and storage under nitrogen. Because the reaction proceeds quickly at room temperature there is no need for the 100°C heating bath with all its inherent problems, and the increased sensitivity permits detection at the picomole level. 

---