Fluorescamine detection limits

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. 

Detection of amino acids is typically by UV absorption after postcolumn reaction with nin-hydrin. Precolumn derivatization with ninhydrin is not possible, because the amino acids do not actually form an adduct with the ninhydrin. Rather, the reaction of all primary amino acids results in the formation of a chromophoric compound named Ruhemann s purple. This chro-mophore has an absorption maximum at 570 nm. The secondary amino acid, proline, is not able to react in the same fashion and results in an intermediate reaction product with an absorption maximum at 440 nm. See Fig. 5. Detection limits afforded by postcolumn reaction with ninhydrin are typically in the range of over 100 picomoles injected. Lower detection limits can be realized with postcolumn reaction with fluorescamine (115) or o-phthalaldehyde (OPA) (116). Detection limits down to 5 picomoles are possible. However, the detection limits afforded by ninhydrin are sufficient for the overwhelming majority of applications in food analysis. 

The spots were located by examination under UV light or spraying with one of following solutions (detection limits in yg) fluorescamine (0.5), ninhydrin (1), NBP followed by heating and treatment with a base (5) [51,63a], and 0.5% iodine in chloroform. Visualization using 4-pyridine-carboxaldehyde 2-benzothiazolyl hydrazone has been described under 5.2.1. 

Saxitoxin has been labeled with fluorescamine, o-phthaldialdehyde (OPA) and dansyl chloride and detection limits as low as 0.1 attomole were reported for the OPA derivative of saxitoxin (26). Labeling, separation, and analysis of saxitoxin was best accomplished using fluorescamine, which produces ionic derivatives that can be separated from other fluorescently labeled marine toxins, such as tetrodotoxin and microcystin. However, the precolumn labeling methods required xM concentrations of analyte, limiting the utility of the technique for trace analysis. 

A protein-binding assay (BA) coupled with hplc provided a highly sensitive post-column reaction detection system for the biologically important molecule biotin and its derivative biocytin, biotin ethylenediamine, 6-(biotinoylamino) caproic acid, and 6-(biotinoylamino)caproic acid hydrazide (71). This detection system is selective for the biotin moiety and responds only to the class of compounds that contain biotin in their molecules. In this assay a conjugate of streptavidin with fluorescamine isothiocyanate (streptavidin—FITC) was employed. Upon binding of the analyte (biotin or biotin derivative) to streptavidin—FITC, an enhancement in fluorescence intensity results. This enhancement in fluorescence intensity can be directly related to the concentration of the analyte and thus serves as the analytical signal. The hplc/BA system is more sensitive and selective than either the BA or hplc alone. With the described system, the detection limits for biotin and biocytin were found to be 97 and 149 pg, respectively. 

A less costly alternative to fluorescamine is o-phthaldehyde (OPT), the derivatives of which are more stable and consequently can be stored overnight if necessary. It is used in a similar manner to fluorescamine the detection limits being about 0.1 ng ca. 4 x g/ml). OPT has been used in the analysis of dopamine, catecholamines and histamines. Other fluorescence reagents that are sometimes used include 4-bromoethyl-7-methoxycoumarin, diphenylindene, sulphonyl chloride, dansyl-hydrazine and a number of fluorescent isocyanates. 

An older method employing fluorescamine may be used if improved detection limits are required. In this method, fluorescamine (Fig. 1.6) reacts with the primary amine groups of the protein, generating flourophores that can be excited at 390 nm to generate emission at 475 nm. Figure 1.6 also shows calibration curves for a variety of proteins this method is capable of detecting as little as 10-ng total protein.10... 

Fluorescamine is employed for more sensitive determination of amino acids by fluorescence spectroscopy. Since it undergoes rapid hydrolysis under aqueous conditions, the reagent is normally dissolved in acetone. It is commonly employed for the detection of amino acids on surfaces, such as thin-layer chromatography (TLC) plates. Amino acids are detected based on emission of light at 470 nm following excitation at 390 nm. Detection limits for amino acids are in the low picomole range. 

In these systems, a high-energy intermediate excites a suitable fluorophore, which then emits its characteristic fluorescence spectrum consequently, they are termed indirect or sensitized chemiluminescence. The most common analytical application has been as a postcolumn reaction detector for liquid chromatography. Various fluorescent analytes (polycyclic aromatic hydrocarbons and polycyclic aromatic amines) and compounds derivatized using dansyl chloride, fluorescamine, or o-phthalaldehyde have been determined with sub-femtomole detection limits. 

The second fluorescent stain, which needs mention, is anilinonaphthalene sulphonate (ANS) which does not fluoresce in water. It does, however, fluoresce when dissolve in organic solvents or when it becomes bound to the hydrophobic regions of proteins.The stain is not as sensitive as fluorescamine and the lowest detection limit Is only 20 mg. The method involves incubation of the gels in 3N HCI for about 5 minutes to denature the proteins. After this the gels are incubated in a buffered solution of the stain. If sensitivity is not such a big limitation, i.e., if the quantity of protein separated is high, one may bypass the acid denaturation step. This allows the final recovery of proteins in the active form. 

The derivatives are used for amino acid analysis via HPLC separation. Instead of mercapto-ethanol, a chiral thiol, e.g., N-isobutyryl-L-cysteine, is used for the detection of D-amino acids. The detection hmit lies at 1 pmol. The very fast racemizing aspartic acid is an especially suitable marker. One disadvantage of the method is that proline and hydroxyproline are not detected. This method is apphed, e.g., in the analysis of fruit juices, in which high concentrations of D-amino acids indicate bacterial contamination or the use of highly concentrated juices. Conversely, too low concentrations of D-amino acids in fermented foods (cheese, soy and fish sauces, wine vinegar) indicate unfermented imitations. Fluorescamine reacts with primary amines and amino acids - at room temperature under alkaline conditions - to form fluorescent pyrrolidones (Aex = 390 nm, Aem - 474 nm). The detection limit lies at 50-100 pmol ... 

ABEI, -(4-aminobutyl)- -ethylisoluminol BSA, bovine serum albumin CL, chemiluminescence DNPO, 6i s-(2,4-dinitrophenyl)oxalate ECL, electrogenerated chemiluminescence EMMA, electrophoretically mediated microanalysis EY, eosine Y FR, fluorescamine HRP, horseradish peroxidase ILITC, isoluminol isothiocyanate LOD, limit of detection RITC, rhodamine B isothiocyanate TCPO, bis-(2,4,6-trichloropheny 1)oxalate TEA, triethylamine TRITC, tetramethylrhodamine isothiocyanate. 

Method 3 (TLC). The amino acids and peptides are separated on silica gel plates with butanol-acetic acid-ethyl acetate-water (1 1 1 1). The chromatogram is dried at 110 °C for 10 min and then cooled to room temperature. The plate is sprayed with a 10% solution of triethylamine in methylene chloride and is dried in air for several seconds. A solution of 0.05% fluorescamine in acetone is then sprayed on to the plate. The plate is again dried in air and is then resprayed with the triethylamine solution before observation under UV light. This procedure was found to be superior to the earlier procedure of using aqueous buffers for spraying the plates prior to fluorescamine reaction [89]. The limit of detection for the modified spray method is 0.5 nmole of the amino acid or peptide. 

VISUAL LIMITS OF DETECTION OF ANILINES WITH FLUORESCAMINE [100]... 

The column packing consists of Vydac reversed-phase material (chemically bonded ODS). The fluorescamine derivatives are separated by gradient elution with 10% methanol in buffer (pH 8.0), followed by a linear increase in proportion to 40% methanol in buffer (pH 8.0). The separation of several diamine derivatives with this system is shown in Fig.4.51. The limits of detection are ca. 2S-S0 pmoles of amine using a fluorimeter with a microflow cell. Both amino groups of the diamines react with fluorescamine so that a minimum of a 2 1 molar ratio of fluorescamine to diamine is required for the best results, a ratio of 3 1 or 4 1 is preferred. 

Phenylcarbamate, phenylurea and many amide herbicides which yield anilines on hydrolysis can be detected after TLC by spraying with fluorescamine [100]. The limits of detection of a number of anilines separated by TLC and detected with fluorescamine are given in Table 4.16. A number of pesticides which yield anilines on hydrolysis or degradation is listed in Table 4.28. The analysis of anilines by this technique is described in Section 4.2.1.2.3. 

For increased sensitivity, reagents that yield fluorescent products are employed. These include OPA, fluorescamine, and 4-fluoro-7-nitro-benzo-2-oxa-l, 3-diazole (NBD-F). Derivatization with OPA provides the best limits of detection for most amino acids. Although the products of OPA derivatization are unstable and decompose fairly rapidly to produce nonfluorescent 2,3-dihydro-lH-isoindol-l-ones, this is not a problem with postcolumn derivatization since the products are detected almost instantaneously. 

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