Fluorescein sodium, reagent

The following are amongst the reagents that have been reported as being added to the mobile phase acids for quinine alkaloids [184], ninhydnn for amino acids [185 — 187], fluorescamine for biogenic amines [188] Fluorescein sodium [189], dichlorofluorescein [190], rhodamine 6G [191], ANS reagent [192] and bromine [193] have all been descnbed as additives to mobile phases... 

Note The full fluorescence intensity usually only develops about 30 min after the dipping process it then remains stable for several days if the chromatograms are stored in the dark (1, 5]. Fluorescein sodium can be employed in the reagent in place of 2, 7 -dichlorofluorescein [5]. The detection limits lie in the lower nanogram to picogram range [1, 5]. 

Figure 9.11 This carbohydrazide-containing fluorescein derivative can be used to modify aldehyde-containing molecules. Glycoconjugates may be labeled with this reagent after treatment with sodium periodate to produce aldehydes.

In Inorganic Chemistry, typical spray reagents for cations include potassium iodine (0.2%, aqueous), hydrogen sulphide (saturated aqueous solution), ammonium sulphide (0.2 N, aqueous), quercetin (0.1%, alcoholic), l-(2-pyridylazo)-2-naphthol (PAN) (0.2%, methanolic), oxine (8-hydroxyquinoline) (1% methanolic, view under visible and UV light), and sodium rhodizonate (0.5%, aqueous). Reaction with dithizone to produce coloured dithizonate chelates of many metals is particularly suitable if quantitative spectrometric analysis (in situ or after elution) is to be carried out. Anions are detected with bromocresol purple (0.1%, alcoholic), 1% ammoniacal silver nitrate + 0.1% alcoholic fluorescein/UV light, zirconium alizarin lake (0.1% in HC1 solution), and ammonium molybdate (1%, aqueous) followed by SnCl2 (1%) and HC1 (10%). Typical detection limits range from 10 ng (10 9g) to several pg (10 6g). 

Chromosomal localization of the axl gene is done by fluorescence in situ hybridization (FISH). The biotin-labeled axl probe is from a cosmid vector containing a 41-kb genomic fragment. The slides with human metaphase cells for chromosomal DNA to be denatured are immersed into a solution of 70 % formamide-x4 in saline sodium citrate buffer (SSC) atpH 7.0 (0.15 M NaCl 0.015 M sodium citrate) at 70 °C for 2 min. For the slides, the hybridization mixture kept at 75 °C consists of 50 %formamide,xl SSC, 10 % dextran sulfate pH 7.0, and 150 mL unlabeled sonicated total human genomic DNA add 50-100 ng labeled probe. The mixture is incubated at 37 °C for 10 min. The slides are washed in 50 % formamide and SSC x3, 5 min each at 40 °C and in SSC x3 at 40 °C. The detection reagent consists of x4 SSC-0.1 % Triton, and 1 % bovine semm albumin for 3 washes 3 min each at 40 °C. The slides are incubated with fluorescein isothiocyanate-conjugated avidin at 37 °C for 30 min. Metaphase cells are counterstained with 4,6-diamidino-2-phenylindole dihydrochloride 200 ng/mL in 2x SSC for 5 min at... 

A layer impregnated with fluorescein (Rgt. No. 121,6) has been used for detection. Pink spots are formed by amounts exceeding 5 (xg after standing for 1—2 h. Dark blue-violet spots on the green fluorescent layer are seen in long-wave UV radiation. The rhodamine B reagent (No. 120) can also be used, followed by spraying with a 10 % solution of sodium carbonate [101] (see also Rgt. No. 77). 

Fluorescein isothiocyanate is an alternative reagent however it requires 0.1 M sodium carbonate buffer pH 9.0, and a reaction time of 8 h at 4 °C. 

Procedure. A small amount of the solid test material or a drop of its solution is treated in a micro test tube with several drops of chromic-sulfuric acid (preparation see Vol. II page 65). The open end of the test tube is covered with a disk of filter paper moistened with 2 N sodium hydroxide. The test tube is kept for 5 minutes in boiling water. The disk of reagent paper is then placed on a glass plate, spotted with several drops of permolybdic acid solution (for preparation see Vol. II page 68) and then dried with a current of cold air. If iodine is present, a brown fleck will appear but it fades within a few minutes. The stain is then treated with a drop of 0.1 % alcoholic solution of fluorescein. A positive response is indicated by the development of a red spot. 

Reagents. Fluorescein solution - dissolve 0.1 g of fluorescein in 25 ml of 0.1 N sodium hydroxide and dilute to 100 ml with water. 

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