Derivatization Reagent for HPLC
Product codeN020 NBD-F
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NBD-F is highly reactive and can label primary and secondary amines under mild conditions (1 min reaction at 60ºC in a weak basic solution). NBD-F is a pre-labeling compound for HPLC analysis of small molecules. NBD-labeled compounds are orange with a maximum wavelength at 470 nm.The excitation and emission of the derivatized compound are 470 nm and 530 nm, respectively.
NBD Labeling Protocol
1. To prepare sample solution, mix or dissolve a sample with 50 mM borate buffer (pH 8.0) containing 20 mM EDTA.
2. Mix 300 μl of the sample solution and 100 μl of 100 mM NBD-F/acetonitrile solution in a reaction vial.
3. Heat the vial at 60ºC for 1 minute and then cool it on an ice bath.
4. Add 400 μl of 50 mM HCl aqueous solution to the reaction mixture.
5. Use this mixture for HPLC analysis to determine NBD-labeled compounds.
1) K. Imai and Y. Watanabe, "Fluorimetric Determination of Secondary Amino Acids by 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole", Anal. Chim. Acta, 1981, 130, 377.
2) Y. Watanabe and K. Imai, "High-Performance Liquid Chromatography and Sensitive Detection of Amino Acids Derivatized with 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole", Anal. Biochem., 1981, 116, 471.
3) Y. Watanabe and K. Imai, "Pre-column Labelling for High-performance Liquid Chromatography of Amino Acids with 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole and Its Application to Protein Hydrolysates", J. Chromatogr., 1982, 239, 723.
4) T. Toyo'oka, Y. Watanabe and K. Imai, "Reaction of Amines of Biological Importance with 4-fluoro-7-nitrobenzo-2-oxa-1,3-diazole", Anal. Chim. Acta, 1983, 149, 305.
5) Y. Watanabe and K. Imai, "Liquid Chromatographic Determination of Amino and Imino Acids and Thiols by Postcolumn Derivatization with 4-Fluoro-7-nitrobenzo-2,1,3-oxadiazole", Anal. Chem., 1983, 55, 1786.
6) Y. Watanabe and K. Imai, "Sensitive Detection of Amino Acids in Human Serum and Dried Blood Disc of 3 mm Diameter for Diagnosis of Inbornerrors of Metabolism", J. Chromatogr., 1984, 309, 279.
7) H. Miyano, T. Toyo'oka and K. Imai, "Further Studies on the Reaction of Amines and Proteins with 4-fluoro-7-nitrobenzo-2-oxa-1,3-diazole", Anal. Chim. Acta, 1985, 170, 81.
8) H. Kotaniguchi, M. Kawakatsu, T. Toyo'oka and K. Imai, "Automatic Amino Acid Analysis Utilizing 4-fluoro-7-nitrobenzo-2-oxa-1,3-diazole", J. Chromatogr., 1987, 420, 141.
9) K. Imai, E. Ueda and T. Toyo'oka, "High-Performance Liquid Chromatography with Photochemical Fluorimetric Detection of Tryptophan Based on 4-fluoro-7-nitrobenzo-2-oxa-1,3-diazole Total Protein Amino Acid Analysis", Anal. Chim. Acta, 1988, 205, 7.
10) K. Imai, Y. Tsukamoto, S. Uzu, S. Kanda, T. Toyo'oka, Y. Tachiiri and S. Fujiwake, "Dynamic Analytical Chemistry: A Trial Study of the Interaction of Fluorogenic Reagents with Living Chinese Hamster Ovary Cells", Anal. Chim. Acta, 1989, 233, 299.
11) C. Nakamura, Y. Inuyama, H. Goto, I. Obataya, N. Kaneko, N. Nakamura, N. Santo and J. Miyake, "Dioxin-binding Pentapeptide for Use in a High-sensitivity On-bead Detection Assay", Anal. Chem., 2005, 77, 7750.
Handling and storage condition
|Appearance：||Pale yellow powder|
|Purity (HPLC)：||≧ 99.0 %|
|Solubility in ethyl alcohol：||To pass test (clear, pale yellow)|
|-20°C, Protect from light|