Application: Amine compound derivatization for HPLC analysis

Chemical Name: 4-Fluoro-7-nitrobenzofurazan
CAS: 29270-56-2

Appearance: Pale yellow powder
Purity: ≥99.0% (HPLC)
MW: 183.10, C6H2FN3O3

Storage Condition: -20oC, protect form light and moisture
Shipping Condition: ambient temperature


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Derivatization Reaction

Product Description
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, et al., Fluorimetric Determination of Secondary Amino Acids by 7-fluoro-4-nitrobenzo-2-oxa-1, 3-diazole. Anal Chim Acta. 1981;130:377-383.
2) Y. Watanabe, et al., High-Performance Liquid Chromatography and Sensitive Detection of Amino Acids Derivatized with 7-fluoro-4-nitrobenzo-2-oxa-1, 3-diazole. Anal Biochem. 1981;116:471-472.
3) Y. Watanabe, et al., Pre-column Labelling for High-performance Liquid Chromatography of Amino Acids with 7-fluoro-4-nitrobenzo-2-oxa-1, 3-diazoleand Its Application to Protein Hydrolysates. J Chromatogr A. 1982;239:723-732.
4) T. Toyo’oka, et al., Reaction of Amines of Biological Importance with 4-fluoro-7-nitrobenzo-2-oxa-1, 3-diazole. Anal Chim Acta. 1983;149:305-312.
5) Y. Watanabe, et al., 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;55:1786-1791.
6) Y. Watanabe, et al., Sensitive Detection of Amino Acids in Human Serum and Dried Blood Disc of 3mm Diameter for Diagnosis of Inbornerrors of Metabolism. J Chromatogr. 1984;309:279-286.
7) H. Miyano, et al., Further Studies on the Reaction of Amines and Proteins with 4-fluoro-7-nitrobenzo-2-oxa-1, 3-diazole. Anal Chim Acta. 1985;170:81-87.
8) H. Kotaniguchi, et al., Automatic Amino Acid Analysis Utilizing 4-fluoro-7-nitrobenzo-2-oxa-1, 3-diazole. J Chromatogr. 1987;420:141-145.
9) K. Imai, et al., 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;205:7-14.
10) K. Imai, et al., Dynamic Analytical Chemistry: A Trial Study of the Interaction of Fluorogenic Reagents with Living Chinese Hamster Ovary Cells. Anal Chim Acta. 1989;223:299-308.
11) C. Nakamura, et al., Dioxin-binding Pentapeptide for Use in a High-sensitivity On-bead Detection Assay. Anal Chem. 2005;77:7750-7757.

Related Categories
Labeling Chemistry


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