Chapter 7

Folin–Ciocalteu Reagent (FC Reagent)

INTRODUCTION

Folin–Ciocalteau reagent was first introduced by O. Folin and D. Ciocalteu in 1927. Chemically, it is a hetero polyacid that is phosphomolybdotugstic acid. It produces blue colour with phenolic group.

STRUCTURE

3 H₂O · P₂O₅ · 13WO₃ · 5MoO₃ · 10 H₂O and

3 H₂O · P₂O₅ · 14WO₃ · 4MoO₃ · 10 H₂O.

MECHANISM OF ACTION

When the organic compound that is drug that reacts with hetero polyacid reagent under alkaline conditions undergoes reduction and is converted to molybdenum blue which may be having either blue (or) green colour with a λ_max of 620–780 nm.

The wavelength of maximum absorption and stability and reproducibility of the reaction are dependent on pit, composition of hetero polyacid complex, nature and concentration of reducing agent, temperature and time.

Drug solution is mixed with the alkali such as NaOH (phenolic 4% w/v) or Na₂CO₃ (amines 20% w/v). The FC reagent solution is added and keep a side for the blue or green colour production based on the nature of the compound. The coloured solution is measured at 620–780 nm.

PREPARATION

Into a 1500 ml flask introduce 25 g of sodium tungestate, 25 g sodium molybdate and 700 ml of water. To this, 50 ml of phosphoric acid and 100 ml of HCL are added. Reflux the mixture gently for about 10 h and then 150 g of lithium sulphate, 50 ml mixture without the condenser for about 15 min (or) until the excess bromine is expelled, cool, dilute with water. Before use, dilute one part of filtrate with two parts of water.

STORAGE

This stock solution was stored at a temperature not exceeding 4 °C.

APPLICATIONS

• Used for the determination of adrenaline and non-adrenaline.
• Used for the determination of polyphenol content excreted in urine.
• Used for the determination of gatifloxacin based on oxidative coupling reaction of reduced gatifloxacin with FC reagent in the presence of NaOH. The blue colour solution is measured at 760 nm.
• Used for the determination of sample without –NH₂ and OH groups.

  Examples: Analgin, amidopine, acetazolamide, aspirin, acetanilide, barbituric acid, diazepam, etc.

• Used for the determination of drugs with NH₂ group.

  Examples: Thiamine HCl, p-amino benzoic acid, procaine HCl, ampicillin, thio aceta zolamine, trimethoprim.

• Used for the determination of samples with –NH₂ and/or –OH groups

  Examples: Folic acid, ascorbic acid, doxycycline, tetracycline, pyridine hcl, allopurinol, glucose, mannitol, sorbitol, etc.

• Used for the determination of samples with NH₂ and –COOH groups.

  Example: Tyrosine.

• Used for the determination of antibiotics.

  Examples: Azithromycin, roxythromycin, clarithromycin.

REVIEW QUESTIONS

1. Give the chemical name and structure for the FC reagent.
2. What are the conditions required for the colour production by the FC reagent?
3. What is the mechanism of the action of the FC reagent on the alcohols?
4. How do you prepare the FC reagent?
5. Give the procedure for the determination of the gatifloxacin by the FC reagent.
6. List out the amino group drugs that are analyzed by the FC reagent.