Chapter 2

Tri Keto Hydrindine Hydrate (Ninhydrin Reagent)

INTRODUCTION

Ninhydrin reagent is chemically known as tri keto hydrindine hydrate which gives positive reaction with α-amino acids, it produces violet colour due to the formation of amine of diketo hydrindylidene (or) diketo hydrin amine.

STRUCTURE

 

Triketo hydrindine hydrate

MECHANISM OF ACTION

• The initial step involves oxidative deamination of amino acids through a transitory iminoacid and α-ketoacid to CO₂, NH₃ and CHO containing one carbon atom less than the original amino acid and ninhydrin as reduced to hydrindantin which is in violet colour.

   

  

   

  Hydrindantin

   

• For stabilising the colour, stabilising agents such as stannous chloride and citrate buffer are used.
• This reagent is mainly used as a spraying agent for the identification of amino acids in TLC and paper chromatography.

PREPARATION

• 4 g of ninhydrin in 100 ml of 2-methoxy ethanol.
• 0.25% w/v ninhydrin in a mixture of 95 ml of 1-butanol, 5 ml of 2M acetic acid gives 0.25% ninhydrin solution.

APPLICATIONS

• Used for the determination of amino acids such as glycine, praline, hydroxypholine. etc. Proline solution is mixed with the ninhydrin reagent in the presence of stannous chloride. Then the produced purple coloured solution is measured at 520–570 nm.
• Used for the determination of pantenol and pantothenate. The hydrolysed solution of pantenol is mixed with the β-alanol and ninhydrin reagent. Then after 15 min, the solution attains the pink coloir which is measured at 570 nm.

  Method: The multivitamin preparation is passed through Doulex column at the rate of 0.5 ml/min. Then eluted with 0.25 N NaOH and then heated for 1 h under reflex. Ninhydrin reagent is added followed by heating at 70 °C. The pink coloured solution is measured at 570 nm.

• Used for the determination of antibiotics.

  Example: Neomycin, polymeric B, bacitracin, novimycin, viomycin.

  An antibiotic gives positive colour with ninhydrin which indicates the amino group. Here pyridine which is an organic base is added to maintain bifidity. This solution is heated on steam bath which gives deep purple colour at 570 mn.

• Used for the determination of piperazine. Ninhydrin gives purple colour with piperazine which is measured at 510 nm.

  Method: Drug is dissolved in water and the solution is mixed with the 5 ml of 2-methoxyethanol and 1 ml ninhydrin reagent. Then heat to boiling for 45 min and immediately cool the solution and make up the volume to 25 ml with isopropyl alcohol. The resulting purple colour solution is measured at 570 nm.

REVIEW QUESTIONS

1. Specify the appropriate reagent for the amino acids analysis.
2. Explain the mechanism of the ninhydrin reagent.
3. Give the procedure for the preparation of 0.2% of the ninhydrin reagent.
4. What is the principle involved in the analysis of proline and glycine by the ninhydrin reagent?
5. What is the procedure involved in the analysis of vitamins by the ninhydrin reagent?
6. What are antibiotics can be analysed by the ninhydrin reagent?