Flavouring agent

VANILLA (Vanilla fragrans)

Vanillin, vanillic acid, 4-hydroxybenzaldehyde and 4-hydroxybenzoic acid by HPLC method

Reagents

•  Ethanol (96% v/v)
•  Methanol
• Dilute phosphoric acid (0.01M)
• Mobile phase : 0.0M Phosphoric acid & methanol (75:25)
•  Prepare standard solutions like Vanilin (0.1 g/L), Vanillic acid (0.008 g/L), 4 hydroxybenzaldehyde (0.0008 g/L) and 4-hydroxybenzoic acid (0.002 g/L) in mobile phase.
•  Internal standard solution (acetyl salicylic acid, 0.6 g/L)

 Procedure

Grind the sample and homogenize it thoroughly. Weigh 20 g of the prepared sample in thimble and extract the test portion in the Soxhlet extractor with about 200 ml of ethanol for 16 h. Make 200 ml of extract in a volumetric flask and make one-in-ten dilution of this solution with mobile phase. Conduct the analysis by HPLC under the following conditions using the mobile phase with a flow rate of 1ml/min.

•  UV detector : wavelength 254 nm
•  Injection volume :10-100 ml
•  Temperature : Ambient
•  Column : C18 bonded silica of 5-10 particle size

 Calibration

Inject the standard and internal standard separately and then as a mixture. Adapt the operating conditions so that the resolution factors of the peaks of all the compounds are at least equal to 1. Inject the prepared sample after filtering through a membrane filter (0.45 mm).