β – Carotene is generally regarded as the most commercially important and widely used carotenoid. It is used as a food coloring agent. It has an anti-oxidant activity. It is currently used incorporated in a wide variety of dietary supplements including multivitamin, Vitamin A and anti-oxidant formulation.


The analysis of beta carotene is determined using both high Performance Liquid Chromatography (HPLC) and Spectrophotometer.

A. Spectrophotometer Method

Spectrophotometer is a still common technique for the analysis of beta carotene in different vegetable oils. The fatty material is dissolved in cyclohexane and in measured at 445 nm. The percentage content of total carotenoids (m/m) is calculated as β – Carotene.


1. Spectrophotometer with visible range.

2. Volumetric flack – 25 ml


1. Cyclohexane – GR

2. Stock Standard β – Carotene: 10 mg of solid beta carotene is dissolved in cyclohexane and the volume make up to 10 ml. This solution contains 1000 ppm of  beta carotene.

3. Working Standard : By diluting the stock standard, 10, 20, 30, 40 & 50ppm of working standard is prepared for calibration curve.


0.05 gm to 0.25 gm of oil sample is taken in 25 ml of volumetric flask. The sample is dissolved by cyclohexane and make up to the mark. The solution is taken in 1 cm of glass cell and measured at 445 nm. A blank sample is performed.



Carotene content ( as β – Carotene) in mg/Kg = Total Vol. x 383 x OD / 100 x Sample Wt.

Fig1fig 2

B. HPLC Method

HPLC analysis is performed using a UV-VIS detector of 450 nm.

Instrumental Condition

Mobile Phase: Hexane: 2-propanol (99.3: 0.7)

Column: Normal Phase C18

Flow: 1.2 ml/min.

Detector: UV-VIS (max 450 nm)

Temperature: 25 oC

Injection Volume: 20 ul.



About 0.5 gm to 1.0 gm of oil is taken into 10 ml volumetric flask. The flask is filled with hexane and the mixture is shaken. The solution is left for 12h in dark. Finally the solution is centrifuged at 500 rpm. The solution is ready for instrument.



The results conclude that the Crude palm Oil (CPO) contained highest amount of β – Carotene compared to other vegetable oils studied. The CPO can be considered as a good source of natural anti-oxidant.