Vitamin-A, Vitamin-E & Vitamin-D Analysis Using HPLC_Part-1 (Sample Preparation)

Summary

This video walks through the detailed procedure for preparing samples to analyze Vitamin A and E using High-Performance Liquid Chromatography (HPLC). It covers six essential steps: reagent preparation, saponification, vitamin extraction, filtration, drying, dilution, and injection. Key equipment such as reflux condensers, separating funnels, and HPLC systems are highlighted, along with the chemicals involved like hexane, ethanol, and potassium hydroxide. The procedure emphasizes accuracy in each step for optimal results in vitamin analysis, with careful attention to sample handling, extraction, and solubilization techniques.

Takeaways

• 😀 Sample preparation for the analysis of Vitamin A and Vitamin E involves six key steps: reagent preparation, sample preparation, saponification, vitamin extraction, filtration, and drying.
• 😀 Proper equipment needed includes a balanced machine, water bath, reflux condenser, saponification flask, separating funnel, hot air oven, measuring cylinders, beakers, funnels, syringes with filters, and various chemicals like n-hexane and ethanol.
• 😀 Step 1: Prepare a 50% potassium hydroxide solution by dissolving 50 grams of potassium hydroxide in 100 mL of deionized water.
• 😀 Step 2: Sample preparation should be done in low light conditions to ensure accurate results, though the video demonstrates it under normal light for clarity.
• 😀 Step 3: Weigh 5 grams of crushed and ground sample and transfer it into a clean, dry saponification flask.
• 😀 Step 4: Add 1 gram of L-ascorbic acid, 50 mL of ethanol, and 15 mL of the potassium hydroxide solution to the flask and mix well.
• 😀 Step 5: Heat the flask at 74°C using a water bath with periodic shaking for 90 minutes, then cool it rapidly in ice-cold water to stop the reaction.
• 😀 Step 6: Extract the vitamins using hexane by transferring the saponified sample to a separating funnel, washing it with ethanol and deionized water.
• 😀 Step 7: Shake the funnel and allow it to cool. Add hexane and shake again, repeating the extraction until a clear hexane layer forms.
• 😀 Step 8: Wash the hexane extract with deionized water multiple times to remove residual potassium hydroxide. Test the washings with a fenugreek indicator to confirm the removal of potassium hydroxide.
• 😀 Step 9: Filter the hexane extract using sodium sulfate anhydrous to remove any remaining water and impurities, and then evaporate the hexane at 37°C.
• 😀 Step 10: Solubilize the extracted vitamins in 10 mL of methanol, ensuring complete dissolution, then inject 20 microliters into an HPLC system for Vitamin A and E analysis.

Q & A

What are the six key steps in sample preparation for vitamin A and vitamin E analysis?

The six key steps are reagent preparation, sample preparation, saponification, vitamin extraction, filtration and drying, and injection for analysis.

Why is it important to prepare samples in low light conditions?

It is crucial to prepare samples in low light conditions to prevent the degradation of vitamins A and E, which are sensitive to light.

What is the purpose of saponification in the sample preparation process?

Saponification is used to break down the sample's fat molecules, which helps release vitamins A and E for extraction.

What should be the temperature during the saponification step?

The saponification should be carried out at a temperature of 74°C using a water bath.

How do you extract the vitamins from the saponified sample?

Vitamins are extracted by adding hexane to the saponified sample in a separating funnel, followed by vigorous shaking and allowing the hexane layer to separate.

Why is it necessary to wash the hexane extract with deionized water multiple times?

The washings with deionized water are necessary to remove any residual potassium hydroxide that might be present in the hexane extract.

What is the purpose of using sodium sulfate anhydrous in the filtration process?

Sodium sulfate anhydrous is used to dry the hexane extract by removing any remaining water after the extraction process.

At what temperature should the hexane be evaporated from the extract?

The hexane should be evaporated at 37°C until it is completely removed, leaving behind only the vitamins.

What solvent is used to dissolve the vitamins after the hexane evaporation?

After hexane evaporation, methanol is added to the beaker to dissolve the vitamins.

What is the final step before injecting the sample into the HPLC system?

The final step is to transfer 1 mL of the dissolved vitamin solution into an amber-colored vial and inject 20 µL into the HPLC system for analysis.