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HPLC Determination of Vitamin A and E

Introduction

Following GB method for Vitamin A and Vitamin E as it is written with a 250 x 4.6 mm Ascentis® Express C30 column gives baseline resolution between the critical pair (gamma-, and beta-tocopherol; peaks 3 and 4). When a 150 x 4.6 mm Ascentis® Express C30 column is used, baseline resolution is still maintained with a separation that is complete in less than half of the time of the original method. Additionally, the flow rate can be doubled with the 250 x 4.6 mm Ascentis® Express C30 column, which gives a separation that is less than ten minutes compared to the original method. This application demonstrates how converting methods to superficially porous particle technology improves throughput.

Chemical structure of retinyl acetate, also known as vitamin A acetate, depicted as a line-angle diagram. The structure consists of a six-membered ring with one double bond, connected to a long chain with four more double bonds and an ester group at the end.

Vitamin A or retinyl acetate

Chemical structure of tocopherol, showing a benzene ring with one hydroxyl group (OH) and three methyl groups (R1, R2, R3) attached, along with a long phytyl tail.

General structure of tocopherol

Chromatogram with a horizontal axis labeled ‘Retention time (minutes)’ ranging from 0 to 30 and a vertical axis labeled ‘Intensity (mAU)’ ranging from 300 to 480. There are five prominent peaks at different retention times, each labeled with numbers from 1 to 5. Adjacent to the graph is a key titled ‘PEAK IDENTITIES:’ which matches the numbers to specific compounds: 1. Retinyl Acetate, 2. δ-tocopherol, 3. γ-tocopherol, 4. β-tocopherol, and 5. σ-tocopherol.
Chromatogram with a graph plotting intensity (mAU) on the y-axis and retention time (minutes) on the x-axis. There are five distinct peaks labeled 1 through 5, corresponding to different substances. A formula “Rg = 1.55” is written above peak number 2. On the right side, there is a box listing peak identities: 1. Retinyl Acetate, 2. δ-tocopherol, 3. γ-tocopherol, 4. β-tocopherol, and 5. α-tocopherol.
Chromatogram displaying five peaks with varying heights labeled 1 through 5. The x-axis is labeled “Retention time (minutes)” ranging from 0 to 30 minutes. The y-axis is labeled with arbitrary units ranging from 0 to 350. There is text indicating a flow rate of “0.8 mL/min” and a resolution (Rs) value of “1.72”. Peak number 2 is the tallest, followed by peak number 5, while peaks number 1, 3, and 4 are shorter.
Chromatogram with a series of peaks corresponding to different compounds. The x-axis is labeled “Retention time (minutes)” and ranges from 0 to 35 minutes. The y-axis is labeled “Response (mV)” and ranges from -20 to 120 mV. There are five prominent peaks, each labeled with a number from 1 to 5. Above the graph, there’s text stating “1.6 mL/min” and “Rs = 1.45,” indicating the flow rate and resolution of the chromatography process, respectively. On the right side, there’s a list titled “PEAK IDENTITIES:” followed by five entries: Retinyl Acetate, β-tocopherol, γ-tocopherol, δ-tocopherol, and α-tocopherol.

Conclusion

Depending on the system capabilities, the separation throughput can be increased by using either a shorter length column or by doubling the flow rate.

Related Materials

The column Ascentis® Express C30 250 x 4.6 mm, 2.7 µm is available as a custom product.

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