Home Small Molecule HPLCHPLC Determination of Vitamin A and E

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.

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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.

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

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