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.

Vitamin A or retinyl acetate

General structure of tocopherol

Tocopherol	R1	R2
Alpha (α)	CH₃	CH₃
Beta (β)	CH₃	H
Gamma (γ)	H	CH₃
Delta (δ)	H	H

Experimental Conditions
Column	Ascentis® Express C30 (2.7µm) 250 x 4.6 mm
Detection	UV @ 294 nm (semi-micro flow cell; 5 µL)
Injection volume	10 µL
Flow rate	0.8 mL/min
Temperature	Column: 20 °C Autosampler: ambient
Pressure drop	237 bar (3437 psi)
Mobile Phase	[A] = Water, [B] = Methanol
Gradient	96% B until 13 min, 96 - 100% B in 7 min, hold at 100% B for 4 min, 100 - 96% B in 0.5 min, hold at 96% B for 5.5 min
Diluent	Methanol:Ethanol:Hexane 6:3:1 (v/v)
Standard Solution	20 µg/mL retinyl acetate, 400 µg/mL δ-tocopherol, 100 µg/mL γ-tocopherol, 1 mg/mL β-tocopherol, and 300 µg/mL α-tocopherol in diluent

Experimental Conditions
Column	Ascentis® Express C30 (2.7µm) 150 x 4.6 mm
Detection	UV @ 294 nm (semi-micro flow cell; 5 µL)
Injection Volume	10 µL
Flow Rate	0.8 mL/min
Temperature	Column: 20 °C Autosampler: ambient
Pressure Drop	153 bar (2219 psi)
Mobile Phase	[A] = Water, [B] = Methanol
Gradient	96% B until 13 min, 96 - 100% B in 7 min, hold at 100% B for 4 min, 100 - 96% B in 0.5 min, hold at 96% B for 5.5 min
Diluent	Methanol:Ethanol:Hexane 6:3:1 (v/v)
Standard Solution	20 µg/mL retinyl acetate, 400 µg/mL δ-tocopherol, 100 µg/mL γ-tocopherol, 1 mg/mL β-tocopherol, and 300 µg/mL α-tocopherol in diluent

Experimental Conditions
Column	Ascentis® Express C30 (2.7µm) 150 x 4.6 mm
Detection	UV @ 294 nm (semi-micro flow cell; 5 µL)
Injection Volume	10 µL
Flow Rate	0.8 or 1.6 mL/min
Temperature	Column: 20 °C Autosampler: ambient
Pressure Drop	243 bar (3524 psi) @ 0.8 mL/min 479 bar (6946 psi) @ 1.6 mL/min
Mobile Phase	[A] = Water, [B] = Methanol
Gradient	96% B until 13 min, 96 - 100% B in 7 min, hold at 100% B for 4 min, 100 - 96% B in 0.5 min, hold at 96% B for 5.5 min
Diluent	Methanol:Ethanol:Hexane 6:3:1 (v/v)
Standard Solution	20 µg/mL retinyl acetate, 400 µg/mL δ-tocopherol, 100 µg/mL γ-tocopherol, 1 mg/mL β-tocopherol, and 300 µg/mL α-tocopherol in diluent

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.