Range of Application and Principle

Extract vitamin K1 from low fat plant samples such as fruits and vegetables with isopropanol and n-hexane. Purify Vitamin K1 by prep column and remove chlorophyll and other interfering substances, then separate vitamin K1 from other impurities with C18 LC column After zinc being reduced, detect with fluorescence detector and quantify with external standard method. (Spinach and apples are selected in this experiment)

Reference standard: GB 5009.158-2016 National Food Safety Standard-Determination of Vitamin K1 in Food

SPE Purification Steps

SPE column: Welchrom® Vitamin K1 Specification: 2g/6mL

Activation: 5mL n-hexane, discard

Sample loading: after injecting all the purified liquid, discard

Rinsing: rinse with 5mL n-hexane and discard.

Elution: elute with 15mL mixture of n-hexane and ethyl acetate, collect it in a 50mL centrifuge tube and drain.

Blow with nitrogen to near-dry, add it to 5mL with methanol, then filter through 0.22 µm membrane for detection of HPLC.

Chromatographic Conditions

Column: Ultisil® XB-C18 4.6×250mm, 5 µm; Ultisil® Zn 4.6×50mm

Mobile phase: 900mL methanol, 100mL tetrahydrofuran and 0.3 ml glacial acetic acid. After mixing well, add 1.5g zinc chloride and 0.5g anhydrous sodium acetate.

Flow rate: 1.0 mL/min

Column temperature: 30℃

Injection volume: 10 μL

Wavelength: the excitation wavelength was 243nm and the emission wavelength was 430nm.

Chromatogram or Results of Spike Recovery Rate

Chromatogram or Results of Spike Recovery Rate
Tab. 1: Related peak information

Fig. 2. Vitamin K1 reference 200ng/mL

Fig. 3. Original apple sample

Fig. 4. Apple sample added a spike of 0.05 µg/gpple sample

Fig. 5. Original spinach sample

Fig. 5. Original spinach sample

If you have any problem or require further information, please contact info@welchmat.com.

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