Hello,
I am doing the sample preparation for gas chromatography for the first time.
Following a protocol available in the lab, but I am facing some difficulties.
The labmates who were experts in this area moved to another places and I am really new to this field. That is why I thought I will ask help from researchers out here.
The details of my experiments are given below:
Sample used- The seeds of a plant, dried at 40 degree overnight, ground under liquid nitrogen.
1. 0.5 g of the powdered sample taken in a glass tube
2. Added 1 ml of deionized water
3. Added 3.75mL of chlorofom:methanol (2:1) and mixed and kept at RT for 2 hours
4. Filtered using whatman #541filter paper
5. Washed the first tube with a solution of chloroform:methanol:water (10:5:4) and matched volume to 4.75 mL
6. Added 1.3 ml of chloroform and mixed well
7. Added 1 ml of 0.74% potassium chloride solution
8. centrifuged at 2000 rpm, 5 min at RT
9. Collect the lower phase and dry under nitrogen gas.
I have to proceed further but the lower phase is not drying up. I have kept the samples at 40 degree Celsius, with a nitrogen flow of 15 psi. (I checked the nitrogen flow by keeping the disposable Pasteur pipette near my ear and I can feel the airflow).
The steps I have to follow afterwards includes
10. Add 1 ml of 0.5 M NaOH in methanol. Flush with Nitrogen and cap it quickly. Vortex for 5 sec. Heat for 10 min at 100⁰C. Then, cool to RT.
11.Add 1 ml of boron trifluoride (BF3) in MeOH (14%, wt/vol).). Flush with Nitrogen and cap it quickly. Vortex for 5 sec. Heat for 5 min at 100⁰C. Then, cool to RT.
12. Add 2 ml of a saturated NaCl solution and 2 ml of hexane. Voretx for 5 sec. Centrifuge at 1000xg for 5 min
13. 1- Transfer hexane layer (top layer) make sure not to disrupt the aqueous phase (lower layer) and place in GC vials. Must completely avoid contamination of lower phase.
What can be the possible problem?
Please share your insights.
Any help on this is greatly appreciated.
See this: Article A simplified method for analysis of polyunsaturated fatty acids
https://www.who.int/docs/default-source/documents/replace-transfats/global-human-surveillance-protocols.pdf?sfvrsn=a99a5265_0
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