Cong T. Trinh:Analysis of fatty acid from total lipid

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Analysis of fatty acid from total lipid

Method 1 (Dionisi et al, Lipids, 34(10),1107, 1999)

Materials

1. Methanolic HCl (cat#: 33355, Sigma, $65.10)
2. Methanol
3. 1-tetradecene (cat#: 87187-5ML, Sigma, $39.7)
4. Methyl pelargonate or nonanoic acid methyl ester (cat#: 76368-5ML, $53.90)
5. Triundecanoin (cat#: T5534-1G, Sigma, 102.5)

Procedures

1. Collect 10 mL of cell cultures (OD600nm > 2). Wash sample twice with (0.9% NaCl).
2. Dry the cell pellet by vacuum centrifugation at 30°C.
3. Suspend the dried cell pellet in 1 mL of methanolic HCl (1.5M) in 10 mL glass tube with teflon-screwed cap. Add 1 mL of methanol. Place the tube in water bath set at 80oC for 10 min.
4. Add 2 mL of water in the tube to quench the reaction. Add 1 mL of hexane for phase extraction.
5. Centrifuge the sample and collect esterified fatty acid in the upper phase. Use samples for GC analysis.

Method 2 (Voelker et al., J Bacteriol, 176(23), 7320, 1994)

Materials

1. Pentadeconic acid, internal standard (cat#: 91446-5G, Sigma, $78.70)
2. GLC-30 FAME Mix (cat#: 1893-1AMP, Sigma, $38.20)
3. CHCl3 (chloroform)
4. Methanol
5. H2SO4 (sulfuric acid)
6. CHCl3:CH3OH (1:1 (v/v))
7. 5% (v/v) H2SO4 in CH3OH
8. 0.9% (w/v) NaCl in water
9. Acetic acid
10. Palmitic acid (C16) (cat#: 76119-5G, Sigma, $71.60)
11. Diethyl ether (cat#: 309966-1L, Sigma, $64.50)
12. Hexane
13. TLC developing tank (cat#: Z204226, Sigma, $64.90, LxHxW: 7.5x15.5x8cm)
14. Diamond glass cutter (cat#: Z169064, Sigma, $159.90)
15. Merck@ TLC plates (cat#:Z292982-1PAK, Sigma, $235, WxL: 2.5x7.5 cm)

Procedures

1. In a 15 mL tube, add 4.5 mL cell culture, 200 uL acetic acid containing 0.1 mg pentadecanoic acid
2. Add 10 mL CHCl3-CH3OH (1:1 (v/v))
3. Remove the aqueous layer. Rotary evaporate the lower layer (organic layer) to dryness. Or introduce the gas stream to evaporate the organic layer to dryness
4. Redissolve the sample in 1 mL of 5% (v/v) H2SO4 in CH3OH
5. Incubate the sample at 90°C for 2 hrs
6. Add 1 mL (0.9% (w/v)) NaCl in H2O and 300 uL hexane
7. Extract the hexane layer to collect sample for GC/MS analysis

GC method

Column

Supelcowax 10 fused silica capillary column (Supelco, Bellefonte, PA), 30m length, 0.25 um diameter, 0.25 nm film thickness)

Method

42.3 mL/min for helium flow, 180°C for starting temperature (3 min), 15 min ramp to 240°C, and holding at 140°C for 7 min.



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