TITLE: Lipid Biomarker Extraction and Elution into different Fractions from sediment
AUTHORS: Lucia Leierer, Antonio V. Herrera-Herrera, Margarita Jambrina-Enríquez

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is a protocol of a method used to obtain the total lipid extract (TLE) from archaeological sediment samples and the further elution of the TLE into different fraction and their preparation for GC-MS analysis.</div></div>

[STEPS]
?. [Preparation]
Oven drying of sediment samples at during and subsampling to of homogenized sediment
5 g
?. [Lipid Extraction]
Add dichloromethane/methanol (DCM/MeOH) 9:1 v/v to the sediment
40 ml
?. [Lipid Extraction]
Mix for in a sonicator (below  )
?. [Lipid Extraction]
Transfer the solvent into a centrifuge tube and centrifugate for at 4700 rpm
?. [Lipid Extraction]
Filter centrifuged solvent through annealed glass wool and evaporate with N2
?. [Lipid Extraction]
Repeat step 2-5 two more times
?. [Prepare silica gel column for SPE extraction]
Add small ball of anniled glass wool to column
?. [Prepare silica gel column for SPE extraction]
Add of sand (50-70 mesh, previously fired at during ) to column
0 g
?. [Prepare silica gel column for SPE extraction]
Add of silica (70-230 mesh, previously fired at during ) to column
1 g
?. [Adding lipids to column]
Add lipids reconstituted in DCM with about 9 drops every 10 minutes
?. [Adding lipids to column]
Leave the column covered in aluminium foil for at least 12 hours
?. [Elution]
Calculate dead volume (DV) using n-hexane
?. [Elution]
Elute n-alkanes with 3/8 DV using n-Hexane
?. [Elution]
Elute aromatics with 2 DV 8/2 v:v n-hexane/DCM
?. [Elution]
Elute ketones with 2 DV DCM
?. [Elution]
Elute alcohols with 2DV of 1/1 v:v DCM/Ethyl acetate
?. [Elution]
Elude acids and diols with 2 DV of Ethyl acetate
?. [Elution]
Elude other compounds with 2 DV of Methanol
?. [Elution]
Evaporate all fractions using N2  at
?. [Adding standard and reconstituting]
Store at until measurement
?. [Adding standard and reconstituting]
Reconstitute n-alkanes with 5α-androstane (8mg/L) and of DCM
150 µl
?. [Adding standard and reconstituting]
Reconstitute aromatics with 5α-androstane (8mg/L) and of DCM
50 µl
?. [Adding standard and reconstituting]
Reconstitute ketones with 5α-androstan-3-ol (8mg/L) and of DCM
50 µl
?. [Adding standard and reconstituting]
Reconstitute acids and diols with Methyl C19:0 (8mg/L)  and of DCM and of Ethyl acetate
40 µl
10 µl
?. [Adding standard and reconstituting]
Reconstitute alcohols with 5α-androstan-3-ol (8mg/L) and of DCM and of Ethyl acetate
40 µl
10 µl
?. [Adding standard and reconstituting]
Reconstitute other compounds with Methyl C19:0  (8mg/L) and of DCM and of Ethyl acetate
40 µl
10 µl
?. [Preparation of alcohols]
Add of N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA) + trimethylchlorosilane (TCMS) 99:1 v/v and of pyridine to the extract
100 µl
100 µl
?. [Preparation of alcohols]
Derivatize at for
?. [Preparation of alcohols]
Dry the sample under N2
?. [Preparation of fatty acids]
Derivatize fatty acids by adding of MeOH, of H2SO4 to the extract
5 ml
400 µl
?. [Preparation of fatty acids]
Heat at for
?. [Preparation of fatty acids]
Neutralize with saturated sodium bicarbonate solution
?. [Preparation of fatty acids]
Extract three times with n-hexane and dry under nitrogen
3 ml