Site Description and Methodology
CHAPTER 2 SITE DESCRIPTION AND METHODOLOGY 64 Extraction by sonication/centrifugation
MeOH, MeOH/DCM, DCM Aminopropyl Bond-Elut separation 2% AcOH in E t,0 2:1 DCM/ isopropanol 'Elash' column chromatography Derivatisation (BSTEA) Acids Neutrals Aromatics Hydrocarbons Polar compounds Total lipid extract
Alcohols and sterols Ketones and wax esters
GC/HT-GC, GC-MS and GC-IRMS Freeze-dried sediment
or
modem catchment vegetation
CHAPTER 2 SITE DESCRIPTION AND METHODOLOGY 65
added. Different concentrations were used for lipids extracted from modem plant material. All vials were sealed with Teflon tape, placed on a heating block at 70°C for 90 minutes, and after cooling the BSTFA was removed on the nitrogen blow-down.
2.2.T.2 Gas Chromatography (GC), Gas Chromatography-Mass Spectrometry
(GC-MS), and Gas Chromatography-Isotope Ratio Mass Spectrometry (GC-
IRMS)
Hexane (20 pi) was added to each derivatised fraction (50-70 pi for hydrocarbons) and 1 pi was taken for GC, GC-MS or GC-IRMS analysis (Appendix C). GC analysis of hydrocarbon, acid, aromatic, and alcohol/sterol fractions used a HP 5890 gas chromatograph with a dimethyl polysiloxane coated phase capillary column (CPSil 5CB; Chrompack; 50 m column length, 0.32 mm internal diameter, 0.12 pm film thickness). A high temperature (HT) column was used for the ketone/wax ester and polar fractions. Helium was used as a carrier gas for the elution of the hydrocarbons, and hydrogen was used for all other fractions. For standard GC mns the oven temperature was programmed to remain at 40°C for 1 min, increase from 40-200°C at a rate of 10°C/min, increase from 200-300°C at 3°C/min, and remain at 300°C for 20 min. HT-GC runs were held at 50°C for 2 min, increased to 350°C at 10°C/min, and held at 350°C for 10 min. The total run lengths were 70.33 and 42.00 min respectively.
GC-MS analysis was performed using a Carlo Erba M ega GC (70 eV El, on-column injection) linked through a direct interface to a Finnigan 4500 MS. The columns and temperature programmes used were the same as for the GC analyses, and helium was used as the carrier gas. Lipids were identified at first by mass spectral analysis, and thereafter by comparison of GC retention times under constant operating conditions (column type, column length, temperature programme, column head pressure, carrier gas, etc.).
GC-IRMS analysis of hydrocarbon, acid, and alcohol/sterol fractions used a Van an 3400 GC coupled to a Finnigan MAT Delta-S isotope ratio mass spectrometer (Matthews and Hayes, 1978). All samples were run in duplicate, and the mean value
CHAPTER 2 SITE DESCRIPTION AND METHODOLOGY 66
calculated. This mean value was used in all subsequent analyses. Acid and alcohol/sterol fractions were derivatised with BSTFA prior to GC-IRMS. This process adds a three-carbon containing TMS group to each protic site in a molecule, and it is necessary to correct values to allow for this addition of exogenous carbon (Rieley, 1994). Derivatised and underivatised samples of a standard were run several times to calculate the of the TMS group (Equation 2.2). Both hexadecanol and cholesterol were used as standards, although the example given shows only the results for cholesterol. It was necessary to repeat the analysis whenever a new batch of BSTFA was used for derivatisation.
Nstd- + 3.0^‘^C:13 tms — (Nstd + 3). 8 Co13/
[2.2]
N;STD u 5‘^Cd -TMS 6 ' "ClCarbon number of standard (16 for hexadecanol, 27 for cholesterol) 5'"C of underivatised standard 5'"C of derivatised standard 8'"C of TMS group carbon Example For cholesterol: Therefore: Ns = 2 7 ;13, 5‘"Cu = -22.73%o S'"Cd =-23.30%o S^^CxMs — -28.43%o
Once the of the TMS group is known, it is possible to correct values of samples accordingly (Equation 2.3). A standard of known was run regularly to check the accuracy of the analyses.
8*^CsD'(Ns + 3) — Ô^^CsU'Ns + 3.8^^Ctms
Ô^^Csu — {8^^CsD (Ns + 3) - 3.8^^C'13 13/ tms}/ Ns [2.3]
N s
ô'"C; 5'"c
Carbon number of sample component 5*"C of derivatised sample component
CHAPTER 2 SITE DESCRIPTION AND METHODOLOGY 67
There are several indications as to the accuracy of compound-specific determinations. Firstly, the C19 standards run at regular intervals during the GC-IRMS analyses were found to have a standard deviation of 0.44%o (N=10), and assuming a normal distribution the 95.4% confidence interval is plus or minus two standard deviations, or dbO.8 8%0. This figure is shghtly misleading as it represents ‘ideal’ conditions - the C19 hydrocarbon was the only component eluting during these standard runs and the isotope ratio can thus be accurately determined according to the method described by Freeman et al. (1990). A better indication of accuracy is provided by analysis of the internal standards added to the lipid extracts. In the case of 5a-cholestane which was added to the hydrocarbon sample, a standard deviation of Q.52%0 (N=32) is calculated. This indicates a 95.4% confidence level of ±1.04%o. However, it should be noted that the hydrocarbon fraction is particularly ‘clean’ in that there are few components present other than the homologous n-alkane series. The acid and alcohol/sterol fractions contain numerous components other than the corresponding «-alkyl series, increasing the chance of co-elution of components. As Freeman et al. (1990) point out, this is a serious problem in compound-specific isotope analysis as there is no reliable procedure for deconvoluting the contributions of co-eluting components. It is likely that the 95% significance limit for these fractions is considerably greater than ±l%o. Furthermore, some of the important components analysed, in particular the C n «-alkane, are present in low concentrations by comparison to the most abundant components. These low concentrations increase the errors associated with GC peak integration and determination.
2.2.8 Dating
No radiometric dating was carried out directly on UACT6. A chronology based on ^^°Pb and dates was transferred from UACT4 to UACT6 by comparison of LOI profiles. The construction of a chronology for UACT6 is described fully in Chapter 3.
2.2.9 Tephra extraction and shard counts
Extraction of tephra shards for counting followed the technique of Rose et al. (1996) given in Figure 2.5. Wet sediment (1.0-1.5 g) was placed in a 12 ml glass centrifuge tube of known weight. The sediment was dried at 40°C and the centrifuge tube was
CHAPTER 2 SITE DESCRIPTION AND METHODOLOGY 68 / \ 0.1-0.2 g dry sediment 3 hours in at 80-90°C
i
3 hours in NaOH at 80-90°C 1 hour in HCl at 80-90°CMake slides and count shards
For tephra peaks, concentrate tephra by sodium polytungstate
density separation
I
Microprobe analysis
Removes organic matter
Removes biogenic silica
Removes carbonates
Identifies tephra peaks
Removes mineral matter with different density from tephra
Identifies tephra on the basis o f geochemistry