Difference between revisions of "OR/12/023 Analytical techniques"

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Wragg, J, Rushton, J, Bateman, K, Green, K, Harrison, H, Wagner, D, Milodowski, A E, and West, J M. 2012. Microbial Impacts of CO2 transport in Sherwood Sandstone. British Geological Survey Internal Report, OR/12/023.

Characterisation of solid materials

X-ray diffraction analyses

Quantitative whole-rock mineralogical analysis and qualitative clay mineral analysis of the post-experimental control materials and biotic residues were determined by X-ray diffraction (XRD) analysis.

Sample preparation

The samples were initially ground in a pestle and mortar. In order to achieve a finer and uniform particle-size for whole-rock XRD analysis, a 2.7 g portion of each ground material was micronised under acetone for 10 minutes with 10% (0.3 g) corundum (American Elements — PN:AL-OY-03-P). The addition of an internal standard allows to validate quantification results and also to detect any amorphous species present in the samples. Corundum was selected as its principle XRD peaks are suitably remote from those produced by most of the phases present in the samples. The samples were then back-loaded into standard stainless steel sample holders for analysis.

Approximately 5 g of each crushed sample was dispersed in deionised water using a reciprocal shaker combined with ultrasound treatment. The suspensions were then sieved on 63 µm and the <63 µm materials placed in a measuring cylinder and allowed to stand. In order to prevent flocculation of the clay crystals, 1 ml of 0.1 M ‘Calgon®’ (sodium hexametaphosphate, Sigma-Aldrich (305553)) was added to each suspension. After a time period determined from Stokes' Law, a nominal <2 µm fraction was removed and dried at 55°C. Only small quantities of <2 μm materials were removed from the samples. The <2 μm materials were re-suspended in a minimum of distilled water and Ca-saturated by adding a few drops of 1M CaCl2.6H2O solution. The Ca-saturated suspensions were then pipetted onto the surface of a ‘zero-background’ silicon crystal.

Quantitative x-ray diffraction analysis

XRD analysis was carried out using a PANalytical X’Pert Pro series diffractometer equipped with a cobalt-target tube, X’Celerator detector and operated at 45kV and 40mA. The micronised samples were scanned from 4.5–85°2θ at 2.76°2θ min-1. Diffraction data were initially analysed using PANalytical X’Pert Highscore Plus version 2.2a software coupled to the latest version of the International Centre for Diffraction Data (ICDD) database.

Following identification of the mineral species present in the samples, mineral quantification was achieved using the Rietveld refinement technique (e.g. Snyder & Bish, 1989[1]) using PANalytical Highscore Plus software. This method avoids the need to produce synthetic mixtures and involves the least squares fitting of measured to calculated XRD profiles using a crystal structure databank. Errors for the quoted mineral concentrations are typically ± 2.5% for concentrations >60 wt%, ± 5% for concentrations between 60 and 30 wt%, ±10% for concentrations between 30 and 10 wt%, ± 20% for concentrations between 10 and 3 wt% and ± 40% for concentrations <3 wt% (Hillier et al, 2001[2]). Where a phase was detected but its concentration was indicated to be below 0.5%, it is assigned a value of <0.5%, since the error associated with quantification at such low levels becomes too large.

The <2 µm oriented mounts were scanned from 2–40°2θ at 1°2θ min-1 after air-drying, after glycol-solvation and after heating to 550°C for 2 hours.


Sampling and sample preparation

The post-experiment control and biotic samples were removed from the flow test apparatus contained within their polytetrafluoroethylene (PTFE) sleeves and with end frits in place. In each case, the contained plugs were then immersed in sterile 0.25M NaCl brine (sterilised by filtration at 0.25 µm), that also contained 0.25 g l-1 of sodium acetate. The PTFE sleeves and end frits were subsequently removed and the plugs re-immersed in the sterile brine. Any prolonged storage was under refrigerated conditions (<6°C).

Sub-sampling for the various solid material analyses was performed by briefly removing the samples from the brine. The biotic sample was split longitudinally with half being retained for petrographic analyses and half taken for microbiology testing and characterisation.

For scanning electron microscope (SEM) analysis, samples were taken as rock chips from carefully noted positions along the length of the cores. For the Biotic core, the chips were taken from central and edge sites at each position.

Scanning electron microscopy procedures

The assessment of the solid experimental materials was carried out using an FEI Company Quanta 600 environmental scanning electron microscope (ESEM) equipped with an Oxford Instruments INCA Energy 450 energy-dispersive X-ray microanalysis (EDXA) system with a 50 mm2 peltier-cooled (liquid nitrogen free) silicon drift detector (SSD) X-ray detector capable of detecting elements from boron to uranium. The EDXA system is used as a guide to mineral phase identification.

The ESEM was used in environmental mode: the pressure of the SEM chamber in this operating mode is held at pressures in the region of 500–750 Pa (4–6 Torr). At these pressures using an H2O atmosphere and with a cooled stage it is possible to keep water in the liquid state at the sample surface. This is illustrated in Figure 3, the phase diagram for water. Adjustment of temperature and pressure with reference to this water phase diagram then allows control of the theoretical sample humidity, with 100% representing the ‘dew point’ and values <100% resulting in evaporation and drying of water in a controlled manner.

To give good control over the chamber and sample conditions, the sample needs to be small so that there is minimal thermal lag, and thermal conductivity between the sample materials and the cooled stage needs to be good. To fulfil these requirements with rock samples, the sample must be small (mm scale) and should be mounted in a saturated state within a small reservoir of liquid. The sample holder used for this is a small aluminium cup (Plate 6a & b). In this study, distilled water was used as the added liquid reservoir.

Typical ESEM operating procedures were as follows. The sample was mounted in the peltier cooled stage Initial pumping of the sample chamber was performed with no purge, with target conditions set to 100% humidity at ~2°C. Initial beam conditions were 7.5–10.0 kV accelerating voltage at spot size 5. Typical optimal working distance was 5–7 mm.

During ESEM analysis, all samples were exposed to a range of carefully controlled temperature and pressure changes to observe the way in which pore-lining fluids changed during drying episodes. Drying episodes were also used to reveal the nature (composition, morphology) of residues in an attempt to identify their origins (mineral, biotic, solution precipitate).

Plate 6    ESEM sample holders and preparation. a: cup holder used for rock chips. b: example cup with mm scale rock chip and pool of liquid.
Figure 3    P/T phase diagram for water in the region relevant to ESEM operation. Tw is the triple point. Source data for the sublimation and melting curves has been derived from Wagner et al. (1994), and for the boiling/saturation curve is IAPWS formulation for industrial use, 1997. Humidity curves have been calculated and superimposed on the gas portion of the diagram.

Microbial analyses

A reference sample of the synthetic groundwater used to fill the syringe pump was taken for comparison to the outflow fluids from the biotic and control columns at the start of each test (day 0) and at approximately 7 day intervals until the end of the experiments. Microbial biomass was evaluated using epifluorescence microscopy. Epifluorescence microscopy uses a short wavelength transmission source to fluoresce a sample stained with the nucleic acid selective cationic fluorochrome. The fluorescent stain, Acridine Orange, or N,N,N',N'–tetramethylacridine 3,6-diamine (C17H19N3), was used to determine total cell counts (Hobbie et al, 1977[3]; Jass and Lappin-Scott, 1992[4]). Acridine Orange is capable of permeating cells and interacting with DNA and RNA by intercalation or electrostatic attractions. When the fluorescent stain interacts with DNA, which is spectrally similar to fluorescein, the excitation maximum is at 502 nm (cyan) and the emission maximum at 525 nm (green), while RNA interactions shift the excitation maximum to 460 nm (blue) and the emission maximum to 650 nm (red). Thus, it is possible to determine if cells are metabolically active as they appear red due to the predominant RNA whereas inactive or slow growing microbes have mostly DNA and appear green. By examination of 20 randomly selected fields of view, the numbers of organisms per ml can be counted.

Chemical analyses

A reference sample of the artificial groundwater used to fill the syringe pump was taken for comparison to the outflow fluids from the biotic and control columns at the start of each test (day 0) and at 7 day intervals until the end of the experiments. Chemical analyses included major anions by ion chromatography, (IC) and cations by Inductively Coupled Plasma — Optical Emission Spectroscopy, (ICP-OES), as well as redox sensitive species (Fe2+/Fe3+), pH and selected microbial nutrients (e.g. C, P, S and N). Non-Purgeable Organic Carbon (NPOC) was also determined, which gives an indication of the degradation rate of organic compounds during the experiments.


  1. SNYDER, R L, and BISH D L. 1989. Quantitative Analysis (Chapter 5). In: Bish, D L, Post, J E. (Eds), Modern power diffraction. Mineralogical Society of America Reviews in Mineralogy, 20, 101–144.
  2. HILLIER, S, SUZUKI, K, and COTTER-HOWELLS, J. 2001. Quantitative determination of cerussite (Lead Carbonate) by X-Ray Diffraction and inferences for lead speciation and transport in stream sediments from a former lead mining area of Scotland. Applied Geochemistry, 16, 597–608.
  3. HOBBIE, J E, DALEY, R J, and JASPER, S. 1977. Use of nucleopore filters for counting bacteria by fluorescent microscopy. Applied and Environmental Microbiology, 33, 1225–1228.
  4. JASS, J, and LAPPIN-SCOTT, H M. 1992. Practical course on biofilm formation using the modified Robbins Device. University of Exeter.