IODP Proceedings    Volume contents     Search



During Expedition 335, chemical analyses of representative rock samples were performed using ICP-AES for major and trace element analyses and gas chromatography for CO2 and H2O analyses. An effort toward evaluation of the microbial populations present in Hole 1256D was envisioned, and appropriate samples were to be collected for shore-based analysis. However, sampling was not possible because of limited recovery. Nevertheless, this methodology is described in this section for completeness.

Sample preparation for geochemistry

Samples were prepared from ~10 cm3 of rock for gabbro and granoblastic basalt and 50 cm3 for Sample 335-1256D-Run02-EXJB, a junk basket basalt (see Table T3 in the “Site 1256” chapter [Expedition 335 Scientists, 2012b]). This sample was prepared from roughly 1 cm size gravel by careful separation from the sand, which made up most of the junk basket material. Other samples were cut from cores or from large rock cobbles sampled by junk baskets during fishing runs, using a diamond saw blade. Whenever possible, a thin section billet was taken from the same rock fragment. All outer surfaces were ground on a diamond-impregnated disk to remove saw marks and altered rinds resulting from drilling. Each cleaned sample was placed in a beaker containing isopropanol and ultrasonicated for 15 min. The isopropanol was decanted and the samples were ultrasonicated twice in nanopure deionized water for 10 min. The cleaned pieces were then dried for 10–12 h at 110°C.

The clean, dry whole-rock samples were crushed to <1 cm chips between two disks of Delrin plastic in a hydraulic press. The rock chips were then ground to a fine powder in a tungsten carbide mill in a SPEX 8510 shatterbox. After grinding, a 5.00 ± 0.05 g aliquot of the sample powder was weighed on a Mettler Toledo balance and ignited at 1025°C for 4 h to determine weight loss on ignition (LOI) with an estimated precision of 0.02 g (0.4%).

The standard shipboard procedure for digestion of rocks and subsequent ICP-AES analysis is described in ODP Technical Note 29 (Murray et al., 2000). The following protocol is an abbreviated form of this procedure with minor changes and additions.

After determination of LOI, 100.0 ± 0.2 mg aliquots of the ignited whole-rock powders were weighed and mixed with 400.0 ± 0.5 mg of lithium metaborate (LiBO2) flux that had been preweighed on shore. Standard rock powders and full procedural blanks were included with unknowns in each ICP-AES run. A check on grinding contamination contributed by the tungsten carbide mills was performed during Leg 206 and was found to be negligible for the elements analyzed onboard (Shipboard Scientific Party, 2003). All samples and standards were weighed on the Cahn C-29 microbalance (designed to measure on a moving platform), with weighing errors conservatively estimated to be ±0.02 mg.

A 10 mL aliquot of 0.172 mM aqueous LiBr solution was added to the flux and rock powder mixture as a nonwetting agent to prevent the cooled bead from sticking to the crucible. Samples were then individually fused in Pt-Au (95:5) crucibles for ~12 min at a maximum temperature of 1050°C in a Bead Sampler NT-2100 (internally rotating induction furnace). After cooling, beads were transferred to 125 mL high-density polypropylene bottles and dissolved in 50 mL 10% dilution of concentrated trace metal grade HNO3 (thereafter referred to as 10% HNO3), aided by shaking with a Burrell wrist-action bottle shaker for 1 h. Samples were then ultrasonicated for ~1 h after shaking to ensure complete dissolution of the glass bead. After digestion of the glass bead, the solution was passed through a 0.45 µm filter into a clean 60 mL wide-mouth high-density polypropylene bottle. Next, 1.25 mL of this solution was pipetted into a plastic vial and diluted with 8.75 mL of 10% HNO3 to bring the total volume to 10 mL. The final solution-to-sample dilution factor for this procedure was ~4000. Dilutions were conducted using a Brinkman Dispensette. During Expedition 335, stock standard solutions were sonicated for 1 h prior to final dilution and analysis to ensure a homogeneous solution.



Major and trace element concentrations of standards and samples were determined using a Teledyne Leeman Labs Prodigy ICP-AES instrument. The analyzed elements and the wavelengths used for sample analysis during Expedition 335 are provided in Table T3. Certified international rock reference materials, calibration and drift solutions, and chemical procedure blanks were included with the unknown samples for each sample run. Detection limits (Table T4) were calculated as three times the standard deviation of the mean for blank solution measurements.

The ICP-AES plasma was ignited at least 30 min before each sample run to allow the instrument to warm up and stabilize. After the warm-up period, a zero-order search was performed to check the mechanical zero of the diffraction grating. After the zero-order search, the mechanical step positions of emission lines were tuned by automatically searching with a 0.002 nm window across each emission peak using the BAS-140 standard (basalt interlaboratory standard created during ODP Leg 140 in Hole 504B; Sparks and Zuleger, 1995; Bach et al., 1996) prepared in 10% HNO3. During the initial setup, an emission profile was selected for each peak, using BAS-140, to determine peak-to-background intensities and set the locations of background levels for each element. The Prodigy data acquisition software uses these background locations to calculate the net intensity for each emission line. The photomultiplier voltage was optimized by automatically adjusting the gain for each element using the standard with the highest concentration for that element.

ICP-AES data presented in “Geochemistry” in the “Site 1256” chapter (Expedition 335 Scientists, 2012b) were acquired using the Prodigy software. The intensity curve for each element is defined by 20 measurements within the designated wavelength window. The user selects the two background points that define the baseline. The Prodigy software integrates the area delineated by the baseline and the intensity curve. Each sample was analyzed four times from the same dilute solution within a given sample run. For several elements, measurements were made at two wavelengths (e.g., Si at 250.690 and 251.611 nm) (Table T3). For each run, the wavelength yielding the best calibration line was identified and used for determining concentrations.

A typical ICP-AES run included

  • A set of five to seven certified rock standards, chosen for their wide range of compositions, in order to calibrate the analyses (basalt JB-1A, BIR-1, and BAS-140; granite JG-1A and JG-2; peridotite JP-1; gabbro MRG-1; and pyroxenite ROA-3);

  • Up to 20 unknown samples;

  • A drift-correcting sample (BAS-140) spiked with Co and analyzed every fourth sample position and at the beginning and end of each run;

  • Blank solutions run near the beginning and end of each run; and

  • 3 “check” standards chosen for their composition similar to that of the analyzed material (basalts BAS-140 and JB-1A and gabbro MRG1) run as unknowns.

A 10% HNO3 wash solution was run for 90 s between each sample analysis. Each sample analysis is the average of four measurements. The check standards were used to test analytical accuracy and reproducibility of the obtained data.

Data reduction for ICP-AES

Following each sample run, raw intensities were transferred to a data file and all analyses were corrected for drift. A drift correction was applied to each element by linear interpolation between drift-monitoring solutions run every fourth analysis. After drift correction, a calibration line for each element was calculated using the results for the certified rock standards. Concentrations used for the calibrations were compiled values from the literature recalculated on a volatile-free basis; compiled values were from Govindaraju (1994) for basalt JB-1A and BIR-1, granite JG-1A and JG-2, peridotite JP-1, and gabbro MRG-1 and from Remaidi (1993) and Sparks and Zuleger (1995), respectively, for laboratory-standards pyroxenite ROA-3 and basalt BAS-140. Element concentrations in the samples were then calculated from the relevant calibration lines.

Estimates of accuracy and precision of major and trace element analyses were based on replicate analyses of check standards, the results of which are presented in Table T4. Run-to-run relative standard deviation by ICP-AES was generally ±1% for major elements and ±2% for trace elements. Accuracy was better than 2% for major elements and better than 5% for most trace elements, with the exception of low concentration data for Ni, Sc, and Zn.

Volatile measurements

Volatile contents were measured using gas chromatography (GC) separations on a Thermo Electron Corporation CHNS analyzer (Flash EA 1112 Series). The procedure was based on methods used during Leg 209 (Shipboard Scientific Party, 2004) and Expedition 304/305 (Expedition 304/305 Scientists, 2006). The samples were splits of powders prepared for ICP-AES and were generally taken near thin section billets. The powders were heated to 110°C for 12 h to remove adsorbed moisture and were stored in a dessicator prior to analysis.

Sample sizes were typically 50 mg. Powders were weighed and packed into tin crucibles and loaded into a carousel autosampler. Samples were then heated at 900°C in a resistance furnace in the presence of oxygen for ~75 s. Nitrogen, hydrogen, carbon, and sulfur released from the sample were oxidized and swept into a separation column (Thermo Scientific Multisep column HaySep-A, P/N 260-079-20) using helium as the carrier gas and then into the thermal conductivity detector. Samples were exposed to a vertically mounted quartz reactor tube containing (in packing order) quartz wool, tungsten(VI) oxide (WO3; 0.6–1.68 mm), quartz wool, and reduced Cu (0.7 mm), all inside the resistance furnace. Sulfite gas originating from sulfide minerals was oxidized to SO2, and SO3 was released from any sulfate minerals present in the sample. A single sample analysis required ~20 min. During this time, signal intensity at the detector was continuously recorded, and nitrogen, hydrogen, carbon, and sulfur separated by the GC were measured sequentially at ~71, 103, 399, and 807 s. Once it was established that the hydrogen and sulfur peaks were not completely separated, the use of V2O5 (catalyst for sulfur oxidation) was discontinued, in order to minimize the effect of the sulfur peak on the hydrogen peak (measured as H2O). All measurements reported here were run without V2O5. Because the hydrogen peak was fairly wide (long in duration) and peak heights were quite low, it was deemed necessary to run a blank crucible between all samples, which limited the number of samples that could be run in one carousel. Nitrogen and sulfur contents were not measured because of low levels and poor peak resolution (for sulfur).

Standards, blanks, and results

Aliquots of 80 mM L-(+)-cysteine hydrochloride ([HSCH2CHNH2COOH·HCl]·H2O at 5, 10, 20, 30, 40, 50, and 60 µL) were used for primary calibration of the instrument. Blanks were determined using empty tin crucibles and were typically run between all samples. Concentrations for each sample were calculated from the integrated peak areas of the respective gases relative to those for the standard using a linear regression. Regressions of peak areas versus standard sizes yielded correlation coefficients >0.98. The certified international rock reference material (peridotite) JP-1 (Imai et al., 1995; ( and the internal laboratory standard BAS-140 were routinely measured to monitor analytical accuracy and reproducibility. Typical analytical sessions included multiple analyses of each of these standard rocks and duplicates of each unknown sample. Because of instrumental drift and the time duration of each run, it was necessary to run a complete standard curve before each set of samples. Results of the GC analyses for BAS-140 and JP-1 during Expedition 335 are presented in Table T5. Based on nine runs, the reproducibility of JP-1 and BAS-140 was better than 8% for CO2 and better than 10% for H2O. Average CO2 concentrations obtained for JP-1 overlap published values (Imai et al., 1995) within 5%, and values for BAS-140 are in reasonable agreement with values reported during Expedition 304 (Expedition 304/305 Scientists, 2006). Average H2O contents for both BAS-140 and JP-1 are higher than reported and accepted values by ~29%. We attribute the anomalously high values to contamination of the hydrogen peaks by sulfur (and the difficulties in separating the two overlapping peaks) and consider the H2O data to be systematically overestimated but include the data in table because the relative abundances may be a guide for future studies. Detection limits, optimistically calculated as three times the standard deviation of all the blank analyses, are 0.01 wt% for CO2 and 0.02 wt% for H2O. In comparing the GC volatile analyses to the LOI results, it is also important to bear in mind that conversion of Fe2+ to Fe3+ during ignition may produce a gain in weight that is 11.1% of the percentage of ferrous Fe contained in the sample. This can lead to LOI analyses that have lower volatile concentrations than those determined by GC analysis.


An effort toward evaluation of the microbial populations present in Hole 1256D was envisioned during Expedition 335, by collecting appropriate samples for shore-based analysis. The goal of these microbiological analyses was to characterize the endolithic microbes associated with the oceanic crust. The specific analytical plan included extraction of DNA and amplification of genes of interest (i.e., 16S rRNA gene and functional genes) for describing microbial community diversity and potential function. The goal was to compare the data to existing studies of microbial life in basaltic oceanic crust on the Juan de Fuca Ridge eastern flank (IODP Expedition 327; Orcutt et al. 2010, 2011). Unfortunately, no samples were collected for microbiology analysis.

Planned sampling procedure

To determine the extent of contamination from drilling fluid, a solution containing 0.5 µm fluorescent microspheres is deployed with the core catcher. The solution is prepared by combining nanopure water with 2 mL concentrated microsphere solution (Fluoresbrite, Carboxylate YG; cat. 15700) in a 40 mL volumetric flask. The solution is placed into a small whirlpack bag and tied to the core catcher with the expectation that it will burst with the first recovered core. Beads on the outside of the core indicate dispersion of the microspheres on the external portion of the core; beads detected on the interior of the core indicate the presence of permeable pathways wider than 0.5 µm, suggesting that contamination of the interior of the core may have occurred.

Upon arrival in the core laboratory, a section of the core is evaluated as a potential microbiological sample by visual inspection with minimal handling and before splitting. Handling of the core is limited to those wearing ethanol-rinsed gloves. The scientist in charge of sampling should collaborate with the petrologists to determine a suitable intact sample for microbiological analysis (ideally a ≥10 cm length piece with at least one vein/fracture or signs of alteration). A photograph is taken of the sample before removing it from the core liner. The sample is picked up with a sheet of baked aluminum foil, wrapped in aluminum foil, placed in a sterile whirlpak bag, labeled, and immediately transferred to the –80°C freezer. Following the cruise, the samples are shipped on dry ice.

Details of microbiological sampling and evaluation of contamination is given in Smith et al. (2000).