Table of Contents

Introduction

The major concern in shipboard microbiological study is whether microbes from the drilling fluid are introduced into the recovered core material during coring. Therefore, it is critical to verify whether recovered cores are contaminated. Perfluorocarbon tracer (PFT) can be used to quantify the amount of contamination due to drilling fluid. It is strongly recommended that this test be routinely conducted when coring for microbiological studies.
PFTs are chemically inert and can be detected with high sensitivity. The JRSO has two chemicals it uses as chemical tracers to monitor potential contamination of sediment and rock samples on the JOIDES Resolution.

Table 1: Physical and chemical properties of perfluorocarbon tracers

Notes on PFMD and PFMCH

Both perfluoromethyldecalin (PFMD) and perfluoromethylcyclohexane (PFMCH) are miscible in each other. The vapor pressure of PFMCH is fairly high so it evaporates readily and quickly at standard room pressure and temperature. The evaporation of PFMD is less significant. The low solubility in water for either compound facilitates gas-phase partitioning and quantitative headspace analysis.
The purity of PFMD purchased from Oakwood Chemical is approximately 80-90%. The predominant contaminants tend to be perfluorodecalin and perfluoro-tert-cyclohexane. The purity of a batch may be found by navigating to Oakwood Chemical's website, searching for "decalin", selecting perfluoromethyldecalin, going to the CofA tab and entering the LOT # located on the bottle label.

https://www.oakwoodchemical.com/

A Note about the Relative Volatility of PFMCH and PFMD

At 70°C, 100% of the PFMCH can be expected to be in the volatile phase, whereas the Antoine-derived curve for PFMD predicts that the partial pressure of PFMD in the 20 mL vial would be approximately 3% of atmosphere (0.03 bar).
It is impossible to heat the vial to the boiling point of the PFMD, however, because it will boil the water in the samples and exceed the pressure capacity of the vial. Even temperatures close to 100°C will liberate significant water vapor, which can be problematic for the gas chromatography conditions.
It is therefore recommended that the samples be heated at 85°C, at which temperature, the PFMD can be expected to have a partial pressure of approximately 6% of atmosphere (0.06 bar). Although the majority of the PFMD is not volatilized in the vial, it is consistently volatilized at a steady temperature in both the standard vials and the sample vials, so a consistent concentration of PFMD will be measured by the GC-µECD.

Introduction of PFT to the Drill Fluid

PFT is continuously fed into the stream of drilling fluid using an Alltech 301 high-performance liquid chromatography (HPLC) pump located within a black cabinet in the Mud Pump room (Figure 1). The tracer is delivered into the drilling fluid stream through a valve on the low-pressure side of the mud charge pump (Figure 2). The rate of the tracer injection is tied to the pumping rate of the drill fluid in order to maintain a final concentration of ~1 mg/L in the drilling fluid through the entire drill string. Operation of the HPLC pumps is usually controlled autonomously by the drill shack. In order to manually operate either pump:

1. Turn off the Ethernet router located on the top rear of Pump A (Figure 3) and use the "Prime" button to prime the pump, or the up/down arrows and "Run" to change its flowrate.

2. If the lines from the tracer bottle to the HPLC pump valve are empty, place an empty plastic syringe into the port labeled "Prime/Purge", twist the black screw valve open and extract until tracer flows into the syringe. Then close the screw valve, remove the syringe and inject the residue back into the tracer bottle.

3. Press "Prime" on the console, then plug the Ethernet back in.

4. Ensure the valves along the flowpath of the tracer to the mud pipe are open and correctly configured (Figure 4).

5. Ensure the lines exiting the cap of the tracer reservoir are sealed with putty to prevent tracer from evaporating away.

   Analytical Overview

   After core retrieval, samples for PFT measurement are immediately taken from selected sections. Headspace vials containing the collected sediment are heated ~30 min in an oven to evaporate and release the tracer, and then an aliquot of the vial headspace is injected onto a gas chromatograph equipped with a micro electron capture detector (GC-µECD), which is extremely sensitive to halogenated compounds.

   APPARATUS, REAGENTS, & MATERIALS

   Laboratory Apparatus

• 20 mL headspace vials (HP 5182-0837) and magnetic metal caps with PTFE/rubber seals

  • Note: the PTFE side should face the vial and sample; the rubber side faces outward when the lid is crimped onto the vial

• Manual vial crimper

• 10 mL, 1 mL, and 200 µL syringes

• 1uL and 10 uL gas-tight syringes

• Oven gloves and metal tray

• GC septa: 11 mm diameter, usable up to 250°C or 400°C

• GC column: Agilent column (15 m x 0.250 mm x 5 µm)

Reagents

• Perfluoromethylcyclohexane (CH0400)

• Perfluoromethyldecalin (CH5029)

• Hexane, Optima Grade (CH0084)

• Helium, ultra high purity (UHP), 80 psi max

• Nitrogen, UHP, 50 psi max

Calibration Standards

Perfluoromethylcyclohexane (PFMCH) Standard Curve

The insolubility of PFMCH creates challenges in creating the dilution curve. In order to deal with this problem, a volatile gas dilution scheme was created as follows:
Stock solution is pure PFMCH. Follow the dilution scheme in Table 1.

1. Using a cemented-needle 10 µL syringe, inject neat PFMCH into calibration level 7.

2. Allow the PFMCH aliquot to completely evaporate (~10 minutes).

3. Use a gastight syringe to extract 0.5 mL of 7 through the septum, and inject it into vial 6.

4. Continue serial dilutions as noted to create all calibration levels as shown in Table 1.

Table 1. Dilution scheme for PFMCH.

The crimp top headspace septa are good for only a few injections; remake standards after five or six injections.

Perfluoromethyldecalin (PFMD) Standard CurveTable 1. Dilution scheme for PFMCH.
The crimp top headspace septa are good for only a few injections; remake standards after five or six injections.

PFMD's ready solubility in hexane (47% w/v) makes serial dilutions of this tracer much more straightforward, but does require some caution on the user's part because the hexane will evaporate at the oven temperature in the incubation oven.
Warning! The user should be careful how much of the hexane-dissolved standard is injected into a headspace vial. At the incubation oven temperature, nearly 100% of the hexane will move into the gas phase. Adding 1 mL of hexane to a 20 mL headspace vial will create nearly 11 atmospheres of pressure at 70°C, which will likely shatter the vial!

Calculation of concentrations of injected standards

Assumptions: All PFMD is volatilized in the vial when heated to 70°C.

Use the following equation to calculate the concentration of the primary standard (PFMD in hexane solvent).

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[STD_1°] = Concentration of PFMD in standard (g/mL hexane)

p_PFMD= Density of PFMD (1.972 g/mL)

Purity = Purity of the PFMD solution (%). Enter the LOT # from the bottle on Oakwood’s website to find the purity of the bottle

V_PFMD = Volume of PFMD pipetted into the hexane solvent (mL)

V_hexane = Volume of hexane solvent (mL)

Use the following equation to calculate the concentrations for secondary standards prepared from the primary standard:

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[STD_2°] = Concentration of PFMD in secondary standards (g/mL hexane)

V_1° = Volume primary standard pipetted into the headspace vial (mL)

V_vial = Volume of the headspace vial (20 mL)

The calibration curve consists of measurements of the secondary standards. While extracting, the additional volume of the connected syringe adds to the total volume of the vial and thus slightly dilutes the PFMD concentrations. This factor is taken into account in the following equation using the ratios of the syringe volume to the vial and syringe volumes. Injected mass would otherwise have an error of 12.5% (for a 2.5 mL extraction of a 20 mL vial).

To determine the mass of PFMD injected in the GC from an extraction of the secondary standard:

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M = mass of PFMD injected on column (g)

V_syringe = Volume of the secondary standard extracted via the autosampler or manual syringe.

PFMD Stock Level A (400,000 ng/mL)

1. Pipette 12.25 ml of Optima grade Hexanes (that is what we currently use as of X395C) into a 20 mL headspace vial.

2. Cap the vial with a PTFE septum (cream-colored "shiny" side facing the vial) and a magnetic crimp cap.

3. Using a 10 µL cemented needle syringe, add 2.5 µL of neat PFMD into the vial. Let the needle remain the vial for a few moments after the injection to ensure all PFMD from the needle tip has evaporated into the vial. 

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Serial Dilutions for working standards

Prepare serial dilutions of stock level A into separate headspace vials. Cap several BLANK vials in advance before working with PFMD to ensure the blanks are free of PFT. Use a cemented needle syringe from SGE Corp. (not a plastic-tip pipettor) to add the specified levels (Table 2) of stock solution A to 20 mL crimp-top magnetic cap headspace vials by injecting through the septum. Use the high precision 1 µL analytical syringe to accurately measure small volumes. Move plunger VERY slowly while drawing and dispensing the solution. You might not be able to see a drop forming at the tip of the needle when dispensing the solution because it evaporates so quickly. Stay in the vial for a few extra moments to ensure the entire injection has evaporated into the vial.

The crimp top headspace septa are good for only a few injections; remake standards from the stock solution after five or six injections.

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Table 2: Serial dilution scheme for PFMD. As of Exp395C, we are only using the Low Level calibration.

Table 3: Dilution scheme for PFD.

To prepare STD A with PFD, pipet 13.72 mL hexane into a GC2 vial, cap the vial, add 0.7 µL PFD through the septum. The dilution follows the traditional PFMD dilution scheme. Concentration of STD A has been adjusted to allow for accurate syringing of extremely low volumes.

Hardware

The GC2 system comprises an HP 6890 gas chromatograph (GC) with a micro-electron capture detector (µECD).
The GC inlet is operated in splitless mode. PFT gas samples obtained using the headspace extraction method may be injected manually after incubation for 30 minutes at 70 deg. C, or can be injected by the Gerstel autosampler (whose incubator oven should be set to 70°C for 600 seconds). The injection port liner assembly is connected to a megabore column (Rt-Alumina BOND/KCl, 50 m, 0.53 mm ID, 10 µm thickness), and then to a µECD detector, which requires both carrier (helium) and makeup gases (nitrogen).

Ensure the syringe installed in the autosampler has the Teflon-tipped plunger (Figure 6).

Nitrogen Supply

Nitrogen gas is used in all three flow lines (column carrier, detector carrier, and makeup gases). Nitrogen suffices as the detector makeup gas for this procedure because chromatographic efficiency is not an issue and it is readily available aboard ship because of the nitrogen generator.
The µECD is designed to operate best with a flow rate of at least 20 mL/min. Carrier flow of capillary columns, typically 10 mL/min, requires make-up gas to ensure the optimum total flow rate for the detector.
Nitrogen supply settings are:

• Line pressure: 50 psi.

• Supply tubing: copper equipped with 1/8 inch Swagelok fitting.

• Flow rate is crucial to prevent damage to the ⁶³Ni foil in the µECD. Do not raise the temperature of the µECD from room temperature without N₂ flow!

• Both the µECD and column are sensitive to oxygen; therefore, an oxygen/moisture trap and oxygen indication trap are highly recommended for the nitrogen supply lines.

Electron Capture Detector

The µECD cell contains ⁶³Ni, a radioactive isotope emitting high-energy electrons (β-particles) with a nominal radioactivity of 10 mCi. These undergo repeated collisions with carrier gas molecules, producing ~100 secondary electrons for each initial β-particle.
Further collisions reduce the energy of these electrons into thermal range. These low-energy electrons are then captured by suitable sample molecules, which reduces the total electron population within the cell. Therefore, with higher sample concentration the conductivity of an existing gas will drop noticeably, which is recorded by the µECD outcoming signal detector.
(Note that the raw signal represents a drop in electron current signal, flipped over to positive peaks through the GC electronics and software.)

Preparing and Running Calibration Standards

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1. Use a dedicated LOW STANDARDS syringe for this step. Do not use labeled syringes for any other purpose.

2. If suspecting contaminated syringes, rinse syringes with methanol (fill syringes with Optima Methanol three times and discard contents, separate plunger and syringe, place segregated on clean foil) and bake them in the oven at 70°C for 12 hours to drive off any possible trace of PFT.  

3. Prepare dilutions of PFT as per the instructions above.

4. In the Agilent Open Lab program, choose Method and Run Control.

5. Choose the latest PFT method file and wait until the Ready message is lit. Do not raise the detector temperature without sufficient nitrogen quality and flow!

6. If injecting manually, incubate the standards in the 70°C oven for 30 minutes beforehand, as if they were samples. The Gerstel oven should be set to the same temperature.

7. Set up a Gerstel autosampler sequence for the calibration standards, or inject each level manually, beginning from the most dilute to the highest concentration. (The samples should be injected by the Gerstel or manually, the same as the standards.)

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Allow the gas chromatograph to return to the Ready state before injecting the next standard; repeat until all of the standards have been run (for both PFMCH and PFMD, if both PFTs are expected to be used).

Approving Calibration

1. Navigate to Calibration > Data Analysis and open the calibration files.

2. Enter required parameters into the Calibration table and view calibration curve and correlation coefficient.

3. If calibration is acceptable, continue with sample analysis.

Sample Preparation & Analysis

PFT is pumped into the drilling fluid during coring. When core is delivered to the deck, small core samples are placed in headspace vials, sealed, and heated before headspace analysis on the GC2. The presence of a PFT peak from a sample from the interior of a core indicates core contamination from drill fluid, which may contain contaminating microbes.

Sample Collection

Sediment samples are collected from the edge and center of the core on the catwalk immediately after cores are retrieved. The sample from the outer edge is used to confirm successful delivery of the tracer to the core, whereas the interior sample is used to estimate the quantity of intrusion of drill water into the core. Because the exterior of the core liner is coated with drilling fluid, contact with the liner should be avoided while collecting core samples for PFT analysis.

Unconsolidated Sediments

1. After cutting the core liner, break up sediment core by pulling sections apart rather than cutting with a knife to ensure that the tracer is not dragged through the core with the knife.

2. Cut the Luer end off 5 mL plastic syringes (one for each sample to be collected).

3. Collect one plug sample (~3 cm³) using a cut-off syringe from the outer edge of the sample along the core liner.

4. Collect another plug sample (~3 cm³) using another cut-off syringe from near the center of the core.

5. Immediately extrude each sample into a 20 mL headspace vial and seal with gas-tight PFTE -lined cap and septa.

Consolidated Sediments

1. After cutting the core liner, place the core on a fresh sheet of aluminum foil.

2. Pare the exterior of the core using hammer, chisel, and tongs. Before using these tools, pass them through a flame torch to remove any PFT contaminant.

3. Collect one sample (~3 cm³) from the outer edge of the sample along the core liner.

4. Collect another sample (~3 cm³) from near the center of the core.

5. Immediately place each sample into a 20 mL headspace vial and seal with gas-tight PFTE-lined cap and septa.

Igneous Rock

1. Immediately after the core liner is split in the core lab, choose pieces of core for PFT analysis.

2. Place several small pieces of rock from the exterior of the core into a 20 mL headspace vial and seal with gas-tight cap and septa.

3. Alternatively, wipe the interior of the core liner with a cotton swab and place the swab into the 20 mL headspace vial and seal with gas-tight cap and septa.

4. Remove PFT from the surface of the rock before sampling the interior. Rinse the exterior with water or methanol, then hold the piece with tongs under the flame of a handheld propane torch until it appears dry.

5. After cleaning, hold the rock on a fresh sheet of aluminum foil and pare away the exterior using a cleaned hammer and chisel or the hydraulic rock splitter. Use cleaned tongs to handle the rock pieces.

6. After each paring, pass tools through the flame of the torch and place rock pieces on fresh aluminum foil.

7. When the entire exterior of the rock is removed, crush the interior of the rock in a mortar.

8. Immediately place an aliquot of the crushed rock into a 20 mL headspace vial and seal with gas-tight cap and septa.

Sample Analysis

Sample analysis includes the following steps:

• Prepare GC and syringes

• Prepare standards and run calibration curve

• Approve calibration

• Run samples

• Analyze results

Nitrogen Gas Purity

Important! The nitrogen gas supply to the Agilent 6890 GC-µECD must be of sufficient purity to protect the ⁶³Ni source, so before the detector is brought to operating temperature, be sure that no significant nitrogen demands are being made throughout the laboratory. For example, if the microbiologists are using the "Berkley bucket" technique to flush nitrogen through a container, do not proceed.
If time-critical measurements must be made without waiting for other usage to go down, talk to the Laboratory Officer about hooking up a UHP nitrogen tank from the reserve tanks in the hold.

Preparing GC and Syringes

1. Check nitrogen gas availability, delivery pressure = 50 psi.

2. Change septum on GC.

3. Clean and bake all syringes at 85°C for at least 10 minutes (the longer the better, up to 24 hours). Place clean aluminum foil on a metal tray. Remove the needles, plungers and valves from each syringe to increase exposure of syringe surfaces. Ensure each syringe and its components are segregated-each should be reassembled exactly as it was disassembled. Verify that Teflon end on syringe plungers are not missing—this is especially important for the 10 µL SGE cemented-needle syringes (Figure 5).

Running Samples (Manual Injection)