...
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
Property | PFMCH | PFMD |
CAS Number |
|
|
Molecular Formula | C7F14 | C11F20 |
Molecular Weight (g/mol) | 350.05 | 512.09 |
Boiling Point °C | 76 | 160 |
Density (g/mL) | 1.788 | 1.972 |
Solubility in Water (mg/L) | ~2 | ~10 |
Solubility in Methanol (mg/L) | 104 |
|
Solubility in Hexane (mg/L) |
| 470,000 |
Vapor Pressure @ 25C (kPa) | 14.11 | 0.29 |
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 85-90%. 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 
and entering the LOT # located on the bottle label.
...
- 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.
- 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.
- Press "Prime" on the console, then plug the Ethernet back in.
- Ensure the valves along the flowpath of the tracer to the mud pipe are open and correctly configured (Figure 4).
Ensure the lines exiting the cap of the tracer reservoir are sealed with putty to prevent tracer from evaporating away.
Anchor _Ref515983560 _Ref515983560 Figure 1: HPLC pumps and tracer bottle.
Anchor _Ref515982275 _Ref515982275
Figure 2: PFMD introduction to mud pump pipeline
Anchor _Ref515983602
anchorFigure 3: Tracer pump ethernet connection for control by drillshack.
_Ref515983057 _Ref515983057 
Anchor _Ref517189599 _Ref517189599
Figure 4: Valve setup for delivering PFMD from tracer pump B to the drilling fluid.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.Anchor RTF31323338333a203248656164 Apparatus, Reagents, & Materials
Laboratory Apparatus
- 20 mL headspace vials (HP 5182-0837) and metal caps with PFTE/rubber seals
- Note: the PFTE 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
- 0.1–1.0 mL 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)
...
- Perfluoromethylcyclohexane (CH0400)
- Perfluoromethyldecalin (CH5029)
- Hexane, Optima Grade (CH0084)
- Helium, ultra high purity (UHP), 80 psi max
- Nitrogen, UHP, 50 psi max
...
...
Figure 5: Different syringe types for preparing PFT standards and for injecting samples. Listed from top to bottom: 1 µL glass analytical syringe from Scientific Glass Engineering (SGE), 0.5 mL Teflon-fitted Pressure-Lok glass syringe from Precision Sampling Corp, 10 µL glass analytical syringe from SGE, 0.10 mL (100 µL) Microliter #710 glass syringe from Hamilton Co.
Calibration Standards
Perfluoromethylcyclohexane (PFMCH) Standard Curve
...
- Using a cemented-needle 10 µL syringe, inject neat PFMCH into calibration level 7 (¿).
- Allow the PFMCH aliquot to completely evaporate (~10 minutes).
- Use a gastight syringe to extract 0.5 mL of ¿ through the septum, and inject it into vial ¿ .
- Continue serial dilutions as noted to create all calibration levels as shown in Table 1.
Calibration Level | Reagent added to 20 mL crimp top headspace vial |
...
|
...
Concentration |
...
(ng/mL |
...
[ppb |
...
]) |
...
¿ | 10 µL PFMCH | 900,000 |
¿ | 0.5 mL Level ¿ (900,000 ng/L) | 22,500 |
¿ | 0.1 mL Level ¿ (900,000 ng/L) | 4,500 |
¿ | 0.5 mL Level ¿ (22,500 ng/L) | 562.5 |
¿ | 0.5 mL Level ¿ (4,500 ng/L) | 112.5 |
¿ | 0.5 mL Level ¿ (562.5 ng/L) | 14.1 |
...
| 0.5 mL Level ¿ (112.5 ng/L) | 2.81 |
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).
...
[STD1°] = 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
...
VPFMD = Volume of PFMD pipetted into the hexane solvent (mL)
Vhexane = Volume of hexane solvent (mL)
Use the following equation to calculate the concentrations for secondary standards prepared from the primary standard:
[STD2°] = Concentration of PFMD in secondary standards (g/mL hexane)
V1° = Volume primary standard pipetted into the headspace vial (mL)
Vvial = 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:
M = mass of PFMD injected on column (g)
Vsyringe = Volume of the secondary standard extracted via the autosampler or manual syringe.
PFMD Stock Level A (400,000 ng/L)
Using a cemented needle syringe, add 5.1 µL of neat PFMD reagent into 25 mL of Optima Grade hexane (mixed hexanes will serve equally well).
Serial Dilutions for working standards:
...
Prepare serial dilutions of stock level A into separate headspace vials. Use a cemented needle syringe from SGE Corp. (not a plastic-tip pipettor) to add the specified levels (Table 1) of stock solution A to 20 mL crimp-top headspace vials by injecting through the septum. Use the high precision 1-µL analytical syringe to accurately pipette small volumes. The crimp top headspace septa are good for only a few injections; remake standards from the stock solutions after five or six injections.
...
...
Table 2: Calibration standards dilution scheme
Batch | Calibration Level | Reagent STD A added to 20 mL crimp top headspace vial | Concentration (ng/ |
...
mL headspace) | |||
Low Level | 4 | 1 µL | 20 |
...
3 | 0.75 µL | 15 |
...
2 | 0.50 µL | 10 |
...
1 | 0.25 µL | 5 |
...
Blank | 0 µL | 0 | |
High Level | 5 | 62 µL | 1,240 |
...
4 | 31 µL | 620 |
...
3 | 6.2 µL | 124 |
...
2 | 0.78 µL | 15.5 |
...
1 | 0.25 µL | 5 | |
| Blank | 0 µL | 0 |
Table 2: Serial dilution scheme for PFMD.
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 85 deg. C, or can be injected by the Gerstel autosampler (whose incubator oven should be set to 85¿C). 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 and makeup gases (nitrogen).
Ensure the syringe installed in the autosampler has the Teflon-tipped plunger (Figure 6).
...
...
Figure 6: Different syringes used be the GerstelAutosampler. The rubber plunger of the syringe shown on top causes significant sample carryover, likely due to tracer penetrating pore spaces within the rubber. It is best to use the syringe with the teflon-tipped plunger shown on bottom.
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 Swagelock 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.
...
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.)
...
...
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.
...
- 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.
...
- Rinse syringes with methanol and bake them in the oven at 70¿C for 12 hours to drive off any possible trace of PFT.
- Prepare dilutions of PFT as per the instructions above.
- In the Agilent Open Lab program, choose Method and Run Control.
- Choose the latest PFT method file and wait until the Ready message is lit.
- Again! Do not raise the detector temperature without sufficient nitrogen quality and flow!
- If injecting manually, incubate the standards in the 85¿C oven for 30 minutes beforehand, as if they were samples. The Gerstel oven should be set to the same temperature.
- 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.)
- Allow the gas chromatograph to return to the Ready state before injecting the next standard; repeat until all of the standards has been run (for both PFMCH and PFMD, if both PFT's are expected to be used).
...
...
Approving Calibration
- Navigate to Calibration > Data Analysis and open the calibration files.
- Enter required parameters into the Calibration table and view calibration curve and correlation coefficient.
- If calibration is acceptable, continue with sample analysis.
...
...
Running Samples (Manual Injection)
- Heat each headspace vial containing sample in an oven at 85°C for ~30 min.
- Using the gas-tight syringe, withdraw 0.25 mL headspace from the heated vial, inject directly into GC injection port A, and press Start on the GC keypad or software dialog box.
- Wait for GC temperature program to cycle and GC to return to ready (~15 min).
- Repeat steps 1–3 for each sample to be analyzed
...
Running Samples (Gerstel Injection)
- Set the incubation oven to 85¿C and program the Gerstel to heat and agitate the vials for 30 minutes prior to injection.
- Set the Gerstel to withdraw and inject 0.25 mL of headspace from the heated vial.
- Wait for GC temperature program to cycle and GC to return to ready (~15 min).
- Repeat steps 1–3 for each sample to be analyzed
...
The area of the PFT peak is integrated and converted to the amount of PFT using values from the standard curve. The amount of sample is determined by weighing each vial and subtracting the weight of an empty vial. The total headspace volume is calculated by subtracting the volume of sample from the total volume of the vial. Total tracer concentration in the sample is corrected to account for the fraction of the headspace that is injected. The amount of drilling fluid in the sample is calculated assuming that the tracer was present at 1 mg/L.
Calculations
...
Use the following equations to determine the amount of drill-water intrusion in a sample: \\
(Drill
...
water,
...
L)/Core
...
material,
...
g)/
...
[(
...
PS – PB)/(
...
CDW x a x W x FI)]
where
PS = integrated peak area of PFT in sample (in arbitrary units),
PB = integrated peak area of PFT in blank (in arbitrary units),
a = slope derived from the calibration curve (in arbitrary units per gram),
CDW = concentration of PFT in drilling fluid (in grams per liter),
W = weight of sample (in grams), and
FI = fraction of the total headspace gas injected:
Vinj/[Vvial – (W/rbulk)]
where
Vinj = volume of sample injected (in liters),
Vvial = volume of vial (in liters),
rbulk = sample density (in grams per liter), and
W = weight of sample (in grams).
...
Quality Assurance/Quality Control
Understanding PFMD Chromatograms
...
Calibrating the instrument produces instrument response factors to absolute component concentrations. To prepare a calibration for quantitation of unknown samples, the retention time(s) for the peak(s) of interest and the amount of component injected must be known.
...
Calibration Cuve
The graphical representation of the amount and response (peak area) for PFT from the calibration samples defines the calibration curve. Because the ECD is not linear across its range of detection, multiple calibration standards are run to calibrate for PFT. Various curve-fit calculations are available to determine optimum regression coefficient including linear, log, power, exponential, quadratic, and cubic.
...
A multilevel calibration is valid over the range of concentrations used in the calibration samples. Extrapolation of a calibration curve, especially if it is not linear, gives at best an approximation result.
...
Health, Safety, & Environment
Safety
Primary safety issues are centered around the electron capture detector, the tracer handling, and GC oven operation
...
- Provide adequate ventilation of the ECD exhaust line to ensure proper evacuation of possible hazardous and radioactive residues.
- Perform a radiation leak wipe test every 6 months of ECD operation. Contact the senior chemistry technician or the Lab Officer.
...
...
Maintenance & Troubleshooting
For procedure or GC operation problems, call a chemistry technician for help.
...
Issues with the chromatograms tend to be due to a poor injection, injecting water or particles, or the degradation of front inlet septa. Particles from the injection or the degradation of the septa cause periodic noisy baselines as the particulates elute the column. Baking out the column or cutting off a small portion of column at the inlet may help in clearing up contamination. Consult Agilent's user guides for walkthroughs. Important: Always note the temperature of the oven to ensure no gases above the ECD's temperature rating are contacting it.
An injection did not occur properly if the peak for the permanent gases is not present within the first minutes of the analysis. Try a second injection. If the second injection fails, change the septum. If this doesn't fix the problem, consult Agilent's user guide about cleaning out the front inlet.
Capillary Column Maintenance
...
Warning: Never rinse or inject the capillary column with inorganic acids or bases!
...
References
Agilent Technologies, Inc., 2007. Agilent Chemstation for GC, LC, LC/MSD, CD, and A/D Systems Revision B.03.01. Hewlett Packard.
Agilent Technologies, Inc., 2008. Understanding Your Agilent Chemstation, Manual G2070-91125. Hewlett Packard.
F2 Chemicals, Ltd. MSDS for perfluoromethylcyclohexane and perfluoromethyldecalin:
PFMCH: http://www.f2chemicals.com/pdf/sds/Perfluoromethylcyclohexane - SDS20122 - ENG.pdf
PFMD: http://www.f2chemicals.com/pdf/sds/Perfluoromethyldecalin - SDS20132 - ENG.pdf
Harvey, R.W., George, L.H., Smith, R.L., and LeBlanc, D.R., 1989. Transport of microspheres and indigenous bacteria through a sandy aquifer: results of natural- and forced-gradient tracer experiments. Environ. Sci. Technol., 23:51.
McKinley, J.P., and Colwell, F.S., 1996. Application of perfluorocarbon tracers to microbial sampling in subsurface environments using mud-rotary and air-rotary drilling techniques. J. Microbiol. Meth., 26:1-9.
Plank, T., Ludden, J.N., Escutia, C., et al., 2000. Proc. ODP, Init. Repts., 185. doi:10.2973/odp.proc.ir.185.2000
Senum, G.I., and Dietz, R.N., 1991. Perfluorocarbon tracer tagging of drilling muds for the assessment of sample contamination. In Fliermans, C.B., and Hazen,T.C. (Eds.), Proc. First Int. Symp. Microbiology of Deep Subsurface. Westinghouse Savannah River Co. Information Service Section Publications Group, 7-145.
Smith, D.C., Spivack, A.J., Fisk, M.R., Haveman, S.A., Staudigel, H., and the Leg 185 Shipboard Scientific Party, 2000. Methods for quantifying potential microbial contamination during deep ocean coring. ODP Tech. Note, 28. doi:10.2973/odp.tn.28.2000
Appendix 1: Agilent GC 6890 PFMD Method
...
Use the GC 6890 parameters listed below to recreate the method for measuring PFMD.
Oven
| FRONT INLET (SPLIT/SPLITLESS) | COLUMN 1 |
FRONT DETECTOR (uECD) | SIGNAL 1 | POST RUN |
PAL SAMPLER AND METHOD | PARAMETERS OF PAL CYCLE |
|