Document ymmqZmkeBd4dLwg6ypLMLnvrD
AR226-2729
E. I. du Font de Hemours I Co. (Inc.)
Polyiaer Products Department Experimental Station Laboratory
Lab Mo. ER-7^9
Page 1 of 11
Determination of Perfluorooctanoic Acid In Vater
Gas Chromatograiphic Method
I. Applicability
This method has been developed for the determination of ammonium
perfluorooctanoate (Cn-APFC) and perfluorooctanoic acid in aqueous solution, at concentrations from 0.01-10 ug/mL. The procedure
described here is for the analysis of aqueous solutions from air impingers. The method has also been used for determination of Cp in
blood samples and, depending on specific interferences, could be applied to other aqueous systems. A nodification which eliminates the drying
step can be used for appropriate solid samples (such as Muelepore filters used in air sampling) and for methanol solutions.
II. Unusual Safety Considerations
The 3M Company has found in preliminary studies that Co caused birth defects vhen fed to rats in a laboratory experiment. Female employees of child bearing capability should be restricted from any portion of this procedure which offers significant chance for exposure. Although no health problems are known for
workers exposed to Cg, it has been found in blood samples and may be very
slowly eliminated from the body (Reference 3). Standard laboratory safety practices for handling toxic, emoroyotoxic, etc. materials and for corrosives
should be used in working -with these perfluorinated acids and their solutions. The electron caputre detector contains a radioactive source ( Hi), and the manufacturer's instructions for safe operation must be observed.
Care should be taken to avoid contact in working with the low temperature baths used for freeze-drying.
III. Principle
Freeze-drying (lyophilization) is used to remove the water from sample aliquots and permit derivatization. Addition of methanolic HC1 to the dried residue, along with perfluorodecanoic acid (C--) internal standard, converts the acids to more volatile methyl esters. These are extracted from the reaction mixture into hexane solution for GC analysis. An electron capture detector (ECD) is used for sensitivity and selectivity.
IV. Fundamental Equations
+
C-F cCOO M + CH-OH
^"(
TTP1
>
C
F
COOGiL + MOH
(M* - RH^ Ha*. H^
(acia not amenable to GC analysis) ------------> (non-epsotlearr, fovrolGaCti)le
Page 2 of 11
V. Interferences
As in any GC analysis, compounds with the same retention time as
the compound of interest will not be distinguished from it. These
can include impurities present in the reagents or other components in the samples themselves.
Interfering peaks from solvents, deriv&tizing reagent, etc., are not generally observed in this laboratory with the reagents specified.
below (Note l), but each new batch should be checked and a blank
included in every analysis. A small peak at the Cn ester position , is found in reagent blanks containing perfluorodecanoic acid internal
standard however, probably from Cn present as an impurity in that
material. This will give an intercept slightly greater than zero in
the calibration plot, but is a significant contribution to the Cn peak area only at the lowest concentrations (ca 1/3 of the total for 0.01 ppm Cp when ca 1 ppm C-- is added).
In the air impinger samples examined to date, no obvious interference or
contribution from compounds other than Cn has been observed. Preliminary analyses of FEP "in-situ" dispersing agent indicate that while one
component of that mixture would co-elute with Cn, it should be accompanied
by other nearby peaks -
VI. Sensitivity, Precision and Accuracy
if lia
The method has been used over the concentration range 0.01-10 vg/mL Cq.
jljjl
Adjustments in the volumes of sample or reagents could probably be maae
to extend this somewhat in either direction, necessary. Due to the
relatively narrow linear range of the ECD, however, the calibration curve must always cover the region of interest.
From the limited data available at this time, precision is estimated to be 10% relative, with quantitative recovery of spikes within this
uncertainty. Accuracy will also be affected by the choice of Cn standard,
which should correspond to the fluorosurfactant composition of the samples (Mote 2).
VII,
Apparatus
Instruments and equipment used in this laboratory are specified here; equivalent apparatus can be substituted.
1- Gas Chromatograph and Supplies
Hewlett-Packard 5830 GC with HP 18803 Electron Capture Detector, equipped for on-column injection with glass packed columns
ft 10 x 2 mm id glass columns (HP configuration 5 with ECD adapter),
packed with 1Q% OV-210 on 70/80 mesh Chromosorb W.AW.DMCS. and conditioned at 200C
Hamilton TOIH 10 pL syringe
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VIII.
2. Lyophilizer
Labconco Bo. 75352 bench-top freeze dryer (12-port, dry ice cooled.)
Labconco No, 75^06 (150 mL) or No. 75^08 (300 mL) Fast-Freeze flasks, -with Bo. 75^76 stainless steel adapters
Vacuum purflp with auxiliary cold trap
McLeod gage (or electronic vacuum gage) reading in the 5-0.005 Torr range.
3. Thermostated Reaction Block
Pierce No. 18802 Reacti-Therm Heating Module, with Beacti-Block to hold 2-dram vials and thermometer (Block C Ho. 18803, has 12 holes
"but they must be enlarged to ca 19 mni to acconodate the 2-dram vials. Block B, No. 18802, can "be used as is, but will hold only 9 vials
and provides poorer thermal contact).
4. Vials and Septum Caps
Wheaton No. 223+884 or Pierce No. 13028 2-dram (ca 7 mL) screw cap
septum vials (borosilicate glass)
Pierce No. 12713 Teflon^-Silicone Septa and No. 13216 open top
screw caps
5. Pipets and Dispensers
Gilson P200 Pipetman, micropipet for quantitative delivery of 50 uL aliquots
1 and 2 mL volumetric pipets for measuring sample and reagent aliquots. (Erinkman Dispensette Bottle-Top Dispensers are a great convenience for repetitive delivery of solvents.)
6. Analytical Balance
7. Common laboratory equipment
Reagents
1. Cp standard, FC-l43 ammonium perfluorooctanoate (3M) or material in use in the area to be monitored (Note 2)
j
2. Perfluorodecanoic acid, PGR Research Chemicals
3. Methanol, Fisber HPLC (A-U52) or Fisher Certified ACS (A-S12) (Note l)
* Reg. U.S. Pat. & Tm. Off-
Page 4 of 11
k. Derivatization Reagent, 3% HC1 in methanol, prepared, from Applied Science Ho. 16053 Instant Methanolic HC1 Kit, but substituting Fisher HPLC or ACS Methanol for the Lipopure
Methanol provided (Note l).
The reagent should Ie stored in the refrigerator, and can
be kept for ca 1 month.
5. Hexane, Phillips Spectrograde or Applied Science Lipopure (Note 1)
6. Water, distilled or deionized
7. Sodium hydroxide, 0.05 M aqueous solution
IX. Procedure
A. preparation of Standard Solutions
Only final solution concentrations are given here -without detailed
preparation procedures, since amounts needed, concentrations of
interest, etc., -will vary "between laboratories. Preparations should "be such that concentrations can be calculated to three significant
figures.
1. Perfluorodecanoic acid in methanol: ea 20 pg/mL
2. Ammonium perfluorooctanoate in water: At least four solutions should be prepared which cover the concentration range of interest. For the example in the chromatograms and calculations below (0.1-1.0 ppm), standard solutions were prepared containing 0.1, 0.25, 0.5, 0.75, and 1.0 ug/mL FC-1^3.
B. Preparation of Samples and Standards for Analysis (Mote 3)
For each sample or standard solution, pipet a 1 fflL aliquot into
a 2-ilran vial. Prepare a blank similarly, using distilled water.
Add 50 pL 0.05 M. KaOH to each and mix gently, keeping the solution
in the bottom of the vial to avoid loss to the cap, sides, etc..
C. Freeze-Ifrying
1. A schematic diagram of the feeeze-drying apparatus is shown in Figure 1. Each of the 12 ports has a separate valve, so that flasks can be attached or removed while the system is under vacuum. The apparatus should be pumped down to an
acceptable vacuum ^Kote k) and the cold traps filled before
attaching the drying flasks".
Wi^'4&^Wtamas^
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2. To prevent lumping, the samples mast Ie frozen 'before they
are placed, under vacuum. To reduce the chance of contamination
or sample loss, cover each vial -with a light filter 'beforehand.;
a small piece of Kimwipe held. in place with a rubber band works well. Freeze the solution in each (Note 5), and. keep the vials
at dry ice temperature until all of them are ready.
3. Place up to four vials in each drying flask and. attach to the
if drying chamber. The pressure should drop "below 0.5' Torr again
within a few minutes no leaks are present, and. the samples should remain frozen as sublimation takes place. Allow them
to dry for ca 4 hours, or until no ice is left in any of the
vials.
D. Derivatization
1. To the dried residue in each vial, add 1 aL methanolic HC1
derivatization reagent and. 50 pL C standard- solution (ca 20 ug/mL in methanol). Close with a sept-ian screw cap and shake well.
2. Thermostat for 1 hour at 65C.
(At this point, the samples can be cooled, to room temperature
and. left overnight to analyze the next day.)
3. Cool to room temperature, ad.d. 1 mL each hexane and. distilled. water, and shake for ca 2 minutes.
U. After the phases separate, the upper hexane layer can be sampled, directly for GC analysis. The solutions are stable in this form for at least several days, although
hexane will gradually evaporate after the septum has been
pierced.
E. &C Analysis
1. GC Conditions - Instrument and. column as above.
Temperatures:
Injection port Detector
Column oven
200C 325C
100C, isothermal
Carrier Gas:
if 90% argon/10^ methane (or nitrogen
recommended for the instrument) flow ca 30 fflL/min
Injection
Volume:
2 uL
Sensitivity:
attenuation as needed to keep peak height measurable,'2' - 2 on BP 5830.
Bun Time:
11-20 minutes, depending on sensitivity
(Mote 6)
P&ge 6 of 11
After installing the column and. establishing the conditions above, check the ECD base frequency and. noise level according to the manufacturer's instructions, to insure that they are stable and. within acceptable limits.
2. Each solution should, "be run twice (or duplicates run once each), bracketing samples with standards of similar concentration in the second, series of injections.
Representative chromatograms are shown in Figure 2 for blanks, standards and samples. Either peak height or peak area can be measured, for quantitation.
X. Calculations (Note 7)
1. Normalized. Cn Peak Values
_________o_________ A tabulation of peak heights for analysis of a series of five
standards and two samples (each prepared in duplicate) is shown in Figure 3.
To correct for any variations in injection volume, reagent volumes,
etc., the raw Cn peak heights (or areas) are first normalized, relative to the internal standard, average
C- average Cn (corrected) = Cp x ---------------
(Calculation of the C average also provides a measure of the precision in sample preparation and GC analysis).
2. Calibration Plot
A calibration curve obtained, by plotting corrected. Cn peak height vs. concentration of the standards is also shown in Figure 3.
For non-linear plots such as this, a smooth curve should, be drawn
through the data points. In some cases, a straight line can be fit
through several points, and. a linear least squares calculation made for the slope and. intercept in that region.
3. Calculation of Sample Concentrations
Using the corrected. Cr> peak height's (or areas) for the samples, corresponding concentrations can be read directly from the calibration plot as shown in Figure 3. Average the values from duplicates for
each sample.
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XI. Notes
1. The solvents specified, in Section VIII vere found preferable to
several tried for this analysis, but others may also be satisfactory,
Even with the same reagents, the appearance of the blank may depend on the condition of the detector. Vith the ECU used here,
contamination results in a significant decrease in selectivity
and. increased, reagent interference.
2. The FC-l43 samples examined to date appear to be a mixture of ca 70-75^ straight chain material with several secondary components (including branched isomers) from 1-9%. (Determined, by HMR and.
capillary GC vith FID peak area). Under the GC conditions used. here, this results in a group of poorly resolved, peaks at the Cn
ester position, as seen in Figure 2. In contrast, commercial perfluoro-n-octanoic acifl (PCB) and. some other ammonium salts
contain ^9 5% straight chain Cn, and. appear more nearly as a
single peak in the analysis. The response factor determined
relative to the C-- internal standard also differs for the tvo
types of material, so it is important to choose the standard
most similar to the samples to be analyzed. All of the aqueous
samples examined thus far closely resemble FC-1U3 standards, but
for chromatograms differing greatly in appearance it may be more
appropriate to substitute a straight-chain standard.
3. At least for initial analyses using this procedure, duplicates
should be prepared as a check on precision.
k. For this freeze-drying application pressures less than 0.5 Torr seem to be satisfactory, although 0.025-0.05 may be achieved. Readings -will also depend on the type of gage used, since the McLeod gage does not read the pressure of -water vapor as electronic
gages do.
5. A small dry ice/acetone bath or the center cold veil of the drying chamber can be used for rapid freezing of the solutions in the
II, bottom of the vials. As noted in Section
to avoid contact with the cold bath.
care must be taken
6. As seen in the chromatograms in Figure 2, some small, broad peaks appear late in the run. (These appear to come from the C-standard, and might be eliminated in purified material), when
running at high sensitivity for lov Cr, concentrations, these can interfere -with the folio-wing run and must be allowed to elute before the next injection.
Page 8 of 11
XII.
7. From the calibration plot in Figure 3, it can "be seen that the
BCD response is not linear with concentration over this range.
Linear plots may be obtained, over other ranges, but still with
concentration-dependent response factors. The calculations described here thus make use of a calibration curve, where the internal standard is used to normalize raw peak values before plotting.
Calculations could be done by a general internal standard procedtire instead for an ECD which -shows a more linear response and constant relative response factors. .(A calibration plot of this sort has been observed with a Varian 3700 GC under similar conditions).
References
1. Research Notebooks E20029, E22306, E22424 (S. Stafford)
2. J. Belisle and D. F. Hagen, "A Method for Determination of Perfluorooctanoie Acid in Blood and other Biological Samples", Analytical Biochemistry 101. 369 (i960)
3. F. A. Ubel, 5. D. Sorenson, & D. E. Roach, "Health Status of Plant Workers Exposed to Fluorochemicals - A Preliminary Report" American Industrial Hygiene Association Journal 111, 58^ (l9fi0)
Prepared by B. S. Stafford, B/8/80 Revised by S. S. Stafford k/3/Ql
Approved by L. J. Papa
FIGURE 1 - APPARATUS FOR FREEZE-DRYING SAMPLES
(a) Prying Chamber and Vacuum Line
To pump
_n
Dry Ice/Isopropanol
Cold Trap (Back-up)
(b) Vacuum Port, Drying Flask, and Samples
.. . ^n
Lid with rubber flanpe for vacuum seal
Dry Ice/Isopropa vacuum seal to
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Drying Chamber
Vacuum (Drying Chamber)
Vacuum Port Adapter (Vacuum port to flask)
2-Drain Vials, tissue over top
Drying Flask - Rubber top fits over glass bottom
for a vacuum seal
Frozen Sample (l mL aliquot)
__ ___ FIGURE 2 - CHROMATOGRAMS FOE Cn DETERMINATION * o _
-7
GC conditions as on p.. 5. Attenuation 2 , chart speed 1 cn/min. Retention "tinie (minutes) indicated, "by each peak.
ra.g,= j.u oi ^.J.
Reagent Blank, solvents and
derivatizing reagent only
Reagent Blank, with 1 ppm C.10 internal standard added
m o>
^LO ester
Sample from Air Inninger
(PBAL Mo. 80-1556)'
*The results shown here are for solutions prepared using 2 mL each derivatizing
reagent, hexane, and va-feer, rather than 1 mL as in the method. Relative peak heights
iri-11 not be af fee-fed, but attenuation is 2x lover. .
'
-
._.
FIGURE 3 - CALCULATION OF CORRECTED PEAK HEIGHTS AND CALIBRATION-PLOT *
(a) Peak Height values for runs of ten standard and four sample
solutions (duplicate preparations
at each concentration)
(b) Calibration Plot, Corrected Cn
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*The results shown here are for solut 2 mL each derivatizing reagent, hexa than 1 mL as in the method. Relativ not be affected, but attenuation is