Automated Solid Phase Extraction(SPE) of 10 Perfluorinated Compounds(PFCs) from Tap Water
APPLICATION NOTE AN1029

ABSTRACTPerfluorinated compounds (PFCs) are a family of molecules
consisting of varying lengths of fluorocarbon chains with afunctional group such as carboxylic or sulfonic acid attached.
Since 2009, PFOs have been classified as POPs (PersistentOrganic Pollutants) and the International Agency for Research
on Cancer (IARC) classified PFOA as a Group 2B possible
carcinogen. The analysis of PFCs can be complex due to theirpresence in multiple items used in the analytical workflow.

This application note describes the automation of the isolation of
10 PFCs from tap water prior to their analysis by LC/MS using aspecific SPE polymer AttractSPE™ WAX-PFC and the Gilson
274 ASPEC® Large Volume (LV) System. The results show good
reproducibility of the method without any contamination fromthe extraction system.

This collaboration study was performed jointly by Gilson, Inc. and Affinisep.

KEYWORDS
Gilson 274 ASPEC® Large Volume System, TRILUTION® LH
Liquid Handling Software, Solid Phase Extraction (SPE),
Perfluorinated compounds (PFCs), AttractSPE™ WAX-PFC.

INTRODUCTION
Perfluorinated compounds (PFCs) are a family of molecules
consisting of varying lengths of fluorocarbon chains witha functional group such as carboxylic or sulfonic acidattached. To achieve the concentration limit, defined by EU
Water Framework Directive and the EPA for perfluorinatedcompounds in water, it is highly recommended to use solid
phase extraction (SPE) to concentrate the sample prior tomass spectrometry analysis.

Figure 1

Gilson 274 ASPEC® Large Volume System

Table 1

List of the tested PFCs sorted from the smallest to the largest.
CompoundChemicalComposition

CAS Number

Perfluorobutanoic acid
(PFBA)

375-22-4

Perfluoropentanoic acid
(PFPeA)2706-90-3

Perfluorohexanoic acid
(PFHxA)

307-24-4

Perfluoroheptanoic acid
(PFHpA)375-85-9

Perfluorooctanoic acid(PFOA)

335-67-1

Perfluorononanoic acid
(PFNA)

375-95-1

Perfluorodecanoic acid
(PFDA)

335-76-2

Perfluorobutanesulfonic acid
(PFBS)

375-73-5

PRECAUTIONS FOR THE ANALYSIS OF PFCs
AND RELIABILITY OF THE METHODPRECAUTIONS FOR SAMPLE HANDLING AND PREPARATIONEPA 537-1[1] method describes numerous precautions for sample
collection, preservation, storage, analysis, and glassware
treatment. It also prohibits the use of materials that can cause
accidental contamination, ultimately resulting in cleaner analyses.
For this study, Gilson HDPE sample bottles and Gilson
PIPETMAN® DIAMOND Tips were used for sample handlingand for standard preparation. The Gilson 274 ASPEC® Large
Volume System was equipped with PEEK tubing to avoid
the use of any fluoropolymer, such as PTFE or FEP, to reduce
contamination risk during the sample preparation process.
Cartridges, and the filters and resins contained in them, canalso be sources of contamination. For this reason, AFFINISEP
has developed a specific AttractSPE™ WAX-PFC dedicatedto the isolation of perfluorinated compounds. AFFINISEP
AttractSPE™ WAX-PFC cartridges will not contaminate any
sample with additional PFCs.
To reduce contamination risk, best practice indicates that all
sampling containers are discarded after use, nitrile gloves
are preferred over other glove materials, the nitrile gloves
are often replaced, and all glass or fluoropolymer containers
are avoided to prevent surface adsorption of PFCs orcontamination from container material.

PRECAUTIONS TO IMPROVE HPLC METHOD RELIABILITY

Perfluorohexane sulfonic acid
(PFHxS)355-46-4

Perfluorooctanesulfonic acid
(PFOS)

1763-23-1

This application note describes the analysis of 10
perfluorinated compounds using a specific AttractSPE™
WAX-PFC. This cartridge chemistry is dedicatedspecifically to perfluorinated compounds. The cartridges
are used in the Gilson Gilson 274 ASPEC® Large Volume
System (Figure 1) to purify and concentrate the sample
prior to analysis by LC-MS/MS.
The analysis of PFCs can be challenging. They are common
material components in analytical systems. Their presencein HPLC systems and solvents, sampling bottles, valves,transfer tubing, etc. can lead to significant chance ofsample contamination and high PFCs background, masking
trace levels of PFCs from detection and/or leading to false
positives.

HPLC devices often contain PTFE parts and tubing. This,
coupled with potential traces of PFCs or other fluorine
containing material in solvents, can hinder analysis at low
concentrations. The fluorine containing materials that arereleased from the HPLC system tend to build-up at thefront of the column creating interference. To minimize
this interference, all PTFE or any other fluorinated orperfluorinated polymers are replaced with non-fluorine
containing materials. Also, despite the difficulty andadded cost, all solvents used must be verifiably PFCs free.
Another approach, which was used in this applicationnote, is the installation of a delay column between theLC pumps and the injector. The diagram below (Figure 2)
demonstrates the proper placement of the delay column
in the HPLC fluid path. The interfering species from thesolvents and LC pumps will concentrate at the front of thedelay column resulting in a shift of retention time. Thisshift in retention time effectively separates interfering
species from the analyte PFCs. This is a simple and costeffective solution.

This application ultimately shows the absence of potential
PFCs contamination through the Gilson 274 ASPEC® Large
Volume System with high recoveries and reproducibility.
Figure 2

Diagram of delay column installation on HPLC.

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PFOA is an interfering species when analyzing PFCs. Twosolutions, a blank consisting of methanol and a 0.5 µg/L
solution of PFOA in methanol were analyzed (Figure 3) todemonstrate the efficiency of the method using a delaycolumn. (Silact™ C18 LC-P 50 x 2.1 cm, 3 μm)

SAMPLE PRE-TREATMENT
The tap water hardness was very high (Ca2+ ~300 mg/L,
NO3- ~20 mg/L, Cl2 ~0.3mg/L). For each sample, 475 mL oftap water was mixed with 25 mL of methanol. The pH wasadjusted to ~4 with 100 µL formic acid. The solution was thenspiked with a mix of 10 PFCs at a concentration of 24 ng/L.
One non-spiked sample blank and four spiked sampleswere processed and analyzed. The blank was analyzed toverify non-contamination.

Figure 3

Injection of methanol blank (left) and injection of 0.5µg/L PFOA
in methanol (right)

The injection of a 0.5 µg/L PFOA solution (Figure 3)
shows two peaks. The first one at 16.69 min is PFOA in theinjected solution at 0.5µg/L, while the second one at
17.59 min corresponds to the delayed PFOA interference
from HPLC lines and solvents. The injection of a blankmethanol showed no interference at the expected
retention time. The delay column allows the elimination ofinterfering PFOA at a concentration estimated between
0.05 and 0.1 µg/L.

The analyses were carried using a manual SPE manifold as acontrol, and on the automated system (Gilson 274 ASPEC®
Large Volume System) to confirm the absence of adsorption
and contamination potentially introduced by the automatedsystem.
SPE PROTOCOL
Step

SPE Protocol

Conditioning/Equilibration

1. 5 mL 0.1% NH4OH in methanol
2. 9 mL methanol
3. 9 mL HPLC grade water with formic
acid (pH = 4)

Loading

500 mL of loading solution (475 mL
tap water + 25 mL methanol pH = 4)

Drying

20 mL air pushed through thecartridge

Elution

1. 2 mL methanol
2. 4 mL 0.1% NH4OH in methanol

ANALYSIS OF 10 PFAS IN 500 mL OF TAP WATERThe SPE protocol was carried out on the Gilson 274 ASPEC®
Large Volume System in tap water using AttractSPE™ WAXPFC 6 mL cartridges. The polymeric WAX phase containedin these cartridges undergoes a proprietary treatment at
Affinisep to ensure it is entirely PFCs free and a specific highloading capacity for PFCs.
The automation of PFCs analysis was carried out usingthe Gilson 274 ASPEC® Large Volume System equippedwith segregated waste lines and VALVEMATE® II sample
selection valve actuator. PEEK tubing was used to reducerisk of PFC adsorption and resulting contamination. Thesolvents and the 500 mL samples of tap water were
contained in Gilson HDPE bottles.

This protocol was easily transferred to TRILUTION® LH
Software for automation:

Figure 4

Gilson TRILUTION® LH SPE method

Figure 5

GX-274 ASPEC® LV Load task

The elution was collected in polypropylene vials,
homogenized, and directly analyzed by LC-MS/MS.
To determine matrix effect, a fraction of the elution of theblank water sample was spiked at 2 µg/L and analyzed.
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HPLC/MSMS PROTOCOL
The analytical conditions are presented in the table below:
Table 2LC-MS/MS conditions for the analysis of the 10 PFCs.
LC Conditions

MS Conditions

LC Dionex U3000

Qtrap 4000 ESI- MS/MSColumn: Silact C18 LC-P 150 x 2.1 cm, 3 μm andpre-column filter at 30°C
Delay column: Silact C18 LC-P 50 x 2.1 cm, 3 μm

Curtain gas: 30Injection volume: 5 µL

IS : -4500 V

T° sampler: 10°CTemperature: 400°C

Flow rate: 0.25 mL/min

GS1/GS2: 50/50

CAD: HighTime (min)Solvent A

Solvent B

AnalyteRetention
time (min)Q1

Q3CE (V)060

40PFBA

4.5

213.0

168.8

-14160

40

PFPeA

8.6

263.0

218.8

-12

20

1090

PFBS

9.4

299.0

79.8/98.9-52/-44

30

1090

PFHxA

12.2

313.0

268.9/119-14/-28

3160

40

PFHpA

14.8

363.0

318.8/168.8-16/-26

3560

40

PFHxS

14.9

399.0

79.9/98.9-74/-56

PFOA

16.7

413.1

368.9/168.8-14/-26

PFOS

18.3

499.0

80.1/98.9-84/-70

PFNA

18.3

463.0

418.9/219.0-16/-24

PFDA

19.6

513.0

469.0/218.8-13/-11

Solvent A: 20 mM ammonium acetate (in water)
Solvent B: methanol

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Table 3

Recovery of 10 PFCs in 500 mL of unspiked tap water and spiked tap water and observed matrix effect after purification with
AttractSPE™ WAX-PFC on Gilson 274 ASPEC® Large Volume System.

CompoundUnspiked Tap Water

Spiked Tap Water at
24 ng/L

RSDr (n=4)

RecoveryBlank water Eluate
spiked at 2 µg/L
Observed matrix effectPFBAND*99%

3%+16%

PFPeA

ND*

99%

1%+12%

PFBS

ND*

101%

3%+10%PFHxA

ND*102%3%+17%PFHpA

ND*100%

4%+8%

PFHxS

ND*101%

2%+1%

PFOA

ND*102%

2%-1%

PFOS

ND*

87%

2%+8%

PFNA

ND*

97%

3%+8%

PFDA

ND*

83%

2%-1%

*ND: Not Detected

RESULTSCONCLUSION

The method linearity was checked against a calibration
curve with PFCs concentrations of 0.5, 1, 2, 4, and 6 μg/L.
The R 2 value was found to be greater than to 0.998 for theten molecules, demonstrating linearity of the method overthis concentration range.

The automation of the isolation of 10 PFCs from tap water
for analysis by LC-MS/MS, was found to be very effectivewith good recoveries from 83% to 102% without any
contamination from the extraction system.

As shown in the Table 3, no detectable contamination was
found using the AttractSPE™ WAX-PFC cartridges on theGilson 274 ASPEC® Large Volume System.
The elution fraction of a blank sample was spiked at 2 μg/L
showing no significant matrix effects, with a maximum of
16% signal enhancement for PFBA by comparison with thecalibration curve. Without an additional concentration stepafter the SPE protocol, good values of LOQ (6 ng/L) andLOD (2 ng/L) were achieved.
Futhermore, AttractSPE™ WAX-PFC showed excellent
recoveries from 83% to 102% and the Gilson 274 ASPEC®
Large Volume System allowed excellent relative standard
deviation from 1% to 4%.

The AttractSPE™ WAX-PFC cartridges have enhanced
selectivity and concentrated the sample more than 80 fold,allowing the direct analysis of the 10 PFCs with a LOQ at6 ng/L without any evaporation step.
The combination of AttractSPE™ WAX-PFC and the Gilson
274 ASPEC® Large Volume System allowed development ofa robust method with excellent repeatability (RSD 1%-4%)that helps eliminate environmental variables and effectively
reduces chances of sample contamination.
Furthermore, the automation greatly reduces laborcost, reduces potential exposure to hazardous solvents,
and increases overall efficiency for high throughput
laboratories.

Figure 6LC-MS/MS chromatogram for the 10 PFCs (see HPLC/MSprotocol (Table 2) for retention times)

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PART NUMBERS OF PRODUCTS USED IN THIS APPLICATION NOTECONSUMABLES
Part Number

Product Name

Quantity

WAX-PFC-50.S.6

AttractSPE™WAX-PFC 6 mL

50/pk

C18LCP-150.2.1

HPLC column: Silact C18 LC-P 150x2.1cm 3μm

1 unitC18LCP-50.2.1

HPLC delay column: Silact C18 LC-P 50x2.1cm 3μm

1 unitGILSON 274 ASPEC LARGE VOLUME SYSTEMPart Number

Product Name

Quantity

2614010LV

GX-274 ASPEC, LARGE VOLUME, WITH Z DRIVE

1Reference
1Method 537.1: Determination of Selected Per- and Polyfluorinated Alkyl
Substances in Drinking Water by Solid Phase Extraction and Liquid
Chromatography/Tandem Mass Spectrometry(LC/MS/MS).
https://cfpub.epa.gov/si/si_public_record_Report.cfm?dirEntryId=343042&Lab=NERL

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NoticeThis application note has been produced and edited using information thatwas available at the time of publication. This application note is subject torevision without prior notice.

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