THE FUNDAMENTALS OF

PFAS ANALYSIS

&

THE ANALYTICAL

LABORATORY

Peter Philbrook

U.S. Environmental Protection Agency

Laboratory Services and Applied Science Division

11 Technology Drive

North Chelmsford, MA

SAMPLE CONTAINER SIZE

250 mL – There is a reason!

❖Specific requirements for

sample containers

❖Extraction technique

requires bottle rinsing (2

steps)

❖Preservation integrity

• Polypropylene or HDPE - PFAS stick to glass!

• Entire water sample is extracted

• Container is rinsed – 2X Water then Methanol

• Sample Preservative Anti-bacterial & Dechlorination

• Trizma (EPA 537) or Ammonium Acetate (EPA 533)

• Multiple bottles required for QC (3) Spike / Dup

• Field Reagent Blanks (FRBs)

• Field contamination can be a real issue

• 14 Day Hold Time (Chilled 40C)

• Most often – the lab is going to supply containers

Aqueous Sampling Requirement Drill Down

SOLID PHASE EXTRACTION (SPE)

Sample preparation technology

Solid particles in a cartridge

Chemically separates different components of a sample

Mobile phase (liquid) Stationary phase (solid)

SAMPLE EXTRACTION

• Vacuum assisted manifold

• Water sample passes through SPE cartridge

• Target compounds are adsorbed

• Target compounds are eluted with methanol

KOOL-AID CHROMATOGRAPHY

Red 40 (Allura Red AC)

MW = 496

Blue 1 (Brilliant Blue FCF)

MW= 793

The Color Purple

ANALYTICAL CHEMISTRY

Chemical Analysis is used on many fields of science

Critical in many aspects of life - medical, pharmacology, geology, oceanography, air and water pollution

QUALITATIVE

ANALYSIS

WHAT?

QUANTITATIVE

ANALYSIS

HOW MUCH?

CHROMATOGRAPHY

The science of

separation

Mobile Phase

Liquid (water, buffer, organic solvents)

HPLC = HIGH PERFORMANCE

LIQUID CHROMATOGRAPHY

Stationary Phase HPLC Column

Autosampler / Vial Tray

High Pressure Pump

15,000 psi

min6.700 6.800 6.900 7.000 7.100 7.200 7.300

%

0

F5:MRM of 1 channel,ES-

413 > 368.8

15OCT2019A008 Smooth(Mn,2x4)

Sigma 1 ng/mL

2.383e+004

PFOA

7.13

1427

COLUMN

CHROMATOGRAPHY

min6.700 6.800 6.900 7.000 7.100 7.200 7.300

%

0

F5:MRM of 1 channel,ES-

413 > 368.8

15OCT2019A008 Smooth(Mn,2x4)

Sigma 1 ng/mL

2.383e+004

PFOA

7.13

1427

min6.700 6.800 6.900 7.000 7.100 7.200 7.300

%

0

F5:MRM of 1 channel,ES-

413 > 368.8

15OCT2019A008 Smooth(Mn,2x4)

Sigma 1 ng/mL

2.383e+004

PFOA

7.13

1427

FLOW

Inject

sample

extractHPLC column separates

PFOS – PERFLUOROOCTANESULFONIC ACID

carbon

fluorine

sulfur

hydrogen

oxygen

C8HF17O3S MW=500.13 g/mol

4

31

27

6

5

8

Peak Target RT Peak

Asymmetry

A PFBA 3.29 0.98

B PFPeA 4.68 1.19

C PFBS 5.10 1.21

D PFHxA 5.67 1.08

E PFHpA 6.41 1.55

F PFHXS 6.58 1.53

G PFOA 6.95 1.67

H PFNA 7.42 1.77

I PFOS 7.54 1.44

J PFDA 7.82 1.38

K PFuNA 8.14 1.33

L PFDS 8.23 1.30

M PFDoA 8.45 1.53

N PFTrDA 8.70 1.62

O PFTeDA 8.89 1.82

HPLC – chromatography

of 15 PFAS compounds

1 drop = 50 uL

Injecting 1/10th of a drop

Mass Spectrometer

Electrospray

Ionization

ESI +/-

Probe

Ion Plume

3-4 K Volts !

LC-MS/MS

Precursor Ion Product Ion

Collision Cell

PFOS 500 g/mol

Precursor = 499

Product = 80

electrospray

negative

--

--

-

-

-

--

--

-

--

-

--

MASS SPECTROMETRY

SPE - LC/MS/MS

Collision CellQuadrupole

CALIBRATION

AND

QUANTITATION

Compound name: PFOA

Correlation coefficient: r = 0.999863, r^2 = 0.999726

Calibration curve: 0.0327837 * x + 0.00290069

Response type: Internal Std ( Ref 6 ), Area * ( IS Conc. / IS Area )

Curve type: Linear, Origin: Include, Weighting: 1/x, Axis trans: None

ng/mL-0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0

Re

sp

on

se

-0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

min6.700 6.800 6.900 7.000 7.100 7.200 7.300

%

0

F5:MRM of 1 channel,ES-

413 > 368.8

15OCT2019A008 Smooth(Mn,2x4)

Sigma 1 ng/mL

2.383e+004

PFOA

7.13

1427

min6.700 6.800 6.900 7.000 7.100 7.200 7.300

%

0

F5:MRM of 1 channel,ES-

413 > 368.8

15OCT2019A008 Smooth(Mn,2x4)

Sigma 1 ng/mL

2.383e+004

PFOA

7.13

1427

BACKGROUND

&

FIELD

CONTAMINATION

Teflon Solvent Line / Reagent Water

Teflon cap liners on solvent bottles

Teflon tipped syringes

Teflon frits

SPE apparatus – lines, solvents, frits, transfer tubing

Weatherproof Anything – Books,jackets

Autosampler Vials

PFAS ANALYSIS – WHY SO MUCH?

BREAKDOWN OF ANALYSIS COST

1. Triple Quad Mass Spectrometer $400K

2. HPLC System $100K

3. Software/Hardware $ 50K

4. Annual Support Plan $ 23K

5. Manual Labor – 2 hours per 10 samples

6. Training

7. Consumables SPE, HPLC Columns, Sampling Containers, Solvents/Reagents

8. Analytical Standards – Isotopically Labeled –Isotope Dilution

9. Throughput – More = Less Cost per Sample

$$$$$$$$ Isotope dilution $$$$$$$$

ISO

TO

PE

DIL

UTIO

N

12C 13C

Carbon 12 Carbon 13

6 protons

6 neutrons

AMU = 12

6 protons

7 neutrons

AMU = 13

13C2 PFOA

PFOA

Native Molecule

414.1 g/mol

PFOA

Isotopically Labeled Molecule

416.1 g/mol

DAILY SODIUM INTAKE = 2,300 MG (~A TEASPOON)

Home Recipe for: 70 ng/L batch of sodium

• Turn shower on full blast (2 gallons per minute)

• Add a level tsp of table salt (NaCl)• Come back in 8 years and 3 months and

turn water off

• Or add tsp salt to 8,674,286 gallons

That’s equivalent to 3 miles of above ground pools!

638 of them * 24’ Diameter @13,594 gallons each

Questions?

THANK YOU!