Separation of dissolved trace elements into ...

Separation of dissolved trace elements into bioaccessibility-based fractions using asymmetrical flow field-flow fractionation (AF4) coupled to ICPMS Chad W. Cuss2, Iain Grant-Weaver2, Marc Cassiede1, Mark Donner2, Lina Du2, Meghan Dueck1, Samineh Kamravaei1, Tommy Noernberg2, Lei Pei1, Nilo Sinnatamby2, Miles Dyck2, Paige Lacy1, Bernadette Quemerais1, and William Shotyk2, Departments of 1Medicine and 2Renewable Resources

MATERIALS AND METHODS Asymmetric flow field-flow fractionation (AF4)

UV-Vis detector

Colloids Flow

200000

60

40

20

Flow 500

600

700

800

56Fe

150000

❹

❶ ❷ 100000

Crossflow shut off

❸

50000

0

900

Time (seconds)

500

1000

1500

Modified from Paquin et al., (2002) (ref. 1)

Agilent 1260 diode array Postnova AF2000 Multi-flow AF4 system • 300-Da PES membrane to separate the smallest colloids by size • Measure 200 ≤ λ ≤ 640 nm in 1 nm increments • 500-μm Teflon spacer, 0.3–0.67 mL sample loop every 0.40 s • Flow program: Channel: 0.7 mL min-1, Tip: 0.2 mL min-1, • Use λ = 254 nm as proxy Crossflow: 2.1 mL min-1, Focus time: 7–10 min., Elution: 29 for [organic matter] min. + 1 min. linear crossflow decrease to 0 + 20 min.

Ultra-clean conditions with advanced QC • AF4-ICPMS in certified class-1000, metal-free clean lab • Leach AF4, plasticware with double-distilled HNO3 • Glass reservoir, autosampler vials replaced with polypropylene • Daily: 2 size calibrations, 2 SRMs, LODs (accounts for membrane drift) • Full blanks analyzed between all samples to minimize and account for carryover

600

800

1000

1200

56Fe

20000

Peak Retention time (s) Area 1 455 1016444 2 594 2782018 3 874 3437919

% area 14.0 38.4 47.5

14

16

18

400

20

600

1000000 800000 600000 400000 200000 0 600

Soil solutions Absorbance (254 nm, mAU)

Absorbance (254 nm, mAU)

80 60 40 20 0 600

Organic matter

1000

1400

1800

2200

2600

3000

Fe

2.0 1.5 1.0 0.5 0.0 600

1400

1800

2200

2600

1000

1400

1800

2200

2600

3000

63

Cu

3000

1000

1400

1800

2200

2600

3000

63

Cu

2000 1000

1400

1800

2200

2600

3000

0 600

3000

2000 1500 1000 500 0 600

137

Ba

1000

1400

1800

2200

2600

Retention time (s)

1500

2000

2500

1000

1500

2000

2500

50 40 30 20 10 0 3000

450 400

3000

40000 400

500

600

700

2000 1000

800

0 3000

0 500

1000

1500

2000

2500

137Ba (CPS)

3000 2000 1000

4000 3000 2000 1000 0 3000

400 500 600 700 800 900 1000

0 500

1000

1500

2000

2500

89Y (CPS)

1000 800 600 400 200 0

400

500

500

600

1000

700

2000 1500 1000 500 0 3000

800

1500

Urine 0.1 1 10 100

350 300 250 1000

3000

238U (CPS)

20000

Absorbance (mAU, log scale)

800

2500

2000

2500

Time (s)

500

200 600

2000

A15

A15

SR

1500

60000

Patient 1

Fe

4000

20000

2500

2500 2000 1500 1000 500 0 3000 8000 6000 4000 2000 0 3000

1000

1200

1400

1600

Patient 2 500

Absorbance (mAU, log scale)

450 400

0.1 1 10 100

350 300 250 200 600

800

1000

1200

1400

1600

1400

1600

1400

1600

1400

1600

56

2000 0 600

2000

500

Time (s)

Organic matter

1000

1500

400 600 800 1000 1200 1400

500

1400

3000

208Pb (CPS)

2500 2000 1500 1000 500 0

1000

40000

6000 4000 2000 0 600

1200

1000

Soil 1, 0.01 M CaCl2 extraction

3000

56

60000

0 600

1000

1000

Retention time (s)

Retention time (min.)

Soil 1, Milli-Q water extraction

800

2500

400 600 800 1000 1200 1400

500

Wavelength (nm)

12

2000

56Fe (CPS)

200000 150000 100000 50000 0

SR

CPS

run #3

1500

0

800 1000 1200 1400

A15

% area 13.4 38.3 48.3

1400

800

63Cu (CPS)

500

400

700

2500 2000 1500 1000 500 0

1400

0

1000

600

600

A15

Peak Retention time (s) Area 1 456 925195 2 593 2649850 3 868 3337576

500

400

300 250 200 150 100 50 0

A15

1200

10000

10

400

SR

1000

10000

Bromophenol blue: 0.69 kDa (8.019 min. 8.024 min.)

8

2

SR

run #2

20000

30000

6

4

SR

56Fe

30000

800

1000

1400

1800

2200

2600

3000

137

Ba

CPS (baseline corrected)

• ICP-QMS and ICP-SFMS instruments

600

0

Counts per second (CPS)

• Quartz duoPUR still for producing ultrapure nitric acid via sub-boiling double-distillation

400

6

Wavelength (nm)

• Contains 14 HEPA-filtered laminar flow air cabinets designed to minimize trace-metal contamination

80 60 40 20 0

55Mn (CPS)

800 600 400 200 0

A15

0 40000

40000

0.0

DOM (mAU, 254 nm)

500

PSS-4: 20.7 kDa (15.776 min. 16.034 min.)

PSS-3: 10.2 kDa (13.062 min. 13.218 min.)

0.4

20000

% area 13.9 39.3 46.8

10000

0.6

• Metal-free facility constructed using virgin polypropylene

• High-pressure ultraclave for digesting recalcitrant material

3000

❶ Void peak: mainly ionic + unfocussed ❷ DOM peak: organic-associated ❸ Mainly inorganic (e.g. oxyhydroxides) ❹ Large mainly inorganic • Statistical fractogram deconvolution2 to deconvolute overlapping peaks

A15

PSS-2: 3.42 kDa (10.147 min. 10.144 min.)

0.8

30000

log(tR-t0) = 0.556·log(MM) – 1.438 log(tR-t0) = 0.564·log(MM) – 1.461 R2 = 0.99

Peak Retention time (s) Area 1 454 874860 2 591 2482475 3 858 2951998

run #1

SR

PSS1: 0.89 kDa (8.623 min. 8.628 min.)

56Fe

SR

Void peak

0.2

• Three separate rooms with different particle count levels are available for separating tasks with multiple levels of cleanliness (classes 10,000, 1000, and 100)

2500

Surface waters

Separation & Deconvolution2

SR

1.0

Thermo-Fisher iCAP Qc • HNO3 added online to 2% conc. with 5 ppb In tracer • Collision cell in KED mode • Monitor 20 elements simultaneously, all measured every 2 s

A15

Repeatability 40000

Calibration

CPS

Bioaccessibility depends on speciation • Free ions/truly dissolved tend to be the most available • Colloid availability differs with physicochemical conditions • Larger dissolved particles tend to be less accessible • Speciation also impacts transport through soils and uptake by plants • For many elements, measuring speciation at natural concentrations requires ultra-clean, metal-free conditions to minimize the risk of contamination and reduce background levels

2000

Time (seconds)

CPS

‘Truly dissolved’ + free ions

Trace element fractograms

Organic matter

80

0 400

Normalized absorbance

Dissolved particles with adsorbed or occluded metals

Quadrupole ICPMS Counts per second

The biotic ligand model

Absorbance (254 nm)

BACKGROUND

30000 20000 10000 0 600

9000

63Cu

6000 3000

800

1000

1200

1400

1600

66Zn

40000

1400

1800

2200

2600

3000

• AF4-ICPMS using the 300-Da membrane provides high-resolution, high-precision separation of dissolved trace elements and organic matter • Statistical fractogram deconvolution2 facilitates resolution into species that have been associated with varying levels of bioaccessibility1 • Next: Relate distribution of trace element species to uptake by fish in controlled toxicity tests, and to transport through soils and uptake by plants • Next: Relate metal species to organics in urine using metabolomics of fractions (GC-MS) • Next: Characterize size fractions using transmission electron microscopy (TEM)

0 600

800

1000

200

1200

1400

1600

0 600

1600

300 200 100 0 600

238U

0 600

800

1000

1200

Retention time (s)

WORKS CITED 1P.R.

800

1000

1200

66Zn

20000

Retention time (s)

CONCLUSIONS AND NEXT STEPS

0 600 10000

100

1000

63Cu

Paquin et al., (2002) The biotic ligand model: a historical overview. Comp. Biochem. Phys. C 133:3-35. 2C.W. Cuss and C. Guéguen (2012) Determination of relative molecular weights of fluorescent components in dissolved organic matter using asymmetrical flow fieldflow fractionation and parallel factor analysis. Anal. Chim. Acta 733:98-102.

1400

800

1000

1200

238U

800

1000

1200

Retention time (s)

ACKNOWLEDGEMENTS