Francine WalkerTechnical Director

Chemical Solutions Ltd.

Acid Leaching MethodsBioaccessibility Bioavailability Exposure

Preparation Types

Digestions ….• Total Impurities• Strong acids

HNO3, HCl, HF, H2SO4, HClO4 , H3PO4

• Other Oxiders H2O2

• Heating Open Vessel vs. Microwave assistedTemps from 95 to 260 deg. C

Extractions….• Bioavailable Impurities• Weaker acids

HNO3, HCl

• Enzymes Pepsin

• Heating Body Temp. 37 deg. C

• Agitation Shaker, Rotator

Toxicity(Oral Exposure)

Total Impurities – Present in any Form

Bioaccessible - Soluble in Digestive Juices

Bioavailable - Reaches Systemic Circulation

Total Impurities ≥

Bioaccessible Bioavailable

≥

Bioaccessibility(Digestive Solubility)

Physical and Chemical Properties Element and Material of interest

For easily solubilized compounds in simple matrices: Pb(NO3)2 in water Total Pb = Bioaccessible Pb

For hard to solubilized compounds in complex matrices: Aluminum in clay Total Al ≥ Bioaccessible Al

Bioaccessibility(Digestive Solubility)

Extraction of trace elements from natural sourced materials can be very dependent on extraction variables – especially strength and type of acid

• minerals (clays and talcs) • mineral derivatives (titanium compounds)

Elemental impurities may be bound within the structure of the compound, in complexes adhered to their surface or as cations that balance negative charges.

Gastric Fluid Analogues have been used since the 1960s to study the bioaccessibility of nutritional elements, particularly iron……

• 0.07 – 1.0 N HCl• Pepsin (digestive protease)

• NaCl, KCl, phosphate buffers, NaHCO3, glycine

• pH 1.4 – 2.8 (can be stepped to/from pH 5 - 6)

• Temperature – 37deg. C • Time – 1 to 6 hours

Simulated Gastric Fluid

EN 71-3 European Standard for Safety of Toys• Measures 17 Elements• Single Step: 1 N HCl at 37 deg. C for 2 hours

ASTM D5517 Extractability from Art Materials• Measures 20 Elements• Single step 0.07 N HCl at 37 deg. C for 1 hour / sit 1 hour• 45 micron filtration post extraction

EPA 9200.2-86 InVitro Bioaccessible Pb in Soil• Validated for Pb but has been used for others• 0.4 M glycine adjusted to pH 1.5 with HCl• Rotation end over end in large water bath• Bioavailability expressed as fraction

Bioaccessibility

References

Sample Digestion Techniques

Open VesselBlock Digestion

Microwave Digestion(closed vessel)

ExtractionTechniques

Incubator Shaker Rotator Shaker

Extraction Apparatus

ExtractionFluid Comparisons

Shaker Methods – Mod. EPA 9200.2-86 500 RPM at 37+/- 2o C for 1 hr Separate/Centrifuge 90 mins 1gram /100ml

1) Water – Deionized (ASTM Type 1)2) Glycine/HCl

0.4 M Glycine adjusted to a pH of 1.5 +/- 0.05 using conc. HCl3) 0.5 N HCl4) 1.0 N HNO3

Microwave Methods – Mod. EPA 3051A 175o C Final Temp. Ramp 5 mins Hold 5mins 0.5 gram / 10ml

5) 1.0 N HNO3

6) Conc. HNO3

0

10,000

20,000

30,000

40,000

50,000

60,000

70,000

Smectite Clay

Glycine/HCL Shaker

0.5N HCl Shaker

1N HNO3 Shaker

1N HNO3 µwave

Conc. HNO3 µwave

Note: Smooth transition with acid strength and temperature

Complicating Issues

Hydrofluoric Acid Usage• Essential for dissolution of siliceous materials

- Minerals/Ores/Soils/Vegetables/Plants• Creates insoluble fluorides (CaF2 MgF2)

Unfiltered Particles• Picked up by autosampler

- Falsely Elevates Results

Internal Standards • Several common I.S. compounds found in minerals

- Indium Germanium Tellurium - Falsely Elevated I.S. = Falsely Lower Result

0

10,000

20,000

30,000

40,000

50,000

60,000

70,000

Smectite Clay

Water

Glycine/HCL Shaker

0.5N HCl Shaker

1N HNO3 Shaker

1N HNO3 µwave

Conc. HNO3 µwave

Note: Aluminum anomaly due to unfiltered particulate

0

2

4

6

8

10

12

Smectite Clay

Water

Glycine/HCL Shaker

0.5N HCl Shaker

1N HNO3 Shaker

1N HNO3 µwave

Conc. HNO3 µwave

Note: Lead anomaly due to unfiltered particulate or Internal Standard

7.9 8.2 8.8

1112.4

0

2

4

6

8

10

12

14

Tellurium Germanium Indium Holmium Gallium

Lead Concentration in ppmmicrowave conc. HNO3

“Quality is never an accident; it is always

the result of intelligent effort”

John Ruskin

Chemical Solutions Ltd.

273 Mulberry Dr. , #9Mechanicsburg, PA 17050

717-697-7536717-697-4800 (fax)

fwalker@chemicalsolutionsltd.com