Accurate elemental speciation by

isotope dilution mass spectrometry with hyphenated techniques

Klaus G. Heumann Institute of Inorganic Chemistry and Analytical Chemistry

Johannes Gutenberg-University Mainz, Germany

Important limitations of hyphenated techniques

These techniques do not prevent and identify possible species transformations during the different analytical steps

Accurate quantification is often not an easy task due to matrix effects, system instabilities etc.

Lack of adequate validation methods

Validation of speciation analysis methods

Difference between precision (statistical error) and accuracy (inaccurate values

are related to systematical errors)

Artifact formation of MeHg in sediments (March 1999)

Concern over possible formation of MeHg artifacts during certain analytical procedures was first expressed at the „1996 Mercury as a Global Pollutant“ conference. Many laboratories now doubt that the CRMs they use are indeed properly certified. The findings on artifact formation are not sufficient to claim that MeHg results are overestimated.

Methyl mercury revisited (September 1999)

Recently, some of our European colleagues involved in the production and certification of environmental reference materials have declared that the potential for artifact MeHg formation during analysis is of little concern. We strongly disagree with this stance, because it undermines the scientist`s obligation to strive for the greatest accuracy possible.

A series of letters to the Editor of the journal ‚Analytical Chemistry‘ were sent in 1999

Who is correct ? Can this be solved by letters to an analytical journal or

should it be better solved by isotope dilution mass spectrometry ?

NS = NSp (hSp2 - R x hSp

1) / (R x hS1 - hS

2)

R isotope ratio (only data to be measured)

NS, Sp number of sample and spike atoms

h1,2 isotope abundance of reference and spike isotope

Isotope 1 Isotope 2

Sample Isotope diluted

sample Spike

Inte

nsity

Isotope 1 Isotope 2 Isotope 1 Isotope 2

The principle of isotope dilution mass spectrometry (IDMS)

Only the isotope ratio of the isotope diluted sample must be measured – and not an absolute amount of the analyte !

Isotope ratio measurement is independent on:

Matrix effects

Signal drifts of the instrument

Sample loss does not affect the result after the isotope dilution step has taken place (determination of recovery not necessary)

Experimental conditions for IDMS

However, detection methods with multi-element capability like ICP-MS have a high risk of wrong results by spectrometric interferences

HPLC/ICP-IDMS system for species-specific and species-unspecific spiking

The species-specific spiking mode

Elemental species must be well defined by composition and structure, e.g. CrO4

2-, IO3-, MeHg+ ……

Best spiking mode if isotope-labeled species is available (spiking prior to separation)

Isotope-labeled species must usually be synthesized

The isotopic composition is constant over the whole chromatographic peak (real-time determination)

Isotope exchange between different species must be avoided

How complicated is synthesis of isotope-labeled species ?

Relatively simple for most inorganic species like iodate:

Na129I Na129IO3

Not too complicated for organometallic species like MeHg+:

Usually too complicated or impossible for large biomolecules

201HgCl2 + Me-Co Me201Hg+

Me-Co = Methylcobalamin

HNO3/HClO3

Determination of inorganic iodine species in a mineral water sample determined by species-specific IC/ICP-IDMS

Spike: 129I- and 129IO3-

3.13 ng I/mL

0.30 ng I/mL

Sum of species analysis 3.43 ng I/mL Total I by ICP-IDMS 3.44 ng I/mL

Commercially available spike compounds

Institute for Reference Materials and Measurements (IRMM), Geel/Belgium:

Isotopically labeled 202HgMe+ spike

ISC Science, Gijón/Spain:

119SnBu3+ / 119SnBu22+ / 119SnBu3

+ (mixed spike)

Chromatogram of a multi-species determination by species-specific GC/ICP-IDMS (mussel tissue, CRM 477)

(N. Poperechna and K.G. Heumann)

20

0 Hg

/ 208 P

b

0 2 4 6 8 1 0 1 2

0

1 0 0 0

2 0 0 0

3 0 0 0

4 0 0 0

Bu3

Sn+

Bu2

Sn2+

BuS

n3+

Me3

Pb+

MeH

g+

0

5 0 0 0

1 0 0 0 0

1 5 0 0 0

2 0 0 0 0

0 2 4 6 8 1 0 1 2

0

1 0 0 0

2 0 0 0

3 0 0 0

4 0 0 0

0

5 0 0 0

1 0 0 0 0

1 5 0 0 0

2 0 0 0 0Bu3

Sn+

Bu2

Sn2+

BuS

n3+

Me3

Pb+

MeH

g+

202 H

g / 20

6 Pb

1

19Sn

12

0 Sn

Retention time (min)

Reference isotopes

Spike isotopes

0

4000

2000

4000

2000

0

Inte

nsity

(cp

s)

20000

10000

0

20000

10000

0

Results of multi-species determination by species-specific GC/ICP-IDMS in mussel tissue (CRM 477) and tuna fish (CRM 463)

Species CRM 477 CRM 463

GC-ICP-IDMS Certified GC-ICP-IDMS Certified

Me3Pb+ (ng/g) 0.34 ± 0.03 0.30 ± 0.02 * 4.10 ± 0.16 4.39 ± 0.16 *

MeHg+ (µg/g) 0.066 ± 0.02 --- 2.95 ± 0.05 2.83 ± 0.15

BuSn3+ (µg/g) 0.93 ± 0.04 1.01 ± 0.19 < 0.0003 ---

Bu2Sn2+ (µg/g) 0.82 ± 0.03 0.78 ± 0.06 < 0.0012 ---

Bu3Sn+ (µg/g) 0.85 ± 0.01 0.90 ± 0.08 < 0.0003 ---

* Indicative value

Excellent agreement between GC-ICP-IDMS and certified/indicative values

HPLC/ICP-IDMS system for species-specific and species-unspecific spiking

The species-unspecific spiking mode

Necessary for all elemental species where exact composition and structure is not known, e.g. metal complexes of humic substances, proteins …..

Addition of spike must be carried out after complete separation of species

Spike may exist in any chemical form

The isotope ratio varies over the whole chromatographic peak (however, real-time concentrations are available)

Signal intensity of the measured element must be independent on the species form (found for normal nebulizer systems like cross-flow, but not for ultrasonic nebulizer with membrane desolvator)

Conversion of an isotope ratio chromatogram for a copper species into a mass flow chromatogram

6

8

10

12

14

0 10 20 30Retention time [min]

65C

u/63

Cu

ratio

spike isotope ratio

Isotope ratio chromatogram

0

1

2

3

4

5

0 10 20 30Retention time [min]

Mas

s flo

w C

u [p

g/s]

Mass flow chromatogram

Distribution of heavy metals in SEC separated fractions of two different waste water samples from a sewage disposal plant by HPLC/ICP-IDMS

In both waste water samples the distribution of different heavy metals is different

Zn prefers a high molecular fraction, Cu interacts with most fractions and Mo only with a small fraction of medium molecular weight

For volatile elemental species

GC/ICP-IDMS

can be used in the species-specific and

species-unspecific spiking mode

Hg0 Hg2+

MeHg+ Me2Hg

Example: Determination of monomethylmercury MeHg+ by

species-specific GC/ICP-IDMS

Hg in environmental samples is present in different species

Air

Biomethylation by algae and bacteria

Soil and rocks

He AFS

AES

drying tube

valve

alkylation and purging vessel

cold trap

+ -

atom emission detector

heating unit

capillary GC

ICP-MS

oven atom fluorescence detector

inductively coupled plasma MS

MeHg+ + NaBEt4 MeEtHg

Hg2+ + NaBEt4 Et2Hg

Ethylation of mercury species by NaBEt4:

Hyphenated GC/atom spectrometric techniques for determination of MeHg+ in water samples

0

40

80

120

160

0 2 4 6 8Retention time [min]

Inte

nsity

[x 1

03 cp

s] 201202

75

90

105

120In

tens

ity [m

V]

GC/ICP-MS:

GC/AFS:

• Quantification usually by external calibration

• Application of IDMS possible

• Additional information on isotope ratios

• 201Hg/202Hg = 0.44 for all species

Gas chromatogram of a mercury species standard solution detected by AFS and ICP-MS

Hgo MeHg+ Hg2+

Spike isotope ratio 201Hg/202Hg = 6.53 (natural = 0.44)

0

40

80

120

0 2 4 6 8Retention time (min)

Inte

nsity

(x 1

03 c

ps) 201

202Me201Hg+

Characterization of a Me201Hg+ spike solution by GC/ICP-MS

A species-pure monomethylmercury spike was obtained

0

50

100

150

200

0 2 4 6 8Retention time (min)

Inte

nsity

(x10

3 cp

s)201202

Hg0 201Hg/202Hg=1.65

MeHg+ 201Hg/202Hg=4.45

Hg2+ 201Hg/202Hg=0.44

MeHg+ analysis in seawater by GC/ICP-IDMS using a Me201Hg+ spike

Transformation of MeHg+ into Hgo

Transformation can only be identified by isotope-labelled species

Determination of MeHg+ in a river water sample spiked with Me201Hg+ prior to alkylation by NaBEt4 and NaBPr4

Inte

nsity

(10

3 cps

)

201202

0

50

100

150

200

250 201202

0 2 4 6 8 10 0 2 4 6 8 10

Retention time (min)

Ethylation Propylation

201Hg/202Hg = 1.69

MeHg+ = 3.8±0.1 pg/mL

Hgo

Hg2+

201Hg/202Hg = 1.61

MeHg+ = 3.6±0.1 pg/mL

Hgo

Species transformation by ethylation but not by propylation

However, in both cases identical results are obtained !

What can we learn from species-specific GC/ICP-IDMS of methylmercury ?

The use of isotope-labeled species identifies species transformations

Even if species transformation takes place, accurate quantification is possible by species-specific GC/ICP-IDMS

(if total mixture between sample and spike species has taken place prior to transformation)

Species-specific ICP-IDMS can best be used for validation of analytical methods for elemental species !

Certified value: (70.3 ± 3.4) ng g-1

GC/ICP-IDMS: (72.6 ± 1.3) ng g-1

Certified value: (2.83 ± 0.15) µg g-1

GC/ICP-IDMS: (2.91 ± 0.07) µg g-1

CRM 580 (sediment) CRM 463 (tuna fish)

Sediment sample + Me201Hg+ in diluted HNO3

Biolog. sample + Me201Hg+ in TMAH

Buffering at pH 4.8 and ethylation by NaBEt4

In-situ extraction of MeEtHg by nonane (5 min)

Microwave extraction (5 min)

Injection of nonane extract into GC 201Hg/202Hg isotope ratio measurement in separated mercury species (6 min)

Determination of MeHg+ in environmental and biological samples by species-specific GC/ICP-IDMS

Isotope exchange between different species must be avoided

Is this precondition always fulfilled ?

Example:

Determination of volatile halogenated hydrocarbons by GC/ICP-IDMS

Iodine isotope chromatogram by GC/ICP-MS of an 129I-labeled ethyl iodide spike

A species-pure ethyl iodide spike was obtained

129I-labeled iodide solution

Addition of NaBEt4 in MQ-water

Addition of acetate buffer

Extraction by nonane 0

2000

4000

6000

8000

10 15 20 25

129I

127I

C2H5I

Retention time (min)

129I/127I = 5.4

Inte

nsity

(cps

)

Retention time (min)

Iodine isotope chromatogram by GC/ICP-MS of natural iodinated hydrocarbons spiked with 129I-labeled ethyl iodide

0

2000

4000

6000

5 10 15 20 25 30

CH

3I

C2H

5I

2-C

3H7I

1-C

3H7I

CH

2ClI

129I

127I

CH

2I 2

Isotope exchange takes place with all iodinated species

Species-specific IDMS not possible !

81Br and 79Br chromatograms of a 79Br-labeled ethyl bromide spike

Species-pure 79Br-labeled C2H5Br spike could be synthesized by ethyl tosylate:

NH479Br + C2H5Ts C2H5

79Br + NH4Ts

0 5 10 15 20 25 30 35 0

2000

4000

6000

8000

10000

12000

79 B

r Int

ensi

ty (c

ps)

Retention time (min)

79 Br chromatogram

0

2000

4000

6000

8000

10000

12000

81 B

r Int

ensi

ty (c

ps)

81 B r chromatogram

81Br and 79Br chromatograms of natural brominated hydrocarbons spiked with 79Br-labeled ethyl bromide

No isotope exchange occurs between C2H579Br spike and other

brominated hydrocarbons:

Species-specific IDMS is possible !

Bond strength: C - I < C - Br < C - Cl < C - F

0

1000

2000

3000

4000

5000

6000

7000

CHBr 3

C 4 H 9 Br

CH 2 BrCl

C 2 H 5 Br

81 B

r Int

ensi

ty (c

ps)

81Br chromatogram

0 5 10 15 20 25 30 35 0

1000

2000

3000

4000

5000

6000

7000

CHBr 3

C 4 H 9 Br CH 2 BrCl

79 B

r Int

ensi

ty (c

ps)

Retention time (min)

79Br chromatogram C2H5Br

Existing experience with the application of species-specific determination of elemental species by ICP-IDMS

Element Isotope Alkylated compounds Inorganic compounds Reference

Br 79Br C2H5Br Br-, BrO3- Heumann

Cr 53Cr CrO42- Kingston,

Heumann

Hg 201Hg CH3Hg+ Donard, Frech, Heumann

I 129I CH3I, C3H7I I-, IO3- Heumann

Pb 206Pb (CH3)3Pb+ Ebdon, Heumann

Se 82Se (CH3)2Se, (CH3)2Se2, SeO32-, SeO4

2- Heumann (CH3)3Se+

Se-methionine Garcia Alonso and Sanz-Medel

Sn 117Sn (C4H9)3Sn+, (C4H9)2Sn2+ Garcia Alonso and Sanz-Medel

Tl 203Tl (CH3)2Tl+ Heumann

Is species-unspecific determination of elemental species necessary by

GC/ICP-IDMS even if volatile compounds are usually

well defined ?

If different species of the same element are to be determined

species-unspecific spiking will avoid synthesis of all spike compounds

Schematic figure of the species-specific and species-unspecific isotope dilution technique for sulfur speciation

(J. Heilmann and K.G. Heumann)

Sample + isotope-labeled analyte for

species-specific spiking technique

GC-Injector

ICP-MS GC capillary

Continuous addition of a species-unspecific spike by

gas cylinder or permeation tube

Units used in the species- unspecific spiking technique

Unspiked sample for species-unspecific

spiking technique

Isotope chromatogram of species-unspecific GC/ICP-IDMS for sulfur species determination in a standard solution using 34S-enriched

dimethyldisulfide

(J. Heilmann and K.G. Heumann)

0

2

4

6

8

10

12

14

16

18

20

9 14 19 24 29

Retention time [min]

34S/

32S

S SCH3

S

S CH3

S

CH3

For quantitative analysis spike flow must be calibrated

Determination of MeHg+ and inorganic Hg2+ in biological materials by species-unspecific ETV/ICP-IDMS

(I. Gelaude, F. Vanhaecke and R. Dams)

Fractionated evaporation of MeHg+ and inorg. Hg by a two-step ETV temperature program

Ar ICP-MS

permeation tube

200Hg (spike)

Isotope dilution of all Hg species by isotope-enriched 200Hg

Water bath

Permeation tube guarantees continuous spike flow into the time-delayed analytes evaporated by ETV

Determination of MeHg+ and inorganic Hg2+ in the certified reference material TORT-2 (lobster) by

species-unspecific ETV/ICP-IDMS

(I. Gelaude, F. Vanhaecke and R. Dams)

Method Concentration (µg Hg/g)

MeHg+ Inorg. Hg Total Hg

ETV/ICP-IDMS

Certified

0.16 ± 0.02 0.12 ± 0.06 0.28 ± 0.05

0.15 ± 0.01 0.27 ± 0.06 -

Summary

Isotope-labeled species must normally be synthesized for species-specific IDMS

Online coupling of ICP-MS with GC and HPLC is technically easy but more difficult for CE

Hyphenated techniques in combination with IDMS are the only possibility to obtain real-time concentrations of species

Because of a lack of alternatives GC/ICP-IDMS and HPLC/ICP-IDMS are the most powerful methods for quantitative elemental speciation and therefore also suitable for routine analysis

Elemental speciation can be carried out by species-specific and species-unspecific ICP-IDMS applying coupling with HPLC, GC and CE

Species-specific IDMS is an ideal tool for method validation

However, spectrometric interferences in ICP-MS can always affect the results

Many thanks for your attention and…..

….. I wish you all successful

investigations in your work on elemental

speciation and many interesting information on this topic during the

following seminars !