IAEA International Atomic Energy Agency

Analysis of Deuterium Enrichment by

Fourier Transform Infrared

Spectrometry (FTIR): Practice

Christine Slater PhD

Nutrition Specialist IAEA

C.Slater@iaea.org

IAEA

Preparation of calibration standard

• Make a large volume of the calibrating standard

• A solution of ~1000 mg/kg (ppm) or 1 g/L should be prepared by weighing 99.8 atom % deuterium oxide (D2O) and diluting in normal drinking water from the region

• Do not used distilled water to make the calibration standard. Distilled water is subject to fractionation

• De-ionised (ultra-filtered) water can be used

• Note that the density of deuterium oxide is 1.105 g/mL

All glassware must be clean and dry before use

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Preparation of calibration standard

• Use a volumetric flask (1 L or 250 mL)

• Transfer to a borosilicate bottle with a PTFE-lined screw cap for storage until required

• Also retain 1 L of the water used to make the dilution (0 standard)

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Storage of the calibration standard

• It is a good idea to store the calibration standards in several smaller, tightly sealed bottles (e.g. 250 mL borosilicate bottles with PTFE-lined screw caps)

• Only one enriched and one natural abundance bottle should be in use at any time, as working standards

• The remainder should be sealed until required

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Storage of calibration standard

• The calibration standards will last for several years if stored in a cool, dark place out of direct sunlight

• Wrapping bottles in aluminium foil helps to protect the contents from light

• The bottles must be well-sealed to prevent ingress of water from the atmosphere

• Do not store the calibration standard in the same place as the deuterium oxide

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Preparation of the calibration standard

• The D2O should be weighed on an analytical balance accurate to 0.0001 g or preferably 0.00001 g

• The standard can be prepared in two stages, but it is important that the weight of deuterium oxide is known to 0.0001 g

• Balances must be levelled and calibrated before use

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Stage 1 using analytical balance

• Weigh a clean, dry 50 mL volumetric flask with its stopper in place on an analytical balance to 0.0001 g

• Alternatively use a clean, dry glass bottle with a cap

• Add a small volume (20-30 mL) of drinking water to the flask, replace the cap and weigh again

• Add 1 g of D2O to the bottle

• 1 g D2O is approximately ~0.9 mL as the density of deuterium oxide is higher than water

(1.105 g/mL and 1.0 g/mL respectively)

• Replace the stopper or cap to avoid losses

by evaporation, and note the weight

• Calculate the weight of D2O in the bottle

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Stage 2

• Weigh a clean dry 1 L volumetric flask with its stopper. At this stage a balance weighing to 0.1 g can be used

• Quantitatively transfer the water from the 50 mL container into the 1 L volumetric flask using a funnel

• Add local drinking water (or de-ionised water) to the smaller container and pour it into the larger container

• Repeat this at least 3 times to ensure that all the D2O is transferred. Be careful not to spill any

• Add local drinking water to the 1 L volumetric flask up to the mark

• Replace the stopper and weigh again

• Calculate the enrichment of D2O in mg/kg

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The FTIR cell

• Calcium fluoride cells with a cell thickness (path length) of 10-4 m (100 m) are recommended for analysis of deuterium in saliva samples

• These cells cannot be used for the analysis of urine samples because they are damaged by the ammonium and phosphate content of urine

• Sodium chloride cells, which are often supplied with the FTIR, are not suitable for analysis of samples containing water

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The FTIR cell: demountable cell

assembly

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The FTIR cell

• When not in use,

store the cells in their

original packaging

• Wipe only with a lint-

free cloth (lens tissue)

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Filling cells

• Fill 1 mL syringe with standard water or saliva

• Firmly press folded absorbent paper over the exit port to absorb excess sample and prevent ingress of air

• Fill the cell by gently pushing the syringe plunger or using firm taps on the plunger with the index finger

• Remove excess/splashes from the outside of the cell window using absorbent paper

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Check for bubbles by holding the cell

up to a light

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Avoid cross-contamination

• If there are visible bubbles in the cell, add more sample until all of the bubbles have been pushed out

• Measure the absorbance from 2300-2900 cm-1

• Remove the sample using the same syringe that was used for filling. Discard the syringe

• Use a new syringe for each sample to avoid cross-contamination

• When all the samples have been analysed, rinse the cell with drinking quality water before storing

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Operation of the FTIR

• Switch on the FTIR 30-40 minutes before use to allow the electronics to stabilise

• If there is a power cut, wait for 30 min after power has been resumed before analysing samples

• Check that both the interface and the mirror are working properly

• Ensure that the following are set:

Measurement mode: Absorbance

Apodization: SqrTriangular

No of scans: 32

Resolution: 2.0

Range (cm-1): Minimum 2300 Maximum 2900

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Operation of the FTIR

• Perform a “Background” scan using the zero standard - the water used to make the calibration standard

• Calibrate the instrument using the 1000 mg/kg (ppm) standard

• The background for body water samples is the baseline (time 0) saliva sample

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Post-installation checks: accuracy

and precision

• Calibration curve

• Within-day reproducibility

• Between days reproducibility

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Standard curve

• The accuracy of deuterium analysis over the range of enrichments likely to be encountered should be checked using gravimetrically prepared standards

• Smaller volumes (e.g. 100 mL) of these standards can be prepared by diluting D2O with local drinking water in a volumetric flask and weighing on an analytical balance

• The enrichment should range from 0 (natural abundance drinking water) to 2000 mg/kg; an enrichment above that likely to be encountered in saliva samples

• Make independently weighed standards, NOT a serial dilution of the calibration standard

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Preparation of gravimetric standards

• Standards should be made (in 100 mL local drinking water or de-ionised water) according to the table

• The deuterium oxide can be pipetted into the volumetric flask but it must be accurately weighed

Target enrichment

(mg/kg) μL D2O

0 0

100 10

200 20

400 40

600 60

800 70

1000 90

1500 140

2000 180

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Preparation of gravimetric standards

• Tare the balance with the volumetric flask and its stopper

• Half-fill the flask with water. Note the weight (A)

• Add the deuterium oxide. Note the weight (B)

• Fill up to the mark with water. Note the weight (C)

• Calculate the weight of D2O (D=(B-A)*1000 mg)

• Calculate the total weight of water (W=(C-D)/1000 kg)

• The enrichment in (mg/kg) is D/W

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y = 0.9891x

R2 = 0.9999

0

500

1000

1500

2000

2500

0 500 1000 1500 2000 2500

Calculated (mg/kg)

Measu

red

(m

g/k

g)

Deuterium calibration curve measured by FTIR

• Analyse the standards in triplicate (3 separate fills)

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Within-day precision

Use standards

• Make repeated scans of the calibration standards 5-10 times

• Fill the cell with the same sample 5-10 times

Precision can be quoted as

the standard deviation (SD)

or coefficient of variation (CV)

CV (%) = (SD/mean) x 100

Each repetition was independently loaded into the cell n times

and read using the same calibration standard (1050 mg/kg)

The cell was loaded once with each standard and scanned 10

times against the same calibration standard (1050 mg/kg)

Data from Mauro Valencia

Data from Mauro Valencia

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Between days precision

• If the precision analysis is repeated on several

days over a period of time, an estimate of

between days precision is achieved

• Between days precision is sometimes slightly

worse than within-day precision

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Acknowledgements 1

• This presentation and the accompanying presentation

on FTIR Theory were originally prepared by Christine

Slater for use in the IAEA Regional Training Course on

deuterium dilution methods for assessment of body

composition, and human milk intake by breastfed

infants (Botswana, 2008) .

• The IAEA is grateful to the late Dr Lesley Bluck from

MRC-HNR, Cambridge, UK who originally developed

and validated the analysis of deuterium enrichment by

FTIR for generously sharing his expertise. Jennings, Bluck, Wright & Elia, Clin. Chem 45:7 (1999) 1077-1081.

• Some of the information presented here does not apply

to the new portable FTIRs.

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Acknowledgements 2

• The IAEA is grateful to staff from the chemistry

department at the Botswana National Food

Technology Research Centre (Juda Bogopa and

Kabo Mosetlha), who are featured in the photos

in this presentation, and made and analysed the

standards shown in the standard curve.

• Data on within-day precision of analysis are from

Mauro Valencia Juillerat, Mexico, who was also

the photographer and fellow lecturer on the

Regional Training Course.

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Advantages of the new portable FTIRs

• Portable: supplied as standard with a

toughened transport case

• Battery driven and free from interruptions in

electricity supply

• No optical mirrors making them much less

prone to damage while being moved

• No optical housing making measurements less

prone to interference from carbon dioxide

• Software control runs under a contemporary 64

bit operating system