Analysis of Deuterium Enrichment by Fourier Transform Infrared Spectrometry (FTIR): Practice. Christine Slater PhD
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1 Analysis of Deuterium Enrichment by Fourier Transform Infrared Spectrometry (FTIR): Practice Christine Slater PhD Nutrition Specialist International Atomic Energy Agency
2 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 (D 2 O) 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 g/ml All glassware must be clean and dry before use
3 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)
4 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
5 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
6 Preparation of the calibration standard The D 2 O should be weighed on an analytical balance accurate to g or preferably g The standard can be prepared in two stages, but it is important that the weight of deuterium oxide is known to g Balances must be levelled and calibrated before use
7 Stage 1 using analytical balance Weigh a clean, dry 50 ml volumetric flask with its stopper in place on an analytical balance to 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 D 2 O to the bottle 1 g D 2 O 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 D 2 O in the bottle
8 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 D 2 O 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 D 2 O in mg/kg
9 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
10 The FTIR cell: demountable cell assembly
11 The FTIR cell When not in use, store the cells in their original packaging Wipe only with a lintfree cloth (lens tissue)
12 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
13 Check for bubbles by holding the cell up to a light
14 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 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 crosscontamination When all the samples have been analysed, rinse the cell with drinking quality water before storing
15 Operation of the FTIR Switch on the FTIR 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 Maximum
16 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
17 Post-installation checks: accuracy and precision Calibration curve Within-day reproducibility Between days reproducibility
18 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 D 2 O 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
19 Preparation of gravimetric standards Target enrichment (mg/kg) μl D 2 O 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
20 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 D 2 O (D=(B-A)*1000 mg) Calculate the total weight of water (W=(C-D)/1000 kg) The enrichment in (mg/kg) is D/W
21 Measured (mg/kg) Deuterium calibration curve measured by FTIR y = x R 2 = Calculated (m g/kg) Analyse the standards in triplicate (3 separate fills)
22 Within-day precision The cell was loaded once with each standard and scanned 10 times against the same calibration standard (1050 mg/kg) Use standards Make repeated scans of the calibration standards 5-10 times Fill the cell with the same sample 5-10 times Data from Mauro Valencia Each repetition was independently loaded into the cell n times and read using the same calibration standard (1050 mg/kg) Precision can be quoted as the standard deviation (SD) or coefficient of variation (CV) CV (%) = (SD/mean) x 100 Data from Mauro Valencia
23 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
24 Acknowledgements 1 This presentation and the accompanying presentation on FTIR Theory were originally prepared by Christine Slater for use in the Regional Training Course on deuterium dilution methods for assessment of body composition, and human milk intake by breastfed infants (Botswana, 2008). The 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) Some of the information presented here does not apply to the new portable FTIRs.
25 Acknowledgements 2 The 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.
26 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
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