Direct Determination of Aluminium in Milk by Graphite Furnace Atomic Absorption Spectrometry
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1 Direct Determination of Aluminium in Milk by Graphite Furnace Atomic Absorption Spectrometry NOTICE: This method has been developed by the EURL-CEFAO using the facilities available in its laboratories. Users have to study the instrumental conditions appropriate for their own instrumentation.
2 CONTENTS PREMISE SCOPE ACRONYMS AND CONVENTIONS METHOD DESCRIPTION REAGENTS SAMPLE TREATMENT INSTRUMENTAL ANALYSIS Al calibration solutions Matrix modifier: Calibration Standards Sample Dilution Drift Check Spectrometer Settings CALCULATION PARAMETERS OF THE EURL CEFAO METHOD FINAL CONSIDERATIONS... 6 Annex 1: Instrumental Settings... 7 Page 2 of 7
3 PREMISE One of the major problem in Al determination is the control of the contamination. In general, there are two potential sources of external Al contamination in a laboratory: migration from the glassware and environmental contamination during sampling and analysis procedures. The use of glassware has to be minimized and all the equipment checked for this element before the use. 1. SCOPE The method describes the procedure for the direct determination of aluminium in milk by GF- AAS. 2. ACRONYMS AND CONVENTIONS GF-AAS Graphite Furnace Atomic Absorption Spectrometry sp Suprapure grade up Ultrapure grade d Density c Concentration expressed in units of milligrams/micrograms per litre RSD r Relative Standard Deviation under repeatability conditions HCL Hollow Cathode Lamp THGA Transversely Heated Graphite Atomizer Tubes LoD Limit of Quantification as defined in CR 333/2007 LoQ Limit of Quantification as defined in CR 333/ METHOD DESCRIPTION The direct determination of Al is performed by GF-AAS. Instrumental quantification is based on the matrix matched calibration approach. The sample is prepared according to the procedure described at point 5. The calibration curve is prepared as specified in section 6.3. WARNING: the application of the method includes the use of hazardous materials, operations and equipment. The user is responsible for the appropriate use of safety tools. APPARATUS and EQUIPMENT - GF-AAS (AAnalyst 800, Perkin Elmer) - Tube THGA - HCL Lamp (Al) 4. REAGENTS The reagents shall have the required adequate purity. Sp or up grade guarantees that the concentration of the analyte in reagents and water is negligible compared with the level of concentration to be determined. 4.1 Up-water, specific resistance 17 MΩ cm for all samples preparation and dilutions. 4.2 HNO 3 sp, not less than 65% mass fraction, d(hno 3 ) ~1,4 g/ml HNO 3 1% from HNO 3 (4.2) with up-water (4.1) HNO 3 10% from HNO 3 (4.2) with up-water (4.1). 4.3 Certified Element Standard Solutions, c = 1000 µg/ml. Page 3 of 7
4 4.3.1 Al 1 mg/l Pipette 0,1 ml of Al stock solution (4.3) and make up to 100 ml with up-water (4.1) in a certified decontaminated volumetric flask Prepare these standard solutions in triplicate, transfer to a plastic decontaminated storage bottle (e.g. Kartell), thoroughly mix. 4.4 Magnesium nitrate: Mg(NO 3 ) 2 6H 2 O (99.99 Sp) Magnesium nitrate (Mg(NO 3 ) 2 ) 6g/l Example: weigh 0.6g of Mg(NO 3 ) 2 (4.5) and make up to 100 ml with up-water (4.1) in a volumetric flask. 4.5 Triton X-100: TX: reagent for analysis Triton X-100 1% (TX1) in water Even if the reagent is liquid, it is easier to prepare the solution by weighting Triton X % (TX2) in water From TX1 (4.6.1) with up-water (4.1). 5. SAMPLE TREATMENT Samples are directly diluted 1:5 with a solution of Triton X 0.05% (4.5.2) in the cuvettes for analysis. 6. INSTRUMENTAL ANALYSIS a) please note: New tips were used and they were rinsed with HNO 3 1% (4.2.2), and up-water (4.1), before the use. Plastic wares (cuvettes, storage bottles, tubes, etc) shall be decontaminated filling them with a diluted HNO 3 water solution (e.g. 10% (4.2.3)), left stand overnight and rinsed at least 3 times with up-water (4.1) before their storage. Before the analysis, the cuvettes shall be rinsed with HNO 3 1% (4.2.2) and up-water. The micropipette used for mixing shall not be the same used for dilution, nor the one used for sample/standard preparation purposes (e.g. declassified micropipette used only for mixing). 6.1 Al calibration solutions These solutions shall be daily prepared c (Al) = 0,25 µg/l, into a 1ml cuvette for instrumental analysis (example): pipette 1 ml of Up-water (4.1) take off 250 µl add 250 µl of Al 1 mg/l (4.3.1) mix the solution by pipetting several times c (Al) = 0, 50 µg/l, into a 1ml cuvette for instrumental analysis (example): pipette 500 µl of Up-water (4.1) add 500 µl of Al 1 mg/l (4.3.1) mix the solution by pipetting several times 6.2 Matrix modifier: Mg(NO 3 ) 2 3 g/l Example: pipette 10 ml of Mg(NO 3 ) 2 6g/l (4.4.1) and make up to 20 ml with up-water (4.1). Page 4 of 7
5 6.3 Calibration Standards Standards for calibration curve and scheme of preparation: Al Standard Milk Al (6.1.1) 250 µg/l (µl) Al (6.1.2) 500 µg/l (µl) Triton X 0.05% (4.5.2) (µl) Blank Base Std 1 (2.5 µg/l) Std 2 (5 µg/l) Std 3 (10 µg/l) Std 4 (15 µg/l) Std 5 (20 µg/l) Sample Dilution Dilution 1:5: into a 1 ml cuvette for instrumental analysis (example): pipette 200µl of milk add 800 µl of triton X 0.05% (4.5.2) and mix thoroughly by pipetting (see 6.1 a). 6.5 Drift Check Drift is checked by analyzing the second and the fourth standard: at the end of the calibration curve (zero point) at the end of an analysis, if the number of samples analyzed is less than 8; after each series of 8 samples; at the end of an analysis, if more than 3 samples have been run after the last check. If the instrumental drift is higher than the pre-fixed value (e.g. 7%), the calibration curve shall be repeated and the previous samples read up to the last acceptable check. 6.6 Spectrometer Settings Optimize the instrument in accordance with the relevant manual and the internal procedures. Set ashing and atomization temperature (step 4 and 5) taking into account the instrument used and the ageing of the spare parts (tubes, contacts, furnace) Furnace programme set in the EURL-CEFAO method: Step T ( C) Ramp Time Hold Time (s) Internal Flow Gas Type (s) Normal Normal Normal Normal Normal Normal Compared to the instrument default method, a further drying step (450 C) has been added in the furnace programme. In this way, the process shows a negligible sample spattering during the ashing step resulting in a scarce production of residual combustion products and therefore in a better method repeatability. Samples and standards volume injected 20 µl; matrix modifier injected 10 µl. Page 5 of 7
6 Two autosampler tip extra-washes and two furnace extra clean-outs are performed. Autosampler tip washing solution HNO 3 1.0% (4.2.1). 7. CALCULATION Calculate the content, w, of the chemical element as mass fraction mg/kg as follows: w = D a V 1000 where: D = dilution of the test solution before the instrumental determination a = is the concentration of the element in the test solution in μg/l V = final volume of the solution (1ml). Considering the dilution, the density of the sample can be approximated to 1. Please note: the concentration of procedure blank is not considered in the final calculation (see 10). 8. PARAMETERS OF THE EURL CEFAO METHOD Commercially available skimmed milk was spiked with a standard solution as to have a sample of about 0.06 mg/kg. LoD and LoQ were determined using 20 aliquots of skimmed milk purchased at the market spiked with a solution containing a small amount (~ 2 µg/l) of the analyte. The parameters obtained during the method validation are summarized as follows: Parameter Al Valitation level (mg/kg) 0.06 s r RSD r (%) 1.6 r (mg/kg) LoD(=3xSD) (mg/kg) LoQ(=10xSD) (mg/kg) FINAL CONSIDERATIONS Care must be taken in the decontaminations of the lab wares. The procedure applied allowed to obtain negligible blank values during the instrumental runs; for this reason these values are not included in the calculation. Page 6 of 7
7 Annex 1: Instrumental Settings The annex is the instrumental report in paper form Page 7 of 7
8 Method Name: Al Milk DD 15 Define Element Method Description : Al Spectrometer Element : Al Wavelength (nm) : Slit Width (nm) : 0.7L Signal Type : AA - BG Measurement : Peak Area Settings Read Parameters Time (sec) : 3.0 Delay Time (sec) : 0.0 BOC Time (sec) : 2 Replicates... Same for All Samples : 2 Lamp Current Use value entered in Lamp Setup window Furnace Program Step Temp( C) Ramp Time Hold Time Internal Flow Gas Type Normal Normal Normal Normal Normal Normal Read Step : 5 Injection Temperature( C) : 20 Extra Furnace Cleanout Steps Run furnace step 6, 2 times: Furnace Autosampler Sample Volume : 20 ul Diluent Volume : 0 ul Diluent Location : 1 Matrix Modifiers #1 Volume : 10 ul Location : 20 Add to calibration blank and standards. Add to reagent blank and samples. #2 Volume : 0 ul Location : 1 Add to calibration blank and standards. Add to reagent blank and samples. Autosampler and Furnace Sequence Step Actions and Parameters A Pipet modifier1 + sample/std Page 1 06/12/ :32:37
9 Method Name: Al Milk DD 15 B C Run furnace steps 1 to end Wash sampler 2 extra times Pipet Speed... Take up : 100% Dispensing : 100% Calibration Equation and Units Equation : Method of Additions Calibrate Maximum Decimal Places : 3 Maximum Significant Figures : 4 Analyst-prepared additions. Calibration Units : ug/l Sample Units : ug/l Calibration Standard Concentrations ID Concentration A/S Loc Stock(uL) Diluent(uL) Calib Blank Bianco 1 20 Addition 1 Addition Addition 2 Addition Addition 3 Addition Addition 4 Addition Addition 5 Addition Initial Calibration Options When opening this method manually: Clear calibration curve(s) and construct a new calib curve. When using this method in a multimethod sequence: Start by constructing new calibration curves. Calibration Check Correlation coefficient checking is not enabled in this method. Recalibration Periodic recalibration is not enabled in this method. Analyze standards at end of analysis is not enabled in this method. Precision Checks Precision checking is not enabled in this method. Beyond Calibration Range Beyond calibration range checking is not enabled in this method. Page 2 06/12/ :32:37
10 Method Name: Al Milk DD 15 Matrix Recovery Matrix recovery checking is not enabled in this method. Automatic Recovery Automatic recovery checking is not enabled in this method. QC Sample Definition A/S Count as Subtract QC Sample ID Loc Sample Reagent Blank QC Sample Concentrations and Limits Schedule for QC Analyses After After QC Sample ID Init Cal Recal Periodic Freq At End Periodic Timing of Analyses Frequency... Same for all QC's : 1 Count : Samples Failure Actions for After-Calibration QC's Times to When All Additional QC Sample ID Retry QC Tries Fail Message Failure Actions for Periodic QC's Times to When All Additional QC Sample ID Retry QC Tries Fail Message Failure Actions for At-End QC's Times to When All Additional QC Sample ID Retry QC Tries Fail Message Maximum Retries After QC Failure After a group of standards or unknowns has been reanalyzed 1 times, then Continue Options Include in Results Display and Printed Log: Page 3 06/12/ :32:37
11 Method Name: Al Milk DD 15 Headers: * Analytical Header * Method Header (Short) * Sample Header (Short) Start each sample on a new page Sample Data Items: Summary Items: * Replicate Data Analysis List * Means and Statistics * Matrix Test Reports Transient Peak Plots (Last) * Calibration Summary Calibration Curves Save with Results: * Transient Peak Profiles Remarks: MM: Mg(NO3)2 3 g/l Sample diluted in Triton X 0,05% Page 4 06/12/ :32:37
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