EnAlgae SOP sheet: Silicate - SFA. 1. Purpose This procedure is to analyse seawater and freshwater for silicate, SiO2.
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1 EnAlgae SOP sheet: Silicate - SFA 1. Purpose This procedure is to analyse seawater and freshwater for silicate, SiO2. 2. Principle Analysis is performed on a segmented flow analyser (SFA), model AutoAnalyser 3 (AA3) from Seal Analytical. This analytical system in general consists of a chemistry module run by a random access XY-2 Sampler, a high precision peristaltic pump, chemistry module trays where actual reaction takes place and dual beam high resolution digital colorimeter which operates within a wavelength range of nm. The pump transmits samples, reagents and air at defined flow rates through flexible pump tubes. Individual sample segments are separated by the insertion of air or nitrogen bubbles. Following the glass mixing coils in the chemistry tray is a heating bath, after which the sample is passed into the colorimeter holding a krypton lamp and the coloured filter for the appropriate method wavelength. The concentration of the sample is determined colourmetrically, as each sample passes through the flowcell the maximum intensity of the colour is measured. The absorbance is then compared to that of known concentration calibration standards and the sample result calculated. Drift samples (the 2 nd highest calibration standard) are analysed from which the software determines if the baseline has changed from the beginning of the run. Based on these the software adjusts sample readings to account for baseline drift. The automated procedure for the determination of soluble silicates is based on the reduction of silico-molybdate in acidic solution to molybdenum blue by ascorbic acid. Oxalic acid is introduced to the sample stream before the addition of ascorbic acid to minimise interference from phosphates (Ehrhardt et al., 1983; Bran+Luebbe, 2004). 3. Requirements 3.1. Equipment and materials Seal Analytical AA3 Segmented Flow Analyser (Pumps, XY Sampler, colorimeters, chemistry module MT19) AACE 6.03 software on PC 820 nm filter Autosampler 5 ml cups (polystyrene) Deionised water Volumetric flasks (various) Pipettes and tips (5 ml and 1 ml) Plastic Pasteur pipettes (1 ml and 3 ml) Analytical balance and weigh boats Stirrer Fume cupboard Beakers (various) Measuring cylinders (various) 250 ml and 100 ml bottles, PE Tube racks Dilution containers, 7 ml Bijou or 30 ml Universal Reagent bottles, glass and plastic ml calibrant tubes (x6) 3.2. Reagents Ammonium molybdate Page 1 of 5
2 3.75 g Ammonium molybdate 1.05 ml Sulphuric acid, conc 1.25 g Sodium dodecyl sulphate (SDS) Solution should be clear and free of precipitate on standing, discard if blue colour exists. Store in an amber polyethylene bottle. Stable for two weeks. Oxalic acid g Oxalic acid Store in an amber polyethylene container. Stable for 1-2 months. Ascorbic acid 12.5 g Ascorbic acid Store in a polyethylene container. Stable for one week. 4. Procedure 4.1. Preparation of stock solutions Standard Stock Solution 1000 µmol/l g ( g) Sodium meta-silicate nonahydrate (freshwater) Or Artificial Seawater 35 g Sodium chloride 0.2 g Sodium hydrogen carbonate Quality Control Stock Solution 1000 µmol/l g ( g) Sodium meta-silicate pentahydrate Or Artificial Seawater 35 g Sodium chloride 0.2 g Sodium hydrogen carbonate Both Stock Solutions are stable for 3 months, store in the fridge Preparation of standards and samples Pipette calibration standards and analytical quality controls (AQC s) into deionised water for freshwater analysis, or into artificial seawater for seawater analysis (see 4.1). Use the following amounts; Table 1 Preparation of working calibration standards and AQC s Standard concentration µmol/l Volumetric flask volume Volume of 1000µmol/L stock solution to pipette 0 N/A Page 2 of 5
3 Pick a mid range value for the analytical quality control (AQC), i.e. the 0-60 µmol/l range requires 6 calibrants including a zero blank, so 30 is mid range and not normally one of the calibrants used. Calibration standards are stored in plastic bottles, and are stable for 1 week if stored in the fridge. Dilute the samples to within the range using deionised water, targeting a concentration between µmol/l. If the concentration of samples is unknown run them direct without dilution and then when the preliminary results are charted add on a sample dilution to the end of the run Measurement of standards and samples Turn on computer and autosampler Connect pump platen, turn on pump and put red toggle switch to Run. Turn on colorimeter and install 820 nm filter Load AACE 6.03 software from computer desktop Click on Charting, click Change and find Silicate-Phosphate2 on list. Select to highlight and then press OK. Can be run as a multi-test with phosphate, if phosphate is not needed then the additional chart can be closed after the baseline has been found. Shake reagents to mix When deionised water has been pumping for minutes then insert labelled reagent probes into reagent bottles (Channel 1 Reagent 1 Ammonium molybdate; Channel 1 Reagent 2 Oxalic acid; Channel 1 Reagent 3 Ascorbic acid) Ensure deionised water containers are topped up Along the top of the main screen click Set Up, and then Analysis. Click onto an existing tray and click Copy run. Give this a new reference name and check that the calibrants are correct (see section 4.2). Click on Tray protocol tab and amend list to reflect current sample references. Insert an AQC at the end of each row (every 11 samples), and also a Null and Drift. Print a copy for reference when loading samples. Click OK when done. Once reagents have been pumping for minutes, in the charting window labelled Silicate right click and Set base Fill a rinsed 14 ml calibrant tube with the top standard (i.e. 60 µmol/l) and place in position 1 on the rack. Double click on XY Sampler and click sample. Time for 2 minutes and then click wash. Click cancel to close. Wait 6-7 minutes for the peak to come through on the charting window, then when it starts to stabilise (level off) right click and Set gain. This should be between 75-85% absorbance. The gain should be around Page 3 of 5
4 Fill the other rinsed calibrant tubes with fresh calibration standards and load into the rack in high to low order, double check baseline is at 5% and click Set base if necessary. Click Run, find the new tray name and double click. Press OK. Load the samples according to the printed tray layout, rinsing the 5 ml cups with a little volume before filling. The calibration coefficient will be displayed before the sample peaks are shown, check that this or better. When run is finished a message will appear. Click OK. Switch reagent probes into deionised water to rinse for 30 minutes 1 hour. If running other analyses then use 1 N HCl to wash out for 15 minutes. Turn off the colorimeter and the autosampler. When finished rinsing put the red toggle switch on the pump to Stop. Disconnect the pump platen and turn off the pump. Top up deionised water containers. Dispose of waste down the sink and dispose of remaining samples as appropriate. Throw away used autosampler cups and rinse calibrant tubes with deionised water. Rinse any pipette tips used for dilutions. Rinse and throw away any dilution containers used. Click Retrieve, and View Chart. Check all peaks are correct and then click on Report. Print the results. Export the results to Excel by clicking on File along the top, then Export to, ASCII file, find the correct run name and double click, click Export and Save, then close. The Excel file will be available in the Data folder on the desktop. When finished turn off computer. 5. Calculation of results Results are expressed as µmol/l as SiO2. If dilution has been used then multiply by the appropriate factor. The AACE software accounts for baseline drift during the run. The range is 0 60 µmol/l, however this can be reduced to 0 40 µmol/l or extended to µmol/l. The detection limit of the method specified by the manufacturer is 0.05 µmol/l. 6. Quality control AQC s are run at the mid range point of the calibration. They are usually analysed 1 per 11 samples, and the results are plotted on Quality Control Charts within Excel. If any QC s exceed + 10% and a cause cannot be determined then a repeat analysis will be required. If a number of QC s exceed + 5%, then prepare an additional AQC solution to check. The QC stock is sourced and prepared separately to the standard stock (see section 4.1). Sample spiking with a known concentration can also be used as a quality control. 7. Errors and interferences Glassware interferes with silicate due to dissolution of silicic acid; all reagents, standards and QC s should be stored in plastic bottles and remain in glass for as little time as possible. Samples should be in plastic containers and connection to glassware should be minimised. Tannin, large amounts of iron, colour, turbidity and sulphide interfere (Bran+Luebbe, 2004). The matrix of the samples and calibration standards can affect the chemistry, therefore as close a match as possible should be made between matrices. For freshwater samples use deionised water standards, but for seawater samples use Artificial Seawater (see section 4.1). Interferences can occur if other chemistries are run on the same manifold, all attempts should be made to completely wash out chemicals before analysis, possibly using 1 N HCl or 1N Page 4 of 5
5 H2SO4. Coloured samples or particulates can interfere with the light path and therefore read higher than actual. Pipettes should be used to dilute samples into deionised water. Samples should be defrosted in the fridge 12 hours before analysis if possible, and should only remain frozen for as little time as possible before analysis. 8. Waste stream and proper disposal All waste can be flushed down the sink with copious amounts of tap water. The sink log should also be completed for any chemicals disposed of. Large amounts of Ammonium molybdate reagent should be separated for disposal over a longer period. Dispose of used autosampler cups and dilution containers. 9. Hazards and precautionary statements Sulphuric acid is corrosive and causes burns, therefore a fume cupboard is necessary for preparation and dealing with acids. Acids are stored in corrosive cabinets. Sodium meta-silicate and oxalic acid are also minor corrosives. All other chemicals are irritants and potentially harmful therefore proper PPE and risk assessment should be adhered to. Consult the MSDS for precautionary measures. 10. References Bran+Luebbe (2004), AutoAnalyser Application, Method No: G Rev 8. Ehrhardt, M., Grasshoff, K., Kremling, K. & Almgren, T. (1983) Methods of seawater analysis, 2nd revised and extended edn, Weinheim, Wiley-VCH Verlag GmbH, ISBN Page 5 of 5
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