Quantification of Total Kjeldahl Phosphorus in Eucalyptus Leaves Using Kjeldahl Digestion and Flow Injection Analysis

doi:10.21203/rs.3.pex-2425/v1

Method Article

Quantification of Total Kjeldahl Phosphorus in Eucalyptus Leaves Using Kjeldahl Digestion and Flow Injection Analysis

https://doi.org/10.21203/rs.3.pex-2425/v1

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This protocol outlines the quantification of total phosphorus concentration in leaf material through Kjeldahl digestion and flow injection analysis. This method utilises a traditional Kjeldahl digestion as an alternative to microwave digestion which is utilised in the protocol “Quantification of Total Phosphorus Content in Plant Material Using Microwave Digestion and Segmented Flow Analysis”. The Kjeldahl digestion is more accessible in most laboratories than microwave digestion. The digestion phase converts phosphorus into orthophosphate which reacts with ammonium molybdate and ascorbic acid during flow analysis to form a blue complex which can be measured spectrophotometrically. Phases of analysis include acid digestion using a Kjeldahl digestion (1.5 days); the preparation of reagents (2 hours) and finally analysis for total phosphorus on the flow analyser (30 minutes plus 90 seconds per sample).

Environmental chemistry

Plant sciences

Eucalyptus

flammability

phosphorus

vegetation

leaf material

plant traits

Kjeldahl

Kjeltec Kjeltabs (this method is based on these tablets containing 1.5 g potassium sulphate and 0.15 g copper sulphate – if this changes, the standards and diluent proportions of potassium sulphate and copper sulphate will need to change accordingly to match the percentage of each in the tab). Contains CAS-No: 7778-80-5 and 7758-98-7
Sulphuric Acid (98%) CAS-No: 7664-93-9
Potassium Sulphate CAS-No: 7778-80-5
Copper Sulphate pentahydrate CAS-No: 7758-99-8
Ammonium molybdate tetrahydrate CAS-No: 12054-85-2
Potassium Antimonyl Tartrate hemihydrate CAS-No: 28300-74-5
L-Ascorbic Acid CAS-No: 50-81-7
Sodium dodecyl sulphate CAS-No: 151-21-3
Anhydrous potassium dihydrogen phosphate (reagent grade) CAS-No: 7778-77-0

Block Digestor for Kjeldahl digestion
75 mL Glass Digestion tubes
Balance (to 4.d.p)
Test tube racks for digestion tubes and 15ml falcon tubes
15 mL and 50 mL falcon tubes
Optifix Dispenser 10 mL
Flow injection analysis equipment for example QuikChem 8500 Series 2 FIA Automated Ion Analyzer (HACH Company, Cat. No. DOC026.53.00808).
Milli-Q water
Variety of volumetric flasks
Anti-bumping granules

1) Kjeldahl Digestion

Complete digestion in a fume hood. Digestion procedure is based on Jones & Bradshaw (1989).

Pre-heat digestion block to 160°C.
To a digestion tube add 0.1 g (+ 0.01 g) plant material, one Kjeltab and 2-4 anti-bumping granules. Include a blank for every ~25 samples.
Using an optifix dispenser add 5.2 mL sulphuric acid (98%). Figure 1 provides a visual aid for what a sample should look like pre-digestion.
Place digestion tubes in digestion block and set temperature to 360°C. Figure 2 provides a visual aid of a sample during digestion.
Digestion will take 2-3 hours – digestion is complete when all samples have turned a turquoise colour. Figure 3 provides a visual aid of samples following a complete digestion.
Remove digestion tubes from digestion block and place in a rack and allow to cool.
Add 70 mL of Milli-Q water – add water slowly and point digestion tube away from you as solution will boil.
Leave tubes in the rack to cool again.
Put in a stopper and invert to mix, leave overnight to stand.
Filter ~30 mL into a beaker through a filter paper to remove any remaining undigested particles.
Decant a 15 mL sample into a falcon tube and store in fridge until ready for analysis.
Clean digestion tubes by soaking in a 2% acid bath overnight then in Decon, rinse with RO water.

2) Preparation of reagents

All reagents can be scaled down to suit the number of samples being analysed. Reagents and standards prepared based on Fishman & Friedman (1985) and Latchat Instruments QuikChem® Method 13-115-01-1-B.

Diluent: To a 1L volumetric flask add 700 mL Milli-Q water then slowly add 70 mL of sulphuric acid (98%) NOTE: Always add acid to water, never the other way around. When solution is cool add 20 g of potassium sulphate and 2 g of copper sulphate pentahydrate. Dilute to 1L with Milli-Q water. Stability 1 month.
Stock Ammonium Molybdate: Dissolve 40 g of ammonium molybdate in 800 mL of water. Add a magnetic stirrer and stir for at least 4 hours or until all the ammonium molybdate had dissolved. Transfer to 1L volumetric flask and dilute to 1L with Milli-Q water. Store in refrigerator. Stability 2 months.
Stock Antimonyl Potassium Tartrate: Add 3g potassium antimonyl tartrate hemihydrate to a 1L volumetric flask. Add ~ 700 mL Milli-Q water and swirl solution to dissolve. Dilute to 1L with Milli-Q water. Transfer to a 1L dark bottle and store in a refrigerator. Stability 2 months.
Molybdate colour reagent: Add 500 mL Milli-Q water to a 1L volumetric flask and add 35 mL sulphuric acid (98%) and leave to cool. When solution has cooled add 213 mL stock Ammonium Molybdate solution and 72 mL stock antimonyl potassium tartrate solution. Swirl to mix. Dilute to 1L with Milli-Q water. Stability 1 week.
Ascorbic Acid Reducing Solution: To a 1L volumetric flask add 500 mL Milli-Q water then add 60 g Ascorbic acid. Swirl to dissolve. Add 1 g Sodium dodecyl sulphate and swirl to mix. Dilute to 1L using Milli-Q water. Stability 2-3 Days.

3) Preparation of standards

Preparation of standards must be done on the day samples are being run. Make new standards each day.

Dry 21.968g of Anhydrous potassium dihydrogen phosphate at 110°C for two hours.
Phosphorus Standard 1 (5000 mg P/L): To a 1L volumetric flask add 800 mL Milli-Q water then add to the dried anhydrous potassium dihydrogen phosphate. Dilute to the 1L mark with Milli-Q water.
Phosphorus Standard 2 (100 mg P/L): To a new 1L volumetric flask add 700 mL of Milli-Q water and 20 mL of the phosphorus standard 1. Add 70 mL of sulphuric acid (98%) and allow the solution to cool. Add 20 g potassium sulphate and 2g of copper sulphate pentahydrate. Dilute to 1L mark with Milli-Q.
Make 100 mL calibration standards A-G in beakers as outlined in Figure 4 and transfer 50 mL to 50 mL falcon tubes to calibrate auto-analyser.

4) Running Auto-Analyser

Run all lines with Milli-Q water to check for leaks. Baseline on computer should be flat.
If using universal dye to calibrate machine timings (e.g. if tubing lengths change) ensure you use a detector that is not 880nm, any other one will do. Make sure you put the 880nm detector back for phosphorus analysis. Use universal dye to calibrate run timings such as ‘time to valve’.
Add the respective reagent lines into the reagents. Again, baseline should be flat after adjusting to reagents.
Add standards and samples into the autosampler and run, as per the manufacturer’s instructions, using the 880nm detector to detect for orthophosphate.
At the end of a run, run Milli-Q water through all lines for ~10 mins and then allow to run dry. Turn off machine.
Assess peaks – if peaks are outside the calibration curve range they need to be further diluted and re-run.

See Figure 5 for troubleshooting advice for this protocol.

Kjeldahl Digestion – total time 1.5 days

Prepare samples for digestion (approximately 2 hours for 40 samples)
Digest samples (3 hours)
Add water to tubes (1 hour)
Leave to stand overnight (16 hours)

Analyse digested samples using flow analysis chemistry – one day

Prepare reagents (approximately two hours)
Place samples into auto-sampler racks
Run flow injection analyser and analyse for orthophosphate (approximately 30 minutes plus 2 minutes per sample).

Dilution of the Kjeldahl digest in 70 mL of water ensured that peaks fell within the calibration curve.

Digested sample P content did decline as time to analysis increased. It is recommended that samples are analysed within a week of digestion (Patton & Truitt 1992). Phosphorus analysis of various understorey plant species yielded results between 0.01-0.08% P.

1. Jones, M. N. & H. D. Bradshaw. Copper: An alternative to mercury; more effective than zirconium in Kjeldahl digestion of ecological materials. Communications in Soil Science and Plant Analysis 20, 1513-1524, DOI:10.1080/00103628909368165 (1989).

2. Fishman, M. J. & L. C. Friedman. Methods for determination of inorganic substances in water and fluvial sediments. US Department of the Interior, Geological Survey, Denver, Colorado, United States. DOI:10.3133/ofr85495 (1985).

3. Latchat Instruments. QuikChem® Method 13-115-01-1-B: Kjeldahl phosphorus analysis on the QuikChem 8500 Series 2 FIA Automated Ion Analyzer.

4. Patton, C. J. & E. P. Truitt. Methods of Analysis by the US Geological Survey National Water Quality Laboratory: Determination of the Total Phosphorus by a Kjeldahl Digestion Method and an Automated Colorimetric Finish that Includes Dialysis. US Geological Survey, Denver, Colorado, United States. DOI:10.3133/ofr92146 (1992).

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Quantification of Total Kjeldahl Phosphorus in Eucalyptus Leaves Using Kjeldahl Digestion and Flow Injection Analysis

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Quantification of Total Kjeldahl Phosphorus in Eucalyptus Leaves Using Kjeldahl Digestion and Flow Injection Analysis

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Anticipated Results

References

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