May 11, 2026
Version 7
An X-HTDC method for estimating particulate phosphorus from phytoplankton and zooplankton V.7
- Yingyu Hu1,
- Zoe Finkel1
- 1Dalhousie University
- Marine Microbial Macroecology LabTech. support email: [email protected]
Protocol Citation: Yingyu Hu, Zoe Finkel 2026. An X-HTDC method for estimating particulate phosphorus from phytoplankton and zooplankton. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg35dq7v25/v7Version created by Ying-Yu Hu
Manuscript citation:
Hu, Y.-Y., Irwin, A.J. and Finkel, Z.V. (2022), Improving quantification of particulate phosphorus. Limnol Oceanogr Methods, 20: 729-740. https://doi.org/10.1002/lom3.10517
Hu, Y.-Y., Irwin, A.J. and Finkel, Z.V. (2026), Chemical stabilization of glass microfiber type F filters for measuring particulate phosphorus using the extra high-temperature dry combustion method. Limnol Oceanogr Methods, 24: e70030. https://doi.org/10.1002/lom3.70030
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 12, 2025
Last Modified: May 11, 2026
Protocol Integer ID: 124269
Keywords: particulate phosphorus, intracellular phosphorus, phosphomolybdenum-ascorbic reduction, orthophosphate, oxalate reagent, adsorbed phosphorus, X-HTDC, High temperature dry combustion, particulate phosphorus from phytoplankton, zooplankton total particulate phosphorus, estimating particulate phosphorus, measuring particulate phosphorus, particulate phosphorus from organic compound, phosphorus recovery from several organic phosphorus compound, phosphorus recovery, tpp recovery from laboratory phytoplankton culture, intracellular phosphorus, intracellular phosphorus in microalgae, laboratory phytoplankton culture, adsorbed phosphorus, marine phytoplankton culture, dry combustion ash, phytoplankton, hydrolysis of the ash, several organic phosphorus compound, used combustion temperature, combustion temperature, polyphosphate, um orthophosphate, particulate sample, high temperature dry combustion, particulate samples from the field, minimum sampling biomass, molybdenum colorimetry
Funders Acknowledgements:
Simons Foundation
Grant ID: 549937
Simons Foundation
Grant ID: 549937
Abstract
Total particulate phosphorus (TPP) is often determined using the High Temperature Dry Combustion (HTDC) method followed by hydrolysis of the ash and then molybdenum colorimetry. Here we show that a higher than traditionally-used combustion temperature, 800 °C vs. 450 - 550 °C, improves phosphorus recovery from several organic phosphorus compounds, marine phytoplankton cultures and particulate samples from the field. In aggregate these improvements to the method double the P recovery from phospholipids to 97%. TPP recovery from laboratory phytoplankton cultures and field samples increased an average of 13%, primarily due to improvements in P recovery from phospholipids, polyphosphates, and nucleic acids. We refer to this new method as the eXtra high temperature dry combustion ash/hydrol method (X-HTDC) and recommend its application for measuring particulate phosphorus from organic compounds in aquatic systems.
The working range of this assay is 1.22 to 500 uM orthophosphate. Minimum sampling biomass is 0.19 ug P/filter.
In order to assess the intracellular phosphorus in microalgae, we recommend an oxalate reagent (Tovar-Sanchez 2003) to wash the microalgae collected on the filter to remove surface adsorbed phosphorus.
An acid-wash step of preparing GF/F filters has been included in this protocol.
Citation
LINK
Citation
LINK
Protocol materials
12 N Hydrochloric acid
Potassium dihydrogen orthophosphateACP ChemicalsCatalog #P-4550
18M sulfuric acid
Ammonium molybdateMerck MilliporeSigma (Sigma-Aldrich)Catalog #09878-100G
Ascorbic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #A5960-100G
Safety warnings
Polycarbonate filter can release toxic gas and smoke during combustion. An exhaust system is required for muffle furnace while using the X-HTDC method.
Before start
We have found that crucibles may lose their temperature resistance after acid-washing or long soaks in alkaline detergent. Crucibles tended to shatter in the oven during the initial increase in temperature from room temperature to 500 °C, even when the ramp rate was carefully controlled at 150 °C/h. We recommend not soaking crucibles in acid but instead we suggest the crucibles be filled with 0.2 M HCl and then incubated at 90 °C for 30 minutes as the acid-washing step. It is necessary to inspect the temperature resistance of newly acquired crucibles by combusting them at 500 °C for 6 h (ramp rate: 150 °C/h) after acid-washing. We found that crucibles that pass this inspection do not usually shatter when heated to 800 °C.
Acid-wash GF/F filters
4h 30m
If GF/F filters are used to collected particulate phosphorus samples, they should be acid-washed and combusted prior to sample collection to minimize background contamination.
0.2 M HCl
In a reagent bottle, dissolve one part of 12 N HCl in 59 parts of Milli-Q water
Place 25 mm GF/F filters into 50 mL Falcon tubes containing 0.2 M HCl, and cap the tubes.
Keep the tubes at 90 °C for 00:30:00
30m
Drain the acid into a beaker containing baking soda for neutralization.
Add Milli-Q water to the Falcon tube, cap it, and shake gently to rinse.
Note
The vigorous shake breaks the filters.
Drain the rinsing water into the beaker containing baking soda. Add Milli-Q water to the Falcon tube, cap it, and shake gently to rinse.
Drain the rinsing water into the beaker containing baking soda. Add Milli-Q water to the Falcon tube, pour GF/F filters and the water into a larger acid-washed beaker, shake for 1 hour.
Measure the pH of the rinse water using a pH strip. If it remains acidic, drain the water into the beaker containing baking soda for neutralization. Add fresh Milli-Q water to the GF/F filter, and shake for 1 hour. Repeat this process until the rinse water reaches neutral pH.
Arrange the GF/F filters on a watch glass and cover them with a second watch glass, to prevent contamination.
Dry at 60 °C overnight.
Using clean filter forceps, gently lift each filter from the watch glass. If a filter adheres to the glass, apply a small amount of 95% ethanol to the edge to help loosen it, then carefully remove it.
Arrange the filters in the base of a borosilicate Petri dish, slightly overlapping, and cover with the lid. Allow the filters to air-dry completely to evaporate the ethanol.
Combust filters at 450 °C for 04:00:00
4h
Sampling
Total particulate phosphorus samples (i.e. intracellular phosphorus and adsorbed phosphorus)
Filter microalgae in liquid media onto polycarbonate filters or 25 mm pre-combusted GF/F filters, using gentle vacuum pressure (5 inches Hg).
Equipment
Filter forceps
NAME
blunt end, stainless steel
TYPE
Millipore
BRAND
XX6200006P
SKU
Rinse samples with saline (35 g NaCl in 1 L Milli-Q water)
Place sample filters in 2 mL Cryogenic Vials.
Equipment
Cryogenic Vials with Closures
NAME
Polypropylene, 2 mL
TYPE
Corning®
BRAND
66021-974
SKU
Filter blank media (without cells) through polycarbonate filter or 25 mm pre-combusted GF/F filter as blank.
Flash freeze filters and store at -20 °C .
Intracellular particulate phosphorus samples
Filter microalgae in liquid media onto polycarbonate filters or 25 mm pre-combusted GF/F filters, using gentle vacuum pressure (5 inches Hg).
Add 5 mL oxalate reagent onto the filter, and let oxalate reagent sit in the filter funnel for 00:05:00
5m
Add 50 mL MilliQ water in a 250 mL beaker.
Weigh 40 g NaOH and slowly pour into the beaker.
Use squeeze bottle to rinse the weighing boat and transfer rinse water into the same beaker.
Use glass rod to gently stir and fully dissolve NaOH.
Note
The solution is very hot and corrosive. It can cause skin burns and eye damage.
Carefully transfer NaOH solution into 100 mL volumetric flask by using glass rod.
Rinse beaker with small amount of MilliQ water three times, transfer rinse water into the flask.
Mix the solution by gently shaking the capped volumetric flask and top to 100 mL with MilliQ water.
Transfer the prepared reagent into a 250 mL PP bottle.
Label the bottle with SDS pictogram.
In a 1000 mL beaker with stir bar, add 600 mL MilliQ water.
Add 18.6 g EDTA, 14.7 g sodium citrate, 0.74 g KCl and 5 g NaCl into the beaker, stir until all ingredients are dissolved. 5.7
10 Mass Percent NaOH is added dropwise to bring pH in between 6 to 7 by using a transfer pipet
Add 12.6 g oxalic acid to the solution, stir the mixture while heating.
After oxalic acid is completely dissolved, stop heating and let it cool to room temperature. A water bath filled with tap water can be used to speed up cooling. 3.3
Add 10 Mass Percent NaOH dropwise to bring pH to 8
Top to 1 L in volumetric flask with MilliQ water.
Filter oxalate reagent by rapid flow to a 1 L PP bottle.
Equipment
Sterile Disposable Filter Units with PES Membrane
NAME
Thermo Scientific™ Nalgene™ Rapid-Flow™
BRAND
5964520
SKU
Label the bottle and keep it at Room temperature .
Drain and then rinse the sample with saline once
Place sample filters in 2 mL Cryogenic Vials.
Equipment
Cryogenic Vials with Closures
NAME
Polypropylene, 2 mL
TYPE
Corning®
BRAND
66021-974
SKU
Filter blank media (without cells) through polycarbonate filter or 25 mm pre-combusted GF/F filter as blank.
Flash freeze filters and store at -20 °C .
Zooplankton samples
Weigh ground and oven-dried zooplankton samples in the crucibles
X-HTDC-ing
Mark with oil-based white sharpie on the lid, log the following information:
(1) The number of crucible
(2) The code of sample in the crucible
Equipment
Porcelain crucibles
NAME
40 mL
TYPE
VWR
BRAND
89037-996
SKU
Equipment
Crucible cover
NAME
VWR
BRAND
71000-146
SKU
Transfer sample to crucible with clean filter forceps and lay filter at the bottom.
Add MgSO4 reagent
Note
Sing-use pipet tip to avoid cross-contamination.
Microalgae samples collected on PC filters
(1) 0.034 M MgSO4 reagent:
Dissolve 1.023 g Magnesium sulfate anhydrous (CAS 7487-88-9) in 250 mL MilliQ water
(2) Add 1 mL of MgSO4 reagent onto each sample
Zooplankton samples
(1) 0.034 M MgSO4 reagent:
Dissolve 1.023 g Magnesium sulfate anhydrous (CAS 7487-88-9) in 250 mL MilliQ water
(2) Add 1 mL of MgSO4 reagent onto each sample
Microalgae samples collected on 25 mm GF/F filters
(1) 2 M MgSO4 reagent:
Dissolve 24 g Magnesium sulfate anhydrous (CAS 7487-88-9) in MilliQ water and top it to 100 mL . Be aware that the dissolving process releases heat, which may cause the water to boil.
(2) Add 1.6 mL of MgSO4 reagent onto each sample
Cover the crucibles and place in the oven at 90 °C until samples are completely dry.
Equipment
Forced air oven
NAME
VWR
BRAND
89511-410
SKU
Note
Remove samples out of the oven as soon as they are dried. If muffle furnace is not available, keep crucibles in the vacuum desiccator.
Combust dried samples at 800 °C for 09:00:00
Equipment
Muffle furnace
NAME
F30428C
TYPE
Thermo
BRAND
10-505-13
SKU
Note
Map the location of crucibles in the oven, in case pencil mark disappears under 800°C.
Note
Ramp rate should be controlled at < 200 °C /hour or follow the instruction provided by manufacture, otherwise the crucibles might shatter.
SP.RAT: 150/PAMPU: hour
Or
SP.RAT: 2.5/PAMPU: minute
9h
Allow samples to gradually cool down in the muffle furnace.
Check sharpie mark on crucibles, remark with general sharpies if necessary.
Digesting
35m
0.2 M HCl reagent:
In a reagent bottle, dissolve one part of 12 N HCl in 59 parts of MilliQ water
12 N Hydrochloric acid
Note
Volume of HCl_0.2M_mL = (5_mL) X (#Sample + #Blank)
Preheat oven to 90 °C
Add 5 mL 0.2 M HCl to each crucible.
Gently swirl the crucible.
Cover the crucibles and place crucibles in the muffin tin pan for easier-handling.
Incubate in the oven for 00:30:00
30m
Cool samples down to Room temperature
To ensure a homogeneous solution, aspirate and dispense with a pipette three times.
Digesting
35m
Transfer 2 mL of the hydrolysate into a microtube, centrifuge at13000 rpm, 00:05:00 to pellet the MgSiO₃ crystal and other debris.
5m
Transfer 500 µL solution to 2 mL microtube. Duplicate each sample and blank.
Preparing standard working solutions
2h
Standard working solutions and reagents can be prepared during sample digestion.
KH2PO4 primary standard stock solution (≈ 1 mM)
Potassium dihydrogen orthophosphateACP ChemicalsCatalog #P-4550
Transfer about 1 g KH2PO4 into a beaker, cover the beaker with foil
Place the beaker into an oven, dry KH2PO4 at 110 °C for at least 02:00:00
2h
Move KH2PO4 into a vacuum desiccator, allow KH2PO4 to cool to room temperature
Dissolve around 0.136 g dried KH2PO4 in 1 L milliQ water.
- Use 1 L volumetric flask
- Take notes of the actual weight of KH2PO4 for final concentration of standard stock solution
Transfer standard stock solution into a 1 L bottle and store in the fridge.
Note
This stock solution lasts quite a long time, unless there is evidence for growth of algae or other extraneous biotic material.
Standard working solution
| KH2PO4 | Primary (ul) | MilliQ (ul) | |
| S1 | 0 | 1000 | |
| S2 | 5 | 995 | |
| S3 | 10 | 990 | |
| S4 | 20 | 980 | |
| S5 | 50 | 950 | |
| S6 | 100 | 900 | |
| S7 | 150 | 850 | |
| S8 | 200 | 800 |
Transfer 500 uL of each standard working solution to 2 mL microtube.
Preparing working reagents
2h
Note
All reagents are freshly prepared before colorimetric measurement.
6 N (3 M) sulfuric acid reagent:
Carefully add 1 part 18 M concentrated sulfuric acid into 5 part MilliQ water
18M sulfuric acid
2.5 Mass Percent ammonium molybdate reagent:
Weigh 0.25 g ammonium molybdate in a Falcon tube and top to 10 g with MilliQ water.
Cap and shake until totally dissolved.
Ammonium molybdateMerck MilliporeSigma (Sigma-Aldrich)Catalog #09878-100G
10 Mass Percent ascorbic acid reagent:
Weigh 1 g ascorbic acid in a Falcon tube and top to 10 g with MilliQ water;
Cap and shake until all dissolved.
Ascorbic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #A5960-100G
Note
Wrap the tube with foil if the reagent is not used right after prepared.
Calculate the volume of molybdate-ascorbic reagent:
Total volume of reagent_mL = (0.5 mL) X (#standard working solution + #samples + #blanks)
Mix the reagents into Falcon tube:
| A | B | |
| Reagent | Part(s) as in volume | |
| MilliQ | 2 | |
| 6N sulphuric acid | 1 | |
| 2.5% ammonium molybdate | 1 | |
| 10% ascorbic acid | 1 |
Note
For fewer than 60 samples, prepare the final reagent in a 50 mL Falcon tube by adding 17 mL Milli-Q water, followed by 1 mL concentrated sulfuric acid (18 M), then 6 mL of 2.5% ammonium molybdate and 6 mL of 10% ascorbic acid reagent. The same pipette tip can be used to transfer ammonium molybdate and ascorbic acid, as they are mixed into the final reagent.
Colorimetric measurement
2h
Preheat incubator/shaker to 37 °C
Equipment
SHAKING INCUBATOR
NAME
71L
TYPE
Corning® LSE™
BRAND
6753
SKU
Add 500 µL reagent to each standard, sample and blank, starting from blanks, including blank for standards and blank for samples.
Equipment
Finntip Stepper Tips
NAME
5 mL
TYPE
Thermo Scientific
BRAND
9404200
SKU
Note
Before dispensing the reagent, wipe or dab the liquid drop on the outside of the tip, avoid wiping the open tip.
Vortex each tube.
Incubate at 37 °C for 03:00:00 while shaking at 200 rpm
3h
Load microplate with 250 uL reactant from each tube, duplicate.
Example of loading the microplate
Equipment
96-Well Microplates, Polystyrene, Clear,
NAME
Greiner Bio-One
BRAND
655101
SKU
Read plate in microplate reader
| A | B | |
| Shake duration | 00:00:05 | |
| Shaking type | Continuous | |
| Shaking force | High | |
| Shaking speed [rpm] | 600 | |
| Wavelength [nm] | 820 | |
| Use transmittance | No | |
| Pathlength correction | No | |
| Measurement Time [ms] | 100 |
Equipment
Varioskan LUX Multimode Microplate Reader
NAME
Thermo Fisher
BRAND
VL0L00D0
SKU
Calculating
Subtract the average absorbance at 820 nm of the blank standard replicates from the absorbance at 820 nm of all other standard working solutions.
Subtract the average absorbance at 820 nm of the blank sample (i.e. blank filter) replicates from the absorbance at 820 nm of all other individual samples.
Prepare a standard curve by plotting the average blank-corrected 820 nm absorbance for each standard working solution versus its concentration in uM.
Use the standard curve to determine the orthophosphate concentration of each unknown sample by using its blank-corrected 820 nm absorbance.
(P per sample)_ug = (orthophosphate)_uM X (V_HCl)_mL X (0.001) X (30.97)
Traditional 500 ºC assay for comparison (optional)
Sample preparation
Phytoplankton samples
Collect on 25mm pre-combusted GF/F filter and transfer into 20 mL scintillation vial, labelled with oil-based white sharpie
Zooplankton samples
Weigh directly into 12 mL clear vials, labelled with oil-based white sharpie
0.034 M MgSO4 reagent:
Dissolve 1.023 g Magnesium sulfate anhydrous (CAS 7487-88-9) in 250 mL MilliQ water
Add 1 mL of MgSO4 reagent onto each sample
Dry samples at 90 °C
Arrange vials in a small foil pan, cover with foil, then combust at 500 °C for 02:00:00 without ramp rate
Allow samples to gradually cool down in the muffle furnace.
Other steps are the same as X-HTDC method.