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Colourimetric determination of phosphorus

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__NOTOC__
{{OR/15/070}}
==Supplied reagents==
'''Solution 1 — '''Buffer (25 mL, pH 5.5) and sodium azide (0.02% w/v) as a preservative. 
'''Solution 2''' — Phytase suspension (1.2 mL). 
'''Solution 3''' — Buffer (25 mL, pH 10.4) plus MgCl2, ZnSO4 and sodium azide (0.02% w/v) as a preservative. 
'''Solution 4''' — Alkaline phosphatase suspension (1.2 mL). 
'''Solution 5''' — Phosphorus standard solution (24 mL, 50 µg/mL) and sodium azide (0.02% w/v) as a preservative. 
'''Bottle 6''' — Oat flour control powder (5g; Phosphorus content displayed on bottle).

==Preparation of reagents==
'''Solution A — Stable for 1 week at 4°C''' 
* In a fume cupboard prepare the 10% ascorbic acid by adding 10g ascorbic acid to 90 mL of de-ionised water (100 mL flask). 
* To this, add 5.35 mL of concentrated (95%) sulphuric acid to dissolve the ascorbic acid powder and make up to the 100 mL volume by adding de-ionised (DI) water.

'''Solution B — Stable for 1 month at 4°C''' 
* Dissolve 1.25g ammonium molybdate to 20 mL of de-ionised water in a 25 mL flask. 
* Make up to volume using DI water.

'''Colour Reagent — Prepare on use''' 
* Mix together solutions A and B in the ratio 5:1, A:B allowing for 0.6 mL per sample. i.e. (0.5 mL solution A and 0.1 mL solution B per sample).

'''Trichloroacetic acid — Stable for 6 months at 4°C''' 
* In a fume cupboard, add 50g trichloroacetic acid to 60 mL of de-ionised water and dissolve. 
* Make up to volume in a 100 mL volumetric flask.

'''Hydrochloric acid''' 
* Add 54.5 mL of 37% HCl to 945.5 mL of de-ionised water in a 1 L volumetric flask (under fume hood).

'''Sodium hydroxide''' 
* In a fume cupboard, add 6g of sodium hydroxide pellets to 180 mL of de-ionised water and dissolve (200 mL flask).

==Equipment needed==

<center>
{|
| ! scope="col" style="width: 325px;" |       UV/Vis spectrometer
| ! scope="col" style="width: 300px;" | Vortex mixer
|-
|       Water bath (stable at 40°C)
| Microcentrifuge and 1.5 mL tubes
|-
|       Glassware
| Micro‐cuvettes (1.5 mL)
|-
|       Analytical balance
| Timer
|-
|       Pipettes (20 µL to 5 mL)
| Megazyme calculation software
|}
</center>

==Assay procedure==

===Phytate extraction===
* Weigh 1g (±0.001g) of sample material into a 75 mL glass beaker. 
* Add 20 mL of 0.66 M HCl, cover with foil and stir vigorously for a minimum of 3 hrs (or overnight for convenience). 
* Transfer 1 mL of the extract to a 1.5 mL microfuge tube and centrifuge for 10 minutes. 
* Immediately after, transfer 0.5 mL of the supernatant to a fresh 1.5 mL microfuge tube add 0.5 mL of 0.75 M sodium hydroxide.

===Enzymatic dephosphorylation reaction===
Add the following solutions to two separate Eppendorf tubes labelled free and total phosphorus.

<center>
{| class="wikitable"
|+ Table 1    First enzymatic dephosphorylation reaction
| ! scope="col" style="width: 175px;" | '''Reagents'''
| ! scope="col" style="width: 175px;" | '''Free phosphorus*'''
| ! scope="col" style="width: 175px;" | '''Total phosphorus*'''
|-
| DI
| 0.62 mL
| 0.60 mL
|-
| Solution 1
| 0.20 mL
| 0.20 mL
|-
| Sample extract
| 0.05 mL
| 0.05 mL
|-
| Solution 2
| ‐
| 0.02 mL
|}
</center>

''* Note both of these are required''

* Mix using the vortex and place in a water bath at 40°C for 10 minutes.
* After 10 minutes add.

<center>
{| class="wikitable"
|+ Table 2    Second enzymatic dephosphorylation reaction
| ! scope="col" style="width: 175px;" | '''Reagents'''
| ! scope="col" style="width: 175px;" | '''Free phosphorus*'''
| ! scope="col" style="width: 175px;" | '''Total phosphorus*'''
|-
| DI
| 0.02 mL
| ‐
|-
| Solution 3
| 0.20 mL
| 0.20 mL
|-
| Solution 4
| ‐
| 0.02 mL
|}
</center>

''* Note both of these are required''

* Vortex and place in a water bath at 40°C for 15 minutes.
* After 15 minutes, add 0.30 mL of trichloroacetic acid to stop the reaction.
* Centrifuge the final solution for 10 minutes. '''DO NOT MIX AFTER CENTRIFUGATION'''.

===Preparation of the calibration curve===

<center>
{| class="wikitable"
|+ Table 3    Calibration preparation
| ! scope="col" style="width: 100px;" |
| ! scope="col" style="width: 135px;" | '''Standard 0 (0 µg)'''
| ! scope="col" style="width: 150px;" | '''Standard 1 (0.5 µg)'''
| ! scope="col" style="width: 150px;" | '''Standard 2 (2.5 µg)'''
| ! scope="col" style="width: 135px;" | '''Standard 3 (5 µg)'''
| ! scope="col" style="width: 150px;" | '''Standard 4 (7.5 µg)'''
|-
| DI water
| 5.00 mL
| 4.95 mL
| 4.75 mL
| 4.50 mL
| 4.25 mL
|-
| Solution 5
| ‐
| 0.05 mL
| 0.25 mL
| 0.50 mL
| 0.75 mL
|}
</center>

===Colourimetric determination of phosphorus===
* Pipette into a 1.5 mL centrifuge tube, 1.0 mL of sample/standard and 0.5 mL of the colour reagent prepared earlier (''see'' Preparation of reagents above). 
* Mix by vortex and place in a 40°C water bath for 1 hour. 
* After 1 hour, mix by vortex and transfer 1 mL into a micro-cuvette for UV/Vis analysis at 655 nm within three hours.

===UV/Vis parameters===
* Wavelength: 655 nm 
* Cuvette: 1 cm light path 
* Temperature: room temperature 
* Final volume: 1.5 mL 
* Sample concentration: 0.5–7.5 µg/mL of phosphorus 
* Read against water

==Calculations==
Determine the absorbance (A655) for each standard. Subtract the absorbance of STD 0 from all other standards hence obtaining ΔA (phosphorus). 
Calculate '''M '''as follows for standards 1 to 4

[[Image:OR15070_equation1.jpg|frameless|center|170px| ]]

Calculate the '''mean M '''as follows

[[Image:OR15070_equation2.jpg|frameless|center|120px| ]]

Use the '''mean M '''to calculate the phosphorus content of tested samples.

'''Phosphorus and phytic acid content''' 
Determine the absorbance (A655) for both the ‘free phosphorus’ and ‘total phosphorus’ samples. Calculate ΔA (phosphorus) using the following.

''LlA (phosphorus) = A (total phosphorus) - A  (free phosphorus)''

The concentration of phosphorus can be calculated using.

[[Image:OR15070_equation3.jpg|frameless|center|300px| ]]

* Mean M = mean value of phosphorus standards
* 20 = original sample extract volume (mL)
* F = dilution factor
* ΔA (phosphorus) = absorbance change of sample
* 10 000 = conversion from µg/g to g/100g
* 1.0 = weight of original material
* ''V'' = sample volume (used in colourimetric determination step)

'''Hence the equation simplifies to:'''

''c = mean M x 0.1112 x LIA (phosphorus)''

'''For Phytic Acid (g/100g)'''

[[Image:OR15070_equation4.jpg|frameless|center|325px| ]]

[[Category: OR/15/070 Quantification of phytic acid in grains | 09]]

OR/15/070 Appendix 1 - Revision history

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Ajhil: /* Colourimetric determination of phosphorus */