Ideally, you must obtain similar results in repeated experiments. But molybdenum blue method is a tricky process. First of all, your glassware should be cleaned with acid to nullify the effect of any phosphorus present previously. No detergent should be present in glassware. In my experiments, I had observed that even distilled water quality affects results badly. So I mostly used distilled water from the same container and distillery.

Secondly, Ascorbic acid intensifies color formation with the passage of time. If you just use blank, you will observe that with the passing time, the color of ascorbic acid solution will become darker. So it also depends how much time earlier you prepared ascorbic acid solution before adding your samples/ standards. And this is the reason the color of the solution becomes darker after reading absorption. Its normal I think

Lastly, spectophotometric analysis varies with the surrounding light. In my case, I used to close all the lights, so that the analysis should yield same results.

I hope these points help you obtain similar results in repeated experiments.

The micro-pumps provided good precision and accuracy of the injected volumes. For the determination of orthophosphates, the molybdenum blue method has been employed. The developed method can be used to detect orthophosphate in the range 0.1 - 0.12 mg L -1 with repeatability about 2.2% at 4mg L-1 and a very high injection throughput of 120 injections h-1.The molybdenum blue method is chosen for the colorimetric determination, using ascorbic acid as ......A determination frequency of 15/h may be achieved, with good repeatability for 12 consecutive injections. For more details consult https://www.ncbi.nim.nih.gov

Good repeatability of research results is the key to validation of our findings. The issue you are having would be due to interference from contamination during the analytical process. As Muhammad Suleiman said, properly wash the glassware, be very sure of purity of your distilled water and also check your standard preparation. Thank you.

Dear Masumeh, the o-PO4 analysis is only working reproducible in very strinct pH conditions between 0.8 and 1.2. You are doing an alkaline extraction and therefore the standards have to be in the same matrix as well. The molar absorption coefficient should be always 22000 mol L-1 cm-1. Best regards, Matthias

Matthias Birkicht

Dear Matthia. Thank you very much for your answer.But, I donot do olsen extraction with bicarbonate alkaline solution. I am working on phosphorous analysis in water not in soil.

Dear Masumeh,

when you are working with water extracts, it is not necessary to acidify your standards. The linear range of the recommended method according to Murphy and Riley with one mixed reagent is from 0.1 - 26 uM. To test the purity of your reagents, the blank should be near to 0 at 810nm or 880 nm in a 1cm cell. I wish you success.

best regards

Matthias

Matthias Birkicht

Thanks alot for your attention and kindness, Matthias. The main question is that the color of blank, standards and samples becomes more intensive quickly ( in a little time )after reading of the absorptions. For this reason, their absorptions change fast. Is it right or not? What should I do to prevent this problem from happening?

Dear Masumeh,

are you so polite, to tell me exactly your calibration range [uM], the number and concentration of your standard [uM], the receipt of your colour reagent and the the amount of sample and reagent [mL]. Also I need the time, which your are waiting, before you read the absorbance. Without that information I can not give you any further advice.

By the way: The blank is always! colorless over time. The reagent have a slightly yellowish color.

Our receipt:

50mL 4.9N H2SO4

best regards

Matthias

Matthias Birkicht

Dear Matthia

Thank you so much for your kindness. I work according to the method in the paper “Test of an Ascorbic Acid Method for Determining Phosphorus in Water and NaHCO3 Extracts from Soil. F. S. WATANABE AND S. R. OLSEN, soil science society 1965”. The range of concentrations are 0.08ppm-1ppm(p) i.e. ( 0.08-0.2-0.4-0.8-1 ppm)which equals to 2.5-32micro molar(p). The cell is 1 cm. wavelength is 880 nm. I read the absorbtions 10 minutes after adding color reagent.

Our color reagent receipt:

REAGENT A Dissolve 12 g of ammonium molybdate in 250 ml of distilled H2O. In 100 ml of distilled H2O dissolve 0.2908 g of antimony potassium tartrate. Add both of the dissolved reagents to 1000 ml of 5 N H2SO4 (148 ml concentrated H2SO4 liter), mix thoroughly and make to 2,000 ml. Store in pyrex glass bottle in a dark and cool compartment.

REAGENT B Dissolve 1.056 g of ascorbic acid in 200 ml of reagent A and mix. This reagent should be prepared as required as it does not keep for more than 24 hours.

After preparation of reagent B( this reagent has a yellowish color), We add 4 ml of reagent B to 5 ml of standards, samples and blank( deionized water). Then, they are diluted to 25 ml and their absorbtions are determined.

Dear Masumeh,

what's about the amount of reagent A? Does your blank is colored as well?

What readings do you have for your standards and which concntration do you use?

cheers

Matthias

Matthias Birkicht

Dear Matthias

AS mentioned in the last post, ascorbic acid is dissolved in reagent A. Sometimes, the blank is slightly blue and it gets intensively blue over time( for example several hours after reading of absorbtions. The absorbtions are as follows:

0.08 ppm 0.0314

0.2 ppm 0.0877

0.4ppm 0.2273

0.8ppm 0.4199

1ppm 0.5276

That s very kind of you for helping me.

Dear Masumeh,

when I am going for photometric readings, I will measure the Blank as well!

I DON'T substract the blank directly from the absorbances of my standard curve.

This is not necessary, because the linear regression will do that anyway and you will loose important information about impurity and contamination.

It can happen, that the reagent will become blue, when it is coming into contact with air.

That is the reason, why some working groups are dealing with a two step reaction.

First they will treat it with with ascorbic acid reagent and afterwards with the molybdate reagent. That complex is stable for hours! It can be read within 30 minutes.

I hope, you can reproduce the method with two reagents I support to you via Excel-File. Please try that out and report to us, if it is functioning.

best regards

Matthias

Matthias Birkicht

Dear Matthias

Thank you so much for your help and answer. I will try this method and inform you. But, I have a question: my sample volume is low( about 5 -10 mL not 50 mL). How should I change the amount of reagent 4( acidified ascorbic acid ) and reagent 6( mixed reagent)?

Sincerely yours.

The problem may be related with the reaction between HCO3 and acidic molibdenum solution which evolves CO2. If you do not have a sparkling free solution your reproducibility will be very low. for this take a certain amount of samples, add some certain amount of distilled water, then add color reagent mix it until the sparkling ends (be careful about the sudden sparkling). Finally make up the volume. Before measuring mix it throughly to remove further evolving gas and homogenous color development.

Dear Masumeh,

when you have only one tenth of sample, divide the amount of reagents by 10.

best regards

Matthias

Matthias Birkicht

Thanks alot, Matthias

sincerely yours.

Hi Masumeh,

If you are working with bicarbonate solutions Veli hit the nail on the head.

Are your standards in bicarb or water?

Molybdate itself can be reduced by ascorbic acid in alkaline solutions, and although the molybdate solution is acidic, if it is mixed with a bicarb solution transient alkaline volumes are present during mixing. So the procedure is more robust if the bicarb is first decomposed to CO2 before adding the molybdate reagent. A colored indicator strip can be added to check that the pH is sufficiently acidic.

At around 25 degrees, colour formation should be complete in ~ 20 min and be stable for at least 30 min. The relation between absorbance and [P] should be linear to an absorbance of ~0.7 using a 1 cm cell at 880 nm.

If your results deviate appreciably from the above something is wrong. For example, there is contamination by P or silicate in the water .

Please let me know what happens so I can assist, Paul.

I agree with Paul Milham