Sodium phosphate buffer (0.2 M)

MATERIALS

Reagents

Na2HPO4•2H2O

NaH2PO4•H2O

METHOD

1. Dissolve 35.61 g of Na2HPO4•2H2O and 27.6 g of NaH2PO4•H2O separately in H2O.

2. Adjust the volume of each solution to 1000 mL.

Store the stock solutions for up to 6 mo at 4°C.

3. To prepare the buffer, mix the stock solutions as follows:

o i. For 50 mL: 38.5 mL of Na2HPO4•2H2O and 11.5 mL of NaH2PO4•H2O.

o ii. For 100 mL: 77 mL of Na2HPO4•2H2O and 23 mL of NaH2PO4•H2O.

o iii. For 500 mL: 385 mL of Na2HPO4•2H2O and 115 mL of NaH2PO4•H2O.

o iv. For 1000 mL: 770 mL of Na2HPO4•2H2O and 230 mL of NaH2PO4•H2O.

4. Check the pH of the solution at room temperature. If necessary, adjust the pH to 7.3

I prepare these solutions for ELISA quite often, I've never used the online tables though. To make a 0.2M solution (for ELISA) I used 28.39g Na2HPO4 and 27.6g NaH2PO4, dissolved in a litre of elgestat water. I then adjusted the pH to 6.5 using a touch of HCL or NaOH depending on what was needed. Hope that helps!

Those look up tables are usually very carefully vetted, and trustworthy. Possible reasons for disagreements with your calculation (other than a trivial error) are that the stock solutions in the look up table are different than those you are using, or are formulated for a different condition (potassium phosphate rather than sodium phosphate, or a different temperature, for example).

thanks to both of you!

Charly, I have obtained the same results (actually, for NaH2PO4 I should use 23,99g. Maybe I am doing something wrong!!)

Daniel, at first sight all the parameters are the same but I will check again.

Thanks again!

The salts used for preparation of the stock solutions may have absorbed (moisture) water from the surrounding air! If you want to be precise with the molar concentrations, you should check this point carefully.

Thanks for the advice!

I worked out 23.99g too when I was looking at it but for some reason that was incorrect.. I can't remember why this was, I think it was because we had the hydrated form in the lab? Anyway that is what I (and the other 20 people in my lab) use and it works fine for ELISA.

Hi, Valeria, there are a lot of different Na-phosphate in the term of water contents: from mono-hydrate to hepta-hydrate. You should consider this in your calculations!

I would suggest to prepare 0.2 M NaH2PO4 and 0.2M Na2HPO4 stocks (but consider water contents in your salt and re-calculate correspondingly). Then you took Na2HPO4 and gradually add NaH2po4 under pH meter before you get pH 6.4.

Here is all possible NaP salt compositions in the term of water contents:

http://www.sigmaaldrich.com/catalog/search?interface=All&term=sodium+phosphate&lang=de®ion=DE&focus=product&N=0+220003048+219853101+219853286&mode=match%20partialmax

Good luck!

Valeria, por ejemplo para preprarar un litro de buffer. Deberías pesar la cantidad de sal de fosfato que tengas que corresponda a 0.2 moles pero respecto al fosfato. Luego, debes disolver esa masa en unos 800 ml de agua destilada y luego ajustar el pH con solucion concentrada de NaOH o de HCl según el pH esté por encima o por debajo de 6.4. Una vez que tengas el pH alcanzado, sólo debes completar con agua destilada hasta el volumen de 1 litro y listo. La solución final será de pH 6.4 coln una concentración de fosfáto de 0.2M. Suerte!

Hi,Valeria u can prepare saturated solution of NaH2Po4 and prepare 0.2M Na2HPo4,take a specific volume of)0.2N Na2HPo4 add drop of NaH2Po4 and check the pH.

Thanks a lot to everybody!!

If you do not want worry about the two different salts just weigh NaH2PO4 for 0.2 M and add NaOH to adjust the pH and it will work well.

Hello, I can recommend the on-line calculator : http://home.fuse.net/clymer/buffers/phos2.html , however, it is always better to check pH of the buffer after preparation.

Calculate the needed grams from the base, dissolve 600 ml DW and then adjust the PH with the acid

Prepare 0.2M solutions using the salts. Then adjust the ph by adding H2SO4 or HCl.

You should not use H2SO4or HCl in preparing phosphate buffer and it is a totally wrong concept

Sorry it should be phosphoric acid.. As you are trying to make acidic pH solution NaOH is not the right choice.

Sodium phosphate buffer (0.2 M)

MATERIALS

Reagents

Na2HPO4•2H2O

NaH2PO4•H2O

METHOD

1. Dissolve 35.61 g of Na2HPO4•2H2O and 27.6 g of NaH2PO4•H2O separately in H2O.

2. Adjust the volume of each solution to 1000 mL.

Store the stock solutions for up to 6 mo at 4°C.

3. To prepare the buffer, mix the stock solutions as follows:

o i. For 50 mL: 38.5 mL of Na2HPO4•2H2O and 11.5 mL of NaH2PO4•H2O.

o ii. For 100 mL: 77 mL of Na2HPO4•2H2O and 23 mL of NaH2PO4•H2O.

o iii. For 500 mL: 385 mL of Na2HPO4•2H2O and 115 mL of NaH2PO4•H2O.

o iv. For 1000 mL: 770 mL of Na2HPO4•2H2O and 230 mL of NaH2PO4•H2O.

4. Check the pH of the solution at room temperature. If necessary, adjust the pH to 7.3

You can make sodium phosphate buffer directly from purity sodium phosphate powder by go to sigma website and search for sodium phosphate buffer. Good luck

Can any one suggest that my method of preparing phosphate buffer is right or wrong?

1. Prepare sodium phosphate dibasic stock (0.5 M) by dissolving 35.5 g of sodium phosphate dibasic in a final volume of 500 mL of H2O.

Some crystallization will occur when the solution is stored at 4ºC. Warm on a hot plate and stir until the crystals dissolve.

2. Prepare sodium phosphate monobasic stock (0.5 M) by dissolving 30 g of anhydrous sodium phosphate monobasic in a final volume of 500 mL of H2O.

3. Prepare 0.1 M sodium phosphate dibasic: Put 80 mL of sodium phosphate dibasic stock (0.5 M) from Step 1 in a beaker and add H2O to give a final volume of 400 mL.

4. Prepare 0.1 M sodium phosphate monobasic: Put 30 mL of sodium phosphate monobasic stock (0.5 M) from Step 2 in a beaker and add H2O to give a final volume of 150 mL.

5. Bring the 0.1 M sodium phosphate dibasic solution from Step 3 to pH 7.2 by adding as much as needed of the 0.1 M sodium phosphate monobasic solution from Step 4.

The resulting solution is 0.1 M phosphate buffer pH 7.2.

Or is it compulsory to use Handerson Hasselbach equation for finding out the exact values.

Hassan M. Awad, thank you for your step-by-step guide on how to prepare the solution. Btwn, may I ask the reason behind using the specific volume of Na2HPO4•2H2O and NaH2PO4•H2O to prepare the buffer? 

Thank you

The answer to your question is that the specific vol of the acid and base will give you the desired pH for the buffer

Dear Colleagues,  

In preparing 0.02M PBS, I would like some help as to how to account for the H2O in NaH2PO412H2O if I need to measure NaH2PO4H2O 27.6g from it. Can anyone suggest some materials or some explanation?

The answer to your question lies in several facts:

1) The HH equation is an "approximation" to reality that ignores many "real" complications.

2) The pKa's listed for many compounds are at infinite dilution, and the pKa's of acids/bases are in fact dependent on the concentration, some more than others, according to the Debye-Huckel relationships. Phosphate is particularly susceptible to pH changes upon dilution, Tris is not. (In fact, the best way to test that your pH meter is responding correctly and reads correct pHs in solutions whose ionic strength is much different than the buffers used to calibrate them, is to take the standard pH 7.00 buffer calibration solution (50mM phosphate) and dilute it exactly 10-fold with degassed DI water... the pH should rise to 7.20!  If it doesn't, then your pH electrode is NOT reading pHs correctly in solutions whose ionic strength of anything other than 50 mM phosphate. I've actually seen them go DOWN sometimes...).

3) Therefore, the HH equation will RARELY give you the exact right ratio...

You have to calculate the M.wt.including the number of water molecules while making the solutions.

How to prepare a 0,2 M phosphate buffer (Na2HPO4-KH2PO4), pH 6.6?

Does anyone konws how to make the 0.2M phosphate buffer of pH 5.5? I have not found the table online. Thank you.

i would like to prepare 20mM phosphate buffer pH 7.2 by using Na2HPO4.

Dear Valeria,

Here is a list of how you can prepare buffer solutions in all possible combinations and pHs from Sigma!!

https://www.sigmaaldrich.com/life-science/core-bioreagents/biological-buffers/learning-center/buffer-reference-center.html

Best regards,

Selim

Dear Valeria,

I totally support the view of Selim Silbir. Nice web page!!!

Best wishes and good luck

Josema

use the online calculator in following link

https://www.aatbio.com/resources/buffer-preparations-and-recipes/phosphate-buffer-ph-5-8-to-7-4

It's not mentioned if the two products are anhydrous ! The molar mass depend on it.

Re#[034] To Azeddine Kabouche:

Honestly:

If you refer to the answers #[031] of Farhad Sabour, or, #[032] of Puni R Bhatt, respectively, with your opinion:

"It's not mentioned if the two products are anhydrous ! The molar mass depend on it", then I only can inform that - within the calculator [ I have not checked the reliablity or properness of this calculator tool ]:

https://www.aatbio.com/resources/buffer-preparations-and-recipes/phosphate-buffer-ph-5-8-to-7-4 the use of:

Sodium phosphate dibasic heptahydrate = H15Na2O11P or - expressed more "normally": Na2HPO4 · 7H2O and

Sodium phosphate monobasic monohydrate = H4NaO5P or - or - expressed more "normally": NaH2PO4.H2O is indicated.

To achieve a more / less physiological pH the relation is ~ 1.33M : 0.67M of both of the phosphates - acidic as well as basic component.

According to the 'molarity' - calculator (found at: ) 20 mM = 0.02 M ... so if you insert those parameters (0.02M, pH 7.2) in the calculator tool as mentioned before, you need (for 1 L) Sodium phosphate dibasic heptahydrate (mw: 268 g/mol)3.569 g 0.0133 M and Sodium phosphate monobasic monohydrate (mw: 138 g/mol)0.922 g 0.0067 M: the 'working instruction' tells you:

What the instruction does not tell is:

Step 2a: Stir sufficiently to dissolve completely the added powder/crystals

Step 3a: Stir sufficiently to dissolve completely the added powder/crystals

Step 4): Depending on the needed versus 'truly' produced molarity of the buffer you will need more/ less concentrated NaOH or HCl-solution. Do not overtitrate!

Step 4a) Don't forget that the higher the molarity of the stock or working solution you make the lesser the (otherwise full) dissociation [below 0.1.5 - 0.1M] of the phosphate ions.

Therefore - if correct tonicity of the buffer solution is needed - this dissociation aspect also has to be taken into consideration and be calculated.

And YES: duplicating diverse buffer-recipes as found in the web may differ in their results (especially when they differ in their composition with regard to molarity, analytic approach, etc. Correct measurement of buffer solution after all (i.e. when all "ingredients" have been added and after those have been given a sufficient time for equilibration] always is mandatory, at least with a suited pH-meter. (Use of) Lackmus / litmus paper/strips (is) are too inaccurate for such task.

Valeria Scagliotti

how to prepared the two solutions with the Henderson-Hasselback equation to obtain pH=6.6

thank you

use HSB eqn

Honestly added: It is quite frustrating to see that serious replies with sufficient information are ignored to be read.

Poor science....or is it too highly sophisticated, scientific education ?? don't know...("....with expertise in: Ethnobotany, Organic Chemistry and Analytical Chemistry")

READ the attached pdf's in my previous answer [034]:

• PO4-phosphate buffers-RECIPES_Preparation of pH buffer solutions,pH1.0-pH13.0_recipes ex web-Delloyd's Lab Tech -hompage(14-02-18pdf).pdf151.83 KB
• GEN_buffers_MILLONIG_Na-PO4_Na-Na-PO4-GEYER(1)(own calculatns,MUSS W,Salzburg_for printg both sides 14-11-21_UD=15-11-05_rev.16-02-18pdf).pdf183.77 KB

Wolfgang H. Muss please I would like to prepare 0.01M pH buffer solution of varying pH using Na2HPO4 • 2H2O and Na2HPO4•12H2O,

I will also like to know what buffer concentration is suitable for pH adjustment in pH experiment of Nanoparticle.