Sodium phosphate buffer preparation - pH adjustment (Jun/05/2008 )

Hi, I need 0.1 M sodium phosphate buffer (pH 6.8). Is there any changes in pH when the buffer is stored in room temperature or in the fridge?

Pls check my buffer preparation protocol below, is it the correct way?

Stock solutions:

0.2M dibasic sodium phosphate (1 liter)
Na2HPO4*12H2O (MW=358.14) --------71.64gm + dH2O to make 1 liter (Solution X)


0.2M monobasic sodium phosphate (1 liter)
NaH2PO4*H2O (MW=138.01) --------27.6gm + dH2O to make 1liter (Solution Y)


Working buffer: 0.1M (1 liter)
pH 6.8 245 ml solution X + 255 ml solution Y ( filled up to 1 liter with dH2O)



I need to use pH6.8 cold buffer for cell lysis buffer, so I should adjust the pH while the buffer is cold?
Or I just need to adjust the pH to 6.8 at room temperature? I add HCl if it is too basic. If it is too acidic, I add NaOH.


The PBS in our lab is the tablet form from Flowlab(1 tablet in 100ml), but I dunno how much is the molarity and the pH of this tablet form PBS...so I cannot use this.

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with any buffer, you should adjust the pH at the temperature at which you intend to use it.

unless you don't care about adding salt to your buffer, the best way to adjust the pH is to use a phosphate solution at the same concentration.

prepare and adjust a high concentration stock of phosphate buffer, making final pH adjustments with the mono or dibasic stock. then dilute to the working concentration, making any additions to the solution that are required by the protocol. unless the additions change the pH there is no need to adjust when diluted.

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Thanks for the information.

So if I need to prepare lysis buffer (0.1M sodium phosphate buffer containing 1% Triton X-100) at pH 6.8, I prepare and adjust a high concentration stock of phosphate buffer, making final adjustments with the mono or dibasic stock until I get pH 6.8, then dilute to the working buffer concentration. Then I add in 1% Triton X-100 into my working buffer, If the pH change, then I need to adjust to pH 6.8 using the mono or dibasic stock again right? And I need to use the lysis buffer when it is cold, so i adjust the pH when it is cold also.

Then how you store your mono or dibasic stock solution? Do u autoclave and store in room temperature? Or need to keep in the fridge?

I should store my lysis buffer (containing 1% triton X-100) in the fridge right?

Thank you so much for replying to my post. Have a nice day.

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add the triton when making the dilution.

no need to further adjust the pH. if you do adjust with the high concentration mono or dibasic stocks then concentration of the buffer and the triton will be off. but phosphate is a strong buffer so the addition of triton to 1% should not have a significant effect on the pH.

it is always better to adjust buffers at the conditions in which they will be used.

we store the mono and dibasic stocks and buffers of naphos at room temperature, they will crystallize at refrigerator temperatures.

kphos solutions can be stored cold.

we don't autoclave the stocks (if we need to sterilize then we use filters).

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Hi!

I have a similar problem. I need to make three NaPi-buffers, 30mM, 50mM and 80mM, all pH 7. I have no idea where to start. Can somebody please help me?! A complete recipe would be great!

Thank you!

/H

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make a higher concentration stock buffer (pH adjusted) and dilute to the concentrations you need (all from the same stock).

for a 1M stock buffer:

39.0 ml 1M monobasic
61.0 ml 1M dibasic
(adjust, as necessary, with 1M mono (to lower pH) or dibasic (to raise pH) stocks).

final volume is not important except to ensure that you have enough for your dilutions.

you can use lower concentration stocks if you want (many use 0.1 or 0.2M stocks) but we always found 1M to be convenient for all of our purposes.

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