You firstly should note the composition of your PBS solution, since it may change. There is a possibility of changing the pH by using Handerson-Hasselbach equations, but since solution would also have NaCl, KCl like salts, it would change the ionic strength of solution, and the results obtained from Handerson Hasselbach may not be the same. But nevertheless, let me put it into words:

You would have to use Handerson-Hasselbach equation:

pKas of Phosphoric acid are 2.3, 7.2, 12.35.

Looking at these values, any pH between 7.2 and 12.35 would require you to use monoprotic (HPO4--) and diprotic (H2PO4-) salts (Na or K salts). Now, what you need to do is to get Handerson-Hasselbach equation, which is

pH = pKa + log ([A-]/[HA])

Now, for A- you put HPO4-- concentration, and for HA you put H2PO4 concentration.

For example, for pH=8, you need:

8 = 7.2 + log(A/HA)

log(A/HA)=0.8

A/HA = 6.31

So, now you know the fold difference between these salts, and the Molarity of the solution would be given to you, and you can calculate the required amount. For example, if molarity is 0.5M:

H+A=0.5

H/A=6.31

from here you can calculate the concentration of each salt, but be careful, don't forget to add the weight of K or Na to your salts while calculating the mass of each salt required to be added.

I wish the calculations are clear, same things are valid for ph=11, where A/HA would be 6309, which is pushing the limits, but still plausable.

But, if these steps would not give the required pH for you, you still can work by having your stock solution, which would be around pH=8. And then, by dropwise addition of high molar NaOH (for ex 10M), you can change the pH of the solution, but still maintain its buffering capability.

Measurements can be done by pH-meter, that gives you instantaneous pH value of solution, not with pH papers.

I wish it was helpful.

Good luck

You firstly should note the composition of your PBS solution, since it may change. There is a possibility of changing the pH by using Handerson-Hasselbach equations, but since solution would also have NaCl, KCl like salts, it would change the ionic strength of solution, and the results obtained from Handerson Hasselbach may not be the same. But nevertheless, let me put it into words:

You would have to use Handerson-Hasselbach equation:

pKas of Phosphoric acid are 2.3, 7.2, 12.35.

Looking at these values, any pH between 7.2 and 12.35 would require you to use monoprotic (HPO4--) and diprotic (H2PO4-) salts (Na or K salts). Now, what you need to do is to get Handerson-Hasselbach equation, which is

pH = pKa + log ([A-]/[HA])

Now, for A- you put HPO4-- concentration, and for HA you put H2PO4 concentration.

For example, for pH=8, you need:

8 = 7.2 + log(A/HA)

log(A/HA)=0.8

A/HA = 6.31

So, now you know the fold difference between these salts, and the Molarity of the solution would be given to you, and you can calculate the required amount. For example, if molarity is 0.5M:

H+A=0.5

H/A=6.31

from here you can calculate the concentration of each salt, but be careful, don't forget to add the weight of K or Na to your salts while calculating the mass of each salt required to be added.

I wish the calculations are clear, same things are valid for ph=11, where A/HA would be 6309, which is pushing the limits, but still plausable.

But, if these steps would not give the required pH for you, you still can work by having your stock solution, which would be around pH=8. And then, by dropwise addition of high molar NaOH (for ex 10M), you can change the pH of the solution, but still maintain its buffering capability.

Measurements can be done by pH-meter, that gives you instantaneous pH value of solution, not with pH papers.

I wish it was helpful.

Good luck

I wonder if you will probably need to add also some deuterated water to this buffer?

Dear Galit,

I am not an expert in NMR area but I frequently play with different buffers for biological experiments for measuring intracellular pH and calcium. Considering the important comments made by experts in this area I guess changing pH of PBS buffer to the extreme alkalinity could create problems in your experiments. When I calibrate the intracellular pH by varying different buffer after permeabilizing the cell with nigericin, I choose a set of buffers which has different pKa and can be a better alternative for a broad spectrum of pH (pH 3.5-8.8).

TES: for pH 3.5-5.0

MES: for pH 5.5-7.0

PIPES for: pH 7.4-8.8

I am sharing a combined pdf file for these chemicals as a ready reference.

Hope that works!!!

What AM said. I wouldn't change pH of PBS to anything higher than 8. Use another buffering agent.

I just want to point out that phosphate buffers are going to have poor buffering capacity at the range of pHs you are examining as the closest phosphate pKas are 7.2 and 12.4. You may want to consider using something with a pKa more at the mean of the range you are studying such as glycine (pKa ~9.6).

Hi Galit,

As noted by the others, PBS will do a poor job as a buffer at these pH values.

However, you can make PBS at different pH values by changing the ratios of the monosodium and disodium phosphate salts you use. This website will do for you the calculations: http://home.fuse.net/clymer/buffers/phos2.html

Good luck,

Yehu

I agree with Elbay

Check this book:

Data for Biochemical Research

Dawson , Elliott, Elliott and Jones

Oxford Science Publications

ISBN 0-19-855358-7

ISBN 0-19-855299-8 (pbk)

All you want to know about buffers and more, but were afraid to ask for.

Phosphate buffer's work best in the range of 5.7-8.0. Beyond this range the working of Phospate buffers is not good. Similar would be the case with PBS as its as Phosphate buffers a major buffering ingredient alongwith NaCl and KCl. In case you wish to use buffer in range of 8-11 you need to use two different buffers. In range of 8-9 Borate would be a suitable option and 9-11 you can use with carbonate buffer or glycine NaOH buffer system

You can adjust the pH of PBS with 6 M NaOH or 6 M HCl. When you prepare the PBS buffer, take not of the various pKa values: pKa1=2.12, pKa2 =7.2, pKa3 = 12.32. This means that if you want the PBS at pH 7 to 8, you have to use the H2PO4-; and for the PBS at pH 11, you need to use the HPO4-. Adjust the pH using 6 M NaOH when you want it to go up and 6 M HCl when you want it to go down. Really simple.

ph  value is 6.5.  Is this value  harmful for vero cells????

 I  want to reduce my ph value of solution from 12 to 10.can any one help me how to rmaintain  ph 10?

Dear Amruta Pattnaik:

pKa of pbs near pH=10 is 12.35. The buffering capacity of solution decreases when |pH-pKa|>1.

I suggest you check the buffers table from web to find a buffer which will be good at pH=10 (i.e. has pKa value between 11and 9).

Cheers