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Offline Dierk

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PBS question
« on: August 16, 2005, 02:50:25 PM »
Hi,

stupid question:

what is 1 x PBS ?

Of course, saline concentration will be 0,9 %, but what about phosphat buffer?

I've seen phosphat buffer ranging from 0,01 mmol to 7 mmol. That's 700 times the concentration!!!

As far as I know, physiologic phosphate would be around 1,1 mmol.

In the protocol on this site, however, 1 x PBS is made like this:

10 X PBS (0.1M PBS, pH 7.2):

       Na2HPO4 -------------------------------------- 10.9 g

       NaH2PO4 -------------------------------------- 3.2 g

       NaCl ------------------------------------------- 90 g

       Distilled water --------------------------------- 1000 ml

       Mix to dissolve and adjust pH to 7.2

       Store this solution at room temperature. Dilute 1:10 with distilled water before use and adjust pH if necessary.

So it gives 10 mmol PB. Is this the "gold standard" for PB concentration?

cu
Dierk

PBS question
« on: August 16, 2005, 02:50:25 PM »

Offline richard03

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PBS question
« Reply #1 on: August 16, 2005, 05:05:59 PM »
Dierk,

I have been working with three different labs in the past 10 years and they all use the same protocol as the one you have posted. My guess is that 0.01M (10mM) PBS is "Gold Standard". I also used several commercial PBS solutions and the concentration is 10mM as well.

I do use PB (no NaCl) alone sometime and the concentration is 0.1M (100mM) for animal perfusion.

My last thought is that the concentration may not be as critical as pH value of the PBS solution.

Richard

Offline discman

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PBS question
« Reply #2 on: August 17, 2005, 03:50:32 AM »
Ok, I think that's a good thread to ask another PBS related question:

In our lab we usually prepare 10x PBS as follows:

NaCl........................87.65g
Na2HPO4x12H2O......28.9g
NaH2PO4xh2O..........2.4g

If you mix this together and adjust with ddH2O to 1l, the pH is around 6.8.

If you dilute it to 1x the resulting pH is around 7.4!!!! WITHOUT adjusting anything.

Does anyone have an idea about this change of the pH value? I always thought that dilution with water shouldn't change the pH in a buffer. (BTW I didn't believe it either until I tried it... ;-) )

GŁnther

Offline richard03

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PBS question
« Reply #3 on: August 17, 2005, 10:17:12 AM »
Hi GŁnther,

Very interesting post. Actually I had similar experience as you do. That was about 6 years ago when I was working in a lab where there was no need to adjust pH when I made 1xPBS. The pH was always 7.2

Now in my new lab in another city, when I make 1XPBS the pH is always 7.0 instead of 7.2 so I have to adjust pH using NaOH.

In summary, I think this has something to do with the pH of distilled water you are using. Sometimes the pH of water changes so I would always check pH everytime when I make buffers since my expericence tells me that pH of buffer is very important for immunohistochemical staining.

Any comments are welcome.

Richard

Offline discman

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PBS question
« Reply #4 on: August 25, 2005, 06:02:57 AM »
Hi Richard,
thanks for your post.

However, I'm a little confused. If water of a different pH changes the pH of a buffer quite significantly, why the heck do we use buffers then  :D

Or, in a different way: I wouldn't have expected 'pure' water to change the pH ~0.5 - 0.6 units.

Do I miss something?

GŁnther

Offline richard03

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PBS question
« Reply #5 on: August 25, 2005, 09:55:22 AM »
Hi GŁnther,

Good question!.

My understanding is that buffer stabilizes the pH value of a solution. It is also the water's resistance to change in pH. However, the buffering capacity is "limited". The following is what I found from other sites about buffering capacity:

"Buffering capacity refers to water's ability to keep the pH stable as acids or bases are added. pH and buffering capacity are intertwined with one another; although one might think that adding equal volumes of an acid and neutral water would result in a pH halfway in between, this rarely happens in practice. If the water has sufficient buffering capacity, the buffering capacity can absorb and neutralize the added acid without significantly changing the pH. Conceptually, a buffer acts somewhat like a large sponge. As more acid is added, the ``sponge'' absorbs the acid without changing the pH much. The ``sponge's'' capacity is limited however; once the buffering capacity is used up, the pH changes more rapidly as acids are added".

Anyway, in theory, the "pure" water should not change pH of a buffer significantly as you suggested and I agree with you at this point. But my personal experience tells me that there are some exceptions out there.

Richard

Offline agenes

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water PH
« Reply #6 on: August 25, 2005, 11:39:34 PM »
You might want to check water ph value. Sometimes it may not be pure water.

Offline frank efterlon

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PBS question
« Reply #7 on: August 30, 2005, 05:52:47 AM »
Hi,

Just two remarks. I think it's better to change the pH of the buffer by adjusting the amounts of the buffercomponents, than adding NaOH or HCl.
Measuring the pH of pure water is not very reliable. You can only get a good reading when there are sufficient ions in the solution.

Frank

Offline KNefeli

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Re: PBS question
« Reply #8 on: October 25, 2016, 10:57:56 AM »
Hi all,

I have the same problem,

So I make PBS 10x and when I dilute it the pH goes up 0.6 points..

We usually use this recipe:
http://cshprotocols.cshlp.org/content/2007/4/pdb.rec10768.full?text_only=true

But I also tried this one to see if I have the same problem:
http://cshprotocols.cshlp.org/content/2007/4/pdb.rec10917.full?text_only=true

However, this does not happen so much to PB, it only increases from 7.5 to 7.7 when I dilute it 10x, using this recipe:
 Phosphate Buffer (0.1M PB, pH 7.4):

 

       Na2HPO4 (anhydrous) -------------------------- 10.9 g

       NaH2PO4 (anhydrous) -------------------------- 3.2 g

       Distilled water ---------------------------------- 1000 ml


Mind that we have Na2HPO4.2H2O & NaH2PO4.H2O, but I adjust the amounts accordingly.

Why does this happen to PBS and not to PB ??? What can I do ?? Not adjust the pH at all??
I'm really confused.

Re: PBS question
« Reply #8 on: October 25, 2016, 10:57:56 AM »