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Phosphate-Buffered Saline precipitate when adding NaOH

Dmitry Bochkariov via methods%40net.bio.net (by dmitryb from advansta.com)
Thu Jul 13 02:38:36 EST 2017


If all salts were weighed correctly and precisely, pH does not need to 
be adjusted. That's the whole point of those PBS recipes.

pH being off target is an indication that amounts of salts were 
incorrect. A common problem is not taking into account the exact 
crystalline form of phosphates - they are available as anhydrous or 
several water-containing forms. The amounts to use depend on water 
content. You may be having bigger problem than just precipitation 
because molar concentrations you have may be incorrect since the pH is off.

The explanation for your precipitation: the 10M concentration of NaOH 
that you use to adjust the pH is too high in case you have Ca or 
especially Mg salts in the solution. No matter how fast you stir the 
solution, the uniformity does not happen instantaneously. It takes at 
least several seconds for the NaOH solution to mix uniformly. While this 
is happening, there are some local areas where pH is still highly 
alkaline (theoretical pH of 10M NaOH, assuming full dissociation, is 
~15, in practice, apparent pH is close to 14, which means alkaline 
concentration is seven orders of magnitude higher than your target!). At 
this level of alkalinity, calcium and, especially magnesium hydroxides 
precipitate. Calcium hydroxide dissolves relatively easy upon mixing, 
magnesium - not so much. It forms very complex forms which dissolve very 
slow at neutral pH. Furthermore, to complicate things even more, 
magnesium phosphate, especially basic phosphate are very insoluble at 
neutral pH, but they are very easily formed in the presence of phosphate 
(regardless where it comes from) and hydroxide (regardless where it 
comes from). And this is your precipitation which at neutral pH is 
irreversible.

So, you have a buffer with wrong concentration of phosphate, wrong pH, 
almost no magnesium, and probably much less calcium that it should be 
(calcium phosphate is also insoluble, same concept as with Mg, but just 
a bit more labile, but don't count on it for practical reasons).

- Check your phosphates first for hydration forms, and if they are 
correct, you should not need to adjust pH, if it's off - your salts are bad.

- Confirm that containers where you keep phosphates are very tightly 
closed, as exposure to air changes composition due to moisture in the 
air, especially if original form was anhydrous.

- If you absolutely need to adjust pH, do NOT use NaOH with 
concentration higher than 1M, and titrate VERY slowly, one very small 
drop at a time and wait till pH stabilizes completely before adding 
another drop.

This is all supposed to be very common basic knowledge in 
inorganic/analytical chemistry which is a prerequisite for buffer 
preparation.

Alternatively, there are plenty of commercial sources of PBS

================================
Dmitry Bochkariov, Ph.D.
Principal Scientist
Advansta Inc.
1505 Adams Drive, Suite B1
Menlo Park, CA 94025
(650) 325-1980 x530
================================

On 7/12/2017 5:21 AM, najib abdellaoui wrote:
> hello
> i was trying to make a PBS as usual and the component of it as following to make 500 ml of it:
>
> -Na2HPO4 0.575 g
> -NaCl 4g
> -KH2PO4 0.1 g
> -KCl 0.1 g
> -CaCl2-2H2O 0.065g
> -MgCl2 0.0235
> normally we dissolve Na2HPO4 first and we add the remaining components.
> we check the pH which was around 6.8 and we added 10 M of NaOH however, we observed formation of cloudy precipitate.
> can someone explain why this formation of white precipitate?
>    
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