If the pK's are well separated, so that only one is titrating at a time ( assume
only two species exist at a time), the number of equiv of NaOH to add would be :
1/(1+10^(pK1-pH)) + 1/(1+10^(pK2-pH)) + 1/(1+10^(pK3-pH))
where each term transitions from 0 to 1 as pH passes through it's pKa
In principle the calculated buffers should be more accurate, especially in the strong
buffering regions where a small error in weighing out doesn't affect pH much. They depend
on accurate pKa values, but these have probably been determined by a physical chemist with
a hydrogen electrode (not glass electrode) in a junctionless system with temperature
control. They are probably more reproducible, since analytical balances don't drift as
much or show as much temperature dependence as pH electrodes. However if you publish just
giving pH and someone else tries to reproduce your results by adjusting a 1M stock
solution at room temperature to the pH and then diluting it to 10 mM in the assay at 4C,
they are likely to get a very different pH.
Also check the label on your NaOH bottle to see the water content and adjust the MW
accordingly- NaOH is often less than 90% NaOH by weight.
Finally if the reason for using a tribasic buffer is to get similar conditions over a wide
pH range, this is a delusion because different buffer species are present at different
pH's. MES^- is a lot more like MOPS^- than citrate^- is like citrate^-2, ionically
speaking. And the ionic strength will vary greatly from one end of the range to the other,
as you go from mostly neutral citric acid to mostly Na2- or Na3-citrate.
eab
Katherine Sippel wrote:
Alternatively you could make a stock solution of citric acid (say 1 M for
example) and
stock solution of sodium citrate (also 1 M). Mix them in the appropriate ratio
to ballpark
the right pH and just adjust up or down with the stock solution. The
concentration of
citrate will be the same no matter the final volume. You can then dilute that
down to
whatever your final concentration of citrate needs to be.
If you are looking for the actual method to do the calculations I would suggest
finding a
chemistry textbook and looking at the chapter on buffering and the
Henderson-Hasselbalch
equation.
Cheers,
Katherine
On Thu, Jan 30, 2014 at 9:31 AM, Daniel Picot <[email protected]
<mailto:[email protected]>> wrote:
But you have to be aware that pH depends on the concentration of the
buffer. This is
especially the case for phosphate and citrate buffer.
Daniel
Le 30/01/2014 15:51, Schnicker, Nicholas J a écrit :
It’s a pain but I usually just make each pH of whatever buffer I’m using
(if you
make it concentrated then you’ll only have to do it once). Also, if you
haven’t
already found it, Hampton has a nice link to calculate volume of components
while
designing a tray as long as you tell it the concentrations.
http://hamptonresearch.com/make_tray.aspx
Nick
From: Roger Rowlett <[email protected] <mailto:[email protected]>>
Reply-To: Roger Rowlett <[email protected] <mailto:[email protected]>>
Date: Thursday, January 30, 2014 at 7:23 AM
To: "[email protected] <mailto:[email protected]>"
<[email protected]
<mailto:[email protected]>>
Subject: Re: [ccp4bb] preparation of citrate buffer pH3-6.5
The easiest way to produce repeatable conditions is to titrate a stock
solution (say
1M) of citric acid with NaOH to the desired pH and use that to mix your
screen.
That's what Hampton does anyway.
If fine sampling pH, you can mix various ratios of pH 3 and 6.5 buffers.
The pH
won't be linear with mixing ratio, but will be easily repeatable. The
actual pH of
the final, magic solution can be directly measured if desired. Calculations
will
never be exactly right; pKa values are ionic strength dependent. Better to
measure.
Roger Rowlett
On Jan 30, 2014 2:37 AM, "sreetama das" <[email protected]
<mailto:[email protected]>> wrote:
Dear All,
We have obtained many tiny protein crystals in a condition
containing
0.1M citric acid pH 3.5, 2M ammonium sulfate. The crystals are too
small for
mounting in loops.
We intend to vary the salt concentration & pH to obtain
larger crystals.
Could anyone direct us to some links, or provide us with a
method
(with calculations) to calculate the amounts of citric acid & trisodium
citrate
required to obtain buffers in a range of pH 3 - 6.5?
I have come across online buffer calculators and links where
the
amounts of the components required are mentioned in grams, but none
explaining
how those values were arrived at.
Thanks & regards,
sreetama
--
"Nil illegitimo carborundum"/- /Didactylos
