buffer preparation confused me

[Ronaldo]

Hi ,

Sorry if i place it in wrong form but i am confused

if i prepared buffer actate (PKa= 4.76 ) ,Volume=1000 (mL) ,ph=7,Concentration=10 (mM)

this is from this web : http://www.liv.ac.uk/buffers/buffercalc.html

my questions are :

1- if i want to control my mobile phase at ph =5.76 , how much do i need to add from the 1000 ml buffer solution above to ( mobile phase 70%methanol water in 500 ml )

2- Does the buffer concentration play important role or it doesn't matter 1 or 10mM ?

3- I have seen in the calculator that they include ( Ionic strength (mM) ) what is this ? because i don't hear about add these number in calculation before

4- is the ph will change with dilution when i dd the some from 1000ml to the mobile phase

i am sure i will find the best help here , so thanks for you all

[tom jupille]

1. First of all, pH of HPLC mobile phases is usually specified for the aqueous part of the mobile, not for the final aqueous/organic mixture (yes,there are exceptions, but they cause lots of calculation problems). The pKa of acetate will be different (probably higher) in the presence of 70% methanol.

1a. If you really insist on knowing the "pH" in 70% MeOH, you will be far better off to measure it (make sure you calibrate your electrode in that same 70% methanol!).

2. For reversed-phase chromatography, once you have sufficient buffer to keep pH under control, adding more makes little difference. How much is "sufficient" depends on your column and sample chemistry. For example, if you are doing dissolution testing and injecting a sample in HCl, you will probably need a more concentrated buffer than injection from DI water. Other techniques such as ion-pair or ion-exchange are more sensitive to buffer concentration.

3. The ionic strength is effectively the buffer concentration. You need to know that in order to calculate how much acid and base to weigh out.

4. When you dilute with organic solvent (e.g., methanol) the pH *will* change, because of the pKa shift I mentioned above. For an aqueous system, the pH is controlled by the ratio of acid to base, so it stays the same regardless of dilution (that's the whole point to using a buffer!) until you get down to a low enough level that the dissociation of water comes into play. As long as you are in the millimolar range, pH will probably vary more as a function of temperature than it will as a function of the buffer concentration.

If you want to get into the subject in more detail, I can recommend these three articles by Bill Tindall:

http://chromatographyonline.findanalyti ... rticle.pdf

http://chromatographyonline.findanalyti ... rticle.pdf

http://chromatographyonline.findanalyti ... rticle.pdf

[Ronaldo]

Well done Tom that's a module answer

[Bryan Evans]

3- I have seen in the calculator that they include ( Ionic strength (mM) ) what is this ? because i don't hear about add these number in calculation before

Referring to the calculator:

For RP chromatography you can leave the "Control of ionic strength?" box unchecked.

The "Control of ionic strength?" box can be used for IEX applications (ex: NaCl gradient at pH 7, ect.)

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[HW Mueller]

If you have to operate a RP column at a pH at which silanolates are prominent, and also if you have cationic anaylytes present you can obliterate the "silanol" effect nicely with a high ionic strength.
Also, HIC is a RP method using high ionic strength for salting out effects.

Ionic strength has a definition, it should be easier to look up than for me to attempt writing the formular here.

[danko]

I’ll just join the choir: Especially in protein separation, the ionic strength has quite a role to play – also in RP mode. Besides, the ionic strength is one of the tools for controlling the pH, or should I say; the buffer capacity.

Best Regards