How to prepare 0.05 M Acetate buffer pH 3.8?

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Hi everyone, I want to prepare 0.05 M Acetate buffer pH 3.8 to use it as a mobile phase with methanol and acetonitrile with the ratio of 87:10:3 for cephalosporin drug separation. Kindly, tell me the method to prepare this buffer and I also need your suggestion, which mobile phase is better to use 0.05 M Acetate buffer pH 3.8/methanol/acetonitrile (87:10:3) or Acetonitrile/water/formic acid (80:120:1). I'm using ZORBAX Eclipse XDB-C18 column. I have a very basic knowledge of chemistry and analytical techniques. Thank you.
2.7 g of acetic acid and 0.41 g of sodium acetate in a 1 Liter flask. Dissolve in water and dilute to the mark. This will get you close but if you want exactly pH = 3.8, you'll need to add some sodium hydroxide or some hydrochloric acid to manipulate the ratio of acetate to acetic acid. Calculations:

In my haste to get this done, I made a mistake on the 4th line. It should be:

10^-0.96 = [CH3COO-]/[CH3COOH]

Image
Why adjust with hydrochloric acid, why not acetic ?

Peter
Peter Apps
You won't have 0.05 M total acetate if you add more acetic acid or more sodium acetate.
...and you'll have a buffer with added sodium-chloride if you use HCl.

This is why preparing buffers is such a messy business. I'm utterly convinced that if you ask 20 chromatographers to prepare a buffer you will get twenty different buffers. They'll probably all give very nearly identical results, and it will be very difficult to find a justification why any particular buffer is "right" compared to the others.

In general, I think the important thing is to establish how you intend to do it, and stick to it. If you set the final pH with HCl, make sure that everyone who does this method does exactly that. At least this maximises the chance of the method being repeatable. Of course when published in a high-flying journal, through lack of space the editor will insist that all these "trivial" details are stripped out, and in consequence it will be impossible for anyone to reproduce the work exactly. I'm getting grumpy as I get older, and worry more about these minor things.

On a more positive note, if you've calculated that the pH of that particular mixture should be exactly 3.8, then you have to think, when writing the SOP, what actually matters, and what is most likely to be wrong. If you trust your chemicals more than your pH meter, there is a case for simply mixing the buffer and ignoring the final pH. If you trust your meter, and the pH matters more than the overall ionic strength, then it makes sense to adjust the pH to the correct value. In this scenario, you have to ask why the pH is off. If you think your sodium acetate is getting wet (ammonium formate etc. are terrible for this), then the sodium acetate solution is likely to be under-strength, and if you have to add a bit more, you're actually just getting back to what it used to be, with a fresh bottle of sodium acetate anyway.

For what it's worth, my personal approach to a 50mM buffer is to start with 50mM of an appropriate base and 50mM of appropriate acid, and blend to pH. For example, 50mM ammonium acetate could be 50mM ammonium acetate + 50mM ammonia (if I want a pH in the range between these), which means I'm guaranteed a final concentration of 50mM acetate, and unknown concentration of ammonia, but known pH. In this particular example, I trust my pH meter more than my ammonia solution, and although I don't much trust my ammonium acetate, it's the best of a bad set of ingredients.
which mobile phase is better to use 0.05 M Acetate buffer pH 3.8/methanol/acetonitrile (87:10:3) or Acetonitrile/water/formic acid (80:120:1)


If you are following an established procedure, then follow that procedure. If you are trying to develop your own procedure, you are waaay out of your depth. Either of those (or neither, for that matter!) could work depending on what you are trying to separate the cephalosporin from.
-- Tom Jupille
LC Resources / Separation Science Associates
tjupille@lcresources.com
+ 1 (925) 297-5374
Thanks banjo, thanks lmh.

I was also wondering about the potential of chloride ions to corrode stainless steel.

Peter
Peter Apps
Point taken lmh. I don't do a lot of HPLC. Can you give me an example of a time where having an extra 35 ppm of chloride ion in the mobile phase made a large difference in the ability of a mobile phase to achieve a separation in HPLC? That's pretty much what you'd get by adding 1.0 mL of 1.0 M HCl to your mobile phase to adjust the pH by 0.1 units. I'd bet that the recipe above gets you to within 0.1 pH units. 1 mL of 1.0 M NaOH moves the pH about 0.1 unit the other direction. You won't have to add a lot of either one to what I described above to get to the target.
Thank you very much everyone. Actually, I'm trying to develop my own method for the detection of cephalosporin residues in the food of animal origin using ATPS for sample preparation. Right now, want to run the standard samples of cephalosporin but confused. Can you suggest me the way I should proceed? I know I have to try different mobile phases with different ratios and etc but I need your suggestions about:

0.05 M Acetate buffer pH 3.8/methanol/acetonitrile (87:10:3) or Acetonitrile/water/formic acid (80:120:1)? (These both mobile phases have been previously used)
Isocratic or Gradient mode?
Should I prepare drug standard solutions in mobile phase, methanol or water? and temperature to store those standards solutions?
Looking forward to your kind suggestions. I have a basic knowledge about these subjects but I'm too much interested in this kind of research.

Regards,
you probably need to get some proper training as it's a lot to learn in one go, and there is a lot that can go wrong.

If you follow an established method, it should (theoretically!) work, so if you've found two different buffer mixes in literature, either should be OK. If in doubt, try both.

Isocratic or gradient is a difficult choice. If you use isocratic, then the buffer must be appropriate (it's harder to do isocratic by guesswork. It's easy to do a gradient by guesswork because you simply run a very wide gradient, 2-95%, and the compound must come out somewhere!). Fortunately you have some starting positions. Isocratic has the advantage that you do not need to keep re-equilibrating the column as you would after a gradient, so the analysis time per sample can be shortened a little. It has the disadvantage that if the samples contain other hydrophobic compounds they can build up on the column, and possibly appear one or two injections later as broad peaks.

It is probably sensible to make up the standards in mobile phase, but do be careful that your standards aren't in a radically different solvent to the samples you will eventually run. It is important not to inject your sample/standard in a solvent that is stronger than the running solvent. This is particularly true of isocratic. For gradient methods things are more flexible. Put crudely, if you have a gradient method starting at 5% acetonitrile, and your peak elutes at 40% acetonitrile, you may get away with injecting in, say, 60% acetonitrile, because it will dilute out rapidly to something less than 40%. Obviously the larger the volume you inject (relative to the size of the column), the more important it is that the solvent in which you inject is appropriately weak. It is also very important that the compound is actually soluble in the solvent you use!

The temperature depends on the compound. You will need to check that (1) it is stable; (2) if it precipitates at low temperature, you can redissolve it effectively on thawing; (3) that you don't experience serious evaporation of the solvent, which will mess up the concentration of your standard.
rb6banjo wrote:
Can you give me an example of a time where having an extra 35 ppm of chloride ion in the mobile phase made a large difference in the ability of a mobile phase to achieve a separation in HPLC?


It definitely will make a large difference when the QA guys stumble upon it and probably want to have that method revalidated because of the extra 35 ppm chloride.
Might sound like a joke but probably is not so far from the truth :( .
rb6banjo wrote:
2.7 g of acetic acid and 0.41 g of sodium acetate in a 1 Liter flask. Dissolve in water and dilute to the mark. This will get you close but if you want exactly pH = 3.8, you'll need to add some sodium hydroxide or some hydrochloric acid to manipulate the ratio of acetate to acetic acid. Calculations:

In my haste to get this done, I made a mistake on the 4th line. It should be:

10^-0.96 = [CH3COO-]/[CH3COOH]

Image


Can you suggest what change shall be made for acetate buffer 0.05 M pH 4.5?
Good evening everyone,

Why not use the buffer calculator either here:

https://www.liverpool.ac.uk/buffers/buffercalc.html

[Simply disable "Control of ionic strength" and set the Prepare and use temperatures o the temperature of the lab].

Result is identical to rb6banjo's: (which makes good sense)

"BUFFER:
To make 1000 ml of 0.05 M Acetate (pKa=4.76) Buffer, pH= 3.8,
Ionic strength = 0.005 M,
(Ionic strength due to the buffer = 0.005M )
Thermodynamic pKa = 4.76, Apparent pKa' = 4.72
Temperature coefficient = -0.0002 per oC
Prepared at 25oC, used at 25 oC

RECIPE:
Dissolve 0.0446 mol of acid component
Dissolve 0.0053 mol of basic component
(No added neutral salts, I due to buffer alone.)
Makeup to 1000 ml with pure water."

Second website:

https://www.zirchrom.com/Buffer.asp

Sign up is free. Both sites work well...Beynon's is a bit more thermodynamically correct for the purist. Beynon also wrote an excellent book for beginners: (shows rb6banjo's method)

Buffer Solutions, by Professor Rob Beynon, J Easterby, June 19, 2003 by Taylor & Francis, Reference - 88 Pages, ISBN 9780199634422 - CAT# RU397
Series: THE BASICS (Garland Science). 65.10 USD.

Worth buying.
MattM
You could also put 4.5 in place of 3.8 in line 3 of my calculations and carry it through from there.
@ rb6banjo,

Quite agreed, your calculation is absolutely spot on. I'm simply very lazy... :P

It is always best to check the work and know how to do the calcs from first principles as you so kindly have shown.
MattM
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