Choice of mobile phase

[mojo]

I am testing a sample that is dissolved in 50 % IPA: 50 % pH 5.5 PO4/Citrate buffer. To analyse the sample, we have tried using the diluent as mobile phase on a Gemini C18 5um 150 mm column. The equilibration time is horrendous, but we don't want to go to a weaker mobile phase as this gave problems when tried previously (MeOH used in the mobile phase instead of IPA). Is there a mobile phase with equivalent solvent strength that could be used? Thanks in advance.

[HW Mueller]

What are you trying to do? Neither phosphate nor citrate are good buffers at pH = 5.5. What is their concentration? What makes you think that IPA is a stronger eluent than MeOH on a Gemini C18?

[Mark Tracy]

Well, acetonitrile and IPA are of similar solvent power, but acetonitrile has a much lower viscosity so your pressure will be lower and your efficiency higher. (Of course if you don't need UV compatibility, there is also acetone.) Citrate (pKa 3.1, 4.8, 6.3) is not a bad buffer at pH 5.5, but I have found it difficult to obtain it clean enough for good HPLC work.

[HW Mueller]

Ok, that´s the problem with adjectives, not good buffer, not bad buffer....
Lets have the figures decide: My refs give a K2 = 4.7, but lets take 4.8. At 5.8 one should consider the buffering as negligible.

[PJ8]

Be careful if moving to acetonitrile though. As Mark says it is generally a stronger eluter than MeOH so may fulfill that criteria. But as an a-protic solvent it can drastically change your chromatography when changing from a previously protic organic component. When I tried to swap from MeOH to MeCN a while back we had to totally re-run the LC-MS impurity identification because the peak order was scrambled by the solvent change.

Regards, Pat.

[Mark Tracy]

OK, at pH 5.5 in water, the distribution of citrate forms is roughly 15:73:12 for H2Cit(-):HCit(2-):Cit(3-). Careful weighing will hit that target. Furthermore, in alcohol-water systems, the pKa for anionic buffers will shift to higher values.

[ym3142]

AS my understanding, citric is perfect good for pH 5.8 buffe since

Citrate (pKa 3.1, 4.8, 6.3)

(thanks to Mark), which is only 0.7 unit from 6.3.

Mark, thank you for the information of

at pH 5.5 in water, the distribution of citrate forms is roughly 15:73:12 for H2Cit(-):HCit(2-):Cit(3-).

but why do I need to care about 5% or 50% Cit(3-) in the system? is this the purpose of buffer that it changes its composite dynamically while keeps the pH constant?

[ym3142]

0.8 unit differece from pH 5.5 buffer to pka 6.3, sorry

[ym3142]

Mark,
sorry, one more question: when you say "acetonitrile and IPA are of similar solvent power" do you mean for both NP and RP? How about comparing MeOH, EtOH, IPA, ACN? how about their solubility (both salt and organic)?

[HW Mueller]

ym3142, it´s 0.8 from the max possible buffering here, but 0.2 from mediocre.

[ym3142]

Hans, you are right about

it´s 0.8 from the max possible buffering here, but 0.2 from mediocre.

so what's wrong with this?

[Mark Tracy]

1- The general idea of a classic buffer (weak acid and conjugate base) is exactly as you said

the purpose of buffer that it changes its composite dynamically while keeps the pH constant?

2- IPA and MeCN have similar solvent strength for reversed-phase, that is hydrophibic elution. MeOH<EtOH<IPA~MeCN<THF is a reasonable first approximation for purely hydrophobic interactions. Solvent strength, like column selectivity is a combination of different interactions, and depends on the analytes in question.

[ym3142]

Mark,
Thanks for your nice reply.
one more question, since IPA and EtOH is between MeOH and ACN but I do not see many develop method on IPA or EtOH. what is the reason other than higer viscosity? I believe there should be some pros and cons. Could you please share with us?

[Mark Tracy]

The biggest advantages for EtOH and iPA are low cost and low toxicity. The biggest disadvantage is viscosity; that leads to higher pressures and lower efficiencies.

[HW Mueller]

ym3142, nothing wrong with this if you don´t mind being much closer to mediocre than ideal.

To get back to mojo: Since one sees the use of IPA mostly in protein RP-HPLC, where the above elution order does not necessarily hold, I just wanted to elicit more info in my first post above: What did mojo do, does he work with proteins.....?