pH of diluent

[amaryl]

How does the pH of diluent (incase its different from the mobile phase ) affects the separation.

Mobile phase example ACN : phosphate buffer 20 mM (50:50)

A different diluent (of different pH) say methanol 100% ...will pH of methanol affect separation apart from the pH of buffer in mobile phase.

Hope my query is clear.

Thanks in advance.

Regards,

Amaryl.

(I just saw a post by pawan ratra and his work on combinations stating the effect of pH of diluent on separation. Looking forward to his explanation on this issue...thanks).

[amaryl]

pH of buffer 6.0

[jitender]

Dont know practically. Theortically, I feel if the diluent is nt strong solvent than your mobile phase, and pH of mobile phase is such that analyte is in unionized state; pH of diluent may or may nt change the RT of analyte depending on the pH of diluent. If the pH of diluent is such that analyte is in unionized state, change in RT is nt expected.

In case at pH of diluent, analyte is in ionized state a slight change in RT may be expected due to equilibration of sample solution in mobile phase.

Considering the large volume of mobile phase compared to sample solution and adequate buffer capacity of mobile phase, dependence of change in RT on pH is expected to be less.

Just a thought from aqueous acid-base equilibria. Dont know about pH and acid-base equilibria in organic or partially organic solvents.

[Uwe Neue]

Your final phosphate buffer concentration is 10 mM, you are working at pH 6, i.e. 1 pH unit away from the pK of the phosphate buffer. These two things together permit you to calculate the buffer capacity.

Otherwise, I agree with jitender: if the pK of your analyte is far away from the pH of your buffer, then one does not expect any sensitivity to minor changes in pH coming from the injection. On the other hand, the pKs of compounds changes with the organic concetration. As a general rule, the pK of acids increases, and the pK of bases decreases with the addition of organic solvent to the mobile phase.

[amaryl]

Your final phosphate buffer concentration is 10 mM, you are working at pH 6, i.e. 1 pH unit away from the pK of the phosphate buffer. These two things together permit you to calculate the buffer capacity.

Otherwise, I agree with jitender: if the pK of your analyte is far away from the pH of your buffer, then one does not expect any sensitivity to minor changes in pH coming from the injection. On the other hand, the pKs of compounds changes with the organic concetration. As a general rule, the pK of acids increases, and the pK of bases decreases with the addition of organic solvent to the mobile phase.

How will be the chromatogram

If the diluent pH affects the separation ( diluent is strong 100% ACN ) and mobile phase pH is close to pKa of analyte or pKa of buffer (50:50 ACN : phosphate buffer pH 6.8 20 mM) i.e inadequate buffer capacity

Broad tailed peak?

what if the diluent pH affects the separation ( diluent is strong 100% ACN and mobile phase 50:50 ACN : phosphate buffer pH 6.0 20 mM i.e. adequate buffer capacity.


Juzt want to know how it affects the peak shape and separation.

Thanks all.

Regards,

Amaryl.

[Uwe Neue]

I do not think that the answer to this is trivial, since you are possibly varying two things at the same time. On one hand, you are injecting the sample in a strong solvent (MeCN). At the same time, since it is dissolved in MeCN it is probably not in a salt form, but in a neutral form.

If a neutral non-ionizable analyte is injected in 100% MeCN, it will usually show peak fronting, if the injection volume is large enough. If an analyte is injected in a non-ionic form, and is converted by the mobile phase to the ionic form, you would usually get tailing, since the non-ionic form is more retained than the ionic form.

So: I think you got potentially two opposite phenomena going on in the proposed case.

[amaryl]

I do not think that the answer to this is trivial, since you are possibly varying two things at the same time. On one hand, you are injecting the sample in a strong solvent (MeCN). At the same time, since it is dissolved in MeCN it is probably not in a salt form, but in a neutral form.

If a neutral non-ionizable analyte is injected in 100% MeCN, it will usually show peak fronting, if the injection volume is large enough. If an analyte is injected in a non-ionic form, and is converted by the mobile phase to the ionic form, you would usually get tailing, since the non-ionic form is more retained than the ionic form.

So: I think you got potentially two opposite phenomena going on in the proposed case.

hmm got a bit. Thanks sir. what if my analyte (glimepiride) is in 100 % methanol. Will it still be neutral (unionised)? i suspect it won't be neutral.

Will the pH of my analyte in diluent ( i checked it was 4.6) here effect the separation.

Mobile phase 20 mM Phosphate buffer (pH 3.0): ACN (40:60).

pKa of glime 6.2.

100 % methanol - less solvent strength than my worked mobile phase conditions. So ideally it should not effect as stated by Jiten.

Sorry to have so much confusion. I just want to know what role does pH of diluent play when we use buffer for mobile phase. ( both are different) How much it effects and what it does to peak.

Regards,

Amaryl.

[amaryl]

I do not think that the answer to this is trivial, since you are possibly varying two things at the same time. On one hand, you are injecting the sample in a strong solvent (MeCN). At the same time, since it is dissolved in MeCN it is probably not in a salt form, but in a neutral form.

If a neutral non-ionizable analyte is injected in 100% MeCN, it will usually show peak fronting, if the injection volume is large enough. If an analyte is injected in a non-ionic form, and is converted by the mobile phase to the ionic form, you would usually get tailing, since the non-ionic form is more retained than the ionic form.

So: I think you got potentially two opposite phenomena going on in the proposed case.

hmm got a bit. Thanks sir. what if my analyte (glimepiride) is in 100 % methanol. Will it still be neutral (unionised)? i suspect it won't be neutral.

Will the pH of my analyte in diluent ( i checked it was 4.6) here effect the separation.

Mobile phase 20 mM Phosphate buffer (pH 3.0): ACN (40:60).

pKa of glime 6.2.

100 % methanol - less solvent strength than my worked mobile phase conditions. So ideally it should not effect as stated by Jiten.

Sorry to have so much confusion. I just want to know what role does pH of diluent play when we use buffer for mobile phase. ( both are different) How much it effects and what it does to peak.

Regards,

Amaryl.

[Uwe Neue]

Well, here is the simple answer: if the analyte is not dissolved in mobile phase or a weaker eluent than the mobile phase, you may get peak distortion. This applies to both the pH and the concentration of organic solvent.

[syx]

Sometime we need to use different pH of diluent in order to dissolve and/or assure stability of the active substances.

[Uwe Neue]

Syx, I am very well aware of this. The point that I am making is that if you do that, and if you inject a sufficiently large sample volume, you may get peak distortion, which may force you to compromise. I am not preaching that one should dissolve the sample all the time in mobile phase.

[syx]

...if you inject a sufficiently large sample volume, you may get peak distortion, ...

I am agree. I have experience with tablet dissolution sample. The media generally used are 0.1N HCl, acetate buffer or phosphate buffer (0.05M KH2PO4). Beside peak distortion, the problem usually occurs in large volume for ionic substance is high variability of retention time, especially if the buffer capacity of mobile phase is inadequate.