Chromatography Forum

Thank you MattM

Actually, I am planning to do it without any modifier. 2 days ago when i ran the analyte without the modifier i could see both indole 3 carbino (I3C) and the condensation product (DIM) but the ratio of I3C was much higher than DIM and no fronting and tailing of the peak could have seen. So 2 peaks were separated with a good resolution.

However, initially when i tested my analyte in an acidic solvents (20 mM acetic acid) the ratio of I3C and DIM was not that different and in addition the fronting of the first peak (I3C) was merged with the tailing of the second peak (DIM).


So that is the reason i am planning to use a shorter C18 column.

Hello again,

Seems reasonable to me, and a better than what I thought of. We know acid is bad (condensation), alkaline is bad (signal suppression), why not intermediate pH and a shorter column? Best wishes for this coming experiment.

Hi again,


Thank you very much. May i ask you a stupid question. I know the concept of pH is when there is an aquas solvent available.

Today, i measureed the pH of 100 % MeOH and it was 8.4. I know methanol is a protic solvent but I couldn’t understand the concept of pH. Why ONLY when we have water we can talk about pH? Can we talk about pH when we have 100 % protic solvent?

Hi again,

Questions aren't stupid--asking questions is a way to learn. ;>)

The pKa of methanol is 15.5 (a weak base...water's pKa is 15.7 to donate OH-), you measured MeOH pH as 8.4. Okay, you used a glass pH probe that is designed to work with aqueous solutions, I assume. It is to say that you used "the wrong tool for the job" in this case. pH is defined for aqueous systems only-though the concept of hydrogen ion activity is still applicable in organic solvents.

https://link.springer.com/article/10.10 ... 002-1455-z

Here's a fun reference if you're suffering from boredom.

Electrode just gave you what it read across the membrane...likely not too valuable. You may need to re-hydrate the electrode, too!! (Someone I know, meaning me, made this error ca. 22 years ago).

People do use the glass electrode to measure the apparent pH of water-aqueous mixtures. There are ways to do this...among others Yuri V. Kazakevich and Rosario LoBrutto have described this.

Sorry for the long-winded response--it's complicated stuff.

Thank you MattM for your reply. I will download the paper and will read it very soon. Yes i used the glass electerod even though i knew it may be harmful for the electrode, i did it fast and i soak it with water afterwards. Is it true that the organic solvent without any water can destroy the glass electrode?

Just you mentioned pka of methanol is 15.5 so it is weak base. Is it not true that in general when the pka value is high like 15 then that compound is weak acid and strong base? Or in comparison to water you said methanol is weak base than water based on pka values?

Hello again,

Methanol won't destroy the electrode, but it will "dry" out the membrane (needs water to work properly) and may affect the junction plug adversely. A soak in dilute HCl may work...or it may be fine just sitting in 4M KCl...hard to tell. The manufacturer may be able to shed some light on a recovery protocol.

Source of data:

http://www.chem.wisc.edu/areas/reich/pk ... lliams.pdf

Yes, that makes MeOH a pretty weak acid (nearly as weak as water's acidity in water itself). This means the conjugate base of MeOH, MeO-, is a strong base--yes once again (organic chemists use potassium methoxide solutions for cleaning glassware for this reason...pretty caustic stuff). But...it takes a LOT to remove that proton from methanol.

Hi again

Thank you for your answer. Have a great weekend.

Kourosh