Different organic solvent in diluent vs. mobile phase

Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.

8 posts Page 1 of 1
Dear All,

is there a downside to use a different organic solvent in the diluent (methanol) than in the mobile phase (acetonitrile). The aqueous part is identical (water with phosphoric acid)
The peak shape is perfect, the base line as well.
Of course, at the front you have a significant solvent peak, but which does not interfere with the active peak to be integrated.
Is there any faster column deterioration etc. to be expected?

Thank you very much!


We've done similar for 30 years, no problems. Our products just dissolve better in methanol or DMF than in acetonitrile or in the mobile phase. The test methods are validated that way.

Just don't use a ginormous (humongus) injection size.
My understanding is that a different sample solvent can cause problems if it is a much stronger solvent compared to the mobile phase, but that wouldn't be an issue if it's methanol vs. ACN. I have had to run injections where the sample solvent was stronger, but they were smaller injections and there were no ill effects.
if you can then it saves time and effort to do it all in the same solvents.
mobile, also diluent is an easier method

when you cannot do it then there is a chance that a strong difference between the solvents will create a peak distortion of either a huge hump at the front or back, or fronting and tailing
the lower the injection volume the better to prevent this problem.
generally also in older HPLCs were tubing if bigger and also volumes the solvents have time to mix and so the effect does not show at all.

better to avoid it if possible, also always less work in prepration
Dear All,

thank you very much for your helpful comments so far.


Sample volume is a huge factor here. Obviously, if you're loading 2 microliters on to an analytical column, the methanol will have little impact.. presumably.

MeCN is a stronger organic than MeOH, so, as long as your sample is loaded in a weaker eluent than the starting mobile phase, you're usually OK.

One other potential area of concern having little to do with eluent strength is viscosity differences between your sample and that at the head of the column.

See "viscous fingering." Once again, seems this ought to be dependent of sample volume, concentration
In your specific case you've answered your own question. If the peak shape is good and you don't have any issues with baseline noise then it's not a problem. You won't be degrading the column excessively unless your sample diluent is concentrated sulphuric acid or something similarly nasty and corrosive.
I cant see a problem with that. As I'm sure others have pointed out methanol is a weaker solvent (we are talking reverse phase) than acetonitrile, and injecting in weaker solvents is fine.

Having said that, it depends on the %ACN in the actual on-column MP composition. Less than approx 70% and the methanol diluent will be stronger than the mobile phase. This could be fixed by adding a similar aqueous fraction to the diluent.... suspect you will say this is not possible.

If it is a gradient method, and the initial conditions are focussing the analyte onto the head of the column then you can probably get away with 100% methanol as the diluent, or even stronger solvents.

If it is isocratic, its probably a case of suck it and see. If the diluent causes problems, reduce the injection volume as far as sensitivity will allow and things may improve.

Hope this helps
8 posts Page 1 of 1

Who is online

In total there are 35 users online :: 1 registered, 0 hidden and 34 guests (based on users active over the past 5 minutes)
Most users ever online was 599 on Tue Sep 18, 2018 9:27 am

Users browsing this forum: MSN [Bot] and 34 guests

Latest Blog Posts from Separation Science

Separation Science offers free learning from the experts covering methods, applications, webinars, eSeminars, videos, tutorials for users of liquid chromatography, gas chromatography, mass spectrometry, sample preparation and related analytical techniques.

Subscribe to our eNewsletter with daily, weekly or monthly updates: Food, Environmental, (Bio)Pharmaceutical, Bioclinical, Liquid Chromatography, Gas Chromatography and Mass Spectrometry.

Liquid Chromatography

Gas Chromatography

Mass Spectrometry