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Which Sovent is Better for Reverse Phase HPLC?

Acetonitrile (AcN) or methanol (MeOH)? This argument has been around since the early days of reverse-phase HPLC.

But it became really big during the Great Acetonitrile Shortage of 2008. In that year, the two biggest AcN manufacturers in the world were shut down.

Globally, prices of AcN went right through the stratosphere.

Sales of UHPLC systems dramatically increased.

And ... methanol became the flavor of the month, so to speak. We were obliged to use MeOH in our HPLC methods, with reluctance.

AcN eventually came back, and the debate flared up again.

So. AcN or MeOH?

In reverse-phase HPLC, the mobile phase has water (that is highly polar) and an organic solvent (that is non-polar).

Therefore, the organic solvent, or modifier as it's called, should have certain properties:

- Freely miscible with water

- Low viscosity, to keep the back-pressure down.

- Low UV cutoff. Below 220nm, most organic solvents absorb UV light strongly, making them useless for many HPLC analyses.

Water has a low uv-cutoff of 190nm. The organic modifier must have an equally low UV cut-off.

 -Strong eluting power. Water is a "weak" solvent.

One organic modifier readily meets all these criteria. Acetonitrile.

AcN is freely miscible, has a cut-off of 190 nm, low viscosity, strong dipole moment and it's aprotic, making it a "strong" modifier.

This results in well resolved peaks and fast analyses, especially in gradient mode. AcN is also compatible with many HPLC buffers.

How about MeOH then?

MeOH is more polar than AcN. Making it slightly "weaker".

Also, MeOH has a higher cut-off at 205 nm, and is more viscous. This rules out MeOH for many HPLC analyses.

MeOH does have a saving grace. It provides a different selectivity, and it can be a complementary modifier to AcN. And, MeOH is cheaper to obtain.

But, and this is a big but - MeOH is not the right modifier for prep HPLC, due to its toxicity.

Then how about other organic modifiers? Well ...

- Ethanol. High cut-off at 210 nm. Not as "strong" as AcN. Four times more viscous. And ... much harder to obtain than MeOH.

- Iso-propyl alcohol (IPA). 205 nm cut-off. Almost as "strong" as AcN. But six times more viscous. Creates really high back-pressures. Useful at elevated temperatures, between 40 to 50 deg C.

- n-propyl alcohol (NPA). As good as IPA. 210 nm cut-off. Slightly less viscous. Can be used at 40 to 50 deg C.

- Acetone. Aprotic, like AcN. As "strong" as AcN. Less viscous. Alas, very high cut-off at 330 nm. But, I've successfully used acetone for analyses at visible wavelengths, like carotenoids at 451 nm.

- THF. 212 nm cut-off. Too reactive. Not worth it.

Bottom-line: Against all contenders ... Acetonitrile wins, comfortably.

I'd use MeOH only if AcN were not available, at all.

Or else, I'd go home, and run my HPLC on another day.

Read also: Analytical Feasibility Checklist in Pharmaceutical Industry

Resource Person: SK Srinivas

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