Solving the chromatography solvent problem

Wouldn’t life be so simple if you could perform your flash chromatography and prep HPLC runs with just one type of solvent? Perhaps it would be simple, but boy, it would also be so…limiting. Still, I feel your pain of having to select a chromatography solvent that can fit your needs, your sample, your budget. To try to alleviate this anguish, I’ve dedicated this post to different solvents used in normal and reversed phase chromatography. I also highlight the solvent properties I find instrumental in achieving good and quick separations. Dig into this chromatography solvent catalogue and start re-solving already!

Our fridge let out its dying breath a few days ago and my wife and I just purchased a new one. I don’t know how your fridge looks like at home, but ours is covered with little notes, art from family and friends, important letters and so on and so on. I had the lovely task of removing all these papers and attaching them to the new fridge. In the process, I realized some of our magnets are really strong and stick like crazy to the fridge door, others are super weak and although I could easily remove them, they could barely hold a piece of paper to the fridge.

Would you be surprised if I told you that this whole experience made me think of chromatography?

Let me explain myself. Take mobile phases. Solvents, just like magnets, have different strengths that make them suitable for different applications. Strength is by far a very important factor. But just like size, shape, price or color might additionally influence your choice of magnet, solvents also have other characteristics we need to examine before deciding which one we want to pick for use in our chromatography system.

What are some of these properties? I would certainly advise you to look at the following:

  • Strength, including polarity, dipolar character, and the ability to form hydrogen bonds
  • UV cut-off values
  • Viscosity
  • Costs

Let us examine some examples of chromatography solvents and their characteristics. In this way, I Hope to show you why I think these factors are the most important for you to obtain a good separation.

Reversed phase chromatography

The most used reversed phase mobile phases are mixtures of water with ethanol, methanol, acetonitrile or tetrahydrofuran. Each chromatography solvent has its own properties:

SolventSolvent StrengthDipole CharacterProton AcceptorProton DonatorUV limit (nm)Costs
H2O0LargeLargeLarge190Low
MeOH++57.57.5210Low
ACN+++82.50200High
EtOH++++455210High
THF++++++430220High

Let me try to summarize what this table is trying to tell you.

  • Water – weakest reversed phase chromatography solvent due to its strong dipole character and its ability to form hydrogen bonds
  • Methanol – relatively weak solvent, due to its polar character and its ability to form hydrogen bonds with the sample and other methanol molecules
  • Acetonitrile – strong organic solvent with highest dipole character; can only form hydrogen bonds with other sample molecules, but not with itself
  • Ethanol – strong organic solvent due to its polar character and ability to form hydrogen bonds with the sample and other ethanol molecules
  • Tetrahydrofuran – very strong organic solvent due to its low dipole character and its ability to form hydrogen bonds with the sample only

Now, another property you should consider is the UV cut-off value of your chromatography solvent. This is important, because you want to prevent the masking of the UV signal of your sample by the solvent signal.

Regarding costs, water and methanol are the cheaper solvents, but they are relatively weak. Ethanol, acetonitrile and tetrahydrofuran are more expensive, but they are also stronger solvents.

A former colleague of mine recently ran a chromatography experiment to demonstrate a reversed chromatography run with several of these solvents. He tried to separate theobromine, theophylline and caffeine. When he used a C18 AQ prep HPLC column with tetrahydrofuran, he was unable to separate the three compounds, due to the strong solvent strength. It’s a bit like having a magnet that is so strong, that it is actually no good, because you can’t remove the magnet from the fridge to replace the picture below it.

When my colleague tried a C18 AQ column with water and methanol, he achieved a good separation in 7 minutes. When he substituted the methanol for ethanol, he reduced the runtime to less than 6 minutes. And when he used acetonitrile, he could separate the three molecules in less than 5 minutes.

Considering that time is money, how quickly your perform your separation might also play a role in your decision, not just the initial cost of the chromatography solvent.

We’ve now discussed all the properties I find important in selecting a chromatography solvent, except viscosity. So, let’s get into it.

Viscosity describes the solvent’s resistance to flow. Solvents with low viscosity are favored as they minimize the back pressure of the cartridge. If you are running a gradient, be aware that as the amount of organic solvent increases overtime, the viscosity of the mobile phase, as well as the back pressure will also increase.

To sidestep the problem of exceeding the maximal pressure of your cartridge or column , you could try using a larger column or cartridge at optimal flow rate.

Normal phase chromatography

Normal phase solvents that are frequently used include n-heptane, n-hexane, dichloromethane (DCM), ethyl acetate and methanol:

SolventSolvent StrengthCostsProton Acceptor
n-heptane0.1High200
n-hexane0.1High200
DCM3.1Medium295
ethyl acetate4.4Low260
MeOH5.1Medium210
H2O10.2Low180

Let me give you a quick run-down on each chromatography solvent as well.

  • n-heptane – very weak solvent strength; has nonpolar properties, has no dipole character and no hydrogen bonding ability
  • n-hexane – only slightly stronger than n-heptane; also has no dipole character, is nonpolar and has no hydrogen bonding ability
  • DCM – moderate strong solvent due to its high dipole character
  • Ethyl acetate – strong organic solvent due to its polar character and ability to form hydrogen bonds with the sample, but not with itself
  • Methanol – very strong organic solvent due to its polar character and ability to form hydrogen bonds with the sample and other methanol molecules

Here, I would like to point out that very often gradients with n-heptane or n-hexane together with ethyl acetate or DCM are used in normal phase chromatography.

Regarding costs, a weak chromatography solvent, such as n-heptane or n-hexane is more expensive, and the costs decrease as chromatography solvent strength increases.

Here is something important for you.

Normal phase solvents have substantially higher UV limits, so you should take these values into account to make sure your sample is not masked by your solvent.

And here is something rather nice. In contrast to reversed chromatography , the pressure generated during normal phase chromatography is not a problem since the viscosity of this type of chromatography solvent is very low.

The colleague I already mentioned tested out several solvents in a normal phase chromatography run too. Specifically, he attempted to separate toluene, octyl phthalate and methyl phthalate in an isocratic run. He found out that a mixture of n-heptane and ethyl acetate offers a good separation in 7.5 minutes. When he substituted n-heptane for n-hexane, he achieved a separation in 4.5 minutes due to the higher solvent strength of n-heptane.

However, he noted that there was poorer separation between toluene and octal phthalate. Just like with tetrahydrofuran in reversed phase chromatography, he could not separate the compounds in a mixture of DCM and methanol, here because of the high polarity of the chromatography solvents.

If you want to see how these runs played out in real life, check out this video:
https://www.youtube.com/watch?v=ohMiSl7VgJc

If this is not enough for you, check out another post I’ve done on selectivity and how to choose the ideal mobile phase as well as a post on how to optimize resolution. And if you would like some more information on selecting mobile phases, stationary phases, selectivity and improving overall resolution, either watch a webinar on chromatography basics, or download a comprehensive chromatography guidebook to enrich your knowledge and solve even more problems…preferably before they even arise.

Till next time,

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