HPLC Solvents, Acetonitrile and Methanol, Key Differences and Properties

Widely used in RP HPLC method development, Acetonitrile (ACN) and Methanol (MeOH) are the two most common solvents you will use with water or aqueous buffers to develop methods. So, besides the fact that Acetonitrile is well know to have a higher elution strength / capacity than Methanol [*but NOT at high organic concentrations (e.g. 95% Methanol vs 95% ACN) where Methanol has a higher elution strength than Acetonitrile does], what other properties should chromatographer's be aware of? Let's discuss a few that all chromatographers should know.

PREPARATIONS of MIXTURES (A/B):
First, a few comments about the preparation of mobile phase solutions. 

• Only the purely aqueous portion can be correctly adjusted for pH. Do not try and measure or adjust the pH of the organic or organic solution mixture. 

     There are two common methods of preparing binary mixtures (V/V) of mobile phase solutions.

• Method #1 is to fill a volumetric flask with a specific volume of the "A" solution, then fill the flask up to the line with the "B" solution.

• Method #2 is to fill a graduated cylinder (or volumetric flask) with a specified amount of "A" solution; fill a second graduated cylinder (or volumetric flask) with a specified amount of the "B" solution and then mix the contents of both together.

Whichever method you use, please fully document it in your HPLC method so anyone reading it will be able to accurately reproduce it. The two methods described above are both correct in design, but will result in solutions with different properties.

ABSORBANCE of UV LIGHT:
For HPLC grade solvent (*we should always use HPLC or LC-MS grade solutions in HPLC analysis), ACN has the lowest absorbance (~ 190 nm) of the two making it well suited for low UV applications. HPLC grade MeOH has a slightly higher UV cut-off, around 205-210 nm, limiting its use in the very low UV ranges. *Methods which require low UV wavelengths (<230 nm) should not use Methanol as the primary solvent.

SOLVENT SOLUBILITY:
There is a significant difference between ACN and MeOH in their ability to dissolve many types of buffer salts AND samples. These differences may be critical during method development as higher salt concentrations could lead to plugs, clogs or precipation. 

Solubility of the Mobile Phase:

• A common reason for gradient runs to show poor reproducibility or to fail may be associated with running high concentrations of buffer combined with high concentrations of organic solvent. Most aqueous / organic solutions containing salt solutions of less than 10 mM concentration are not likely to precipitate under most gradient conditions (running to a max of 95% organic, not 100%). If high percentages of organic solvent are mixed with more concentrated buffer solutions, then the higher salt concentrations may precipitate out of solution during the analysis (resulting in clogs, leaks, plugs and/or inaccurate results). Be cautious when mixing organic solvents and buffers together for gradient analysis. Make sure the solutions used will stay in solution and be stable at all concentrations used. Also verify that the buffering capacity is still present when high organic concentrations are used (as your buffer will be diluted). *Not sure if the salt will stay in solution? Just mix up a sample at the same concentration for a test. Look at it. Is there any turbidity or particulate visible? You should have your answer.

• Methanol's overall better solubility characteristics (better than ACN) mean that it often does a better job of dissolving most salts (esp NH4, K and Na) at higher concentrations resulting in better performance and less precipitation.

Solubility of the Samples (changes to Peak Shape, Selectivity & Retention):

• A fundamental requirement of liquid chromatography is that the sample fully dissolves in the mobile phase (initial mobile phase). Dissolve the sample in the mobile phase or in a slightly weaker strength solution (not a stronger solution) before analysis. This insures it will be loaded onto the head of the column as a concentrated slug improving peak shape and RSD. If the sample does not fully dissolve in the mobile phase then you are not in fact analyzing the whole sample. Another area where Methanol may be superior to ACN can be found in its ability to fully dissolve more types of samples. This improved solubility may result in better overall peak shape. Methanol also has different selectivity, often better than ACN (not just the elution strength) which may result in peaks eluting at different retention times than expecting. This is another reason why we always try different mobile phase mixtures containing either ACN or MeOH when developing RP methods. Please never assume that one solvent will be better than the other. Too many novice chromatographer's use only ACN as their main organic solvent for method development. Please don't make their mistake as such a strategy indicates a lack of practical experience and knowledge. You must first try them both separately (ACN & MeOH) to evaluate the results with your own sample (best to start with comprehensive gradients at different pH values, as applicable). You will be rewarded for putting in the initial time to test both types of solutions as no simulator has yet been developed which can predict a truly accurate result with your own sample(s). You may be surprised to learn how many samples show better peak shape and performance using MeOH solutions. If no improvement is seen, document it and move forward with more confidence.

BACKPRESSURE & OUTGASSING:

• ACN is less viscous than MeOH ( 0.34 vs. 0.54 viscosity, respectively) and if used alone will result in lower column and system back-pressures overall. Less gas will dissolve into ACN vs MeOH. Mixtures of ACN and Water will also exhibit an endothermic reaction (cooling the solution) which can trap gas inside the solution. If you pre-mix your mobile phase, let it rest for several minutes after preparation.Mixtures of ACN and Water will show a pressure max around 70% ACN (*This is an unusual characteristic well worth learning).
  

• MeOH is more viscous than ACN alone. It also has an unusual property where a 50/50 mixture of MeOH and Water will result in a much higher system and column back pressure than either MeOH or Water alone will (*ACN has a similar property, but the peak pressure occurs between 60-70%). The effect with methanol is very Gaussian with a peak pressure observed with a 50/50 mixture. An exothermic reaction results from an initial mixture of the two solutions (MeOH and Water) releasing some gas. When preparing solutions it is best to allow the solution to rest for a few minutes to out-gas before topping off or using in the HPLC system.

I hope that this short discussion about some of the differences between these two popular HPLC solvents will aid you in developing better quality HPLC and LC-MS methods.

Reference: Table of HPLC Solvent Properties

CHIRAL HPLC / SFC METHOD DEVELOPMENT (Alcohols):

We are experts in chiral HPLC and SFC method development of pharmaceutical samples and have operated a contract separations labs for nearly two decades. We have learned a great deal about developing fast and reliable racemate separation methods, often where other companies have failed. The knowledge we have gained has allowed us to develop over ten thousand new chromatographic methods for our clients. This has made our company the leading expert in the chiral separations field. 

I would like to share a tip with you regarding the use of different alcohols in chiral method development. That tip is to experiment with different alcohols during the method development process (*Please make sure the alcohol is compatible with your column!). Many of the normal and reversed phase chiral columns can be used with some unconventional alcohols to achieve excellent separations. These alcohols are often used isocratically at 100% concentration for HPLC methods and at levels ~ 10 to 20% for many SFC methods. We have had a great deal of success using 100% pure Methanol for HPLC methods on normal phase style chiral columns (though 100% Ethanol is still one of the best alcohols to initially choose). For SFC methods, Methanol, Ethanol and Butanol (plus mixtures of these) are still some of our favorite co-solvents.Note: SFC needs these alcohols to make the compressed CO2 more polar. Since most chiral drug compounds are resolved using Normal Phase chromatography, SFC is still limited in what it can resolve chromatographically vs HPLC. SFC will never replace conventional HPLC as SFC is far more limited in application (restricted in its range of polarity), but SFC is a worthwhile and important technique to supplement HPLC separations. 

• Here is a list of some popular alcohols (HPLC grade) worth using in your chiral method development: 

Methanol; Ethanol;1-Butanol; 2-Propanol; 2-Butanol and Acetonitrile (I know this last one is not an alcohol, but it is often overlooked in chiral method development. It works where other solvent systems fail for both HPLC (100%) and less so for SFC (10% as a co-solvent with Methanol). 

Many of the above liquids can be used at 100% concentration, but others require mixing with Hexane or Heptane to yield lower concentrations of alcohol. *Remember to always consult with the column manufacturer first to determine which solvents are safe to use.

• A note about acids. Weaker is often better in chiral method development. Examples: TFA at 0.01% and Acetic or Formic Acids at 0.1% concentration are often strong enough to ionize most compounds.