Translator for HPLC HINTS and TIPS for Chromatographers

Saturday, September 2, 2017

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 to develop methods. So, besides the fact that Acetonitrile is well know to have a higher elution capacity than Methanol, what other properties should chromatographer's be aware of? Let's discuss a few that all chromatographers should know.

First, a few comments about 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 mixture. 
There are two common methods of preparing V/V 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 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.

For HPLC grade solvent (*we should always use HPLC grade solutions in HPLC analysis) ACN has the lowest absorbance (~ 190 nm) of the two making it well suited for low UV analysis. MeOH has a higher UV cut-off around 205-210 nm slightly limiting its use in the very low UV ranges.

There is a significant difference between ACN and MeOH in their ability to dissolve many types of buffer salts AND samples. These differences are critical in method development. 

Solubility of the Mobile Phase:
  • A common reason for gradient runs to show poor reproducibility or to fail can be associated with running high concentrations of buffer with high concentrations of organic solution. While 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 98% organic, not 100%), most buffer solution used with HPLC applications will have higher salt concentrations which may precipitate out of solution (resulting in clogs, leaks, plugs and inaccurate results) when the analysis conditions contains high percentages of organic solvent. Be cautious when selecting organic composition values in RP methods. Make sure the solutions used will be stable under all concentrations used. Also verify that the buffering capacity is still present when high organic concentrations are used (as your buffer is 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 have your answer.
  • Methanol's overall better solubility characteristics (better than ACN) mean that it 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 (effect on Peak Shape & 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 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. 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 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, well at least you now know this because you tried it and can move forward with confidence.

  • ACN is less viscous than MeOH so will usually result in lower column and system back-pressures overall. 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.
  • 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. The effect is very Gaussian with a peak pressure observed with a 50/50 mixture. An exothermic reaction also results from an initial mixture of the two solutions giving off some gas. When preparing solutions it is best to allow the solution to rest for a few minutes to out-gass 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

Saturday, July 8, 2017


When selecting standards for use in testing an HPLC column OR for evaluation of an HPLC system’s performance (e.g. System Suitability, Performance Verification, Retention), in addition to selecting special high purity chemical compounds, consider using the actual sample which is specific to the method or application. Characterize the sample’s retention, peak shape and spectra (if applicable) and use the data to measure, compare and detect changes in  performance over time. 

Running Sugars on an Amino Column? Use simple and complex sugars as standards. Example: D-Fructose; D-Glucose; Lactose; Maltose; Sucrose.

NP (prepare the test solution in a mixture of Hexane/Ethanol, as appropriate)
Diethyl phthalate; Dimethyl phthalate; Toluene; Benzene.

RP (prepare a test solution in ACN/Water or Methanol/Water, as applicable)
Select 4 or 5 of these compounds for use in a mix. Uracil; Benzene, Acetophenone; Toluene; Naphthalene; N,N-Diethyl-m-toluamide; Phenol;  diethyl phthalate; diamyl phthalate; di-n-hexyl phthalate; dioctyl phthalate.

Include a Void Marker in your Test Solution:
Always measure the actual void volume of your specific HPLC column with a compound which is un-retained by your column. For RP applications which utilize at least 20% organic, Uracil or Thiourea are often used, but some inorganic salts (e.g. sodium nitrite and sodium nitrate) have also been shown to work as well.

Saturday, May 20, 2017

Chromatography Basics - Coffee Filter Chromatography

This month I would like to promote sharing and learning about chromatography with others. As scientists, sharing and passing on information to others is key to introducing new people to how we use science and the scientific method to explore what things are made of. Examples of chromatography are often seen in every-day life. For example, a spilled liquid drink on a napkin often reveals different colored or toned rings radiating out, hinting at the many compounds present within the one solution.

One of the simplest examples of a paper chromatography method can be set up and safely run at home. A jar with some water serves as the mobile phase and a coffee filter as the "Column" or support. Ordinary ballpoint pen ink (i.e. blue) or a magic marker can provide "samples" to evaluate. If available, various pure alcohols can replace the water and used as mobile phase solution(s) which may improve resolution. 

Over the past decades I have used these simple setups to demonstrate both what the analytical technique of chromatography is and how it works to hundreds of people of all ages and backgrounds. Many of examples can be found on the web and I have included a link below to one of the more recent published methods from Scientific American magazine. Please demonstrate and share it with others. Perhaps in doing so you will unleash the curiosity of someone to delve deeper into understanding what things are made of or the scientific fields?

  Link to an article illustrating how to run some paper liquid chromatography experiments at home. "Chromatography: Be a Color Detective"