Popular LC/MS and HPLC Volatile Mobile Phase Modifiers

For applications which utilize an Evaporative Light Scattering Detector (E.L.S.D.), Charged Aerosol Detector (CAD) and/or Mass Spectrometer Detector with Electrospray Ionization source (e.g. LC/MS, MSD or LC/MS/MS), a fully volatile buffering system is usually required. Many of the common HPLC buffers such as sodium or potassium phosphate are not compatible.Use the smallest amount of buffer which provides buffering under the analysis conditions (e.g. 10mM). *Select a buffering agent (or modifier) which are within 2 pH units (+/- 1) of the sample's pKa and 2 pH units away from any acid's pKa. 

• For LC/MS applications: Positive ion mode favors acidic mobile phases and Negative ion mode favors basic mobile phases. However, feel free to experiment using both ionization modes and don't forget about using adducts (e.g. ammonium and sodium) with all types of samples to improve signal response. *Maintain these buffers at or below 10 mM. Adjust the pH of the mobile phase to be 1 to 2 units away from your sample's pKa.

Table 1:  Popular examples of useful volatile mobile phase buffers, modifiers and/or additives.

BUFFERING/MODIFIER AGENT                                   USEFUL pH RANGE

• Ammonium formate                                 2.8 - 4.8; 8.2. - 10.2
• Formic Acid                                            3.3 - 4.3
• Pyridine/Formic Acid                               3.3. 4.3, 4.8 - 5.8
• Trimethylamine/Formic Acid                     3.3 - 4.3, 9.3 - 10.3
• Ammonium Acetate                                  3.8 - 5.8; 8.2 - 10.2
• Acetic Acid                                              4.3 - 5.3
• Trimethylamine/Acetic Acid                      4.3 -5.3, 9.3 - 10.3
• Ammonia/Formic Acid                              3.3 - 4.3, 8.8 - 9.8
• Ammonia/Acetic Acid                               4.3 - 5.3, 8.8 - 9.8
• Ammonium Bicarbonate                           5.9 - 6.9,  8.8 - 9.8
• Ammonium Carbonate                              5.9 - 6.9, 8.8 - 9.8  
• Carbonic Acid                                            6 - 8 (pKa 6.37/pKb 7.63)
• 1-Methylpiperidene                                   10.0 - 12.0  

• Trifluoroacetic Acid (TFA)                        pKa = 0.3 (WARNING when used with MS

                                                                                     systems!).  See notes #2 and #4 below.           

*Notes: (1) Formic acid (3.75) is slightly stronger and more volatile than Acetic acid (4.75). Formic acid is often available in higher purity grades and absorbs less in the UV region making it a better choice for most chromatography applications. It works well in positive mode LC/MS analysis, esp at 0.1%. (2) Trifluoroacetic acid (TFA, pKa = 0.3) is very strong and volatile, but we do not recommend its use in LC/MS applications as it can increase the background signal levels (esp. in Negative Mode) LC/MS (m/z 113), be very hard to remove from the source and result in long term instrument contamination. Difluoroacetic acid (DFA) and ammonium formate are other alternatives as they offer good ion pairing capacity with less ion suppression problems. (3) Triethylamine (TEA, pKa 11) is volatile, strong and very stable, but causes similar contamination problems resulting in high background signals when used in Positive Mode LC/MS (m/z 102). (4) Many ion-pairing reagents suppress ionization, bind to the plastics and metals used and contaminate the flow path. If you must use them, please do so using the lowest possible concentrations levels and thoroughly decontaminate the entire flow path of the system after use (or dedicate the MS system to use with them only). Minimize further contamination by labeling and using a dedicated column for the application (Do not use that same column exposed to ion pairing compounds for any other methods or applications). (5) Acids and bases alone provide little "buffering" so should be used with a secondary buffering species to resist change in pH.

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.