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.
