Considering the more complex products analytical scientist needs to develop a specific, accurate, robust stability-indicating analytical method. At the time of method development, analytical scientists need to focus on the development of the mass-compatible method. At the start of method development, it is important to have systematic information about the sample.
Selection of Buffers for HPLC:
It is essential to separate all probable impurities from the degradation sample with minimum run time. Samples are categorized as neutral and ionic while ionic includes acid, base, ampholytic and organic salts. The acidic and basic nature of samples requires buffer content mobile phase and the neutral buffer is usually not required. If there is no resolution between 2 closely eluted peaks then need to vary the solvent strength of the mobile phase. Solvent strength and solvent type impact the selectivity of sample peaks. Following are the solvents which control the retention.
| HPLC Method | Solvents |
| Reversed Phase | Water, Methanol, Dimethyl sulfoxide, Acetonitrile |
| Normal Phase | Hexane, Toluene, THF, Ethyl acetate, Propanol, Ethanol |
| Ion Pair | Water, Methanol, Dimethyl sulfoxide, Acetonitrile |
| Ion Exchange | Buffered aqueous solution with salt |
During HPLC method development selection of a proper buffer is important for the separation of peaks and their symmetry. Following are the common different types of organic/inorganic buffers:
- Phosphate Buffers: Potassium phosphate, Di-Potassium phosphate, Monosodium phosphate, Disodium phosphate, Phosphoric acid, etc.
- Acetate Buffers: Ammonium acetate, Sodium acetate, etc. (Phosphate and acetate buffers are most common because they can be used at wavelengths below 220 nm)
- Di-ethyl amine/Tri-ethyl amine buffers
- Ion Pairing reagents buffers like tetra butyl ammonium hydrogen sulfate, Butane sulfonic acid, Pentane sulfonic acid, Hexane sulfonic acid, Heptane sulfonic acids, etc. Ideally 0.0005 M to 0.02 M conc. is recommended with a dedicated HPLC column with a proper cleaning method after every usage to improve the life of the column.
If the buffer concentration of the mobile phase is minimum then the sample may partially ionized and retention time will vary and a distorted peak observed. If the buffer concentration of the mobile phase is higher then there are chances of precipitate formation when it mixes with mobile phase B and the residue is damaging the parts of HPLC. Hence ideally 25 mM buffer concentration is a good proportion for the mobile phase.
Knowing the pKa value of the sample is very much important as the pKa need to adjust the mobile phase pH. The pH of the mobile phase should be adjusted over the range of pKa ± 2.
Selection of Buffers for LC-MS:
In some cases, unknown degradation impurities were observed by the HPLC method which needs to be identified and quantified hence need to transfer the same method to LC-MS or develop the mass-compatible method i.e. it should be volatile. Volatile buffers do not carry residue which deposits on the cone and source. Due to this reason inorganic buffers, like phosphate buffers not suitable for LCMS application. Trifluoroacetic acid offers a good substitute in the case of polar compounds with bad peak shape and poor resolution.
Trifluoro acetic acid (TFA) Buffers are volatile and can be used in LCMS applications. The TFA conc. must be minimum because due to the ion pairing effect of TFA, ionization efficiency is impacted due to part charge masking of the sample. The alternative is to use the different volatile chemical as Formic acid. The alternate method is the addition of Propionic acid, Acetic acid, or Formic acid as 0.1 % to 1 % v/v to the post column. Volatile electrolyte additives were regularly added to LC-MS buffers to improve the peak shape.
Following are the most common buffers used for HPLC and LC-MS:
| Name of Buffer | Range of pH | Compatible to Mass |
| Phosphate: pK-1 | 1.1 – 3.1 | No |
| Phosphate: pK-2 | 6.2 – 8.2 | No |
| Phosphate: pK-3 | 11.3 – 13.3 | No |
| Sodium acetate | 3.8 – 5.8 | No |
| Ammonium acetate (< 50 nM) | 3.8 – 5.8 | Yes |
| Citrate: pK-1 (20 mM) | 2.1 – 4.1 | No |
| Citrate: pK-2 | 3.7 – 5.7 | No |
| Citrate: pK-3 | 4.4 – 6.4 | No |
| Trifluoro acetic acid (0.1 %) | 2.0 | Yes |
| Phosphoric acid (0.1%) | 2.0 | No |
| Formic or Acetic acid (0.01 % to 1 %) | 2.7 | Yes |
| Ammonium formate (< 50 nM) | 2.7 – 4.7 | Yes |
| Ammonium bicarbonate | 6.6 – 8.6 | Yes |
| Borates | 8.3 -10.3 | Yes |
| Bis/Tris Propane | 5.8 – 7.8 | Yes |
| TRIS (tri-hydroxy methyl-amino methane) | 7.3 – 9.3 | Yes |
The selection of buffers for mobile phases is the most important stage of analytical method development. It is most important to know the sample characteristics and importance of buffer selection for the mobile phase which will be used for formulation products analysis. Good laboratory practice must be followed while preparing buffers for mobile phases to ensure the obtained results must be reproducible within and between laboratories.
References:
- L. R. Snyder, J. J. Kirkland, and J. L. Glajch, Practical HPLC Method Development, Wiley-Interscience, New York,
- Wysocki VH, Resing KA, Zhang Q, Cheng G, Mass spectrometry of peptides and Proteins, Methods,
- TL Constantopoulos, GS Jackson, CG Enke, Effects of salt concentration on analyte response using electrospray ionization mass spectrometry, J Am Soc Mass Spectrom,
- A. Cappiello, G. Famiglini, L. Rossi and M. Magnani, Buffers in LC-MS, Anal. Chem,
- A Hutchaleelaha, J Sukbuntherng, HH Chow, M Mayersohn, Practical guidelines of preparing mobile phases in LC-MS/MS, LC/GC,
