Developing HPLC Methods

Column Screening: The Ascentis & Discovery Method Development “Tool Kit”
Choosing an Ascentis or Discovery Phase
Selecting the Right Buffer
Guidelines for Preparing Mobile Phases

Column Screening: The Ascentis & Discovery Method Development back to top
“Tool Kit”  

We recommend every HPLC method developer have these four columns in their arsenal.

  • Ascentis C18 – Classic C18 selectivity will achieve most reversed-phase separations
  • Ascentis RP-Amide – For enhanced retention and performance of polar compounds, especially bases (uncharged) and compounds with H-bond potential
  • Ascentis Phenyl – For enhanced retention and performance of polar compounds, especially ring systems, dipoles, and nitroaromatics
  • Discovery HS F5 – For enhanced retention and performance of polar compounds, especially bases (charged) and when the sample contains a mixture of non-polar and polar compounds

Simply screen these four columns in your desired mobile phase, using your preferred column.

Choosing an Ascentis or Discovery Phase Based on Compound Class back to top
and Separation Challenge or Objective  

Typically, Ascentis C18 is the first choice for starting a new method. However, when a C18 doesn’t give the desired separation or your sample contains compounds that are known to be difficult to retain or resolve on a C18, consider changing the stationary phase. The range of selectivity provided by Ascentis and Discovery phases makes this easy. The flow chart below helps guide the selection of Ascentis or Discovery phase based on the particular compound type or separation challenge.

The column selection guide below gives recommendations for improving retention or resolution, based on compound class and separation challenge on C18.
Analyte Type Observation on C18   Recommendation
Non-Polar Compounds Too Much Retention Ascentis C8
Polar & Non-Polar
Poor Peak Spacing Discovery HS F5
Polar Compounds Retention Too Short or Inadequate Separation Basic Compounds Charged
Uncharged     Ascentis RP-Amide
H-Bonding Phenolic, Acidic or Hydrated Compounds
π Electron-Acceptors Ascentis Phenyl
Nitro-Aromatics, Hetero-aromatics
Strong Dipoles
π Electron-Donors Discovery HS F5
Poor Peak Shape Basic Compounds Ascentis RP-Amide
Very Polar Compounds Not Enough RP Retention, LC-MS (HILIC Mode) Ascentis Siica
Geometric Isomers Not Enough Separation (Normal Phase)
(Reversed-Phase) Discovery HS F5

Selecting the Right Buffer back to top

A partial list of common buffers and their corresponding useful pH range is supplied. Perhaps the most common buffer in HPLC is the phosphate ion. Although, with the growth of LC-MS, volatile buffers such as TFA, acetate, formate, and ammonia are becoming more frequently used. Remember, the purpose of a buffer in the mobile phase is to inhibit a pH change in the mobile phase after the introduction of a sample. When developing a method, it is important to select a mobile phase with a final pH at least one pH unit away from any analytes pK value. As a rule of thumb, one should work within a ±1 pH unit of the buffer pKa. Typical buffer concentrations for HPLC tend to be 10-100 millimolar level.

Buffer  pKa @ 25 °C   Useful pH Range 
Trifluoroacetic acid (TFA)   0.5 <1.5
Phosphate 1 2.1 1.1 - 3.1
Formate 3.8 2.8 - 4.8
Acetate 4.8 3.8 - 5.8
Phosphate 2 7.2 6.2 - 8.2
Ammonia 9.2 8.2 - 10.2
Phosphate 3 12.3 11.3 - 13.3

Guidelines for Preparing Mobile Phases back to top


It should be understood that slight variations in pH and buffer concentration could have a dramatic affect on the chromatographic process; consistent and specific techniques should be a regular practice in the preparation of mobile phases. A common practice is to place a sufficient amount of pure water into a volumetric flask and add an accurate amount of buffer. The pH of the solution should be adjusted, if necessary, and then dilute to final volume of water prior to adding or blending of organic solvents. Then, add a volumetrically measured amount of organic solvent to obtain the final mobile phase. Thorough blending, degassing, and filtering prior to use is also recommended.

To view a listing of suitable HPLC and LC-MS additives and solvents, visit sigma-aldrich.com/lc-ms-solvents 

Additional Topics:

Harnessing the Power of Chromatographic Selectivity

Functionalized Stationary Phases for Alternate Selectivity