Investigating the Effects of Chromatographic Parameters on Column Equilibration in Isocratic and Gradient HILIC Separations
? Hydrophilic Interaction Liquid Chromatography (HILIC) is a powerful technique for the separation of hydrophilic and polar compounds.
? HILIC utilises a polar stationary phase and high-organic containing mobile phase.
? The HILIC retention mechanism is complex and includes electrostatic, polar and partitioning interactions.
? The key to HILIC retention is formation of a water-enriched layer at the particle surface.
? Column equilibration to form a stable water-enriched layer is vitally important for reproducible chromatography.
? Long and variable equilibration times are often cited as a negative aspect of the HILIC technique.
? This work aims to assess key isocratic and gradient method parameters and how they affect column equilibration.
? The three ACE HILIC phases (100 x 3.0 mm, 3 μm) were assessed in this study:
? HILIC-A (An acidic stationary phase showing high cation exchange capability)
? HILIC-B (A basic character phase with reasonable anion exchange capacity)
? HILIC-N (A neutral bonded phase with low anion and cation exchange capabilities)
? Isocratic equilibration: The following parameters were assessed:
|Fresh vs used column||Freshly packed column vs previously used column|
|Mobile phase ionic strength||5, 10 and 20 mM ammonium formate pH 3.0|
|Percentage water in the mobile phase||6, 10, 15 and 20 % water|
? Gradient equilibration: The effect of post gradient re-equilibration time was investigated (re-equilibration times equivalent to 10, 20, 30 and 200 column volumes).
For all experiments, a test mix containing 4-hydroxybenzoic acid (acid), salbutamol (base) and 2’-deoxyguanosine (polar neutral) was injected.
3. Fresh vs Used Columns
? Freshly packed columns were equilibrated with mobile phase for 400 column volumes. Sample injected every 10 mins.
? The buffer was removed from the column (MeCN:H2O 1:1) and stored for 48 h in IPA.
? Columns were then re-equilibrated with mobile phase for 400 column volumes and sample injected every 10 mins.
? Used columns were found to equilibrate faster than freshly packed columns, potentially due to the presence of residual non-aqueous packing solvents in pores of freshly packed columns.
? Equilibration times for used ACE HILIC columns were similar to the ACE C18.
? Under these conditions, fresh bonded phases appear to equilibrate faster than bare silica.
Mobile phase: 10 mM Ammonium formate pH 3.0 in MeCN/H2O 90:10 (v/v)
Flow rate: 0.6 mL/min Temp: 35 °C, Detector: 214 nm Injection volume: 5 μL
4. Ionic Strength
? All three phases were equilibrated with 5, 10 and 20 mM ammonium formate pH 3.0 in MeCN:H2O 9:1 (v/v) for 400 column volumes. Sample injected every 10 mins.
? All three phases were >95% equilibrated after 20 column volumes with 5 and 10 mM buffer concentrations.
? The 20 mM ionic strength mobile phase required slightly longer to equilibrate (up to 50 column volumes on the HILIC-A).
Mobile phase: 5, 10 or 20 mM Ammonium formate pH 3.0 in MeCN/H2O 90:10 (v/v)
Flow rate: 0.6 mL/min Temp: 35 °C
Detector: 214 nm Injection volume: 5 μL
5. Percentage Organic
? All three ACE phases were equilibrated with 10 mM ammonium formate pH 3.0 in MeCN:H2O at different volume fractions for 400 column volumes. Sample injected every 10 mins.
? 20 column volumes was sufficient to equilibrate (>95%) all ACE phases under most conditions.
? 94% MeCN took >100 column volumes to equilibrate on the ACE HILIC-B and HILIC-N.
? This implies that formation of a stable water layer takes longer when using low-aqueous containing mobile phases in HILIC mode for bonded phases, possibly due to the presence of a more substantial water-enriched region at the particle surface.
6. Isocratic: Observations
? Similar trends were observed for acidic(4-hydroybenzoic acid), neutral (2’-deoxyguanosine) and basic (salbutamol) analytes.
? Stable retention was achieved on all ACE phases once equilibrated.
? Fresh columns took longer to equilibrate than used columns
? Minimum 20 column volumes for used columns
? Minimum 60-80 column volumes for fresh columns
? Higher ionic strength mobile phases may require longer to equilibrate.
? Formation of a stable water-enriched layer may be substantially slower when using mobile phases containing ~5-6% water.
7. Gradient Equilibration
? A HILIC gradient was run on each phase and the post-gradient equilibration time varied:
? 10, 20, 30 and 200 column volumes
? 1 column volume = 0.75 minutes (100 x 3.0 mm column, 0.6 mL/min)
? 10 injections performed, 1st injection discarded
? The length of the re-equilibration stage was found to affect analyte retention.
8. Gradient Equilibration
? Replicate injections (n=9) for each re-equilibration experiment showed excellent reproducibility (%RSD typically <0.3).
? This implies that re-equilibration times of 10-20 column volumes should be generally applicable to obtain reproducible chromatography.
? It is essential to accurately control gradient re-equilibration times in HILIC mode. Care should also be taken to account for differing system dwell volumes.
? Important to carry out a blank run before injecting or discard the first injection.
? It is essential to fully and consistently equilibrate HILIC columns to obtain reproducible chromatography.
? With adequate equilibration, ACE HILIC columns show similar reproducibility to reversed-phase columns.
? Fresh columns should be equilibrated for at least 60-80 column volumes before injecting.
? Equilibration times for used columns are shorter, 20 column volumes is usually sufficient.
? Under certain conditions, column equilibration may take longer than 20 column volumes.
? High buffer concentrations
? Low percentage of water in the mobile phase
? For HILIC gradients, varying the post-gradient re-equilibration time affects analyte retention and potentially selectivity. It is crucial to accurately control and record re-equilibration times.
? When transferring HILIC gradients, instrument dwell times may need compensating for to maintain consistent re-equilibration times.