Exsil PolarP
Very High Polarity Reversed Phase
For Direct Serum Analysis
In line with our drive to introduce novel chromatographic media with extended user benefits, we have developed a new phase containing highly polar reversed phase groups that give the phase distinctive selectivity. Working in a highly aqueous environment, this phase gives unique selectivity compared with conventional reversed phase. Unlike standard C18 phases, the PolarP does not adsorb proteins and this allows the phase to be used for direct plasma analysis.
Features:
- High Polarity/High Purity Reversed Phase
- Unique Selectivity
- Direct Serum Analysis
- Water Wettable
- MS Compatible
- Water Wettable
- Direct Serum Analysis
- Unique Selectivity
High Purity Exsil Pure Base Silica
Exsil PolarP uses a neutral mixture of hydrophobic and hydrophilic groups bonded onto our high purity Exsil Pure silica to generate a highly polar reversed phase. This has three main effects
- It reduces the amount of organic component needed for elution by approximately a third compared with a standard C18 phase.
- Retains small molecules but not proteins allowing for direct analysis of drugs in plasma
- Leads to significant differences in selectivity compared to conventional reversed phases.
Direct Serum Analysis
The PolarP phase does not adsorb proteins under reversed phase conditions making it suitable for the direct analysis of drugs in serum. The low levels of organic needed for elution also help to reduce the risk of protein precipitation in the column.
The retention of basic molecules can be controlled by adjustment of pH. For example, the amount of organic needed to elute Amitriptyline increases from 16% at pH 3 to 50% at pH 7.
Amitriptyline was dosed into human Serum Albumin at a level of 0.05% w/w and 5ml samples containing 185mg of Albumin and 100 ng of Amitriptyline were analysed at pH3 on a 3cm column of 5 mm Exsil PolarP. Protein elutes at the void volume with Amitriptyline cleanly resolved
A total of 121 samples were analysed in 38 minutes at a flowrate of 3 ml/min (Equivalent to 190 samples/hour).
The chromatograms show the start and finish chromatograms, in this case run at 1 ml/min, and showed no change in retention, peak shape or backpressure.
This demonstrates the potential of Exsil PolarP for cost effective, very high-speed direct serum analysis.
Selectivity
In addition to the novel behaviour with proteins, Exsil PolarP shows some potentially useful differences in selectivity when compared to standard C18 phases.
The compounds Uracil, Phenol, Diethyltoluamide (DEET) and Toluene elute in that order on a C18 phase but the elution order of the Phenol and DEET are reversed on the PolarP phase.
150 x 4.6 mm Exsil PolarP, 5µm
16.5% MeCN/16.5mM KH2PO4 pH3, 1 ml/min 350C
Uracil, NN Diethyltoluamide, Phenol and Toluene
Peak shape is reasonable at low pH as is shown by the strong bases Psuedoephedrine, Diphenhydramine and Amitriptyline.
150 x 4.6 mm Exsil PolarP 5µm
16,5% MeCN/16.5mM KH2PO4, pH3
1ml/min 35°C Pseudoephidrine, Diphenhydramine and Amitriptyline
Performance at pH 7 with difficult bases is also strong with USP asymmetries typically in the 1.2 region and again at much reduced organic levels (50% rather than 80%).
150 x 4.6 mm Exsil PolarP, 5µm
50% MeOH/50% 25mM K2HPO4, pH7, at 1ml/min 35oC
Procaine, Lidocaine, Diphenhydramine and Amitriptyline
The phase shows increased steric selectivity in methanol compared with a C18 with the geometric isomers of Doxepin being well resolved.
150 x 4.6 mm Exsil PolarP, 5µm
50% MeOH/50% 25mM K2HPO4, pH7, at 1ml/min, 35°C
Doxepin
The hydrocarbons Acenaphthene and Fluorene are also baseline resolved a feat normally only achieved with a polymeric C18 phase. Interestingly, this selectivity is suppressed if the methanol is replaced by acetonitrile.
As a final point, the phase shows low bleed characteristics indicating good LC/MS compatibility.