The Essential CHROMacademy Guide Mobile Phase Optimization Strategies for Reversed Phase HPLC
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Speakers Kevin Schug HPLC Dept. Dean CHROMacademy Tony Taylor Technical Director CHROMacademy Moderator Dave Walsh Editor In Chief LCGC Magazine
Mobile Phase Optimization Strategies for Reversed Phase HPLC
Aims & Objectives 1. Review of Reversed Phase Retention Mechanisms 2. Methanol or Acetonitrile - which one is best? 3. Eluotropic Strength - quick ways to reach the ideal! 4. Changing Solvent - useful tools and approaches 5. Mobile phase ph - understand the effects 6. Optimising ph vs. retention 7. Which buffer - what strength? 8. Strategies for when all else fails
Reversed Phase Retention Mechanisms 1. Mobile Phase - mixture of organic and aqueous solvents 2. Stationary phase - hydrophobic moiety chemically bonded to silica 3. Mobile phase MORE POLAR than Stationary Phase 4. Analyte partitions between the two phases depending upon its chemistry (hydrophobicity) 5. Increasing the % organic in the mobile phase increases the elution power of the mobile phase 6. Retention and Selectivity are altered by changing the chemistry of the stationary phase mobile phase and the temperature
Controlling Retention and Selectivity Retention and Selectivity are altered by changing: Stationary Phase - chain length and chemistry, inclusion of polar moieties, exposure of silanol surface, polar end capping reagents Mobile Phase - organic solvent type, % organic, ph, buffers and other additives Temperature - especially with ionisable analytes
Solvents for Reversed Phase HPLC (I) 1. Acetonitrile has lower viscosity - reduces back pressure and often results in slightly better peak shape 2. Acetonitrile has lower UV cut-off - advantage for UV detection 3. Methanol is less expensive and less toxic 4. Methanol is more polar - reducing the risks of solid buffer precipitation
Solvents for Reversed Phase HPLC (II) 1. Acetonitrile forms binary mixtures with water 2. Methanol is protic and can undergo polar polar / ionic interactions with solutes 3. Usually results in better selectivity for more polar compounds - at the expense of longer run times and increased peak asymmetry 4. Acetonitrile shows good wetting properties when low % B gradients are required - better early peak retention time reproducibility MeOH MeCN
Effect of Organic Modifier on Retention Neutral HPLC Test Compound Mixtures - Selectivity also changes
Selecting Optimum Eluotropic Strength (1) 1. Carry out separation at high %B (80%) 2. This saves time vs. starting at low ph 3. Reduce by 5-10% B in steps to assess retention 4. Look out for changes in selectivity 5. Really only works for neutral compounds 6. Ionisable species need to employ ph control
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Scouting Gradient Methods If Dt g < 0.25 t G then isocratic analysis is possible! Isocratic composition: tr(avg) = ti + tf /2 tr(avg) = 12.8 + 21.2 / 2 tr(avg) = 17.0 mins. At 17.0 mins. eluent composition = 80% B (Note: account for dwell volume!) That s the BEST ESTIMATE isocratic composition for this separation We will discuss the ph of the mobile phase and choice of buffer shortly
Simulation to Optimise %B 1. Use modelling for predicting separation 2. This DryLab model was achieved using 2 injections at 30% B and 50% B Mixture of 6 Neutral Compounds
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Altering Selectivity 1. What if separation isn t achieved with the OPTIMUM isocratic composition from the scouting gradient? 2. Adjust %B either side of optimum by 5-10% B 3. Use an alternative solvent to adjust selectivity
Iso-Eluotropic Mobile Phases 1. Iso-eluotropic - same elution power 2. Iso-eluotropic solvents elute analytes in the same time frame with different selectivity 3. Can use an iso-eluogram (nomogram) to find iso-eluotropic solvent compositions
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Mobile phase ph effects 1. ph reflects the hydrogen ion (or hydroxonium ion) concentration in solution 2. Adding acid (proton donor), increases the hydrogen ion concentration (lowers ph) of the solution 3. Adding base (proton acceptor), lowers the hydrogen ion concentration (increases ph) of the solution
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Extent of Analyte Ionisation 1. Extent of analyte ionisation changes with mobile phase ph 2. Le Chateliers principle applies to the equilibrium when adding acidic or basic species to the mobile phase 3. Ionised form is more polar - less well retained under reversed phase conditions 4. Non-ionised (ion-suppressed form) is less polar - retained longer under reversed phase conditions pka = 50% Ionised (ph 4.6)
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Retention Control using Mobile Phase ph
Optimising Separations using ph What ph? Nicotine pka? Robustness?
Simulation to Optimise ph 1. Use modelling for predicting separation 2. This DryLab model was achieved using 5 runs injections at: ph 2.9 / 3.0 / 3.5 / 5.0 / 6.5 Mixture of Acidic and Neutral Analytes
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Buffers for Reversed Phase HPLC (I) 1. A buffer - an aqueous solution of a mixture of a weak acid and its conjugate base or a weak base and its conjugate acid. ph of a buffered solution changes very little when a small amount of strong acid or base is added to it. 2. Where on the HPLC system do we expect ph to change? 3. Buffer capacity is critical - work within 1pH unit of the buffer pka!! 20% of maximum buffer capacity is critical!
Buffers for Reversed Phase HPLC (II) 4. 25-50mM is a good starting point for buffer solution concentration 5. Chose appropriate conjugate acid or base! 6. Always buffer the aqueous component of the mobile phase separately 7. ph of the mobile phase and pka of analyte will change in the presence of organic solvents - consistency is the key!
Buffers for Reversed Phase HPLC (III) 8. Increase buffer concentration or capacity if peak shape is poor - note this may affect selectivity! 9. Consider UV-Cutoff 10. TEA and TFA degrade and their UV cut-off increases 11. Citrate buffers corrode stainless steel 12. Solubility of salts NH 4 < K< Na
Effects of Buffer Concentration on Retention 1. Buffers change the polarity and ionic strength of the mobile phase 2. Doesn t usually effect analyte retention except where secondary effects are taking place with ionisable analytes (i.e. Silanol interactions) 3. Beware that some species used as buffers (i.e. TFA) are excellent ion-pairing agents and can drastically alter analyte retention times at the wrong concentration!
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Working at Low ph with Basic Analytes! Amphetamine peak 7 Amphetamine peak 7 ph 2.5 ph 7.0
Using a sacrificial base 1. Triethylamine (TEA) and Dioctylamine (DOA) can be used to end cap the column on the fly 2. Improves peak shape 3. Can be used in conjunction with other buffers at lower concentrations (0.1%)
Using Ion Pair Reagents 1. How do we control retention with amphoteric moleculeswhich cannot be rendered neutral using ph control? 2. Some modern reversed phase HPLC column chemistries are suitable 3. More usual solution is to use ion pairing reagents 4. Ionic / Hydrophobic reagents (e.g. Sodium Dodecyl Sulphonate) are used to pair with the analyte ionised moiety in solution
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Using Ion Pair Reagents 1. Actual mechanism is a mixture of ion-pairing and ion-exchange 2. Ion Pair Concentration is important and has an optimum concentration 3. More on Ion Pairing Chromatography in a future Essential Guide!
When all else fails!!
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