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The Essential CHROMacademy Guide

Mobile Phase Optimization Strategies forReversed Phase HPLC

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Speakers

Kevin SchugHPLC Dept. DeanCHROMacademy

Tony TaylorTechnical DirectorCHROMacademy

Moderator

Dave WalshEditor In ChiefLCGC Magazine

Mobile Phase Optimization Strategiesfor Reversed Phase HPLC

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

Aims & Objectives

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

Reversed Phase Retention Mechanisms

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

MobilePhase_01.flv

Scouting Gradient Methods

If Dtg < 0.25 tG 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

MobilePhase_02.flv

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

MobilePhase_03.flv

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

MobilePhase_04.flv

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)

MobilePhase_05.flv

MobilePhase_06.flv

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

MobilePhase_07.flv

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 NH4 < 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. Silanolinteractions)

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!

MobilePhase_08.flv

Working at Low pH with Basic Analytes!

pH 2.5

Amphetamine – peak 7 Amphetamine – peak 7

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 amphotericmoleculeswhich 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 DodecylSulphonate) are used to ‘pair’ with the analyte ionised moiety in solution

MobilePhase_09.flv

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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