Best Broken into four categories

Theoretical Background

Gas Chromatography

HPLC Columns and Interactions

HPLCHigh Performance Liquid Chromatograpy

Use for: Small molecules

o Charged, Neutral, Hydrophillic, Hydrophobic Large molecules

o Synthetic polymers, proteins, carbohydrates Thermolabile molecules

Must be soluble in mobile phase

Not used for: Gasses Very small simple molecules

o Alkanes

HPLC Setup

1

• Mobile Phase Reservoir• Degasser

2• Pump

3

• Injector• Autosampler

4

• Column• In optional oven

5• Detector

6

• Recorder • PC

Mobile Phase Reservoir

Mixture of Different SolventsExamples: Organic, Buffers, Water

Methanol, Ethanol, Acetonitrile, Hexane, DCMBuffers: Phosphate, Acetate, Formate

All solvents must be HPLC quality grade (Extremely pure)

Before use the mobile phase must be Filtered through fine filters to remove any particles from solvents Degassed

o Remove dissolved gas from the solution• Vacuum Degasser• Helium as a degassing gas

1• Mobile Phase Reservoir

• Degasser2• Pump

3• Injector

• Autosampler4

• Column• In optional oven

5• Detector

6• Recorder

• PC

Pumps

Pumps for HPLC need to be: Able to pump at high pressure Very precise control of flow rate

Isocratic ElutionUse of single solvent or mixed solvent of constant compositionGradient ElutionTwo pumps, or one pump and proportioning valve, used to deliver changing mixture of two or more solventsUseful to achieve better separation and shorter elution times by creating mobile phase gradients

1• Mobile Phase Reservoir

• Degasser2• Pump

3• Injector

• Autosampler4

• Column• In optional oven

5• Detector

6• Recorder

• PC

Injectors

Introduces sample to systemDifficult as under high pressure

Direct injection is difficult

Two types of injectors: Manual Auto Sampler

Sample loop fixes the exact quantity of sample to be injected each time

1• Mobile Phase Reservoir

• Degasser2• Pump

3• Injector

• Autosampler4

• Column• In optional oven

5• Detector

6• Recorder

• PC

Column

Contains stationary phase particlesUsually made from stainless steel or plastic

Pre-Columns, Guard Cartridge, used either to: Protect column

o Same stationary phase Pre-separate sample

o Different stationary phase

Once activated:Washed after use, and kept in solvent

1• Mobile Phase Reservoir

• Degasser2• Pump

3• Injector

• Autosampler4

• Column• In optional oven

5• Detector

6• Recorder

• PC

Column

Stationary Phases

Normal Phases NP Polar Stationary Phases

o Silica, Alumina Adsorption Chromatography

Reversed Phases RP Non-polar Stationary Phases Polar Stationary Phases

Bonded silica, C2, C4, C8, C18

Absorption / Partition Chromatography

pH Sensitive (Between pH 2 to 8)Silica is slightly soluble at alkaline pH

1• Mobile Phase Reservoir

• Degasser2• Pump

3• Injector

• Autosampler4

• Column• In optional oven

5• Detector

6• Recorder

• PC

Requirements Column must be equilibrated

before separation process Shorter Narrower tubing

reduces dead volume (extra column volume)

OvensRetention time temperature dependantTwo injections of same sample can be significant in temperature changesOven maintains temperature

Detectors

ExamplesUltraviolet and Visible absorptionDifferential refractive indexElectrochemical: AmperometricElectrochemical: ConductometricFluorescenceMass SpectrometrySolution Light ScatteringEvaporative Light Scattering

1• Mobile Phase Reservoir

• Degasser2• Pump

3• Injector

• Autosampler4

• Column• In optional oven

5• Detector

6• Recorder

• PC

Detectors

UV/VIS DetectorsDiode Array Detector (DAD)

1• Mobile Phase Reservoir

• Degasser2• Pump

3• Injector

• Autosampler4

• Column• In optional oven

5• Detector

6• Recorder

• PC

Detectors

UV/VIS DetectorsPhoto Diode Array Detector

Peak Purity Comparison of spectra

at three different points of peak elution time (start, middle, end)

If identical three spectra identical

1• Mobile Phase Reservoir

• Degasser2• Pump

3• Injector

• Autosampler4

• Column• In optional oven

5• Detector

6• Recorder

• PC

Detectors

Refractive Index Detectors

UniversalBulk Property Good for polymersGood for analytes with no chromophores

1000 times less sensitive than UVUseless in gradient elutionSensitive to temperature and pressureNot good for trace analysis

1• Mobile Phase Reservoir

• Degasser2• Pump

3• Injector

• Autosampler4

• Column• In optional oven

5• Detector

6• Recorder

• PC

Sample PreparationSample must be soluble in mobile phaseOverload of sample may damage columnConcentration of key analytes should be in linear range of detectorQuantity of sample introduced to system depends on sample loop sizeSeparated compounds can be collected and isolated after detection point (if not destroyed)

Separation Process - Parameters

Column LengthLonger Column, Better Separation, Longer Retention TimeParticle Size of Stationary PhaseSmaller Particles, Narrower Peaks, Higher PressuresTemperatureHigher Temperature, Faster Diffusion, Better Separation, Shorter Retention Times, Column DegradesCombination of Mobile PhasesGradient Elution, Best Separation, Shortest TimepH of Mobile PhaseIonised compounds not retained well on RP-Columns

Best Broken into four categories

Theoretical Background

Gas Chromatography

HPLC Columns and Interactions

Effect of Particle SizeSmaller Particles increases Efficiency due to Sharper Peaks!More theoretical plates N, smaller HETP h.Smaller Particles need Higher Pressure to force mobile phase through columnMaximum flow rates are limited by high pressures

Silica SurfaceDominated by Polar Functional Groups

Reversed – Phase SilicaAcid condensation of silica forms RP silicaHydrophobic molecules bonded to silica surfaceSwitches to a non-polar environment

Analyte Interactions

Silica SurfacePolar

NP

Silica SurfaceNonPolar

RP

What do you expect to happen when red line breaks?

Blue solvent: Water

RP vs NP

Like Attract LikeOpposite Phases

Repel

NP vs RP

Like Attracts Like

Solvents with similar functionalities to the analytes will adsorb in the same way and compete for the same interaction sites. This will effect the binding equilibrium

Reversed Phase Silica is more common

Why?Better Peak ShapeLess Tailing due to strong interactionsAqueous content of mobile phase allows bufferingControl analyte ionisation with pHBetter Control of mobile phase gradientsComposition varies more widely

LastFirst

Predicting the Order of Elution

Identify key points of differencePolarityMore Polar = Stronger Interaction in Normal PhaseMore Polar = Weaker Interaction in Reversed Phase

What is eluted first in a Normal Phase Column?Opposite applies for Reversed Phase

OH

OH

O

N

O

OH

O

N

O

O

O

N

O

O

Morphine Codine Heroin

Effect of Stationary Phase

For reverse phase silicaLonger chain lengths result in longer elution times and better resolution

Effect of Mobile PhaseFor reverse phase silica

Increasing the proportion of ‘weak ‘ solvent reduces competition for adsorption – analytes are retained longer and resolved better

Optimising Mobile Phase

If some analytes are insufficiently retained while others are excessively retained, vary strength of mobile phase!

Use of gradients allow peaks at certain intervals to spread or narrow giving good resolution and improve overall run time

Effect of Mobile Phase pH

General RuleAnalytes should be uncharged

Low pH for acidsHigh pH for bases

BUT! Remember silica has limited stability at high and low pH.