Chromatography:
                       Practical Aspects

Forms of PLANAR chromatography (see Section 3), usually involving a PARTITION mechanism in which the stationary phase is polar (eg water) and mobile phase is non-polar (eg butanol) (see 3.4.2).
This type of retention is termed a conventional or FORWARD PHASE system.
During chromatography must keep atmosphere around paper or plate sealed and saturated with solvent vapour - evaporation of the more volatile mobile phase will distort chromatogram.

Advantages of TLC vs paper chromatography

• QUICKER (solvent equilibrates and moves faster
• Can use strongly acidic or oxidative reagents in detection --> MORE SENSITIVE
• MORE ADAPTABLE to wide variety of samples by using different support matrix (silica gel, alumina, calcium sulphate etc)

2-Dimensional Chromatography
Resolve complex mixtures of constituents by 2-stage chromatography (2 solvent systems used ; plate turned through 90° in between).

Gives a very distinctive 'map' of particular mixture applied - identify, eg, blend of drugs.

Modify using other separation method, eg  electrophoresis + chromatography ---> peptide map after trypsin digestion to ID protein.

Liquid Chromatography (LC)

LC refers to use of a column containing a stationary phase through which a liquid mobile phase is passed.

Minimise peak broadening -> better resolution

Peaks broaden by DIFFUSION (effect increases with time), HYDRODYNAMIC EFFECTS (EDDYING) effect (increases with faster flow), SLOW EQUILIBRATION OR MASS TRANSFER between phases (effect increases with faster flow), or because APPARENTLY HOMOGENEOUS SAMPLES BEGIN TO RESOLVE (eg yellow dextran on Sephadex G-100).

In PARTITION and (especially) SIZE-EXCLUSION systems - minimise peak broadening by applying sample in minimum possible volume.

In ADSORPTION, ION-EXCHANGE and AFFINITY systems (& sometimes partition) - can "concentrate" sample at top of column by applying in strong retention solvent. Then increase eluting power of mobile phase. Eluate gradients can minimise broadening (trailing edge subject to stronger eluant).

"Dead volumes" give rise to mixing; always try to minimise.

High Performance Liquid Chromatography (HPLC)

Overcomes most peak-broadening effects in LC associated with fast flow by using finely divided stationary phase particles (less eddying, faster mass transfer), and relatively pulse-free pumping systems.
Transforms LC into much FASTER technique --> very wide applications for almost all types of biochemical and environmental analytes.
Pump might have to deliver high pressure with some column types (original abbreviation HPLC).
Wide variety of stationary phases, allow separations based on any of the retention mechanisms (least suitable for size-exclusion using conventional gels, but still applicable using materials that resist compression and clogging)

HPLC Instrumentation

Basic HPLC system

A,B,C = De-gassed solvents in reservoirs D = Mixing valve/gradient former E = Pump F = Pulse damper G = Injection valve H = Column I = Flow cell J = Detector K=Recorder/integrator/computer

Importance of introducing sample in small volume without undue spreading by diffusion
---> design of special sample injection loop

HPLC SAMPLE INJECTION

HPLC Detectors and Sample Detection

Detectors can make use of spectroscopic properties of the sample molecules, eg UV or visible absorption (nucleotides, flavins), or fluorimetric detection.

Sometimes DERIVATISATION used first to convert analyte molecules into spectroscopically detectable form, eg amino acids may be subjected to DANSYLATION (reaction with diethylaminonaphthalene-5-sulphonyl chloride) --> fluorescent dansyl-amino acids (high sensitivity detection).

If no spectral properties, electrochemical detection usually by voltammetric principle, might be possible for oxidisable or reducible species.

If all else fails consider refractometric detection . All solutes increase refractive index of solvent. Need rigorous temperature control, no gradients, pulse-free pumping.

Reverse Phase HPLC

Conventional forward phase partition systems (see 4.1) suitable for many HPLC separations.

But many samples separate better by making stationary phase non-polar and mobile phase more polar = reverse phase partition mechanism.
Supporting matrixes used in HPLC columns are intrinsically polar eg silica gel, alumina.
For REVERSE PHASE HPLC, HYDROPHOBIC GROUPS HAVE TO BE CHEMICALLY BONDED ON TO MATRIX.
octadecylsilane(ODS) group widely used - C₁₈H₃₇ chain gives non-polar (hydrophobic) interaction with sample molecules and eluants.
Note: chemically bonded stationary phases also used for many forward phase separations (use attached POLAR GROUPS (eg cyano, amino)
In reverse-phase chromatography, polar sample molecules interact least with stationary phase --> eluted first.
Non-polar - retained by stationary phase --> eluted later.

Comparison of Forward and Reverse Phase HPLC

Ion-pairing Reagents allow reverse-phase HPLC to be applied to polar analytes.
eg (Sample)^- + (C₄H₉)₄N⁺.HPO₄^- ¤ (Sample)^{- +} (N(C₄H₉)₄) <-- hydrophobic ion pair product, retained by ODS.

Gas Chromatography

Mobile phase is a gas, called the CARRIER GAS, usually nitrogen, sometimes hydrogen or helium.

The stationary phase on the column can be a solid, in which case retention is by adsorption.

In GLC, liquid stationary phase is of HIGH BOILING POINT and GOOD THERMAL STABILITY - to withstand prolonged T > 200° without evaporation or decomposition.
Stationary phase is held as a coating on inert finely powdered solid support. Allows carrier gas to percolate through freely, while exposing large surface area of liquid for equilibration (see 3.4.1)

Instrumentation for GLC

Basic GLC setup

A=Carrier gas supply B=Injection oven C=Injection syringe D=Column oven and column E=Oven controls F=Detector oven & vent G=H2 & air supply for detector (FID) H=Detector electrometer I=Recorder/integrator/computer

Oven Unit in three parts:

• Injector Oven: Temperature high enough to volatilise all components of sample. Picked up by carrier gas ---> column.
  Samples (Usually dissolved in volatile liquid) are injected using a 1-10 microlitre syringe. Needle penetrates silicon rubber septum that re-seals when withdrawn.
  Gas samples (eg atmospheric contamination test) require larger injection volume.
  
• Column Oven: May be held at constant temperature for isothermal chromatography. More commonly temperature caused to increase at a programmed rate = temperature programmed chromatography.
  
• Detector Oven maintains sample in gas phase at suitable temperature for operation of detector.

Temperature Programming in GLC

Temperature programming, like use of a gradient in LC, enhances peak sharpness (trailing edge of peak encounters higher T ---> favours partition into mobile phase), and hence resolution. Shorter run time as temperature increase during run moves each component in turn through column.
Typical comparison shown below:

Linear temperature programs are used most, but more complicated variations possible.

Detectors for GC

Thermal Conductivity Detector (TCD)
Heated filament balances electrical circuit.
Vapour-phase component eluted from column transfers heat from filament --> change in temperature --> change in conductivity of wire --> circuit imbalance. Not very sensitive (~10^-7 mole)

Flame Ionization Detector (FID)

Sample burns in flame --> ion current More sensitive (~10-9 mole). Can't recover sample

Electron Capture Detector (ECD)
Ionization of carrier gas by radioactive [beta] source. Eluted species capture [beta] emission (electrons).

GC Columns

Packed Columns
As described in 4.4. Column packed with finely powdered inert support particles. Stationary phase coats surface of particles.
Original form of GLC, still used for 75%+ of applications.

Capillary Columns
Superior resolution but have very small sample capacity. Two main types:

Low capacity may be advantage for biological samples. If difficult to inject very small volume, use pre-column splitter so most of sample will bypass column.

Post-column splitter in GC - allows part sample to be quantified by detector, part to go to preparative vial or to MS for identification.

Sample Derivatisation for GC

Sample for GC must be in vapour form at column temperature (usually < 200°C), and stable at that temperature.
Most important biochemical species are non-volatile because of polar groups ---> dipolar interactions between molecules.
Hence derivatisation for GC = chemical modification to block polar groups with a stable covalent link ---> more volatile species.