Chromatography

Chromatography is the most important separation method for biomolecules.

The outcome of a chromatography experiment is a CHROMATOGRAM

Column chromatography
 

Planar chromatography
 

Why Chromatography?

• Adaptable to wide range of compounds, because variety of :
    separation principles (retention mechanisms), and
    types of experimental setup (planar or column - gas and liquid phase elution)
  
• Separated analyte is immediately available for identification or quantification
  
• Can be scaled up for preparative use.

  and WHY NOT?

• Some instrumentation expensive and not easily portable
  
• Often need preliminary bench "work-up" of sample to avoid column contamination

Examples of analytical applications

• Drug analysis - therapeutic monitoring or abuse detection
• Vitamins, hormones, specific peptides and proteins
• Environmental pollution, pesticide residues etc

Basic Chromatographic Principles

All chromatographic systems contain:
A stationary phase
A mobile phase
Sample molecules (mixture for separation)
 

Movement of molecules determined by the balance between two forces :   
Impelling force of mobile phase carries with it molecules for which it has affinity -
favoured by solubility (LC), volatility (GC) 

Retarding force of stationary phase holds back molecules with which it interacts

Balance between forces differs for different molecules ---> different MOBILITIES, hence SEPARATION OF COMPONENTS.
NB: Solute molecules not decomposed by chromatographic separation

Retention Mechanisms

Overview of five common retention mechanisms
 

Partition Chromatography

Stationary phase = sorbed solvent
held on the surface, or within the grains or fibres of an inert solid supporting matrix
 

Sample molecules equilibrate (PARTITION) between liquid stationary phase and mobile phase. (Mobile phase is liquid in LC and HPLC systems and gaseous in GLC systems).
Retention depends on a sample molecule's escaping tendency into the mobile phase versus its solubility in the stationary phase.
Quantitatively given by the PARTITION COEFFICIENT, K_D, the ratio of solubilities in the two phases 

Examples of Partition Chromatography Systems

Gas liquid chromatography - see later

Liquid chromatography systems based on partitioning include paper chromatography, and thin-layer chromatography (TLC) on polar matrixes such as silica gel and alumina.

The mobile phase is always a solvent mixture
- one component is polar (eg H₂O) --> taken up by polar matrix to form the stationary phase. Sample molecules soluble in this solvent will be retarded - this component of solvent is the retarder.

- one component is non-polar (eg butanol)
---> travels around stationary phase. Sample molecules soluble in this solvent don't spend time in stationary phase -this component of mixture is the mover.

- sometimes a third component to maintain miscibility and adjust pH (eg acetic acid).

Q. Will polar or non-polar solutes elute faster in this type of system?

Adsorption Chromatography

Solute in liquid (or gas) phase interacts with adsorption sites on solid surface (finely divided particles for maximum surface area).

Suitable solids include HYDROXYAPATITE (Ca₃(PO₄)₂.Ca(OH)₂), ALUMINA (Al₂O₃), MAGNESIUM CARBONATE. Often applied to glass plate for TLC.

Polar groups on solid form dipolar interactions (eg hydrogen bonds) with sample dissolved (usually) in organic solvent.
Elute by increasing polarity of the solvent (eg if using acetonitrile CH₃CN, add methanol (CH₃OH)) --> competing bonds with adsorption sites.
Gradient elution useful (also for ion-exchange and partition chromatography) 

Ion-Exchange Chromatography

 Retention by attraction between groups on stationary phase with opposite charge to sample molecules. Stationary phase = insoluble, but solvent permeable polymer matrix (eg cellulose) chemically modified to introduce ionizable groups (eg -COOH). 

Elute by

• Change of pH to neutralise charged group on either solute or stationary phase.
• Increase [salts] (especially polyvalent) in eluant buffer --> Displace by competing ions.
• pH or salt gradient to enhance separation.

Ion-exchange media are classified according to whether the attached ionizable group is strongly or weakly acidic or basic --> determines the usable pH range
 

Note DEAE-cellulose and CM-cellulose popular for chromatographic separation of proteins - mild, non-denaturing procedure.

Size-Exclusion Chromatography

Also called GEL PERMEATION CHROMATOGRAPHY
Separation principle
 

giving chromatogram as below

Mild, non-denaturing conditions - very suitable for separating proteins of different molecular masses.

Also used for:

• De-salting or buffer exchange of, eg, protein solutions
• Determination of Molecular mass of biological macromolecules - calibrate column with similar molecules of known molecular mass

Quaternary structure usually remains intact

Types of matrix for forming stationary phase:

• Cross-linked dextran polymer (Sephadex G-10 to G-200)
• Cross-linked polyacrylamide (Biogel P-2 to P-300)
• Agarose - the largest pore size

Affinity Chromatography

Retention due to biospecific interaction using a ligand molecule chemically coupled to a dextran or cellulose matrix - (see 3.4 above for diagram). Hence may be able to isolate analyte from complex mixture.
Examples
 

Elution can be by displacement with ligand molecules in free solution. But analyte then eluted as complex with ligand.
Better to elute by change of pH to weaken binding.

Chemistry of ligand coupling to matrix using cyanogen bromide.
 

3.5 Indexes of Column Efficiency & Chromatographic Separation

In a chromatogram, important parameters to consider are retention time (t_R) and peak width (W) - measured in the same units.
 

• The efficiency of separation of any two components is given by the resolution (R)
   

  R =

  [2(tR(B) - tR(A))]

  /

  [W(A) + W(B)]

  The efficiency of a column in chromatographing each individual component is measured in terms of the theoretical plate number (N)
   

  N

  =

  16(

  tR

  /

  W

  )2

•  

Theoretical plate can be thought of as length of column allowing one complete equilibration of sample and stationary phase.

If measure peak width at half-height (W_0.5h) rather than at base, then for Gaussian peak:
 

For comparing two columns, that might have different lengths, or comparing the same column under different running conditions, the Height Equivalent to a Theoretical Plate (HETP) is most often used 

  where L is the column length in cm. A larger N, or a smaller HETP = more efficient

Resolution, Theoretical Plate Number, and HETP are widely used in Gas Liquid Chromatography (GLC) and in High Performance Liquid Chromatography (HPLC).

Elution Parameters in Size- Exclusion Chromatography

To assess suitability of size-exclusion column for sample, need parameters that show extent to which sample molecules enter the internal volume of the gel vs the void volume (see 3.4.4)
In theory, the elution volume (V_e) is related to void volume (V_o) and internal volume (V_i) 

 In practice, approximate V_i by V_t - V_o, where V_t is the total bed volume (this neglects that a small part of V_t is space occupied by matrix).
Instead of the true K_D, this gives an "available" distribution coefficient, K_av: 

K_av usually between 0 and 1; larger difference between solutes indicates easier separation.
If K_av >1, mechanisms additional to size-exclusion may be operating, eg ion-exchange.

Qualitative Chromatographic Analysis

Paper and Thin Layer Chromatography

RETARDATION FACTOR,      RF = sample spot distance            solvent front distance  Compare to standards run alongside.
VARIATIONS IN COLOUR of spots when sprayed (developed) with reagent (eg ninhydrin for amino acids.)

GLC and HPLC

• RETENTION TIME. Compare to standards run under identical conditions in same column.

  Components emerging from column flow to MASS SPECTROMETER for identification.

Quantitative Chromatographic Analysis

Paper and Thin Layer Chromatography
(Difficult to quantify accurately)

• Extract stained spot with solvent & analyse in spectrophotometer
• Scan plate with DENSITOMETER. Generates chromatogram similar to monitoring column eluate, then treat as for GC, HPLC.

GLC and HPLC

Chromatogram = plot of component concentration (or proportional parameter eg absorbance) vs eluate volume or elution time. 

Calibrate with standards of known mass; measure peak areas.

Measurement of Peak Areas
Rough calculation of area by approximating peak to a triangle.

Modern GC and HPLC instruments allow electronic or computerised integration of output signal.

Poorly resolved peaks and/or sloping baselines present complications.
Therefore sometimes use Peak heights - less theoretically correct, but may be OK when calibrated with standards run under identical conditions.

Internal Standard
To counteract random error in injection volume (sample size applied to column), or in pre-column manipulations.
Internal standard is CHEMICALLY SIMILAR (not identical) SUBSTANCE to analyte - resolved as separate peak in chromatogram.
 

Express area relative to known constant area of internal standard.