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Learning Outcomes
When you have mastered this topic, you will:
What is “chromatography”?
Chromatography is a process whereby a mixture of solutes may be resolved into its components by exploiting differences in affinity of the solutes for particles of an insoluble matrix over which a solution of the components is passing. The insoluble matrix is called the stationary phase, while the solution which passes through it is called the mobile phase. We can illustrate thiis by looking at the technique of column chromatography, as used in a hypothetical example.
For example, suppose we have a mixture of two solutes, A and B, dissolved in a suitable buffer solution. A column is filled with beads of an insoluble substance that is expected to bind differentially and reversibly to A and B. The column is initially equilibrated by running a certain volume of the buffer through it, and the mixture applied to the top of the column as a narrow layer. If the stopcock of the column is carefully opened, the layer of solutes will pass into the column, and the solutes can be washed out slowly by pouring more buffer into the column. This process is called elution, and the solution emerging at the bottom of the column is called the eluate.
Let us assume, for the purpose of this illustration, that solute B binds more strongly to the matrix than solute A. Then, as the mixture travels down the column, the molecules of B will be retarded with respect to the molecules of A. In time, they will separate into bands, and be eluted at different times.
If we have a system for collecting constant volumes of the eluate (which are called fractions), the separated solutes will be distributed in the different fractions, as shown in the graph below:
If fractions 4-9 are combined (“pooled”), a solution of pure A will be obtained. Equally, if fractions 12-18 are pooled, a solution of pure B will be obtained.
In this example, the solid matrix is placed in a column, whence the term column chromatography. In related techniques, the solid matrix may be a sheet of suitable material, such as paper (paper chromatography) or a layer of matrix on a glass, aluminium or polymer sheet (thin layer chromatography).
Types of chromatography
The nature of the molecules that need to be separated dictate the type of solid matrix that is used. In structural biology, we are largely concerned with the separation of mixtures of proteins or nucleic acids. The available methods are tabulated below, and are discussed in separage pages, accessed from the indicated links:
Technique | Based on | Suitable for |
---|---|---|
(GF) | Differences in size | Proteins, peptides, polynucleotides |
(IEX) | Differences in charge | Proteins, peptides, polynucleotides |
(AC) | Recognition of specific ligands | Proteins |
(HIC) | Differences in hydrophobic residues | Lipoproteins |
Resolution
The resolutionof a column chromatography system is a measure of the relative separation between two peaks. The better the resolution, the better the purity of the separated components. In the diagram above (left) the peaks overlap to a large extent, an we say therefore that the resolution of the system is poor. In the case depicted on the right, we have a good separation (the concentration betwen the peaks is the baseline value).
The resolution of a system may be quantified in the following way:
The resolution, Rsis a dimensionless constant which relates the elution volumes VR1and VR2of two components as shown in the above diagram with the width of the respective peaks, Wb1and Wb2(measured as volumes of eluted liquid) at the base of the peaks. Then,
The significance of Rsmay be appreciated by looking at the following figures:
For baseline separation(the ideal case), Rs31.5. In this case, the two component peaks are completely resolved, and each peak contains 100% pure component.
If Rs= 1.0, then 98% purity has been achieved at 98% of peak recovery, provided the peaks are symmetrical and approximately equal in size.
Efficiency
As a solute travels down a gel filtration column, the peaks broaden, and this reduces the capability of the column to fractionate component solutes. A column is said to have a high efficiency if conditions are such as to reduce peak broadening.
It is always desirable to increase the efficiency of a system. This is achieved by:
- Well-packed columns, evenly packed, neither to tight or too loose, with no air bubbles;
- Small matrix particle size, bearing in mind that small particle sizes decrease the flow rates, and hence increase the run times;
- Even buffer flow.
References on Chromatographic and Related Techniques
General
Affinity chromatography
- Affinity Chromatography – Principles and Methods (Amersham Handbook)
- Amersham HiTrap™ Blue HP Affinity Columns
- Purification of (His)6-tagged proteins using HiTrap Chelating HP columns charged with different metal ions (Amersham Application Note)
- Reactive dye affinity chromatography matrices
Gel filtration chromatography
- Gel filtration chromatography – Principles and methods (Amersham Biosciences)
- Desalting and buffer exchange with Sephadex® G-25 (Amersham BioSciences Application Note)
- Sephacryl™ High Resolution chromatography media
- Sephacryl™ High Resolution chromatography instructions
- Size exclusion chromatography of water-soluble polymers with Tosoh polymer-based TSK-GEL TW and TSK-GEL PWXL columns
Ion-exchange chromatography
- Ion Exchange Chromatography Principles and Methods
- CM Sepharose Fast Flow, DEAE Sepharose Fast Flow, Q Sepharose Fast Flow and SP Sepharose Fast Flow ion exchangers
- Q- and SP-sepharose
- Q Sepharose XL, Q Sepharose XL virus and SP Sepharose XL
- Instructions for Q Sepharose Fast Flow anion exchanger
- ANX Sepharose 4 Fast Flow (high sub) – Information
- ANX Sepharose 4 Fast Flow (high sub) – Instructions
- Instructions for the use of HiPrep 16/10 Q FF, HiPrep 16/10 SP FF and HiPrep 16/10 ANX FF (high sub) prepacked columns
- Instructions for the use of HiPrep 16/10 SP XL and Q XL prepacked ion-exchange columns
Equipment
- All about fittings. By John W Watts
- Column K 9. Amersham Bioscience.
- Superdex™ 75 10/300 GL and Superdex 200 10/300 GL
- Instructions for HR 16/5, HR 16/10, HR 16/50 columns
- Amersham C Columns for Standard Chromatography
- Amersham C Column Adaptors
- Amersham C Column Packing Reservoirs
- Instructions for XK-columns and AK-adaptors
- HiTrap ion exchange columns
- Amersham HiTrap™ Blue HP Affinity Columns
- HiPrep 16/10 CM FF and DEAE FF prepacked ion-exchange columns
- Instructions for the use of HiPrep 16/10 SP XL and Q XL prepacked ion-exchange columns
- HiTrap ion exchange columns
- Instructions for Rheodyne manual sample injectors
- 305-306 HPLC Pumps (Technical Specifications) [Gilson]
- 305 HPLC Piston Pump User’s Guide (Gilson)
- Gilson 155/156 UV/VIS Detectors User’s Guide
- Gilson 151/152 UV/VIS Detectors User’s Guide
- Gilson FC 203B Fraction Collector Users Guide
- Gilson FC 203B Fraction Collector Quick Reference Guide
- Tubing Properties
- Minipuls 3 Peristaltic Pump User’s Guide
- Selecting Tubing and Connectors for Minipuls 3 Peristaltic Pump
- Gilson SC-type Piston Pump Heads User’s Guide
- Selecting Tubing and Connectors for Minipuls 3 Peristaltic Pump