Chromatography and NMR

Topics: Chromatography, High performance liquid chromatography, Gas chromatography Pages: 20 (2953 words) Published: May 26, 2014
Binding curves:

CD can be used to monitor the binding if s substrate to a protein. The substrate can give a very different CD spectrum when free in the solution relative to when bond in solution.

Outside of farUV: 180-240nm.
1. Near UV CD: 240n-320nm, Aromatic amino acids and disulphide bonds. 2. Visible CD: d-d transition in some metal protein complexes for eg Cu (II) prion.

Principles of Chromatography

Substances present in a mixture are allowed to distribute themselves between two phases: the stationary phase (fixed) and the mobile phase. As the mobile phase flows over the stationary phase, components of the mixture experience many transfers between the two phases. Depending on the properties of the molecule to be separated and that of the mobile phase and stationary phase, the molecule with distribute between the two phases with a particular partition coefficient. Partition coefficient: [stationary phase]/ [mobile phase].

Components of the mixture then interact strongly with the station phase will travel more slowly down the column.

Column chromatography:
The mixture is injected at one end of the column and the mobile phase is passes through at constant velocity. All components of the mixture must travel the full length of the column before they are eluted at the outlet of the column and are detected. The fastest moving components takes less time to pass through and are eluted first and they have the shortest retention time.

Good features of chromatography:
1. There are many possible combinations of stationary and mobile phases. These can be selected taking into account the physical and chemical properties of the materials that are needed to be separated. 2. Most substances that can be dissolved are amenable to some from of chromatography. Like biological molecules such as peptides and proteins that are often temperature sensitive, water soluble and not easy to purify by traditional chemical methods such as distillation, solvent extraction and recrystallization.

Typical chromatography setup:

Equipment:
Pumps: the flow of the mobile phase through the column this is produced by gravity, pumps or gas pressure. Column: the station phase might be packed in a glass or steel column. Detector: UV absorption, florescence, refractive index, electrochemical combustion, or even mass spectrometer.

Example of a chromatogram:

Here different proteins are separated, smaller protein travel slower through this column.

Chromatography terminology:
Chemically different substances have different retention time but the observations that two sample give identical retention times on column chromatography (or Rf values on TLC) does noe constitute proof that they are chemically identical. Additional evidence for their identity may be obtained by using detection methods that provide structural information or by comparing their behaviours on several different mobile phase/ stationary phase combinations. The retention time for a particular compound will increase if in a longer column are lower mobile phase velocity it used but tR also shows some selectivity to the temperature and age of the column and to the concentration of the sample being analysed. It is important to ensure that identical experimental conditions are used when sample are compared. If two sample’s each gives a single peak when run individually but two peaks when injected simultaneously then this is clear proof that they are chemically different. Vr = Retention volume and tR = Retention time.

The time or volume of liquid that is takes for the sample to elute from the column after being loaded on to the column: Vr (ml) = (flow rate) (ml/min) x tR (min).

Retention time of non-retained substance (to) & void volume (Vo). Substances that are not retained at all by the stationary phase will have the same retention...
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