Medical Lab Technology, Chromatography
Chromatography is a technique used to separate and identify the components of a mixture. Works by allowing the molecules present in the mixture to distribute themselves between a stationary and a mobile medium. Molecules that spend most of their time in the mobile phase are carried along faster.
1- Thin layer chromatography
2- Gas chromatography
3- Column chromatography.
4- High performance liquid chromatography
5- Ion exchange chromatography
- Thin layer chromatography (TLC) is a method for identifying substances and testing the purity of compounds.
TLC is a useful technique because it is relatively quick and requires small quantities of material. Separations in TLC involve distributing a mixture of two or more substances between a stationary phase and a mobile phase.
The stationary phase:
This is a thin layer of adsorbent (usually silica gel or alumina) coated on a plate.
The mobile phase:
This phase is a developing liquid which travels up the sta
tionary phase, carrying the samples with it. Components of the samples will separate on the stationary phase according to how much they adsorb on the stationary phase versus how much they dissolve in the mobile phase.
Preparing the Chamber:
To a jar with a tight-fitting lid add enough of the appropriate developing liquid so that it is 0.5 to 1 cm deep in the bottom of the jar. Close the jar tightly, and let it stand for about 30 minutes so that the atmosphere in the jar becomes saturated with solvent.
Preparing the Plates for Development:
With a pencil, mark two small dots into the adsorbent about 2 cm
from the bottom of the plate. The dots should be on the edges of the plate, and each dot should be the same distance up from the bottom of the plate. The dots must be farther from the bottom of the plate than the depth of the solvent in the jar. Using a drawn-out capillary tube, spot the samples on the plate so that they line up with the dots marked.
Developing the Plates:
After preparing the development chamber and spotting the samples, the plates are ready for development. Be careful to handle the plates only by their edges, and try to leave the development chamber uncovered for as little time as possible. When the plates are removed from the chamber, quickly trace the solvent front (the highest solvent level on the plate) with a pencil.
- Gas Chromatography
- Separation of gaseous & volatile substances
- Simple & efficient in regard to separation
- GC consists of GSC (gas solid chromatography)
- GLC (gas liquid chromatography)
Gas → Mobile phase
Solid / Liquid → Solid phase
GSC not used because of limited no. of Solid phase
GSC principle is ADSORPTION
GLC principle is PARTITION
The organic compounds are separated due to differences in their partitioning behavior between the mobile gas phase and the stationary phase in the column.
- Carrier gas
- Flow regulators and flow meters
- Injection devices
- Temperature control devices
- Recorders and Integrators
- Column chromatography
- Column of stationary phase is used
- » Solid – P
- » Liquid – M.P
- ◊ Adsorption
- Mixture of component components dissolved in the M.P is introduced in to the column.
- Components moves depending upon their relative affinities.
Selection of Stationary Phase:
- Success of chromatography → proper selection of S.P, it depends on the following.
- Removal of impurities
- of components to be separated
- Length of the column used
- Affinity differences b/w components
- Quality of adsorbent used
Selection of mobile phase:
They act as Solvent, developer and Eluent.
» Bottom portion of the column – packed with glass wool/cotton wool or may contain asbestos pad,
» Above which adsorbent is packed
» After packing a paper disc kept on the top
Two types of packing techniques are there.
- Dry packing
- Wet packing
Dry Packing Technique
Adsorbent is packed in the columbine dry form
Fill the solvent, till equilibrium is reached
DEMERIT: Air bubbles are entrapped b/w M.P & S.P→ cracks appear in the adsorbent layer.
Wet Packing Technique
Ideal & common technique
Adsorbent + M.P in a beaker & poured in to column
S.P settles uniformly & no crack in the column of adsorbent
Advantages of C.C:
» Any type of mixture can be separated
» Any quantity of mixture can be separated
» Wider choice of M.P
» Automation is possible
Disadvantages of C.C
» Time consuming
» ↑ amounts of M.P required
» Automation makes techniques expensive.
HPLC is a form of liquid chromatography used to separate compounds that are dissolved in solution
A mixture sample is separated into components for identification, quantification and purification of mixtures.
The heart of a HPLC system is the column
- The column contains the particles that contains the stationary phase
- The mobile phase is pumped through the column by a pump
- Solvents must be degassed to eliminate formation of bubbles.
The role of the pump is to force a liquid (mobile phase) through the liquid chromatograph at a specific flow rate.
- The injector serves to introduce the liquid sample into the flow stream of the mobile phase.
May be auto-sampler or manual.
- Ion exchange chromatography
- Ion-exchange chromatographyis a process that allows the separation of ions and polar molecules based on their affinity to the ion exchanger.
- It can be used for almost any kind of charged molecule including large proteins, small nucleotides and amino acids.
- Cations or Anions can be separated using this method.
» Glass, stainless steel or polymers
- Packing the column
» Wet packing method:
A slurry is prepared of the eluent with the stationary phase powder and then carefully poured into the column. Care must be taken to avoid air bubbles.
- Application of the sample
- After packing, sample is added to the top of the stationary phase, use syringe or pipette.
- This layer is usually topped with a small layer of sand or with cotton or glass wool to protect the shape of the organic layer from the velocity of newly added eluent.
- Mobile phase
Acids, alkalis, buffers…
- Stationary phase
The ionic compound consisting of the cationic species (M+) and the anionic species (B-)
Components of mixture separate & move down the column at different rates depending upon the affinity of the ion for ion exchanger.
- The elutes are collected at different stage
- Analysis of the elute
Spectrophotometric, flame photometry polarography…