Chromatography Daheeya Alenazi

Definition Technique for separating the components or solute of a mixture on the basis of the relative amounts of each solute distributed between a moving fluid stream called the mobile phase, and a fixed stationary phase.

Mobile phase Solid phase ( Column) Definition consists of the sample being analyzed + the solvent that carries the sample through the column or paper. Fixed substance. Either: 1-paper paper chromatography 2- Silica layer  thin layer chromatography 3- Column GC or HPLC status Liquid or Gas Liquid, solid or Gas Retention time: is the characteristic time it takes by analyte to pass through the solid phase to the detector under set conditions.

Principle of chromatography Mixture of sample separated when it injected into stream of mobile phase to get into stationary phase. The components of the sample move through the column at different velocities, which are a function of specific physical interactions with the adsorbent(silicon) in column . The velocity of each component depends on its chemical nature, on the nature of the stationary phase (column) and on the composition of the mobile phase.

A-Thin layer chromatography Advantage of thin layer chromatography ( TLC) faster runs, better separations, adsorbents, better resolution and to allow for quantification.

After put in solvent Under UV

B-Gas chromatography (GC) Separation technique in which the mobile phase is an inert gas (helium) , and stationary phase is a liquid ,(silicone) with very high boiling point ( oven).

Components OF GC 1-Auto samplers: automatically penetrate the sample. 2-Inlets: the Injectors are responsible for the sample to enter column 3-Column: actual separation of the mixtures takes place in it. The columns are enclosed in an oven ,there for it is the material used for the columns should be able to hold up the heat pressure. Columns made from inert materials ,with very narrow diameter( 0.53 – 0.18mm). The column can be: A-packed made from Stainless steel and glass B-capillary that made from quartz or fused silica glass . ( more common)

4-Oven : give high temperature to ensure that the sample remains in gas form. 5-Detectors: can be the flame ionization detector, flame photometric detector and the electron capture detector. 6-Chromatogram: is a Read out device which is the visual output of the chromatograph. Different peaks or patterns on the chromatogram correspond to different components of the separated mixture. where the signal is proportional to the concentration of the specific analyte separated. https://www.youtube.com/watch?v=uSG8ANBTaN0

Application of GC Forensic investigations. Food analysis Oil analysis Drug analysis

C-High-performance liquid chromatography (HPLC) Technique used in analytical chemistry used to separate, identify, and quantify each component in a mixture. It relies on pumps to pass a pressurized liquid solvent containing the sample mixture through a column, leading to the separation of the sample components. 1-Stationary phase: is a granular material made of solid particles (e.g. silica, polymers), 2–50 micrometers in size. 2-Mobile phase: is pressurized liquid is typically a mixture of solvents (e.g. water, acetonitrile or methanol)

The deference between liquid chromatography(LC) & HPLC The main deference between LC &HPLC is: LC has low pressure so it relies on the force of gravity to pass the mobile phase through the column ,whereas in HPLC high pressures.

Components of HPLC

Solvent (M.P) Column Pump Injection Column (S.P) Read out

1-Inject the sample mixture into the mobile phase stream which carries it into the column. 2-The pumps deliver the desired flow and composition of the mobile phase through the column. 3-Column oven that allows for adjusting the temperature at which the separation is performed ( 5-45 C) 4-The detector generates a signal proportional to the amount of sample component emerging from the column, hence allowing for quantitative analysis of the sample components. 5-Digital microprocessor and user software control the HPLC instrument and provide data analysis

Application of HPLC During the production process Research (e.g. separating the components of a complex biological sample Medical (e.g. detecting vitamin D levels in blood serum) https://www.youtube.com/watch?v=IUwRWn9pEdg