Outline

Definition of Chromatography History of Chromatography Chromatography terms Classification of Chromatography Chromatograph Applications of Chromatography

Definition
Is the collective term for a family of laboratory techniques for the separation of mixtures Involves passing a mixture which contains the analyte through a stationary phase, which separates it from other molecules in the mixture and allows it to be isolated
‡ Analyze
Separate

‡ Identify ‡ Purify

Mixture

Components

‡ Quantify

who used columns of calcium carbonate for separating plant pigments During the 1940s and 1950s.History 1903. They established the principles and basic techniques of partition chromatography Since then. the technology has advanced rapidly . Mikhail Semyonovich Tsvet.

either a liquid in LC or gas in GC . Can be a particular solid or gel-based packing (LC) or a highly viscous liquid coated on the inside of the column (GC) Mobile phase: Solvent moving through the column.Chromatography Terms The analyte is the substance that is to be separated during chromatography Stationary phase: Phase that stays in place inside the column.

Linear velocity: Distance passed by mobile phase per 1min in the column (cm/min) . Chromatogram: Graph showing detector response as a function of a time.Eluent: Fluid entering a column Eluate: Fluid exiting the column Elution: The process of passing the mobile phase through the column. Flow rate: How much mobile phase passed / minute (ml/min).

Classification Column Paper Planar Thin layer Chromatography Supercritical fluid Separation mechanism physical state of mobile phase Ion exchange Size exclusion Liquid Gas .

column chrotography .Column Chromatography The stationary phase (column packing) in the column is very polar One starts will a less polar solvent to remove the less polar compounds. and then you slowly Increase the polarity of the solvent to remove the more polar compounds Figure 1. solvent column Bands of separated consistuents .

.larger Rf .Thin Layer Chromatography Here the mobile phase is a liquid Flowing past a thin layer of powder on a solid support Substances that are less attracted to the solid or are more soluble in the liquid move faster And so move further up the plate by the time that the process has been stopped by taking the plate out of the liquid.

principles in TLC.Figure 2. .

.

Paper chromatography jar .Paper Chromatography Figure 3.

Rf = distance moved by substance distance moved by solvent front For substances that are very soluble in the liquid Rf will be close to ...... 1 For substances that are rather insoluble in the liquid Rf will be close to . 0 ..

. retained longer than large molecules. therefore.Size exclusion Chromatography GEL-PERMEATION CHROMATOGRAPHY Gel-Permeation Chromatography is a mechanical sorting of molecules based on the size of the molecules in solution. Small molecules are able to permeate more pores and are.

Ion. .exchange Chromatography Separation in Ion-exchange Chromatography is based on the competition of different ionic compounds of the sample for the active sites on the ion-exchange resin (columnpacking).

Liquid Chromatography Liquid chromatography (LC) is a separation technique in which the mobile phase is a liquid. use high performance liquid chromatography (HPLC) Figure 4 . HPLC 1525 . Liquid chromatography can be carried out either in a column or a plane Present day.

Gas Chromatography Principals of Separation Column is selected. packed with Liquid Phase. and installed. Sample vapor becomes partitioned between Moving Gas Phase and Stationary Liquid Phase The time the different compounds in the sample spend in the Vapor Phase is a function of their Vapor Pressure. argon). The more volatile (Low Boiling Point / Higher Vapor Pressure) compounds arrive at the end of the column first and pass into the detector . Sample injected with microliter syringe into the injection port where it is vaporized and mixed into the Carrier Gas stream (helium. nitrogen.

chromatograph .Chromatograph Figure 5.

chromatographic peak is fronting Figure 7. chromatographic peak is tailing .Figure 6.

HPLC Figure 6. Gas chromatography .Applications Figure 5.

its components.Chromatography is used by scientists to: Analyze ² examine a mixture. and their relations to one another Identify ² determine the identity of a mixture or components based on known components Purify ² separate components in order to isolate one of interest for further study Quantify ² determine the amount of the a mixture and/or the components present in the samplecxxc .

Pharmaceutical Company ² determine amount of each chemical found in new product Hospital ² detect blood or alcohol levels in a patient·s blood stream Law Enforcement ² to compare a sample found at a crime scene to samples from suspects Environmental Agency ² determine the level of pollutants in the water supply Manufacturing Plant ² to purify a chemical needed to make a product So on .

net/__chomatography-free-book_ebook_./index. .org/wiki/Chromatography www./library.com/.thin%20layer%20chomatography www..ebookpdf.shtml?.wikipedia..htm.References http://en.docjax.waters....org www.html www....chromatography-online.com/.

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