College of Veterinary Medicine & Animal Resources Department of Clinical Studies Toxicology & Forensic Medicine Chromatography By S Al-Shokair
Chromatography (from Greek: chroma, colour and: graphein to write). http://en.wikipedia.org/wiki/Chromatography
They all have a stationary phase (a solid, or a liquid supported on a solid) and a mobile phase (a liquid or a gas).
Chromatography is used to separate mixtures of substances into their components. All forms of chromatography work on the same principle.
The mobile phase flows through the stationary phase and carries the components of the mixture with it. Different components travel at different rates.
Types of chromatographyPaper chromatography Thin layer chromatography Gas chromatography Liquid chromatography
High Performance Liquid chromatogrphy (HPLC)Affinity ChromatographyColum Chromatography
In paper chromatography, the stationary phase is a very uniform absorbent paper. The mobile phase is a suitable liquid solvent or mixture of solvents.
AimsTo demonstrate separation of amino acid by paper chromatography To use chromatography to identify amino acids
UsesClinical research hospitals Manufacturing industries Forensic science studies.
DisadvantagesThe disadvantage of paper chromatography is that many a times complex mixtures can’t be separated by using this method.
Chromatography tank and lid • Chromatography paper • Capillary tubes • Amino acid samples • Pencil & metric ruler
Unknown samples Solvent (BAW): Butan-1-ol : Acetic acid : Water 60 : 15 : 25 (FLAMMABLE, TOXIC) Hairdryer or heating tray
Spray can of Indanetrione (Ninhydrin) 1% in acetone butan-1-ol (FLAMMABLE, TOXIC) Latex gloves Access to fume cupboard Drying oven at 100°C
Touch the tip of the capillary to the first mark and pull it awayLet this dry and apply the sample againRepeat for each sample or mixtureLet the paper dry.
A. The eluting solution for this experiment is 1-butanol + H2O + Acetic Acid: 60 ml :15 ml : 25 ml. • B.Develop using Ninhydrin • (triketohydrindene hydrate) • : acetone (0.2 mg ninhydrin dissolves in 100 ml).
Draw a line 1.5 – 2cm above the bottom of the chromatography paper Make small marks at 0.5cm intervals along the line. Fill a capillary tube by capillary action with your first sample
Standthe chromatography paper in the tank so that the bottom edge is in the solvent but the remaining paper does not touch the tank. Place the lid on the tank and leave for about 1 hour. Wearing gloves, remove the damp paper and mark where the solvent has reached
Dry the paper in the fume cupboard. Still in the fume cupboard, spray the whole of the paper with ninhydrine in butanol. Dry the paper and transfer it to the drying oven for 1-2min.
B.Develop using Ninhydrin • (triketohydrindene hydrate) • : acetone (1g ninhydrin dissolves in 100 ml).
Mark the position of each spot that develops. Note the colour and Rf (or travel distance) of each standard and hence find the amino acid composition of the unknown samples.
Quantitative analysis of chromatographic data To calculate the Rf (retention factor or retardation factors ), we use the equation: Note that an Rf value has no units because the units of distance cancel. Rf= distance traveled by component from application pointdistance traveled by solvent from application point
Rf = distance traveled by component from application point distance traveled by solvent from application point In our example, this would be: Note that an Rf value has no units because the units of distance cancel.
determination of the components a plant contains analyzing ceramides and fatty acids detection of pesticides or insecticides in food and water analyzing the dye composition of fibers in forensics, or assaying the radiochemical purity of radiopharmaceutica
Analytical Conditions Column : Zorbax SIL Mobile phase : Toluene-ethylacetate-formic acid-methanol (89+7.5+2.0+1.5) Flow rate : 1.0ml/min Column temp. : 40°C Detection : Shimadzu spectrofluorophotometer (Em=365nm, Ex=425nm)