Identification of Sugarsby Paper ChromatographySection A4

WHAT IS CHROMATOGRAPHY?a method of separating and identifying the components of a complex mixture

differential movement through a two-phase system, in which the movement is affected by a flow of a liquid or a gas (mobile phase) which percolates through an adsorbent (stationary phase) or a second liquid phase

WHAT IS CHROMATOGRAPHY?Components of a mixture may be interacting with the stationary phase based on charge, relative solubility or adsorption

Basic Chromatographic Principles All chromatographic systems contain: A stationary phase A mobile phase Sample molecules

PAPER CHROMATOGRAPHYPaper Partition Chromatography

to emphasize the partitioning of solutes between a mobile phase and water adhering to the filter paper during chromatography

PAPER CHROMATOGRAPHYa method for testing the purity of compounds and identifying substances

a useful technique because it is relatively quick and requires small quantities of material

PAPER CHROMATOGRAPHYCarried out by:

placing samples to be analyzed on sheets of filter paperallowing an organic solvent to develop the chromatogram

PAPER CHROMATOGRAPHYMobile phase: ORGANIC SOLVENT

Stationary phase: THE WATER WHICH IS HELD TO THE CELLULOSE FIBERS OF THE PAPER BY HYDROGEN BONDING

PROCEDUREPrepare the developing chamberOrganic solvent: Butanol:Ethanol:Water (52:32:10)

Prepare the Stationary PhaseSpotting the SamplesDeveloping the ChromatogramSpray the ChromatogramHeat the ChromatogramIdentify Spots

PROCEDUREPrepare the developing chamberOrganic solvent: Butanol:Ethanol:Water (52:32:10)

Prepare the Stationary Phase

Spotting the SamplesDeveloping the ChromatogramSpray the ChromatogramHeat the ChromatogramIdentify Spots

PREPARATION FOR THE STATIONARY PHASEDraw a line, 2cm away from the edge of the filter paper using a pencil.

Divided the baseline into 6 spots.

3. Marked each spot and labelled according to the name of the standard sugars.

PROCEDUREPrepare the developing chamberOrganic solvent: Butanol:Ethanol:Water (52:32:10)Prepare the Stationary Phase

Spotting the Samples

Developing the ChromatogramSpray the ChromatogramHeat the ChromatogramIdentify Spots

SPOTTING THE SAMPLESUsing a capillary pipette, 4l each of the given sugar solutions were applied.

Application is done 6 times for each spot (drying spot in each applications).

PROCEDUREPrepare the developing chamberOrganic solvent: Butanol:Ethanol:Water (52:32:10)Prepare the Stationary PhaseSpotting the Samples

Developing the Chromatogram

Spray the ChromatogramHeat the ChromatogramIdentify Spots

DEVELOPING THE CHROMATOGRAMThe paper was placed in the chamber containing the solvent. The spots should not touch the solvent.

PROCEDUREPrepare the developing chamberOrganic solvent: Butanol:Ethanol:Water (52:32:10)Prepare the Stationary PhaseSpotting the SamplesDeveloping the Chromatogram

Spray the ChromatogramAniline acid oxylate spray

Heat the ChromatogramIdentify Spots

PROCEDUREPrepare the developing chamberOrganic solvent: Butanol:Ethanol:Water (52:32:10)Prepare the Stationary PhaseSpotting the SamplesDeveloping the ChromatogramSpray the Chromatogram

Heat the ChromatogramHeat in a hot plate until spots become visible.

Identify Spots

PROCEDUREPrepare the developing chamberOrganic solvent: Butanol:Ethanol:Water (52:32:10)Prepare the Stationary PhaseSpotting the SamplesDeveloping the ChromatogramSpray the ChromatogramHeat the Chromatogram

Identify Spots

CHROMATOGRAM (1A4-1)

CHROMATOGRAM (1A4-2)

RESULTSDistance traveled by farthest sugar = 5.5 cmDistance traveled by farthest sugar = 5.1 cm1A4-11A4-2

COMPUTING THE Rf VALUEi.e. Galactose (1A4-1)Distance traveled by the sugar = 2.6 cmDistance traveled by farthest sugar = 5.1 cm

RESULTS1A4-11A4-2

DISCUSSIONMobile phase: Organic solvent systemButanol: Ethanol: Water (52:32:10) solvent systemStationary phase: Water molecules embedded in Whatman filter paperColor visualization: Reaction of reducing sugars with aniline acid oxalate and the addition of heatDehydration which produced fulfural from hydroxymethyl-fulfural

DISCUSSIONProblem: Solvent ran off the paper

Solution: Change the denominator to the distance traveled by the farthest sugar

Unknown: Sucrose

SOURCES:Jack, R. (1995). Basic Biochemical Laboratory Procedures and Sampling. New York: Oxford University Press, pages 84-85.Furfural. Accessed on 07/08/09 at http://en.wikipedia.org/wiki/FurfuralHMF. Accessed on 07/08/09 at http://en.wikipedia.org/wiki/Hydroxymethylfurfural

word chromatography means "color writing" which is a way that a chemist can test liquid mixturesa method used for separating organic and inorganic compounds (so that they can be analyzed and studied)by analyzing it, we can figure out what makes up that compoundFrequently referred to as paper partition chromatography (to emphasize the partitioning of solutes between a mobile phase and water adhering to the filter paper during chromatography)

Carried out by placing samples to be analyzed on strips or sheets of filter paper and allowing an organic solvent, saturated with water, to develop the chromatogram (to flow slowly up or down over the paper by capillary action so as to separate components of the mixture)The spot is as small as possible not exceeding 3mm in diameter...Too big spots will cause the samples to overlap and influence the movement of solute...

If R value of a solution is zero, the solute remains in the stationary phase and thus it is immobile. If R value = 1 then the solute has no affinity for the stationary phase and travels with the solvent front.Aniline acid oxalateGlucose: brownGalactose: yellow to brownFructose: brownMaltose: brownSucrose: no reactionNon-reducing sugars do not react.