Experiment number 6 Paper chromatography OBJECTIVES To separate and identify the components of an indicator dye mixture using chromatography

APPARATUS 400-ml Beaker Glass Tubing (8 mm x 30 cm) Iron Stand Aspirator Bulb 10-ml Graduated Cylinder Watch Glass Iron Clamp Cork (for glass tubing) 250-ml Erlenmeyer Flask 100-ml Beaker

REAGENTS dye solution (12 mg F + 12 mg MB +25 mls Distilled Water) Methylene Blue (MB) (25 mg + 25 mls Distilled water) Erythrosine (E) (25 mg + 25 mls Distilled Water) (60 mg sodium indigo disulfonate + 25 mls Distilled Water) Methyl orange (MD) (10mg +25mls Distilled Water) Fluorescein (F) (25 mg + 25 mls Distilled Water) white man #1 Filter Paper (14cm x 14cm) Aluminum oxide or Calcium carbonate fluorescein (F) - methylene blue (MB) Indigo Curmine (I) 95% Ethanol MIXTURES Equal Volumes of MD-MB F-MB, I-MB, E-I

Experimental Set-Up:

PART A: PAPER CHROMATOGRAPHY

PROCEDURE A. Paper Chromatography 1. In the 14 x 14 cm filter paper, draw a pencil line 1 cm from one edge. To the pencil line, mark X with pencil 9 spots 1.3 cm apart from each other and from the edge of the paper.

2. Label below the pencil mark X the following using pencil :MO(methyl orange),F(fluorescein), MB (methylene blue),E(erythrosine), I(Indigo carmine), MO/MB (mixture), F/MB (mixture), I/MB (mixture),E/I (mixture).

3. Use a capillary tube or toothpick to place a drop of the sample in the pencil mark. The drop should be 4-5-mm in diameter. After the first

application, let dry for a few seconds, and make the second application of the same sample on the same spot. Proceed to the next pencil mark and proceed with the same procedure until the entire sample is spotted. 4. Staple the vertical ends of the filter paper such that the spots are facing outward horizontally. Let the filter paper stand upright on a clean sheet of paper.

5. Pour 10-ml of the solvent system (95% ethanol=water=1:1) into a 400ml beaker.

6. Drop the spotted filter paper into the beaker making sure that the filter paper does not touch the sides on the beaker.

7. Cover the beaker with a piece of paper and watch glass.

8. Let the beaker sit quietly on the table for the chromatogram to develop.

9. Remove the chromatogram from the beaker when the solvent front is about 1 cm from the top edge of the filter paper.

10. Let the chromatogram stand upright on a clean piece of paper, and let dry for 5-10 minutes.

11. Measure the distance between the center or highest density of each spot from the starting point. Measure also the distance between the solvent front and starting point.
12. Compute and tabulate the

values.

Observation and Results: A. Paper Chromatography

Pure Component

Color of Spot blue purple yellow light yellow

Distance traveled (cm) 1.9 2.2 5.4 4.2

Rf Value

Methylene blue (MB) Eriochrome-black (EBT) Bromothymol blue (BB) Bromocresol green (BG) Nitrobenzeneresorcinol (NR) Methyl orange (MO)

0.352 0.407 1 0.778

pink

2.8

0.519

golden yellow red violet colorless

2.1

0.389

Methyl red (MR) Solvent

2.7 5.4

0.500 1

Mixture

Color of Spot blue blue red violet purple purple light yellow

Distance traveled (cm) 3 1.3 1.6 3.4 2 1.5

Rf Value

MB / MR MB MR EBT / BG EBT BG

0.556 0.241 0.296 0.630 0.370 0.278

MB/NR MB NR

blue blue pink

4.4 1.6 2.9

0.815 0.296 0.537

DISCUSSION Chromatography is a method of separation in which the components to be separated are passed over a stationary phase by a moving solvent. The components of the mixture move over the stationary phase by capillary action at different rates defending on their affinity towards the moving and stationary phase. If the moving phase is less polar than the stationary phase, if the moving phase is less polar component. This will effect a separation of the components based on the differences in polarity of the components in the mixture and on the solvent system. In paper chromatography, the solvent system is ethanol and water. The filter paper absorbs the water and water is the stationary phase. The moving phase is ethanol. Since ethanol is less polar than water, the moving phase is less polar than the stationary phase. For analytical purposes, it desirable to obtain the rate of flow ( ) values. This is the ratio of the distance traveled by the component to the distances traveled by the solvent. The values are obtained from the chromatogram by measuring the distances from the starting material to the estimated point of greatest density of each moving spot. The distance traveled by the solvent is found by measuring the distance travelled by the solvent from the starting material up to the point reached by the solvent (solvent front). In column chromatography, a glass tubing is packed with an absorbent (aluminum oxide r calcium carbonate) and placed in an upright position. The column is first passed with the solvent (ethanol) and thoroughly wet. The mixture sample is added all together, then eluted by components are eluted from the column. The stationary phases the calcium carbonate and the moving phase is the ethanol.

CONCLUSION: As the water containing the dye molecules flows past the fibres, those molecules which are strongly attached to the fibres will take much longer to

flow than those of which a much greater proportion that tends to stay in the water resulting in different speed the dye moved. So the fibres exert a different drag on the different components of the ink. None of the components travels as fast as the water itself, we can see that the paper is damp much further than any part of the dye has reached.