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Dr. Mohammed N. Sabir, BSc. Pharm, MSc., Ph.D. Pharmacog.

Lecturer, Dept. of Pharmacognosy and Pharmaceutical Chemistry
College of Pharmacy, University of Sulaimani
mohammed.sabir@univsul.edu.iq

Experimental pharmacognosy I
First semester, third-year pharmacy, 2021 – 2022

Experiment No. 4

Separation of ink mixture using paper chromatography technique.

The paper chromatography (PC) is a chromatographic separation technique used for separation of polar
substances from their mixtures. The stationary phase is a cellulose paper, and the mechanism of the separation
is partition (why?). The mobile phase is chosen based on the physicochemical nature of the targeted compounds
in the sample mixture. In general, medium polarity solvent are more productive.
Despite the limited use of this technique, the PC is a cheep, quick, simple, and beneficial for chromatogenic
polar substanes.
This experiment aims to separate the various components of an ink mixture.

Extraction procedures

1. Prepare an ink mixture from at least three dyes (yelloo, red, and blue).
2. Assebmle the chromatography jar using a suitable glass beaker with a glass petridish cover.
3. Prepare the solvent system (mobile phase), using ethyl acetate: distilled water: acetic acid (5:4:1) v:v:v.
4. Saturate the jar (beaker) medium with the solvent vapur by pouring 10 mL of the mobile phase into the
beaker, and leave for 15 minutes.
5. Prepare the chromatogram by drawing the baseline (1 cm above the lower paper margin, should be above
the mobile phase, and use a graphite pen), and 0.5 cm bellow the upper margin (solvent front).
6. Apply the mixture as a spot using pinpoint cappilary tube on the baseline of the chromatogram.
7. Apply color spots (one spot for each, the yellow, red, and blue colors) on the chromatogram baseline.
8. Place the chromatogram inside the jar. Allow solvent development.
9. Calculate the time required for the mobile phase to reach the solvent fron.
10. Calculate the Rf-values for each of the applied colors and the separated colrs from the mixture. For each
identical color, the same Rf-value should be obtained from the separated mixture.
11. Prepare your report..

Materials
1. Yeallow, red, and blue inks.
2. Cappillary tubes.
3. Pipettes (5 mL).
4. Glass beaker (400 mL).
5. Glass petridishs.
6. Glass conical flasks (250 mL).
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7. Glass funnel (100 mL).

8. Glass rod.
9. Measuring cylinder.
10. Deionized distilled water.
11. Ethyl acetate (analytcal grade).
12. Acetic acid (analytical grade).
13. Graphite pen.
14. Ruler.
15. Notebook.
16. Nitrile gloves.
17. Mask.