QUALIFICATION AND QUANTIFICATION OF

ARTIFICIAL FOOD COLORING IN THAI TEA LEAVES

By Pnotporn Jantarakolica (u5861115) Pichayathida Siriwechdaruk (u5861105)
Supervised by Dr.Manchuta Dangkulwanich and Ms.Natamon Rittilertnapa

Materials and Equipments

SUBJECT CHEMICALS LAB EQUIPMENTS OTHER EQUIPMENTS
- Tea samples - Isopropyl alcohol - Hot plate - Permanent markers
- Ammonia - Filter paper - Scanner
- Ethyl acetate - Silica gel 60G F254 plates - ImageJ software
GLASSWARES - Methanol
- Beakers - Micropipette - Pencil
- Ethanol - Micropipette tip - Rulers
- Glass funnel
- Butanol - Microsyringe - Cutting mat
- Stirring rod
- Acetic acid - Fume Hood - Box cutter
- Graduated cylinder
- Distilled Water - Gloves - Cling film
- Watch glass
- Carmoisine - Pipette bulb - Tooth picks
- Volumetric Flask
- Ponceau 4R - Cork - Scissors
- Pipette
- Sunset yellow - Electric balance - Timer
- Tartrazine - Spatula - Hair dryer
- Forceps

Procedure
SAMPLE PREPARATION
1. Measure the tea samples to approximately 7 grams each
2. Boil the tea using preheated water, 30 ml of water per tea sample
3. Let the sample cool down
4. Prepare another set of beakers and a set of glass funnel
5. Fold the filter paper and put it into the funnel in order to perform filtration
6. Filter each of the cooled tea samples

STANDARD PREPARATION
1. Measure the standard dye pigments to approximately 0.1 grams each
2. By pouring some water into the volumetric flask then adding the standard dyes into it, mix well until the dye is completely dissolved and
the solution is homogeneous.
3. Add more distilled water until the solution reaches the 100 ml label of the volumetric flask.
4. To make a standard mix, create a 1:1:1:1 ratio solution. Each of the standard added to the mix should be equal in terms of volume.
5. Make each of the dilution of the standard mix in order to be used to create the linear regression line by:
DILUTION PREPARATION SPOTTING
50% 1 : 1 standard mix: water 0.6 μl, twice
75% 3 : 1 standard mix: water 0.6 μl, twice
100% standard mix 0.6 μl, twice
125% standard mix 0.6 μl, twice; then followed by 0.3 μl once
150% standard mix 0.6 μl, three times

PROCESS OF TLC
1. Prepare the Silica gel 60G F254 (8.5 x 6 cm)
2. Make a line on the TLC plate by using pencil → start = 1 cm from edge, end = 7cm from the start line
3. Prepare the beaker by cutting the filter paper into strips, and put in on the inner side of the beaker
4. By using two toothpicks, create a holder for the TLC plate in the beaker
5. Cover the spout of the beaker by using cling film to ensure that the system would be closed.
6. Prepare the mobile phase according to the table
7. Spot the dilution of the sample mix prepared beforehand onto the most left hand side of the silica plate in order to be used as a reference.
Dry the sample mix spots between each time of spotting by using the hair dryer
8. Spot the teas onto the silica plate, 0.6 μl for each spot twice. Dry the tea spots in between each time of spotting by using the hair dryer.
9. Prepare the mobile phase as shown in the table. Pour the mobile phase into the beaker and label the beaker by using marker. Cover the
beaker after the mobile phase is poured by using watch glass.

MIXTURE NO. CHEMICALS USED RATIO

I Isopropyl alcohol (C3H8O): Ammonia (NH3) : Distilled Water (H2O) 10 : 1 : 1
II Ethyl acetate : Ethanol: Distilled water 15 : 7 : 6
III Ethyl Acetate : Ethanol : Ammonia 30% 5:3:1
IV Ethyl Acetate : Ethanol : Water 15 : 7 : 6
V Isopropyl alcohol : Distilled Water : Ammonia 30% 10 : 1 : 1
VI Isopropyl Alcohol : Ammonia : Distilled Water 10 : 3 : 1
VII Isopropyl Alcohol : Ammonia : Distilled Water 10 : 1 : 3
VIII Isopropyl Alcohol : Ammonia : Distilled Water 12 : 1 : 1
IX Isopropyl Alcohol : Ammonia 5:1
X Isopropyl Alcohol : Ammonia 12 : 3
XI Isopropyl Alcohol : Ammonia 2:1
XII Isopropyl : Ammonia: Distilled water 10 : 1 : 1
XIII Isopropyl Alcohol : Ammonia 4:1
XIV Ethyl Acetate : Methanol : Distilled Water : Acetic Acid 65 : 23 : 11 : 1
XV Butanol : Distilled Water : Acetic Acid 20 : 12 : 5

10. Leave the mobile phase for about 10-15 minutes in order to let the vapor of the solvent to be saturated within the TLC chamber
11. Place the prepared TLC plate into the chamber straightly and wait until the solvent front meets the finish line.

PROCESS OF QUANTIFYING
1. Select image, open it in ImageJ program
2. Convert image to grayscale, by selecting image → type → 32 bit
3. Adjust brightness by selecting image → adjust → brightness contrast
4. Select the rectangular tool, draw around the band (make sure that it covers the whole thing)
5. Select analyze → gels → select first lane (command 1)
6. Then draw the box from the first lane to second band of the gel
7. Select analyze → gels → select next lane (command 2)
8. Repeat steps 6 & 7 until all of the bands are completed
9. To finish select analyze → gels → plot lanes (command 3)
10. Then select straight line tool from the tool menu and draw the line from a side of a peak base to another.
11. Repeat step 10 until all is completed
12. Select wand tool from the tool menu, select each band peak (they will outline peaks into yellow color)
13. Another window called results will appear, showing the peak area
14. Copy and paste results into a spreadsheet
15. Plot a line graph by using the band intensity obtained from the ImageJ software as the y-axis and use the concentration of the solution
spotted in the unit μg/μl as the x-axis
16. In order to obtain the concentration of the dilution of the standard sample, use the formula:
grams of standards x volume of the substance
mL of water total volume of mixture
6
17. Convert g/mL to g/mL by multiplying 10 to the value.
18. Obtain a linear equation by using the linear regression line from the plotted information. The equation should be in the form of y=mx+c.
19. By using the linear equation obtained in step 16, calculate the concentration of the tea sample spotted by substituting the band intensity
as the y value.