Introduction: Paper Chromatography/UV-Vis Experiment With Arduino

This experiment uses an Arduino microprocessor, along with household items, to perform a paper chromatography experiment and analyze the results using a technique similar to Ultraviolet-Visible (UV-Vis) spectroscopy. This experiment is meant to replicate several aspects of an HPLC (High Performance Liquid Chromatography) instrument, such as the chromatographic separation and the UV-Vis detection. You will learn many scientific techniques with this experiment, as well as learning about the Arduino microprocessor.

Step 1: Video Demonstration

Step 2: Purpose

The purpose of this experiment is to replicate some of the functions of an HPLC instrument. HPLC separates compounds through liquid chromatography and uses UV-Vis as a detector. In this experiment, these two functions will be performed separately. Paper chromatography will represent the liquid chromatography within HPLC and will be used to separate mixtures of food dyes. The separated dyes will then be used to create samples that will be analyzed using a technique similar to UV-Vis spectroscopy. A simplified version of a UV-Vis instrument will be created, and this will represent the detector of the HPLC. With this experiment, you will learn about chromatography, UV-Vis spectroscopy, HPLC instrument functions, and the Arduino Uno microprocessor.

Step 3: Gather These Supplies

Paper Chromatography Supplies:

  • Paper towels (~$1-2 per roll)
  • Toothpicks (~$3 per box)
  • Food colors (~$4 per box)
  • Isopropyl (rubbing) alcohol (~$3 per bottle)
  • Stapler
  • Pencil
  • Ruler
  • Cup
  • Water
  • Scissors
  • Plastic wrap

Arduino Supplies:

  • Arduino Uno or similar microprocessor (~$15)
  • Photoresistor
  • Resistor (10 K ohms)
  • Wires (male-male)
  • Breadboard (~$5)

Instrument Supplies:

  • Flashlight
  • Some type of clear glass tube - glass syringe used in this example
  • Styrofoam piece with hole in middle
  • Toilet paper roll
  • Duct tape

Step 4: Perform Paper Chromatography & Create Samples

Paper chromatography:

  1. Cut a roughly 4x6 inch rectangle out of a paper towel.
  2. Using a pencil and ruler, draw a straight line parallel to the longer edge of the paper towel 1 inch from the bottom.
  3. Using pencil, draw Xs along this line roughly 1/2 to 3/4 inches apart.
  4. Create mixtures of food coloring (blue+yellow, blue+red, red+yellow).
  5. Using a toothpick, dot the food color mixtures and the pure food colors onto the drawn Xs. Each color or mixture will be dotted onto its own X. Allow to dry.
  6. Roll the paper towel into a cylinder, bringing the shorter sides together. Staple this cylinder together, leaving a small gap between the two sides of the paper towel.
  7. Add roughly a 1/4 inch of water to a cup that will fit the cylinder you created.
  8. Put the cylinder in the cup with the dotted side closest to the water.
  9. You will see the water being absorbed into the paper towel, and the food colors will begin to travel up the paper towel.
  10. When the water line on the paper towel reaches about 3/4 inches from the top, remove the paper towel from the cup. Remove the staples and allow to dry flat on another paper towel.

Creating the samples:

  1. Once the paper towel is dry, cut out the different colored spots from both the mixtures and the pure food coloring.
  2. Add these cut out spots to isopropyl (rubbing) alcohol.
  3. Cover this with plastic wrap and allow to soak until the majority of the color has been removed from the paper towel.
  4. These will be the samples that will be analyzed using UV-Vis spectroscopy.

Step 5: Assemble the Electronics

Following the circuit diagram and the board setup image, wire the bread board to the Arduino.

You will use the following on the Arduino:

  • 5 V output
  • Ground
  • A0 output

You will use the following parts:

  • Male-male wires
  • 10 K ohm resistor
  • Photoresistor

Step 6: Assemble the Instrument

  1. Create a sample holder
    • Use a piece of styrofoam with a hole in the center large enough to hold your sample.
    • Poke holes across from one another in the sides of the styrofoam large enough to house the photoresistor. The other hole will be the light input.
    • Set this on the board with photoresistor in one of the holes.
  2. Create a tube to block out ambient light
    • Use a toilet paper roll and duct tape the top end closed.
    • This will sit over the sample holder when taking measurements to reduce the amount of unwanted light.

Step 7: Program the Instrument

  1. Use the code provided (UV_Vis_readings).
  2. Verify the code.
  3. Upload the code to the Arduino.
  4. Check that the serial monitor function is working by seeing if larger numbers are present when the photoresistor is exposed to light and smaller numbers when the resistor is in dark.

Step 8: Test the Instrument

  1. Put isopropyl alcohol into glass tube or syringe.
  2. Put tube into sample holder, making sure it lines up with the holes in the styrofoam.
  3. Position flashlight with light entering one of the holes.
  4. Place toilet paper roll over top to block extra light.
  5. Turn on SerialMonitor and record the measurement once stable.
  6. This value is transmittance, but needs to be converted.
  7. Multiply value by (5/1024) to get the actual transmittance (T).
  8. Perform the following calculation to get absorbance: Absorbance = log (1/T).
  9. This is the value of the blank.
  10. Repeat steps 1-8 for each separated sample.
  11. Subtract the absorbance of the blank from these values to account for background light.
  12. Compare the absorbances - Do you see any trends? Were the more intense spots higher or lower in absorbance?

Step 9: Improvements

Different materials:

  • Coffee filters would be a good replacement for paper towels.
  • An LED bulb could be programmed into the code to use as a source light, instead of a flashlight.
  • Test tubes could be used in place of the glass syringe.

Improving separation:

  • Different solvents could be used during paper chromatography to improve the separation of the food colors. This could be tested by seeing what solvents made the separation of colors in the food color mixtures more obvious. Different ratios of solvent mixtures could also be tested.

More applications:

  • A similar experiment can be performed by separating pigments from plants.
  • Other colored substances could be tested, as well.

Step 10: References