Paper Chromatography

Purpose:

The purpose of the paper chromatography lab is to learn how to use paper chromatography to decipher what dyes were in a mix of dyes by using an Rf value to find what other dyes looked like then compare it to the unknown dye. This improves our problem solving skills and logical reasoning to create a reasonable conclusion.

Hypothesis:

I predict that Unknown 1 is Yellow 5 and Blue 1. And Unknown 2 is Blue 1 and Green 3. I think this because U2 looks like a mix between a green and a blue because its dark blue but a little earthy, and I think U2 is that because it looks like a more green but not the right shade to be green 3.

Pre-Lab Questions:

Materials:

- Filter Paper

- Sample sized paper (3"x4.5")

- 400Ml glass (any size works)

- Tape

- Pencil

- Unknown dye 1 (Made of blue1 and yellow5)

- Unknown dye 2 (Made of Blue1 and Red3)

- Blue 1

- Blue 2

- Green 3

- Yellow 5

- Yellow 6

- Red 3

- Red 40

Procedure:

Cut out filter paper based on the 3"x4.5" template
Fill a beaker with a small amount of water
Hold the filter papers next to the water and make a small pencil mark along the water line
Put 5 drops of dye on one filter paper on the line
Put 4 drops of dye on the other filter paper on the line
Label the drops with pencil
mark where the dye starts with pencil
Put two pieces of tape on the top of each paper
Put both papers into the water with the dye above the water
Tape it into position with the two pieces of tape on the top
Wait until the water line reaches 3/4ths of the way up, or whatever your desired wait time is. Don't let it get to the tape.
Pull one paper out of the water and use a pencil to mark where the water reached and where each dye reached
Pull the other paper out of the water and use a pencil to mark where the water reached and where each dye reached
UNKNOWNS WILL HAVE TWO PLACES TO MEASURE FROM
Wait to dry (After drying the dyes and water marks are arbitrary because the dye shifts a little while being still, ONLY use the marks from here out)
To calculate the Rf value measure the distance from the start line of the dye to the finish line
Measure the distance from the start line to end line of the dye
Divide the dye distance by the total distance and that is your Rf value
Repeat steps 15-17 for every dye
Compare the results
Find what the unknown values are closest too and you have your dyes!

Observations:

Fermentation:

It was hard to perfectly cut out the paper perfectly, I wonder if that will have an effect
Very small amount of water works best
Hard to make sure it was perfectly straight across like the water, so I made a mark on either side and then used a ruler across
This worked really well but you can only do a max of 5 on a paper at once, I tried doing all of them at once and they darted in different directions during the experiment and it didn't work
This one had better results because I let it sit for longer and because they were more separated
I used a piece of tape when placing my dyes on the paper to remember where was where, I didn't have it on the paper, I read it left to right to make sure I wouldn't do it upside down and mess up the dyes.
I marked the bottom of each individual drop for better accuracy. The line at the bottom just for reference when placing the drops
I put tape to keep it in the glass
I put both papers in the glass making sure the dye was above the water
I used tape to secure it, on one test that I didn't use I put it a little crooked and it messed up the results
When I didn't let my water go up as much as I should I had hard to read results
You have to do this quick because it dries quickly and moves the dye, but be careful because the paper is wet and it is very ripable
Do this one quick for the same reason
Do not forget to measure it because its got more than one dye in it
It dries pretty quick
Try to be as precise as possible
I would always choose the furthest point
Calculator
It goes pretty quickly
Some things were a little off comparing to other people in class
Unknown dyes were not perfect matches, just closes in number + hue

Data Collection and Analysis:

Paper Chromotography

Data Analysis Questions:

Examine the structures of the FD&C Red No. 40, Blue No.1 and Yellow No.5 dyes. What are the similarities and differences in the structures of the three dyes?
- They all have NaSO3
- Red 40 and yellow 5 have a N=N bond
- They all have at least one side group that is a single hexagon
- Blue 1 has compounds neither yellow 5 or red 40 have
- Yellow is the only one with a N-N bond
In the Introductory Activity, the developing solvents were 2% sodium chloride aqueous solution and 2% isopropyl alcohol aqueous solution. Draw separate molecular diagrams of how sodium chloride and isopropyl alcohol would interact in water. Identify the types of intermolecular attractions within each diagram.
Based on the diagrams and intermolecular attractions identified in Question 2, predict and compare the nature of intermo-3Iecular attractions experienced by the FD&C Red No.40, Blue No.1 and Yellow No.5 dyes with the two solvents.
Chromatography paper, and paper in general, is highly hydrophilic. Paper is made from a natural polymer called cellulose, which is a long chain of glucose molecules. Glucose is a cyclic structure with a number of-OH groups around the ring.

a. Predict and explain the types of intermolecular forces that would occur between paper and water. How do these interactions account for the hydrophilic nature of paper?

Paper is super hydrophilic because its cellulose fibers have lots of -OH groups that form strong hydrogen bonds with water. This makes it great at soaking up and spreading water through capillary action.

b. Explain the types of intermolecular interactions that would occur between the FD&C Red No.40, Blue No.1 and Yellow No.5 food dyes and the paper.

Food dyes stick to paper because their polar groups form hydrogen bonds with the -OH groups in cellulose. Their polarity is the main reason they cling to the paper.

Successful test and calculations.

I should have let the test run for longer, but the results were still accurate.

I cut my paper slightly off so the results were tilted making my measurements have a lot of room for human error. I did not use these results because they differed way too far from my first test.

Conclusion:

In this lab I utilized my understanding of biology and chemistry to research and understand a lab thoroughly before starting because this lab happened very quickly and if you didn't know what your next step was you would throw off the whole experiment. After doing calculations for each dye in the lab and comparing my results I say with confidence that Unknown 1 is made of Blue 1 and yellow 5. According to my data Unknown two is made of Blue 1 and Red 3. Comparing to my hypothesis my data supported 3/4 of my guesses so i got 75% correct, however the guess I made wrong was Green 3 for unknown 2 as my data does not support that.

Reflection:

This lab was really fun! It was quick and painless, the only difficulty I ran into was cutting out the paper to run the test on, I had a hard time making it perfect and it would show on some of my test when they curved off direction. I wish we had one or two more days to run more tests so I could get an average for the dyes rather than just one test because human error in extremely present in many ways like cutting out the paper to dipping it in the water at a slight angle or securing it a little off or even measuring slightly off completely altering results. But I really did have fun with this lab and I cant wait for more techniques in the coming weeks like this one because this was fun!

Lab Technique:

The title of this lab and the technique—Paper Chromatography— this technique is very simple yet tricky to get right. You need to cut out a perfect rectangle in the filter paper to have the dyes move properly in the distilled water. The next hard part is to get all of the dyes onto a straight line all starting at the same point. After that you have to carefully set the paper into the distilled water but make sure the water stays below the dyes and stays perfectly perpendicular with the water because if it goes in at an angle the data becomes void and doesn't work. Although you might have thought the hard work was done, it's not. The next step is too use pencil on he wet paper and make marks where everything stopped right then including the solvent front because after it dries out the dyes will move to different places but you have to be wary you don't press too hard with the pencil because the paper is wet.

20250227_135334.mp4

Lab Sheets:

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Lab sheet for the experiment

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