Body : This is one combination of stains that has worked using this set-up:
1. Safranin
2. Congo Red
3. Methylene Blue
4. Crystal Violet
5. Nigrosin
6. Methyl Orange
7. Methyl Green
8. Indigo Carmine
UNKNOWNS
9. Indigo Carmine & Methyl Orange
10. Nigrosin & Methyl Orange
11. Crystal Violet & Indigo Carmine
12. Methyl Orange & Nigrosin
13. Methylene Blue & Congo Red
Here is a sample script that may be useful as an introduction.
[Write on board: PAPER CHROMATOGRAPHY LAB
Take Notes - Staple - 4 points]
?In this lab you will be using the technique of paper chromatography to analyze a few dyes. I will be explaining what you?ll be doing in this lab for the next few minutes. EVERYONE, take notes. Staple your notes to your lab report. YOUR NOTES WILL BE WORTH 4 POINTS.?
Paper chromatography is one way to help identify what kinds of components are present in a solution that contains unknown substances. For example, if you have a water solution of two different dyes dissolved in water, and one of them is yellow and the other dye is
blue, the mixture might look as if it contains a green dye, but running a paper chromatogram might reveal that the solution actually contains a mixture of two dyes, a yellow and a blue one.
Let?s go over the actual steps that you?re going to be following to run your chromatograms. First of all, you are going to be running chromatograms using two different solvents, water and ethanol - so you?ll need to start with TWO pieces of this round 11-cm filter paper; one for the ethanol, and the other for the water.
In the middle of each filter paper, use a toothpick or a sharp pencil to make a hole, like this [demonstrate how this is done]. This hole doesn?t have to be in the exact geometric center, the approximate center will work out fine. The next step is to draw IN PENCIL a
circle of about 3 cm diameter centered as close as you can get it so that the hole ends up about in the middle of the circle. It turns out that if you trace around the the open top of a 250-ml Erlenmyer flask - just turn it upside down, like this, and trace around it - this will
give you just about the right sized circle.
Somewhere close to the edge, label one filter ?ethanol? and write your name on it, and label the other one ?water.? Again, make sure that you use pencil for writing on your chromatography paper, otherewise the ethanol will drag the ink from your pen along your chromatogram and turn your chromatogram into a big ugly mess.
Now you?re going to be making a total of 13 spots all around the circle. You will be told the identity of eight of the 13 stains; the other five stains each contains a mixture of two of the eight stains.
Your objective in this lab is to identify, for each of the five unknown solutions, which two dyes have been mixed together to make that particlar mystery solution. Since you?ll be making thirteen spots all around the origin, in other words thirteen spots that will be roughly evenly spaced along the circle that you?ve just drawn in
pencil....we?re going to draw a short little line in thirteen places all around the origin, and when we spot our spots, we?re going to aim the toothpick so that it touches right here, where the lines cross, in all of these thirteen places around the circle. Before we spot the
spots, I?m going to write down on the inside of the circle, just below where each spot is going to be, the numbers 1-13, IN PENCIL, so that if the spot migrates, I?ll be able to remember where the spot was at the start of the chromatogram.
Use a toothpick to make a spot of each of the 13 stains. Remember, you need to make two spots for each stain, one that you?re going to run in ethanol, and the other in water. [Demonstrate how to make a spot] After you have all 13 spots made for each filter paper, you?re ready to run the chromatogram.
You?ve probably been wondering what the hole is for, the hole in the middle of the filter paper that we made right at the start of this lab...and this is where you find out. Before we get to that though, I?m going to take two of these petri dishes, and fill one about half-way up with water, and fill the other one about half-way up with ethanol. Then I?m going to take my two chromatograms, with 13 spots on each one, and I?m going to take a small piece of paper towel here, and roll it between my fingertips like so, so I end up with a nice
thin wick that?s made of rolled paper towel, about 2 or 3 inchs long, and pointed and thin enough to be able to slip through the hole in the filter paper....then take a pair of scissors, and if you need to, cut off the excess home-made wick so that it?s not too long, so that
your filter paper sits on the edges of the petri dish like this; if the wick is too short, on the other hand, the wick won?t touch the solvent, and you?ll be wondering why your chromatogram is taking so long to develop....I?m also going to cut the excess ?home-made
wick? from the front side too, so that the solvent spreads evenly and goes where we want it to go, rather than up into the paper towel. When you drop the chromatogram onto the petri dish, and the wick is touching the solvent, you?ll see the solvent creep up the wick
and then start to spread out from the center ofthe filter paper in a nice widening circle.
For the ethanol run - the chromatogram that you develop using the ethanol solvent - to help the solvent along, take another petri dish and use it, holding it upside-down like this...and cover the filter paper with it. This will keep more of the ethanol vapor from
evaporating off the paper, so it won?t take as long to develop.?
In practice I have found that most students do very well with this lab aand are able to run very good-quality chromatograms and to correctly identify the unknown mixtures.
Another variation on using toothpicks for spotting is to use home-made glass capillaries, which presents a very colorful demonstration of the cohesiveness of water to glass, and they?re more fun to use than toothpicks.
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