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Lab2 Chromatography(1) - Tagged
General Chemistry Laboratory I (CH 153)
Sacred Heart University
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Department of Chemistry
Sacred Heart University
CH153-LAB-
Chromatography
Pre-lab Questions:
- Draw a sketch of a sample piece of chromatography paper as described in step 2 in the procedure in your lab notebook. Make sure to note the distances of all lines. You should be able to use this diagram to set up the paper without referring to the lab write up.
- After reading the lab, you will see that ammonia (NH 3 ) is used two times in this experiment. How is it different in each case (for what purpose is it used each time)?
- You will be using acetic acid in this lab. What is the chemical formula for acetic acid?
- What will you do with the eluting solution after you are done with the experiment?
- What is the Rf value and how it is calculated?
- From the formula for Rf, do you think Rf should have a unit. Why or why not?
Introduction:
The United States Food and Drug Administration control the colors which can be used in our foods. At present, there are seven synthetic colors which are allowed. FD&C blue #1 and #2, red #3 and #40, yellow #5 and #6 and green #3 (fast green). All foods which have coloring added are colored with one or more of these dyes.
One way to separate the dyes is with paper chromatography. Paper chromatography works because of differences in capillary action, the interaction of compounds with the paper and the solubility of the compounds in solvents. Paper chromatography takes advantage of capillary action by allowing a solvent to rise along a piece of paper. The solvent will carry with it any compounds that are dissolved in the solvent. The distance the compounds are carried depends on how soluble the compound is in the solvent and how much interaction or attraction the compound has for the paper. The more soluble the compound is in the solvent, the further it travels. The more interaction a compound has with the paper, the less distance the compound travels.
In order to quantify how far the compound travels, we measure something called an Rf value. This value is the distance traveled by the compound divided by the distance traveled by the solvent (measured at the center of the spot)
Rf = distance traveled by compound/distance traveled by solvent
When the dye is used in a food, sometimes the other component of the food can interfere with the chromatography test. To insure a correct determination, the dye needs to be separated from the other components.
Identification of the components of a mixture is aided if each substance in the mixture exhibits a unique color (as with most food dyes). However, for dyes with similar colors identification may be aided by examining the fluorescence exhibited under illumination with ultraviolet light. Fluorescence is the phenomenon in which molecules absorb and immediately re-emit light. The emitted light is usually a different color than the absorbed light.
In this lab you will use a chromatography system to look at food dyes. You will then run a second chromatogram to separate dyes used in the coatings of candies. By comparing Rf values, color and fluorescence characteristics of the dyes, you will identify the individual dyes used to create the candy coatings.
NOTE: this is a long experiment. In order for you to finish in three hours, it is necessary for you to plan ahead and work on other parts while you are waiting for your chromatography paper. Do not do any measurements or calculations on your first chromatography paper until the second piece is in the 600 mL beaker.
Procedure:
Obtain a piece of chromatography paper. The paper is 10 cm by 20 cm. Notice that it has a long side and a short side. Be careful at all times to keep the paper clean and dry. Touch the paper only at the edges. If you make mistakes marking the paper, DO NOT ERASE, simply redraw over your mistake. Place the paper so the long edge is horizontal to you. With a PENCIL, lightly make a line across the lower portion (the long side) of the paper 1 cm from the bottom. Make seven X marks along this line evenly spaced from each other and at least 2 cm from the outer edges. Label the X marks B1, B2, Y5, Y6, R3, R40, and G. Make a second line across the long side of the paper 7 cm from the bottom of the paper. Write your initials in an upper corner of the paper.
Use a toothpick to place the appropriate food dye (on top of each bench) on the corresponding X (the diameter of the dye spot should not exceed 3mm). Make each spot dark by applying dye to the same spot several times. Allow the spot to dry between applications.
Into a 600 mL beaker add from the dispensing bottles in the hood 15 ml of butanol, 5 ml ethanol, and 5 ml ammonia (2 M). The dispensing bottles already set to dispense these amounts. Make sure you are using 2M ammonia to make the solution. The level of the eluting solution in the beaker must be BELOW the line on your paper and any dots which go below the line. (i. the eluting solution in the beaker should not touch any of the dye spots) Hold your paper up next to the beaker to check that the eluting solution is at an appropriate level.
On the second run, some of the colors may have separated into several spots. You need to record an Rf value for EACH spot. Consequently, a single color may have one, two or even more Rf values.
Observing Fluorescence characteristics
- Place the chromatogram on the edge, in the drying oven for 5 minutes or more until dry. Place your dry chromatogram under the long-wavelength UV light on the side bench. Record the intensity of the observed fluorescence for each spot.
Post-lab Questions:
- Make a table of the seven FD&C dyes and their Rf values and their fluorescence color and intensity. Make a table of the food and candy colors and their Rf values and their fluorescence color and intensity. Using all the information available to you, for each of the food colorings and candy color determine which of the dyes were used to create the coloring. Explain your reasoning in each case.
- Why is the Rf value alone inadequate to make an identification of a food dye?
- What is the difference between the FD&C dyes and food coloring?
- Why is it so important that you don't erase mistakes on the chromatography paper?
- What purpose do you think the wool served in this experiment?
- Why did we use a pencil instead of a pen to mark the chromatography paper?
- Which FD&C dye seems to have the greatest affinity (interaction) with paper? Which seems to have the greatest affinity for the solvent?
- Why did you make a fresh batch of eluting solution for your second run?
- Make sketches of the two chromatography papers in your lab notebook.
Waste Disposal:
Dispose the eluting solution in the waste beaker in the hood.
All solid waste and chromatography papers may be thrown into the trash can.
All other solutions may be dumped down the sink with water.
If you have any questions concerning this site, please e-mail alkhatibe@sacredheart
Updated August 14, 2019
Lab2 Chromatography(1) - Tagged
Course: General Chemistry Laboratory I (CH 153)
University: Sacred Heart University
