Analía Bellizzi – Chemistry Classes

Ronald Reagan Senior High School

LAB SEPARATION METHODS #2 Chromatography

Background

Almost all substances we encounter everyday are impure; that is, they are mixtures. That’s why a major focus of research in chemistry is
designing methods of separating and identifying components of mixtures.
Many separation methods depend on the different physical properties of the components in a mixture.

  • Filtration relies on the different states of the substances (solid vs. liquid or dissolved in a liquid)
  • Centrifugation depends on differences in density. The denser substances will precipitate faster using a centrifuge.
  • Distillation makes use of differences in boiling points of the various components.
  • Chromatography is based on the differences in solubility and adsorption.

The word chromatography, which comes from two Greek words “chroma” = color and “graphy” = to write, was used first to separate colored components of plant leaves. Today, we can also separate substances which are colorless and then use a locating agent, which will make visible to the naked eye or in presence of UV light substances that are colorless. An example of this separation is used to identify a mixture of aminoacids using ninhydrin as locating agent.

There are several types of chromatography are commonly used, among which are

  • Paper chromatography
  • Thin-layer chromatography or TLC
  •  Liquid-liquid chromatography,
  • Gas chromatography,
  • High performance liquid chromatography or HPLC.

Chromatography is so useful that some form can be found in most scientific laboratories around the world. It is widely used in forensic chemistry crime laboratories; the FBI maintains a library of chromatograms of inks that are used commercially. The first time that chromatography of inks were used, a man in Miami falsified travel and expense vouchers. However, the pen he used had ink that wasn’t available commercially until 3 years after the trips had taken place.

In paper chromatography

 
  • A drop of the mixture is placed on a pencil line near the bottom of a rectangular piece of chromatography paper
  • The paper is the stationary phase and the line is called the origin.
  • The lower edge of the paper is placed in a specific solvent chosen based on the solubility of the substances to be separated.
  • The chamber (container with the solution) must be closed at all times to allow the vapors of the solvent to concentrate inside.
  • Capillary action causes the solvent to flow up the paper at a uniform rate creating a “wet” line across the paper.This line is called the solvent front.
  • As the solvent front moves over the stationary phase, it passes through the mixture and the pigments are carried along at different rates, and so, they get separated into discrete (individual) zones.
  • The distance travelled by each pure substance will depend on:
    • Solubility: The more soluble a component is in the mobile phase the faster it will be transported along the stationary phase.
    • Adsorption: It refers to the ability of a substance to ‘stick’ (or be adsorbed) to a surface. The stronger a component is adsorbed to the stationary phase, the slower it will be transported by the mobile phase.
  • The ratio between the distance travelled by one pigment and the distance travelled by the solution is called Rf (Retardation Factor)
  • Rf is an intensive property. If the Rf values are the same, most likely we will have the same compound

Important Parts of a Chromatogram:

Table Header Table Header

STATIONARY PHASE

The surface where the chromatography takes place; in our case: Paper

MOVING PHASE

A solvent chosen according to the properties of the components in the mixture (eluting solution). In our case, we use a 0.1% NaCl solution - 0.1 g of salt dissolved in 100 cm3 of distilled water. 

ORIGIN

The pencil line near the bottom your chromatography paper

SOLVENT FRONT

The wet / dry line that shows how far the solvent travelled.

The ratio between the distance travelled by one pigment and the distance travelled by the solution

Separation Method #2: Chromatography of M&M Candies
LAB INSTRUCTIONS

Purpose of the lab:

Materials:

(Verify that you have all the materials and notify me if you are missing anything)

  • Chromatography paper
  • M&M Candies (One of each color)
  • 1 Piece of Paper Towel
  • Eluting Solution (0.1% NaCl solution)
  • Capillary tube or micro-pipette (BE CAREFUL, VERY FRAGILE!!!)
  • Chamber (600 mL Beaker)
  • Square glass (To cover the chamber)
  • 50 mL labelled distilled Water
  • plastic pipette
  • Pencil, Ruler, stapler

Procedures

A. Preparation of the pigments

  1. Place every candy you got and order them in line on a paper towel with the “m” mark facing down.
  2. Use a plastic pipette to place ONE SMALL DROP of distilled water on each candy you have.
  3. Do not move the candies so the water does not get spilled.
  4. Put 1 cm high of NaCl solution in the chamber (beaker) and cover tight with aluminum foil. This prevents the evaporation of the water vapor inside the bea
  5. ker and helps to accumulate enough vapor to help your chromatography to be successful.

Procedures

B. Preparation of the Chromatogram

  1. On the chromatography paper, use a PENCIL to draw a “spotting line”. This line should be at 1.5 cm from the base (look at the graph before doing it)
  2. Divide the paper into sections. Label each section, using pencil with the color of the candy you will 
  3. use in that zone (see the table below) The number of sections should equal the number of colors you have.
  4. Place a spot of each M&M color ON the pencil line, you drew using the capillary tube.
  5. Rinse the capillary tube using distilled water and repeat the process for the other colors
  6. Dry the paper using the hair dryer and repeat the process three times for each color to concentrate the pigments. BE SURE THAT THE SPOT IS AS SMALL AS POSSIBLE.

C. The Eluting or Mobil Phase

  1. Roll chromatography paper so that ends may be taped together or stapled. DO NOT OVERLAP!!!
  2. Measure the height of the liquid in the chamber an
    d the height of your pencil line. The color dots should not touch the solution.
  3. Place the paper cylinder in the chamber with eluting solution very fast and cover the chamber using the aluminum foil (lid) as soon as possible. The paper should NOT touch glass on the side of the chamber.
  4. Allow the eluting solution to move until the moving face is near the top end of the paper.
    Be careful. The solution front does not have to reach the top of the paper!
  5. Remove the paper from the chamber and place it on a paper towel, mark the maximum level reached by the solution. (be careful not to break your paper)
  6. Dry your paper so you can mark the results and perform calculations.

Note:
Since you will have only one “chromatogram” per group, you can take a picture and use that
as part of your lab report.

IMPORTANT: MARK THE DISTANCES FOR EACH PIGMENTON THE PAPER SO I CAN SEE THEM AND COMPARE THE RESULTS WITH MINE.

D. Calculating your Rf values.

Rf= distance spot moved divided by the distance the solvent moved. Each color will have an Rf value

  1. To get the “distance spot moved” measure from spotting line to center of spot. (the center of the strongest color)
  2. To get the “distance solvent moved” measure from spotting line (pencil line) to solvent front
    Distance moved by the solvent front = ……..…….cm (2 DECIMALS)
  3. Show the calculations for every Rf  in the table above. SHOW UNITS

E. Data Table

Complete the table below

F. Analysis Questions

  1. Which m&m candy sugar coats contain only one pigment?
  2. Which are the units of Rf?
  3. Why do colors move at different rates?
  4. Why do you measure from the center of the strongest color spot?
  5. Which is the color that moved the most?
  6. Which is its Rf value?
  7. Which is the color that moved the least?
  8. Which is its Rf value?
  9. Choose one color and compare the distance you recorded with the distances recorded by other groups. Are they the same?
  10. For the color you choose in question 9, compare the Rf value you obtained
    with the Rf values from the other groups. Are they the same?
  11. How can you explain these results?
  12. Which are the real-life applications for this lab?
  13. We are investigating a crime. The killer left a message using pen X. We got
    three suspects, A, B, and C. We performed a chromatography paper to discover who is the killer. Who is the killer?
  14. Use the graph to calculate the Rf value for the dot near the solvent front. 
    Use the following graph to answer questions 15 and on.

  15. What does “dw” represent in the Figure?
  16. Which pigment is not present in the sample mixture?
  17. Which is the Rf value of W?
  18. Which is the Rf value of X?
  19. If a sample moves together with the solvent front. Which will be the
    value of Rf?
  20. If the substance does not move at all during the chromatography, what
    can we say about the substance?

Lab report must include the following items in the following order: 

  1. Title

  2. Purpose of the lab

  3. Materials

  4. Procedures

  5. Lab Setup (Preparation of pigments and beaker with chromatogram inside)

  6. Chromatogram, showing the stains with the distances covered by each pigment and the distance for the solvent front. (the chromatogram should be an exact copy of the original used in your group. 

  7. Calculations of Rf for each color present in each candy. Show your calculations clearly with correct amount of significant figures and units (if applicable)

  8. Data table. (Showing the distances and the Rf for each candy).

  9. Analysis questions (all of them) 
    You can print the questions and then answer them or copy the questions and resolve. You can use pencil to resolve the word problems.