CHE106: C h e m i c a l S c i e n c e C o n c e p t s 8

Chromatography Name

Partners

Date

Objectives

o  To separate a water-soluble ink into it component colors using paper chromatography

o  To separate and identify the components of a mixture using thin layer chromatography (TLC)

o  To calculate the Rf value (ratio to front) of the spots on a developed plate and paper

o  Interpret a TLC plate to determine if a “white powder”is an illegal substance or an over the counter analgesic

Chromatography and Forensic Science

Thin layer chromatography (TLC) is a separation technique frequently used in a forensic laboratory to screen for controlled substances, identify explosives and to analyze inks from forgeries and ransom notes. TLC by itself is not a confirmation technique, however, by comparing unknown samples with standard reference samples, compounds can be identified. TLC data must be supported by other analytical techniques. Paper chromatography is generally not used in a crime lab.

Introduction

Chromatography is a method used to separate, purify and identify the compounds in a mixture. This is accomplished by distributing (partitioning) the mixture between two different phases called the stationary phase (a fixed phase) and the mobile phase (moving phase). The stationary phase is usually a solid; the mobile phase is usually a gas or liquid.

Most chromatography methods work because of a difference in polarity. The stationary phase holds back the components of a mixture of similar polarity causing them to move along more slowly than the mobile phase. Because of differences in solubility of compounds in the mobile phase, and because of the strength of attraction to the stationary phase, some components move faster than others. This results in separation of the compounds. In this experiment you will use paper and thin layer chromatography methods to separate mixtures.

Chromatography methods are classified according to the physical states of the stationary and mobile phases. The term “paper chromatography” is used for the practice of separating mixtures by partitioning the compounds between a mobile liquid phase (the solvent) and a stationary solid phase (the paper).

Thin Layer Chromatography (TLC) is used for identifying compounds and determining their purity. The process is carried out on a plate that is covered with a thin layer of adsorbent (stationary phase). In both processes, the liquid solvent (mobile phase) creeps up the plate or paper by capillary action. Small amounts of samples are spotted in a row near one end of the plate or paper, which is placed, sample end down, in a developing chamber containing the solvent. As the solvent rises, the compounds in the samples move to varying heights on the plate. The individual compounds appear as spots although often either a UV light or an iodine chamber is needed to visualize the spots.

The relative intensity of the spots is not an accurate indication of the amount of compound present. The distance the spot travels on the plate is expressed as “ratio to front” or Rf value given in the equation below.

Rf = ¾¾¾¾¾¾¾¾¾¾¾¾¾¾

The Rf value can be affected by many experimental conditions. The only way to be reasonably sure that a TLC of an unknown is identical to a known compound is to spot a solution of the known compound on the same TLC plate. Figure1 illustrates how to determine the Rf values of the spots.

Rf = ¾¾¾¾¾¾¾¾¾¾¾¾¾¾

Rf = ¾¾¾ = 0.5

Figure 1. Measuring The Rf Value Of A Spot On A Developed Chromatogram

The objective of part A is to develop a strip of chromatography paper that has been spotted with a water-soluble ink, and to calculate the Rfvalue of each spot.

Analgesic drugs are compounds that relieve pain. Some nonprescription analgesics are aspirin, ibuprovin and acetaminophen. Sometimes caffeine is added to these compounds to alleviate drowsiness.

The objective of part B of this experiment is to determine if a white powder found at a crime scene is an analgesic drug by comparing the TLC of your unknown to solutions of aspirin, acetaminophen and caffeine.

Crime Scene

Several packages of mysterious white powders were found on a table in the student center, in the bookstore, library and in some classrooms. A crime scene investigation team documented and packaged the evidence. Preliminary tests were performed on the samples and the team narrowed down the identification to either heroin or some type of over the counter analgesic compound. Your job is to analyze the powders and determine the identity of the compounds. Write the name of each compound under the structure given below. The compound names are acetaminophen, caffeine, ibuprofen, heroin, and aspirin.


EXPERIMENTAL

A. Paper Chromatography: Separation of the Dyes in Fiber Tipped Pens.

Equipment:

·  Developing solvent: 5 mL distilled water

·  Developing chamber: gas collecting bottle plus a 5cmx5cm piece of cardboard

·  Chromatography paper: 3 cm x 12 cm

·  Pencil

·  Ruler

·  Fiber tipped pen

Procedure:

Spotting

Draw a “starting line” in pencil, about 2 centimeters above the bottom edge of a 3 cm x 12 cm piece of precut chromatography paper. Apply two different colored ink spots (about 2 mm in diameter) from the fiber tipped pen on the starting line about one centimeter from the edges of the paper. (See Figure 2.) While the spots air dry, prepare your developing chamber.

Figure 2. Preparation of Chromatography Paper


Developing Chamber

Add enough water to a gas-collecting bottle to give a liquid depth of about 1 cm. Slosh the solvent around for about one minute.

Development

Support the spotted chromatography paper in the slit in the square piece of cardboard. Make sure the paper just barely touches the bottom of the bottle, and does not touch the sides of the bottle. Also make sure the solvent line is below the starting line.

When the solvent front moves to approximately 3 cm from the top of the paper, remove the strip from the developing chamber. Mark the solvent front with a pencil before the paper has time to dry. Let the strip air dry and calculate the Rf of the dye spots. (See Figure 1.)

Attach your chromatogram here. Calculate the Rf value of each spot on your chromatogram. Show your work for credit.


B. TLC of an Unknown White Powder

Equipment:

·  Developing Chamber - 250 mL beaker

·  Developing solvent: petroleum ether, methylene chloride ethyl acetate (1:1:2)

·  watch glass

·  Filter paper

·  Capillary micropipets

·  Pencil

·  Ruler

Procedure:

Sample Preparation

Dissolve half of your unknown in 1 mL of methanol. Three reference solutions (acetaminophen. aspirin and caffeine) are provided. Draw a starting line, in pencil, about one centimeter above the bottom edge of a 5 cm x 8 cm TLC plate. Draw four evenly spaced dots, in pencil, on the starting line about a half of a centimeter from the edge of the plate. Spot your unknown on the third dot. Spot the reference solutions on the remaining dots using a clean micropipet for each spot.

Developing Chamber

Place a piece of folded filter paper (Figure 3) and about one centimeter (depth) of developing solvent in a 250 mL beaker. Cover the beaker with a watch glass, and slosh the solvent so that the filter paper is wet.

Figure 2. Preparation of Chromatography Paper

Development

When the spots are dry, place the spotted plate in the developing chamber. Make sure the solvent level is below the starting line. When the solvent front is about one centimeter from the top of the plate, remove it from the developing chamber. Immediately mark the solvent front height with a pencil. Let the plate air dry. Hold the chromatogram under a UV lamp and circle the spots. Identify the spots in the unknown lane by comparing the unknown spots with the known spots. Identify your unknown from the following list of brand name drugs.

Brand Name / Ingredients
Anacin® / aspirin, caffeine
Bayer® Aspirin / aspirin
Excedrin® / acetaminophen, aspirin, caffeine
Tylenol® / acetaminophen
NoDoz / caffeine

Unknown number: ______

Draw your TLC plate and identify the spots. Your TLC plate should be discarded when your analysis is complete.

List the ingredients in your unknown.

Identify the name brand of your unknown: ______

Explain how you determined the identity of the white powder.


Questions

1.  What does the technique of chromatography allow you to do?

2.  Explain the importance of the following statements.

a. The level of the solvent must be below the starting line.
b.The spots should be concentrated yet small in diameter.

3.  Why is the starting line drawn for positioning the spots drawn in pencil, never in ink?

4.  The identity of a suspect material is not considered definitive. Explain this statement.

5.  Often a forensic scientist has to explain the TLC technique to a jury. Discuss how you would present the following chromatography concepts so that everyone would understand.

a.  Capillary action

b. Visualization using a UV light