NAME ____________________________________ PERIOD _________
LAB PARTNER ____________________________________
FORENSIC SCIENCE – MS. STARK-HOUCK
INTRODUCTION
Chromatography separates suspect drug mixtures into their individual components. In the field, forensic investigators will frequently use simple color tests to give a very general identification of suspect drugs. In the laboratory, more specific analysis is necessary.
Paper chromatography separates a sample on specialized paper (solid phase) using a variety of liquid solvents (liquid phase). The solvent carries the mixture up the paper by capillary action.
The more soluble a component is in the solvent, the further it will travel up the paper with the solvent front. Less soluble compounds travel much more slowly through the paper and remain closer to the sample origin.
In this two-day laboratory, your group will separate four drugs into their components and measure the retention time (Rf value) for each component.
MATERIALS (per lab group)
STEP ONE
12 x 14 cm rectangular chromatography paper metric ruler number 2 pencil black marker (amphetamine) purple marker (barbiturate) green marker (GHB) orange marker (heroin)
4 paperclips
2 pencils
2 beaker with 15 ml liquid phase (methanol/acetone)
Protective eye wear
STEP TWO
4 completed chromatograms metric ruler number 2 pencil
PROCEDURE
STEP ONE - CHROMATOGRAPHY

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Divide chromatography paper LENGTHWISE into 4 even long strips. USE CAUTION!
Chromatography paper will absorb oils from your hands – handle by sides or top edge only!!! DO NOT USE PEN ON CHROMATOGRAPHY PAPER IT WILL
SEPARATE!
Use a number 2 pencil to write your lab group’s initials and the class period in the upper right corner of each strip of chromatography paper. Cut bottom of each strip into the shape of a point.
Measure 2cm up from the bottom (pointed end) of the each strip and draw a line lightly with pencil across the entire length of the chromatography paper.
Measure 2cm in from left edge of filter paper and place a dot of black marker along the light pencil line.
Leave 2 cm of space and place a dot of purple marker along the light pencil line.
Leave 2cm of space and place a dot of green marker along the light pencil line.
Leave 2cm space and place a dot of orange on the last strip of chromatography
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Allow sample dots to air dry (30 sec), then gently roll TOP of strip over a pencil until the tip of the point just comes in contact with the bottom of the beaker.. Trim any excess at top so that you have just enough to paper clip to the pencil.
Use a paper clip to keep the strip in place closest to the pencil. Attach a second strip to the pencil but do NOT let either strip touch each other or the sides of the beaker.
This will be your chromatogram.
Bring ONE beaker up to teacher to obtain mobile phase. CAUTION methanol fumes should be avoided – USE HOODS AT ALL TIMES
Carefully pour HALF of the mobile phase into each beaker – DO NOT pour over samples!!!
Observe samples as they migrate with the solvent front through the paper.
Allow the mobile phase to run until either 5 minutes before the end of the period or until solvent reaches near paper clip.
Remove the two pencils from beakers. LEAVE beakers in hoods to evaporate. Place chromatography strips on clean paper. Carefully remove paperclip, flatten strips.
Label paper with group names and period. Put the plain paper in hood to dry and return all equipment EXCEPT BEAKERS for next class.
STEP ONE - OBSERVATIONS
1. Which drug sample contained the most components (pigments)?
2. Which drug sample contained the least components (pigments)?
3. Which drug sample component (pigment) moved the greatest distance up the filter paper?
4. Which drug sample component (pigment) moved the shortest distance up the filter paper?

STEP TWO – CALCULATING Rf VALUES
Chromatography works because different substances, (such as different pigment molecules) when dissolved in a specific solvent, will migrate through paper at different rates. The rate at which each specific substance moves on the chromatography paper is determined by:
1) The solubility the unknown substance in the particular solvent.
2) How well the unknown substance adheres to the chromatography paper.
Once the individual types of molecules have been separated by chromatography, they can be identified by calculating "Rf" (Relative front) values.
The relationship of the distance moved by a pigment to the distance moved by the solvent front is specific for a given set of conditions.
We call this relationship the Rf and define it as follows:
Rf = DT/SF
Distance moved by the pigment
Distance from pigment origin to solvent front
Thus, simple paper chromatography can be used to identify substances both qualitatively
(by color) and quantitatively by its characteristic Rf value.
Calculating the Rf value:
If the solvent front is 6 cm then the Rf value for the pigment at 3 cm would simply be 0.5
Calculate the Rf values of the four drug samples on your chromatogram.
Use the metric ruler to measure the distance that the solvent front traveled.
Then use the metric ruler to calculate the distance each pigment contained in each drug sample traveled.
Write in your answers in the Data Table under the appropriate drug samples you tested while making the chromatogram.

COMPLETE THE ENTIRE DATA TABLE, ATTACH YOUR CHROMATOGRAMS TO ONE OF
YOUR COMPLETED LABS AND TURN IN FOR GRADE.
CHROMATOGRAPHY DATA TABLE
SAMPLE
BAND #1
Distance traveled in cm
BAND # 1
Rf value =
DT/SF
BAND #2
Distance traveled in cm
BAND #2
Rf value =
DT/SF
BAND #3
Distance traveled in cm
BAND #3
Rf value =
DT/SF
BAND #4
Distance traveled in cm
BLACK
Amphetamine
BAND #4
Rf value =
DT/SF
CALCULATION WORK SPACE:
PURPLE
Barbiturate
GREEN
GHB
ORANGE
Heroin