STEM Introduction to Chromatography

What is Chromatography? Derived from the Greek word Chroma meaning color, chromatography provides a way to identify unknown compounds and separate mixtures

3 Chromatography is a method of physically separating mixtures of gases, liquids, or dissolved substances. Chromatography can be used to identify drugs, poisons and many other substances. Separation is determined by the molecular size and/or charge

Mixtures & Compounds Mixture – Two or more substances that are mixed together, but not chemically combined. Examples of mixtures... Air – mixture of gases Soda pop – mixture of soda syrup, water, and CO2 gas Kool-Aid – mixture of water, sugar, and flavor crystals Examples of compounds... Salt –Sodium and chlorine combined chemically Water –Hydrogen and oxygen combined chemically Carbon Dioxide – Carbon & oxygen combined chemically Compounds – Two or more elements that are chemically combined.

Applications of Chromatography Forensics Research Pharmaceutical industry

All forms of chromatography involve Two Phases 1. stationary (absorbent) phase –the material on which the separation takes place 2. mobile phase -the solvent transports the sample Types of Chromatography - 5 of them

5 Types of Chromatography… Paper HPLC Gas Thin Layer Column

Chromatography Application: Matching of Ink Types Chromatography can be used to physically separate the components of inks. There are many types, such as:  HPLC—high-performance liquid chromatography  TLC—thin-layer chromatography  Paper Chromatography

Gas Chromatography -a gas separates the components -they are then ionized and an electrical signal is recorded -mobile phase - carrier gas -stationary phase - thin film of liquid

HPLC High Performance Liquid Chromatography (HPLC) - done at room temp. - advantageous for chemicals that are heat sensitive or volatile - example LSD - a liquid (mobile phase) is pumped through a column containing fine particles (stationary phase)

TLC Thin - Layer Chromatography (TLC) -A plate is coated with a granular gel - usually silica gel or aluminum oxide (stationary phase) - the substance to be separated is carried up the plate by capillary action - the substance with most affinity for the plate will rise the farthest.

Paper Chromatography Same as TLC but paper is used as stationary phase

Electrophoresis - Similar to TLC but the substance is separated through a gel by electric current - Due to different size and charge substances will move across the plate at different speeds.

14 Paper Chromatography Reflects Differences in Components such as:  Raw material  Weight  Density  Thickness  Color  Watermarks  Age  Fluorescence (radiation emitted by exposure to ultraviolet light)

15 Chromatography as Evidence  Class characteristics may include general types of pens, pencils or paper.  Individual characteristics may include unique, individual handwriting characteristics; trash marks from copiers, or printer serial numbers.

16 Paper Chromatography of Ink Two samples of black ink from two different manufacturers have been characterized using paper chromatography.

Process of Paper Chromatography (Click to advance)

Retention Factor (R f )  A number that represents how far a compound travels in a particular solvent  It is determined by measuring the distance the compound traveled and dividing it by the distance the solvent traveled.

19 Solve for R f

Calculations Black Ink Blue Dye Red Dye Orange Dye

Your mission, should you choose to accept it, is to……..  Analyse several ink samples to connect them to a suspect

Paper Chromatography  Sample: writing implements (pen, marker, etc.  Stationary Phase: chromatography paper  Mobile Phase: water, alcohol, acetone

So what will happen?  Each dye will travel up the paper at different speeds  The speed depends on the solubility of the dye in the regent and its interaction with the paper  The dyes are composed of different molecules with different characteristics

Example: Calculation of Results

Calculation of Results Analysis = Calculate an R f value for each spot. R f = Distance from start to the middle of a spot Distance from start to finish point of the regent