Introduction to Chromatography STEM

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

Separation is determined by the molecular size and/or charge Chapter 15 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 Kendall/Hunt

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 Chromatography is a technique for separating mixtures of compounds identifying unknown compounds establishing the purity or concentration of compounds monitoring product formation in the pharmaceutical and biotechnology industries Chromatography is widely used by forensic teams to analyse blood and urine samples for drugs, for paint analysis and testing for the presence of explosives. Most chromatography uses modern instrumentation and involves placing the sample to be analysed on a support (paper or silica) and transporting it along a mobile phase. The mobile phase can be a liquid (liquid chromatography) or a gas (gas chromatography). Pharmaceutical industry Research

All forms of chromatography involve Chapter 15 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 Kendall/Hunt

5 Types of Chromatography… Thin Layer Paper Most chromatography uses modern instrumentation and involves placing the sample to be analysed on a support (paper or silica) and transporting it along a mobile phase. The mobile phase can be a liquid (liquid chromatography) or a gas (gas chromatography). HPLC Column Gas

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 Chapter 15 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 Kendall/Hunt

HPLC High Performance Liquid Chromatography (HPLC) Chapter 15 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) Kendall/Hunt

TLC Thin - Layer Chromatography (TLC) Chapter 15 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. Kendall/Hunt

Same as TLC but paper is used as stationary phase Chapter 15 Paper Chromatography Same as TLC but paper is used as stationary phase Kendall/Hunt

Chapter 15 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. Kendall/Hunt

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

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.

Paper Chromatography of Ink Chapter 15 Paper Chromatography of Ink Two samples of black ink from two different manufacturers have been characterized using paper chromatography. Kendall/Hunt

Process of Paper Chromatography (Click to advance)

Chapter 15 Retention Factor (Rf) 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. Kendall/Hunt

Chapter 15 Solve for Rf Kendall/Hunt

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 Each marker contains food dyes. Your challenge is to separate the food dyes and identify them. You will be given samples of the following E numbers: E133 (blue) and E122 (pink) E124 (red), E110 (yellow) E numbers are codes for food additives and are usually found on food labels throughout the European Union You will be given samples of known food dyes to compare the ones in the marker to. You must generate data for the food dyes in each marker by comparing the Rf values.

Paper Chromatography Sample: writing implements (pen, marker, etc. Stationary Phase: chromatography paper Mobile Phase: water, alcohol, acetone The principle is that the inks are water soluble and travel up the paper. Each marker is composed of more than one dye so each dye will separate as it moves up the paper with the water. The inks used in the dye are soluble in water and so will travel up the paper. The sample refers to what you want to test which in this case are colour markers. The standard refers to the reference material you will compare the sample to. In this case it is Goodalls food dye. Each food dye contains E numbers. The stationery phase is the support you apply your sample to – in this case chromatography paper. You spot each sample on the paper and allow it to dry. The mobile phase is water and the paper is placed in the water. Since each E number is a different chemical with its own characteristics and properties it will behave differently when you run it on the chromatography paper.

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 First you spot the food dye. Check on the bottle and confirm which E numbers are contained in the food dye. Make a note of it in your workbook. Then you spot your marker. Make sure you note down which spot is which because once they move along the paper you won’t be able to recognise them. The key point is that you don’t just visually compare the two. You must calculate a value for each spot and compare them so you can conclusively say whether the E numbers in the food dye are the same as the ones in the marker.

Calculation of Results Analysis = Calculate an Rf value for each spot. Rf = Distance from start to the middle of a spot Distance from start to finish point of the regent The Rf value is a measure of how far each spot has moved relative to the solvent front. Each dye will have its own Rf value so you can compare Rf values and confirm whether food dyes were used in making these markers.