1. Introduction to Chromatography
STEM

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

3. 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

4. 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.

5. 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

6. 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

7. 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

8. 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

9. 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

10. 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

11. 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

12. 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

13. 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

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

17. Process of Paper Chromatography
(Click to advance)

18. 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

19. Chapter 15
Solve for Rf
Kendall/Hunt

20. Calculations
Black Ink
Blue Dye
Red Dye
Orange Dye

21. 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.

22. 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.

23. 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

24. 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.

25. 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.