Introduction to Chromatography Science@CSCB Introduction to Chromatography The lab session that forms part of the Science@CSCB workshop will take about an hour and a half. During this time you will each get a chance to try out paper chromatography and to take part in some demonstrations involving an underwater crystal garden and the chemical snake. As you all know scientific experiments are based on making observations. When chemists set up a chemical reaction in the lab to make something new they don’t know what they have made by just looking at the liquid or crystals in the round bottom flask. They must set on the quest to identify what they have made. They build up data to support their hypothesis and to discover what they have made. Sometimes it’s enough to look and take measurements but not everything is visible…….

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

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). Research Pharmaceutical industry

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).

Chromatography is a technique used 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).

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 Gas Column

Your mission should you choose to accept is to…….. Analyse coloured markers 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. Test whether they contain E133 (blue), E122 (pink), E124 (red) or E110 (yellow)

In the animation below the red molecules are more soluble in the liquid (or less volatile) than are the green molecules.

Thin Layer Chromatography Sample – marker Standard – food dyes Stationary phase – chromatography paper Mobile phase - water 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.

Structures of E numbers….. E122 pink E110 yellow E124 red E133 blue

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

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 You must now calculate an Rf value for each spot. Rf = Distance from the start to the middle of a spot Distance from start to finish point of the water 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.

DNA Profiling Examining DNA for a pattern to compare two or more samples or individuals.

All human chromosomes have sections of DNA with no known functions Principles of DNA Profiling https://www.twig-world.com/film/forensics-dna-profiling-1484/ All human chromosomes have sections of DNA with no known functions These sections have short base sequences These sequences repeat over and over They are inherited from parents Their length and position are unique to each person

DNA Profiling Steps of DNA Profiling Cells are broken down and DNA is released. (2) If DNA sample too small Can be expanded by polymerase Chain reaction (PCR).   (3) The DNA is cut into fragments using restriction enzymes (cut at particular base sequences). (4) The fragments are separated on the basis of their size using electrophoresis.  (5) The pattern of DNA fragments is analysed and compared between individuals.

(1) Cells broken down by centrifugation

(2) DNA cut into fragments using enzymes from bacteria

(3) Fragments separated on the basis of size – Gel Electrophoresis

(4) A DNA profile is created and patterns of individuals are compared

Application 1 : Forensic Science Jake and Emma's four children were grown and had families of their own. Jake was murdered, and the police suspected his oldest child (#1) as the culprit, who had unexplained scratches on his arms and face. DNA was extracted from under the victim's fingernails, and that was compared with those of the other members of the family. Be a real Sherlock Holmes: was the culprit really the oldest child?

Application 2 : Paternity Tests

What are these? How do they work? KS4/5 starter Boots are now selling home paternity testing kits in all their stores. What are these? How do they work? © Snapshot Science, 2011

The mother, father and child all have saliva taken from their mouth. KS4/5 Activity The mother, father and child all have saliva taken from their mouth. This gets sent to a laboratory where DNA profiling takes place. Click on the equipment to try it out. © Snapshot Science, 2011

The results are studied and the parents informed. KS4/5 Activity The results are studied and the parents informed. © Snapshot Science, 2011

What do the results show? KS4/5 Activity Family A What do the results show? Mother Child Father © Snapshot Science, 2011

What do the results show? KS4/5 Activity Family B What do the results show? Mother Son Daughter Father © Snapshot Science, 2011 Image: Steve Polyak at wikimedia.org