Hands-on Chromatography Sheryl K. Hoffmann Concordia College CONASTA 60 Wednesday 11.35-1.40 then LUNCH

Definitions Definition 1: Chromatography is a technique for separating similar compounds from one another by using differences in the strength of their adsorption on an inert material. Adsorb: hold (molecules of a gas or liquid or solute) to its surface Absorb: swallow up, incorporate, take in Definition 2: Chromatography is a technique which can be used to separate the components of a mixture. The method depends upon differences in the abilities of the components to adhere to a porus solid such as silica gel, called the stationary phase differences in the abilities of the components to be dissolved in a stream of liquid or gas, called the mobile phase, which is passed over the solid.

Main Types Paper Chromatography Thin Layer Chromatography (TLC) Column Chromatography High Performance Liquid Chromatography (HPLC) Gas-Liquid Chromatography

Basic Method Basic Method for Paper Chromatography Take a piece of chromatography paper and mark a pencil line 2 cm from the bottom. Mark the positions of the samples evenly across the paper with a cross, leaving at least 2cm at each side. Spot the samples on the positions marked, and allow to dry. Pour < 1cm of solvent into a beaker, test tube or chromatography tank. Lower the chromatography paper into the solvent in a beaker & support. Allow the run until the solvent nearly reaches the top of the paper. Mark final solvent height if Rf values are to be calculated.

Spotting Technique Spotting Methods To get good separation of bands, it is important to keep the spot as small as possible & as concentrated as possible. Capillary Tubes, Toothpick & Pasteur Pipettes can all be used to spot chromatography paper

Tips If the spot is large, components start from different origins and will thus blur any separation that would normally occur. Try a quick dabbing motion, the longer the liquid is in contact with the paper the larger the spot If the spot is not very concentrated the resulting bands, once spread over a wider area, will be even fainter. If the solution to be spotted is not very strong, spot several times, being sure to allow the spot to dry in-between, a hair drier will speed up the drying process.

2. Comparison of 1M NaCl & Water in Separating Food Colour Hands On Activities 1. Practice Spotting 2. Comparison of 1M NaCl & Water in Separating Food Colour 3. Sticks of Chalk 4. Counterfeit Smarties? 5. Comparing Black Inks Go through & explain each practical (because they never read the notes) We will be using a plastic ice-cream container as a Chromatography tank FIRST - everyone practice spotting technique on a filter paper look at the TLC plate & try spotting on it NOTES: 4 activities to try – don’t all start at #1 At least try washing a Smartie with 2 drops water Towards the end we will look at everyones’ results + Activity 5 & 6 + watch a short video

Activity 2 – Food Dyes LEFT RIGHT 1M NaCl vs water (Blue & yellow) and (blue & red) separate more completely in 1M NaCl

Activity 3 - Chalk

Activity 4 - Smarties

Activity 5 – Black Pen Ink Clearly the pen third from the right matches the ink sample on the right hand side

Activity 6 – Geranium TLC Paper LEFT RIGHT TLC vs Paper Clear TLC gives better separation than paper TLC Paper

Activity 6A – Frozen Spinach

Activity 6A –Spinach

Activity 7 – 2 Columns Large column in a Quickfit separating funnel & a small column in a Pasteur pipette