1. 1/17/2015 1 Thin-Layer Chromatography References: Pavia:Experiment 5A; pp. 36 – 39 Technique 2.0; pp. 801 – 812 Note:The above reference material is not currently in the GMU subset (ISBM-13: 978-1-111-40114-6) edition of the main Pavia text (ISBN-13: 978-1-4390-4392-7), but is being E-Mailed to students until a new subset edition is printed

2. 1/17/2015 2 Thin-Layer Chromatography Background Thin-Layer Chromatography is an important technique used to separate non-volatile compounds in a mixture It is one of several types of chromatographic techniques, ranging from thin-layer chromatography and column chromatography, which are relatively simple and inexpensive, to high-performance liquid chromatography, which is very sophisticated and expensive Thin-layer Chromatography involves the distribution of the compounds (or ions) between two phases, one of which is stationary and the other moving It is similar to solvent extraction, which depends on the relative solubilities of the compounds in two solvents

3. 1/17/2015 3 Background (con’t) In chromatography, the separation process depends on differences in how strongly the components in the mixture (analytes) are adsorbed to the stationary phase and how soluble they are in the moving phase These differences depend on the relative polarities of the components Thin-Layer chromatography is performed on a sheet of glass, plastic, or aluminum, which is coated with a thin layer of adsorbent material, usually silica gel, aluminum oxide (alumina), and is referred as the stationary phase This experiment will utilize Polyester Support Silica Gel Plates (5 – 10 cm) Thin-Layer Chromatography

4. 1/17/2015 4 Background (con’t) After the sample solution has been applied on the plate adsorbent stationary phase, a solvent or solvent mixture (known as the mobile phase) is drawn up the plate via capillary action Separation of the analytes (compounds or ions), occurs because different analytes ascend the TLC plate at different rates The mobile phase has different properties than the stationary phase For example, silica gel, a very polar substance, would be used with a non-polar mobile phase, such as heptane After the solvent phase has moved to the top of the plate via capillary action, the spots, representing the analytes are visualized by one of several techniques Thin-Layer Chromatography

5. 1/17/2015 5 Background (con’t) In this experiment, Iodine (I 2 ) will be used to visualize the compounds (or ions) Quantification of the results is represented by the: Retention Factor, R f A substance whose structure resembles the stationary phase will have a low Rf A substance that has a similar structure to the mobile phase will have a high Retention Factor The Retention Factor will change whenever either the mobile or stationary phase changes Thin-Layer Chromatography

6. 1/17/2015 6 Procedure The detailed instructions from the Pavia text have been E-mailed to you Obtain a pre-cut plastic backed Polyester Support Silica- Gel Thin-Layer-Chromatography plate (5 cm – 10 cm) Note:Do not excessively bend these flexible plates nor touch the surface as they could crack causing the adsorbent to flake off Very lightly draw a line with a pencil across the plate about 1 cm from the bottom (see slide 7) Starting about 1.0 cm from the left edge of the plate, mark off five 1 centimeter intervals (see slide 7) These are the points at which the samples will be spotted, i.e., where the sample dissolved in the solvent will be deposited using an open-ended capillary tube Thin-Layer Chromatography

7. 1/17/2015 7 Thin-Layer Chromatography Polyester Support Silica Gel Plate (5 cm - 10 cm) Sample Spotting Locations 1 - cm 9-Fluoronone 9-Fluorenol Fluorene Unknown Mixture Solvent Front

8. 1/17/2015 8 Procedure 9 (con’t) The sample matrix for this experiment is as follows:  (A) 1% 9-Fluorenone- A Ketone  (B) 1% 9-Fluorenol- An Alcohol  (C) 1%Fluorene- A Hydrocarbon  Unknown (two of the above)  Mixture (1:1:1) of all 3 compounds above The compounds are dissolved in Acetonitrile In a hood, moving left to right on the plate, use a micropipet to spot a drop of sample from each solution above on the indicated spot on the plate Allow the solvent to evaporate until you cannot see the wet spot on the silica gel Thin-Layer Chromatography

9. 1/17/2015 9 Procedure (con’t) Development Chambers  Glass jars with a plastic screw top  Add development solvent Methylene Chloride (Dichloromethane) to the chamber  Note:The development solvent level in the jar must be below the origin where you spotted the compounds  Do not insert a filter paper liner into the chamber as suggested in the Pavia Notes Once the plate has been inserted into the development chamber, the solvent will begin to travel past the spots via capillary action to separate the compounds chromatographically Thin-Layer Chromatography

10. 1/17/2015 10 Procedure (con’t) Solvent development always takes place with the plastic caps screwed onto the chamber to ensure the gas phase is saturated with solvent molecules and the solvent in the silica gel does not evaporate during development The solvent development process usually takes about 10 minutes At the conclusion of the development process remove the plate from the jar and use a pencil to mark the point where the development solvent front has stopped Set the plate on a piece of paper towel to dry When the plate is dry, place the plate in a jar containing a few crystals of iodine (I 2 ) Thin-Layer Chromatography

11. 1/17/2015 11 Procedure (con’t) Cap the jar and warm it gently (setting ~ 3) on a hot plate until the spots begin to appear Remove the plate from the jar and lightly outline all the spots that became visible with the iodine treatment Note:Very gently circle each spot with a pencil as the spots can bleach out Using a ruler marked in millimeters, measure the distance that each spot has traveled relative to the solvent front Calculate the Rf values for each spot Explain the relative positions of the three known compounds in terms of polarity Identify the unknown compound Thin-Layer Chromatography

12. 1/17/2015 12 Procedure (con’t) Lab Report  From the information on the plate, use Powerpoint to create a picture of the plate showing the initial level of the solvent, the initial locations where the samples were deposited, the final position of the spots after development, and the point where the solvent advance stopped (See example on slide 7)  Convert Powerpoint file to “PNG” format and insert picture into the MS Word lab report  Do not submit the plate to instructor  The MS Word template for the data sheet has been E-Mailed to you. Insert this file into your lab report  Enter the data into the data sheet Note:Be sure to include Molecular & Structural formulas of the compounds  Insert data sheet into lab report Thin-Layer Chromatography

13. 1/17/2015 13 Thin-Layer Chromatography Data Sheet for TLC Experiment Unknown Number: Known Comp #1: Structure: Known Comp #2: Structure: Known Comp #3: Structure: Solvent and Concentration for Spotting: Solvent: Conc: 1 % Solvent for Development: R f Values: Spot 1 = Spot 2 = Spot 3 = Spot 4 (Unk) = Spot 5: Comp #1 Comp #2: Comp #3: Acetonitrile Methylene Chloride (Dichloromethane) Molecular Formula Structural Formula 1234 1234 1234 1234