Chemistry 212 Fall Experiment 6: Column Chromatography and TLC of Spinach Evaluation Summary

Transcription

1 Experiment 6: Column Chromatography and TLC of Spinach Evaluation Summary Last Name: Atique Date: 12/3/2013 Lab Partner(s): First Name: Anika Lab Day/TA/Group: Wednesday, Patrick Julien, Group C Labs reports must be typed and chemical structures must be drawn with ChemDraw. Report must not exceed five pages (including this page). Page limit does not include any attached spectra. ** Deductions for hand written report/structures and exceeding page limit ** Report Breakdown Intro/Results: /10 Discussion: /10 Report Total: /20 Other Lab Marks Performance: /6 Prelab Quiz: /3 TA Comments on Report/Performance Report assessment TA (name): TA (signature): Performance assessment TA (name): TA (signature): 1 of 6

2 Experiment 6: Column Chromatography and TLC of Spinach Lab Report Guidelines Include the following in your report (in the following order, use provided table): 1. Title, Date, Your Name, Your Day/Time/TA Atique Anika. Lab day: Wednesday, TA: Patrick Julien, Group: C 2. Objective: (1 mark) What is the purpose of this experiment? The purpose of this experiment is to extract and separate the different pigments of the spinach extract. 3. Introduction: (2 marks) Brief description of the concept/reaction studied extraction, column chromatography and why it is important The extraction process is efficient but is indiscriminate so we end up with a mixture of pigments. This is why separation by column chromatography is important to be able to identify the different pigments present. Extraction is done by grinding the spinach leaves to obtain a pigment mixture and then using a separating funnel to isolate only the pigment extract from the water present. Column chromatography is used to separate the pigments based on their polarity. The more polar pigments tend to associate more with the polar silica stationary phase of the column while the less polar components associate more with the non polar solvent. This difference in the degrees of association separates the pigments into clear layers. 4. Procedure: (2 marks) Summary of lab procedure what YOU did, include KEY observations be concise! Preparing Column: Added a cotton blob and some sand to the column. Dissolved silica in eluent and added this to the column in turns while draining the eluent to make the stationary phase. Add a final top layer of sand to keep the silica in place while always ensuring there was enough eluent present to keep the silica wet. Stopped the stopcock once the eluent level reached the top sand layer. Development and elution of column: Added the pigment extract to the column using a pipette and used eluent until the green layer of pigment dropped below the sand. The addition of eluent was continued to rinse the column until it was colorless. The eluent was added in turns and drained off until all the colored band appeared. Different bands were observed- cloudy white just below the level of the top sand layer, then in descending manner, bluish green, dark green, blue green, cloudy white and finally yellow. The 3 different pigment were then drained into individual beakers (blue-green, yellow and green). TLC: 3 different TLC plates were set up using different ratios of acetone and ether for the mobile phase. 9:1, 8:2 and 7:3. The three different pigments along with the crude sample was spotted onto the plate and the components were allowed to separate. In the 9:1 plate The crude separated into one spot and only the yellow pigment gave a spot. In the 8:2 plate the crude gave three spots 2

3 and the 3 pigments each gave an individual spot. In the 7:3 plate The crude gave 3 spots and the individual pigments each gave their own spot. 5. Results: (5 marks) a. Table 1: Column and TLC Plate Observations Fraction 1 Fraction 2 Fraction 3 b. Figures: TLC Plates Colour R f Value (TLC) Pigment Identity Yellow Blue green Green 9: : : :1-0 8: : :1-0 8: : β-carotene Chlorophyll b Chlorophyll a i. TLC plates diagram of each TLC plate, label spots/solvent front/mobile phase/rf values 3 of 6

4 6. Discussion: (10 marks) a. Discussion of Physical Properties i. Analysis of Column/TLC Plates Effectiveness of column chromatography? Rationalize R f values and order of elution from column. Column chromatography is effective for easy separation of the pigments but the results obtained are not as concrete as TLC. We can observe some zones of mixed colors between the bands where the different pigments are not entirely separated. Also, if the pigments are drain too quickly they tend to run into each other. The Rf value for yellow pigment is the highest, this is because it is the least polar and associates more with the non polar solvent and so is the first one to be eluted from the column. The BG pigment has the second highest Rf and is eluted second while the Green pigment has the smallest Rf value and is eluted last. b. Discussion Questions i. Rationalize the decision to use the 8:2 pet ether:acetone as the solvent system in the column. 4

5 Acetone is more polar than ether, by using a high ether ratio we increase the non-polar degree of the solvent so that the pigments can be separated more effectively. This happens because the non polar pigments easily associate with the solvent and are eluted and as the degree of polarity of the pigments increases the association with the solvent decreases and the pigment hold on to the polar silica for a longer period of time to ensure effective separation. ii. Column chromatography is also very useful for separating mixtures of organic compounds that are not coloured. Briefly explain how you would use column chromatography for the separation of a colourless mixture. If the compounds are colorless then small fractions of the eluent from the column can be collected in labelled test tubes can the components present in these fractions can be identified by TLC. TLC carried out will let us know what and how many components are present. By doing this repeatedly we can identify which test tube contains mixtures of compound and which ones contains individual compounds. iii. Rationalize the observed colour of each isolated pigment β-carotene, chlorophyll-a and chlorophyll-b. Colored pigments absorbs visible light of different frequencies. B-Carotene absorbs light from the blue/violet spectrum and reflects light from the red/ yellow section and so appears yellow. Chlorophyll a absorbs light from the red/ yellow and blue/ violet section but doesn't absorb green light and so appears green. Chlorophyll b absorbs light from the two extremes - red/yellow and dark blue/violet and not from the mid green/blue region and so it appears bluish green 5 of 6