Report-sheet-chromatography PDF

Preview

Summary

Download Report-sheet-chromatography PDF

Description

CHEM 212Report Sheet2018-2019

Column Chromatography Group Experiment 3 – Individual Lab Report Last Name:

Nguyen

First Name:

Lucy

Lab Partner(s):

Tapi Sambabure

TA Name:

Lida

Date Lab Performed:

2018-11-23

Group:

☒A

☐B

☐C

Comments for Grading TA: IMPORTANT: If you performed your lab on a day other than your scheduled day, and/or with a TA other than your regular TA, please provide details here.

REPORT MUST NOT EXCEED FIVE PAGES This page limit includes the cover page, but does not include spectra or references. Deductions will be applied for handwritten report/structures/diagrams, submitting non-pdf files, and exceeding the page limit LATE PENALTY: 2 MARKS PER DAY LATE Reports will not be accepted after 7 late days

This template is designed to work with the desktop version of Office 365, available to McGill students free of charge at https://www.mcgill.ca/it/o365 Please see Appendix B: Lab Report Guidelines for detailed descriptions of report requirements, grammar guidelines, and template tips.

1

CHEM 212Report Sheet2018-2019 Objective (3 marks): The purpose of the experiment is to extract and separate 3 pigments in spinach: cholorophyll a, chlorophyll b and B-carotene using column chromatography and become familiarised with techniques of column and thin layer chromatography. Introduction (3 marks): Extraction is used to obtain the crude product from the spinach. The general purpose of extraction is to obtain organic compounds from a more complex mixture (spinach leaves in this case). Extraction method utilises two types of solvent – organic solvent (petroleum ether) and another solvent (methanol –water-soluble). The general criteria is that the 2 solvents must not mix – they are immiscible. The organic compound that needs to be extracted (from spinach leaves) will be soluble in the organic solvent (petroleum ether) forming hydrophobic species, which will not mix with the aqueous layer- hydrophilic species. The organic layer can then be separated and collected as crude product. The crude product contains various organic compounds of different polarities. This leads to different retention to the stationary phase and different solubilities in the mobile phase. Hence, chromatography is performed to separate these organic compounds. The stationary phase is silica gel – which is highly polar, while the mobile phase – 8:2 petroleum ether/acetone is less polar. Non-polar or less polar compound will dissolve better in the mobile phase and travel down the column faster and can be collected first. The most polar compound will have high affinity to stationary phase so will travel down slowest and will be collected last. Hence, the organic compounds will different polarities can be separated and collected (which are B-carotene first, chlorophyll a and then b) (1) Procedure (3 marks): Procedure begins with one partner carrying out the preparation of column, while the other performs extraction of pigments from spinach. For the extraction of pigments, 3-4 spinach leaves, mixed with 8 ml of methanol and a little of sand were grinded using pestle and mortar. Mixture was then decanted into a clean separatory funnel. This was followed by the addition of 5 ml methanol and 15 m of petroleum ether to spinach leaves in the mortar and the mixtures were grounded for another 1 minute. The liquid was decanted into the separatory funnel. Final grinding was performed as 7 ml of methanol and 15 ml petroleum ether were added and decanting into separatory funnel was repeated. The bottom layer in the funnel was allowed to run down into a 125 ml Erlenmeyer flask. 15 ml of water was added to contents in the funnel. The funnel was then inverted a few times with occasional opening of stopcock to release pressure. The bottom layer was then drained and added to the same 125ml Erlenmeyer flask. The top layer was then collected into a clean 125 ml Erlenmeyer flask with the addition of 1 scoop of anhydrous of sodium sulfate. After a few seconds of swirling, the green liquid was decanted and added to a clean 250 ml beaker. The green liquid was then 2

CHEM 212Report Sheet2018-2019 concentrated using air in fume hood to evaporate off the solvent in excess. For the preparation of column, a small piece of cotton was added to bottom of column, along with addition of 1 cm of sand. 20 ml of silica gel was mixed with 35 ml of petroleum ether:acetate 8:2 (eluent) in a 125 ml of Erlenmeyer flask and poured into the column. A small amount of eluent was used to rinse the flask to remove the remaining silica and add to the column. Wooden stick was used to tap on column with the aid of air tube to pack the silica and allow faster process. About 1 cm of sand was added to top of gel and eluent was drained until eluent was just 6 cm above the sand. Column chromatography was then performed. About 1 ml of concentrated green liquid was added to column. Stopcock was open and eluent was added on top of column; the previous step was repeated until the eluent was colourless. Then eluent was added regularly to make sure the sand was not dried out. A yellow band was developed at bottom and collected in a clean 50 ml Erlenmeyer flask. Similarly, the blue green and green band start to separated and was collected respectively in the same fashion. After 3 pigments had been separated, three TLC plates were obtained for 3 different solvent system: 7:3, 8:2 and 9:1 pet ether/acetone. For each TLC plate, a baseline was drawn roughly 1 cm from the edge of TLC plate. 4 spots were marked lightly on the baseline, labelled Cr (Crude), Y for Yellow, BG for blue green and G for green. Small drops of corresponding pigment were then added to their spots. Each TLC was then placed to their appropriate chamber (7:3, 8:2 and 9:1). Results were observed and Rf were calculated. Results from TLC showed that Crude split into 3 different levels, while each pigment reached its own specific level, with yellow spot reaching furthest, followed by blue-green and finally green pigment. Then clean up was carried out and waste was disposed appropriately.

Results (9 marks): Table 1. Column and Corresponding TLC Plate Observations Colour Rf value (8:2 TLC)

Pigment Identity

Fraction 1

Yellow

0.950

B-Carotene

Fraction 2

Turquoise

0.450

Chlorophyll a

Fraction 3

Green

0.0300

Chlorophyll b

Figure 1. TLC Plates

3

CHEM 212Report Sheet2018-2019

Discussion (12 marks): 1. Discussion of physical properties: Comment on the effectiveness of column chromatography, and rationalize Rf values and the order of elution of the compounds from the column. There was good separation between the fractions so column chromatography was effective at separating the pigments. B-carotene has the highest Rf for TLC (0.95), followed by cholorophyll a (0.45) and chlorophyll b (0.050) , which corresponds to the order of elution in column chromatography as B-carotene is collected first, followed by chlorophyll a, and lastly cholorophyll b. This can be rationalized based on the polarity of each molecule. B –carotene is a non-polar molecule, thus dissolves well in the eluent as the mobile phase (8:2 petroleum ether/acetone) which is less polar than the stationary phase of silica gel. Hence, B-carotene runs down fastest and gets collected first, which also corresponds to the highest Rf value (highest distance travelled from baseline to solvent front) . The next fraction collected was chlorophyll a, followed by b; Chlorophyll a has similar structure to that of b, except that instead of –CH3 group like in a, b has a –COH group. This makes chlorophyll a less polar than b and this allows a to dissolve better in the eluent (eluent is less polar) and has lower affinity to silica gel (more polar phase). This means chlorophyll a runs down and gets collected second and has the second highest Rf value. Chlorophyll b has the highest affinity to the stationary phase (more polar siica gel) since it is the most polar molecule and gets collected

4

CHEM 212Report Sheet2018-2019 last, which corresponds to its lowest Rf value.

2. Rationalize the decision to use 8:2 petroleum ether/acetone as the solvent system for the column. As TLC and column give corresponding results, the most effective solvent system in column is the one that provides the clearest separation in TLC between 3 pigments. In this case, it is 8:2 pet ether/acetone. For 7:3, the solvent system seems excessively polar, hence all 3 pigments moved up the TLC fast and hence will not allow sufficient time for fractions to separate clearly. For 9:1, the acetone amount is insufficient to allow the more polar pigment move up TLC and this makes Rf for chlorophyll a and b almost 0. Hence, it will not be an effective solvent system for column. 8:2 solvent system gives appropriate polarity solvent that allows 3 pigments to separate clearly in both TLC and column, hence it is the most effective solvent system to be used. (2)

3. 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 organic compounds in mixture are colourless, the experiment will be carried out differently. When eluent runs down, test tubes are used to collect eluent such that each test tube contains the same volume of eluent; for example, 20 tubes were used to collect 200 ml of eluent, with each tube contains 10 ml. TLC will then be performed, compared with the crude product. A stain will be added to visualise the spot, such as phosphomolybdic acid or iodine chamber. Test tubes that have the same Rf based on TLC results can then be combined and dried off using Rotovap or by blowing air to obtain the desired compound. If the desired compound contains pi system or is aromatic, UV light can be used to visualise the bands in column and TLC will not be needed.

4. Rationalize the observed colour of each isolated pigment (β-carotene, chlorophyll-a, and chlorophyll-b) based on structure. Coloured pigments from spinach absorb visible light of different range of frequencies. The colour that is perceived by eyes will be the frequencies that are reflected/ not absorbed by the pigments. Specifically, B-Carotene absorbs blue/violet light from spectrum, and reflects red/yellow light and thereby appearing yellow. Chlorophyll a and b have similar structure, but chlorophyll a has –CH3 group while chlorophyll b has –CHO group. Chlorophyll b absorbs mostly yellow and blue light in spectrum and reflects green so chlorophyll b appears green. 5

CHEM 212Report Sheet2018-2019 Chlorophyll a absorbs purple and orange mainly, and reflects green/blue. Hence, chlorophyll a appears blue green. Chlorophyll a and b have ring system of conjugated double bonds that form a large cloud of delocalised electrons, and B-carotene contains extended pi conjugation. Hence, all 3 pigments produce visible light and absorb photons of visible light range. As different pigments have different polarity and structure, they have different energy line spectra and hence absorb photons of different energies. Less polar group (B-carotene) will absorb higher energy photon, and reflects light of lower frequency (red/yellow). (3) End of Counted Page Limit (5 Pages)

References: (1) Column Chromatography Theory . https://www.utsc.utoronto.ca/webapps/chemistryonline/production/column.php (accessed Nov 30, 2018). (2) Kevin. Notes by Kevin. https://mycourses2.mcgill.ca/d2l/le/content/356676/viewContent/4107232/View (accessed Nov 30, 2018). (3) Biological pigments. http://www.webexhibits.org/causesofcolor/7I.html (accessed Nov 30, 2018).

6...