Lab 2 – Chromatography of Photosynthetic Pigments
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What was the purpose of this lab?
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To extract photosynthetic pigments from spinach leaves by paper chromatography and then measure their absorbances at different levels using spectrophotmetry
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What are the photosynthetic pigments that were looked at?
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chlorophyll A, chlorophyll B, beta-carotene, lutein, and violaxanthin
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What are lutein and violaxathin both known as?
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xanthophylls
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Where are the photosyntehtic pigments found?
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inside of the chloroplasts embedded in the thylakoid disc membranes
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beta-carotene is a precursor to what vitamin?
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vitamin A
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Where do the light and dark rxns take place?
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light rxns = thylakoid membranes dark rxns = stroma
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What is more polar chlorophyll A or B?
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B because it has a carbonyl group
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Chlorophyll B structure is similar to what? why?
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hemoglobin because both have the porphyrrin ring
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Which is more pre-dominant, chlorophyll A or B?
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A
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What is the metal ion on the chlorophyll B ring?
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magnesium
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Since chlorophyll A is non-polar, we would also call it _________, thus where is it located?
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hydrophobic; in the interior of the thylakoid membrane
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What is the order from the highest to the lowest in terms of where the pigments end up on the chromatography paper?
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beta carotene, xanthopylls (lutein and violaxathin), chlorophyll A, and chlorophyll B
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Why is beta carotene at the top?
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The chromatography paper is made out of cellulose (long chains of glucose) making it polar, so the more polar substances have a strong attraction and don't move as far, while the more non-polar molecules travel the farthest since they do not like to interact with polar molecules
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When light strikes an object what are three things that can happen?
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it can be reflected, absorbed, or transmitted
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The amount of light absorbed when light of a specific wavelength passes through a solution depends on what?
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the length of the light path through the solution and the concentration of the solute responsible for absorption
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The relationship between these components is described as what?
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The Beer-Lambert Law: A = (sigma)dc
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Define all the variables in the beer lambert law?
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sigma = extinction coefficient of the solute (constant), A = absorbance, d = distance of the light path, c = solute concentration
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What does the Beer-Lamvert law state?
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the amount of light absorbed by a solution (absorbance) is directly proportional to the concentration of the solution
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What could be some errors introduced during spectrophotmetry?
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other reagents present than the solute, test tube was dirty, etc.
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Thus how do we correct for small divergences from the Beer-Lambery law?
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internal blanks
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The amount of monochromatic light absorbed by a solution can be measured by what?
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a spectrophotometer
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Define spectrophotmeter.
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device that includes a source of monochromatic light, a cell to hold the absorbing solute, and a sensitive device to measure the amount of light absorbed or transmitted by a sample
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When you insert blank, what should the transmittance/absorbance be?
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100/0
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What needs to happen everytime you change the wavelength?
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blanking
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What is the solvent used for the chromatography jar? The petroleum ether is highly _____ - _____.
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9 volumes of petroleum ether: 1 colume acetone; non-polar
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Do you start the chromatography right away when the solvent is added to the jar?
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no, you wait 30 minutes for the vapor to saturate the jar with solvent vapor
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Why is all of the glass-ware used here have to be cold?
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it helps preserve the integrity of the molecules
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What is added to the mortar in this experiment with the spinach leaves?
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acetone and sand
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Why acetone? Why sand?
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acetone is going to help solubilize the plasma membrane/liberate pigments while the sand helps to bust the cell wall open to get access to the pigments
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How the chromatography paper prepared?
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after you make the spinach solution and it's settled on ice for 10 minutes, you draw a line about 1-2 cm from the bottom of the paper making sure it's above the level of the solvent in the chromatography jar, and use a capillary tube to apply 10 streaks of pigment extract to the line
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The line drawn should be ______ and _______. It should be no thicker than what?
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thin and concentrated; 6-7mm
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How long do we let the chromatogram develop? When do we want to remove the paper?
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10-15 minutes; before the solvent front reaches the end of the paper
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Once the chromatogram is removed, what should be done immediately?
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mark the location of the solvent front with a pencil and let it dry and mark the location of each of the five visible bands with the pencil
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Once the distances from the pigments to the solvent front are calculated, what is calculated?
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Rf values = distance traveled by solute/distance traveled by solvent
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How do we get these separated pigments now into a test tube for spectrometry?
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cut the bands where the pigments are and dissolve them in test tubes filled with acetone
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What are some errors that could have contributed to off-data from spectrophotometry?
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mixture of the pigments in the bands that were dissolved in the acetone, error w/ machine, and genes weren't expressed properly
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Do any pigments absorb in the green/yellow wavelengths?
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no because they reflect this light which is why we see them as that color
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Tables should usually report what type of data?
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summary-level data such as means, standard deviations, etc. rather than raw data
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Which is preferred, graph or table?
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graphs: most people understand graphs better than tables
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Where does aerobic respiration take place?
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mitochondria
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What are the two peaks of chlorophyll A? Where is its minimum?
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440 and 690 nm; around 480nm
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What are the two peaks of chlorophyll B? Where is its minimum?
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460 and 660 nm; 530-540 nm
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What are the two peaks of beta-carotene? Where does it lag off?
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460 nm and 520 nm where it starts to decrease and loses any absorbane past 550 nm
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None of the photosynthetic pigments absorb after what wavlenght?
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700nm = infrared
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Where do the xanthophylls peak? where is it's minimum?
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around 440nm; around 540nm
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What two wavelengths show the highest relative photochemical efficiency for the pigments? are these the same wavelengths of the relative light absorption? where are the minimums around?
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around 430nm and 660nm; yes; 540nm and 700nm
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Review structures of the pigments
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