Lab 7- Photosynthesis pigments, chromatography, and spectrophotometry

-Measure the distance from the midpoint of the band to the solvent front -If the band are curved, chose an arbitrary and consistent way to measure

After the chromatography experiment, how do you measure the bands?

-The most abundant carotene in plants, carried along the solvent front because it is very soluble and forms no H-bonds with cellulose

Describe Beta-carotene

-Water-soluble pigment, reddish in color, often trails behind or is left at the starting line

Describe anthocyanins

-Chlorophylls contain oxygen and nitrogen and are more tightly bound to the paper, however it is soluble -Chlorophyll a is bluish-green, primary photosynthetic pigment in plants; two molecules of chlorophyll a are located at the reaction center of photosystems -Chlorophyll b is yellowish-green

Describe chlorophyll (a and b)

-Contain oxygen, found further from the solvent front than beta-carotene because they are less soluble and have some H-bonding with the cellulose

Describe xanthophylls

About 10 minutes

How long will it take for the chromatography liquid to reach the top of the paper?

-Use a different solvent, run the experiment several times with different solvents, use a different type of paper, use a longer strip of paper

In some cases, two or more pigments may overlap each other on the chromatogram, making it difficult to isolate and identify them. What are some things you could do to modify the method to improve pigment separation (distance between pigments)?

Beta-carotene, xanthophylls, chlorophyll a, chlorophyll b, anthocyanins

What are the five pigments discussed in this lab?

It is highly flammable and can be dangerous if inhaled; make sure to avoid inhaling fumes as much as possible and keep away from flames, sparks, fire, or other ignition sources

What are the safety precautions related to the solvent?

-Measure to the top of the solvent level in the tube with the ruler -Get a strip of chromatography paper -The pigment must be placed on the paper such that it is above the solvent level by about 1 cm, so measure this amount out and mark with a pencil

What are the steps from placing the pigment on the chromatography paper?

-We must ensure that our extracts are prepared in the same way (ex: same solvent and same quantity)

What do we need to do if we want to compare the light absorbing properties of pigments contained in different plant tissues?

Blank the spectrophotometer with a solution containing only the solvent to eliminate absorbance due to molecules that make up the solvent

What do we need to due if we are interested in measuring the light absorbing properties of the solute ONLY?

-Avoid picking up solid pieces because they will clog the capillary tube -When dotting pigment on, make sure to let the ethanol dry in between lines, otherwise the pigment will spread too muc

What do you need to be careful of when placing the pigment on the chromatography paper?

-Allows us to select a single wavelength of light, pass it through the solution, and determine the total amount of light absorbed by the solution (and all molecules in it) -Does not tell us which molecules are accounting for the light absorption

What does a spectrophotometer allow us to do and what does it NOT tell us?

They start to smell bad, which tells you that you have ground them enough

What happens as the leaves are ground?

-Paper chromatography is a useful technique for separating and identifying pigments and other molecules from cell extracts that contain a complex mixture of molecules -The solvent moves up the paper by capillary action, which occurs due to attraction between solvent molecules and the paper and the solvent molecules and each other. When the solvent moves up the paper, it carries the solute. Pigments are carried at different rates because they have varying solubilities and because they have different attraction levels to the paper through H-bonds

What is paper chromatography used for and how does it work?

-The Rf value is the rate that the pigment moved -It is calculated using this equation: Rf= Distance moved by pigment/ Distance moved by solvent

What is the Rf value? How do you calculate it?

-9:1 solution of petroleum ether and acetone

What is the solvent used in this lab?

-Beta-carotene, xanthophylls, chlorphylls, anthocyanins -OR beta-carotene, carotene, chlorophyll b, chlorophyll a, xanthophyll (?)

What order should the solvents run (from solvent front to starting point)?

-Mortar and Pestle= grind the leaves -Spinach leaves= source of the pigments -Sand= used as an abrasive to grind the leaves in the mortar and pestle -Ethanol= used to help grind leaves, used to soak leaves and retrieve pigments for spectrophotometry -Chromatography solution= used as the solvent to separate the pigments -Ruler= for measuring where the place the starting pigment -Pencil= for marking where to place the starting pencil and labelling tubes -Capillary tube= used to "paint" the pigment on the chromatography paper -Chromatography paper= used to separate the pigments

What supplies do you need for the experiment? What are they for?

Lima bean leaves

What type of leaves were used in the video?

Right before the liquid reaches the end of the paper

When should you stop the chromatography experiment?

-Chlorophyll a is primary; chlorophyll b, beta-carotene, xanthophylls, and anthocyanins are accessory -Two molecules of chlorophyll a is located in the reaction center of photosystems. Other chlorophyll a molecules, chlorophyll b, carotenes, and xanthophylls capture light energy and transfer it to the chlorophyll a molecules at the reaction center; carotenoids protect the photosynthetic system from UV light

Which pigments are primary and which are accessory? Describe what they do and how they work together

Chlorophylls mask the presence of other accessory pigments

Why do we not see these pigments in the leaf?

-You must stop it because you must be able to measure the final distance the solvent travelled in order to calculate the Rf -If it runs off the paper, you can't calculate the Rf

Why is it important to stop the chromatogram before the solvent front reaches the top of your chromatography paper? What would happen to your chromatogram if you let it run too long?

No, it can change based on the type of plant

Will the ratio of chlorophyll a to chlorophyll b always be the same?

No because the solvent would travel at a different speed through the paper and the pigments would have different solubilities

Would you expect the Rf values to remain the same for each pigment if a different solvent had been used?