{"id":1078,"date":"2020-03-18T17:33:59","date_gmt":"2020-03-18T21:33:59","guid":{"rendered":"https:\/\/wordpress-projects.wolfware.ncsu.edu\/bio-183-lab-yhnn8gz\/?page_id=1078"},"modified":"2026-02-20T13:55:57","modified_gmt":"2026-02-20T18:55:57","slug":"photosynthesis-hill-reaction-experiment","status":"publish","type":"page","link":"https:\/\/wordpress-projects.wolfware.ncsu.edu\/bio-183-hybrid-lab\/photosynthesis-hill-reaction-experiment\/","title":{"rendered":"Unit 3: Photosynthesis Make Up Module"},"content":{"rendered":"\n
\"Chloroplast\"<\/figure>\n\n\n\n

Pigments and Photosynthesis<\/strong><\/h2>\n\n\n\n

Read along in your lab handout\/lab manual with the information in this webpage.  Fill in and answer all questions in Activity 1, Activity 2 and at the end of the lab unit accordingly.  Your lab handout will be collected and graded later.<\/em><\/h4>\n\n\n\n

Photosynthesis Lab Handout<\/a>\u00a0\u2013\u00a0 Includes areas for filling out your tables and answering questions.<\/p>\n\n\n\n

\"Photosynthesis<\/figure>\n\n\n\n

(Balanced equation 6CO2<\/sub> + 6H2<\/sub>O \u2013> C6<\/sub>H12<\/sub>O2<\/sub> + 6O2<\/sub>)<\/strong><\/h3>\n\n\n\n

Leaf Anatomy and Photosynthesis:<\/strong><\/h3>\n\n\n\n
\"Photosynthesis<\/figure>\n\n\n\n

Leaf Cross section<\/strong><\/p>\n\n\n\n

\"leaf<\/figure>\n\n\n\n

Structure and Working of Stomata<\/a>  (3:50) \u2013 animation<\/h3>\n\n\n\n

Stomata from BIO 183 student prepped Zebrina<\/em> epidermal whole mount and leaf peels (A, B, and C)<\/h3>\n\n\n\n
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\"Zebrina
A.<\/strong><\/figcaption><\/figure>\n<\/div>\n\n\n\n
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\"Stomata
B.<\/strong><\/figcaption><\/figure>\n<\/div>\n\n\n\n
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\"Lower
C.<\/strong><\/figcaption><\/figure>\n<\/div>\n<\/div>\n\n\n\n

A. Zebrina<\/em> lower epidermis, 40X magnification.<\/strong> B. Zebrina<\/em> lower epidermis, 100X magnification, note guard cell shape creating the stoma\/opening.<\/strong> C. Zebrina<\/em> lower epidermis, 400X magnification, note chloroplasts in the guard cells.<\/strong><\/p>\n\n\n\n

What types of cells can you see inside the leaf?  Once in a cell, what organelles do you see?  Once in the chloroplast, what structures can you see?  What rotating molecules are embedded in the surface of the grana?  Where else have we seen this special molecule?<\/p>\n\n\n\n

Visible Wavelengths of Light and Absorbance Spectra of PS Pigments<\/h3>\n\n\n\n
\"Plant<\/figure>\n\n\n\n
<\/div>\n\n\n\n

Activity 1: Paper Chromatography<\/strong><\/h2>\n\n\n\n

Plant Pigment Chromatography<\/strong> \u2013 review your lab handout\/lab manual for the key concepts of the procedure that produced the following images. <\/p>\n\n\n\n

\"\"<\/figure>\n\n\n\n

Photosynthetic Pigments in Spinach<\/strong> \u2013 separation by a similar technique that we used with magnolia leaves.<\/p>\n\n\n\n

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A. <\/strong><\/figcaption><\/figure>\n<\/div>\n\n\n\n
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\"Plant
B. <\/strong><\/figcaption><\/figure>\n<\/div>\n\n\n\n
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C. <\/strong><\/figcaption><\/figure>\n<\/div>\n<\/div>\n\n\n\n

A<\/strong>.  A coin is used to crush the Magnolia leaf and embed the chromatography paper with leaf tissue.  <\/p>\n\n\n\n

B.<\/strong>  The chromatogram placed into a chamber with a 9:1 Ether Acetone solvent.   Within 5-10 minutes you should start to see 4 distinct photosynthetic pigments separating as the solvent is absorbed up the paper.  <\/p>\n\n\n\n

C.<\/strong>  The same chromatogram after 30 minutes and dried.  You can see 4 distinct photosynthetic pigments.  Can you tell which pigment is the primary pigment, chlorophyll a? Which are the accessory pigments chlorophyll b, xanthophyll and beta-carotene?  What is the bottom brownish line composed of?  Sketch and name all of these colored compounds in your lab handout. <\/p>\n\n\n\n

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D.<\/strong><\/figcaption><\/figure>\n<\/div>\n\n\n\n
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E.<\/strong><\/figcaption><\/figure>\n<\/div>\n<\/div>\n\n\n\n

Above (D<\/strong>) is a new chromatogram that only ran for 15 minutes.  You can see the pigments have not separated as much form each other as in the 30 minute chromatograms above in C.  D is a picture taken with regular room lights (white light).  <\/p>\n\n\n\n

E<\/strong>.  Is the same chromatogram as seen under UV light.  The lower green pigment above the origin line seems to be glowing a red color (it is fluorescing!).  What does this infer about the green pigment, light energy, and the pathway of the photosynthetic reactions?<\/p>\n\n\n\n

<\/div>\n\n\n\n

Activity 2: Light Reaction Measurements \u2013 Background information:<\/strong><\/h2>\n\n\n\n

Light Reactions of Photosynthesis<\/a> \u2013 a nice animation explaining how ATP and NADPH are produced.<\/p>\n\n\n\n

A new Animation of Light Reactions<\/a>  \u2013more in depth than above or needed for lab, but may be helpful for lecture.<\/p>\n\n\n\n

Light Reactions Diagram below<\/strong><\/p>\n\n\n\n

\"Thylakoid<\/figure>\n\n\n\n

We will study the light reactions of photosynthesis by using an artificial electron acceptor (DPIP) with isolated spinach chloroplasts and a source of light.  As electrons move along in the Light Reactions of Photosynthesis, DPIP can be used to capture electrons.  DPIP is dark blue, but will become more clear as it accept\/intercepts electrons moving along in the Light Reactions.  By measuring the change in color (from blue to more clear) we can quantify the rate of the Light Reactions of photosynthesis.  We will use a spectrophotometer to monitor this reaction by monitoring the Transmittance<\/em> of light passing through the DPIP\/chloroplast solution.  This experiment is referred to as the Hill Reaction.<\/p>\n\n\n\n

View this video for the Hill Reaction procedure<\/a>  being done for an AP Biology class.  The whole video is 12:54, time 0-7:10 minutes provides good background for the experiment and set up.<\/h3>\n\n\n\n