Olgga A. Hara Post Lab. Discussion by: Group 1 Lozano, Tweela Magpantay, Marielle Mendigorin, Kristine Mercado, Jerra Miyata, Michiko W An organelle found within the cells of green plants and eukaryotic algae which contains the membranes, photosynthetic pigments, and enzymes necessary for photosynthesis. < A pigment that is present in chloroplasts that captures the light energy necessary for photosynthesis. < Pigments are "molecules that absorb specific wavelengths (energies) of light and reflect all others." < Any light that does not have enough or has too much energy can not be absorbed and is reflected. < Green plants have six closely-related photosynthetic pigments (in order of increasing polarity): 4 ,7otene - ,n o7,nge pigment 4 X,nthophyII - , yeIIow pigment 4 hIo7ophyII , - , bIue-g7een pigment 4 hIo7ophyII - - , yeIIow-g7een pigment < hlorophyll a is the most common of the six, present in every plant that performs photosynthesis. < The reason that there are so many pigments is that each absorbs light more efficiently in a different part of the spectrum. < hlorophyll a - 400-450 nm and at 650-700 nm < hlorophyll b - 450-500 nm and at 600-650 nm. < Xanthophyll - 400-530 nm. < However, none of the pigments absorbs well in the green-yellow region, which is responsible for the abundant green we see in nature. `Tuba-tuba (,9745, .:7.,8 L.) leaves used as sample (group 1) Leaves washed with distilled water and were cut into small pieces. Leaves were placed in a pre-cooled mortar with 20ml cold pure acetone used as a solvent. Leaves were grinded using the pestle in subdued lighting. ote: Conducting the activity in subdued lighting is needed to prevent pigment degradation. Chlorophyll oI plants are extremely light sensitive and easily destroyed by photobleaching or exposure to light. They are also Iragile and very unstable. Light, in the presence of molecular oxygen (O 2 ), degrades pigments (photo-oxidation) Homogenate was filtered using cheesecloth. The collected filtrate was placed in a 100ml beaker. Green Suspension was transferred to a cold 50ml centrifuge tube. t was centrifuged at 1500 rpm for 1 minute at 4. *Filtration is done to remove large debris (e.g., cell walls), unbroken cells and fragments. * The low-speed centrifugation will sediment remaining large bodies from the filtrate. Supernatant was decanted into a clean centrifuge tube. The remaining suspension was recentrifuged. But now in 4000 rpm for 10 minutes. *Moderate-speed centrifugation will sediment chloroplasts, leaving mitochondria and ribosomes and soluble components in the supernatant. Supernatant was discarded. hloroplast pellet was resuspended in 5.0 ml of cold 0.35 M Nal. Vortex was used to disrupt the packed pellet. hloroplast suspension was observed in microscope (OO) Mark a llghL llne on each chromaLography paper aL 13 cm from Lhe boLLom end Carefully and evenly apply Lhe crude chlorophyll suspenslon uslng caplllary Lube along Lhe llne Allow Lo dry 8epeaL Lhe appllcaLlon wlLh drylng 10x unLll Lhe llne Lurns dark green Obtain 5 strips of 2x15 chromatography paper Jrap Lhe LesL Lubes wlLh alumlnum foll our 20mL of 91 peLroleum eLher aceLone lnLo Lhe LesL Lube (uLvLLClnC SL1u) Carefully lower Lhe boLLom edge (wlLh exLracL) Lowards Lhe developlng soluLlon lnslde Lhe Lube Allow Lhe chromaLogram developmenL Lo occur unLll Lhe solvenL has reached 031 cm from Lhe Lop of Lhe paper P,pe7 h7om,tog7,phy W hromatography is a technique used to separate the components of a mixture. There are various types of chromatography (column, paper, thin-layer, gas). W but in all cases the separation is achieved by distribution of components between a fixed or stationary phase (Filter Paper) and a moving or mobile phase (Solvent) by polarity of the substances used. n paper chromatography, the components of a mixture are separable into discrete zones on a sheet of filter paper. With a capillary tube, the mixture is streaked on the chromatography paper: enough sample is applied so that there will be an adequate amount for subsequent extraction and spectrophotometric analysis. rude chlorophyll suspension applied on the chromatogram ellow, a lighter yellow, and an almost clear bands of pigments were formed. These bands of pigments were not readily seen in our sample leaves. Only green coloration of the leaves was observed. Bands of Pigments Produced: ellow Light yellow Light yellow (almost clear) The bands of pigment formed in the chromatogram are cut and then grouped according to color. n separate tubes, 4 mL of pure acetone was added. Each group of pigments were added to each test tubes. The tubes were shaken for 10 minutes. &sing a transfer pipette, eluted epigments were transferred to a cuvette. To another cuvette, pure acetone was added. Absorbance was read from 350 to 700 nm (at 25 nm interval) Absorbance vs. avelength was plotted Consists oI two instruments: spectrometer Ior producing light oI any selected color (wavelength), and a photometer Ior measuring the intensity oI light The instruments are arranged so that liquid in a cuvette can be placed between the spectrometer beam and the photometer. The amount oI light passing through the tube is measured by the photometer. Spectrophotometer (photometer) (spectrometer) display The colours displayed by different pigments are result of the light that is being reflected or transmitted by the molecules. The wavelength of light that is most ABSORBED would be complimentary to the wavelength that is best TRANSMITTED . They would be found on opposite sides of the &# WHEE. Thus, a green pigment would be expected to show the greatest ABSORBANE in the red region of the spectrum (640 700 nm). Spectrophotometer in calculation of pigment concentration: Pigment concentrations are calculated by taking the absorbance reading of the highest peak in a pigment spectrum, as characterized by a spectrophotometer, and dividing that absorbance value by the pigment's specific absorption or molar absorption coefficients. Specific Absorption oefficient (L g-1 cm-1) oncentration of Pigment (mg/)
Absorbance &nits (Au)
x 1000 *SPEF ABSORPTON OEFFENT A standard universal value (for each pigment) usually given in volume per weight with respect to path length of the cuvette used. -0.02 -0.01 0 0.01 0.02 0.03 0.04 0.05 0 100 200 300 400 500 600 700 800 Pigment 1 W,;eIength ;s. Abso7b,nce Absorbance of pigment: at 00 00- -500 nm 500 nm 1. PGMENT ELLO Possible dentity of the Pigment: Xanthophyll Xanthophyll WXanthophyll is a yellow pigment from the carotenoid group. Xanthophylls absorb well at a wavelength of 400-530nm. WThere are so many pigments present in chlorophyll because each of them absorbs light more efficiently in a different part of the spectrum. WHowever in the experiment, only yellow pigment (xanthophyll) was observed in chromatogram. -0.25 -0.2 -0.15 -0.1 -0.05 0 0.05 0 100 200 300 400 500 600 700 800 Pigment 2 W,;eIength ;s. Abso7b,nce 2. PGMENT LGHT ELLO Absorbance of pigment: at 50 50- -700 nm 700 nm -0.02 -0.015 -0.01 -0.005 0 0.005 0.01 0.015 0.02 0 100 200 300 400 500 600 700 800 Pigment 3 W,;eIength ;s. Abso7b,nce 3. PGMENT LGHT ELLO TO ALMOST LEAR Absorbance of pigment: at 50 50- -700 nm 700 nm Possible dentity of Pigments 2 & 3: nthoxanthin nthoxanthin WAnthox,nthins are a type of flavonoid pigments in plants. Anthoxanthins are water-soluble pigments which range in color from white or colorless to a creamy to yellow, often on petals of flowers. WThese pigments are very susceptible to color changes. Thus, explains why the leaves of Jatropha curcas are still green even though Anthoxanthin is generally present. < hloroplast. The Encyclopedia of Science. Available from: http://www. daviddarling.info/encyclopedia//chloroplasts.html. < Parts of hloroplasts. Retrieved on 3 Aug 2011 from http://micro.magnet.fsu.edu/ cells/chloroplasts/chloroplasts.html. < Photosynthetic Pigments. Retrieved on 3 Aug 2011 from http://www.ucmp .berkeley.edu/glossary/gloss3/pigments.html. < Jatropha curcas L. image. Retrieved 3 August 2011 from http://chalk.richmond.edu/flora- kaxil-kiuic/j/Jatropha_curcas_&_02s.JPG < Separation of plant pigments by paper chromatography. Available from http://www.chemtopics.com/unit0/pchrom.pdf < Photosynthetic pigments. Retrieved 3 Aug 2011 from http://www.tutorvista.com/content/biology/biology-iv/photosynthesis/photosynthetic- pigments.php < Action and absorption spectra. Available from http://users.rcn.com/jkimball.ma.ultranet/BiologyPages//ctionSpectrum.html < Absorbance application notes: pigment concentration. Retrieved 5 August 2011 from http://www.turnerdesigns.com/t2/doc/appnotes/S-0075.pdf Pet7oIeum Ethe7 will serve as the polar part of the solution Acetonewill serve as the non-polar part of the solution Remember: "Like dissolves like Photosynthetic pigments are composed of polar and non-polar substances. Therefore, polar and non-polar solvents should also be used so that all components of the pigments can travel the chromatogram.
Ultraviolet/Visible Spectroscopy Purdue University Instrument Van Project Analysis of Plant Pigments Using Paper Chromatography and Visible And/Or Uv Spectros