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Proced re

Today2s laboratory has 4 phases#1) solation of chloroplasts from fresh spinach lea es$) Determination of chlorophyll concentration in the chloroplast suspension

/) *bser ing the effects of light intensity on the rate of the Hill Reaction4) erforming an experiment of your choice to explore another factor affecting the Hill Reaction

"afety notice#cetone is a flammable organic sol ent.cetone will 567T any type of plastic, and also parafilm.8se only glass tubes, glass pipets, and glass cu ettes when wor!ing with acetone.f you get concentrated acetone on your s!in, flush the area generously with cold water.

hloroplast isolation1) 9ash and weigh $: g spinach or other green plant from which the petioles and ma;or eins

ha e been remo ed. ut into small pieces and place in a cold blender container.$) dd 100 m7 cold chloroplast isolation media and homogeni&e lea es with three : second bursts at full speed. -ilter the blended mixture through cheesecloth./) Transfer $0 m7 ali acetone. o er with a small piece ofaluminum foil and ortex carefully to mix.$) entrifuge at 1/00 x g for : min to pellet precipitated protein and debris./) Transfer approximately / m7 of the supernatant to a glass cu ette and record the absorbance(?:$ nm). The blan! cu ette should contain a similar olume of a 1#100 dilution of chloroplastisolation buffer in 0> acetone.4) alculate the chlorophyll concentration in mg'm7 using the formula below#

:) Determine the olume of chloroplast suspension re

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/) 8se a light meter to measure the light intensity (fc) deli ered to the reaction cu ette.Readings are approximate and should be made

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Lab Report )rite*+p , ide Results Section

1) repare a graph of absorbance ersus time for tubes 1%:. nclude a legend which labels each

trace with the proper light intensity.

$) 8se the abo e graph to determine G o for each reaction, in terms of ?00 nm 'min. 8se only theinitial linear portion of the relationship.

/) repare a 5aster "ummary Table to show the effect of light intensity (fc) on photosyntheticrate ( ?00 nm 'min). f tube A: demonstrated any incidental reduction of D , subtract this ratefrom the rates calculated for tube 1%4. "uggested column headings for this table are# Tubenumber, G o (initial elocity, ?00 nm 'min), and G o (initial elocity, molecules of Dreduced 'min'=g chlorophyll, see below for the calculation).

4) repare a graph showing the effect of light intensity on reduction of D (molecules of Dreduced 'min'=g chlorophyll). alculation approach#a) on ert ?00 nm alues to molarity of D , using the molar extinction coefficient for Ddetermined in the I ntroduction to 7aboratory Techni