Human Anatomy & Physiology (11th Edition)
11th Edition
ISBN:9780134580999
Author:Elaine N. Marieb, Katja N. Hoehn
Publisher:Elaine N. Marieb, Katja N. Hoehn
Chapter1: The Human Body: An Orientation
Section: Chapter Questions
Problem 1RQ: The correct sequence of levels forming the structural hierarchy is A. (a) organ, organ system,...
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Create a schematic diagram of the experimental procedure down below
Transcribed Image Text:B. Determination
of Chlorophyll Concentration
1. Dilute 0.1 mL of the prepared chloroplast suspension to 20 mL of 80% acetone
(1:200 dilution) to extract the chlorophyll.
Zero the
2. Set the wavelength selector of the spectrophotometer at 652 nm.
instrument with a blank containing 80% acetone. Measure and record the
absorbance of the chlorophyll solution. After measuring the absorbance, discard
this 1:200 dilution.
3. Calculate the amount of total chlorophyll per mL (concentration) of the chloroplast
suspension:
4.
mg chlorophyll/mL
C = A x DF
ab
= absorbance x 200
34.5
= 5.8 x absorbance
Note: At 652 nm, the molar extinction coefficients of chl a and b are both equal to 34.5.
Dilute the original suspension with the proper amount of cold PBS solution to
obtain a concentration of 0.05 mg chlorophyll/mL.
Example: After the addition of acetone, a 1:200 dilution of chlorophyll extract
registered an absorbance of 0.06. (a) Calculate the amount of total
chlorophyll/mL of suspension. (b) Calculate the volume of PBS needed
to obtain 25 mL of 0.05 mg/mL chlorophyll concentration.
(a) mg chlorophyll/mL = 5.8 (0.06) = 0.348
(b) C₁V₁ = C2V2
(0.348 mg/mL) (V₁) = (0.05 mg/mL) (25 mL)
V₁ =
3.59 mL of the original chloroplast suspension
Volume of PBS needed = 25 mL - 3.59 mL
= 21.41 mL PBS to be added
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