Download BIO 120 Exer 5 Spectrophotometric Analysis v4 (1)

BIO CELL
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SPECTROPHOTOMETRY
A particular substance only absorbs light of certain wavelengths and transmits the rest due to its molecular
structure. For instance, chlorophyll absorbs a significant amount of red light and transmits green light, thus,
appears green (assuming other pigments are absent or in negligible amounts). Moreover, although two
substances may absorb light of the same wavelengths, it is very probable that their light absorption degrees will
be different because of intrinsic variation in molecular structures. These are the bases of spectrophotometry,
the quantitative measurement of the absorption and transmission of light by a substance.
By measuring the amount of light absorbed (or absorbance) at different wavelengths, an absorption spectrum
can be constructed. This is a graph showing the amount of light a substance absorbs at different wavelengths.
A substance has a unique absorption spectrum because of the unique molecular structure. Hence, an unknown
substance can be identified using this graph. For example, the absorption spectrum below shows that isoprene
absorbs light maximally at 222 nm, demonstrated as a peak in its absorption spectrum.
Figure. 5.1. Absorption spectrum of isoprene. (Image from Reusch 2013; reprinted by permission of author)
At the wavelength of maximum absorption (λmax), a substance can absorb light even at a very low
concentration. This λmax can also be utilized to determine the concentration of a substance in a mixture by
interpolation in a standard curve (SC) or by using the Lambert-Beer Equation.
The spectrophotometer measures the absorption or transmission of light by a solution. Figure 5.2 diagrams its
basic components. Light from the source is collected and focused by the collimator to the monochromator that
separates the light beam into component wavelengths. Only light of wavelengths to which the selector is preset will pass through the aperture and strike the solution contained in a cuvette in the cell compartment. Some
of the light will be absorbed by the solution, the rest will be transmitted. A photoelectric device detects the
amount of transmitted light and converts it into an electrical signal. A readout system then displays the
magnitude of the electrical signal.
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Figure 5.2. Essential components of a spectrophotometer. (Image from Vo 2015. CC BY-NC-SA 3.0 US)
PHOTOSYNTHESIS
Photosynthesis is the process by which chlorophyll in the chloroplast of green plants capture light energy and
use it to form carbohydrates and free oxygen from carbon dioxide and water. It has the overall equation:
nCO2 + nH2O → (CH2O)n + nO2
This whole process takes place in two distinct stages: (1) using light energy to make ATP and reducing power in
the form of NADPH; and (2) using the ATP and NADPH to power the synthesis of organic molecules from
atmospheric CO2. The first stage, occurring in the thylakoid membrane within the chloroplast, happens in the
presence of light, hence, known as the light reactions. The second stage, occurring in the stroma, is called the
Calvin cycle and as long as ATP and NADPH are available, this may occur in the absence of light (Figure 5.3).
Figure 5.3. The light reactions and Calvin cycle of photosynthesis. (Image from: Avissar Y et al., 2019.
OpenStax Biology, CC BY 4.0)
In the light reactions, O2 is also photochemically released from water, not from CO 2, together with ATP and
NADPH. This is known as the photochemical or Hill reaction. Robert Hill showed that isolated chloroplasts can
generate O2 when they are illuminated in the presence of an artificial hydrogen acceptor even in the absence of
CO2. The Hill reaction is defined as the reduction of an electron acceptor (A) by electrons and protons from
water, with the evolution of O2, when chloroplasts are exposed to light. This can be expressed as:
2H2O + 2A → 2AH2 + O2
Light
The artificial hydrogen acceptor is A and AH2 is the reduced form. In vivo, the final electron acceptor is NADP.
The Hill reaction can be observed either by measuring the O2 evolved, or by measuring the rate of reduction of
the hydrogen acceptor A. If A is a dye that changes color as it accepts electrons (i.e. reduction) then the change
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in color intensity can be conveniently determined spectrophotometrically. Several artificial electron acceptors
can be used to study Hill reaction in vitro. DPIP or 2,6-dichlorophenolindophenol is suitable for the
measurement of the rate of Hill reaction because it changes color in solution depending on its oxidation state.
DPIP is blue in the oxidized form and becomes colorless when reduced (Figure 5.4).
oxidized (blue)
reduced (colorless)
Figure 5.4. The structure of DPIP in its oxidized and reduced forms.
(Image from Mills 2007. Public Domain)
CELLULAR RESPIRATION
A metabolic pathway so fundamental to the maintenance of a cell is respiration, the process through which
energy in the form of ATP is extracted from food stuffs (e.g., carbohydrates, fats, and proteins). Cellular
respiration may be visualized to consist of five processes:
1. Conversion of food materials to pyruvate (the process of glycolysis for carbohydrates);
2. Oxidation of pyruvate to acetyl CoA;
3. Complete oxidation of acetyl CoA to CO2 and H2O with reduction of FAD and NAD+ to FADH2 and
NADH,H+, respectively (tricarboxylic acid (TCA) cycle or Krebs cycle);
4. Oxidation of FADH2 and NADH,H+ and transfer of electrons to a series of electron carriers resulting to a
relatively large release of free energy (electron transport system or ETS); and
5. Generation of ATP using the free energy released in some steps of the ETS by an ATPase enzyme
(oxidative phosphorylation).
The tricarboxylic acid cycle, electron transport system and oxidative phosphorylation are localized in the
mitochondrion in eukaryotes (Figure 5.5). Thus, the mitochondrion serves as the cell’s energy transducing
system and can be aptly called the “powerhouse” of the cell.
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Figure 5.5. A summary of energy-generating metabolism in the mitochondria.
(Image from San Pascual JCK 2018; generated using Perkin Elmer
ChemDraw® Professional version 16.0.1.4(77))
Enzyme Activity and Inhibition
Respiration, like all other metabolic reactions occurring in the cell, requires a group of proteins to act as catalysts
for the reactions. These special proteins are called enzymes. Enzymes bind specific substrates accelerating their
reaction until equilibrium is established. Like all catalysts, enzymes provide an alternative route with a lower
activation energy, thus speeding up a reaction.
The substrate specificity of an enzyme may be either absolute (when it only acts on a specific substrate) or
relative (if it acts on several closely related compounds). The specificity of enzymes depends on its threedimensional molecular configuration especially that of the portion of the enzyme known as the active site (to
which the substrate structurally and chemically fit). The activity of enzymes may be inhibited in different ways.
The inhibition of enzymes refers to the decrease in the extent and/or the rate of enzymatic activity or to its
complete abolition in the presence of specific ions or molecules (inhibitors) which combine with the enzyme.
Enzyme inhibitors may be classified as reversible or irreversible. Reversible inhibitors bind to enzyme
noncovalently and upon its separation from the enzyme, the full activity of the enzyme is restored. In contrast,
an irreversible inhibitor binds to the enzyme covalently and permanently modifies its functional group making
it inactive.
Reversible inhibitors may be classified as competitive, uncompetitive, or noncompetitive. A competitive
inhibitor is a substance that binds to the free enzyme but not to the enzyme-substrate complex. It is structurally
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similar to the substrate and, thus, is capable of binding at the active site of the enzyme. It competes with the
substrate for the same binding site. It can be detected experimentally because increasing the substrate
concentration decreases the percent inhibition at a fixed inhibitor concentration.
An uncompetitive inhibitor is a substance that binds to the enzyme-substrate complex but does not bind to the
free enzyme. It does not bind at the active site of the enzyme and usually bears no structural similarity to the
substrate. The enzyme-substrate inhibitor complex produced is often inactive. Merely increasing the substrate
concentration cannot reverse this type of inhibition.
A noncompetitive inhibitor is a substance that can combine with either free enzyme or enzyme-substrate
complex, interfering with the action of both. Noncompetitive inhibitors bind to a site other than the active site,
often to deform the enzyme, so that it does not form the enzyme-substrate complex at its normal rate, and
once formed, the enzyme-substrate complex does not decompose at the normal rate to yield products. Merely
increasing the substrate concentration does not reverse these effects.
In competitive inhibition, the affinity of the enzyme for the substrate and the inhibitor can be determined by
obtaining the competitive ratio of the inhibitor to the substrate. This is the ratio at which the competition
between the two is equal and the enzyme shows only 50% of its maximum activity. For instance, it may be found
that with a proportion of five substrate molecules to one inhibitor molecule the enzyme can be spoken of as
having five times as great affinity for the inhibitor as for the substrate.
Succinate Dehydrogenase Activity
Succinate dehydrogenase (SDH) has a molecular weight of 97,000 Da and is the only enzyme of the Krebs cycle
bound to the inner mitochondrial membrane. It is a flavoprotein that consists of a large subunit (with FAD as
the prosthetic group) and a smaller subunit containing iron and sulfur atoms.
SDH catalyzes the oxidation of succinate to fumarate (Figures. 5.6 and 5.7). The prosthetic group, FAD, accepts
a pair of electrons from the substrate succinate; FAD, is thus reduced to FADH2.
Figure 5.6. Oxidation of succinate to fumarate. (Image from San Pascual JCK
2018; generated using Perkin Elmer ChemDraw® Professional
version 16.0.1.4(77))
In a normal functioning cell, FADH2 then passes these electrons directly to the iron cations of ubiquinone, which
pass them to the other carriers in the ETS. Ultimately, the electrons are transferred to oxygen, the final electron
acceptor of the ETS in an aerobic condition.
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reposted, or replicated in any form without permission from the Genetics and Molecular Biology Division, Institute of Biological Sciences, CAS, UPLB
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Figure 5.7. The Tricarboxylic Acid (TCA) Cycle emphazing succinate dehydrogenase
activity. (Image from San Pascual JCK 2018; generated using Perkin Elmer
ChemDraw® Professional version 16.0.1.4(77))
LEARNING OUTCOMES:
At the end of the exercise, the student should be able to:
● explain the principle of spectrophotometry;
● demonstrate Hill reaction and its significance in photosynthesis;
● discuss the role of chloroplast and light in the Hill reaction;
● enumerate the major pathways involved in cellular respiration;
● measure the activity of isolated mitochondrial succinate dehydrogenase spectrophotometrically using
DPIP; and
● determine the effects of malonate and cyanide on cellular respiration.
SUGGESTED OPEN EDUCATIONAL RESOURCES:
● These videos discuss in a very comprehensive way the principle of spectrophotometry, absorbance and
transmittance, and the use of spectrophotometry in determining the unknown concentration of a
substance through Lambert-Beer Law.
Khan
Academy.
2021.
Spectrophotometry
and
the
Beer-Lambert
Law.
https://www.khanacademy.org/science/ap-chemistry-beta/x2eef969c74e0d802:intermolecularforces-and-properties/x2eef969c74e0d802:beer-lambert-law/v/spectrophotometry-introduction
Khan Academy. 2021. Worked example: Calculating concentration using the Beer-Lambert Law.
https://www.khanacademy.org/science/ap-chemistry-beta/x2eef969c74e0d802:intermolecular-
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reposted, or replicated in any form without permission from the Genetics and Molecular Biology Division, Institute of Biological Sciences, CAS, UPLB
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forces-and-properties/x2eef969c74e0d802:beer-lambert-law/v/spectrophotometry-example
Creative Commons Attribution-NonCommercial-ShareAlike 3.0 United States License.
● This online resource provides further details about photosynthesis, especially on Hill reaction, and a
laboratory experiment to investigate Hill reaction. It is under the BISSC 110 course at Wellesley College.
Wellesley College. 2009. Series 3 – Lab 9 Photosynthesis – The Hill Reaction.
https://openwetware.org/wiki/BISC110:_Series_3_Experiment_9_Hill_Reaction
Creative
Commons Attribution-ShareAlike 3.0 Unported license.
ACTIVITY: PHOTOSYNTHESIS
A. Isolation of Chloroplasts
1. Devein 10 g of fresh papaya leaves and wash with distilled water. Place
the leaves and 50 mL cold phosphate buffered saline (PBS, 0.2 M
phosphate buffer, pH 7.0 and 0.35 M NaCl solution), in an ice-cold
blender.
2. Blend at high speed for 30 sec, then cool the jar in an ice bucket for 30
sec. Do this three times.
3. Strain the extract through two layers of cheesecloth into an ice-cold
beaker. Transfer the suspension into two cold 50-mL centrifuge tubes.
Ensure that they are of equal weights.
4. Centrifuge at 200 xg for 5 min. (Compute for rpm value using r = 8 cm.)
Decant the supernate into two clean 50-mL centrifuge tubes and discard
the pellet.
5. Centrifuge at 1400 xg for 15 min. Discard the supernatant and resuspend the pellet in 5 mL PBS for
each tube. Collect all suspensions in a small beaker immersed in an ice bath.
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.
2. Set the spectrophotometer at 652 nm. Zero the instrument with 80% acetone as blank. Measure
and record the absorbance of the chlorophyll solution. Afterwards, discard this 1:200 dilution.
3. Calculate the amount of total chlorophyll per mL (concentration) of the chloroplast suspension:
A
c = × DF
ab
mg chlorophyll/mL =
absorbance
× 200
34.5
= 5.8 × absorbance
Note: At 652 nm, the molar extinction coefficients of chl a and b are both equal to 34.5.
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4. Dilute the original suspension with the proper amount of cold PBS solution to obtain a
concentration of 0.05 mg chlorophyll/mL.
Ex. 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
C. Spectrophotometric Measurement of the Hill Reaction
1. Place 15 mL of the 0.05-mg chlorophyll/mL suspension in a test tube. Wrap the tube with aluminum
foil and chill in an ice bucket. This is the source of unheated chloroplasts.
2. Place the remaining 10 mL of the 0.05-mg chlorophyll/mL suspension in a test tube and heat until
boiling using an alcohol lamp. Heating inactivates the chloroplasts. Stand the heated mixture for 5
min. to allow the debris to settle.
3. Prepare the contents of each setup listed in Table 5.1. Pipet solutions into cuvettes in the order
given. For the “Unheated (Dark)” setup, immediately wrap the cuvette with aluminum foil even
before the first reading is made.
Table 5.1. Contents of tubes for spectrophotometric determination of the Hill reaction.
Volume Added (mL)
Contents
PBS
Heated chloroplasts
Unheated chloroplasts
0.1% DPIP
(to be added immediately before
the first reading)
Blanks
Experimental set-ups
Heated
Unheated
Heated
Unheated
(Light)
Unheated
(Dark)
3.5
1.0
-
3.5
1.0
2.5
1.0
-
2.5
1.0
2.5
1.0
-
-
1.0
1.0
1.0
4. Set the spectrophotometer at 605 nm. Zero the spectrophotometer using the appropriate blank –
“Heated Blank” for “Heated” experimental tube, “Unheated Blank” for both “Unheated (Light)” and
“Unheated (Dark)” experimental tubes.
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5. Add 1 mL 0.1% DPIP to experimental tubes “Heated”, “Unheated (Light)”, and “Unheated (Dark)”
prior to the initial readings. Cover the mouth of the cuvette with a piece of aluminum foil and thumb
then invert the cuvette several times to mix its contents. DPIP must be added to experimental tubes
when about to be placed in spectrophotometer. Do not delay reading if DPIP was already mixed.
6. Take the initial readings of the experimental set-ups. For the “Unheated (Dark)” setup, immediately
wrap the cuvette in aluminum foil after taking the initial reading. Measure the absorbance again
after 30 min. Uncover only upon reading the absorbance.
7. After the initial readings, place all the cuvettes in a beaker placed in an ice bath and illuminate the
cuvettes with a 100-watt bulb placed 18 inches away. Measure the absorbance of the “Heated” and
the “Unheated (Light)” tubes at 5-min intervals up to 30 min. Mix the contents before reading.
8. Record all the data as Table 5.2 in your laboratory notebook.
9. Construct the most appropriate graph for your data as Figure 5.10 in your worksheet.
10. Provide a picture of the “Heated”, “Unheated (Light)”, and “Unheated (Dark)” solutions after 30
minutes as Figure 5.11 in your worksheet.
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ACTIVITY: CELLULAR RESPIRATION
(NOTE: This will be a data analysis task that will be completed online rather than on-site.)
In this exercise, respiration will be observed by monitoring the color change of DPIP, which would reflect SDH
activity.
A. Homogenization of Liver Tissue from Mouse
1. Sacrifice a mouse by pulling its tail firmly and quickly while securing the mouse at its neck region.
2. Open the abdominal and thoracic cavities with an incision at the midline, taking care not to damage the
liver.
3. Rapidly remove the liver. Weigh the liver and immerse in cold 0.05 M phosphate buffer, pH 7.3. (For
every 1 gram of liver, use 50 mL cold phosphate buffer.)
4. Grind using cold mortar and pestle or any available tissue homogenizer. (If volume permits, blend at
high speed in a cold blender for 30 seconds then cool the jar in an ice bucket for another 30 sec. Do this
three times.)
B. Purification of the Liver Mitochondria
1. Decant the homogenate into two 50-mL centrifuge tubes taking care not to include the debris that
settled.
2. Spin the homogenate at 600 xg for 10 min. at 0-4o C to sediment the debris and nuclei. Decant the
supernatant into chilled centrifuge tubes. Discard the pellet.
3. Spin the supernatant at 10000 xg for 15 min. at 0-4o C. Discard the supernatant.
4. Add 5 mL of 0.05 M phosphate buffer to each of the mitochondrial pellet. Resuspend and collect all into
a small beaker.
C. Measurement of Respiration by Dye Reduction
1. In each of 6 test tubes, place 1 mL of liver homogenate and appropriate reagents as indicated in Table
5.3. Tube 4, which contains only the homogenate, is to be heated for 5 min and then cooled before the
other reagents are added. For each tube, a specific blank is to be used.
Figure 5.8. Test tube contents for the blanks of each set-up.
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Table 5.3. Test tube contents for the measurement of respiration by dye reduction.
Tube/
Blank No.
1
Blank 1
2
Blank 2
3
Blank 3
4
Blank 4
5
Blank 5
6
Liver
Homogenate
1
1
1
1
1
1
1 (heated)
1 (heated)
1
1
1
Blank 6
1
Volume of Contents (mL)
Succinate
0.1 M
Solution
NaCN
1
0
1
0
1
1
1
1
0
0
0
0
1
0
1
0
1
0
1
0
1 (+0.1 g solid
0
succinate)
0
1 (+0.1 g solid
succinate)
dH2O
1
2
0
1
2
3
1
2
0
1
0
Malonate
Solution
0
0
0
0
0
0
0
0
1
1
1
DPIP
Solution
1
0
1
0
1
0
1
0
1
0
1
1
1
0
2. Note that 0.1% DPIP is added just prior to reading the absorbance of each of the mixtures. Do not forget
to shake the mixture and wipe the cuvette before reading.
3. Read the absorbance at 605 nm at one-min interval for five min. Read one tube at a time. Start with
Tube 1. Only after the final reading will DPIP be added to Tube 2, and so on. Results are presented in
Table 5.4 in the worksheet. Construct the most appropriate graph for Table 5.4 and label it as Figure
5.12
.
Figure 5.9 shows the sample solutions before and after the absorbance measurements.
before reading
after reading
Figure 5.9. Different sample solutions before and after spectrophotometric measurements.
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4. Compute for the average respiration rate in each tube using the formula below and record them in
the worksheet (Table 5.5):
R=
Af - A i
t
Where, R = average rate of respiration based on DPIP absorbance changes
Ai = initial DPIP absorbance
Af = final DPIP absorbance
t = total observation time
REFERENCES
Photosynthesis:
Arnon DI. 1949. Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiol. 24: 1–15.
Atkins P, de Paula J. 2011. Physical Chemistry for the Life Sciences. 2nd edition. W.H. Freeman and Company.
Beer-Lamber Law. 2009. Edited by Frederic P. Miller, Agnes F. Vandome, John McBrewster.
Bregman A. 1990. Laboratory Investigations in Cell and Molecular Biology. 3rd ed. New York: John Wiley and Sons, Inc.
Hall
DO,
Rao
KK.
1999.
Photosynthesis.
6th
ed.
Cambridge
https://books.google.com.ph/books?id=6F7yuf1Sj30C&lpg=PP1&pg=PA22#v=onepage&q&f=false.
University
Press.
Hill R. 1937. Oxygen evolved by isolated chloroplasts. Nature 139: 881-882.
Ostrovsky DS, Steucek GL. 1981. Photosynthesis: A Simple Demonstration of the Hill Reaction. The American Biology Teacher 43 (7): 391.
Oxford Cambridge and RSA. 2016. The Hill Reaction. https://www.ocr.org.uk/Images/170050-the-hill-reaction-activity-teacherinstructions.pdf
Reece JB, Urry LA, Cain ML, Wasserman SA, Minorsky PV, Jackson RB. 2014. Campbell Biology. 10 th ed. Boston: Pearson Education, Inc.
Smith CH, Wood EJ. 1992. Cell Biology: Molecular and Cell Biochemistry. London: Chapman and Hall.
Spectrophotometry: accurate measurement of optical properties/materials. 2014. Edited by Thomas A. Germer, Joanne C. Zwinkels,
Benjamin K. Tsai. Series: Experimental methods in the physical sciences v.46. Elsevier
Vo
K.
2015.
Spectrophotometry.
Chemistry:
Libretexts.
https://chem.libretexts.org/Bookshelves/Physical_and_Theoretical_Chemistry_Textbook_Maps/Supplemental_Modules_(Physical_an
d_Theoretical_Chemistry)/Kinetics/Reaction_Rates/Experimental_Determination_of_Kinetcs/Spectrophotometry
This course material is intended solely for the personal use of the student enrolled in BIO120. No part of this course material can be reproduced,
reposted, or replicated in any form without permission from the Genetics and Molecular Biology Division, Institute of Biological Sciences, CAS, UPLB
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Cellular Respiration:
Alberts B, Johnson A, Lewis J, Morgan D, Raff M, Roberts K, Walters P. 2015. Molecular Biology of the Cell. 6th Edition. New York:
Garland Science. 1464 pp.
Bregman A. 1990. Laboratory Investigations in Cell and Molecular Biology. 3 rd ed. New York: John Wiley Sons, Inc.
Cassimeris L, Lingappa VR, Plopper G. 2011. Lewin’s Cells. 2nd ed. Jones and Bartlett Publishers, LLC.
Conn EE, Stumpf PF. 2009. Outlines of Biochemistry. 5th ed. Wiley India Pvt. Limited.
Nelson DL, Lehninger AL, Cox Mm. 2013. Lehninger Principles of Biochemistry. 6th ed. New York: W.H. Freeman.
Smith CH, Wood EJ. 1996. Cell Biology: Molecular and Cell Biochemistry. 2nd ed. London: Chapman and Hall.
Young JK. 2010. Introduction to Cell Biology. World Scientific Publishing Co. Pte. Ltd.
FIGURE REFERENCES
Figure 5.1. Reusch W. 2013. Visible and Ultraviolet Spectroscopy.
https://www2.chemistry.msu.edu/faculty/reusch/VirtTxtJml/Spectrpy/UV-Vis/spectrum.htm.
Figure 5.2. Vo K. 2015. Spectrophotometry. Chemistry: Libretexts.
https://chem.libretexts.org/Bookshelves/Physical_and_Theoretical_Chemistry_Textbook_Maps/Supplemental_Modules_(Ph
ysical_and_Theoretical_Chemistry)/Kinetics/Reaction_Rates/Experimental_Determination_of_Kinetcs/Spectrophotometry.
Creative Commons Attribution-NonCommercial-ShareAlike 3.0 United States.
Figure 5.3. Avissar Y, Choi J, DeSaix J, Jurukovski V, Wise R, Rye C. 2019. OpenStax Biology. Creative Commons Attribution 4.0
International License. https://cnx.org/contents/[email protected]:W7ctJeSI@8/Overview-of-Photosynthesis.
Figure 5.4. Mills J. 2007. Reaction scheme for the reduction of DCPIP. Public Domain.
https://upload.wikimedia.org/wikipedia/commons/a/aa/DCPIP-reduction-2D-skeletal.png.
Figure 5.5. San Pascual JCK. 2018. Image generated using Perkin Elmer ChemDraw® Professional version 16.0.1.4(77).
Figure 5.6. San Pascual JCK. 2018. Image generated using Perkin Elmer ChemDraw® Professional version 16.0.1.4(77).
Figure 5.7. San Pascual JCK. 2018. Image generated using Perkin Elmer ChemDraw® Professional version 16.0.1.4(77).
This course material is intended solely for the personal use of the student enrolled in BIO120. No part of this course material can be reproduced,
reposted, or replicated in any form without permission from the Genetics and Molecular Biology Division, Institute of Biological Sciences, CAS, UPLB