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Bio 120 Lab Ex 4 Postlab

This document discusses two parts of an experiment involving photosynthetic pigments and standard curves. In part A, paper chromatography was used to separate and identify photosynthetic pigments in a plant sample based on absorption spectra. A yellow band indicated the presence of chlorophyll-b, which absorbs most at 450nm. Part B describes constructing a standard curve of methylene blue absorbance at different concentrations to determine an unknown sample's concentration. The unknown sample absorbed at 43% concentration according to the curve, and 42.74% by calculation, showing good agreement between methods. Spectrophotometry can be used to measure various biological substance concentrations.

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266 views

Bio 120 Lab Ex 4 Postlab

This document discusses two parts of an experiment involving photosynthetic pigments and standard curves. In part A, paper chromatography was used to separate and identify photosynthetic pigments in a plant sample based on absorption spectra. A yellow band indicated the presence of chlorophyll-b, which absorbs most at 450nm. Part B describes constructing a standard curve of methylene blue absorbance at different concentrations to determine an unknown sample's concentration. The unknown sample absorbed at 43% concentration according to the curve, and 42.74% by calculation, showing good agreement between methods. Spectrophotometry can be used to measure various biological substance concentrations.

Uploaded by

Sunflower
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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PART A.

PHOTOSYNTHETIC PIGMENTS

In the first part of the experiment, we identify photosynthetic pigments present on a sample
based on their absorption spectra using preparative paper chromatography in order to separate pigments
present in the homogenate. Figure 4.1 is a representative chromatogram of the photosynthetic pigments
present in Golden Duranta which illustrates the band of yellow pigment that is formed.

In order to detect the pigment present, 450nm wavelength of light should be used since based on
the absorption spectrum in figure 4.2, the highest absorbance was detected at 450nm wavelength which
we can use in identifying what photosynthetic pigment is present since the error in concentration is the
lowest. In the activity, we only obtained one photosynthetic pigment. However, a study conducted by
Schoefs (2005), plants have three major types of photosynthetic pigments present – Cholorophyll,
Carotenoids, and Phycobilins.

According to Mahmoud et al (2014), chlorophyll-b, a photosynthetic pigment, has the highest


absorption at 450nm and 640nm. These wavelengths correspond to the blue and red parts of the
spectrum. The combination of red-blue light increases chlorophyll content, photosynthetic rate, and leaf
area and number. However, chlorophyll, both chlA and chlB, absorb small amounts of light such as green
and yellow which enhances plant growth when combined with the red-blue light. Based on the data
gathered on the experiment, we can infer that the separated photosynthetic pigment is chlorophyll-b
which in Figure 4.2 has the highest absorbance at 450nm and showed yellow band in Figure 4.1.

PART B. STANDARD CURVE

In this activity, we used standard curve in order to determine unknown concentration present.
Based on the constructed standard curve for the absorbance of four known dilutions of 1.40 x 10-5 M
Methylene blue at 650nm in Figure 4.3, the lowest absorbance was obtained in 10% dilution concentration
of Methylene blue while the highest was observed in the undiluted sample. By interpolating the
absorbance of the unknown in the standard curve using Microsoft excel, the value obtained for the
unknown’s dilution concentration was 43%. While the concentration of the unknown was 42.74% using
the Lambert-Beer Law formula.

The obtained interpolated value of 43% is closely related to the computed value which is 42.74%.
The Lambert-Beer Law defines the relationship between the absorbance and the concentration of a
solution where it states that the absorbance of light absorbing matter is directly proportional to its
concentration.

In order to determine the exact concentration of a substance it is extremely high and initially
registers a reading of 100% absorbance, one should dilute the sample first. With this, the number of
particles detected by the spectrophotometer can be reduced and the initial reading would decrease (Jove,
2019).

As a biology student, spectrophotometry can be used in measuring substance concentration such


as protein, DNA and RNA, growth of bacterial cells, and enzymatic reactions. It can also be applied I
biological researches such as measurement of cell density and determination of rate of chemical reactions
REFERENCES:
JoVE Science Education Database., 2019. General Laboratory Techniques. Introduction to the
Spectrophotometer. JoVE, Cambridge, MA.
Mahmoud, G.S., Hassan, T., Othman, R., Messikh, A., 2014. Simulation of Absorption Spectrum of
Photosynthetic Pigments of Chlorella Vulgaris B Algae. Malaysian Journal of Mathematical Sciences 10 (S),
pp. 123-130.

Schoefs, B., 2005. Plant Pigments: Properies, Analysis, Degradation. Advances in Food and Nutrition
Research, Vol. 49, pp. 41-89.

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