Exercise 4

Spectrophotometry
B. Discussion

Figure 4.1. Paper chromatogram of plant pigments found in Ipomoea batatas leaves.

0.3 LG
G
0.25 LY
Y
0.2 O
Absorbance

0.15

0.1

0.05

0
400 425 450 475 500 525 550 575 600 625 650 675 700
Wavelength (λ)

Figure 4.2. Absorbance spectra for the different pigments obtained from Ipomoea batatas.
 0.7
Known Concentrations
0.6 Unknown Concentration
Linear (Known Concentrations)
0.5

0.4
Absorbance

0.3

0.2

0.1

0
10 25 50 75
-0.1

-0.2
Concentration

Figure 4.3. Standard curve for the absorbance values of different concentrations of methylene
blue.

For extraction of photosynthetic pigments, 10 g fresh potato leaves (Ipomoea batatas)

were washed, deveined, shredded, placed in a precooled mortar, and added with 20 mL cold pure
acetone. The leaves were thoroughly ground in subdued lighting and the resulting homogenate
was labelled the crude extract. For preparative paper chromatography and elution of pigments,
six ¾” × 6” strips of chromatography paper were marked 2 cm from one end with a pencil line,
handled with forceps, and placed on top of a clean piece of paper. The crude extract was
carefully and evenly applied over the pencil line, dried, and reapplied ten more times until the
stripe is visibly green and no thicker than 5 mm. While the strips were dried, the 20 mL 9:1
petroleumether:acetone developing solution had been prepared, and 2 mL of this developing
solution was dispensed in each of six large test tubes individually wrapped in carbon paper and
covered with a rubber stopper. The ends of the strips were submerged in the developing solution.
Chromatogram development was permitted for 30 minutes, after which the chromatograms were
removed, dried, and noted in Figure 4.1. The resulting bands were cut from the strips and were
grouped according to colour. Each group of pigments were eluted and shook in 4 mL pure
acetone for two minutes, yielding partially purified pigments. For the spectrophotometric
characterization of pigments: determination of the absorption spectra, the absorbances of a blank
containing pure acetone and the decanted eluents in individual cuvettes were read in a
spectrophotometer from 400–700 nm at 25-nm intervals. Next, the absorbances were plotted
against wavelengths for all pigments in Figure 4.2, and smooth lines were drawn to show the
estimated absorption spectra. The obtained spectra were compared with literature values and
were identified using the known spectra of plant pigments. For the determination of unknown
concentration based from a standard curve, 5 mL concentrations of 1.4 × 10-4 %(w/v) methylene
blue (MB) namely the blank, 10%, 25%, 50%, 75%, and an unknown concentration (UK) were
placed in cuvettes. All absorbances of the concentrations were read with a spectrophotometer at
650 nm against the blank and plotted in Figure 4.3. The concentration of the unknown was
determined using interpolation and also using the Lambert-Beer Law equation. The obtained
values from both methods of determining the unknown concentration were compared.

Figure 4.1 shows the paper chromatogram of plant pigments obtained from Ipomoea
batatas leaves. From the stationary phase to the tip of the mobile phase, the pigments were
coloured light green (LG), green (G), light yellow (LY), yellow (Y), and orange (O). Comparing
the five photosynthetic pigments, LG and G must almost be similar in structure, for where a
methyl group is bound to the chlorin ring of LG, an alkanal group is in its place for G, making
Pigment 2 more polar; whereas Pigment 3is highly hydrophobic.Since the developing solution is
semipolar, the supposed order of separation from the stationary phase is Pigment 3, Pigment 1,
then Pigment 2, because more petroleum ether is present in the solution. However, one study
states that extraction of Pigments 1 and 2 is slow yet more effective in diethyl ether—a solvent
similar to petroleum ether—and quite effective in acetone; and yet the extraction is time-
dependent: extraction slows down as time progresses. Extraction of Pigment 3 is rapid in acetone
and slower in petroleum ether regardless of time (Sumanta, Haque, Nishika, & Suprakash, 2014).
The rationale of why two solvents were used for the developing solution was that different
pigments will be soluble in one solvent but not another, which is why combining two solvents
will yield better separation of pigmentsInvalid source specified..

Figure 2 shows the absorbance spectra for the various plant pigments obtained from
Ipomoea batatas leaves. Pigment 1, maximally absorbing light at approximately 425 nm and 650
nm, is possibly chlorophyll a because according to literature it maximally absorbs light at
430nm(blue) and 662nm(red), giving it its characteristic grass green colorInvalid source
specified.. Pigment 2, maximally absorbing light at approximately 450 nm and 650 nm, is
possibly mostly chlorophyll b because according to literature it maximally absorbs light at 453
nm(blue) and 642nm(red), giving it its characteristic blue-green colorInvalid source specified..
However, Pigment 2 appears pale yellow-green, which can be accounted for by the unevenly
distributed homogeneous mixture of the developing solution, creating smudges of pigments in
the strip.Pigment 3, maximally absorbing light at approximately 450 nm, 550 nm, and 650 nm, is
possibly mostly carotenoids specifically β-carotene because according to literature it maximally
absorbs light at 462nm(blue) and 550nm(yellow), giving it its characteristic orange colorInvalid
source specified.. However, Pigment 3 is observed to be yellowsince β-carotene may appear
yellow when mixed with other carotenoids and some chlorophyll pigments. Chlorophyll a
absorbs light in the blue-violet range and chlorophyll b absorbs red and blue light, thereby
reflecting green and appearing as green because their structures are similar. Carotenoids absorb
light in the green-violet range and reflect the longer wavelengths in thered-yellow rangeInvalid
source specified..Of course, to detect any photosynthetic pigment, the logical choices of
wavelengths to use for their detection are ranges where the highest maximum absorbance values
are obtained i.e. 430-470nm and 640-680 nm to allow the two main chlorophyll pigments a & b
to gather the maximum light energy. Blue light (460-480 nm) may also be intensified to allow
carotenoids and xanthophylls to absorb more light as wellInvalid source specified..

Interpolating the concentration based on the obtained absorbance value of the unknown,
its value is 50%. The interpolation was done by simply drawing a horizontal line corresponding
to the obtained absorbance value and determining the point of intersection with the standard
curve of the known absorbance values of methylene blueInvalid source specified.. When using
Lambert-Beer’s Law equation however, the concentration is approximately 56.55%. This was
obtained by calculating the linear regression for the slope of the standard curve (absorbance
index of the unknown solution) and then using the value obtained in the equation to solve for the
concentration. Graphically, this involves determining the point of intersection with the trend line
of the standard curve which should ideally be linear. Lambert-Beer’s Law shows that the
concentration of a substance and its absorbance are directly proportional.If the concentration is
increased, then the light will have more molecules to hit when it passes through, blocking more
light thus yielding a higher absorbance valueInvalid source specified..

To determine the exact concentration of a substance if it is extremely high and initially

registers a reading of 100% absorbance, dilute the concentrated solution carefully with the same
solvent that the substance is dissolved in while noting the dilution until a reliable reading is
achieved, and then multiply the obtained absorbance value by its dilution factor.

I will be a cardiosurgeon in the far future, so spectrophotometry may help me determine

the seriousness of heart attacks by taking enzymes from the blood of patients and comparing
their standard curves to the standard curve produced by enzymes from the blood from normal
heartsInvalid source specified.. When I do research on , spectrophotometry can determine the
identity of gases that allow light to pass through them while sealed in containers: whether they
are poisonous or not and how they behave and how they affect living organisms.

C. References

Bibliography
Sumanta, N., Haque, C. I., Nishika, J., & Suprakash, R. (2014). Spectrophotometric Analysis of
Chlorophylls and Carotenoids from Commonly Grown Fern Species by Using Various
Extracting Solvents. Research Journal of Chemical Sciences , 4 (9), 63-69.