PREPARED BY

Muhammad Zain Latif_3625

LAB
REPORT
BB

Submitted to:
Dr. Abdullah Ijaz

Zainlatif5242@gmail.com
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Experiment # 1:

Determine the lambda max of β-Carotene by Spectrophotometer

Introduction / background:

Beta-carotene is a naturally occurring pigment found in many fruits and vegetables, including carrots,
sweet potatoes, and spinach. It is a member of the carotenoid family, which are organic pigments
that are responsible for the bright colors of many fruits and vegetables. Beta-carotene is a precursor
to vitamin A, which is essential for vision, growth, and immune function. In this lab report, we will be
analyzing the absorbance of beta-carotene at different wavelengths using a spectrophotometer. The
spectrophotometer measures the amount of light absorbed by a sample at different wavelengths,
which can be used to determine the concentration of the sample. The maximum absorbance of beta-
carotene occurs at a wavelength of approximately 450 nm, which is known as the lambda max.

A spectrophotometer is an instrument used to measure the amount of light absorbed by a sample at

different wavelengths. It works by passing a beam of light through a sample and measuring the
intensity of the light that passes through the sample. The amount of light absorbed by the sample is
proportional to the concentration of the sample.

Single-beam spectrophotometers measure the intensity of light before and after it passes through a
sample. The sample is placed in a cuvette, which is a small container that holds the sample

Double-beam spectrophotometers measure color in a sample by using two beams. One beam passes
through the sample side, and the other beam passes through the reference side, so the reference and
the sample can be read simultaneously with no need to recalibrate the instrument.

The basic principle behind the spectrophotometer is based on the absorption of light by molecules.
When a beam of light passes through a sample, some of the light is absorbed by the sample, while the
remaining light passes through it. The spectrophotometer measures the amount of light that a sample
absorbs at a particular wavelength, which can be used to determine the concentration of the sample.
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Materials:

 Spectrophotometer
 glass cuvettes
 beta carotene
 volumetric flask
 solvent (n-hexane)
 Vortex

Glass cuvettes Spectrophotometer Vortex

Method:

1. A one ppm solution of beta carotene was prepared in a volumetric flask.

2. The spectrophotometer along with computer was turned on and allowed to warm up for 10 to
15 minutes before starting the experiment.
3. The solution was mixed thoroughly by shaking it on a vortex mixer.
4. A cuvette was filled with the solution.
5. The cuvette was inserted into the spectrophotometer.
6. The absorbance of the solution at different wavelengths was recorded using the
spectrophotometer.
7. The wavelength at which the absorbance was maximum was identified. This wavelength
corresponded to the lambda max of beta carotene.
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Result:

The lambda max of beta carotene is noted at 449nm.

Wavelength(nm) Absorbance
800 -0.0122
780 -0.0138
760 -0.0123
740 -0.0048
720 -0.0055
700 -0.0077
680 -0.0048
660 -0.0053
640 0.0007
620 0.0037
600 0.0076
580 0.0159
560 0.0279
540 0.0538
520 0.1065
500 0.2086
480 0.5301
460 2.7107
449 2.9832
440 2.9824
420 2.3786
400 2.2443
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Graph: Wavelength on X-axis and Absorbance of β-Carotene on Y-axis

Discussion:

In this experiment, we have determined the lambda max of β-Carotene by most known instrument
spectrophotometer. The significance of the lambda max of beta carotene is that it provides
information about the electronic structure of the molecule. Beta carotene has a system of 11
conjugated double bonds, which gives rise to its characteristic orange color. The lambda max of
beta carotene occurs at 449 nm, which corresponds to the blue region of the visible
spectrum. When beta carotene absorbs light with this wavelength, one of its electrons is excited
from a lower energy level to a higher energy level. The relationship between the lambda max of
beta carotene and its structure is that the conjugated double bonds in the molecule allow for the
delocalization of electrons, which results in the absorption of light at specific wavelengths. This
phenomenon is known as the “conjugated pi-electron system.”
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Conclusion:
Beta carotene is important pigment and precursor of vitamin A. The lambda max of beta carotene is
approximately 450nm which is in blue region. To determine the lambda max of beta carotene we have used
spectrophotometer. The significance of the lambda max of beta carotene is that it provides information about
the electronic structure of the molecule.

References:
1. https://www.studocu.com/en-us/document/university-of-nebraska-lincoln/organic-chemistry-i-laboratory/experiment-
5-isolation-of-b-carotene-from-spinach-leaves-lab-report/52248589
2. https://academic.oup.com/jaoac/article/87/5/1070/5657163
3. https://www.hunterlab.com/blog/single-beam-vs-double-beam-spectrophotometer/
4. https://byjus.com/chemistry/spectrophotometer-principle/
5. https://chem.libretexts.org/Bookshelves/Organic_Chemistry/Map%3A_Organic_Chemistry_%28Bruice%29/13%3A_Mas
s_Spectrometry_Infrared_Spectroscopy_and_Ultraviolet_Visible_Spectroscopy/13.19%3A_The_Effect_of_Conjugation_
on_max
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Experiment # 2:

Determine the β-Carotene Content from carrot juice by spectrophotometer

Introduction:
Beta-carotene is a carotenoid pigment that is responsible for the orange color of carrots. It is also a
precursor of vitamin A, which is essential for vision, growth, and development. Beta-carotene is a
type of nutrient known as a carotenoid, which means it’s a natural food coloring. Foods high in beta-
carotene and other carotenoids tend to have deep, rich colors. But beta-carotene does much more
than make your plate more rainbow-esque. Beta-carotene is a special kind of carotenoid known as
a provitamin A carotenoid. That means it’s essentially a precursor to vitamin A.
“When it’s absorbed in your body, beta-carotene transforms into vitamin A in your intestines,”
explains registered dietitian Carly Sedlacek, RD, LD.
Beta-carotene is a great source of antioxidants and full of anti-inflammatory properties, too.

Carrot juice is a rich source of beta-carotene, which is good for your vision and overall eye support
and protection from ultraviolet light. It may help in cancer prevention and reduce heart
complications. The fiber in the juice can lower blood glucose levels and may aid weight loss. The
other important nutrients in the beverage may promote immunity and skin health.

Materials:

 Carrot Juice
 Spectrophotometer
 Solvent(n-hexane)
 Test tubes
 Distilled water
 Quartz Cuvette
 Vortex
 Micron filter
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Method:
The procedure of this experiment is given as:

1. Prepared stock solution of beta carotene of 2000ppm.

2. Took four test tubes and labelled them as 1, 2, 3 and 4.
3. Added beta carotene stock solution and hexane as solvent in test tube 1 and
made up the volume upto 5ml the concentration of the solution become
250ppm.
4. Shifted 1ml of solution from test tube 1 to 2 and made up the volume upto 5ml
by adding n-hexane (solvent) and solution of test tube 2 become 500ppm.
5. Shifted 0.5ml of beta carotene solution from test tube 2 to test tube 3 solution
become of 1000ppm.
6. Shifted 0.25ml of beta carotene from test tube 3 to test tube 4 solution become
2000ppm.
7. Filled each solution from test tube to cuvette for further calculations.
8. Then noted the absorbance of these solution.
9. Took 1ml carrot juice, filtered it, and made a solution of 10% by adding 9ml of
water.
10. Made further dilutions of it, noted the absorbance of each, and plotted the
graph.
Formula:
y=mx+c

Result:

The beta carotene in carrot juice is 1.8mg/ml

Solution (ppm) Absorbance

250 (test tube-1) 0.168
500 (test tube-2) 0.399
1000 (test tube-3) 0.860
2000 (test tube-4) 1.56

Y (absorbance of carrot juice) diluted 10 times = 1.45

Y= 0.790x + 0.005

1.45 = 0.790x + 0.005

x = 1.829 ppm

X = 1.829 * 10

X = 18.29 ppm
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Conclusion:
The result of 18 ppm indicates that the beta-carotene content in the juice is 18 parts
per million. So 18 ppm of beta-carotene in juice is equivalent to 1.8 mg of beta-
carotene per ml of juice.

Discussion:
Determination of beta carotene in carrot can also be done with TLC. TLC is a simple,
rapid, and cost-effective technique that separates compounds based on their polarity
and size.