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Problem Statement:: 30g KOH Was Dissolved in

This document describes a proposed experiment to compare the glycogen levels in normal and cancerous cervical smears using UV spectroscopy. The hypothesis is that normal cells contain more glycogen than cancerous cells. Tissue samples will be digested and treated to extract glycogen, which will then be quantified colorimetrically using anthrone reagent. Glycogen levels will be determined by comparing absorbance values to a standard glucose curve generated via UV spectroscopy. This will test the hypothesis that cancerous smears contain less glycogen than normal smears.

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128 views4 pages

Problem Statement:: 30g KOH Was Dissolved in

This document describes a proposed experiment to compare the glycogen levels in normal and cancerous cervical smears using UV spectroscopy. The hypothesis is that normal cells contain more glycogen than cancerous cells. Tissue samples will be digested and treated to extract glycogen, which will then be quantified colorimetrically using anthrone reagent. Glycogen levels will be determined by comparing absorbance values to a standard glucose curve generated via UV spectroscopy. This will test the hypothesis that cancerous smears contain less glycogen than normal smears.

Uploaded by

annmarie
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as DOCX, PDF, TXT or read online on Scribd
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Problem Statement: Cervical cancer is traditionally diagnosed by microscopic examination of a

smear test for the presence of abnormal cells. Using spectroscopy, scientists have found that the
amount of glycogen is dramatically reduced in cancerous smears when compared to a normal smear.
Suggest a spectroscopic method of analysis for a smear based on the amount of glycogen present and
design an experiment to compare a normal smear with a cancerous smear.

Hypothesis: Normal cells have more glycogen than cancerous cells.

Aim: To compare the glycogen level in a normal cell and a cancerous smear

Introduction: The tissue sample containing glycogen is digested with 30% potassium hydroxide, and
then precipitated with ethanol. The precipitate is treated with anthrone reagent and glucose in the
hydrolysate is determined colorimetrically as reduced sugars by the method of Hassid and Abraham,
1957.

Reagents:

1. Potassium hydroxide (KOH) 30%: 30g KOH was dissolved in


100ml of double distilled water.
2. 95% Ethanol (v/v): 94.905ml of 99.9% ethanol was made up to 100ml with 5.095ml double
distilled water.
3. Sulphuric acid (H2SO4) 95%: 95ml of concentrated sulphuric acid was made up to 100ml
with 5ml of distilled water.
4. Anthrone reagent 0.2%: 0.2g anthrone was dissolved in 100ml of 95% sulphuric acid.
5. Standard glucose (Stock standard): 100mg d-glucose was dissolved in 100ml distilled water.
6. Working standard: 0.025, 0.05, 0.1, 0.15, 0.2ml stock solutions were made up to 5ml to get
concentrations of 25, 50, 100, 150, 200μg, respectively.
7. Distilled water
8. Sample tissue

Apparatus:

1. Laboratory Centrifuge with transparent tubes


2. Water bath
3. Filter paper
4. Measuring cylinder
5. Hot plate
6. Beakers
Procedure:

1. 5 mg tissue was digested with 1ml of 30% KOH for 20min in a boiling water bath.
2. The contents were cooled in an ice bath and 1.25ml of 95% ethanol was added, thoroughly
mixed and gently brought to boil in a hot water bath.
3. The mixture was then cooled and centrifuged for 15 min at 750 x g.
4. The supernatant was decanted and the tubes were allowed to drain on a filter paper for few
min.
5. The precipitate was redissolved in 1ml of distilled water, reprecipitated with 1ml of 95%
ethanol, centrifuged and drained as stated before.
6. The precipitate was dissolved in 5ml distilled water and 10ml of 0.2% anthrone reagent was
added under ice-cold conditions.
7. 5ml of distilled water and series of standards with a final volume of 5ml were treated with
anthrone reagent and subjected to the same procedure.
8. The tubes were covered with glass marbles and heated for 10min, in a boiling water bath. The
contents were cooled immediately and then placed in the UV spectrometer.
The amount of glycogen is expressed as mg/g wet tissue.

Ultraviolet Spectroscopy

1. A beam of light is separated into its component wavelengths by a prism or diffraction grating.
2. Each monochromatic (single wavelength) beam in turn is split into two equal intensity beams by
a half-mirrored device.
3. One beam, the sample beam (colored magenta), passes through a small transparent container
containing a solution of the compound being studied in a transparent solvent.
4. The other beam, the reference (colored blue), passes through an identical cuvette containing only
the solvent.
5. The intensities of these light beams are then measured by electronic detectors and compared.
6. The intensity of the reference beam, which should have suffered little or no light absorption, is
defined as I0.
7. The intensity of the sample beam is defined as I.
8. Over a short period of time, the spectrometer automatically scans all the component wavelengths
in the manner described.
9. The ultraviolet (UV) region scanned is normally from 200 to 400 nm, and the visible portion is
from 400 to 800 nm.

Diagram:

DIAGRAM SHOWING THE UV SPECTROSCOPY

Variables:
Independent Variable: Standardized solutions

Dependent Variable: Wavelengths

Controlled Variable: Mass of sample used

Expected Results:

If the sample compound does not absorb light of a given wavelength, I = I0. However, if the sample compound
absorbs light then I is less than I0, and this difference may be plotted on a graph versus wavelength, as shown on the right.
Absorption may be presented as transmittance (T = I/I0) or absorbance (A= log I0/I). If no absorption has occurred, T =
1.0 and A= 0. Most spectrometers display absorbance on the vertical axis, and the commonly observed range is from 0
(100% transmittance) to 2 (1% transmittance). The wavelength of maximum absorbance is a characteristic value, designated
as λmax.
Different compounds may have very different absorption maxima and absorbance. Intensely absorbing compounds
must be examined in dilute solution, so that significant light energy is received by the detector, and this requires the use of
completely transparent (non-absorbing) solvents. The most commonly used solvents are water, ethanol, hexane and
cyclohexane. Solvents having double or triple bonds, or heavy atoms (e.g. S, Br & I) are generally avoided. Because the
absorbance of a sample will be proportional to its molar concentration in the sample cuvette, a corrected absorption value
known as the molar absorptivity is used when comparing the spectra of different compounds.

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