BIO506P

Assignment 01,Spring
2025
By M.Kashif(03064963933)

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 Note : Students are required to make and upload Handmade
diagrams of all the Diagrams given in this assignment + Text
answers within the given bordered spaces + Al & plagiarism must
be less than 15 percent as per VU Biosciences Department Policy

Inbox for paid LMs Work & Project Handing at M.Kashif(03064963933)

 VIRTUAL UNIVERSITY OF PAKISTAN
Semester: Spring 2025
Course Code: BIO506P Course Name: Biochemistry-II
Total Marks: 25 Due Date: 21st May 2025
Name:______________________________________ VU ID:_________________________________
Assignment 1: Analytical Techniques in Biochemistry
Assignment Duration: 21st April 2025 – 21st May 2025
Assignment Instructions: This open-book assignment is based on core practicals from your Biochemistry II
lab manual. It must be completed individually within the assigned practical schedule. Read the following
instructions carefully to ensure your submission meets the academic and technical requirements:
1. Time Frame: The duration for submitting your assignment is 31 days from 21st April 2025. Submissions
after the deadline will not be considered under any circumstances.
2. AI Prohibition: Usage of artificial intelligence or any machine-assisted writing tools is strictly forbidden.
Discovery of AI assistance will lead to an automatic score of zero. There is zero tolerance for violations of this
rule. Any violation will result in a zero score with no appeals considered.
🔐 AI Policy – Strict Prohibition on AI-Generated Content
The use of Artificial Intelligence (AI) tools for completing this assignment is strictly prohibited. This
includes but is not limited to:
a) ChatGPT / GPT-4 / GPTZero
b) QuillBot, Grammarly AI, Writesonic, or any paraphrasing or content-generation tools
c) Any text, diagram, or reference suggestion that originates from machine learning or AI-based
platforms
All submissions will be screened using Turnitin, GPTZero, and other AI-detection software.
If your submission is found to contain AI-generated content, it will receive a score of zero and be reported
for academic misconduct.
You are strongly advised to rely on your own critical thinking, analysis, and lecture-based
understanding when completing this assignment.

3. Originality and Plagiarism: Submissions must be the student's own original work. Plagiarism, including
copying from peers, will result in a zero score. The originality of assignments will be verified using Turnitin.
4. Word Limit: Do not exceed the specified word count for responses to each question. Points will be deducted
for overages.
🔴 Word Limit Enforcement:
If any answer exceeds the word limit by more than 25%, a 50% deduction in marks will be applied
for that question.
If the response exceeds the word limit by more than 50%, a score of zero marks will be awarded for
that answer.
Please adhere strictly to the stated word limits to avoid penalties.

5. Marks Allocation: Each question carries an equal weight of 5 marks.

6. Effort and Critical Thinking: Evaluation will be based on the effort and critical thinking demonstrated in your
responses. Answers should reflect comprehensive understanding and analytical depth concerning the subject
matter.
7. Formatting: All responses must be typed in Times New Roman, 12-point font, and double-spaced
within the designated space in this document. Failure to comply will result in a 20% penalty.
8. Inquiries: For questions or clarifications, please contact the course instructor at
waheed.ahmad@vu.edu.pk.
9. Submission: Your typed responses and any hand-drawn illustrations must be compiled within this
document, then converted to a PDF and submitted via the Learning Management System (LMS).
Email submissions will not be accepted.

Please follow these instructions carefully to ensure that your work meets the requirements for this assignment.
Questions

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Q1. (Critical Scenario: Buffer Preparation Challenge)_________________________________________
Your lab receives an unstable plant extract requiring precise pH conditions for carbohydrate isolation.
You are asked to prepare 200 mL of 20 mM sodium phosphate buffer at pH 7.0.
a) Explain how you would calculate and prepare the buffer using appropriate salt combinations (150
words).
b) Discuss why buffering is essential in the isolation of amylose and amylopectin, and how pH
fluctuation could affect your results. (150 words)
a) Buffer Preparation (150 words):
To prepare 200 mL of 20 mM sodium phosphate buffer at pH 7.0, we use a mixture of NaH2PO4
(monobasic) and Na2HPO4 (dibasic). The Henderson-Hasselbalch equation is:

pH = pKa + log ([Na2HPO4] / [NaH2PO4])

7.0 = 7.2 + log ([Na2HPO4] / [NaH2PO4])
log ([Na2HPO4] / [NaH2PO4]) = -0.2
[Na2HPO4] / [NaH2PO4] = 10^(-0.2) ≈ 0.63

With total concentration of 20 mM:

Na2HPO4 = 7.73 mM
NaH2PO4 = 12.27 mM

For 200 mL:

Na2HPO4·7H2O = 0.00773 mol × 200 mL = 1.546 mmol = 0.427 g
NaH2PO4·H2O = 0.01227 mol × 200 mL = 2.454 mmol = 0.340 g

Dissolve both salts in about 180 mL distilled water. Adjust pH to exactly 7.0 using dilute NaOH or HCl.
Finally, make the volume up to 200 mL.

b) Importance of Buffering in Carbohydrate Isolation (150 words):

Buffering is essential in isolating amylose and amylopectin to prevent changes in their structure caused by
pH fluctuations. These carbohydrates are sensitive to pH, especially under acidic or basic conditions. At
low pH, glycosidic bonds may hydrolyze, breaking down the polymers. At high pH, the branched
structure of amylopectin can unfold or precipitate. Using a buffer like sodium phosphate at pH 7.0
maintains a stable environment, ensuring that the molecules stay in their native forms. This is especially
important if enzymes are used during extraction, as enzymes work best at specific pH ranges. Also, tests
like iodine-binding color reactions give inaccurate results if pH shifts. A stable buffer prevents
unexpected chemical reactions, helps maintain solubility, and avoids degradation of the sample. Without
buffering, the isolation process would yield poor-quality or misleading results, affecting both separation
and final characterization of amylose and amylopectin.

Q2. (Diagram-Based Fermentation Setup – Hand-Drawn Only)__________________________________

Draw a labeled diagram of a controlled fermentation setup using five flasks (A to E), each representing
different sugar or yeast combinations. Ensure your drawing shows balloon placement, labeling of flask
contents, and heat-treated conditions. Briefly describe the purpose of each flask based on what they are
expected to demonstrate about fermentation. (150 words + diagram)

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 This experiment investigates the impact of sugar type and yeast activity on fermentation by
observing balloon inflation in five different flasks. Each flask is fitted with a balloon to visually
capture carbon dioxide production, indicating fermentation.

• Flask A contains glucose and heat-treated yeast. The balloon remains deflated, showing no
fermentation as the yeast enzymes are inactive.
• Flask B contains glucose and active yeast. The balloon is fully inflated, indicating strong
fermentation due to readily fermentable glucose.
• Flask C contains sucrose and yeast. The balloon is moderately inflated, showing fermentation
after enzymatic breakdown of sucrose into glucose and fructose.
• Flask D contains lactose and yeast. The balloon is slightly or not inflated, as standard yeast
cannot ferment lactose effectively.
• Flask E contains glucose and heat-killed yeast. The balloon is deflated, confirming that active
enzymes are essential for fermentation.

Q3. (Carbohydrate Reaction Pathways)/ Carbohydrates Testing_________________________________

Using your theoretical understanding, describe the chemical interaction of iodine with different
carbohydrate types (monosaccharides, disaccharides, and polysaccharides). Why does iodine selectively
produce a blue-black complex with starch but not with glucose or sucrose? Use your understanding of
molecular structure to justify your answer. (250 words)

Iodine and Carbohydrate Interaction: A Molecular Perspective (250 Words)

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 1. Introduction
Iodine’s interaction with carbohydrates is widely used in biochemical testing to distinguish between
different carbohydrate types. The reaction is especially notable for its ability to produce a blue-black
complex with starch but not with simpler sugars like glucose or sucrose.

2. Interaction with Monosaccharides and Disaccharides

Monosaccharides (e.g., glucose) and disaccharides (e.g., sucrose) are small, non-helical molecules. Their
structures do not allow the formation of inclusion complexes with iodine. As a result, when iodine is
added to these sugars, no significant color change is observed. Iodine molecules (I₃⁻ in solution) remain
unbound and retain their natural yellow-brown appearance.

3. Interaction with Polysaccharides (Starch)

Starch is a polysaccharide composed of two main components: amylose and amylopectin. The amylose
fraction is a linear polymer of α-D-glucose units that naturally coils into a helical structure. Iodine
molecules can fit inside this helix, forming a charge-transfer complex. This alters the electronic
environment of the iodine, resulting in the absorption of visible light and the appearance of a blue-black
color.
Amylopectin, though branched, may allow partial iodine interaction in its linear regions but contributes
less to the intense coloration.

4. Structural Justification
The iodine test is selective due to the presence of a helical cavity in amylose. This unique structural
feature is absent in glucose and sucrose, hence the lack of complex formation or color change.

5. Conclusion
The iodine-starch reaction highlights how molecular geometry, rather than simple composition,
determines the outcome of chemical interactions.

Q4. (Lab Equipment – Hand-Drawn Identification and Usage)__________________________________

Hand-draw and label any four instruments used across the following procedures:
➢ Solution preparation
➢ Amylose/amylopectin isolation
➢ Fermentation
➢ Iodine-based testing
For each, explain:
a) Its core function,
b) The reason for selection in that particular experiment, and
c) Any handling precautions if applicable.
Drawings must be original and hand-drawn. (300 words + diagrams)

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Lab Instruments Across Four Biological Procedures

Procedure Instrument Core Function Reason for Handling

Selection Precautions
Solution Preparation Beaker Holding/mixing Wide mouth; Avoid heating
solutions good for unevenly; use
dissolving and gloves for hot
stirring reagents liquids
Measuring Measures liquid Accurate liquid Read at eye
Cylinder volume measurement level; dry before
before solution use
formulation
Stirring Rod Manually stirs Ensures solute Handle with
solutions dissolves evenly care to avoid
in solvent glass breakage
Weighing Measures solid Ensures correct Calibrate before
Balance reagent mass solute amount for use; avoid
precise airflow on pan
concentrations
Amylose/Amylopectin Centrifuge Separates starch Sediments Balance tubes;
Isolation fractions amylose and don’t open
amylopectin from while spinning
plant extract
Mortar & Pestle Grinds plant Breaks down Clean
material tissue for starch thoroughly
extraction between
samples
Water Bath Maintains constant Controls temp Check water
temp during starch level; avoid
gelatinization or electric contact
enzymatic steps
Filter Paper Separates solids Helps remove Use with
from liquid plant debris funnel; avoid
before starch tearing
separation
Fermentation Conical Flask Culture vessel Easy to seal; Sterilize before
allows gas use; handle
collection during gently
fermentation
Balloon Gas Visual indicator Don’t
collection/indicator of CO₂ overinflate;
production in check for leaks
fermentation
Glucose Substrate for Provides Prepare fresh;
Solution fermentation fermentable sugar use correct
for yeast concentration
Thermometer Measures Ensures optimal Handle gently;
incubation temp yeast don’t submerge
fermentation above
conditions immersion line
Iodine-Based Testing Test Tube Holds test samples Suitable for Avoid tipping;
small-scale label clearly

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 reactions like
iodine test
Dropper/Pipette Delivers iodine Adds controlled Clean after each
drops reagent volume use; avoid air
to observe color bubbles
change
Watch Glass Flat surface for Used for Handle by
spotting observing iodine- edges; avoid
starch reaction on scratching
solid samples
Iodine Reagent Stores iodine Essential reagent Store in dark
Bottle solution for starch place; wear
detection gloves when
handling

Q5. (Process Flow Diagram – Amylose and Amylopectin Isolation)_______________________________

Create a hand-drawn flow diagram representing the entire procedure of amylose and amylopectin
extraction from sweet potato. Include key steps such as blending, buffering, centrifugation, and storage.
Label all stages clearly. Briefly describe how temperature and solvent conditions are critical at each stage.
(250 words + flow diagram)

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1. Washing & Peeling
o Sweet potatoes are rinsed under running water to remove soil and surface microbes.
o Peeling eliminates skin and external contaminants, improving starch purity.
2. Blending
o Peeled tubers are diced and blended with chilled distilled water (4–8 °C) to form a uniform
slurry.
o Keeping the temperature low minimizes endogenous amylase activity and prevents
premature gelatinization of starch granules.
3. Filtration
o The slurry is strained through multiple layers of muslin cloth to separate fibrous debris
from the starch-rich liquid.
o Efficient removal of solids ensures a clearer filtrate and higher downstream yield.
4. Buffering
o The clear filtrate is adjusted to pH 5.2 by adding cold sodium acetate buffer at 4 °C.
o This pH optimum preserves granule integrity and further inhibits enzymatic breakdown of
starch.

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 5. Centrifugation
o The buffered suspension is centrifuged at 4 °C, 4 000 × g for 10 minutes.
o Cold centrifugation pellets intact starch granules while preventing gelatinization or
hydrolysis.
6. Solvent Washing
o The starch pellet is resuspended and washed with 75 % ethanol at 25 °C.
o Ethanol selectively solubilizes linear amylose; branched amylopectin remains insoluble.
o Maintaining moderate temperature avoids heat-induced structural changes.
7. Separation of Fractions
o After a second centrifugation, the supernatant—rich in dissolved amylose—is decanted
into a clean vessel.
o The remaining pellet comprises mainly amylopectin.
8. Drying
o Each fraction is dried under reduced pressure at temperatures below 40 °C to prevent
polymer degradation.
o Gentle drying preserves molecular weight and functional properties.
9. Storage
o Dried amylose and amylopectin are stored in airtight containers at 4 °C with low humidity.
o Cool, dry conditions prevent retrogradation, moisture uptake, and microbial growth,
ensuring long-term stability and purity.

Throughout these stages, precise control of temperature, pH, and solvent polarity is essential to maximize
yield, maintain polymer structure, and achieve clear separation of amylose and amylopectin.

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