Assignment Code: BBCS-183/TMA/2021-22

Course Code: BBCS-183

Assignment Name: Tools and Techniques in Biochemistry

Year: 2021-2022

Verification Status: Verified by Professor

Note: Attempt all questions. The marks for each question are indicated against it. Write all answers in your own words.

Q1. Define the following terms: [2x5=10]

i) Normality

Ans) The number of gramme equivalents weight of solute present in 1 litre (L) of solution is referred to as the solution's normalcy. Normality is denoted by the letter N. One litre of normal solution (1N) contains gramme equivalent weight of solute reagent. The gramme molecular weight of an acid or a base that contains 1 mole of replaceable hydrogen (H+) or hydroxyl ion (OH) ions is called the gramme equivalent weight.

ii) Molarity

Ans) In biochemical work, ‘molarity' is the most prevalent means for expressing the concentration of a solution. A concentration word is referred to as molarity. The number of moles of a substance per litre of solution is referred to as moles. A "mole" is a unit of measurement for a material containing 6.02 x 1023 atoms (Avogadro's number).

iii) Electromagnetic spectrum

Ans) The electromagnetic spectrum refers to the variety of frequencies (the spectrum) of electromagnetic radiation, as well as the wavelengths and photon energies associated with each frequency. Scientists use the phrase electromagnetic spectrum to define the complete range of light that exists. Most of the light in the universe is invisible to us, from radio waves to gamma rays.

iv) Zwitterion

Ans) A zwitterion, also known as an inner salt or dipolar ion in chemistry, is a molecule with an equal number of positively and negatively charged functional groups. In the case of amino acids, a chemical equilibrium will be achieved between the "parent" molecule and the zwitterion in solution.

v) Stock shift

Ans) The difference (in energy, wavenumber, or frequency units) between the positions of the band maxima of the absorption and emission spectra of the same electronic transition (fluorescence and Raman are two examples). Stokes shifts are sometimes expressed in wavelength units, however this is less useful than energy, wavenumber, or frequency units because the absorption wavelength varies.

Q2. (a) Distinguish between the followings: [5+5=10]

(i) Glass pipette and Micropipette

Ans) The differences between Glass pipette and Micropipette are:

1. Micropipette is an extremely small pipette, while pipette is a small glass tube, generally having an expansion or bulb in the middle, and usually graduated, used for transferring or delivering measured quantities of a liquid.

2. Pipettes and micropipettes are essential laboratory tools for drawing up, measuring, and delivering precise liquid quantities. The distinction between the two is that micropipettes range from 1 to 1000 litres, whereas pipettes typically begin at 1 millilitre.

(ii) Beer's Law and Lambert's Law

Ans) Beer's law indicates that the amount of absorbed light is proportional to the solution concentration, whereas Lambert's law states that the absorbance and route length are directly related.

(b) What is distilled water? Describe methods of water purification with suitable diagram. [20]

Ans) Water that has been heated into vapour and then condensed back into liquid in a separate container is known as distilled water. Impurities in the original water that do not boil below or near water's boiling point stay in the container. As a result, distilled water is a pure water. The following are some water purifying methods:

Distillation Method: One of the oldest ways of water purification is distillation. To remove pollutants from tap water, it is evaporated and condensed in this procedure. To distil water, it is first heated to boiling point, then conveyed as vapour into a cooled condenser coil, where it condenses into liquid form. Distilled water is a liquid form of water that has been purified.

Water pollutants such as organic and inorganic chemicals, sediments, and heavy metals can be removed using the distillation process. Some germs can also be disinfected with it.

The distillation assembly is seen in the diagram above, and it consists primarily of two parts: the distillation flask and the condenser coil. The tap water is heated to boiling point in a distillation flask, then evaporated and transferred into the inner coiling of the condenser, where it is cooled before becoming liquid water. After introducing the oxidising agent, potassium dichromate (K2Cr2O7), which oxidises particles to solids, water impurities remain in the distillation flask. Although distillation is a slow and low-cost method of purifying water. Although distillation may separate many contaminants from water, it cannot entirely remove all toxins, therefore water purifying procedures have been utilised.

Ion exchange Method: Ion exchange is a common method for eliminating ionic contaminants from water, such as sodium and calcium. Ion exchange resins are cationic and anionic resins that are made up of a matrix of cationic and anionic charged bead resins. Cationic resin is exchanged with positive ions (Na+) and anionic resin is exchanged with negative ions in this process (Cl-) The hydrogen and hydroxyl ions combine to produce water molecules when these resins displace opposing ions. Deionized water is water that is free of ions. Ionic impurities, on the other hand, frequently produce erroneous laboratory results. As a result, deionized water is required in laboratories for enzymatic analysis.

Filtration Method: It removes particles from tainted liquids using a membrane. The membrane's unique pore size (0.2m) prevents contaminations such as bacteria and other dirt particulate dissolved in water from passing through. A hollow sheet of membrane filter (pore size 0.025 micron) is used in the Ultra-filtration process to filter out water contaminants using external force. Membrane ultrafilters are commonly employed to filter bacteria, endotoxins, and other pathogens from water in cell culture and molecular biology facilities.

Reverse Osmosis (RO) Method: It is based on the osmosis concept, which in this procedure acts in the opposite direction. Water molecules migrate from a high concentration solution to a low concentration solution over a semipermeable membrane under applied external pressure in this method water filter system. It can remove the bulk of inorganic dissolved contaminants from water (95-98 percent).

However, to obtain good quality pure water for scientific purposes, a mix of methods is used. Each method of water filtration has its own set of advantages and disadvantages. Some can remove a substantial percentage of many contaminants, while others specialise in eliminating a single type of pollutant. Because they are equipped with reverse osmosis and ultrafiltration technologies, advanced water purification equipment is quite expensive.

Water distillation device is one of the most common methods for preparing distilled water in laboratories. It's the slowest, least energy-efficient, least pure (the best is), and most difficult to maintain. The following are some of the most popular uses of distilled water in laboratories:

1. Buffer solution and reagent preparation;

2. For chromatography procedures, making polar and non-polar solvents;

3. In cell culture, preparation of culture medium;

Q3. (a) Write the roles of following component in a spectrophotometer: [2.5X4=10]

i) Light source

Ans) A spectrophotometer's light sources are two types of lamps: a Deuterium lamp for measuring in the ultraviolet range and a tungsten lamp for measuring in the visible and near-infrared ranges. It emits a continuous spectrum ranging from 300 to 3,000 nm.

ii) Monochromator

Ans) A monochromator produces a single-color light beam with a very narrow bandwidth. When adjustable monochromatic light is required, it is employed in optical measuring tools. A monochromator produces a single-color light beam with a very narrow bandwidth.

iii) Cuvette

Ans) Cuvettes are used to store samples for spectroscopic measurements, which involve passing a beam of light through the sample to determine its absorbance, transmittance, fluorescence intensity, fluorescence polarisation, or fluorescence lifespan.

iv) Detector

Ans) The spectrophotometer's reaction is provided by a detector, which turns light into a proportional electrical signal. The human eye is a sensitive detector of colour changes and has been successfully used in colorimetric colour matching equipment.

(b) Define the pH meter with the schematic diagram. Explain how does it work. [20]

Ans) A pH metre is a typical scientific equipment for determining the precise pH (acidic or alkaline) of a liquid solution. The potential difference between the glass electrode and the reference electrode, as well as the temperature, form the basis of its operation.

The pH metre works by measuring the activity of hydrogen ions in a solution. It detects the difference in electrical potential between the pH selective electrode and the reference electrode (electromotive force-EMF). The electric potential in a pH metre is created when ions pass through a porous membrane from the solution to the pH glass electrode. Occasionally, the pH metre is referred to as a "potentiometric pH metre."

A standard pH metre comprises of an electric amplifier (voltmeter), a glass electrode, and a temperature-compensated reference electrode. A combination electrode (both glass and reference electrodes) is housed in a single glass or plastic tube in today's pH metres.

The glass electrode is the most significant component of a pH metre that measures the pH of an aqueous solution. It's a sodium ion-selective silicon dioxide-based thin glass bulb. Silver wire coated silver chloride (Ag-AgCl2) with a fixed voltage was immersed in dilute HCl (0.1M). This glass electrode is extremely sensitive to the concentration of H+ ions in an aqueous solution. As a result, it's also called a pH electrode or a pH-dependent electrode.

Reference Electrode: It is made up of an Ag-AgCl2 wire that is in contact with liquid mercury (Hg) in a saturated potassium chloride (KCl) solution housed in a glass or plastic tube. It is the pH-independent reference calomel electrode. The primary function of the reference electrode is to maintain a continuous electrical circuit between the reference electrode and the sample being measured.

Before measuring the pH of an unknown solution, the pH metre must be calibrated using a known pH standard buffer solution, e.g. (pH 4.0, pH7.0 and pH 9.2). Calibration aids in making modifications to measuring electrodes and maintaining accurate pH readings.

To calibrate pH meter, the following steps include:

1. Turn on the pH metre and wait at least 10 minutes for it to stabilise. Remove the glass electrode from the 3M HCl solution and rinse it completely with deionized water before drying it with tissue and adjusting the temperature.

2. Carefully place the glass electrode in the beaker holding the pH 4 standard buffer solution and adjust the pH to 4.0 using the pH meter's calibration control knob.

3. For a pH 7.0 standard buffer solution, repeat step 4.

4. Remove the glass electrode from the buffer solution with care and wash the glass bulb with de-ionized water. Next, insert the glass electrode in a solution containing standard pH 9.2 and set the pH 9.2 in the pH metre.

5. Rinse the glass electrode in deionized water after removing it from the buffer.

6. Now that the pH metre has been calibrated, it may be used to determine the pH of any unknown solution.

7. Place the glass electrode in the unknown solution and record the pH value in your notebook.

A glass electrode or a combination electrode is commonly used to measure the pH of an unknown solution in a beaker. The amount of hydrogen ions in the solution is measured by the pH metre.

The pH metre measures the concentration of hydrogen ions in a solution and displays the corresponding pH value. The presence of a large concentration of hydrogen ions (H+) in the solution raises the voltage yet lowers the pH reading on the pH meter's display. At low H+ concentrations, on the other hand, the voltage drops as the pH measurement rises. As a result, the pH meter's display is indirectly proportional to the voltage.

Q4. (a) Calculate how much quantity of substance would be needed to make following solutions: [2.5X4=10]

(i) 100mL of 0.1M NaOH

Ans)

Given,

Na = 23 g

O = 16 g

H = 1 g

To make 1N NaOH solution = dissolve 40 grams of NaOH in 1L of water.

To make 0.1N NaOH solution = dissolve 40 grams of NaOH in 1L of water.

To make 0.1N NaOH solution in 100 ml of water:

1000 ml of water = 4 g of NaOH

1 ml of water = (4/1000) g of NaOH

For 100 ml of water = (4/1000) × 100 = 0.4 g of NaOH.

Thus, the amount of NaOH required to prepare 100ml of 0.1N NaOH solution is 0.4 g of NaOH.

(ii) 500mL of 0.1N H2SO4

Ans) The following formula can be considered to calculate the actual quantity (in mL) of concentrated H2SO4 required for 0.5 litre of 0.1N H2SO4 solution,

Required Quantity (mL) = Gram equivalent weight x Required normality x Required volume (in L) x 100 / Specific gravity x %purity

To put the values in given formula:

= 49 x 0.1 x 0.5 x 100 / 1.84 x 96

= 245 / 176.64

= 1.38

Thus, 0.1N H2SO4 solution for 0.5 litre, 1.38 ml of concentrated H2SO4 is required.

(iii) 50 ml of 5% Sugar solution

Ans) Table sugar is the common name for a sugar known as sucrose. It is a type of disaccharide made from the combination of the monosaccharides glucose and fructose. The chemical or molecular formula for sucrose is C12H22O11, which means each molecule of sugar contains 12 carbon atoms, 22 hydrogen atoms and 11 oxygen atoms.

(iv) 70% ethanol

Ans) Take 70 mL ethanol and mix with 30 mL of distilled water to prepare 70% ethanol solution.

(b) What is virtual labs? describe its importance and application in modern science education. [10]

Ans) Virtual Lab (VL) is a web-based platform that allows students to conduct virtual experiments. It was created as an alternative way for improving scientific teaching in today's educational system. The virtual science lab allows students to conduct laboratory experiments at their own pace and leisure in a safe and interactive environment. It is extremely useful for teaching and learning science experiments in situations where there is no access to an actual lab.

Importance of Virtual Labs

The virtual lab's goal is to provide students at all levels, from undergraduate to graduate, with remote access to laboratories in a range of science and engineering fields. The Ministry of Human Resource Development of the Government of India has launched a programme to establish virtual labs. The National Mission on Education via Information and Communication Technology oversees this programme. In India, almost 100 virtual labs have been created. The virtual lab concept and development are low-cost to operate and maintain. Virtual Labs, on the other hand, are an effective tool for learning science experiments that may be made enjoyable, fascinating, and inspiring. In science teaching, the virtual is extremely important.

As a result, science students can utilise the virtual lab to:

1. Learn and experiment whenever and wherever you want.

2. Allows you to simulate tests and is completely free to use.

3. Easily assessable on a laptop, a computer system with internet access, or virtual lab software.

4. Provides a versatile and user-friendly learning environment.

5. Allow students to repeat experiments at their own pace and time using excellent virtual aids in the laboratory.

During the COVID-19 epidemic, the role of virtual lab/online learning has expanded for lab-based subjects/programs, particularly in science. The virtual labs, on the other hand, provide an additional digital platform for practising and performing science experiments without having to visit a lab.

Applications

The use of ICT transforms real-world lab experience into a moving lab in the form of an online format or web resources.

The main function of an ICT-based virtual lab is to:

1. Information must be communicated, created, disseminated, stored, and managed.

2. To enable remote access to labs in a variety of science and engineering disciplines;

3. To assist educators in bringing novel and compelling learning experiences into the classroom.

4. Students at all stages of education, including undergraduates, postgraduates, and research experts;

5. To encourage students to attend virtual labs; to encourage students to learn at their own speed and on their own time; to encourage students to learn at their own pace and on their own time; to encourage students to learn at their own pace and on their

6. To develop a comprehensive learning management system.

Q5. Write short notes on the following: [5+5=10]

(i) Safety measures in laboratories

Ans) The Safety measures in laboratories are as follows:

1. Wear personal protective equipment such as a lab coat, disposable gloves, and safety eyewear at all times.

2. In the lab, avoid eating, drinking, and mouth pipetting.

3. Do not eat or drink from lab glassware.

4. Long hair, scarves, and ties must all be tied back.

5. In the laboratory, do not rush around or perform practical pranks.

6. In the laboratory, wear open-toed shoes.

7. Food should not be kept in the laboratory refrigerator.

8. In the laboratory, never work alone.

9. After removing gloves, before leaving the laboratory, and especially after handling potentially dangerous materials or microorganisms, wash your hands with water and soap.

10. In the event of an accident or a chemical leak, contact your teacher or lab instructor right once.

11. Before starting work, any abrasions, cuts, or open sores in the skin should be treated with an adhesive plaster.

12. While working in the laboratory, do not use your cell phone.

13. Please notify your lab instructor/teacher right away if you are pregnant, uncomfortable, or unwell.

(ii) Properties of Good buffers system

Ans) The important properties of a goods buffer are:

1. Because most biological reactions take place in an aqueous environment, a buffer should be extremely soluble in water.

2. A buffer should not be permeable through the plasma membrane.

3. pKa value: Because most biochemical experiments have an ideal pH range of 6-8, a buffer should be very efficient in maintaining a pKa range between 6 and 8.

4. Ionic strength: The ionic composition of the medium, temperature, and concentration should have the least impact on a buffer. They should keep their ionic strength at a physiological level.

5. UV absorption: The buffer must not absorb visible or ultraviolet light.

6. Nonreactive: Buffer components should interact with biological components as little as possible, such as DNA, metal ions, or enzymes.

7. Non-toxic: No buffer components should be hazardous to the test system (cells).

8. Purity: A buffer should be supplied in high purity or be devoid of any impurities that could interfere with the analysis.

9. Preparation of buffers: They should be simple to make in the lab.