Practical Spectral Analysis

Podcast

Play an AI-generated podcast conversation about this lesson

Questions and Answers

Which of the following factors does NOT affect the rate of migration of an ion in an electrical field?

Strength of electrical field

Net charge of molecule

Color of the particle (correct)

Solvent temperature

How is mobility of particles affected by the shape and size of the particle?

Size and shape have no effect on migration

Larger particles migrate faster than smaller ones

Shape does not influence migration velocity

Smaller particles migrate faster due to less friction (correct)

What happens to the mobility of a molecule at its zwitterion state?

Mobility increases significantly

Mobility is doubled

Mobility becomes negative

Mobility is retarded to zero (correct)

Which type of electrophoresis involves the migration of charged particles on supporting media?

Zone electrophoresis Signup and view all the answers

Which of the following types of electrophoresis relies on the pH of the solvent to influence migration?

Isoelectric Focusing Signup and view all the answers

What does the strength of the electrical field in electrophoresis determine?

The velocity of particle movement Signup and view all the answers

Which of the following describes the behavior of ions in electrophoresis?

They move towards opposite charges. Signup and view all the answers

In the context of mobility in electrophoresis, what role do buffers play?

To set the pH, influencing the charge of molecules Signup and view all the answers

What is one application of molecular cloning mentioned in the content?

Creating new genetic circuits Signup and view all the answers

Which technique can be utilized for DNA fingerprinting?

Electrophoresis Signup and view all the answers

What type of gel is more robust than agarose gels?

Polyacrylamide gel Signup and view all the answers

How does PAGE separate proteins during the electrophoresis process?

By charge to mass ratio and molecular size Signup and view all the answers

What effect does the electric field have in the PAGE procedure?

Drives macromolecular anions towards the anode Signup and view all the answers

What is a benefit of polyacrylamide gels mentioned in the content?

They maintain stability over a wide range of pH and temperature Signup and view all the answers

What is the purpose of placing the sample over the gel column during the PAGE procedure?

To allow for electrophoretic mobility Signup and view all the answers

Which characteristic differentiates polyacrylamide gels from agarose gels?

Covalent cross-linking structure Signup and view all the answers

What is the typical pressure range that the pump can achieve?

4000 psi Signup and view all the answers

What is the purpose of the sample injector in the HPLC system?

To introduce the liquid sample into the flow stream Signup and view all the answers

Which factor does NOT affect the sensitivity of an HPLC column?

Flow rate of the mobile phase Signup and view all the answers

What is a common particle size range for stationary phases used in HPLC?

3–10 µm Signup and view all the answers

What is the significance of maintaining a constant temperature during HPLC analysis?

To enhance the stability of the mobile phase and column Signup and view all the answers

What is the typical internal diameter range for standard HPLC columns?

2 - 5 mm Signup and view all the answers

Why are smaller particle sizes often preferred in traditional HPLC?

They provide more surface area and better separations Signup and view all the answers

How does the pore size of the stationary phase influence analyte interaction?

Pore size determines the ability of analyte molecules to penetrate and interact with the stationary phase Signup and view all the answers

What is the main advantage of using Direct ELISA over other methods?

Fewer steps and elimination of cross-reactivity from secondary antibodies Signup and view all the answers

In an Indirect ELISA, what is added to the plate after washing away unbound primary antibodies?

A secondary antibody conjugated to an enzyme Signup and view all the answers

What role does the substrate play in an Indirect ELISA?

It is converted by the enzyme to produce a colored product Signup and view all the answers

Which of the following statements reflects a feature of Competitive ELISA?

The antigen-antibody mixture is filled into an antigen-coated well Signup and view all the answers

Why is Blocking important in the Indirect ELISA procedure?

To prevent false positive results Signup and view all the answers

What is a key advantage of Indirect ELISA compared to Direct ELISA?

It has increased sensitivity by using multiple labeled antibodies Signup and view all the answers

What is primarily measured using a spectrophotometric plate reader in Indirect ELISA?

The colored product produced from enzyme-substrate reaction Signup and view all the answers

Which feature is NOT an advantage of using Indirect ELISA?

Ability to conduct the assay at low temperatures Signup and view all the answers

What is the primary function of the fluidics system in a flow cytometer?

To transport the sample to the flow cell Signup and view all the answers

Which component of a flow cytometer is responsible for measuring Forward Scatter (FSC)?

A detector positioned in line with the light beam Signup and view all the answers

What role does the electronics system play in a flow cytometer?

It digitizes and processes the photocurrent Signup and view all the answers

How does Side Scatter (SSC) contribute to the analysis of particles in flow cytometry?

It reflects the complexity of the cell's nuclear structure Signup and view all the answers

What is the purpose of using fluorescent labels in flow cytometry?

To detect different cell types based on emitted light Signup and view all the answers

What aspect of a cell can the Forward Scatter (FSC) measurement indicate?

The cell's volume Signup and view all the answers

What is the result of scattering light by suspended particles in a flow cytometer?

It helps in identifying and analyzing cell properties Signup and view all the answers

Which of the following components is NOT part of the optical system in flow cytometry?

Fluid transport tubes Signup and view all the answers

What is the main purpose of isoelectric focusing in protein analysis?

To determine the isoelectric point (PI) of proteins Signup and view all the answers

At what pH does a protein stop migrating during isoelectric focusing?

When the protein reaches the isoelectric pH Signup and view all the answers

In HPLC, what role does the stationary phase play?

It separates components based on their affinity Signup and view all the answers

What is the primary characteristic of compounds in HPLC during the separation process?

They are attracted by both the mobile and stationary phases Signup and view all the answers

What happens to a protein at a pH below its isoelectric point?

It migrates towards the cathode due to positive charge Signup and view all the answers

What is a chromatogram in the context of chromatography?

The visual output representing the separated components Signup and view all the answers

Which of the following statements about proteins in isoelectric focusing is true?

Proteins stop moving after reaching their isoelectric charge Signup and view all the answers

Which parameter is essential for HPLC to effectively separate components?

The differential affinities for the phases Signup and view all the answers

Study Notes

Practical Part of Spectral Analysis and Applied Spectroscopy (CP508)

Course for Biochemistry Diploma Students
First Semester, 2024-2025

Biochemical Lab Instruments

Contents:
- Electrophoresis Techniques
- HPLC
- Spectrophotometer
- ELISA
- Flow Cytometry

Electrophoresis Techniques

Electrophoresis is the migration of charged particles (DNA, RNA, proteins) in a solution under an external electric field.
Rate of ion migration is influenced by:
- Net charge of the molecule
- Size and shape of the particle
- Strength of the electric field
- Properties of the supporting medium
- Temperature
Particle mobility depends on particle size and shape, and the medium
Different types include:
- Zone electrophoresis (paper, gel, thin layer, cellulose acetate)
- Moving boundary electrophoresis (capillary electrophoresis, isotachophoresis, isoelectric focusing)

Paper Electrophoresis

Filter paper (Whatman No. 1 and 3) is used in strips (3mm or 5cm wide).
Separation takes 12-14 hours.
Advantages: economical, easy to use.
Disadvantages: Certain compounds (proteins, hydrophilic molecules) cannot be resolved due to the adsorptive and ionogenic properties of paper

Gel Electrophoresis

Separation based on molecular sieving (molecular size).
Gel material is a colloid (mostly water, Agar, Agarose, Polyacrylamide, Starch).
Gel is electrically neutral.
Different gels suitable for different molecules.
Macromolecule separation, allowing smaller molecule migration is possible.

Agar and Agarose Gels

Agar is a mixture of polysaccharides (seaweeds).
Agarose is a highly purified polysaccharide.
Agarose dissolves in boiling liquid and gels upon cooling.
Advantages: easy preparation, high resolution, good protein separation.
Disadvantages: electroosmosis is high resolution is lower than polyacrylamide.

Polyacrylamide Gel Electrophoresis (PAGE)

Polymerization of acrylamide monomers in the presence of methylene-bis-acrylamide (cross-linking).
Covalent cross-links forms a more rigid gel.
Suitable for smaller molecules.

Capillary Electrophoresis

Charged molecules migrate towards opposite poles.
Separates molecules based on characteristics.
High voltage across buffer filled capillaries.
Capillary can be filled with a gel, eliminating electroosmosis.
High resolution and sensitivity.
Suitable for analysis of mixtures and detection of small differences.

Isotachophoresis

Based on the development of a potential gradient.
Higher mobility leading electrolyte (e.g. chloride) and lower mobility trailing electrolyte (e.g. glycinate) are used.
Sample ionic components stack into discrete zones.
High resolution.

Isoelectric Focusing

Based on isoelectric point (pI) of proteins.
Proteins migrate to the pH gradient based on net charge.
pH of the protein equals the pH of the gradient.
Separation of proteins in sharp bands.

Starch Gel Electrophoresis

Suspension of granular starch, boiled in a buffer to give a clear colloidal suspension.
Cools to form a semisolid gel (amylopectin).
Advantages: High resolving power, yields especially proteins.
Disadvantages: Electro osmotic effect, variation in pore size.

Thin Layer Electrophoresis

Studies done in a thin layer of silica or alumina.
Advantages: Less time consuming, good resolution.
Applications: combined electrophoretic-chromatographic studies (proteins, nucleic acids)

Cellular Acetate Electrophoresis

Contains 2-3 acetyl groups/glucose unit; lesser adsorption capacity than paper.
Advantages: No tailing of proteins, good for hydrophilic material, sharp bands, wide range of particle sizes, and high voltage.
Disadvantages: Expensive, presence of sulphonates/carboxylic residues cause electroosmosis.

High Performance Liquid Chromatography (HPLC)

Separates mixtures of components based on differential affinities with mobile and stationary phases.
Mixture is placed on the stationary phase and mobile phase passes through.
Mobile phase dissolves the components, carrying them through stationary phase.
Components separate based on attraction to each phase.
Chromatograph: the equipment; Chromatogram: output display
Stationary phase: fixed inside column hardware.
Mobile phase: moves in a definite direction.
Analyte/Sample: the substance to be separated
Retention time: characteristic time for sample to pass system (from injection to peak maximum)
Eluate: analyte + eluent (from column to detector)
Elution: analyte migration through system and detection.
- Isocratic elution: mobile phase constant
- Gradient elution: eluent mixture changes over time
HPLC Principle: separation based on hydrophobic interactions between the polar eluent, the relatively nonpolar analyte, and the nonpolar stationary phase

Spectrophotometer

Measures intensity of electromagnetic energy at various wavelengths.
UV-visible-NIR spectrophotometers operate in UV, visible, and near infrared regions.
Light source, monochromator (separates light), and detector.
Non-destructive technique.
Based on Beer-Lambert law showing relationship between absorbance/amount of absorbed light, path length, and concentration
Factors influencing absorbance:
- sample thickness (path length)
- concentration
- chemical nature of the compound

ELISA (Enzyme-linked immunosorbent assay)

Biochemical technique detecting/quantifying proteins/antibodies/enzymes/hormones
Principle: antigen-antibody interaction
Components:
- Antigen: substance stimulating immune response
- Antibody: protein made in response to antigen
- Enzyme Conjugate: antibody joined with enzyme (e.g., HRP, ALP)
- Substrate: compound converted by enzyme for color change
Types: Direct, Indirect, Sandwich, and Competitive.
Advantages/Disadvantages vary depending on the specific type.

Flow Cytometry

Passes cells/particles in single file through laser beam.
Uses fluorescent labeling.
Detects cell components/properties based on light scattered/emitted.
Measures cell health; identifies and characterizes distinct cell subsets.
Uses fluidics, optics and electronics.
Analyzes sample with a laser and detectors.
Generates graphs showing cell characteristics.

Studying That Suits You

Use AI to generate personalized quizzes and flashcards to suit your learning preferences.

Description

This quiz covers the practical aspects of spectral analysis and applied spectroscopy for biochemistry diploma students. Focus areas include electrophoresis techniques and several biochemical lab instruments like HPLC and spectrophotometers. Dive into the fundamental principles that govern these techniques.

Practical Spectral Analysis - CP508

Choose a study mode