Ion Exchange and Gel Filtration Chromatography

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Questions and Answers

What is the primary purpose of using ion exchange chromatography?

To remove all impurities from a solution.

To enhance the temperature stability of enzymes.

To separate molecules based on the attraction between different charges. (correct)

To filter based on size and molecular weight.

Which ion form is preferred for stability in the supply of ion exchange materials?

Carbonate form.

Sulfate form.

Chloride form. (correct)

Hydroxide form.

What is a common application of ion exchange chromatography in water treatment?

Removal of trace metals only.

Removal of multivalent ions causing water hardness. (correct)

Addition of alkaline substances to improve pH.

Filtration through a layer of hydroxide resin.

What sequence is typically used in water demineralization using ion exchange?

First cation exchange in [H+] form, followed by anion exchange in [OH-] form. Signup and view all the answers

Which statement is true regarding size exclusion chromatography?

It is also known as gel filtration chromatography. Signup and view all the answers

The process of neutralization using ion exchange involves which of the following preferences?

Cationic exchanger to neutralize alkali hydroxide and anionic exchanger for acidity. Signup and view all the answers

What is the role of borate in the separation of carbohydrates using ion exchange?

To assist in the ionization of the sugar molecules. Signup and view all the answers

What defines the size exclusion chromatography process?

It allows smaller molecules to pass while larger ones are retained. Signup and view all the answers

What is the main purpose of plotting elution volumes against the log molecular weight of proteins?

To create a calibration curve for determining unknown protein molecular weights. Signup and view all the answers

Which term describes the volume occupied by the pores in the gel used for protein separation?

Vo (Void volume) Signup and view all the answers

What is a significant drawback of gel filtration chromatography?

It may dilute the sample throughout the separation process. Signup and view all the answers

What does the term Kd represent in the context of protein elution from a column?

The distribution coefficient for the protein. Signup and view all the answers

How is the elution volume (Ve) calculated for a protein that partially enters the gel pores?

Ve = Vo + Kd (Vs) Signup and view all the answers

Why can oxidizing agents not be used with Sephadex during separation processes?

They interfere with the stability of the gel and separated components. Signup and view all the answers

What relationship can be inferred from the idealized elution profile for proteins?

Larger proteins generally elute at lower volumes than smaller proteins. Signup and view all the answers

In the context of the described elution process, which component primarily dictates Ve?

The proportion of porous matrix available for the molecules. Signup and view all the answers

Which type of gel is specifically noted for its capability to separate very large molecules up to 600,000 Daltons?

Sephadex G-200 Signup and view all the answers

What is the main limitation of chromatography as mentioned?

It can only resolve substances with a 10% molecular mass difference. Signup and view all the answers

What determines the pore size in polyacrylamide gels?

The concentration of acrylamide used Signup and view all the answers

For which type of gel would you use boiling water to prepare it for gel filtration chromatography?

Agarose Signup and view all the answers

Which factor has the greatest impact on the transition temperature (Tm) of phospholipids?

Length of the fatty acid chain. Signup and view all the answers

Which type of gel is mainly used for the separation of small peptides and globular proteins with small to average molecular mass?

Dextran Signup and view all the answers

How does the existence of unsaturated bonds affect the transition temperature of phospholipids?

It decreases the Tm because of reduced energy needed for melting. Signup and view all the answers

What role does cholesterol play in relation to phospholipids?

It can either increase permeability or reduce interference based on distribution. Signup and view all the answers

What is the primary factor that affects the pore size of agarose gel?

Concentration of agarose in the gel Signup and view all the answers

Which of the following exclusion limits corresponds to the Sephadex G-50 gel?

30,000 Daltons Signup and view all the answers

What is measured using differential scanning calorimetry (DSC) in phospholipids?

Transition temperature (Tm) and melting points. Signup and view all the answers

What is true about the binding capability of phosphotidyl ethanol amine compared to phosphotidyl choline?

Phosphotidyl ethanol amine has an enhanced binding ability due to hydrogen bonds. Signup and view all the answers

What type of functional group is mainly involved in the bonding that holds agarose gel together?

Hydrogen bonds Signup and view all the answers

What is described as a solid physical characteristic of solids?

Melting point. Signup and view all the answers

Which type of gel is characterized by the smallest pore sizes, ideal for separating very small biomolecules?

Sephadex G-10 Signup and view all the answers

Which type of bonds are significant in determining the melting behavior of phospholipids?

Hydrogen bonds. Signup and view all the answers

What is a disadvantage of disc gel electrophoresis using capillary methods?

It uses one sample per tube, making comparisons difficult. Signup and view all the answers

In isoelectric focusing (IEF), what is the significance of the isoelectric point (pI)?

It is the pH where a protein has no net charge. Signup and view all the answers

What role do ampholytes play in isoelectric focusing?

They create a pH gradient necessary for separation. Signup and view all the answers

What is the typical size of gel strips used in slab (thin layer) gel electrophoresis?

20 × 10 cm Signup and view all the answers

Which of the following accurately describes the design of a vertical gel electrophoresis system?

It allows for simultaneous multiple-strip analysis. Signup and view all the answers

What is a common characteristic of proteins separated by isoelectric focusing?

They are distinctly separated based on charge differences. Signup and view all the answers

Which component is NOT commonly associated with the preparation of isoelectric focusing gel?

Cellulose fibers Signup and view all the answers

In a vertical electrophoresis setup, what is crucial for maintaining the stability of the gel?

Proper gel stabilization in position. Signup and view all the answers

Study Notes

Ion Exchange Chromatography

Ion exchange chromatography separates molecules based on their charge using a stationary phase with opposite charges.
Ion exchangers are charged molecules bound to a matrix, like a framework.
The stationary phase is usually supplied as a chloride instead of hydroxide because the chloride form is more stable.
The process can be regulated with increasing pH or ionic strength of the solution to enhance efficiency and speed.
Ion exchange chromatography is used for various applications:
- Water softening by removing calcium, magnesium, and other multivalent ions.
- Water demineralization by removing cations and anions.
- Neutralization using cationic exchangers for alkali hydroxides and anionic exchangers for acids.
- Separation of electrolytes from non-electrolytes.
- Separation of carbohydrates and their derivatives.

Gel Filtration Chromatography

Gel filtration chromatography separates molecules based on their size and molecular weight using a porous gel matrix.
The stationary phase is a gel matrix, like a sieve, that allows smaller molecules to enter and elute later, while larger molecules are excluded and elute earlier.
Different types of gels are used, each with a specific pore size and range of separation:
- Dextran: Used for separating small peptides and globular proteins with small to average molecular mass.
- Polyacrylamide: Similar to dextran, available in a wide range of pore sizes.
- Agarose: Used for separating large globular proteins or long linear molecules like DNA.
Gel filtration chromatography has several applications:
- Purification of compounds, especially enzymes and proteins.
- Isolation and quantification of components in a mixture.
- Estimation of molecular weights, primarily for globular proteins.

Differential Scanning Calorimetry (DSC)

DSC is used to determine the transition temperature (Tm) of phospholipids, which is the temperature at which acyl chains in the phospholipid become disordered.
Tm is influenced by several factors:
- Length of fatty acid chains: Longer chains increase interactions, requiring higher energy to melt.
- Unsaturation (double bonds): Unsaturation reduces interactions, lowering the melting point.
- Proportion of phosphotidyl ethanol amine to phosphotidyl chlorine: Phosphotidyl ethanol amine forms hydrogen bonds, increasing interactions and Tm.
- Cholesterol: Cholesterol can increase or decrease membrane permeability depending on its distribution.

Slab and Disc Gel Electrophoresis

Slab (thin layer gel) electrophoresis
- Uses gels in strips with different thicknesses (0.1-0.25 mm).
- Gels can be run vertically or horizontally.
- Allows for analysis of multiple samples simultaneously.
Disc (capillary) gel electrophoresis
- Uses gels in tubes with a diameter of 7 mm and a length of 10-20 cm.
- Allows for easy sample addition and recovery.
- Limits analysis to one sample per tube and makes comparisons difficult.

Isoelectric Focusing (IEF)

IEF separates proteins based on their isoelectric point (pI), the pH at which they have no net charge.
A pH gradient is established using ampholytes, which migrate to their corresponding pI position, creating a pH gradient.
Proteins migrate in the electric field until they reach their pI, where they stop moving.
This technique can be used to separate proteins with similar molecular weights but different charges.

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Description

Explore the principles and applications of ion exchange and gel filtration chromatography in this quiz. Learn how these techniques separate molecules based on charge and size, respectively. This quiz covers essential concepts and methods used in biochemical applications.