Column chromatography - gel filtration chromatography lecture

Summary

This tutorial video from 'Somos Fallacy' delves into the principles of Size Exclusion Chromatography, also known as Gel Filtration Chromatography. It explains how proteins and nucleotides are separated based on size and hydrodynamic volume, using agarose as the stationary phase to create a porous network. The video clarifies misconceptions about molecular weight separation and highlights the technique's advantages for identifying protein states and its limitations in resolution and separating closely sized molecules.

Takeaways

• 🔍 Size-exclusion chromatography, also known as gel filtration chromatography, is a technique used to separate molecules based on their size and molecular weight.
• 🧬 The stationary phase in size-exclusion chromatography is typically composed of agarose, a polymer that forms a network with pores of varying sizes.
• 🚫 Unlike other chromatographic techniques that may involve charge-based separation, size-exclusion chromatography relies solely on the gravitational force for the movement of molecules.
• 🕰 Large molecules cannot enter the small pores of the agarose network and thus elute faster, while smaller molecules get trapped and take longer to elute.
• 📊 The elution profile of a size-exclusion chromatography run typically shows larger molecules coming out first, followed by smaller ones, creating a characteristic curve.
• 🔬 Hydrodynamic volume, the volume a molecule occupies in solution, is a key factor in separation, with folded proteins having a smaller hydrodynamic volume compared to unfolded proteins.
• 🔬 The technique can be used to differentiate between folded and unfolded proteins based on their hydrodynamic volumes, which are approximately 14 angstrom for folded and 36 angstrom for unfolded proteins.
• 👍 A significant advantage of size-exclusion chromatography is its ability to separate large molecules and identify the state of protein folding.
• 🚧 The resolution of size-exclusion chromatography is limited, meaning it may not effectively separate molecules with very similar sizes or hydrodynamic volumes.
• 🚫 It is a common misconception that the technique separates molecules strictly by molecular weight, but it actually separates based on size and hydrodynamic volume.
• 📉 The technique has disadvantages including low resolution and difficulty in separating molecules of very close sizes, making it less suitable for high-resolution separations.

Q & A

What is size exclusion chromatography also known as?

Size exclusion chromatography is also known as molecular exclusion chromatography, molecular permeation chromatography, gel filtration chromatography, and sometimes just molecular exclusion chromatography.

What is the basis for the separation of molecules in size exclusion chromatography?

In size exclusion chromatography, molecules are separated based on their size and molecular weight, with larger molecules typically eluting before smaller ones due to their inability to enter the pores of the stationary phase.

What is the stationary phase in size exclusion chromatography?

The stationary phase in size exclusion chromatography is typically agarose, a polymer network that creates a structure with small pores through which molecules can pass.

How does the process of size exclusion chromatography differ from gel electrophoresis?

Size exclusion chromatography relies on gravity as the driving force for the separation of molecules, whereas gel electrophoresis uses an electric current to drive the movement of charged molecules through a gel matrix.

What is the concept of void volume in size exclusion chromatography?

Void volume in size exclusion chromatography refers to the volume of the column that is not occupied by the stationary phase, allowing larger molecules to pass through quickly without entering the pores.

Why is size exclusion chromatography particularly useful for protein separation?

Size exclusion chromatography is useful for protein separation because it allows for the separation of proteins based on their size and hydrodynamic volume, which can be indicative of whether the protein is folded or unfolded.

What is the hydrodynamic volume and why is it significant in size exclusion chromatography?

The hydrodynamic volume is the volume or area occupied by a molecule in a solution, which influences how it is separated in size exclusion chromatography. It is significant because it can indicate the folded or unfolded state of a protein.

What are some disadvantages of size exclusion chromatography?

Some disadvantages of size exclusion chromatography include its low resolution, meaning it is not very effective at separating molecules of very similar sizes, and its reliance on size rather than molecular weight, which can sometimes lead to inaccurate separations.

How does the percentage of agarose in the stationary phase affect the separation process?

The percentage of agarose in the stationary phase affects the pore size of the agarose network. A higher percentage of agarose results in smaller pores, which can influence the separation of molecules based on their size.

What is the principle behind the entrapment of smaller molecules in the pores of the stationary phase?

The principle behind the entrapment of smaller molecules is that they can enter and pass through the pores of the stationary phase, taking a longer path and thus eluting later than larger molecules, which cannot enter the pores and therefore travel a shorter distance through the void volume.

How can size exclusion chromatography help in identifying the native state of a protein?

Size exclusion chromatography can help identify the native state of a protein by separating it based on its hydrodynamic volume. A folded protein has a smaller hydrodynamic volume (14 angstroms) compared to an unfolded protein (36 angstroms), allowing for the determination of its state.

Outlines

00:00

🔍 Introduction to Size Exclusion Chromatography

This paragraph introduces the concept of size exclusion chromatography, also known by various names such as molecular exclusion chromatography or gel filtration chromatography. The technique is used for separating molecules based on their size and molecular weight, primarily for proteins and nucleotides. The stationary phase is agarose, which forms a network with pores that allow for the separation process. The process is likened to gel electrophoresis but operates differently, using gravity instead of an electric field as the driving force. The stationary phase preparation and the basic principles of separation are discussed, highlighting the differences from other chromatographic techniques.

05:02

🧬 Separation Mechanism in Size Exclusion Chromatography

The second paragraph delves into the mechanism of separation in size exclusion chromatography. It explains how large molecules are unable to enter the small pores of the agarose stationary phase and therefore travel through the void volume, exiting the column faster. In contrast, smaller molecules enter the pores and are trapped, leading to a slower elution. The concept of void volume is introduced as the space through which large molecules travel, while column volume represents the total solvent volume. The paragraph also describes the process of elution, where molecules are collected in different chambers based on their size, resulting in a separation curve that shows larger molecules eluting first and smaller ones later.

10:03

📊 Advantages and Disadvantages of Size Exclusion Chromatography

The final paragraph discusses the advantages and disadvantages of size exclusion chromatography. It is highlighted as a common technique for separating larger molecules based on their hydrodynamic volume, which is the volume a molecule occupies in solution. The technique can distinguish between folded and unfolded proteins based on their hydrodynamic volumes. However, the method has limitations, including low resolution, which affects the ability to separate molecules of closely similar sizes. Additionally, it is noted that the technique does not strictly separate based on molecular weight and struggles with differentiating between molecules of nearly identical size. The paragraph concludes with a call to action for viewers to like and subscribe for more educational content.

Keywords

💡Size Exclusion Chromatography

Size Exclusion Chromatography (SEC), also known as gel filtration chromatography, is a separation technique that separates molecules based on their size in solution. It is a crucial method in biochemistry for protein and nucleotide separation. In the video, the speaker explains that SEC uses a stationary phase, often agarose, which forms a network with small pores that allow smaller molecules to enter while larger ones are excluded and travel through the void volume, resulting in separation based on the size of the molecules.

💡Molecular Exclusion Chromatography

Molecular Exclusion Chromatography is another term for Size Exclusion Chromatography. It emphasizes the exclusion of larger molecules from the pores of the stationary phase, which is a key principle in the separation process. The video script mentions this term as one of the many names for the technique, highlighting the concept that larger molecules are excluded from the pores of the gel matrix.

💡Agarose

Agarose is a type of polysaccharide used as the stationary phase in gel filtration chromatography. It forms a network with pores of specific sizes that allow for the separation of molecules based on their ability to enter these pores. In the video, agarose is described as creating a network with small pores that are crucial for the size-based separation of molecules like proteins.

💡Stationary Phase

The stationary phase in chromatography refers to the medium that is fixed in place, through which the mobile phase and sample mixture pass. In the context of the video, agarose forms the stationary phase in SEC, providing the porous network that facilitates size-based separation of molecules.

💡Mobile Phase

The mobile phase is the fluid that carries the sample through the stationary phase in chromatography. In the video, the mobile phase is the buffer solution that is used to elute the sample through the column packed with agarose, leading to the separation of molecules based on their size.

💡Column

A column in chromatography is the tube or vessel that contains the stationary phase. The video explains that the column is filled with agarose to create the stationary phase for SEC. The column's structure, including the size and distribution of pores, is critical for the separation process.

💡Void Volume

Void volume in chromatography is the volume of the column that is not occupied by the stationary phase but by the interstitial spaces between the packing material. In the video, it is explained that large molecules, which cannot enter the pores of the agarose, travel through this void volume and are eluted from the column first, which is a key aspect of how SEC separates molecules by size.

💡Hydrodynamic Volume

Hydrodynamic volume refers to the volume a molecule occupies in solution, which is influenced by its shape and interactions with the solvent. The video discusses how SEC separates molecules based on their hydrodynamic volume, which is the effective volume a molecule presents to the solvent, rather than just its molecular weight.

💡Folded Protein

A folded protein is one that has a specific three-dimensional structure, which is often compact and has a smaller hydrodynamic volume. The video script mentions that SEC can be used to differentiate between folded and unfolded proteins, as the latter have a larger hydrodynamic volume and are eluted later in the SEC process.

💡Unfolded Protein

An unfolded protein lacks a defined three-dimensional structure and has a larger hydrodynamic volume due to its more extended conformation. In the video, the speaker explains that SEC can separate folded from unfolded proteins based on their different hydrodynamic volumes, with unfolded proteins eluting later because they are trapped more within the agarose pores.

Highlights

Size exclusion chromatography is also known by various names including molecular exclusion chromatography and gel filtration chromatography.

This technique separates molecules based on size and molecular weight, particularly useful for protein and nucleotide separation.

The stationary phase in size exclusion chromatography is agarose, which forms a polymer network with pores for molecule separation.

Agarose's ability to create pores is demonstrated by its use in gel electrophoresis for DNA separation.

Unlike gel electrophoresis, size exclusion chromatography is driven by gravity rather than an electric current.

The separation principle involves large molecules moving through voids while small molecules get trapped in the pores.

The void volume is the space within the column that large molecules can easily pass through without entering the pores.

Small molecules take longer to elute because they are trapped inside the agarose pores.

The elution process results in a curve where larger molecules elute first, followed by smaller ones.

Size exclusion chromatography is advantageous for separating larger molecules and identifying protein folding states.

The technique can distinguish between folded and unfolded proteins based on their hydrodynamic volumes.

The hydrodynamic volume for a folded protein is approximately 14 angstrom, and for an unfolded protein, it's 36 angstrom.

The resolution of size exclusion chromatography is not high, limiting its ability to separate molecules of very close sizes.

The technique does not perfectly separate molecules based on molecular weight, contrary to some beliefs.

Difficulties arise when trying to separate molecules of very similar sizes due to the technique's limitations.

Transcripts

friends welcome back to another video

from somos fallacy and in this video

tutorial we'll be talking about size

exclusion chromatography we've been

talking about different types of

chromatography lately and in this video

I'll be talking about the size exclusion

chromatography size-exclusion

chromatography is also known as

molecular exclusion chromatography or

molecular permeation chromatography it

is also known as molecular exclusion

chromatography so all these names

molecule exclusion chromatography gel

filtration chromatography size-exclusion

chromatography molecular exclusion

chromatography all of them are the same

this is the first thing you should know

all of them are the name of the same

technique that we know so here I will

talk it about this process now it is

also known as gel filtration I told you

gel filtration chromatography now if you

heard this name gel filtration

chromatography it's much more clear to

understand about the technique so why

it's called as gel filtration gel

filtration means here filtration means

the filtering of molecules because you

know chromatography means separation of

molecules from each other it may be

depending upon their charge it may be

depending upon their size and mass and

all the stuff now we already talked

about the ion exchange chromatography

where the molecules are separated based

on that charge in this case of gel

filtration chromatography or it's also

known as molecular exclusion

chromatography in this case we separate

proteins based on their size and

molecular weight the molecular telling

molecular weight is not perfectly okay

here usually they separate molecules

based on their size okay and so this is

the parameter based on the size of the

molecule so as its size based molecule

we mostly use this technique for protein

separation as well as nucleotide

separation mainly for the protein

separation like any other any other

chromatographic techniques we need to

know the basic things that is what is

the stationary phase

of this what is the mobile phase of this

and what is the column means and how its

prepared the idea here the stationary

phase for this size expression

chromatography is agarose you know

polymer network that can produce network

between each other you know agarose can

have small pores prepared between them

like this small pores are created now if

I draw the column the column is the

chamber where we put all the stationary

phase molecules there this is the column

and this column is filled with filled

with all those mash networks throughout

let us say throughout this network is

present okay now here this networks

contains pores in it so we need to

prepare this column or stationary phase

with some polymer molecules that can

create this net-like structure now the

molecule here we use is agarose so

agarose can create this type of pores we

know that because we can separate DNA

using agarose using gel electrophoresis

and stuff so the situation of this the

process of size exclusion chromatography

is very familiar with the process of

electrophoresis but there is a

significant amount of difference while

in electrophoresis we drive energy we

create an energy barrier with which a

voltage gradient with which when the

molecule is moving the force is created

and provided by the change in charge

that we apply the current flow that we

apply but in this case of the size

exclusion chromatography we do not need

to apply any voltage it is entirely

based on the gravitation force that the

molecules will come that is a

significant amount of difference between

the size exclusion chromatography and

the gel electrophoresis remember that so

here it is by the force the driving

force by the gravity okay so now let us

say this is the column and we prepared

the stationery phase with agarose now

what we do we load this

with the molecules the mixture from

where we want to separate our molecules

now the mixture contains various size of

molecules let us say the mixture is

homogeneous it's made with proteins only

but some proteins are smaller some

proteins are larger so here the idea the

principle of separation is that as they

are creating the pores the pores are

very small so the large protein

molecules cannot enter inside the core

okay they cannot enter inside the pol as

they are unable to insert inside the

pore they can move through other regions

other regions through this because

remember in this case production of this

column is not rigid like that this

column has many blank spaces in it and

this whole volume of the column is a

column volume so column volume is

nothing but the solvent volume okay

whatever solvent we apply that is the

volume of the column that is the idea

but there is another volume some regions

are blank through which the large

molecules can pass easily because the

large molecules cannot enter inside each

of the pores because pore sizes are

smaller only small molecules can insert

inside each of the pores

so as the small molecule will insert

inside the pore and it will move through

the pore it will take more time for the

small molecules to come out while the

large molecules will come out pretty

fast because they will be traveling less

distance because they are which are very

less volume here so the volume which is

covered by the large covered by the

large molecules is known as void volume

void volume that is the volume which is

the volume where the smaller molecules

never enter the volume consisting of the

large molecules moving through the

column that is the void volume while the

column volume is so

volume the total solvent volume so this

is the idea if I draw you another

picture it will be much more clear let

us say here this is one specific pour if

i zoom into one of the pores it will

look something like this and in this

pour let us say there is a small

molecule and there is a large molecule

so what will happen this large molecule

cannot enter into the pore so it can

easily pass through this but the small

molecule will enter and it will pass

through this whole chamber so it will be

entrapped smaller molecules will be

entrapped inside those pores inside

those chambers so as they're in trapped

inside it will take long time for them

to come out and the large molecules are

not entrapped so they will come out

pretty easily earlier okay that is how

you separate things you might think that

small molecules will travel fast large

will travel later but no here it is not

the case yet the case is small will be

trapped so it will come later large will

will not trap so it will come earlier in

the illusion stage now the illusion

means after you load this column with

those mixtures then you run buffers and

after some time you start taking out

molecules this because the solutions

will slowly come down using the force

gravitation gravitation it will come

down to the bottom and then slowly you

start collecting all these molecules in

chambers okay collecting living chambers

now you start collecting larger

molecules first so if I draw the chamber

of collection in this way let us say we

have 1 2 3 3 different chambers let us

say at the very first chamber we get the

larger molecules coming out the second

chamber we get the moderate-sized in the

third chamber we get smaller size so

that is the idea if you look at the time

duration in the time gap you will get a

curve like this

you'll get a curve

let me draw it this this is time the

x-axis this is the rate of Illusion the

y-axis so how it will look like it will

look like this this red is the big one

so big one will elute first while the

smaller one will in later so this is for

the small this is for the big this is

how the illusion take place okay this is

the graph it will look like so this is

the idea of size exclusion

chromatography or gel filtration

chromatography or whatever you say the

molecular exclusion chromatography is

the entrapment technique that we use

very similar with the technique called

gel electrophoresis but the difference

is the driving force here is gravity

where the driving force in in case of

the gel electrophoresis is the current

flow so that I hope this is helpful so

let me talk about a little about the

advantage and disadvantage before

closing the advantage is that we

vigorously use this technique for

separation of larger molecules all the

time is very common technique to use and

the separate things based on their you

know based on their size and another

thing which I should tell you here is

about the hydrodynamic volume they

actually separate molecules based on

their hydrodynamic volume hydrodynamic

volume is the area or the volume taken

by a molecule which is present in water

or solution that is known as the

hydrodynamic volume so as they can

separate molecule based on their

hydrodynamic volume it is this technique

is very very important to think of

separation of an identification of both

folded and unfolded protein it can get

an idea of whether the protein you are

dealing with is folded

or unfolded because the hydrodynamic

volume let me write the hydrodynamic

volume completely filled so let's erase

some of the stuff hydrodynamic volume

the hydrodynamic volume that we are

talking about hydrodynamic volume for a

folded protein and unfolded protein for

a folded protein it is 14 angstrom for

an unfolded protein it is 36 angstrom so

you know degree so this is the idea the

hydrodynamic volume for a folded protein

14 for the unfolded 36 because you know

the unfolded protein will occupy more

volume because it's scattered it is

unfolded but a folded we are Q less area

so this is the idea so we can easily

separate folded and unfolded protein

using this technique so we can tell

whether the protein we are dealing with

is in its native state or its unfolded

state that is another very important

advantage now some disadvantages as this

process applies only separation based on

the size and hydrodynamic volume it is

not very good and the resolution of this

process is not very good though the

resolution depends on resolution means

how we separate molecules and how

closely the size of the molecules could

be to separate them that will be known

as resolution in this case now the

resolution for this is very it's not

good because here for this column to run

it depends on the type the percentage of

agarose that you take the more

percentage of agarose that will take the

more smaller the pore size will be and

so the separation will be different so

these things matter for the separation

so this process is not very high

resolving process with low resolution

that's one disadvantage another

disadvantage is they cannot separate

proteins based or their molecular weight

it's not always true you

read that it can separate by molecular

weight but it is not actually based on

the molecular weight it is not

completely true and the third thing is

that for same type of molecules it's

very difficult if you have same very

very close size difference it's very

difficult for for this technique to

separate them from each other so these

are the advantages and disadvantages of

size exclusion chromatography or

molecule exclusion chromatography or gel

filtration chromatography whatever name

you want to say and I hope this video

helped you if you like this video please

hit the like button hit the subscribe

button that is present here in the top

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that thank you