Column chromatography - gel filtration chromatography lecture
Summary
TLDRThis 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
🔍 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.
🧬 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.
📊 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.
Mindmap
Keywords
💡Size Exclusion Chromatography
💡Molecular Exclusion Chromatography
💡Agarose
💡Stationary Phase
💡Mobile Phase
💡Column
💡Void Volume
💡Hydrodynamic Volume
💡Folded Protein
💡Unfolded Protein
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
as well as in the bottom and subscribe
to my channel to get more videos like
that thank you
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