Nucleophilic Substitution Reactions – Sn1 & Sn2 Stereochemistry

– Nuclephilic Substitution Reactions

The viability of nucleophilic substitution over a single bond is determined by the bond polarity. A nucleophile (Nu-) will attack the δ+ atom in a polar bond and replace the existing δ- atom.

A good example of this is the haloalkanes, where the halogens are more electronegative than the Carbon atom. As the the halogen has a higher affinity for -ve charge, the bonding electrons are found closer to the halogen than the carbon, shifting the dipole charges in the molecule.

Nuleophilic Substitution of Iodine with Cyanide in Iodomethane.

Nuleophilic Substitution of Iodine with Cyanide in Iodomethane

As you can see the nucleophile (which likes +ve charge) attacks the δ+ carbon atom, and this essentially severs the C-I bond, releasing I-.

There are a large number of other suitable nucleophiles, including the following. I’ve included products too, excuse the lack of correct punctuation. This allows conversion of an Alkyl Halide into many different compounds.

Starting Material Reacts With Produces AKA
RX PPh3 RP+Ph3X- Phophodium Salt
RX R’S- RSR’ Thioester
RX Na2S RSH Thiol
RX NC- RCN Nitrile
RX EtO- ROEt Ether
RX HO- ROH Alcohol
RX N3- RN3 Alkyl Azide
RN3 H2 / PdC RNH2 Amide
RX NH3 RNH3+X- Ammonium Salt
RNH3+X- HO- RNH2 Amide

The ones in GREY at the bottom are the amide chain – there are two routes to an Amide. One is through an alkyl azide and the other through an ammonium salt.


A special note: Hydride (H-) reducing agents such as LiAlH4 can be used as sources of nucleophilic hydride ions which will replace the halogen group. This allows conversion of an alkyl halide into an alkane.

Nucleophilic Substitution of Halide with Hydride via LiAlH4

Nucleophilic Substitution of Halide with Hydride via LiAlH4


A stable molecule is a good leaving group, such that H2O is better than HO-. In haloalkanes, reactivity goes from RI > RF (travelling down the group).

This can be explained better when we look at the basicity and nucleophilicity of the atom/molecule. Note that while Nucleophilicity is a kinetic property determining the rate of reaction with a Carbon atom (how fast the reaction progresses), Basicity is a thermodynamic property determining an atom/ion/molecul’s ability to accept a proton.

Nucleophilicity (rate of reaction): NC- > I- > RO- > HP- > Br- > Cl- > ROH > H2O
Basicity (ability to accept proton): RO- > HO- > NC- > H2O > ROH > Cl- > Br- > I-

– The Reaction Pathway – Sn1 and Sn2 Reactions

There are two main pathways that a nucleophilic substitution reaction can follow:

Sn1 (Substitution, Nucleophilic, Unimolecular):

  • Substrate ionises to form a planar intermediate carbocation in the rate determining step.
  • The intermediate cation then rapidly reacts with the nucleophile. This means there are two transition states.
  • This is a 1st order reaction as rate = k[substrate]. It is a unimolecular process.
  • Favoured in polar solvents – this aids ionisation.
  • Favoured Tertiary > Secondary > Primary as the two state process allows access to the carbon centre without steric hindrance (see Sn2 below).
Sn1 Substitution

Sn1 Substitution

Sn1 creates a racemic product (an equal amount of left and right enantiomers) which as a result is optically inactive. This means it will not rotate polarised light.

Sn2 (Substitution, Nucleophilic, Bimolecular):

  • Reaction occurs completely within one transition state.
  • This is a second order reaction as rate = k[substrate][nucleophile]
  • Reaction favoured in polar aprotic solvents (solvents which have high polarity but cannot dissociate a H+) such as DMF (Dimethylformamide) and DMSO (Dimethyl Sulfoxide).
  • Steric hindrance slows or stops reaction progression in tertiary systems as steric crowding stops attack by the nucleophile (aka there isn’t room!) and tertiary cations are quite stable. In this case we would expect Sn1. Note that the “back route” must be clear else the reaction will proceed by Sn1.
  • Favoured Primary > Secondary > Tertiary.
Sn2 Substitution

Sn2 Substitution

Sn2 creates a product with an inverted stereo structure to that of the substrate. Essentially the Nucleophile attaches to the opposite side from the leaving group, inverting the molecule’s original stereochemistry.

– Alcohols

Alcohols are extremely important for synthesising new molecules:

Synthesising new molecules from Alcohols (In this case Propanol)

Synthesising new molecules from Alcohols (In this case Propanol)

It is especially useful when you consider that we can already use the Alkyl Halide table from above to form a variety of molecules that way.

Hybridisation – Mixing Up Orbitals with sp, sp2, sp3

Essentially, hybridisation is the mixing of standard atomic orbitals to form new orbitals – which can be used to describe bonding in molecules.

Most importantly we have sp3, sp2 and sp hybridisation.

sp3 Hybridisation in Methane (CH4):

The best way I can describe sp3 hybridisation is in Methane (also the most basic choice!). This is simplified for expression. Remember that Carbon has 6 electrons.

  • In methane (CH4), 1 Carbon binds with 4 Hydrogens. The carbon atom itself has only 2 electrons available for bonding in the 2p subshell.

    Carbon - Ground (normal) electron states. 1s2, 2s2, 2p1 2p1.

  • In order for 4 hydrogens to bind there need to be 4 electrons available for bonding, which cannot be achieved at the moment. The pull of a hydrogen nucleus results in an electron being excited from the 2s subshell into the 2p subshell, where it is available for bonding.

    Carbon - An electron has been excited to the 2p orbital.

    Carbon - An electron has been excited to the 2p orbital.

  • This excitation changes the forces on the valence (bonding) electrons as the nucleus now exerts a stronger effective core portential upon them. This and other factors leads to the creation of a new ‘hybridised orbital’, called sp3.
    Carbon - Hybridisation forms SP3 orbital.

    Carbon - Hybridisation forms sp3 orbital.

    This leaves 4 valence electrons which will each overlap with the s orbital of a Hydrogen to form a σ (sigma) bond. These hydrogens space themselves as far apart as possible, leading to the tetrahedral structure of methane.

    3D animation of methane.

    3D animation of methane. Produced on ChemSketch.

    Each of the bonds in the image above are σ-bonds.

    Methane Hybridisation. Shows the S orbits of H overlapping with SP3 orbitals of C. Note 2 electrons in each bond, one from carbon and one from hydrogen. Image by K. Aainsqatsi, released into public domain.

    Methane Hybridisation. Shows the S orbits of H overlapping with sp3 orbitals of C. Note 2 electrons in each bond, one from carbon and one from hydrogen. Image by K. Aainsqatsi, released into public domain.

sp2 Hybridisation in Ethene (C2H4):

This is similar to sp3 hybridisation, except there are only 2 hydrogen nuclei pulling on the bonding electrons (which need an electron each) and the other 2 electrons are required for the π (pi) bond (double bond) between the two Carbons.

A molecule of Ethene.

A molecule of Ethene.

The electron configuration in carbon starts the same:

Carbon - Ground (normal) state of electrons


Carbon - An electron has been excited to the 2p orbital.

Carbon - An electron has been excited to the 2p orbital.

but the resulting spread is different:

Carbon hybridisation in Ethene (sp2)

Carbon hybridisation in Ethene (sp2)

Only 2 of the 2p orbitals are used in sp2 hybridisation; in contrast to the 3 used in sp3 hybridisation (you should be seeing where the numbers come from!).

This leaves us with 3 sp3-orbitals and 1 p-orbital to bond with. 2 of the sp3 orbitals are used for forming σ-bonds with the 2 hydrogens, while the remaining sp3 orbital binds with the other carbon to form a σ-bond and the p-orbital bonds with a p-orbital from the other carbon to form a π-bond.

Every double bond (regardless of what atoms it joins) consists of a π-bond and a σ-bond.

Shows seperate carbon atoms in sp2 hybridisation, then combined to form ethene.

Shows seperate carbon atoms in sp2 hybridisation, then combined to form ethene.

sp Hybridisation in Ethyne (C2H2):

This can occur on an atom with a triple bond such as the alkynes. Ethyne is the simplest.

Ethyne. Triple bonded Carbon with 2 hydrogens.

Ethyne. Triple bonded Carbon with 2 hydrogens.

In this case we only have 1 hydrogen attached to a carbon, and three bonds between each carbon.  That’s 1 hybridised bond between the carbon and hydrogen with another hybridised bond between the carbons. The other two p-orbitals form two more bonds between the carbons.

Ethyne - sp Hybridisation

Ethyne - sp Hybridisation

This essentially means that the triple bond consists of 1 σ-bond and 2 π-bonds.


Essentially, the hybridisation of the carbon atom is based on the number of bonds to other carbons or identical atoms.
sp3 = single bond
sp2 = double bond
sp = triple bond

Drawing Chemical Formulae on your PC

You will undoubtedly need to draw an equation out on your computer at some point, and there are several ways to do this.

  1. Load up Paint and spend hours perfecting a drawing.
  2. Use an Office suite and spend equally long hours trying to get all the lines in the right place.
  3. Use a specialist application to do it in seconds.

Assuming you chose option 3, we’d like to introduce you to MDL ISIS DRAW 2.5. Best of all, it’s free for personal use!

A couple of the templates provided by ISIS DRAW

A couple of the templates provided by ISIS DRAW

You must register first but you can then download ISIS draw through their site here. I believe they are working on replacing it as MDL has now become Symyx, so I am unsure whether it will stay free when they release the next version. The current version (just updated) now works better with Vista.

You could also use an application called ChemSketch. This can be downloaded from their website here.

These applications are very useful for drawing accurate molecules, checking them (for over bonded atoms etc) and naming. They can also generate 3D images of the molecules you make.

I’m not going to go into how to use them here, I’m just introducing you!

How about 3D?

There are quite a few notable molecular visualisation applications and I’ve just included some of the easier to use here.

You can also use the above programs to render in 3D, the easiest to use (I think) is ChemSketch. Simply draw a molecule in the standard view and click copy to 3D – done!

Another choice is Jmol. Jmol is a java applet which means it runs in your browser without installing (there is a stand-alone downloadable version also available). You can check that out here if you need added functuality over ChemSketch. There’s also a host of demonstrations and guides through the link. Like the rest of the applications on this page, it’s free!

The final example is Polyview – you simply fill in a form here and it pumps out a very nice 3D image or animation that can be put into presentations etc easily.

For more information on any of this and more I would suggest checking out this site, there is a huge number of resources for Jmol and other tools.

Chemical & Mathematical Equations in MS Word & LateX

When writing chemical or mathematical equations in Microsoft Word it is often easiest to use the equation editor.

This editor allows you better formatting than entering a formula with the standard tools. It’s also supplied with most copies of Microsoft Office, which you will probably find installed on institution/corporate computer facilities.

I have prepared this using Microsoft Office 2007. If your Office looks different, you should be able to find Equation Editor in “Insert > Object > Microsoft Equation 3.0”. If you can’t find it at all then you will need to install it from your Microsoft Office CD (this site shows you how to install).

If you don’t have MS Office then you could use a free editor such as this LateX Equation Editor which will render your equations as images which you can then copy into your work. They have a working trial at the top of the page, click the center image to load it.

The equation editor contains quite a few pre-set equations which you can enter automatically and gives you access to all the symbols you will need.

Quite simply, find out more through practice! There are more in depth guides available on this tool, including this one for older versions of Office. A simple google search will reveal more information.

Close Packing of Atoms & Metallic Elements

Close packing of atoms is simply close packing of spheres. There is only one way to efficiently arrange circles in 2D…and we can visualise the stacking with 2D drawings…

…each sphere (seen from above) is surrounded by 6 other spheres. This is a single layer, the next step is to stack the layers.

The most efficient place to put the next spheres is in the depressions between each sphere on layer one. So, we have one of two options.

We can either have rows in A or rows in B. Due to the size of the spheres it is not possible to fill ALL holes (A and B).

In position A:

In position B:

The fact is that the two sketches above are mirror images. This means it doesn’t matter which holes the second layer fall into! The important thing here is the creation of a new hole…directly above the spheres in layer 1. So for layer 3, we either have a repeat of layer 1 or a new layer in a new position.

Making 3 different layers with a new position:

Matching layer 1:

There are no spheres/atoms in the same position on the first 3 layer spread, but in the second we can see that layer 3 is in the same position as layer 1.

So, we have several possible outcomes, including:

  1. 1..2..3..1..2..3..1..2..3……. Three different layers, could be cubic close packing (ccp).
  2. 1..2..1..2..1..2..1..2..1……. Two different layers, could be hexagonal close packing (hcp).

HCP and CCP are the simplest and most common close packing structures. Each atom is surrounded by 12 other atoms – giving both these structures co-ordination numbers of 12.

So while these are close packed, a body centered cubic arrangement would NOT be, as it only has a co-ordination number of 8. This can also be illustrated with packing densities, where HCP and CCP have a density of 74.1% while BCC (body centered cubic, I) has a density of only 68% (although it is still a common metallic structure).

– Structures of Metallic Elements

In 1883 William Barlow (the curator of the science museum) suggested that the metals would each take one of three structures. These were hcp (hexagonal close packing), ccp (cubic close packing) and bcc (body centered cubic structure – which is NOT close packing).

Note that both hcp and ccp follow a face centered structure and as such have the same packing density, of 74.1%.

This image shows the structures of the metals in the periodic table at room temperature & pressure. I have it correct as far as I know, if you see any problems let me know.

Metal Elements of the Periodic Table - Structures

Metal Elements of the Periodic Table - Structures


Some metals hold different structures at different temperatures and pressures. This is called Polymorphism and can occur in any compound.

Iron (Fe) is one such metal, and at atmospheric pressure a simple change in temperature will change it’s structure:

Above 1809K – Liquid – (No crystal struture)
1809K – 1665K – Delta-Fe – Body Centered Cubic (bcc)
1665K – 1184K – γ-Fe – Face Centered Cubic (fcc)
Under 1184K – α-Fe – Body Centered Cubic (bcc)

Note – Polymorphism of elements is known as allotropy. An example of allotropy is how carbon forms as diamond, graphite, nanotubules etc.

– Interstitial Holes

Close packing leads to two different types of hole between layers. One has a co-ordination number of 4 and the other of 6 (the number of surrounding spheres/atoms).

These are Tetrahedral (Td) with the co-ordination number of 4 and Octahedral with the co-ordination number of 6.

More to come… **UPDATE ME**

Crystal Structure Studies

Crystalline solids are composed of regular repeating patterns over relatively long distances ( >1000 Å). This is known as having a long range order.

ONLY crystalline solids produce macroscopic crystals (visible crystals).

Crystals have flat faces which can vary in size from crystal to crystal. The angle between similar faces is constant, and they break (cleave) in preferred directions (a feature exploited by diamond cutters).

Crystal Structure Studies tell us:

  • Chemical Characteristics
    • How are atoms connected?
    • What are the bond lengths and angles between them?
    • What does this say about the bonding?
  • Inorganic Solids
    • Structural features which may lead to an important property.
    • How changing a metal coordination sphere may alter the property (eg. in superconductors and electrical materials).
  • Organic Compounds
    • Which points of stereochemistry are important.
    • How solubility is affected by the way the molecules pack together.
    • How many ways the molecules can pack together.
  • Solution vs Solid State Structure
    • Structures may differ between different phases.

A couple of things about Molecule Packing:

  • Atoms, ions or molecules always try to pack into the lowest energy configuration.
  • This configuration can then be repeated for a large number of units.
  • As the configuration is repeated, a regular pattern forms and a lattice emerges through the crystalline as a whole.
  • This pattern may interact with certain wavelengths of radiation and lead to diffraction (constructive interference) which provides a means of studying the pattern.

3D Lattices and Translations – Unit Cells:

We can use three translations (a, b and c) to illustrate distance between atoms, ions or molecules; and three angles between each of these translations (α, β and γ). These parameters define the size and shape of the unit cell.

*Note that angle α corresponds to the translation a, angle β to translation b…etc.*

The unit cell is:

  • A small volume defined by 6 faces, consisting of 3 identical pairs.
  • A small unit of the larger structure which is then repeated.

“The smallest repeating unit that shows the full symmetry of the crystal structure.”

There are 7 Crystal Systems

  • Triclinic – a≠b≠c – α≠β≠γ (ALL Different!)
  • Monoclinic – a≠b≠c – α=γ=90°, β
  • Orthohombic – a≠b≠c – α=β=γ=90°
  • Tetragonal – a=b≠c – α=β=γ=90°
  • Hexagonal – a=b≠c – α=β=90°, γ=120° (Need a and c)
  • Rhombohedral – a=b=c – α=β=γ≠90°
  • Cubic – a=b=c – α=β=γ=90° (ALL Equal, Only need a)

Let’s look at some unit cells. Try and work out the number of atoms in each 2D cell:

You should have:
i) 1
ii) 1
iii) 2

Why? This is something we’ll explore a little more later, but for now consider this. i) has 4 dots (atoms, ions or molecules) in it’s shape. Each of these four dots can participate in a total of four unit cells.

Look at the red dot. It is shared between four unit cells and so only contributes 1/4 to the contents of that cell. The same can be said for each other dot at the corner of a cell, as we assume that it is only attributing 1/4.

So we know that i) has 4 corners, each worth 1/4. That totals 1!

ii) is just the same, but iii) is different. It has 4 corners each worth 1/4 BUT it also has a single dot in the middle that is not shared with any other cell…meaning it is worth a whole 1. So (4×1/4)+1 = 2.

– Bravais Lattices

Although there may be an infinite number of chemical structures, there are only 14 3D lattice types, known as Bravais lattices.

We’re just going to look at the cubic system to start with. Remember, a=b=c – α=β=γ=90°. In the cubic system there are 3 lattice types, P (primitive), I (body centered), and F (face centered).

  • Primitive cubic cells contain only corner dots, which can be shared between 8 different unit cells, meaning each dot is worth 1/8. 8*(1/8)  is 1 lattice point.
  • Body Centered (I) cubic cells have the primitive structure but feature a central dot, worth 1 (as it is exclusive to that cell.  This gives 8*(1/8) + 1 = 2 lattice points.
  • Face Centered (F) cubic cells once again follow the primitive structure but this time feature a dot on each of the 6 faces. Each of these dots can be shared between two cells and so each is worth 1/2. This gives 8*(1/8) + 6*(1/2) = 1+3 = 4 lattice points.

So, when counting particles in a unit cell:

  • An atom at a corner counts for 1/8
  • An atom on an edge counts for 1/4
  • An atom on a Face counts for 1/2
  • An atom within the cell counts for 1

Technically this is only true for cells with 90 degree angles, but it does actually work for all cells.

– Projection Drawings:

Although we could draw the 3D shape of a cell every time, it is easier to draw a projection – a top down sketch of the cell.

Each atom’s height within the cell is indicated as a fraction of the cell height. Note the cell heights in the drawing below.

Here are the projection drawings for each cubic lattice:

– Packing Densities:

To find the structure offering the maximum density of atoms, we can calculate the atomic volume and packing density per unit cell. This involves some fairly basic maths which I have recapped here. We’ll start with Primitive cubic cell.

Volume of cubes = a³ (a*a*a)

Volume of a Sphere/Atom = 4/3 x π x r³
(r = atomic radius)

Packing Density = n x p / a³
(n = number of lattice points per cell, p = Volume of Atom)


There is 1 lattice point per cell – and the atomic radius is half the distance between the closest atom points. In this case, r = 1/2a.


  • Volume of atom = 4/3 x 3.142 x (1/2a)³ = 0.524a³

The Packing Density of these atoms is then simply the volume of one atom (which we have found to be 0.524 a³) divided by the volume of the cube (which is a³):

  • Packing Density = (0.524a³) / a³ = 0.524

Body Centered:

There are 2 lattice points per cell, and in this case the radius is not simply 1/2a as the central atom is the closest point. More trigonometry!

The distance between the 2 corner points is √3a, as shown in the first image. Thus the distance between the two closest atoms is half of this…√3a/2. This means the atomic radii must be √3a/4.


  • Volume of Atom = 4/3 x 3.142 x (√3a/4)³ = 0.34a³
  • Packing Density = (2 x 0.34a³) / a³ = 0.68

(Note the doubling of the atomic volume in the packing density calculation – this is because Body Centered cubes contain 2 lattice points.)

Face Centered:

4 lattice points per cell, with more trigonometry.

The distance between a corner atom and the corner atom diagonally across the same face is √2a. This means the distance between a corner atom and a face atom is half of this, √2a/2. This means the atomic radii must be √2a/4.


  • Volume of Atom = 4/3 x 3.142 x (√2a/4)³ = 0.185a³
  • Packing Density = (4 x 0.185a³) / a³ = 0.74


Primitive Density (P) = 0.524 = 52.4%

Body Density (I) = 0.68 = 68%

Face Density (F) = 0.741 = 74.1%

You can see the density increasing as you move down the list.

– Using this knowledge

  1. Finding the metallic radius of α-tungsten. We know the unit cell is cubic (so we can use what’s above), a = 3.15Å, Atomic Mass = 183.85g mol^-1 and it’s measured density is 19.25g cm^-3 near room temperature and pressure. Step by step:
    1. We need to calculate the number of atoms in the unit cell (the number of lattice points). We know:
      Density = (Mass of 1 atom x Number of Atoms in Cell) / Volume of Unit Cell
      19.2 = ((183.85/6.023×10^23)* x N) / (3.15×10^-8)*^3
      and Number of Atoms = 2 (rounded to leave an integer).

      We now know that there are 2 lattice points, or atoms per unit cell. Looking above we know that this unit cell is Body Centered.

      (* Finding the mass of 1 atom is done by taking the molar mass of an element and dividing it by Avogadro’s number, AKA the number of atoms per mole.)
      (** This value has been converted into Metres from Angstroms. 1Å = 1×10^8 Metres.)

    2. Calculate the metallic radius – we have learnt that the unit cell is body centered, so we already know the distances involved.
      The atomic radius in this lattice is √3a/4, and we know that a for α-tungsten = 3.15Å. Therefore the metallic radius is (√3 x 3.15)/4 = 1.36Å.

Some Basic Trigonometry

It seems that doing maths for as long as I can remember didn’t make it stick in my mind so I’m having a little refresh as I go. I’m starting with some basic trig.

– Pythagorus’ Theorum – Find the length of a third side on a Right angled Triangle.

Very simple. Say we have a right angled triangle with sides a, b and c – where c is the hypotenuse…

Crucially, a² + b² = c²

AKA the sum of the areas of the two squares on the legs (a and b) equals the area of the square on the hypotenuse (c).

So how about this triangle?

5² + 9² = c²
25 + 81 = c²
c² = 106

Now square root 106…

c = ~10.30 (4sf) or √106 in surd form.

Protein Purification

– Protein Identification

Protein purification begins with the need to identify the protein we want to purify! There are several methods that can be used to rapidly identify the protein:

  • Enzyme Assay (by catalytic activity) – with certain enzymes we can use colorimetry to detect a product as a reaction progresses. The higher the more enzyme present, the faster the colour or light absorbance will change. An example is testing for Alcohol Dehydrogenase, which will lead to a change in the levels of NADH and NAD+ as Ethanol is converted to Ethanal. This change can be detected by colorimetry at ~340nm.
  • SDS-PAGE Electrophoresis (by size) – this method seperates protein chains by size by electrophoresis. This method denatures the proteins.
    The sample is run at the same time as a molecular mass marker sample, containing proteins of known mass. The marker sample will provide a scale for the mass of your sample. Once you’ve run the gel you will be able to plot the results as above, draw a best fit line and read off the Molecular Mass of your sample protein.
  • Immuno-Assay (by specific antibodies) – Antibodies that fit specifically to the protein you are looking for are added to the sample. When these bind to the target protein they will instigate a colour change or some other noticable change. The presence and concentration of the target protein can be assessed by the extent of the changes – if it was a colour change then the darker the colour goes, the more target enzyme must be present.
  • Western Blotting – A combination of electrophoresis and immuno-assay. The immuno-assay technique is run by electrophoresis. This will be useful if your sample contains several different proteins and you need to identify the target protein. The band of colour (or change) will show you the correct protein, and then you simply need to calculate the approximate molecular mass using the molecular mass markers.

– Protein Purification

I’ve broken the purification methods here into 8 different headers, each a physical-chemical property or biological activity.

  1. Stability (Heat). Some proteins are more heat tolerant than others and can survive heating while others denature. If your target protein is heat stable at above 60C and your contaminants are not, then simply heating your mixture to 60C for 30 minutes will denature most of the contaminents. This will leave you with a much higher concentration of your target protein in your mixture.
  2. Solubility (Seperate by pI). Proteins are least soluble at the pH equal to their isoelectronic point. When helped by the addition of salts to the solution this can lead to their precipitation. As the salt concentration increases, different proteins will precipitate.
  3. Size. Proteins can be seperated by Gel Permeation Chromatography (Gel filtration). The proteins are run through a buffered, porous, cross linked resin. While small molecules are able to fit into the pores in the resin, the larger proteins cannot and so travel ahead, with the small molecules lagging behind.
    This is due to a larger volume of buffer available to the smaller molecules, meaning more buffer must pass down the column for them to elute, compared to the relatively smaller volume of buffer required to elute the larger, excluded proteins.
  4. Density (Centrifuge). By centrifuging the sample in a test tube containing a sucrose density gradient, the centrifugal forces will force the proteins down the tube until they reach a concentration where the density of the sucrose solution is the same as their own. This level is known as it’s isopycnic level.
  5. Charge. There are several different methods of purification by charge:
    1. Gel Electrophoresis – Based on movement of a protein through a cross linked gel called polyacrylamide. This would occur at a pH where the protein has a charge (not at it’s pI). The size of the pores can be altered by changing the concentration of cross linking reagent, and the speed at which a protein travels is equal to its charge:mass ratio. (This method does not tell us anything about the protein’s molecular weight).
    2. SDS PAGE – This cannot really be used for purification because SDS detergent (Sodium Dodecylsulphate) is used which denatures the protein. It unfolds the protein and surrounds it with -ve charge sulphate groups which means all the proteins have a uniform charge:mass ratio. SDS has a 12 carbon hydrocarbon chain, and then a hydrophilic sulphate group. The Sulphate groups surrounding the protein form a miscelle.
      The sample is now allowed to run on a gel, from -ve to +ve, and as they all have the same mass to charge ratio, their rate is determined only by their size. The smaller protein molecules move faster and the larger molecules move slower through the gel.
    3. Isoelectric Focusing – Very similar to (1) above, but instead of an electric charge, there is a pH gradient along which the proteins can move until they are at a point where they  have no net charge (at their pI).
    4. Ion Exchange Chromatography. Essentially, both columns and proteins become charged at different pH’s, and by altering the pH we can hold on to some proteins while others are eluted.
      Diethylaminoethyl-Cellulose (DEAE-Cellulose) has a +ve charge below pH 9.5 wheras CarboxyMethyl-Cellulose (CM-Cellulose) has a -ve charge above pH 3.0. Therefore:
      – Proteins with a +ve charge at pH7 will bind to a column of CM-Cellulose, while
      – Proteins with a -ve charge at pH7 will bind to a column of DEAE-Cellulose.
      We can then alter the pH of the solution to release certain proteins or to pick up others. Another way of dispersing ionic interactions between the column and proteins is to increase the salt concentration.
  6. Hydrophobicity. Proteins nearly always feature hydrophobic areas or side chains and these allow the proteins to bind to resins with hydrophobic groups attached. This means the proteins can be eluted with a gradient of buffer (eg an organic solvent such as ethanol). The proteins forming the strongest interactions with the resin column will require higher concentrations of ethanol to elute.
  7. Biological Function. If a protein has a high affinity for a substrate (eg. ADH has a high affinity for NAD+) then we can use affinity chromatography. If we immobilise the substrate (eg. NAD+) then the protein will bind to that substrate, immobilising itself – allowing other proteins to run free of the column. By releasing free NAD+ throught he column the substrate will gradually release the immobilised NAD+ in favour of the free NAD+ and run free of the column.
    This method can purify a protein in one step, and works best if the protein has a high affinity for the bound ligand.
  8. Fusion Proteins. This involves the addition of a gene to a protein that essentially ‘tags’ the protein. An example would be a tag containing histidine residues, which would bind to metal ions in the column.
    Here, the imidazole rings on the histidine residues stick to the immobilised metai ions allowing other proteins to elute the column. Then, like the method above, add free imidazole to release the fusion proteins and then use a protease to cut the tag away. Run the column again and only the tags will bind, allowing the protein of interest to run free.

Proteins – Quaternary Structure & Overview

The quaternary structure of a protein involves the association of folded polypeptide chains into a mature, active protein.

  • This can be a single polypeptide chain (monomer), 2 chains (dimer), 3 chains (trimer), 4 chains (tetramer) and so on…
  • The associated chains can be identical or different.

Some quaternary structure require additional polypeptide chains (which were removed during production) in order to achieve a working protein state (eg. Mature insulin). There are also structures which will revert to their original shape once broken, as the order is set in the primary structure of Amino Acids.

With Insulin, a helper amino acid strand is used to ‘hold’ two sequences in place, allowing the formation of disulphide bridges. I’ve tried to illustrate before and after:

S is the signal chain, while B acts as a support structure during disulphide bridge formation between A and C.

– An Overview

  • Primary Structure – The sequence of Amino acids on a chain.
  • Secondary Structure – The 3D relationship between Amino Acids – leading to α helix, β pleated sheet etc.
  • Tertiary Structure – The 3D relationship between parts of the above structure.
  • Quaternary Structure – The number of and relationship between amino acid chains (seperate tertiary structures).

Proteins – Stabilising Forces

There are several different types of forces acting on/within a protein molecule. These include:

  1. Covalent Bonds:
    1. Peptide bonds between Amino Acids (C-N). Can be broken down into individual amino acids by hydrolysis with 6M acid/alkali, or by proteases/proteolytic enzymes.
    2. Disulphide bridges form between cysteine to form cystine. (Cysteine has -SH which forms disulphide bridge -S-S- with another HS-). Bridges are broken down by reduction with β-mercaptoethanol to form cysteines once again.
  2. Non-Covalent Forces/Bonds:
    1. Hydrogen Bonds – these bonds are throughout the protein. The bonds in the middle of the protein structure contribute most to stability as they are furthest away from water (which would disrupt them). These can also be disrupted by heat.
    2. Van Der Waals forces/interactions – short range dipole-dipole (δ+ & δ-) interactions between close atoms. Easily disrupted by heat or denaturing agents.
    3. π-π overlap – π electron clouds delocalised over rings & bonds. Are disrupted by heat.
    4. Electrostatic bonds, Ionic interactions and Salt bridges between residues. All broken by changes in pH or high ionic strength. (Eg, positive residues include Lys, Arg, His while negative residues include Asp, Glu, Tyr & Cys).

– Zwitterions

Zwitterions are amino acids in free solution that are doubly charged. Their net charge will depend on the pH of the solution. Each amino acid has an isoelectric point at which it has no net charge.

Below the isoelectric point (also known as pI), they have a net positive (+ve) charge and above the pl they have a net negative (-ve) charge.

When amino acids become part of a polypeptide/protein, they lose their NH2 and OH groups so only the side chains can carry charges.

Proteins themselves can have isoelectronic points – and this will depend on the number and type of different amino acid residues.

– Hydrophobic Interactions

This is the prime driving force for protein folding (AKA hydrophobic collapse).

Essentially the protein chain will fold in such a way as to minimise the exposure of hydrophobic residues within the chain. This leads to the residues with hydrophilic (polar) side chains being situated on the outside of the molecule.