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Separation & Chromatography Cheat Sheet (DRAFT) by

Separation & Chromatography notes

This is a draft cheat sheet. It is a work in progress and is not finished yet.


Pure substances
Contain only ONE thing - have fixed melting and boiling points
Contain MORE THAN ONE thing, do not have exact proper­ties, formed by a physical change so they can be easily separated

Purifi­cation as part of drug synthesis

Weigh up starting materials
Set up a reaction
Monitor reaction
Work up reaction to start purifi­cation
Solid-­phase extraction
Melting point

Separation techniques

1. Distil­lation
Used to separate the components of a LIQUID mixture by vapour­ising, condensing vapours and then collecting the liquid conden­sate.
Separation is a result of different boiling points
2. Filtration
Physically separated solids from liquids
Especially used for insoluble solids in liquids
3. Centri­fug­ation
Uses centri­fugal force that spins the samples fast
Separated even fine solid matter form liquids
4. Recrys­tal­lis­ation
1. Dissolve impure crude material in minimum volume of solvent
2. Filter any insoluble material
3. Allow solution to cool slowly
4. Crystals will form


Definition of solubility
Ability of a solvent to dissolve in a solute, depending on the nature of the forces acting between the solute and solvent
e.g. Ionic compounds are more soluble in polar solvents
Covalent compounds are more soluble in non-polar solvents

Solvent mixtures

Immisc­ible: if the mixtures are too different, they will NOT mix, and will form separate layers or 'phases'

Partially miscible: mixtures are not too different but not too similar, they may mix at determ­inate propor­tions depending on the compos­itions

Miscible: if they are similar, they mix in EVERY propor­tion, forming one layer or 'phase'

Liquid­-liquid Separation

What is liquid­-liquid separa­tion?
A method in which 2 molecules initially present in one sample can be separated by giving them the choice of different immiscible solvents
- Very common when isolating or purifying a product
- Can be used to extract natural products
- Normally done in workup stage after a reaction
Criteria of organic solvent for liquid­-liquid extraction
SHOULD readily dissolve the substance to be extracted
SHOULD NOT react with the substance to be extracted
SHOULD NOT react with or be miscible with water (usual second solvent)
SHOULD have a low boiling point so it can be easily removed from the products
Common extraction solvents are dichlo­rom­ethane or ethyl acetate


What is chroma­tog­raphy?
Physical method of separation where the components to be separated are distri­buted between 2 phases: the stationary and mobile phase
Common types:
- TLC (thin layer)
- Column
- HPLC (high perfor­mance liquid)
- GC (gas)
- SEC (size exclusion)
- Ion exchange
- Chiral

Chroma­tog­raphy - defini­tions

Substance being separated (or analysed)
Mobile phase
Phase that moves in a definite direction - consists of sample being separated and the solvent that moves the sample through the column
Stationary phase
The substance fixed in place for the chroma­tog­raphy procedure
Solvent entering column
Solvent leaving column
Process of passing liquid through chroma­tog­raphy


1. Adsorption chroma­tog­raphy
Separation is based on differ­ences between the adsorption affinities of the sample analytes for the surface of a solid-­sta­tionary phase
Based on non-co­lavent intera­ctions
Used for organic molecules
2. Partition chroma­tog­raphy
Separation based on differ­ences between the solubility of the sample analytes in the mobile and stationary phases → stationary phase = immobi­lised liquid
Based on non-co­valent intera­ctions
Used for organic polar molecules
3. Exclusion chroma­tog­raphy
Separation based on exclusion effects e.g. differ­ences in size and shape
used for proteins and nucleic acids
4. Gel electr­oph­oresis
Specia­lised example of size-e­xcl­usion chroma­tog­raphy which uses agaros­e/p­oly­acr­ylamide and passes electr­icity through
Separates based on size and shape
Used for proteins and nucleic acids
Visualised by using UV and stains
5. Ion exchange chroma­tog­raphy
Separation based on differ­ences in ion exchange affinities → e.g. differ­ences in charge size and type
Based on ionic forces
Used for cations, anions, proteins, peptides, amino acids and nucleic acids
- Cation exchangers = negative stationary phase
Anion exchangers = positive stationary phase
6. Thin layer chroma­tog­raphy
Separation based on Kx values
Polar stationary phase = thin layer of silica spread over a glass plate
Stationary phase is placed in the mobile phase
Analyte moves up the plate and components are spread based on their Kx values
Lower Rf = compound is MORE polar
Higher Rf = compound is LESS polar
Increased eluent polarity can increase the Rf

More polar = stronger intera­ctions in the normal phase
Less polar = stronger intera­ctions in the reverse phase
7. Flash column chroma­tog­raphy
Used for large amounts of material (5-25mg) when TLC is not effective


1. High perfor­mance liquid chroma­tog­raphy
Analytical use: identiry multiple analytes in complex mixtures
Prepar­ative use: purifi­cation
2. Gas chroma­tog­raphy
Separated by their volatility
Forces the analyte through a column of the stationary phase by the gas mobile phase at a high pressure and temper­ature

Quanti­tative analysis

Single point calibr­ation
Multiple point calibr­ation
Internal standard
1. A solution containing a known concen­tration of the compound to be measured in the sample is injected → relation concen­tration obtained =AUC
1. Several solutions containing different concen­tra­tions of compound to be measured are injected → AUC obtained
1. One solution containing a known concen­tration of the compound to be measured AND a known concen­tration of internal standards are injected to find a Response factor
2. The sample with unknown concen­tration is injected → new AUC is compared with previous to find concen­tration in the sample
2. The sample with unknown concen­tration is injected → new AUC is compared with previous to find concen­tration in the sample
2. The sample and the same known concen­tration of internal standard are injected to find the AUC and Response factor

Separation in pharmacy

Drugs in the clinic must be pure:
1. Impurities can be harmful and cause side effects
2. Impurities can alter the ability to formulate a drug correctly
3. Impurities can also affect the stability and shelf-life of the drug
Drugs in develo­pment MUST be pure:
This is to ensure that it is the drug that brings about a therap­eutic effect and not any impurities
- Before any new compound is tested, it is purified and charac­terised
Separation is also part of the quality control and monitoring process

Importance of ionisation

Some functional groups can be charged, and this depends on:
- whether the molecule is an acid or a base
- the pH of the molecule
Physio­logical pH = 7.4
we need to know what groups are charged at this pH as it will help us determine:
- types of drug-t­arget binding intera­ctions
- solubility (uptake and distri­bution)
- potential salt forms
e.g.1 - Carboxylic acids
e.g. 2 - Aliphatic amines
Acids react with water:
Bases react with water:
HA + H2O ⇌ H3O+ + A-
B + H2O ⇌ BH + OH-
pKa of carboxylic acids ~ <5
pKa of aliphatic amines ~ >8
These functional groups are almost ALWAYS ionised at physio­logical pH
pKa & pH = measures of dissoc­iation
If the pKa = pH, then the functional group is 50% ionised


Distri­bution of a solute between 2 solvents
Compound is present in BOTH phases according to its relative solubility in both
Solute distri­butes itself between the 2 liquids in accordance with its partition coeffi­cient
Partition coeffi­cient
Ratio of the concen­tration of the solute in one liquid OVEr the concen­tration in the other
Dynamic equili­brium exists between the 2 liquids, temper­ature dependent

Partit­ioning in pharmacy

Relative hydrop­hob­ic/­hyd­rop­hilic properties of a drug are crucial as it influe­nces:
- solubility
- adsorption
- distri­bution
- metabolism
- excretion
Drug too polar? It will not cross the cell membranes across the gut wall
Drug too lipoph­ilic? Drug will be poorly absorbed, so will likely be taken up into the fat tissue and not circulated


LogP: used as a measure of hydrop­hob­icity of a drug

Hydrop­hob­ic/­hyd­rop­hilic character is measured using partition
- Hydrop­hobic molecules prefer the octanol layer → high P
- Hydrop­hilic molecules prefer the water layer → low P

LogP measures only unionised forms of the drug between octanol and water
LogD is sued to represent relative distri­butions of all species, charged or uncharged

Distri­bution Coeffi­cient (Kx)

C(stat) = conc of X in stationary phase
C(mob) = conc of X in mobile phase

Each compound will have a different Kx
Chroma­tog­raphic separa­tions can be altered by changing the nature of the stationary and/or mobile phase(s)

Solid-­phase extraction

Used in sample prepar­ation to remove matrix interf­erences such as proteins
Concen­trates sample of interest
Solid phase in SPE is similar to the stationary phase used in chroma­tog­raphy
Active substances can be retained or unretained
SPE vs. Chroma­tog­raphy
- Column is much smaller for SPE
- Specia­lised columns are more common
- Analytes are typically strongly retained on the SPE column, then impurities are washed away
↳ In chroma­tog­raphy, both the analyte and impurities are passed through at different rates
SPE is used for sample prepar­ation, then chroma­tog­raphic methods follow

Stationary phases

Normal phase
Reverse phase
Stationary phase = polar
Mobile phase = non-polar
Stationary phase = non-polar
Mobile phase = polar
Components elute in order of increasing polarity
Components elute in order of decreasing polarity
Used in TLC and flash column
Used in HPLC
More polar compounds are retained stronger
More polar solvents increase elution
Less polar compounds are retained stronger
Less polar compounds increase elution (e.g. methanol, ethanol)
Solvents = the complete range but rarely greater than 20% for very polar solvents
Solvents = very polar solvents (e.g. water, methanol)
A strong solvent for the normal phase is a weak solvent for the stationary phase

Component retention

For TLC, retention is expressed in terms of Rf
For column, retention is expressed in terms of column volumes (CV)
The relati­onship between Rf and CV are reciprocal
Lower Rf = greater CV → Low RF if preferred as it increases analyte contact time and improves chances of component separation or resolution
To separate adjacent compounds, a large ∆CV is desired

Order of elution

TLC and HPLC depend on polarity
- Normal phase: polar retained more, non-polar elutes first
- Reverse phase: non-polar retained more, polar elites first
Gas chroma­tog­raphy depends on volatility or molecular weight
- Highly volatile or low MW elutes first
Size exclusion depends on molecular size
- Low size retained more, largest size elutes first
Ion exchange depends on charge
- Comple­mentary charges are retained more