Separation
Pure substances |
Contain only ONE thing - have fixed melting and boiling points |
Mixtures |
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
Synthesis |
Purification |
Characterisation |
Weigh up starting materials
Set up a reaction
Monitor reaction
Work up reaction to start purification |
Extraction
Recrystallisation
Distillation
Chromatography
Solid-phase extraction |
NMR
IR
MS
RF
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 |
Polarity
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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
- LIKE DISSOLVES LIKE
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e.g. Ionic compounds are more soluble in polar solvents
Covalent compounds are more soluble in non-polar solvents
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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
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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
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- Very common when isolating or purifying a product
- Can be used to extract natural products
- Normally done in workup stage after a reaction
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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
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Common extraction solvents are dichloromethane or ethyl acetate |
Chromatography
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What is chromatography? Physical method of separation where the components to be separated are distributed between 2 phases: the stationary and mobile phase
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Common types:
- TLC (thin layer)
- Column
- HPLC (high performance liquid)
- GC (gas)
- SEC (size exclusion)
- Ion exchange
- Chiral
Chromatography - definitions
Analyte |
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 |
Eluent |
Solvent entering column |
Eluate |
Solvent leaving column |
Elution |
Process of passing liquid through chromatography |
DIFFERENT METHODS & RETENTION OF ANALYTES
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 |
INSTRUMENTAL CHROMATOGRAPHY
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 |
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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 |
Partitioning
Partition |
Distribution of a solute between 2 solvents |
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Compound is present in BOTH phases according to its relative solubility in both
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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 |
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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
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 |
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