Chromatographic Methods Of Analysis Flashcards
(45 cards)
What two phases do chromatographic systems consist of?
Mobile phase (either liquid or gas) Stationary phase (adsorbent solid or a liquid surrounding and adhered to a solid 'backbone')
If both SP and MP are liquids, they must NOT be miscible.
When does sorption and desorption occur?
Sorption= MP moving into SP Desorption= SP coming out of SP and into MP
What does retention result from?
Adsorption
Partition (liquids adhering onto solid backbones)
Explain Adsorption chromatography
SP is always a solid but MP can be a gas or a liquid. In GC the MP is NON-INTERACTIVE. In LC the MP will also compete with the analyte for adosorption sites on the SP Silica gel most widely used material in adsorption chromatography as it can be used for the SP or the backbone.
Define normal phase and reverse phase chromatography in liquid chromatography
NPC= SP is more polar than MP. Polar analyses will retain the longest. RPC= SP is less polar than MP. Polar analyses will retain the least.
How does the MP work in adsorption GC?
It is non-interactive therefore normal and reverse terminology does not apply.
Explain Partition chromatography
Differences in solubility of analytes in the SP. Typically the SP is a bonded phase. The liquid SP is chemically bonded onto a silica support. Polar, ionic and ionisable molecules are easily analysed. SP’s of varying polarities are available.
What are the two common modes of partition LC?
Ion suppression chromatography
Ion pairing chromatography
What is ion suppression chromatography?
Ionisation analytes (weak acids/bases) can be suppressed and retention manipulated.
What is ion pairing chromatography?
Used to separate weak acids/bases. Can be manipulated by MP. Pairing ion may be a surfactant for example. Ion pairing controlled by varying chain length of pairing agent, concentration of the pairing ion, pH of the MP, concentration of organic solvent in MP.
What are typical ion pairing reagents for cations?
Alkyl sulphonic acids
What are typical ion pairing reagents for anions?
Dibutylamine ammonium salts
In TLC what is the SP and MP?
SP= cellulose, silica gel or alumina MP= solvent that moves up the paper/plate
What variables might influence the Rf?
Thickness of SP
Moisture content of SP and MP
Temp at which TLC is performed
Sample size
To minimise these effects, spot reference markers/ standards onto each plate and calculate the relative retention factor (Rx)
How could you identify analytes using TLC?
Compare Rf and Rx values
Visualisation techniques-
1. Iodine vapour- turns brown with organic compounds
2. Alkaline tetrazolium blue- specific for corticosteroids, produces blue spots
3. Ethanol/sulphuric acid spray- used for corticosteroids that fluoresce at 365nm.
Explain how Silica gel G254 is used
UV light is used to illuminate the plate, if the analyte absorbs UV it can be seen as a black spot on a yellow background. This method is used as an identity test for methyl prednisolone
What are known impurities?
Ones originating from the manufacturing process come from degradation pathways are known impurities.
What are the advantages and disadvantages of TLC?
Adv- robust and cheap
Applicable in pharmaceutical industry
Allows simultaneous analysis of batches
Gives both quantitative and qualitative analysis
Disadv- not suitable for analysing volatiles
Requires great operator skill
Sensitivity may be limited
What is HPTLC?
An automated form of TLC. Has greater resolving power, consumes less solvent. Plates can be characterised by smaller particles, thinner layers and smaller plates. Particle size distribution is narrower than for normal TLC. Requires band broadening to be minimised.
How may you improve the system of sample application in TLC?
By using a universal capillary holder. Instead of a spot being made on the TLC plate, it is more common to apply a band. Bands are more distinct, takes the retardation factor more precisely.
What are the advantages of HPTLC?
Provides a chemical profile Requires only basic skills Universally applicable Generates a fingerprint (looks at number, position, colour and intensity of bands) Enables qualitative identity
How does Gas Chromatography work?
Gas/liquid is inected into a heating chamber at top of column. Heating chamber causes rapid vaporisation which is carried onto column. Condensation occurs at the top of the column. Vaporised mix is carried through column by a continuous flow of inert gas. Components migrate at different rates. Detector monitors as each component emerges.
What are packed GC columns?
External surface= stainless steel, glass or copper. Packed with solid particles or a solid support coated with a liquid.
What are capillary GC columns?
Smaller diameter than packed but much longer. SCOT columns= support coated open tubular columns. Made of fused silica with polyamide coating on outside for flexibility.