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.
Why is resolving power in capillary GC higher than packed GC?
As when increasing length, it significantly increases the number of theoretical plates in capillary columns as compared to packed.
How can you quantify the polarity of a GC stationary phase?
McReynolds constant. Higher the constant the more polar.
It determines the retention of certain analytes.
How is overloading of a capillary column avoided?
‘Splitting’= majority going through column is carrier gas, small amount is analyte. Split 90% off so most will not reach column
Split valve must be integrated into GC hardware for this.
How is flame ionisation detection used in GC?
Universal for all organic compounds. It is a destructive detector but you can still collect analytes by splitting the effluent stream before the FID. Sample is burned and ions produced, ions are measured and used to make a chromatogram.
What are the disadvantages of FID in GC?
It will NOT produce a signal for water. Major disadvantage is that it requires 3 gases (carrier, air and hydrogen) to operate and won’t work for inorganics.
What other detectors can be used in GC?
Electron capture detector- selective for halogenated chemical derivatives or environmental pollutants
NPD- nitrogen or phosphorous levels
FT-IR- good for structure elucidation. Qualitative tool. Expensive.
What is GC-MSD?
A hypenated technique as it combines chromatographic and spectroscopic techniques. It provides both qualitative and quantitative information.
Why is chemical derivatisation needed in GC?
It is used as another method of manipulating separation.
Increases volatility and decreases polarity of analyses
Decreases thermal degradation
Increases detector response
Improves separation and decreases band broadening
What are the disadvantages of derivatising in GC?
It might interfere with analysis. Increase analysis time. Might cause extra expense. May cause unintended chemical changes in analyte.
What are the 3 groups of derivatisation reagents that get used?
Silylation- uses TMCS.
Acylation
Alkylation
What does temperature programming in GC achieve?
Separation of complex samples. Enables separation of a sample with both high and low volatility components through selected increases in temperature at specific points.
How can separation of analytes be estimated?
By calculating the RESOLUTION (Rs).
Rs= difference in retention/ mean of peak base widths.
Usually acceptable if equal to or above 1.5
Factors that affect resolution include selectivity and efficiency of the column.
How may you define the efficiency of a chromatography column?
By its number of theoretical ‘plates’ it has. The higher the number, the greater the efficiency. High efficiency= well resolved peaks and good shape (reduced band broadening).
N = 5.54 x ( retention time / width of peak at half height) 2
Or
N = 16 x ( retention time / width of peak at base) 2
HETP = Length of column / Number of theoretical plates
What does rate theory consider?
A term= eddy diffusion
B term= longitudinal molecular diffusion
C term= resistance to mass transfer in liquid SP.
Van Deempter used rate theory to express the height equavalent of H as a function of average linear flow velocity (u)
H = A + B / μ + Cμ
Optimal conditions require A, B and C to be as small as possible.
Explain the A term
Caused by inhomogeneities in particle size or packing of column. Independent of the MP flow rate. A carefully packed column with small, spherical particles will reduce the effects of eddy diffusion.
It is increased by increasing column length.
Explain the B term
Diffusion strongly related to viscosity of the medium. Diffusion results from tendency of molecules to diffuse away from a source of high conc towards low conc.
Larger molecules diffuse slower
Longitudinal diffusion increases as the flow rate decreases
Diffusion less in liquids
Explain the C term
SP mass transfer. If long time in or on SP then band broadens.
In adsorption, it is important to ensure an ‘energetically’ homogenous surface to reduce H.
In partition, the liquid film thickness should be minimal and the liquid stationary phase should have a high analyte diffusion rate.
Explain liquid chromatography
Requires really high pressure to push the liquid MP through the column. In HPLC a mixture is filled into an injection loop, loop switched by a valve into flow of MP which takes it onto column. Components separate according to their polarity/solubility.
How does normal phase HPLC work?
Polar SP (e.g. silica) and less polar MP (e.g. hexane). Normally the less polar analytes would elute first and most polar last.
A non-polar MP would cause very few analytes to elute.
A very polar MP (methanol) would cause most analytes to elute.
How does reverse phase HPLC work?
Non-polar SP (e.g. C8 octylsilica) and a more polar MP (e.g. methanol and water) Normally most polar analytes elute first and the least polar last.
A polar MP (water) would cause very few analytes to elute.
A less polar MP would cause most analytes to elute.
How could you manipulate separation in HPLC?
Change composition of MP Change polarity of the column Heat the column Ion suppression Ion pairing Gradient elution Derivatisation
