MAAC: Chromatography 2 Flashcards
Briefly describe what a TLC is composed off and how it is done.
What mechanism is this?
- The sample is dissolved in suitable solvent/ liquid and is then spotted using a glass capillary tube onto paper (cellulose) or a plate attached to silca gel or alluminium oxide.
- The cellulose, Alumunia and Silica gel are the stationary phases.
- Once spotted and dried, the plate is placed upright in a tank with a small amount of solvent in the bottom - needs to be below spot level - and tank sealed.
- The solvent moves up the TLC plate via capillary action and picks up the analytes and enables them to interacted with both the SP and MP. It is these interactions which enables the seperation of analytes. This is because each analyte will interact differently
- The plate is left in the tank for a pre-set time period (make sure solvent doesnt reach top) and then removed. Using a pencil the distanve the solvent and analytes travelled should be marked
As the TLC is composed of a solid SP and liquid MP it is an adsorption mechanism
Describe the analysis of TLC
- The Retardation factor (Rf) Should be calculated for each analyte. This is specific for each analyte under these specific conditions:
Rf = distance travelled by analyte/ distanced travelled by solvent
SHOULD ALWAYS BE BELOW 1
- To analyse the Rf values it is important to compare with a reference standard of knonwn identity. This standard should be run on the same TLC plate to ensure conditions are exactly the same
What are variables that influence Rf?
- Thickness of stationary phase
- Temperature of mobile phase
- Higher temp = analytes more sol in MP therefore travel further
- Moisture content (MP and SP)
- TLC plates absorb moisture therefore need to be in sealed container
- Sample size
- Too large and will form large banf moving up MP rather than small well defined spot so will be difficult to measure distance travelled
How can these effects be minimised?
By analysing a standard on the same TLC plate and calulating the relative retention factor (Rx):
Rx = Distacne travlled by analyte/ Distance travelled by Ref marker
The standard would be a known compound but different to analyte being analysed. Rx can be greater or less than 1.
What are the different ways to identify analytes by TLC (qualatiative)
- •Compare Rf values (between known compounds and those under analysis).
- •Compare Rx values (more reproducible than above).
- Unlikey to give 100% positive identification
- •Use visualisation techniques specific for each functional group
- •Compare analyte retention in a selection of different mobile phases.
- 2 analytes may travel similar difference so by using other MP and comparing Rx and Rf will help with positive indentificayion
- •Scrape spot from TLC plate, solvate and extract analyte and concentrate/dry down. Analyse concentrated fraction by spectral techniques.
Describe the use of visualisation techniques
Before one can be used the plate must be dried. Leave the plate in fume cupboard for solvent to evapporate off. Once died agent can be sprayed
1. Iodine vapour:
- Produces brown spots with many ORGANIC compounds
- Not a particularly specific reagent!
- BP application: Fixed oils, Cetrimide
2. Alkaline tetrazolium blue
- Specific for CORTICOSTEROIDS, produces blue spots on a white background under alkaline conditions.
3.Ethanol/sulphuric acid spray
- Used for corticosteroids which fluoresce at 365 nm.
- BP application: dexamethasone; prednisolone.
What are commonly used SP (visualisation)?
1. Silica gel G254
- Silica gel with a fluorescent agent
- UV light is used to illuminate the plate (l= 254 nm) and if the analyte absorbs UV it can be seen as a black spot on a yellow background.
- The flurescent material absorbs light at 254nm and emmits light in the visiable region. Analytes present block light reaching these areas so appear as black spot. Use penicl to mark these , remove from light souorce and calculate Rf and Rx
- BP application: identity test for methyl prednisolone
2. Cellulose powder:
- Cellulose powder of < 30µm particle size
- BP application: identity test for penicillins.
What are the qualatative and quantative applications of TLC?
Qualatative:
- Identity tests
- Screening of cleanliness of equipment used (ensures safety of product)
Quantatiative:
- To perform limit tests for determination of impurities (product safety)
When carrying out limit tests what applies?
For both types of impurities the following applies:-
- IDENTICAL volumes of stds (reference solutions) and unknown samples must be applied to the plate.
- If only visual appraisal is to be used then must assume:
INTENSITY of spot is equivalent to the CONCENTRATION and thus the size/shape of spots must be uniform.
i.e. Process requires skilled personnel!
Advantages and disadvantages of TLC
Advantages:
- Fast, simple, no specialist equipment required
- Robust and cheap method
- Detection by chemical reaction with a localisation reagent is easily performed.
- Using an appropriate localisation reagent all components in the chromatographic system can be seen. GC/HPLC some compounds may not be seen.
- Very applicable to pharmaceutical industry allows simultaneous analysis of batches of samples.
Lends itself readily to the retrieval of quantitative and qualitative data.
Disadvantages:
- More labour intenisive
- Limited/ poor sensitivity therefore not good for analysiing small quantities
- Not suitable for analysis of volatiles
- Requires more operator skill than GC or HPL
- . Sensitivity may be limited.
- Number of theoretical plates and thus resolving power of technique is limited.
- Scanning densitometer required for accurate quantitation.
Advantages of HPTLC
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