D L3: Thin layer Chromatography Flashcards
where was TLC first developed and when?
Ukraine 1937
what samples can be sued for TLC?
Amino acids, drugs, lipids
what do samples in TLC need to be in?
need to be in a solution with an organic solvent
In TLC samples may have to be?
dissolved in a solvent or extracted from a substrate
what materials re TLC plates?
glass, plastic or foil
what are TLC plates coated in?
fine grade silica (SiO2)
How big are TLC plates?
purchased at 200x200 mm2
cut to 20x50 mm2 or 50x80 mm2 rectangles
what is important to make sure the TLC is run accurately?
important make sure that the silica at the edges of the plates is not extensively chipped
what does the silica on TLC plates need to be like?
robust enough to write on using a soft pencil
how is a sample appleid to a TLC plate?
The sample is applied using a TLC spotter which is a fine glass capillary
what size should the sample spots me?
4-5mm in diameter
How should you increase the amount of solvent applied?
repeat spot, waiting for the solvent dry in between applications
where does the TLC plate need to be placed to run it?
in a sealed developing tank.
what is filter paper used for during TLC?
plate in a sealed developing tank.
where do the spots of sample need to be ?
above the level of the solvent
How does the solvent move up the TLC plate?
by capillary action
when should the plate be removed from the tank?
when the solvent front is 3-4 mm from the top of the plate and the position of the solvent front marked
what ways can a TLC plate be viewed?
- under a UV light
- Coloured spots
- Liquid developers
what wave are used when suing a UV light to look at a TLC plate?
short wave (254 nm) and long wave (365 nm)
Why can a UV light be used to develop a TLC plate?
TLC plates contain fluorescent compounds which are bright under UV.
shows up as a dark purple spots on a white background
When using a UV light what does a compound nee dot be visible?
a chromophore which absorbs the UV light
How can you tell which compound is more present when using UV light on a TLC plate?
The relative intensity of a spot when compared to other spots of the same compound gives an indication of the amount of compound present
what is chromophore?
This is the part of the molecule which is responsible for the light absorbing properties of a molecule
what does chromophore consist of?
consist of a series of conjugated double bonds
How is a liquid developer used to develop a TLC plate?
- sprayed on or the plate can be dipped in
- plate heated to initiate the reaction between developer and compounds
are stains formed form liquid developers permanent?
not always, can fade overtime
what is the retention factor?
distance moved by compound
if two spots have the same Rf what can this mean?
they are likely to be the same compound
what is adsorption?
interaction between the analyte and the surface of the stationary phase
The more strongly the analyte is adsorbed the _________ it travels through the stationary phase
slower
what is silica surface?
its polar
When using a Silica stationary phase the solvents chosen are usually ______ in nature
organic
if the analyte is polar what does the solvent need to be
polar
when is optimal separation achieved?
when all the spots are separated
what happens when the solvent is too polar ?
all the spots are crowded at the top of the plate
what happen when the solvent is too non-polar?
the spots are crowded at the bottom of the plate
what should you do when analysing basic molecules?
add a small amount of base to solvent system such as NH3 or Et3N
what should you do When analysing acidic molecules ?
add an acid to the solvent mixture
what dictates polarity of an organic molecule?
functional groups
what do functional groups contain that make a compound polar?
polarised bonds
what does it mean if The compound runs as a streak rather than a spot ?
The sample was overloaded
what does it mean if the The sample runs as a smear or a upward crescent ?
Compounds which possess strongly acidic or basic groups tend to smear
what does it mean if the sample runs as a downward crescent ?
The adsorbent was disturbed during the spotting
what does it mean if the plate solvent front runs crookedly ?
Either the adsorbent has flaked off the plate or the sides of the plate are touching the paper
what does it mean No spots are seen on the plate ?
Not spotted enough compound, or use a different developer or the solvent is over the baseline
what is 2D-TLC?
- sample is run in one direction and then the plate is turned around 90o and run in the other direction
- Each time the plate is run a different solvent system is used
- Spots that overlap in the first run may separate out in the second
what are some advantages of TLC?
- High through-put
- Low cost
- Easy sample preparation
- Requires the minimum amount of equipment
- Limited training of analyst required
- An excellent ‘look-see’ technique which gives you an idea of what is in the sample
- Good for a whole variety of compounds, though basic compounds can be problematic
- Range of visualisation techniques
what are disadvantages of TLC?
- Poor chromatographic resolution
- Constant need to run standards alongside analytes on the same plate
- Quantification difficult and may not be reproducible
