D L3: Thin layer Chromatography

Question 1

where was TLC first developed and when?

Answer 1

Ukraine 1937

Question 2

what samples can be sued for TLC?

Answer 2

Amino acids, drugs, lipids

Question 3

what do samples in TLC need to be in?

Answer 3

need to be in a solution with an organic solvent

Question 4

In TLC samples may have to be?

Answer 4

dissolved in a solvent or extracted from a substrate

Question 5

what materials re TLC plates?

Answer 5

glass, plastic or foil

Question 6

what are TLC plates coated in?

Answer 6

fine grade silica (SiO2)

Question 7

How big are TLC plates?

Answer 7

purchased at 200x200 mm2

cut to 20x50 mm2 or 50x80 mm2 rectangles

Question 8

what is important to make sure the TLC is run accurately?

Answer 8

important make sure that the silica at the edges of the plates is not extensively chipped

Question 9

what does the silica on TLC plates need to be like?

Answer 9

robust enough to write on using a soft pencil

Question 10

how is a sample appleid to a TLC plate?

Answer 10

The sample is applied using a TLC spotter which is a fine glass capillary

Question 11

what size should the sample spots me?

Answer 11

4-5mm in diameter

Question 12

How should you increase the amount of solvent applied?

Answer 12

repeat spot, waiting for the solvent dry in between applications

Question 13

where does the TLC plate need to be placed to run it?

Answer 13

in a sealed developing tank.

Question 14

what is filter paper used for during TLC?

Answer 14

plate in a sealed developing tank.

Question 15

where do the spots of sample need to be ?

Answer 15

above the level of the solvent

Question 16

How does the solvent move up the TLC plate?

Answer 16

by capillary action

Question 17

when should the plate be removed from the tank?

Answer 17

when the solvent front is 3-4 mm from the top of the plate and the position of the solvent front marked

Question 18

what ways can a TLC plate be viewed?

Answer 18

1. under a UV light
2. Coloured spots
3. Liquid developers

Question 19

what wave are used when suing a UV light to look at a TLC plate?

Answer 19

short wave (254 nm) and long wave (365 nm)

Question 20

Why can a UV light be used to develop a TLC plate?

Answer 20

TLC plates contain fluorescent compounds which are bright under UV.
shows up as a dark purple spots on a white background

Question 21

When using a UV light what does a compound nee dot be visible?

Answer 21

a chromophore which absorbs the UV light

Question 22

How can you tell which compound is more present when using UV light on a TLC plate?

Answer 22

The relative intensity of a spot when compared to other spots of the same compound gives an indication of the amount of compound present

Question 23

what is chromophore?

Answer 23

This is the part of the molecule which is responsible for the light absorbing properties of a molecule

Question 24

what does chromophore consist of?

Answer 24

consist of a series of conjugated double bonds

Question 25

How is a liquid developer used to develop a TLC plate?

Answer 25

- sprayed on or the plate can be dipped in | - plate heated to initiate the reaction between developer and compounds

Question 26

are stains formed form liquid developers permanent?

Answer 26

not always, can fade overtime

Question 27

what is the retention factor?

Answer 27

distance moved by compound

Question 28

if two spots have the same Rf what can this mean?

Answer 28

they are likely to be the same compound

Question 29

what is adsorption?

Answer 29

interaction between the analyte and the surface of the stationary phase

Question 30

The more strongly the analyte is adsorbed the _________ it travels through the stationary phase

Answer 30

slower

Question 31

what is silica surface?

Answer 31

its polar

Question 32

When using a Silica stationary phase the solvents chosen are usually ______ in nature

Answer 32

organic

Question 33

if the analyte is polar what does the solvent need to be

Answer 33

polar

Question 34

when is optimal separation achieved?

Answer 34

when all the spots are separated

Question 35

what happens when the solvent is too polar ?

Answer 35

all the spots are crowded at the top of the plate

Question 36

what happen when the solvent is too non-polar?

Answer 36

the spots are crowded at the bottom of the plate

Question 37

what should you do when analysing basic molecules?

Answer 37

add a small amount of base to solvent system such as NH3 or Et3N

Question 38

what should you do When analysing acidic molecules ?

Answer 38

add an acid to the solvent mixture

Question 39

what dictates polarity of an organic molecule?

Answer 39

functional groups

Question 40

what do functional groups contain that make a compound polar?

Answer 40

polarised bonds

Question 41

what does it mean if The compound runs as a streak rather than a spot ?

Answer 41

The sample was overloaded

Question 42

what does it mean if the The sample runs as a smear or a upward crescent ?

Answer 42

Compounds which possess strongly acidic or basic groups tend to smear

Question 43

what does it mean if the sample runs as a downward crescent ?

Answer 43

The adsorbent was disturbed during the spotting

Question 44

what does it mean if the plate solvent front runs crookedly ?

Answer 44

Either the adsorbent has flaked off the plate or the sides of the plate are touching the paper

Question 45

what does it mean No spots are seen on the plate ?

Answer 45

Not spotted enough compound, or use a different developer or the solvent is over the baseline

Question 46

what is 2D-TLC?

Answer 46

- 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

Question 47

what are some advantages of TLC?

Answer 47

- 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

Question 48

what are disadvantages of TLC?

Answer 48

- Poor chromatographic resolution - Constant need to run standards alongside analytes on the same plate - Quantification difficult and may not be reproducible