TLC

Question 1

Equation for solving Retardation factor

Answer 1

Rf = distance travelled by analyte / distance travelled by solvent front
* No units

Question 2

Influences on the Rf

Answer 2

1. Thickness of stationary phase - affects affinity a compound has to the stationary phase
2. Mositure content of both phases -
3. Temperature at which TLC performed -
4. Sample Size - Harder to measure when too much as the spot will be big

Question 3

How to overcome influences on the Rf?

Answer 3

1. Thickness - use comercially prepared tlc plates with standardized, uniform thickness
2. moisture - perform TLC in a controlled enviroment with low humidity
3. Temperature - conduct

Question 4

Formula for Relative Retention Factor?

Answer 4

Rx = distance travelled by analyte / distance travelled by reference

Question 5

What infromation can be achieved using TLC?

Answer 5

1. Compare Rf values (between known compounds and those under analysis)
2. Compare Rx values (more reproducable than Rf)
3. Spraying the paper to visualize different functional groups
4. Altering the mobile phase can allow us to differentiate any coelution
   5.

Question 6

List suitable dyes and sprays in TLC

Answer 6

1. Iodine Vapour - produces brown spots with many organic compounds
2. Akaline tetrazolium blue - specific for corticosteroids
3. Ethanol/sulphuric acid spray - used for corticosteroids which fluoresce at 365nm (dexamethasone, prednisolone)

Question 7

List Of stationary phases used in TLC

Answer 7

1. Silica gel - UV light is used to illuminate the plate and if the analyte absorbs UV it can be seen as a black dot and a yellow background
2. Cellulose powder - identity test for penicillins

Question 8

Qualatitive applications of TLC

Answer 8

1. Identity tests -
   Various identity tests have BP applications
2. Screening for multiple samples
   Rapid screening of multiple samples - Manufacturing/validation plant - identify impurities, contamination

Question 9

Quantative applications of TLC

Answer 9

1. Limit testing - determination of impurities
   * Known impurities e.g. degredation prducts
   * Unknown impurities

Question 11

What is required when doing TLC limit tests?

Answer 11

1. Identical volumes of standards and unknown samples must be applied to the plate
2. If only visual appraisal is to be used then must assume that the intensity of the spot is equivalent to the concentration and size of spots must be uniform

Question 12

Formula for % Limit

Answer 12

% Limit = (conc std / conc test) x100

Question 13

Advantages of TLC

Answer 13

1. Robus and cheap method
2. Easy to perform
3. Using an appropriate localisation reagent all components can be seen on the plate. this is not true for GC/HPLC.
4. Simulataenous analysis of batch samples in industry
5. Can run multiple samples for comparision in one go

Question 14

Disadvantages of TLC

Answer 14

1. Not suitable for analysis of volatiles
2. requires more operator skill than GC/HPLC
3. Sensitivity may be limited
4. Scanning densitometer required for accurateb quantitation

Question 15

What is HPTLC?

Answer 15

1. High Performing TLC
2. Automated form of TLC

Question 16

Advantages of HPTLC

Answer 16

1. Greater resolving power per unit distance
2. Develop faster
3. Consume less solvent