chromatography

Question 1

explain what chromatography is

Answer 1

method of separating components in a mixture that are distributed between a mobile phase and stationary phase, based on their interaction with the stationary phase.

Question 2

give some examples of POLAR stationary phases

Answer 2

1. silica
2. alumina
3. paper

Question 3

example of a non-polar stationary phase

Answer 3

charcoal

Question 4

explain partition chromatography

Answer 4

• both phases are liquid
• ## a liquid stationary phase is bonded onto silica support

Question 5

why is chromatography needed in pharmacy

Answer 5

• drug design and manufactures
• quality assurance
  -clinical trials

Question 6

list some quality issues with chromatography in pharmaceuticals

Answer 6

1. quality of actives (purity, identity, content)
2. quality of excipients (raw materials)
3. cleanliness of manufacturing plant.
4. stability testing. including prediction of degradation pathways.

Question 7

draw out a diagram of a gas chromatography instrument

Answer 7

COMPONENTS:

• gas cylinder on the left with regulator on top
• autosampler/injector
• inlet
• two columns with a modulator inbetween
• the gc oven
• detector feeding info to a computer

Question 8

briefly explain the process of gas chromatography.

Answer 8

• a sample mixture heated 50degree C higher than its BP
• top of column is lower than heating block to encourage concentration condensation
• mixture carried through column by a continuous flow of inert gas.

Question 9

what are GC columns packed with?

Answer 9

solid particles or a solid support coated with a liquid either by adsorption or chemically bonded to it

Question 10

difference between capillary and packed columns?

Answer 10

capillary much smaller diameter but a lot longer.

Question 11

3 types of capillary columns?

Answer 11

1. wall coated open tubular(WCOT)
2. Support-coated open tubular(SCOT)
3. porous-layer open tubular (plot)

Question 12

what effect does mcreynolds constant have on the stationary phase?

Answer 12

higher the constant, the more polar the stationary phase

Question 13

why is derivatization used in GC

Answer 13

1. increases volatility, decreases polarity of analytes
2. decreases thermal degradation of analytes
3. improves detector response
4. improves separation and decreases band broadening
5. improve extraction efficiency

Question 14

what are some disadvantages to derivatization in gc

Answer 14

1. derivatizing agent can be difficult to remove and interferes with analysis
2. can cause unintended chemical changes to analyte
3. increases analysis time
4. can cause extra response

Question 15

what are the three types of derivatization in gc

Answer 15

1. alkylation
2. acylation
3. silylation

Question 16

how do you calculate resolution

Answer 16

difference of retention divided by mean of peak base widths

Question 17

explain plate theory

Answer 17

says that there are theoretical plates along a stationary phase. the more plates the more efficient the chromatography column is

Question 18

over what digit, does resolution be considered ‘valid’

Answer 18

over 1.5

Question 19

what three principles are considered for Rate theory

Answer 19

1. eddy diffusion (A term)
2. longitudinal molecular diffusion(B term)
3. resistance to mass transfer in the liquid stationary phase (C term)

Question 20

draw out a diagram of a liquid chromatography apparatus

Answer 20

1. mobile phase reservoir
2. leading to pump beside injection valve.
3. column followed by detector
4. detector leads to waste and data recorder

Question 21

what force is required for LC

Answer 21

very high pressure

Question 22

what is the difference between normal phase and reverse phase HPLC

Answer 22

• normal has a polar stationary phase(silica) and less polar mobile phase
• reverse phase is, you guessed it, the reverse.
  reverse would use water or methanol as mobile.
  90% of time reverse phase is used

Question 23

suggest how you would manipulate the separation of HPLC

Answer 23

1. change the composition of mobile phase (increase water content, increase organic modifier.)
2. change polarity of the column
3. heat the column
4. derivatisation

Question 24

why is HPLC detection useful

Answer 24

• used for quantitative and qualitative analyses.
• analyzing peaks on a chromatogram
• finding the identity of analyte(s) within each chromatogram peak. (detector selectivity)

Question 25

list three types of detectors and rank their selectivity

Answer 25

1. mass spectral detection (MSD), the greatest selectivity 2. UV & Fluorescence detectors, much better selectivity. 3. Refractive index detectors. (RID) low selectivity

Question 26

how would you modify a system to enhance detector selectivity

Answer 26

derivitisation of analytes

Question 27

explain what a chromophore is

Answer 27

region in a molecule where the energy difference between 2 different molecular orbitals falls within the UV or visible spectrum

Question 28

explain pre-column derivatisation and why its done

Answer 28

- analyte/sample undergoes derivatisation before separation on column - any reaction conditions can be made (time, temp, reagents) - however it might alter chromatographic properties like resolution or elution order of analytes

Question 29

explain post column derivatisation and possible disadvantages

Answer 29

- occurs on-line between column and detector so doesnt affect resolution *disadvantages are: - might need to alter HPLC system to introduce reaction chambers - reaction conditions employed must be kept moderate - reaction must complete within 30 secs

Question 30

explain the layout of a CZE setup

Answer 30

- capillary with tube diameter less than 1mm - filled with a running buffer - one end has a 'destination vial' - other end has a source vial with an anode

Question 31

give a brief description of Electro Osmotic Force. ie. buffer, silica, interactions, layer

Answer 31

1. capillary is made of fused silica so have Si-OH at inner surface 2. running buffer above Ph 3 has OH minus ions 3. these interact causing this eqn through de-protonation: Si-OH + -OH -> SiO- + H2O 4. SiO- groups attract postive cations ie Na+ from buffer. 5. this then forms a fixed layer held through non-covalent interactions, but is negative. 6. a 2nd layer of cations form on fixed layer, which is deemed the mobile phase.

Question 32

why is CZE used as a form of chromatography

Answer 32

- an electrically driven system is uniformly distributed along the capillary. - thus there is no pressure drop and velocity of flow is uniform. - this cause a lot less band broadening when viewed after detection