Chromatography

Question 1

What is quality control

Answer 1

Checking that the right ingredient is present in the qaunity

Question 2

What is QA

Answer 2

Checking SOP such that the quality of the product can be assured

Question 3

What is the BP

Answer 3

a book that contains info of on the acceptable parameters for the quality of formulated drugs

Question 4

What is Chromatography

Answer 4

It is a process which a chemical mixture is carried around by a liquid or gas over

Question 4

What is Chromatography

Answer 4

It is a process which a chemical mixture is carried around by a liquid or gas over a stationary phase

Question 5

What is the stationary phase and what is it comprised of

Answer 5

A solid or liquid material this is coated onto thin layer if inert material.

Question 6

What is the mobile phase and what is it comprised of

Answer 6

A gas of liquid that is allowed to flow over and through the stationary phase

Question 7

What is Colum chromatography

Answer 7

It is when the stationary phase is held in a tube and the mobile phase is forced through the column under pressure or gravity

Question 8

What are the two types of column chromatography

Answer 8

Liquid

Gas

Question 9

What are the 4 different types of liquid chromatography

Answer 9

-Absorption: 
       > S phase is an adsorbent solid- charcoal or silica
-Partition
       > S phase is a liquid coated solid 
-Ion exchange
       > S phase has ionic bonded phase 
-Gel permeation 
       > S phase is porous

Question 10

What is the S phase of Absorption chromatography made of?

Answer 10

Made of a polar groups
> SiO2, Al2O3
>Molecules are attracted by Dipole-dipole force, Hydrogen bonding or weak Van der waals forces.

Question 11

What is the S -phase of partition chromatography made of

Answer 11

Made of an inert solid, coated with a liquid SiO/H2O

Question 12

What is a reverse phase chromatography

Answer 12

This when the stationary phase is non-polar and the mobile phase phase is polar

Question 13

Why does separation occur in partition chromatography

Answer 13

Due to the difference in solubility of the solutes

>The solute is partitioned between the two phases, the amount of time spent in each is dependant on solubility.

Question 14

What is the S phase of ion exchange chromatography

Answer 14

coating of solid of resin ions (cations and anions) is covalently bonded to it.

> Ions of opposing charges bind electrostatically to it.

Question 15

How dose Ion exchange chromatography work

Answer 15

When eluent containing ions is eluted through the column, the electrostatically bound ions are released as others are preferentially bound.
>the smaller and more highly charged an ion is, the stronger it will be retained in the column

Question 16

How do Gel permeation chromatography work

Answer 16

-Works without equilibrium between the solutes and stationary phase

-The stationary phase is passed through a porous gel
> Depending on size the molecules will, the larger molecules elute first

Question 17

Preparative of liquid column chromatography (driven by gravity)

Answer 17

This is when the column is packed with large stationary molecules such as Silica or alumina.

Sample mixture is coated unto the top and Eluent is flushed down by the liquid mobile phase

Question 18

Describe HPLC

Answer 18

It is an extension of liquid column chromatography

> particles are Small and passed through column by an eluent pumped under pressure of by gravity

Question 19

What type of stationary phase can be used in HPLC

Answer 19

It can use all sorts
>Adsorption
>Partitions
> Ion exchange
> gel permeation

Question 20

What are the options for the HPLC packing material

Answer 20

1. Microspheres
   - Particulate with thin SA
2. Porous microspheres
3. Bonded phase: When silica has different groups bonded to it.

Question 21

Which is the most common material used as packing material in reverse phase chromatography

Answer 21

ODS silica

Question 22

Which factors affect retention time

Answer 22

The nature of the mobile phase
> More polar phase means more quickly eluted in Silica
> More lipophilic = quicker elution in ODS

Question 23

What are the ideal factors of a detectors for HPLC

Answer 23

• Rapid and reproducible response to solutes
• A linear response
• Stability of operation

Question 24

What are the 2 classes of detectors

Answer 24

Optical Electrochemical

Question 25

Name the types of optical detectors and the Advantages vs disadvantages

Answer 25

1. Refractivity index: Monitor difference between a reference stream and the eluent. >Advantage: it is universal >Disadvantage: It is not very sensitive -Sensitive to temperature change 2. UV/VIS a) fixed wavelength: A fixed wavelength (at which you expect the solute to absorb) is selected >Advantages: Response is linear providing the concentration is not too high >Disadvantage: can only use for species that absorb at a set know wavelength in the UV/Vis region. You can’t really analyse unknown samples. b)Diode array spectrometer: measures absorbance of the sample coming from the column over all wavelengths simultaneously >How it works: >Results in a 3D graph 3. Detects light fluorescing from natural fluorescent solutes (fluoresces in the current wavelength of light )

Question 26

What are the two types of electrochemical detectors

Answer 26

Conductivity: Used in ion exchange chromatography. > Samples are ionic and mobile phase had low conductivity Redox electrochemical: These detectors are useful for solutes than can be oxidised or reduced.

Question 27

What are the structural factors that effect the rate of elusion in HPLC in neutral compounds?

Answer 27

The balance between polarity and lipophilicity Altering the pH of the mobile phase has no influence on this

Question 28

What are the structural factors that effect the rate of elusion in HPLC in ionisable compounds?

Answer 28

pH - Usually in reverse phase where the mobile phase is aqueous >drugs are ionisable, and the degree of ionisation is dependant on the pH

Question 29

What is liquid chromatography-mass spec (LCMS)

Answer 29

It has 2 separate analytical techniques : chromatography and mass spec

Question 30

What is gas chromatography

Answer 30

mobile phase; Gas stationary phase: >Liquid adsorbed to a solid (GLC) >organic species bonded to a solid surface Separation is based on volatility

Question 31

What are the 2 factors that effect the desperation of GLS

Answer 31

>The volatility of the component at column temperature | >The polarity (solubility) of the component in the stationary phase

Question 32

What are the 2 different types of GC columns

Answer 32

Packed column: Capillary column: Longer (up to 500m) and thinner > Require small sample size so has very sharp peaks

Question 33

Types of stationary phases of GC and their properties

Answer 33

All based on silica polymers > Methyl silicate: Non polar separation >Cyanopropyl phenyl methyl silicate: food for moderate polarity eg Aromatic compounds >Triflurosilicone: Good for polar separation eg Alcohol

Question 34

Good properties of a GC detectos

Answer 34

>Must respond rapidly | >Response must be linear (steep slope)

Question 35

How can the sensitivity of the detector be measured

Answer 35

1. Measure the slope on a detector response versus amount of sample graph 2. Look at the limit of detection, i.e. the smallest peak one can actually say with confidence is a peak and not just background noise For a 95% confidence of a peak being an actual component the limit of detection (L.O.D) is equal to the mean background + 2 standard

Question 36

An ideal GC detector is:

Answer 36

``` Sensitive Linear response temp ranges to at least 400 C Quick response High reliability Does not destroy the sample Respond similarly to all solutes ```

Question 37

What is a flame ionisation detector and how does it work?

Answer 37

A detector used in GC The column gas is mixed with air and H2(g) then burnt using a plasma to get to very high temperatures (>3000oC). compounds are ionised (+ve) and detected by the cathode Electrons flow to anode

Question 38

Advantages of Flame ionisation detector

Answer 38

- Sensitive - Quick response time (<1 millisecond) - Peaks are proportional to amount of solute 

Question 39

disadvantages of Flame ionisation detector

Answer 39

- Can only detect combustible materials (mainly organic – carbon containing) - Destroys the sample

Question 40

What are chromatograms

Answer 40

A detector that responds to solute concentration which give different peaks which represent a separated component of mixtures

Question 41

What is retention time

Answer 41

The time it takes a solute to reach the detector after injection

Question 42

What determines a good chromatographic separation

Answer 42

- Resolution | - Efficiency

Question 43

What is resolution

Answer 43

It is the measure of how well a peak separated and is needed for quantitative calculations. R= 2(tR2-tR1)/ (wb1+wb2) wb = with of base of each peak measured The greater the value of R, the better the resolution of the two compounds

Question 44

What is an alternative calculation for the resolution if the base with is unclear

Answer 44

1.18 * (tR2 - tR1)/ wh1+ wh2

Question 45

What are the ways to improve resolution

Answer 45

Increase the column length but this affects efficacy - Alter mobile and stationary phase - Anything that increases efficacy and leads to narrow peaks

Question 46

How is efficacy calculates

Answer 46

n= 5.54*(tR)^2/ L x (Wh)^2 Wh = width at half height of the peak

Question 47

What are the factors that affect efficacy in a column

Answer 47

The length of the column > The molecules of a single compound, despite having the same properties, take different lengths of time to travel through a column. 1. The multiple pathway affect 2. Non equilibrium mass transfer 3. Sample size 4. Diffusion 5. Time on column

Question 48

1. The multiple pathway affect

Answer 48

Packaging of column is not uniform so solutes can take different pathways and so travel at different speeds

Question 49

2. Non equilibrium mass transfer

Answer 49

Some molecules may travel further into the stationary phase or be too far away for the surface

Question 50

3. Sample size

Answer 50

if you increase the solute concentration too much, the stationary phase may become saturated

Question 51

4. Diffusion

Answer 51

Diffusion occurs mostly in the mobile phase and is grater in gas chromatography, compare to liquid chromatography.

Question 52

5. Time on column

Answer 52

longer a compound is on the column the more opportunity the molecules have had to diffuse

Question 53

Overall factors required for high efficiency (narrow bands)

Answer 53

``` Small sample size Small particle size Thin layer of stationary phase Optimum mobile phase velocity Low dead volume ```