PH1125 - HPLC

Question 1

what does hplc stand for?

Answer 1

• high performance liquid chromatography

Question 2

what is column chromatography?

Answer 2

• components separate due to differential affinities for both stationary and mobile phases

Question 3

How does HPLC work?

Answer 3

Separation of the unknown onto a column (stationary phase) by eluting liquid (mobile phase). To pass through detector and obtain quantitive analysis.

Question 4

What is the normal phase of HPLC?

Answer 4

Stationary phase is more polar than the mobile phase.

Question 5

What is the reverse phase of HPLC?

Answer 5

Mobile phase is more polar than the stationary phase.

Question 6

What is the difference between using a water or buffer?​

Answer 6

Water has no control over the ionic state, pKa dependant. Buffers have control of ionic state.

Question 7

What is the purpose of a buffer?

Answer 7

Resists changes in pH and improves the chromatography to eliminate multiple peaks.

Question 8

What is the purpose of organic Modifiers in reverse phase HPLC.

Answer 8

Must have high miscibility in water to buffer and cannot cause the buffer to precipitate.

Question 9

Give an example of an organic modifiers. in reverse phase HPLC?

Answer 9

methanol- viscous when mixed with water with high back pressure and has inductive effect and trans methylation.

Question 10

What is the function of the pump in HPCL?

Answer 10

Solvent delivery system- maintains precise mobile phase delivery.

Question 11

What is the function of the column in HPCL?

Answer 11

Stationary phase- longer column better retention.

Question 12

What does column performance depend on in HPLC?

Answer 12

Proportional (>) to theoretical plates > surface area > particle size performance- greater resistance to flow.

Question 13

What is the equation used for HPCL for P?

Answer 13

Solubility of X in non-aqueous medium/ Solubility of X in aqueous medium.

Question 14

What is end capping?

Answer 14

Conversion of free silanol units to increase lipophilicity, improve band spreading and tailing.

Question 15

How are chiral compounds separated in HPLC?

Answer 15

Using Chiral stationary Phase (CPS).

Question 16

What is the 3 point rule in HPLC?

Answer 16

All CPS have to interact with 3 points of an enantiomer.

Question 17

What does the detection method depend on?

Answer 17

Nature of analysis and Anticipated drug level.

Question 18

what are the limitations of UV spectrophotometry? (5)

Answer 18

• low sensitivity
• assumption; absorbance arising from a single compound
• absorbances additive
• overlapping
• separation needed

Question 19

what do small particles result in in the column? (3)

Answer 19

• smaller gaps (for particles to move in the mobile phase)
• greater resistance to flow
• back pressure

Question 20

can enantiomers be separated easily?

Answer 20

• same physiochemical properties make them hard to separate

- cannot be resolved on regular HPLC columns

Question 21

what does the separation of racemates make?

Answer 21

• two peaks with the same areas

Question 22

how do you calculate the absorbance? (5)

Answer 22

• A = ε c l
• A is the absorbance (amount of light lost at detector)
• ε is a constant
• c is concentration (eg ng ml-1)
• l is path length (usually 1cm)

Question 23

why is it important to know the precise purity level?

Answer 23

• assuming 100% purity will lead to inaccuracy an reduces precision

Question 24

what is the capacity factor? (2)

Answer 24

• k’ (kay prime)

- analyte retention time normalised in terms of solvent front (aids identification)

Question 25

how do you calculate capacity factor?

Answer 25

• (t1 - t0) / t0

Question 26

what does a long base line in a chromatogram suggest?

Answer 26

• waste of solvent in the process

Question 27

what is the retention controlled by?

Answer 27

• controlled by adjusting mobile phase polarity

Question 28

if the retention is too long what should be added to the normal phase?

Answer 28

• add miscible polar solvent

Question 29

if the retention is too long what should be added to the reverse phase?

Answer 29

• add miscible non-polar solvent

Question 30

what is α?

Answer 30

• quantification of separation

Question 31

what is the relationship between α and a peak?

Answer 31

• the bigger the α the greater the distance between peak apices

Question 32

how do you work out α?

Answer 32

• k’2 / k’1

Question 33

what does an α of more than 1 mean?

Answer 33

• separation

Question 34

what does an Rs of more than 1.5 mean?

Answer 34

• no merging (baseline resolution)

Question 35

how do you calculate Rs? (5)

Answer 35

• resolution = 2 (t2 - t1) / w1 + w2
• t1 is the time to apex of first peak
• t2 is the time to apex of second peak
• w1 is the time interval of first peak (base width)
• w2 is the time interval of second peak (base width)

Question 36

what does an Rs of less than 1.5 mean?

Answer 36

• merging (no baseline resolution)

Question 37

what does it mean when P is small and large? (3)

Answer 37

• approx. 1; amphilic (both hydrophobic and hydrophilic)
• small; more hydrophilic
• large; more hydrophobic/lipophilic