PH1125 - HPLC Flashcards
what does hplc stand for?
- high performance liquid chromatography
what is column chromatography?
- components separate due to differential affinities for both stationary and mobile phases
How does HPLC work?
Separation of the unknown onto a column (stationary phase) by eluting liquid (mobile phase). To pass through detector and obtain quantitive analysis.
What is the normal phase of HPLC?
Stationary phase is more polar than the mobile phase.
What is the reverse phase of HPLC?
Mobile phase is more polar than the stationary phase.
What is the difference between using a water or buffer?
Water has no control over the ionic state, pKa dependant. Buffers have control of ionic state.
What is the purpose of a buffer?
Resists changes in pH and improves the chromatography to eliminate multiple peaks.
What is the purpose of organic Modifiers in reverse phase HPLC.
Must have high miscibility in water to buffer and cannot cause the buffer to precipitate.
Give an example of an organic modifiers. in reverse phase HPLC?
methanol- viscous when mixed with water with high back pressure and has inductive effect and trans methylation.
What is the function of the pump in HPCL?
Solvent delivery system- maintains precise mobile phase delivery.
What is the function of the column in HPCL?
Stationary phase- longer column better retention.
What does column performance depend on in HPLC?
Proportional (>) to theoretical plates > surface area > particle size performance- greater resistance to flow.
What is the equation used for HPCL for P?
Solubility of X in non-aqueous medium/ Solubility of X in aqueous medium.
What is end capping?
Conversion of free silanol units to increase lipophilicity, improve band spreading and tailing.
How are chiral compounds separated in HPLC?
Using Chiral stationary Phase (CPS).
What is the 3 point rule in HPLC?
All CPS have to interact with 3 points of an enantiomer.
What does the detection method depend on?
Nature of analysis and Anticipated drug level.
what are the limitations of UV spectrophotometry? (5)
- low sensitivity
- assumption; absorbance arising from a single compound
- absorbances additive
- overlapping
- separation needed
what do small particles result in in the column? (3)
- smaller gaps (for particles to move in the mobile phase)
- greater resistance to flow
- back pressure
can enantiomers be separated easily?
- same physiochemical properties make them hard to separate
- cannot be resolved on regular HPLC columns
what does the separation of racemates make?
- two peaks with the same areas
how do you calculate the absorbance? (5)
- 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)
why is it important to know the precise purity level?
- assuming 100% purity will lead to inaccuracy an reduces precision
what is the capacity factor? (2)
- k’ (kay prime)
- analyte retention time normalised in terms of solvent front (aids identification)
how do you calculate capacity factor?
- (t1 - t0) / t0
what does a long base line in a chromatogram suggest?
- waste of solvent in the process
what is the retention controlled by?
- controlled by adjusting mobile phase polarity
if the retention is too long what should be added to the normal phase?
- add miscible polar solvent
if the retention is too long what should be added to the reverse phase?
- add miscible non-polar solvent
what is α?
- quantification of separation
what is the relationship between α and a peak?
- the bigger the α the greater the distance between peak apices
how do you work out α?
- k’2 / k’1
what does an α of more than 1 mean?
- separation
what does an Rs of more than 1.5 mean?
- no merging (baseline resolution)
how do you calculate Rs? (5)
- 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)
what does an Rs of less than 1.5 mean?
- merging (no baseline resolution)
what does it mean when P is small and large? (3)
- approx. 1; amphilic (both hydrophobic and hydrophilic)
- small; more hydrophilic
- large; more hydrophobic/lipophilic
