HPLC

Question 1

What is HPLC?

Answer 1

• High Performance (Pressure) Liquid Chromatography
• Widely used method of analysis for quantification of drugs. Involves chromatographic separation
• Uses a mobile phase flowing over a stationary phase (fine silica particles) packed in a column (stainless steel). The injection of sample and flow (pumping) of mobile phase (liquid) are precisely controlled by the instrumentation.

Question 2

Why are the solvents used degassed?

Answer 2

To avoid bubbles which can interfere with analysis and detection

Question 3

What does the mixing valve do?

Answer 3

Control the proportion of each solvent used depending on gradient

Question 4

What is the pump control?

Answer 4

set at flow rate (e.g. 1mg/ml) to deliver mobile phase. Creates back pressure typically 1000 psi (max 5000 psi)

Question 5

How is the instrumentation set out?

Answer 5

Pump -> injection valve -> column -> detector

Question 6

How is separation performed?

Answer 6

 Column equilibrated with starting solvent
 Analyte sample injected using injection valve e.g. 20 µL (20µg)
 Solvent composition can be changed with time (gradient method)
Or it can be kept constant (Isocratic method)- depends on choice.
 Analytes are separated by interaction with stationary phase
packed inside the column.

SEPARATION VIA NORMAL PHASE OR REVERSE PHASE

Question 7

How is detection achieved?

Answer 7

As analytes pass through the detector

Question 8

What does the area under the peak reflect?

Answer 8

The amount of particular analyte eluted

Question 9

What does the NORMAL phase use?

Answer 9

Uses polar stationary phase and non-polar mobile phase

Question 10

What is the adsorption process for normal phase?

Answer 10

• The analyte (solute) is retained by the interaction of its polar functional groups with the polar groups on the surface of the stationary phase.
• Stationary phase: majority use microporus silica.e.g. unmodified silica or chemically modified (with polar groups) e.g. cyanopropyl or diol.
• Mobile phase: a non-polar solvent e.g. n-hexane, heptane , chloroform, dichloromethane, isoporopanol

Question 11

What is normal phase chromatography useful for?

Answer 11

• Analytes elute from the column starting with the least polar a (most lipophilic) followed by others in order of their increasing polarity.
• Water-soluble analytes are retained too strongly so no good for this.
• Normal-phase chromatography is useful in the separation of analytes with low polarity and high solubility in low-polarity solvents such as hexane

Question 12

Why is reverse phase more commonly used i pharmaceutical sciences?

Answer 12

As most drugs are moderately polar and not suitable for normal phase

Question 13

What does reverse phase use?

Answer 13

In Reverses phase (RP) the stationary phase is non-polar, and the mobile phase is polar. The analytes interact with the surface by partitioning.
• The most polar analyte elutes from the RP column FIRST followed by others in order of decreasing polarity.

Question 14

How can the stationary phase be made less polar in reverse phase?

Answer 14

The stationary phase can be made less polar by using more alkyl groups i.e. more carbon chains. –> INCREASES RETENTION TIME (elutes slower)

Question 15

What molecules are used in RP?

Answer 15

Molecules < 3000 Molecular weight.

Question 16

How does RP work?

Answer 16

• Works by partitioning of the lipophilic (hydrophobic) portion of the drug molecules into the bonded (e.g. n-alkyl) stationary phase.
• Involves van der Walls interactions.
•

Question 17

In what ways can resolution/performance be improved?

Answer 17

 Column length – longer
 Flow rate: Slower can be better
 Stationary phase particle size: smaller better
 Gradient: less steep gives better resolution (but avoid peak broadening)
 Mobile or stationary phase composition & type, pH, temperature, etc , degassing

Question 18

How long will molecules remain associated with the stationary phase?

Answer 18

Molecules remain associated with stationary phase till the concentration of the organic modifier is high enough to break off interactions at which stage it partitions into the mobile phase and elutes.

Question 19

What is included in quantitative analysis?

Answer 19

 By measuring peak heights
 By measuring peak areas (preferred)
Both require: Calibration and Standards

Question 20

What is the external standard method?

Answer 20

Concentrations calculated from the ratio of sample peak areas to the corresponding standard peak areas. Usually used in drug manufacture & stability testing.

Question 21

What is the internal standard method?

Answer 21

More precise method. Less chance of error from reproducibility &sample preparation concerns. Often used in pharmacokinetics where trace levels of drugs or metabolites are present in a biological matrix

Question 22

What are the 2 types of gas chromatography?

Answer 22

Gas Solid   (GSC) –  involves adsorption of analytes 
	Gas Liquid  (GLC) – involves partitioning of analytes
     GLC most common form we will focus on. Partition of molecules between gas (mobile phase) and liquid (stationary phase)

Question 23

What are the components of Gas chrom?

Answer 23

Mobile phase : inert/unreactive gas to transport molecules
e.g. He, Ar, N2,CO2, H2
Pressure regulators control the gas flow – 10 to 50psi
Gas flow rates can be controlled (1 to 150ml/ml)

Question 24

How is separation achieved in GC?

Answer 24

o Column is packed with liquid Phase.
o Sample introduced into injector block where it is vaporized and carried by flowing gas stream
o Sample vapor partitions between Gas and Stationary Liquid phases.
o The time different components spend in the column depends on their Vapor Pressure (low Boiling Point = Higher Vapor) and ability to interact with the liquid phase
o Low volatile (high b.Pt.) = longer retention time
o Greater solubility in the liquid phase = longer retention time

Question 25

GC instrumentation?

Answer 25

•	Injector (Heated) – 0.1-10l
•	Column – inert support, range of polarity stationary phases
•	Oven – RT -400ºC
•	Detectors;
o	FID (Flame Ionisation Detector)
o	ECD (Electron Capture Detector) 
o	NPD (Nitrogen Phosphorous Detector)
o	Mass Spectroscopy
•	Chart Recorder/Integrator

Question 26

GC Chromatogram?

Answer 26

Chromatogram interpreted similar to HPLC for identification and quantitative analysis using same parameters
 Each volatile component produces a peak with characteristic retention time
 Area under the peak reflects amount of component present