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Flashcards in HPLC Deck (26)
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1
Q

What is HPLC?

A
  • 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.
2
Q

Why are the solvents used degassed?

A

To avoid bubbles which can interfere with analysis and detection

3
Q

What does the mixing valve do?

A

Control the proportion of each solvent used depending on gradient

4
Q

What is the pump control?

A

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

5
Q

How is the instrumentation set out?

A

Pump -> injection valve -> column -> detector

6
Q

How is separation performed?

A

 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

7
Q

How is detection achieved?

A

As analytes pass through the detector

8
Q

What does the area under the peak reflect?

A

The amount of particular analyte eluted

9
Q

What does the NORMAL phase use?

A

Uses polar stationary phase and non-polar mobile phase

10
Q

What is the adsorption process for normal phase?

A
  • 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
11
Q

What is normal phase chromatography useful for?

A
  • 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
12
Q

Why is reverse phase more commonly used i pharmaceutical sciences?

A

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

13
Q

What does reverse phase use?

A

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.

14
Q

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

A

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

15
Q

What molecules are used in RP?

A

Molecules < 3000 Molecular weight.

16
Q

How does RP work?

A

• 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.

17
Q

In what ways can resolution/performance be improved?

A

 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

18
Q

How long will molecules remain associated with the stationary phase?

A

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.

19
Q

What is included in quantitative analysis?

A

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

20
Q

What is the external standard method?

A

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

21
Q

What is the internal standard method?

A

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

22
Q

What are the 2 types of gas chromatography?

A
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)
23
Q

What are the components of Gas chrom?

A

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)

24
Q

How is separation achieved in GC?

A

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

25
Q

GC instrumentation?

A
•	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
26
Q

GC Chromatogram?

A

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