High performance liquid chromatography (HPLC)
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Principal of HPLC:-
HPLC is separation technique based on a solid stationary phase and a liquid mobile phase.Separation is achieved by adsorption partition or ion exchange process.
Principal:
- Adsorption
- Partition
- Ion exchange.
- Injector/Auto injector
- Pump
- Detector
- Column
Advantage of HPLC:-
- Analysed compound is dissolved in a suitable solvent.
- Most of analysis of room temperature.
- Most drugs being non volatile thermally unstable compound.
- Chromatography always used for hydrocarbon soluble compound of molecular weight less than 1000.
- Compound elutes from the column they passes through cell and absorb the radiation resulting in measurable energy level change.
- U.V. Visible (Multi-Wavelength detector)
- P.D.A. (Photo-diode array detector)
- R.I. (Refractor index detector)
- Fluorometric detector
- Electrochemical detector
Normal phase:
Polar stationary phase and non polar mobile phase are describe as normal phase.
Revers phase:
Non polar stationary phase and polar mobile phase are describe as revers phase.
Column work:
Separation is achieved by partition of compound in the test solution between the mobile phase and stationary phase.
Pump work:
Pump system deliver metered amount of mobile phase from the solvent reservoirs to the column through high presser.
Check the column performance or Qualification:
- Number of theoretical plates
- Tailing factor
- Capacity factor
- Resolution
Number of theoretical plates(N) is a measure of column efficiency for Gaussian peak.
N=16(T/W)2
N=5.54(T/W h/2)2
Where,
T=Retention time of peak.
W=Width of the peak.
Tailing factor:
Tailing factor(T) a measure of peak symmetry is unit of perfectly symmetrical peak.
T=W 0.05/2f
W 0.05=Width of peak at 5% height
F=Half distance peak maximum to the loading edge of peak distance being measured at a point 5% of the peak height from the baseline.
Capacity factor:
Time spent of substance in stationary phase
K= ------------------------------------------------------
Time spent of substance in mobile phase
Resolution: (Grater than=1.5)
The separation of two component in a mixture.
2(t2 - t1)
R=---------------------
W2 + W1
t2 and t1 are the retention time of two component.
W2 and W1 are the corresponding width at the base of the peak.
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