High performance liquid chromatography (HPLC)

High performance liquid chromatography (HPLC)

High performance liquid chromatography (HPLC)

High performance liquid chromatography
Image Source-Google | Image by -QC/QA Methodology

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.

  • 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.
Detector work:
  • Compound elutes from the column they passes through cell and absorb the radiation resulting in measurable energy level change.
Type of detector:
  • U.V. Visible (Multi-Wavelength detector)
  • P.D.A. (Photo-diode array detector)
  • R.I. (Refractor index detector)
  • Fluorometric detector
  • Electrochemical detector
Type of mobile phase:

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: (Not less than =3000)
Number of theoretical plates(N) is a measure of column efficiency for Gaussian peak.

N=5.54(T/W h/2)2
          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
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)
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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