High performance liquid chromatography (HPLC)

High performance liquid chromatography (HPLC)

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.

Principal:

• Adsorption
• Partition
• Ion exchange.

Parted:

• 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=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.