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.

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.

Comments

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