HPLC – High Performance Liquid Chromatography – Calibration Procedure

 

HPLC Calibration

 

SCOPE :

This Procedure is extended to the High Performance Liquid Chromatograph instruments.

 

PROCEDURE :

 

Cleaning of flow cell :

To achieve satisfactory noise and drift characteristics of the detector a flow cell of the detector is cleaned by passing solvent through the flow cell in the following order :

Column should be replaced by union during this treatment.

SOLVENTS USED	VOLUME
Deionised water	20 ml
10 % NH4OH/ Water Solution	20 ml
Deionised water	20 ml
Nitric Acid ( 20 % v/v)	20 ml
Deionised water	20 ml
Isopropyl Alcohol	20 ml
Deionised water	20 ml

Pump Calibration/ Flow Rate Accuracy:

 

Connect Inert sill ODS, 250 mm X 4.6 mm HPLC Column and calibrate the pump flow at  1 ml per minute flow rate.  

To check the flow rate accuracy at 5 ml per minute, disconnect the column and connect the guard column 10GLC4- C8, 10mmX 4 mm and calibrate the pump.

Pump Flow	Composition of Mobile Phase	Time Limit	Collection Volume
1.0 ml/ min	Water: Methanol (1:1)	25 min	24 to 26 ml
5.0 ml/ min	Water: Methanol (1:1)	10 min	48 to 52 ml

Detector Calibration/ Wavelength Accuracy :

 

Prepare a 0.02 mg/ ml (0.002 % i.e. 20 ppm) solution of caffeine in Mobile phase. 

 

Run a chromatogram of above caffeine solution, using following Chromatographic Conditions.  

Column	Inert sill ODS, 250 mm X 4.6 mm (5 micron)
Mobile Phase	Water: Methanol (70:30)
Flow Rate	1 ml/ min
Injection Volume	20 micro lit.

Chromatogram should be recorded at each of the following wavelength

Sr. No.	Wave Length ( nm )
1	238
2	240
3	242
4	244
5	246
6	268
7	270
8	272
9	274
10	276