CALIBRATION OF HIGH PRESSURE LIQUID CHROMATOGRAPHY

1.0. PURPOSE:
To provide procedure for Calibration of High Pressure Liquid Chromatography.
2.0. SCOPE:
Applicable to HPLC
Make : Shimadzu
Model : LC-2010AHT
Pump : LC-10Atvp Detector : SPD-10Avp
System Controller : SCL-10Avp
Auto Injector : SIL-10Advp
3.0. RESPONSIBILITY:
3.1. Officer -QC
3.2. Executive-QC
4.0. ACCOUNTABILITY:
Manager-Quality Control
5.0. DEFINITION:
Nil
6.0. PROCEDURE:
6.1. Start the HPLC pump as per IOP and check the flow rate by collecting water in a 10ml measuring cylinder. Using a stop watch set the time for 5 minutes and measure the volume collected for 1.0ml/ minute 1.5ml/minute and 2.0ml/minute flow rates respectively. Note the readings in duplicates.
I
Time in minute 5min
Flow rate 1ml/min
Volume of water collected in ml Pump A: ____ Pump B: _____
Limit in ml 4.95-5.05
II
Time in minute 5min
Flow rate 1.5ml/min
Volume of water collected in ml Pump A: ____ Pump B: _____
Limit in ml 7.45-7.55
III
Time in minute 5min
Flow rate 2ml/min
Volume of water collected in ml Pump A: ____ Pump B: _____
Limit in ml 9.95-10.056.2.

CALIBRATION OF COLUMN
Chromatographic system:
Column : C18 Or C 8 (25cmx 4.0mm), (5micron
Column No. to be mentioned in reports.
Detector : 254nm
Flow rate : 1ml/min
Mobile phase : Degassed and filtered solution containing
Acetonitrile : Water ( 70 : 30 )
Injection volume : 20 ml.
Standard preparation : Transfer 1ml of Toluene and 1ml of Benzene into a 100ml volumetric flask and make upto volume with methanol, shake well, further dilute 5ml to 50ml with methanol. Inject 20ml of standard solution for five times.
Record the following
1. RT of Toluene and Benzene : RSD NMT 2.0%
2. Area of Toluene and Benzene : RSD NMT 2.0%
3. Theoretical plates of Toluene and Benzene : NLT 1500
4. Resolution between Toluene and Benzene : NLT 2.0
6.3. CALIBRATION OF DETECTOR/INJECTOR
To a clean, dry 100ml volumetric flask, add 0.1ml of Benzene and 0.1ml of Toluene. Mix by adding mobile phase and make up to volume. Inject equal quantities three times and record the ratio of the peak area.
Chromatographic conditions
Detector : 254nm
Flow Rate : 1.0 ml/minute
Mobile Phase : Methanol : Water = 80 : 20
Absorbance Scale : 0.02
Attenuation : 5
Injection Volume : 20ml
Column : C-18 (Shim-pak)
Calculation
Area of first peak
Area of second peak
6.4.
Injection No. Ratio % deviation from the average (limit±2%)

1
2
3
Dedicated columns are used as mentioned in the monograph.
6.4.
FREQUENCY OF CALIBRATION: Once in three months
7.0. REFERENCES:
Instrument Manual
8.0. ENCLOSURES:
Nil
9.0. ABBREVIATIONS:
Nil