Calibration of HPLC

Calibration of High Performance Liquid Chromatography

Calibration of HPLC

•       To perform calibration of HPLC following parameters should be considered.

•       Leakage Test/Pressure Test

•       Flow Rate Calibration

•       Drift and Noise Calibration

•       Column Oven Calibration

•       Linearity and Reproducibility of Injection volume

•       Detector Linearity response

Leakage Test/Pressure Test

•       Switch on the instrument and check it if the instrument is ready for calibration proceed further for Leakage/Pressure test.

•       Place the suction tube in HPLC grade water and turn on the pump.

•       Allow the flow of mobile phase at the rate of 0.1ml/min for 5 minutes.

•       Now block the outlet of the pump with a screw.

•       Set the maximum pressure usually 300 bar.

•       Record the gradual increase in pressure till it reach the maximum limit.

•       The pressure rise should be smooth if it shows any fluctuation or didn’t rise up to the set limit there should be possible leakage in the system.

•       Repeat the procedure for 2 more times.

•       Record the data in the calibration log book.

•       In case of any failure of the test inform the concern authoritative person.

Flow Rate Calibration

•       When the system passes the pressure/leakage test proceed for the Flow Rate Calibration.

•       Connect stainless steel union instead of column with the instrument.

•       Properly degas mobile phase (Water) and kept in the reservoir.

•       Purge the system at flow rate of 2ml/min for 5 minutes so that any bubble present in the lining is removed.

•       Take 10ml clean and dried glass calibrated cylinder and keep at the outlet tube in such a way that drops from the outlet falls directly into cylinder without touching walls of the cylinder.

•       Set the flow rate 0.5 ml/minute and start the pump and allow to run for 15 minutes.

•       Now place the 10ml cylinder at the outlet tube and start pre-calibrated stopwatch simultaneously.

•       Observe the volume rise in cylinder when the volume reach up to the 10ml mark stop the stopwatch and remove the cylinder.

•       Record the data on calibration sheet.

•       Repeat the same procedure for flow rate of 1.0 ml/minute and 2.0 ml/minute.

Acceptance Criteria

•       Time observed should be ±2.0% of the theoretical time.

S.No	Flow rate	Volume delivered	Time observed	Limit
1	0.5 ml/minute	10 ml		1176—1224 seconds
2	1.0 ml/minute	10 ml		588—612 seconds
3	2.0 ml/minute	10 ml		294—306 seconds

 

Drift and Noise Calibration

Chromatographic Conditions

Detector                UV

Wavelength          254

Column                  C18 (250×4.6) 5 micron

Flow rate               1.0 ml/minute

Injection volume  0.0

Mobile Phase       Water : Methanol

                                    70   :   30

•       Preparation of Mobile Phase

•       Thoroughly mix 700 ml of HPLC grade water and 300 ml of Methanol HPLC grade.

•       Now filter mobile phase through 0.45 micron filter paper and degas it properly with the help of sonicator.

Procedure

•       Run the system as per the approved SOP.

•       Purge the system at flow rate of 2 ml/minute for 5 minutes.

•       Set flow rate 1.0 ml/minute and allow it to run for 15 minutes to equilibrate the system.

•       Run blank injection for 15 minutes, and complete 6 replicates.

•       Process the data and record the results.

Acceptance Criteria

•       Noise should less than or equal to 60 micro AU

•       Drift should be less than 10 milli AU/Hour

Column Oven and Sample Cooler calibration

•       Calibration of column oven can be carried out at temperature of 15◦c, 30◦c and 60◦c.

•       Pre-calibrated digital thermometer should be used during calibration of column oven.

•       Set the temperature of the column oven at 15◦c and put the probe of calibrated thermometer inside the oven.

•       Observe the temperature display for at least 15 to 20 minutes so that the temperature stabilize.

•       After stable readings record the data on calibration sheet.

•       Repeat the same procedure for 30◦c and 60◦c and record the data in the same way.

•       Calibration of sample cooler can also be carried out in the same way.

Acceptance Criteria

•       The set temperature should be within the range of ±2◦c.

Linearity and Reproducibility of Injection Volume

Chromatographic Conditions

Detector                UV

Wavelength          254

Column                  C18 (250×4.6) 5 micron

Flow rate               1.0 ml/minute

Injection volume  20.0 micron

Mobile Phase       Water : Methanol

                                    70   :   30

Preparation of Mobile Phase

•       Thoroughly mix 700 ml of HPLC grade water and 300 ml of Methanol HPLC grade.

•       Now filter mobile phase through 0.45 micron filter paper and degas it properly with the help of sonicator.

Preparation of standard solution

•       Take 100 ml clean and dry calibrated volumetric flask and add to it 0.5ml Benzene and 0.5 ml Toluene.

•       Make up volume with methanol and mix well.

Procedure

•       Check the instrument that is it ready for operation.

•       Flush the column at a flow rate of 1.0 ml/minute.

•       Check the set parameters required for calibration of injection volume.

•       Allow the mobile phase to run for 20, 30 minutes to equilibrate the column and steady baseline is obtained.

•       For reproducibility of injection volume checking inject 6 replicates of the standard solution at injection volume of 20 micron.

•       Process the method and record the data on the calibration table.

•       Find out the RSD of the 6 replicates.

•       For linearity of injection volume inject 3 replicates of injection at different injection volume i.e 10, 15, 20, 15 and 30 micron.

•       Fine out the correlation coefficient “r2” for 5 levels of injection volume.

•       Record the data on the calibration sheet.

Acceptance Criteria

•       For reproducibility RSD should be less than 2.0%

•       Ratio of the average of two areas should be less than 3.0%

•       For linearity of injection volume correlation coefficient “r2” should be greater than 0.999

Injection	Area of Benzene	Area of toluene	Ratio of the two areas
1
2
3
4
5
6
Average of the ratio
Mean of areas
RSD of area

 

Injection Volume	Injection 1 Area		Injection 2 Area		Injection 2 Area		Mean	RSD%
	Benzene	Toluene	Benzene	Toluene	Benzene	toluene
10 ml
15 ml
20 ml
25 ml
30ml

 

Detector Linearity Response Calibration

Chromatographic Conditions

Detector                UV

Wavelength          254

Column                  C18 (250×4.6) 5 micron

Flow rate               1.0 ml/minute

Injection volume  20.0 micron

Mobile Phase       Water : Methanol

                                    70   :   30

Preparation of Mobile Phase

•       Thoroughly mix 700 ml of HPLC grade water and 300 ml of Methanol HPLC grade.

•       Now filter mobile phase through 0.45 micron filter paper and degas it properly with the help of sonicator.

Preparation of standard solution

•       Solution 1

•       Take 100 ml clean and dry calibrated volumetric flask and add to it 0.5ml Benzene and 1.0 ml Toluene.

•       Make up volume with methanol and mix well.

•       Solution 2

•       Take 100 ml clean and dry calibrated volumetric flask and add to it 1.0 ml Benzene and 1.0 ml Toluene.

•       Make up volume with methanol and mix well.

•       Solution 3

•       Take 100 ml clean and dry calibrated volumetric flask and add to it 1.5ml Benzene and 1.0 ml Toluene.

•       Make up volume with methanol and mix well.

Procedure

•       Check the instrument that is it ready for operation.

•       Flush the column at a flow rate of 1.0 ml/minute.

•       Check the set parameters required for calibration of injection volume.

•       Allow the mobile phase to run for 20, 30 minutes to equilibrate the column and steady baseline is obtained.

•       Operate the instrument as per approved SOP

•       Inject the standard solutions one by one in triplicate.

•       Process the method and record the data in calibration sheet.

•       Calculate the ratio of benzene and toluene area and find out the average value.

•       Calculate the correlation coefficient “r2” of the average values in response to three levels

•       Plot the curve of average area ratio of benzene to toluene vs  concentration.

•       Find out the RSD of the retention time.

Acceptance criteria

•       RSD of the retention time should not be more than 2.0%

•       Correlation coefficient should NLT 0.999

Solution No.	Benzene Area and RT		Toluene Area and RT		Ratio of Benzene to Toluene Area
	RT	Area	RT	Area
Solution 1
Mean
RSD		NA		NA

 

Solution No.	Benzene Area and RT		Toluene Area and RT		Ratio of Benzene to Toluene Area
	RT	Area	RT	Area
Solution 2
Mean
RSD		NA		NA

 

Solution No.	Benzene Area and RT		Toluene Area and RT		Ratio of Benzene to Toluene Area
	RT	Area	RT	Area
Solution 3
Mean
RSD		NA		NA

 

•       If All the parameters were found within the acceptance range the Instrument should be tagged as “calibrated”

•       In case of any failure the concerned person should be informed.