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.
2 Comments
tumbs up for you pharmacytimess
ReplyDeletewell explained. thanks for sharing such kinda stuff
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