TITLE: CALIBRATION of HPLC
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SOP NO.: | QC00X-01 | EffectiveDate: | DD/MM/YYYY | Company Logo |
Supersedes : | NA | Page No.: | 01 of end | |
Department: | Quality Control | Review Date: | DD/MM/YYYY |
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I OBJECTIVE
- To provide a procedure for calibration of the HPLC (High-Performance Liquid Chromatography).
II SCOPE
- This procedure is applicable for the HPLC series, used in the Quality Control department of ABC Ltd, Location.
III RESPONSIBILITY
- It is the responsibility of all the QC personnel involved in the analysis using the system.
IV DOCUMENT REFERENCE(s)
SOPs : Policy on calibration (Current version of QA
0XX)
Forms :
|
S.No |
Details |
Format No. (Current version) |
|
1 |
Calibration of the HPLC system |
QC0XX/F0X-XX |
V SCHEDULE:
- Calibration shall be carried out once every 6 months.
VI PROCEDURE
1.0 Calibration Procedure
1.1 To Perform the
Calibration of HPLC as follows.
1.2 Calibration shall
be carried out with the below parameters.
1.2.1 Pump flow calibration
1.2.2 Wavelength accuracy
1.2.3 System precision
1.2.4 Carryover
1.2.5 Detector linearity
1.2.6 Injector linearity
1.2.7 Temperature verification for oven and sample
cooler.
1.2.8 Gradient Performance Check
1.2.9 Signal Noise and Drift
1.3 Enter the calibration details in the tag and appear the
same to the respective instrument
1.4 Calibration procedure shall be carried out as follows
Conditions:
Column :
Zorbax SB-C8 150mm x 4.6mm, 3.5u or equivalent
Wavelength :
273 nm
Flow
Rate : 1.0ml/minutes
Injection
Volume : 20 μl
Runtime : 10 min
Temperature : Ambient temperature
1.5 Mobile Phase Preparation
A
filtered and degassed mixture of Methanol and Water in the ratio of 60:40V/V.
1.6 Preparation of Caffeine stock solutions
Weigh accurately 100 mg of Caffeine into 100 volumetric flasks and make up to the mark with the mobile phase.
1.7 Sample Solutions Preparation:
1.7.1 Sample
solution-1 (0.02mg/ml concentration) preparation: Take 2.0 ml of Caffeine stock
solution in a 100 ml volumetric flask and make it to the mark with the mobile phase.
1.7.2 Sample
solution-2 (0.04mg/ml concentration) preparation: Take 4.0 ml of Caffeine stock
solution in 100ml volumetric flask and make it to the mark with the mobile phase
1.7.3 Sample
solution-3 (0.06mg/ml concentration) preparation: Take 6.0ml of Caffeine stock solution
in a 100 ml volumetric flask and make it to the mark with the mobile phase.
1.7.4 Sample solution-4 (0.08mg/ml concentration) preparation: Take
8.0ml of Caffeine stock solution in a 100 ml volumetric flask and make it up to the
mark with the mobile phase.
1.7.5 Sample solution-5 (0.1mg/ml concentration) preparation: Take
10.0ml of Caffeine stock solution in a 100 ml volumetric flask and make it to the
mark with the mobile phase.
1.8 Pump flow rate calibration
1.8.1 Measure the flow rate ua sing 10 and ml, 25 ml volumetric flask, and calibrate the ted stop clock. Measure the flow
rates of 1.0ml/min, 1.5 ml/min, 2.0 ml/min, and 2.5 m/min.
Volume in ml X 60
Actual time taken to collect the volume in seconds
1.8.2 Acceptance
Criteria : ± 2.0%.
1.9 Wavelength Accuracy
1.9.1 Chromatographic
conditions
Column :
Zorbax SB-C8 150mm x 4.6mm, 3.5 micron or equivalent
Flow
rate : 1.0 ml/min.
Injection volume : 20 ml
Run
time : 10 min
1.9.2 Preparation of
1.9.3 Prepare 0.1 mg/ml caffeine solution in the Mobile phase. Inject caffeine solution at each length rise from 200nm – 210nm, from 239nm – 249nm, and from 268nm-278nm (increment of 1nm), record the details in HPLC calibration format record as per format No: QC0XX/F0X-0X
1.9.4 Acceptance Criteria
- Maximum peak response must be at wavelength: 205 ± 2 nm for200 nm-210 nm
- Maximum peak response must be at wavelength: 273 ± 2 nm for 268 nm-278 nm
- Minimum peak response must be at wavelength: 245 ± 2 nm for 239nm-249nm
1.10 Detector Linearity
1.10.1 Inject Sample solution-1 to Sample solution-5
into the chromatographic system.
1.10.2 Record the area of Caffeine in each sample
solution and calculate the Correlation coefficient.
Draw the linearity graph with area vs. concentration.
1.10.3 Acceptance Criteria: The
Correlation coefficient should be not less than 0.99.
1.11 System Precision:
1.11.1 Inject 5 times sample solution-5 into the
Chromatographic system and calculate the %RSD
for Retention time and %RSD for Areas of the sample solution in 5 injections.
1.11.2 Acceptance Criteria: %RSD for Area and
Retentions times should be less than 2.0.
1.12 Carryover:
1.12.1 Applies only if
carry-over is run immediately after precision.
1.12.2 Inject
diluent as a blank. And record the chromatogram.
1.12.3 Calculate the carryover percent using the fifth injection area of precision.
Blank area/height
Carryover (%)
= ------------------------ x 100
Sample area/height
1.12.4 Acceptance Criteria: NMT 0.2 % for the area and NMT 0.4 % for height.
1.13 Temperature verification for Oven and Sample cooler:
1.13.1 Check the oven temperature at different set
temperatures at 30°C, 40°C, 50°C, 60°C using
calibrated Digital thermometer.
1.13.2 Check the Sample cooler temperature at 5°C and 10°C
1.13.3 Acceptance Criteria: ±1°C
1.14 Injector Linearity:
1.14.1 Inject Sample solution-5.
10µl, 20µl, 30µl, 40µl, 50µl into Chromatographic
system.
1.14.2 Record areas of Caffeine in
each injection and draw the linearity graph with area vs injection volume.
1.14.3 Calculate correlation
coefficient.
1.14.4 Acceptance Criteria: The correlation Coefficient should not be less than 0.99.
1.15 Gradient performance check:( GPV Test)
1.15.1 Preparing the Instrument:
1.15.2 Connect with union
1.15.3 Filter and degas
methanol, 0.5% acetone in methanol
1.15.4 Method Parameters:
1.15.5 Detector wavelength of 254 nm
1.15.6 Binary gradient
with a total flow of 2.0 ml/min
1.15.7 Gradient table as per below table for pumps A and B.
1.15.8 Running the Gradient proportionate Valve test:
|
Time |
A Methanol |
B |
|
0.01 |
100 |
0 |
|
4.01 |
90 |
10 |
|
8.01 |
100 |
0 |
|
12.01 |
75 |
25 |
|
16.01 |
100 |
0 |
|
20.01 |
50 |
50 |
|
24.01 |
100 |
0 |
|
28.01 |
25 |
75 |
|
32.01 |
100 |
0 |
|
36.01 |
0 |
100 |
|
40.01 |
100 |
0 |
|
44.0 |
100 |
0 |
.
1.15.9 Purge
all the pumps with a flow rate of 2 to 5 ml/min for about 2 to 5 minutes.
1.15.10 Set the total flow rate at 2.0ml/min and wait
until the baseline is stable.
1.15.11 Set the gradient profile for Pumps A and
B.
1.15.12 Record the height of peaks.
1.15.13 Consider height at
B conc. At 100% 100 calculate the other peaks' heights.
1.15.14 For Quaternary
systems, repeat the study for other channels of C and D.
1.15.15 Consider height at D conc. At 100% 100 calculate the peak heights.
Calculation:
Height of B/D peak at different concentrations
Height % of Peak B/D = ---------------------------------------------------------- X100
Height of full-scale peak (100% B/D peak height)
1.15.16 Acceptance
criteria: The difference in the proportion of B or D concentration should not vary by more than ± 1.5% for the
set concentration.
1.15.17 then select the Setup Detector signal option enter the required values and click OK.
1.15.18 to her that if already method already was saved we have to open it, and if it any necessary to modify
the method we can change it.
1.15.19 now run the
sample, the sampler will pick the sample after the sample is picked the needle is washed from the additional vial kept for purposes
5.20 Check the flow on
running and we can get the result.
1.15.21 here select printer and press OK. Again select the Report/print option. The report is printed and filed.
1.16.0 Signal noise and Drift:
1.16.1 Fix the union as a column.
Mobile phase: Water.
Flow rate : 1.0 ml/min
Wavelength : 254 nm
Run Time : 24 min
Start Time : 03 min, End Time: 20 min
1.16.2 Run the chromatogram and
record the data.
1.16.3 Evaluation of the noise
and drift between 3 min to 20 min.
1.16.4 Determine the noise and
drift using software.
1.16.5 Acceptance criteria: Should be less than 40µV for noise and Should be less than 500µV/Hz 8.3µV/min.
2.0 CARE AND MAINTENANCE:
2.1 Ensure the details are documented in the column usage log after the analysis & washing activity are completed
2.2 Use
85:15 Acetonitrile and water for the plunger wash.
2.3 Use mobile phase/diluent vial for syringe
needle wash.
2.4 Verify the performance of the system as per
the schedule/after a major breakdown of the instrument.
2.5 Replace the inlet filters
based on their performance.
2.6 Enter the breakdown details & spares utilized in the Instrument malfunction record.
* All mobile phase solutions shall be prepared as per the analysis requirement. However mobile phase solutions stored beyond 48 hrs shall not be used. And document the details in the mobile phase and mobile phase log
REFERENCE:
* Instrument Manual of HPLC
REVISION HISTORY
Version | Effective | Summary of Revision |
0.0 | DD/MM/YYYY | New SOP |
END O F THE DOCUMENT
Prepared By Checked By Approved By
Sign & Date
Name & Dept QA Executive QC Manage QA Manager.
#HPLC #Calibration, #GPV #Performance
