Standard Operating Procedure (SOP) and format/Protocol of HPLC system calibration as per newly released Indian Pharmacopoeia (IP) recommendations. This Procedure is applicable for Shimadzu make HPLC and UPLC Systems, Agilent make HPLC Systems, Thermo make UHPLC & UPLC, Waters make HPLC Systems etc.
HPLC Calibration Parameters are –
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- Flow rate accuracy – HPLC Pump Calibration
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- Temperature accuracy of HPLC column oven
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- Calibration of HPLC Detector – Detector Linearity Test
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- Wavelength Accuracy Test
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- Injector Linearity Test
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- System Precision
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- Carryover Test
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- Gradient Composition Calibration
Procedure for HPLC Calibration
1.0 Purpose
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- The objective of this SOP is to describe the procedure for the calibration of High-Performance Liquid Chromatography (HPLC) instruments in the Quality Control (QC)/Department / Testing Laboratories.
2.0 Scope
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- This SOP is applicable for HPLC instrument calibration being used in the Quality Control / Testing Laboratories;
3.0 Responsibility
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- Chemist / Executive / whoever is operating the instrument: To follow the laydown procedure.
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- Head – QC or Designee: To assure the implementation of SOP.
4.0 Procedure for HPLC Calibration
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- Cleaning of HPLC System prior to Calibration :
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- Ensure that the power supply to the instrument is switched ‘OFF’ during cleaning.
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- Clean the instrument with fiber-less tissue paper or dry cotton cloth.
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- Before starting the analysis flush the system with degassed purified water.
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- Clean solvent inlet filter.
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- After every analysis wash the column, then the system with water /appropriate solvent.
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- Empty the waste before it overflows.
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Calibration of HPLC System:
- Record all calibration details and data in an issued controlled copy of the calibration record, format no. 1.
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- Flow rate accuracy ( HPLC Pump Calibration) :
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- Disconnect the column and attach the outlet tubing of the pump to the inlet tubing of the detector.
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- Prime all channels of the pump & needle wash with purified water.
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- Take purified water as mobile phase, filtered, and degassed. Connect the union at the place of the column.
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- For 0.5 ml/min, 1.0 ml/min and 2.0 ml/min.: Take a 10 ml class A volumetric flask and ensure that it is thoroughly dry. Prime solvent line A with 100% concentration and prepare the stopwatch to begin
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- When the flow rate and pressure are stable, simultaneously insert outlet tubing into the flask and start the stopwatch.
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- Stop the stopwatch when the bottom of the meniscus reaches the 10 ml mark of the flask, and record the time in seconds.
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- Calculate the flow rate using the following formula;
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- Flow rate measured value = Volume of flask / (measured time in seconds / 60).
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- Repeat the flow accuracy test for B, C, and D channels of the Quaternary pump.
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- The tolerance limit for the flow rate measured value is 2% of the set flow rate value.
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Temperature accuracy of column oven :
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- Place the thermometer inside the column oven, ensure that the sensor does not contact any surface and close the chamber door properly.
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- Set the temperature of the column oven at 30° C. Wait till the set temperature is attained and the temperature display on the instrument is stable.
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- Note down the temperature displayed by the instrument and the temperature of the thermometer.
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- Repeat the above procedure for calibration of the oven at 40.0° C, 60° C, and 80° C.
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- The tolerance limit for set temperature accuracy should be ± 2.0° C (Set temperature – Actual temperature).
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Temperature Accuracy of Sample Cooler :
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- Temperature accuracy of sample cooler calibration is not applicable for HPLC instruments where instruments do not have an inbuilt provision for sample cooling.
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- Place the thermometer inside the auto-sampler chamber in the center of the sample tray.
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- Make a. method for each of temperatures 5° C, 10° C, and 25° C and run one by one. Allow sufficient time for sample compartment to attain and stabilize at the set temperature.
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- Record the observation on the thermometer.
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- The tolerance limit for set temperature accuracy should be ± 2°C (Set temperature -Actual temperature).
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Calibration of HPLC Detector :
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- Detector Linearity Test :
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- Chromatographic conditions;
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- Mobile Phase: Water : Methanol (60:40 v/v)
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- Column : Inertsil ODS, 100 mm x 4.6 mm, 5μ.
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- Wavelength: 272 nm
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- Oven Temperature : 40° C
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- Flow rate : 1.0 ml / min
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- Injection Volume – 20 µl
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- Run Time – 05 Minutes
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- To perform detector linearity test prepare 5 PPM, 25 PPM, 50 PPM, 125 PPM and 250 PPM solution of caffeine in mobile phase.
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- Preparation of 500 PPM caffeine standard stock solution:
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- Dissolve and dilute about 50 mg caffeine standard in 100 ml volumetric flask with mobile phase.
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- Preparation of 250 PPM caffeine standard solution:
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- Dilute 25 ml of 500 PPM Caffeine standard stock solution to 50 ml with mobile
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- Preparation of 125 PPM caffeine standard solution:
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- Dilute 25 ml of 250 PPM caffeine standard solution to 50 ml with mobile phase.
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- Preparation of 50 PPM Caffeine standard solution:
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- Dilute 10 ml. of 500 PPM caffeine standard stock solution to 100 ml with mobile
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- Preparation of 25 PPM caffeine standard solution:
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- Dilute 10 ml, of 125 PPM caffeine standard solution to 50 ml, with mobile phase.
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- Preparation of 5 PPM Caffeine standard solution:
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- Dilute 10 ml of 25 PPM Caffeine standard solution to 50 ml with mobile phase.
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- Inject blank and sample in single, record the response and then calculate the correlation Coefficient (r) for concentration against
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- The correlation coefficient (r) should not be less than 0.99.
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Wavelength Accuracy Test :
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- The wavelength accuracy test verifies that programmed wavelengths are within accuracy specifications.
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- Set the mobile phase and chromatographic condition as that of the detector linearity test except for wavelength.
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- Prepare sample set with methods for wavelength from 266 to 277 nm.
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- Check the wavelength accuracy using a 25 PPM solution of caffeine prepared in the mobile phase. Inject 20 µl. of 25 PPM caffeine solution in single at wavelengths from 266 nm to 277 nm and record the
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- In the case of Shimadzu Prominence – I, LC- 2030 C 3D Series, inject a single injection of 25 PPM solution of caffeine by creating a method with a range of 200 to 400 nm and extract the chromatograms in 266 nm to 277 nm with single wavelength number difference as above.
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- The peak area of caffeine peak should be obtained at 272 ± 2 nm
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Calibration of Auto-Injector in HPLC Calibration:
- Injector Linearity Test :
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- Set the mobile phase and chromatographic condition as that of the detector linearity test except for the injection volume.
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- Inject various injection volumes of 125 PPM caffeine e. 5.0 µl, 10.0 µl., 20.0 µl, 50.0 µl, and 100.0 µl in single and record the response of peak due to caffeine.
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- Inject blank and sample in single, record the response, and then calculate the correlation coefficient (r) for concentration against
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- Calculate the correlation coefficient (r) for injection volume against peak response.
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- The Correlation coefficient (r) should not be less than 0.99.
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System Precision in HPLC Calibration:
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- Set the mobile phase and chromatographic condition as that of the detector linearity test.
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- Inject blank and six replicates of 125 ppm caffeine solution and record the response for the peak due to caffeine.
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- The relative standard deviation for retention time and peak area of six replicate injections should not exceed 2.0%.
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Carryover Test :
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- Set the mobile phase and chromatographic condition as that of the detector linearity test.
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- Preparation of 250 ppm caffeine standard solution: Dilute 25 mL of 500 ppm caffeine standard stock solution to 50 mL with mobile (Use solution prepared in detector linearity).
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- Inject samples as blank, caffeine 250 PPM, and blank in single and record the response of peak due to caffeine.
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- Calculate the carryover using the following formula;
% Carryover = (A x 100)/B
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- Where, A = Area of Caffeine peak in blank Injection. B = Area of Caffeine Peak in Standard Injection.
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- The carryover should not be more than 0.01%.
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Gradient Composition Calibration :
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- This procedure uses acetone as a tracer to determine the accuracy of the gradient composition of the Waters & Shimadzu autosampler series HPLC instrument
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- Chromatographic Conditions:
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- Flow : 2 0 ml/Minute
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- Detector : 265 nm
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- Stop Time : 27 Minute
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- Solvent A : HPLC Grade Water
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- Solvent B, C, D : 05% Acetone in HPLC Grade Water
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- Injection Volume : 0 μl
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- Gradient Program:
| Time | A% | B%/C%/D% |
| 0.01 | 100 | 0 |
| 4.00 | 100 | 0 |
| 4.01 | 80 | 20 |
| 8.00 | 80 | 20 |
| 8.01 | 60 | 40 |
| 12.00 | 60 | 40 |
| 12.01 | 40 | 60 |
| 16.00 | 40 | 60 |
| 16.01 | 20 | 80 |
| 20.00 | 20 | 80 |
| 20.01 | 0 | 100 |
| 24.00 | 0 | 100 |
| 24.01 | 100 | 0 |
| 27.00 | 100 | 0 |
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- Prior to the test gradient, a 10-minute pre-purge is to be performed.
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- Run the sequence with an empty vial at the required position for Port A and Port B, Port A and Port C, Port A, and Port D with respect to Port A. Calculate the gradient profile % as below;
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- Gradient Profile in % : Each Height Peak × 100 / Full Peak Height
- Acceptance Criteria :
Step 1 of 20 % = 18.0 – 22.0 %
Step 2 of 40 % = 38.0 – 42.0 %
Step 3 of 60 % = 58.0 – 62.0 %
Step 4 of 80 % = 78.0 – 82.0 %
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Refractive Index Detector (RI Detector) Calibration :
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- Start the instruments as described above, and take water & methanol as mobile phase in the ratio of 60:40 v/v mix, degas, and use.
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- Wash the flow channel with the mobile phase at this stage using a coupling with no column connected.
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- Pump the mobile phase and then replace the coupling with the column. Take 150 × 4.6 mm C 18, 5µ column for calibration place the column in the column oven & set the temperature to 40˚C.
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- Saturating the column with a mobile phase up to a stable baseline is not observed.
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- Prepare 250 ppm of caffeine solution in water and use it as a sample solution.
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- Inject 20µL each of water as blank followed by the mobile phase as blank for 5 minutes and check that there is no peak in the detector on the chromatogram.
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- Inject 20µL of 250 ppm caffeine solution for 5 minutes and note the peak area and retention time of caffeine.
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- Repeat the process for a further 5 injections (a total of six replicate injections of 250 ppm caffeine solution).
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- Check the % relative standard deviation of peak area and retention time. The % RSD for peak area should not be more than 2.0% and for retention time should not be more than 0.5 %.
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Conductivity detector calibration. Chromatographic condition.
Column : waters IC-Pak Anion, 4.6 × 50mm
Mobile Phase : Borate/Gluconate Eiuent Mixture
Injection Volume: 20µL
Column Temp. : 33˚C
Cell Temp. : 35˚C
Run Time : 9 Min
Cell Constant : 16.7
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Preparation of Mobile Phase:
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- Weigh accurately 9.06 g of sodium gluconate in a 1000 ml volumetric flask and dissolve it in 500 ml of purified water.
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- Add 7.2 g of lithium hydroxide monohydrate and 25.5 g of boric acid mix well.
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- Add 94 ml of 95 % glycerol mix well and dilute up to the Mark with purified water.
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- Transfer 20 ml of this solution to a 1000 ml volumetric flask. Add 500 ml of purified water.
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- Add 120 ml of Acetonitrile, mix well adjust the volume up to the mark with purified water.
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- Preparation of 1000 ppm potassium bromide standard stock solution.
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- Dissolve and dilute about 100 mg potassium bromide standard in 100 ml volumetric flask with water.
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- To perform detector linearity test prepare 2 ppm, 5 ppm, 10 ppm, 25 ppm, 50 ppm, 100 ppm solution of Potassium bromide in water.
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Preparation of 100 ppm potassium bromide standard solution:
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- Dilute 10 ml of 1000 ppm potassium bromide standard stock solution to 100 ml with water.
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- Preparation of 50 ppm potassium bromide standard solution:
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- Dilute 25 ml of 100 ppm potassium bromide standard solution to 50 ml with Water.
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- Preparation of 25 ppm potassium bromide standard solution:
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- Dilute 25 ml of 50 ppm potassium bromide standard solution to 50 ml with Water.
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- Preparation of 10 ppm potassium bromide standard solution:
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- Dilute 10 ml of 100 ppm potassium bromide standard solution to 100 ml with Water.
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- Preparation of 5 ppm potassium bromide standard solution:
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- Dilute 10 ml of 25 ppm potassium bromide standard solution to 50 ml with Water.
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- Preparation of 2 ppm potassium bromide standard solution: Dilute 2 ml of 100 ppm potassium bromide standard solution to 100 ml with Water.
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- Check the linearity with 2 ppm, 5 ppm, 10 ppm, 10 ppm, 25 ppm, 50 ppm, & 100 ppm of bromide solution.
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- Inject blank and sample in single, record the response, and then calculate the correlation coefficient (r) for concentration against response.
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- The correlation coefficient (r) should not be less than 0.9. Record all data in the worksheet.
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System Precision Test:-
- Chromatographic Condition :
Column : waters IC-Pak Anion, 4.6 × 50mm
Mobile Phase : Borate/Gluconate Eiiuent Mixture
Injection Volume : 20µL
Column Temp. : 33˚C
Cell Temp. : 35˚C
Run Time : 9 Min
Cell Constant : 16.7
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- At the above chromatographic condition inject 20 µL of six replicate injections of 50 ppm of bromide standard solution.
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- The % RSD if the area should be NMT 2.0 % and for RT should be NMT 2.0 %.
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- Calibration Frequency: Half Yearly
Calibration record of RI detector
Instrument No. : ____________ Instrument Name : _________
Make : ____________ Model : _________
Calibration Date : ____________ Calibration Due On :__________
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System Precision :
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- Mobile Phase : ______ml: _______ml. (Water: Methanol 60:40 v/v).
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- Preparation of 500 ppm Caffeine standard stock solution:
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- Dissolved and diluted ______ mg (50 mg) caffeine standard in _______ ml (100 ml) volumetric flask with water.
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- Preparation of 250 ppm caffeine standard solution:
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- Diluted ______ ml (25 ml) of 500 ppm caffeine standard stock solution to ______ ml (50 ml) with water.
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- Injected blank and six replicates of 250 ppm caffeine solution and recorded the response for the peak due to caffeine.
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Chromatographic Condition:
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Column : _____________________ (Column ID:______________)
Oven Temperature : __________˚C (40˚C)
Flow Rate : __________ ml (1.0 ml/min)
Injection Volume : ________µl (20.0 µL)
Run Time : ______ min (5 Min)
| Sr. No. | Caffeine 250 ppm | Retention Time of Caffeine | Area of Caffeine | Tolerance |
| 01 | Injection – 1 | The relative standard deviation for retention time should not be more than 0.5 % and peak area of six replicate injections should not be more than 2.0 %. | ||
| 02 | Injection – 2 | |||
| 03 | Injection – 3 | |||
| 04 | Injection – 4 | |||
| 05 | Injection – 5 | |||
| 06 | Injection – 6 | |||
| Average | ||||
| % RSD | ||||
Remark:______________________________________________________
Calibrated By:___________ Checked By:___________
Calibration of Conductivity detector
Instrument No. : ____________ Instrument Name : _________
Make : ____________ Model : _________
Calibration Date : ____________ Calibration Due On :__________
Potassium Bromide Lot : ___________ Use before :__________
- Mobile Phase: Borate / Gluconate Eiiuent Mixture :
1.0 Detector Linearity Test:
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- Preparation of 1000 ppm potassium bromide standard stock solution.
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- Dissolve and dilute about ______ mg (100 mg) potassium bromide standard in _____ ml (100 ml) volumetric flask with water.
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- Preparation of 100 ppm potassium bromide standard solution:
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- Dilute ____ ml (10 ml) of 1000 ppm potassium bromide standard stock solution to _____ ml (100 ml) with water.
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- Preparation of 50 ppm potassium bromide standard solution:
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- Dilute ______ ml (25 ml) of 100 ppm potassium bromide standard solution to _____ ml (50 ml) with Water.
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- Preparation of 25 ppm potassium bromide standard solution:
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- Dilute ______ ml (25 ml) of 50 ppm potassium bromide standard solution to _____ ml (50 ml) with Water.
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- Preparation of 10 ppm potassium bromide standard solution:
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- Dilute ______ ml (10 ml) of 100 ppm potassium bromide standard solution to ____ ml (100 ml) with Water.
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- Preparation of 5 ppm potassium bromide standard solution:
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- Dilute _____ ml (10 ml) of 25 ppm potassium bromide standard solution to 50 ml with Water.
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- Preparation of 2 ppm potassium bromide standard solution:
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- Dilute ______ml (2 ml) of 100 ppm potassium bromide standard solution to ______ ml (100 ml) with Water.
Chromatographic Condition:
Column : _____________________ (Column ID):____________)
Mobile Phase : Borate/Gluconate Eiiuent Mixture
Injection Volume : ________µL (20µL)
Column Temp. : ________˚C 33˚C
Flow Rate : ________ ml (1.5 ml/min)
Cell Temp. : ________˚C (35˚C)
Run Time : ______ min (9 Min)
Cell Constant : _______ (16.7 1/cm)
| Sr. No. | Concentration of Bromide in ppm | Area of Pot. Bromide | Tolerance (˚C) |
| 1 | 2 | Correlation Coefficient (r) should not be less than 0.99 | |
| 2 | 5 | ||
| 3 | 10 | ||
| 4 | 25 | ||
| 5 | 50 | ||
| 6 | 100 | ||
| Correlation Coefficient (r) | |||
2.0 System Precision:
Injected blank and six replicates of 50 ppm bromide solution and recorded the response for the peak due to potassium bromide.
Chromatographic condition:
Column : _____________________ (Column ID):____________)
Injection Volume : ________µL (20µL)
Column Temp. : ________˚C 33˚C
Flow Rate : ________ ml (1.5 ml/min)
Cell Temp. : ________˚C (35˚C)
Run Time : ______ min (9 Min)
Cell Constant : _______ (16.7 1/cm)
| Sr. No. | Bromide 50 ppm | Retention time of Bromide (min) | Area of Bromide | Tolerance |
| 01 | Injection – 1 | The relative standard deviation for retention time and peak area of six replicate injections should not be more than 2.0 %. | ||
| 02 | Injection – 2 | |||
| 03 | Injection – 3 | |||
| 04 | Injection – 4 | |||
| 05 | Injection – 5 | |||
| 06 | Injection – 6 | |||
| Average | ||||
| % RSD | ||||
Remark:__________________________________________________________________
Calibrated By:___________ Checked By:___________
Recording format for HPLC Calibration record
- Instrument No. : ____________ Instrument Name : _________
- Make : ____________ Model : _________
- Calibration Date : ____________ Calibration Due On :__________
1.0 Flow Rate Accuracy (HPLC Pump Calibration):
Stop watch ID: _________________ Calibration Done: ______________
Calibration Due: ______________
Channel A:
| Sr. No. | Set Flow (ml/min) | Volume of flask (in ml) | Measured time in sec. To fill the flask (in ml) | Flow rate measured value in sec. | Tolerance |
| 01 | 0.5 ml/min | 10 | 0.49 to 0.51ml/min | ||
| 02 | 1.0 ml/min | 10 | 0.98 to 1.02 ml/min | ||
| 03 | 2.0 ml/min | 10 | 1.96 to 2.04 ml/min |
Channel B:
| Sr. No. | Set Flow (ml/min) | Volume of flask (in ml) | Measured time in sec. To fill the flask (in ml) | Flow rate measured value in sec. | Tolerance |
| 01 | 0.5 ml/min | 10 | 0.49 to 0.51 ml/min | ||
| 02 | 1.0 ml/min | 10 | 0.98 to 1.02 ml/min | ||
| 03 | 2.0 ml/min | 10 | 1.96 to 2.04 ml/min |
Channel C:
| Sr. No. | Set Flow (ml/min) | Volume of flask (in ml) | Measured time in sec. To fill the flask (in ml) | Flow rate measured value in sec. | Tolerance |
| 01 | 0.5 ml/min | 10 | 0.49 to 0.51ml/min | ||
| 02 | 1.0 ml/min | 10 | 0.98 to 1.02 ml/min | ||
| 03 | 2.0 ml/min | 10 | 1.96 to 2.04 ml/min |
Channel D:
| Sr. No. | Set Flow (ml/min) | Volume of flask (in ml) | Measured time in sec. To fill the flask (in ml) | Flow rate measured value in sec. | Tolerance |
| 01 | 0.5 ml/min | 10 | 0.49 to 0.51 ml/min | ||
| 02 | 1.0 ml/min | 10 | 0.98 to 1.02 ml/min | ||
| 03 | 2.0 ml/min | 10 | 1.96 to 2.04 ml/min |
Remark:_______________________________________________________________
2.0 Temperature Accuracy of Column Oven:
Thermometer Id:_________ Calibration Done:____________ Calibration Due:_____________
| Sr. No. | Set Temperature (˚C) | Temperature Displayed on Thermometer (˚C) | Set Temperature Accuracy | Tolerance |
| 01 | 30 | 28.0 to 32.0 | ||
| 02 | 40 | 38.0 to 42.0 | ||
| 03 | 60 | 58.0 to 62.0 | ||
| 04 | 80 | 78.0 to 82.0 |
Remark:_______________________________________________________________
3.0 Temperature Accuracy of Sample Cooler:
Thermometer ID:___________ Calibration Done:_________Calibration Due:_____________
| Sr. No. | Set Temperature (˚C) | Temperature Displayed on Thermometer (˚C) | Set Temperature Accuracy | Tolerance |
| 01 | 5 | 2.0 to 8.0 | ||
| 02 | 10 | 7.0 to 13.0 | ||
| 03 | 25 | 22.0 to 28.0 |
Remark:___________________________________________________________________
4.0 HPLC Detector Linearity Test:
Mobile Phase: _________ml :_________ ml. (Water:Methanol,60:40 v/v)
Preparation of 500 ppm caffeine standard stock solution:
Dissolved and diluted ________ mg (50 mg) caffeine standard in _______ ml (100 ml) volumetric flask with mobile phase.
Preparation of 250 ppm caffeine standard stock solution:
Diluted ________ ml (25 ml) of 500 ppm caffeine standard stock solution to_______ ml (50 ml) with mobile phase.
Preparation of 125 ppm caffeine standard stock solution:
Diluted ________ ml (25 ml) of 250 ppm caffeine standard stock solution to_______ ml (50 ml) with mobile phase.
Preparation of 50 ppm caffeine standard stock solution:
Diluted ________ ml (10 ml) of 500 ppm caffeine standard stock solution to_______ ml (100 ml) with mobile phase.
Preparation of 25 ppm caffeine standard stock solution:
Diluted ________ ml (10 ml) of 125 ppm caffeine standard stock solution to_______ ml (50 ml) with mobile phase.
Preparation of 5 ppm caffeine standard stock solution:
Diluted ________ ml (10 ml) of 25 ppm caffeine standard stock solution to_______ ml (50 ml) with mobile phase.
Chromatographic Conditions:
Column : _________________ (Column ID:______________)
Wavelength : __________nm
Oven Temperature : __________˚C (40˚C)
Flow Rate : __________ ml (1.0 ml/min)
Injection Volume : ________µl (20.0 µL)
Run Time : ______ min (5 Min)
Injected blank & sample in single, recorded the response to calculate the Correlation Coefficient (r).
| Sr. No. | Concentration of Solution | Area of Caffeine | Tolerance |
| 01 | 5 ppm | Correlation Coefficient (r) should not be less than 0.99 | |
| 02 | 25 ppm | ||
| 03 | 50 ppm | ||
| 04 | 125 ppm | ||
| 05 | 250 ppm | ||
| Correlation Coefficient (r) | |||
Remark:_____________________________________________________________________
5.0 Wavelength Accuracy Test:
Injected 20µl of 25 ppm caffeine solution in single at different wavelengths from 266 nm to 277 nm and recorded the response. Injected 20 µl of 25 ppm caffeine solution in single for Shimadzu Prominence – I, LC – 2030 C 3D Series I.E. at wavelengths from 200 nm to 400 nm and recorded the response at wavelength 266 to 277 nm.
Chromatographic Conditions:
Column : _________________ (Column ID:______________)
Wavelength : __________nm
Oven Temperature : __________˚C (40˚C)
Flow Rate : __________ ml (1.0 ml/min)
Injection Volume : ________µl (20.0 µL)
Run Time : ______ min (5 Min)
| Sr. No. | Wavelength | Area of Caffeine | Tolerance |
| 01 | 266 nm | The maximum peak area of caffeine peak should be obtained at 272 ± 2 nm. | |
| 02 | 267 nm | ||
| 03 | 268 nm | ||
| 04 | 269 nm | ||
| 05 | 270 nm | ||
| 06 | 271 nm | ||
| 07 | 272 nm | ||
| 08 | 273 nm | ||
| 09 | 274 nm | ||
| 10 | 275 nm | ||
| 11 | 276 nm | ||
| 12 | 277 |
Observation: Maximum peak area of caffeine peak observed at _______ nm.
Remark:_________________________________________________________________
6.0 HPLC Calibration – Injector Linearity Test:
Injected various injection volumes 125 ppm caffeine i.e. 5.0µl, 20.0µl, 50.0µl and 100.0µl in single and recorded the response of peak due to caffeine.
Chromatographic Conditions:
Column : _________________ (Column ID:______________)
Wavelength : __________nm
Oven Temperature: __________˚C (40˚C)
Flow Rate : __________ ml (1.0 ml/min)
Injection Volume : 2.0 µL, 5.0 µL, 10.0 µL, 20.0 µL, 50.0 µL, 100.0 µL (As per loop size)
Run Time : ______ min (5 Min)
| Sr. No. | Wavelength | Area of Caffeine | Tolerance |
| 01 | 2.0 | The maximum peak area of caffeine peak should be obtained at 272 ± 2 nm. | |
| 02 | 5.0 | ||
| 03 | 10.0 | ||
| 04 | 20.0 | ||
| 05 | 50.0 | ||
| 06 | 100.0 | ||
| Correlation Coefficient (r) | |||
Remark:_____________________________________________________________________
7.0 System Precision:
Injected blank and six replicates of 125 ppm caffeine solution and recorded the response for the peak due to caffeine.
Chromatographic Conditions:
Column : _________________ (Column ID:______________)
Wavelength : __________nm
Oven Temperature : __________˚C (40˚C)
Flow Rate : __________ ml (1.0 ml/min)
Injection Volume : ________µl (20.0 µL)
Run Time : ______ min (5 Min)
| Sr. No. | Bromide 50 ppm | Retention time of Bromide (min) | Area of Bromide | Tolerance |
| 01 | Injection – 1 | The relative standard deviation for retention time and peak area of six replicate injections should not be more than 2.0 %. | ||
| 02 | Injection – 2 | |||
| 03 | Injection – 3 | |||
| 04 | Injection – 4 | |||
| 05 | Injection – 5 | |||
| 06 | Injection – 6 | |||
| Average | ||||
| % RSD | ||||
Remark:_______________________________________________________________________
8.0 Carry over test:
Injected blank, caffeine 250 ppm and blank in single and recorded the response of peak due to caffeine.
Chromatographic Conditions:
Column : _________________ (Column ID:______________)
Wavelength : __________nm
Oven Temperature : __________˚C (40˚C)
Flow Rate : __________ ml (1.0 ml/min)
Injection Volume : ________µl (20.0 µL)
Run Time : ______ min (5 Min)
| Sr. No. | Injection | Area of Caffeine |
| 01 | 250 ppm caffeine standard solution | |
| 02 | Blank |
% carry over = _________________ × 100 =__________ % (should not be more than 0.01 %)
Remark:_______________________________________________________________________
9.0 Gradient Composition Calibration:
Preparation of 0.05 % Acetone:
Mixed _____ ml (0.5 ml) of acetone in ________ ml (1000 ml) of water, mixed and degassed.
Chromatographic Conditions:
Flow : ______________ ml/min (2.0 ml/min)
Detector : ______________ nm (265 nm)
Stop time : ______________ minutes (27 minutes)
Solvent A : _______________ ml of Water
Solvent B,C,D : ________________ ml of 0.05 % acetone in water
Injection Volume : ________________ µl (0.0 µl)
Gradient Calibration for Port A and Port B
Step 1 of 20 % = __________________ × 100
= __________________ (18.0 – 22.0 %)
Step 2 of 20 % = __________________ × 100
= __________________ (38.0 – 42.0 %)
Step 3 of 20 % = __________________ × 100
= __________________ (58.0 – 62.0 %)
Step 4 of 20 % = __________________ × 100
= __________________ (78.0 – 82.0 %)
Gradient Calibration for Port A and Port C
Step 1 of 20 % = __________________ × 100
= __________________ (18.0 – 22.0 %)
Step 2 of 20 % = __________________ × 100
= __________________ (38.0 – 42.0 %)
Step 3 of 20 % = __________________ × 100
= __________________ (58.0 – 62.0 %)
Step 4 of 20 % = __________________ × 100
= __________________ (78.0 – 82.0 %)
Gradient Calibration for Port A and Port D
Step 1 of 20 % = __________________ × 100
= __________________ (18.0 – 22.0 %)
Step 2 of 20 % = __________________ × 100
= __________________ (38.0 – 42.0 %)
Step 3 of 20 % = __________________ × 100
= __________________ (58.0 – 62.0 %)
Step 4 of 20 % = __________________ × 100
= __________________ (78.0 – 82.0 %)
Remark:____________________________________________________________________
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