Calibration of an instrument is the demonstration that the instrument or device produces results within specified limits by comparison with those produced by reference or traceable standard over an appropriate range of measurement. HPLC Calibration is the most critical activity in the laboratory, HPLC is the most sophisticated instrument in the Pharmaceutical Laboratory.
This article is published into 3 parts to make it more detailed as the procedure of HPLC Calibration is itself a big subject.
HPLC Calibration Parameters and recommended frequency –
A) Quarterly Calibration Parameters
- Pressure Test.
- Drift and Noise
- Column oven and sample cooler
- Pump by flow rate accuracy measurement.
- Pump by gradient flow measurement.
- UV-Vis / PDA detector by reference energy check.
B) Half Yearly Calibration Parameters
- UV-VIS/PDA detector by linearity measurement.
- Auto sampler by carry over check.
- Auto sampler by linearity check.
- UV-VIS/PDA detector by wavelength accuracy measurement.
- Autosampler for RI detector by linearity measurement.
C) Yearly Calibration Parameters
- RI Detector by linearity measurement.
- Fluorescence detector by linearity measurement.
- Fluorescence detector by wavelength accuracy measurement.
First of all,
- Before start, the Calibration activity, Ensure your system is neat and clean,
- Clear the surroundings of your HPLC system,
- Check the system Power connections, Reservoir Bottles tray, etc.
- Ensure the required consumables like Ferrules, Peak nut, Tubing are available.
- Make sure your drain collection container is placed and properly attached to drain tubing.
1. Pressure Test.
- The pressure test shall be performed before pump calibration as per the procedure is given below.
- Purge the system for each flow line with the mobile phase and use any of the flow lines for the pressure test.
- Ensure that the tubing from the reservoir to the column inlet shall be removed.
- Plug the outlet of the pump using a dead nut.
- Set the flow rate of the pump to 0.1 ml/min.
- Observe the pressure until it reaches the maximum psi/kgf.
- Repeat the same procedure for further two times and observe the same.
- In case the pressure does not reach the maximum and fluctuate at some psi/kgf, it implies poor check valve performance or leaks within the pumping system.
- After satisfactory completion of the pressure test, proceed for further calibration of other parameters.
- For observation Table – Refer SOP for HPLC Calibration
2. Drift and Noise.
Procedure for HPLC Calibration:
- Mobile Phase Preparation- Mix 700 ml of HPLC grade water and 300 ml of methanol and filter through 0.45-micron nylon filter, degas for 10 min by sonication.
- Flush the system with hot water for 15 minutes.
- Ensure that, the column is conditioned before injecting the sample.
- After conditioning, the column equilibrates the system for 15 min and then run the blank injection as per test condition for 15 min.
- After blank injection run completion, process the data using the processing method.
- Record the results in the observation table
System Requirement for HPLC Calibration:
- Column: C18 (25 cm x 4.6 mm), 5 micron or equivalent.
- Flow rate: 1.0 ml/min
- Wavelength For UV: 254 NM
- Wavelength For PDA: 254 nm (2D data collection at 3.6 nm bandwidth)
- Injection volume: 0.0 ml
- Run time: 15 min
Observation Table :
- For observation, Table Refer SOP for HPLC Calibration
- Noise should be less than or equal to 60 micro AU for a UV detector and less than or equal to 80 micro AU for PDA detector.
- Drift should be less than 10 milli AU/Hr for UV detector and less than 10 million AU/Hr for PDA detector.
3. Column oven and sample cooler.
- Carry out the calibration for the temperature point at 30°C, 60°C and 15°C using digital temperature indicator.
- Set the temperature of the column oven at 30°C, keep the temperature probe in the center of the column oven and allow stabilizing the set temperature for at least 15 to 20 min or more requirements.
- Check the displayed temperature of the column oven and temperature indicator device and record the reading in the observation table.
- Similarly, carry out the calibration for 60°C temperature calibration in the same manner and note all the reading in the observation table.
- If column oven is available with cooler, set the temperature of column oven at 15°C, keep the temperature probe in the center of column oven and allow stabilizing the set temperature for at least 15 to 20 min and noting the reading in observation table-1.
- Similarly set the sample cooler temperature at 4.0°C and 25.0°C and repeat the procedure as described in sec 5.2 to 5.4 and record the results in the observation table.
- The location shall decide the calibration range for the column oven and sample cooler temperature based on there working range. Sample cooler temperature calibration shall be applicable for only that system which has a sample cooler facility.
Observation Table :
- For observation, Table Refer SOP for HPLC Calibration
- Observed temperature reading should be within ± 2.0°C of set temp.
4. Pump by flow rate accuracy measurement.
- Where four inlet lines (A, B, C & D) are mixed into GPV assembly, before the pump (system operated with a single pump). The mixing accuracy of lines is decided by GPV functionality, this is normally measured by GPV test in such case only one-time flow rate accuracy shall be performed. In the case of a binary system where two pumps exist for each line then individual pump flow rate calibration shall be done.
- Mobile Phase: HPLC grade water
- The location shall decide the calibration range based on there working range for the calibration of instruments.
- Testing Condition : Flow Rate = 0.5 ml/min, 1.0 ml/min, and 3.0 ml/min.
- Sample / Standard preparation : None.
- Connect the stainless steel tubing, or other restriction devices, in place of column connections with the instrument.
- Set the vacuum degasser (if installed) to run continuously, or set the purge rate to 100%.
- Keep the mobile phase in the respective reservoir.
- Purge the system for each flow line with filtered HPLC grade water and allow it to run the water for about 15 min before checking the flow.
- Ensure that the tubing from the reservoir to column inlet shall be free from the air bubble and system pressure should be constant.
- Take 10 ml clean and dried volumetric flask and keep at the restricted capillary’s outlet in such a way that when the pump is on the water drops fall into the volumetric flask
- Set the pump for 0.5 ml/minute flow-rate, start the pump and allow to run about 10 min. Then keep the flask at restricted capillary’s outlet and then simultaneously start a stopwatch.
- Stop the Stopwatch when the volume of water reached the mark in the volumetric flask. Derive the time taken and record the observation in the observation table.
- Repeat the same procedure for the flow rate of 1.0 ml/min and 3.0 ml/min in 10 ml volumetric flask and record the observations in below table
|Set Flow rate (ml/min)||The volume delivered by the pump (ml)||Theoretical Time
|Limits (± 2% of the theoretical limit, seconds)||
Actual time (seconds)
Acceptance criteria :
- The actual time observed should be within ± 2.0% of the theoretical time.
5. Pump by gradient flow measurement.
- HPLC grade water or equivalent
- Restricted capillary (5-meter Length x 0.17 mm ID)
Mobile phase preparation
- Mobile phase A (A=B if the pump is Quaternary): Take HPLC grade water 1000 ml filter with 0.45-micron nylon filter is cleaned and dried mobile phase bottle and sonicate it for about 10 min
- If the pump is Quaternary, mobile phase A is to be kept in reservoir A and B both.
- Mobile phase B (C=D if the pump is Quaternary): Take acetone 5 ml in clean and dried 1000 ml volumetric flask, dilute up to mark with HPLC grade water. Filter it with 0.45-micron nylon filter paper and degas by sonication for about 10 min.
- If the pump is Quaternary, mobile phase B is to be kept in reservoir C and D both.
|Testing conditions||Applied conditions|
|Column: Restricted capillary||ID No.:|
|Flow Rate: 2.0 ml/min.|
|Wavelength: 265 nm|
|Injection volume: 10 ml|
|Runtime: 30 minutes for binary (20 min if the pump is quaternary)|
|Column oven/Sample cooler temperature||NA|
- Take HPLC grade water or equivalent as a standard/sample
- Ensure the column should not be attached with the instrument If it is there then remove it and connect with the restricted capillary tube.
- Purge the system with HPLC grade water, which is previously filtered through 0.45 m
- Take the HPLC grade water previously filtered through 0.45 m nylon filter and use as mobile phase for Pump-A ( Use for Pump A and Pump-B if the pump is quaternary)
- Prepare 0.5% v/v solution of Acetone in HPLC Water and filter in 0.45 m nylon filter and use as mobile phase for the Pump – B (Pump–C and Pump-D if the pump is quaternary).
- Set the system gradient flow program as per table-1 for binary and table-2 for the quaternary pump.
- Inject HPLC grade water or equivalent as a sample for five times and record peak height/area in the respective table.
- Plot a linearity curve of peak height % (Theoretical) Vs observed peak height (µv) using the least square method. Calculate the squared correlation coefficient (r2) and RSD of peak height and record the observations in the observation table for the binary pump.
- Table: Gradient flow programming table for the binary pump.
|Flow||Time in minutes||% Channel –A||% Channel – B|
Table: Gradient flow programming table for the Quaternary pump.
Table: Observation table for the binary pump.
For binary pump, Inject three standard trial before starting the actual sequence for system equilibration and also observe the pressure graph. If it is found satisfactory then start the sequence.
|Sr. No.||Peak Height
Between 5.0 to 6.0 minutes
Between 11.0 to 12.0 minutes
Between 17.0 to 18.0 minutes
Between 23.0 to 24.0 minutes
the squared correlation coefficient (r2)
( r2 = NLT0.99)
Table Observation table for the Quaternary pump.
For Quaternary Pump, Inject three standard Injection run as a trial injection before running the actual sequence for HPLC system equilibration and also observed pressure graph. if it is found satisfactory than start the sequence.
|Sr. No.||Peak Height (A + B)
(100%) at Approx. 6 minutes
|Peak Height (C)
(10%) at Approx. 12 minutes
|Peak Height (D)
(10%) at Approx. 16 minutes
|%RSD Þ (NMT 2.0%)|
- (Mean of second peak height/Mean of first peak height) X 100 =______% (Limit: Between 9.5% and 10.5%)
- (Mean of third peak height/Mean of first peak height) X 100 =______% (Limit: Between 9.5% and 10.5%)
Linearity – squared correlation coefficient (r2 )= NLT 0.99 for binary.
%RSD of peak height obtained for five replicate injections of the sample should be NMT 2.0% for Quaternary.