HPLC (High-Performance Liquid Chromatography) calibration means checking and confirming the instrument's performance to obtain reliable, precise results. How to Calibrate HPLC: Essential Tips for Reliable Analytical Results. Calibration parameters typically include the following:
A. Pump
- 1. Flow Rate Accuracy
- 2. Flow Rate Consistency
- 3. Compositional Accuracy (Gradient Profile)
- 4. Delay Volume of the System
B. Autosampler
- 5. Injection Volume Accuracy
- 6. Injection Volume Precision
- 7. Injection Linearity
- 8. Autosampler Temperature Accuracy
C. Column Compartment
- 9. Column Oven
D. Detector
- 10. Detector Linearity
- 11. Wavelength Accuracy
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1. Flow Rate Accuracy:
- Materials required are HPLC-grade water, a 10 mL volumetric flask, and a calibrated stopwatch.
- At first, remove the column from the system.
- Put all channel inlets in reservoirs of HPLC-grade water and start purging all ports to remove air bubbles.
- Set the flow rate at 1.0 ml/min. Keep the composition of all channels at 25% (A-25%, B-25%, C-25%, D-25%)
- Allow the system to saturate.
- After some time, collect the HPLC-grade water from the column inlet in a dry 10.0 mL volumetric flask.
- Record the time taken to fill up the volumetric flask up to the mark or lower meniscus of the flask.
- Perform the same procedure 3 times and also note the time in minutes.
- Then calculate the flow rate by using the formula (flow rate = volume in ml/time in minutes).
- Again perform the above process at flow rates of 2.0 ml/min and 3.0 ml/min. Calculate flow rate.
Acceptance criteria: The limit for flow rate accuracy should be within ±2.0%.
For a flow rate of 1.0 ml/min, the value should be within 0.98-1.02 ml/min.
For flow rate 2.0 ml/min, value should be within 1.96-2.01 ml/min.
For a flow rate of 3.0 ml/min, the value should be within 2.94-3.06 ml/min.
2. Flow Rate Consistency:
- Materials required are HPLC-grade water, HPLC-grade methanol, a C-18 or C-8 column, caffeine IPRS, an analytical weighing balance, 50 mL and 100 mL volumetric flasks, a 10 mL pipette, a beaker, and a measuring cylinder.
- At first, prepare the mobile phase by mixing HPLC-grade water and methanol in a 1:1 ratio. Filter and sonicate for 10-15 minutes.
- Now weigh about 50 mg of caffeine IPRS in a 50 mL volumetric flask. Dissolve by adding 10 mL of methanol and make up volume with mobile phase, which gives a stock solution of 1000 ppm.
- To prepare a 10 ppm solution, dilute 1.0 mL of 1000 ppm into a 100 mL volumetric flask with mobile phase.
- Fill a vial with mobile phase as a blank, and prepare a 10 ppm standard solution in another 6 vials and label them.
- Set the chromatographic system as described in the table:
|
Mobile Phase |
Methanol: Water
(1:1 ratio) |
|
Flow rate |
1.0 ml/min |
|
Column |
C-18 or C-8 |
|
Injection volume |
20 µL |
|
Detector wavelength |
272 nm |
|
Run time |
10 minutes |
|
Caffeine peak |
RT at about 5
minutes |
- Now allow the system to stabilize. Inject the blank solution and then 6 replicates of the 10 ppm caffeine standard solution.
|
Injection-01 |
Blank solution |
|
Injection-02 |
10 ppm caffeine standard solution-01 |
|
Injection-03 |
10 ppm caffeine standard solution-02 |
|
Injection-04 |
10 ppm caffeine standard solution-03 |
|
Injection-05 |
10 ppm caffeine standard solution-04 |
|
Injection-06 |
10 ppm caffeine standard solution-05 |
|
Injection 07 |
10 ppm caffeine standard solution-06 |
Acceptance criteria: The % RSD of the retention time of the caffeine peak should not be more than 1.0%.
|
Caffeine
10 ppm std. |
Retention
time of caffeine peak |
|
Injection-01 |
5.103 |
|
Injection-02 |
5.109 |
|
Injection-03 |
5.108 |
|
Injection-04 |
5.104 |
|
Injection-05 |
5.101 |
|
Injection-06 |
5.111 |
|
Average |
5.106 |
|
%RSD |
0.08 |
3. Gradient profile:
In isocratic mode, the same composition of mobile phase remains during the complete analysis. In gradient mode, the composition of the solvent changes over time during analysis. It checks the ability of the system to deliver the correct mobile phase composition at the correct time.
- Materials required are a mobile phase reservoir, a graduated pipette, a beaker, and a dead volume connector.
- Remove the column from the system and connect the dead volume connector.
- Prepare a 0.25% v/v solution of acetone in water by adding 2.5 ml of acetone stoution in 1000 ml of HPLC-grade water.
- Put channels A and B in HPLC-grade water and channels C and D in 0.25% v/v solution of acetone in water.
- Purge all ports to remove the air bubbles from all tubes.
- Set the flow rate at 1.0 ml/min. Keep 25% composition for each channel.
- Allow the system to stabilize for a few minutes.
- Flush all the channels at a flow rate of 1.0 ml/min for 20 minutes using the composition as shown
|
Time
(minute)
|
Channel A&B in HPLC-grade water |
Channel
C & D in 0.25%
v/v acetone in water
|
|
0 |
25%+25% |
25%+25% |
|
10 |
25%+25% |
25%+25% |
|
12 |
50%+50% |
0+0 |
|
20 |
50%+50% |
0+0 |
- Set the chromatographic system as shown
|
Mobile phase: A
& B |
HPLC-grade water |
|
Mobile phase: C
& D |
0.25% v/v solution
of acetone in water |
|
Flow rate |
1.0 ml/minute |
|
Column |
Dead volume
connector |
|
Detector wavelength |
254 nm |
|
Run time |
30 minutes |
|
Injection Delay |
15 minutes |
- Inject 0 µL or the minimum volume of HPLC-grade water and record the gradient profile.
Run the gradient using channels A and C.
|
Time (Minute)
|
HPLC
Grade Water (Channel
A)
|
0.25%
v/v acetone in water (Channel
C)
|
|
0 |
100% |
0% |
|
4 |
100% |
0% |
|
6 |
80% |
20% |
|
10 |
80% |
20% |
|
12 |
60% |
40% |
|
16 |
60% |
40% |
|
18 |
20% |
80% |
|
22 |
20% |
80% |
|
24 |
0% |
100% |
|
28 |
0% |
100% |
|
30 |
100% |
0% |
- Repeat the same gradient using channel combination B and D.
- Print the overlay plot of gradient profile A/C and B/D.
Acceptance criteria: The gradient profile of A/C and B/D should overlay with each other.
Limit for Difference in Absorbance NMT 0.01 AU
Absorbance difference=152.5-151.3 mAU=1.2 mAU=1.2/1000=0.0012 AU
Limit for difference in time: NMT 20 sec
Time difference=15.263-15.253 minutes=0.01 minutes=0.01*60 sec=0.6 seconds
4. Delay volume:
- Re-check the gradient profile performed under compositional accuracy.
- Record the time in minutes taken for the actual first change in absorbance.
- The delay volume of the system can be calculated in terms of ml by subtracting 5 minutes from the actual time in minutes taken for changes in absorbance.
Delay Time=5.950 min
Delay volume= delay time in minutes x flow rate in ml/min=0.9x1.0=0.9 ml delay volume.
Acceptance criteria: The limit for delay volume is NMT 1.0 ml.
5. Injection Volume Accuracy:
- Materials required are HPLC-grade water, an analytical weighing balance, and vials. ls.
- Purge the instrument with HPLC-grade water. Set the chromatographic system as
|
Mobile Phase |
HPLC-grade water |
|
Flow rate |
1.0 ml/minute |
|
Run time |
1.0 minute |
|
Injection volume |
20 µL |
- Fill the HPLC vial with HPLC-grade water. Weigh the initial weight of the vials in grams as W1.
- The density of water is 0.99982 g/ml at 20⁰ C and 0.9970 g/ml at 25⁰ C. So the volume of water is equivalent to the mass of water.
- Inject 20 µL (10 replicates) from the same HPLC vials.
- After completion, remove the vial and weigh the final weight as W2.
- Calculate the average volume by using the formula (W1-W2)*1000/10/10
Acceptance criteria: The average volume of injection should be 20 µL ± 0.4 µL.
6. Injection Volume Precision:
- For injection volume precision, use the same material and chromatographic system as described in flow rate consistency.
- Prepare a 10 ppm solution of caffeine by using the same procedure.
- Inject 6 replicates of the 10 ppm caffeine standard and calculate % RSD.
|
Caffeine
10 ppm std. |
Peak
Area of Caffeine |
|
Injection-01 |
0.3921 |
|
Injection-02 |
0.3945 |
|
Injection-03 |
0.3985 |
|
Injection-04 |
0.3968 |
|
Injection-05 |
0.3974 |
|
Injection-06 |
0.3959 |
|
Average |
0.3921 |
|
%RSD |
0.65 |
Acceptance criteria: The RSD for peak area NMT 1.0%
7. Injection Volume Linearity:
- For injection volume linearity, the materials are the same: 10 ppm solution. Use the same chromatographic system used in injection volume precision and flow rate consistency.
- Inject blank solution first.
- In this process, a 10 ppm standard solution is injected by varying injection volumes such as 5 µL, 10 µL, 20 µL, 50 µL, and 100 µL. The concentration of the standard caffeine is constant, and the injection volume is changed.
- Plot the linearity graph and calculate the value of R-squared.
Acceptance criteria: R-square NLT 0.999
8. Autosampler temperature accuracy:
- Manage one calibrated digital thermometer.
- Set the sample compartment temperature at 40°C. Allow the system to stabilize for 10 minutes.
- After 10 minutes, record the observed temperature using a calibrated probe with a digital thermometer.
- Repeat the same procedure and record the temperature observed at 40 °C, 30 °C, 15 °C, 10 °C, and 5 °C.
Acceptance criteria: The observed temperature should be within ±2°C of the set temperature.
9. Calibration of column oven:
- Take a digital thermometer.
- Set the oven temperature at 60°C. Allow the system to stabilize for 10 minutes. Record the temperature observed.
- Repeat the same procedure and record the temperature at 60°C, 50°C, 30°C, 20°C, and 10°C
Acceptance criteria: The observed temperature should be within ±2°C of the set temperature.
10. Detector Linearity:
- Materials required are HPLC-grade water and methanol, C-18 or C-8 Column, weighing balance, volumetric flask, 50 ml volumetric flask, 3 volumetric flasks of 100 ml, beaker, Caffeine IPRS.
- Prepare mobile phase by mixing a 1:1 ratio of HPLC-grade water and HPLC-grade methanol. Filter and sonicate to remove air bubbles...
- Set the chromatographic conditions as
|
Mobile Phase |
Methanol: Water
(1:1 ratio) |
|
Flow rate |
1.0 ml/min |
|
Column |
C-18 or C-8 |
|
Injection volume |
10 µL |
|
Detector wavelength |
272 nm |
|
Run time |
10 minutes |
|
Caffeine peak |
RT at about 5
minutes |
- Weigh about 50 mg of caffeine IPRS in a 50 mL volumetric flask. Add 10 mL of methanol to dissolve and make up the volume to a 0.01 ppm solution.
- To prepare 1.0 ppm (0.001 mg/ml) Caffeine Standard solution: Dilute 0.1 mL of 1000 ppm stock solution in 100 mL V.F. with mobile phase.
- To prepare 10.0 ppm (0.01 mg/mL) Caffeine Standard Solution: Dilute 1.0 mL of 1000 ppm stock solution in 100 mL V.F. with mobile phase.
- To prepare 100.0 ppm (0.10 mg/ml) caffeine standard solution: Dilute 10.0 ml of 1000 ppm stock solution in 100 ml V.F. with mobile phase.
- Fill the vial with mobile phase as the blank, and prepare 1 ppm, 10 ppm, and 100 ppm solutions in different vials and label them.
- Allow the system to saturate
- Inject a blank solution and then inject a replicate of 10 µL of each standard solution.
- Record the chromatogram and plot the graph.
Acceptance criteria: R-square value NLT 0.999
11. (A) Wavelength Accuracy for Photodiode Array Detectors (PDA):
- Use the same chromatographic system as used in detector linearity.
- Prepareee 10 ppm caffeine standard solution.
- Set the PDA detector wavelength at 200 nm to 400 nm.
- First inject a blank solution, and then inject 20 µL of 10 ppm standard solution for the entire range.
- Record the spectrum and report maxima and minima.
Acceptance criteria:
The maxima should be obtained at 273 nm within ±2 nm...
The maxima should be obtained at 205 nm within ±2 nm
The minima should be obtained at 245 nm within ±2 nm
11. (B) Wavelength accuracy for variable wavelength detectors (VWD):
- Here also a 10 ppm caffeine standard solution is used. Here tests are performed at different standards, i.e.,e.,e., 205 nm, 245 nm,
- Chromatographic conditions are the same as for detector linearity.
- Create 32 acquisition programs with the same parameters but changing the wavelength at intervals of nm each.
- Inject 20 µL of 100 ppm solution of caffeine.
|
Wavelength |
Range |
|
205 nm |
200,201,202,203,204,205,206,207,208,209,210
nm (200 nm to 210 nm) |
|
245 nm |
239,240,241,242,243,244,245,246,247,248,249
nm (239 nm to 249 nm) |
|
273 nm |
269,270,271,272,273,274,275,276,277,278
nm (269 nm to 278 nm) |
- Run the sequences for each wavelength.
- Record the chromatogram and report maxima and minima.
Acceptance criteria are the same as
