HPLC (High-Performance Liquid Chromatography) calibration means checking and confirming the instrument's performance in order to obtain reliable, precise results. How to Calibrate HPLC: Essential Tips for Reliable Analytical Results. Calibration parameters typically include:
A. Pump
- 1. Flow Rate Accuracy
- 2. Flow Rate Consistensy
- 3. Compositional Accuracy (Gradient Profile)
- 4. Delay Volume of the System
B. Auto sampler
- 5. Injection Volume Accuracy
- 6. Injection Volume Precision
- 7. Injection Linearity
- 8. Autosampler temperature Accuracy
C. Column Compartment
- 9. Columnn Oven
D. Detector
- 10. Detector Linearity
- 11. Wavelength Accuracy
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1. Flow Rate Accuracy:
- Materails required are HPLC grade are, 10 ml volumetric flask, calibrated stop watch.
- At first remove the column from the system.
- Put all channel inlets in reservoirs of HPLC grade water and start purging of all port to remove air bubbles.
- Set the flow rate at 1.0 ml/min. Keep the composition of all channel as 25% (A-25%, B-25%,C-25%,D-25%)
- Allow the system for saturation.
- After sometime collect the HPLC grade water from the column inlet in dry 10.0 ml of volumetric flask.
- Record the time taken to fill up the volumetric flask up to the mark or lower meniscus of flask
- Perform the same procedure 3 times and also note the time in minutes.
- Then calculate the flow rate by using formula (Flow rate= volume in ml/time in minutes).
- Again perform above process at flow rate 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 flow rate 1.0 ml/min, value should be within 0.98-1.02 ml/min.
For flow rate 2.0 ml/min, value shoud be within 1.96-2.01 ml/min.
For flow rate 3.0 ml/min, value should be within 2.94-3.06 ml/min.
2. Flow Rate Consistency:
- Material required are HPLC grade water, HPLC grade methanol, C-18 or C-8 Column, Caffeine IPRS, analytical weighing balance, 50 ml, 100 ml volumetric flask, 10 ml pipette, beaker, Measuring cylinder.
- At first prepare the mobile phase by mixing HPLC garde water and methanol in 1:1 ratio. Filter and sonicate for 10-15 minutes.
- Now weigh about 50 mg of caffeine IPRS in 50 ml volumetric flask. Dissolve by adding 10 ml of methanol and make up volume with mobile phase which give stock solution of 1000 ppm.
- To prepare 10 ppm concentration of solution, dilute 1.0 ml of 1000 ppm into 100 ml volumetric flask with mobile phase.
- Fill mobile phase in a vial as blank and 10 ppm standard solution in another 6 vial and label.
- 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 replicate of caffeine 10 ppm 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 |
|
Injection07 |
10 ppm caffeine standard solution-06 |
Acceptace criteria: The % RSD of retention time of 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 same compostion of mobile phase remain during complete analysis. In gradient mode the composition of solvent changed over the time during analysis. It check the ability of system to deliver the correct mobile phase composition at correct time.
- Materials required are mobile phase reservoir, graduated pipette, beaker, dead volume connecter.
- Remove the column from the system and connect the dead volume connecter.
- Prepare 0.25% v/vsolution of acetone in water by adding 2.5 ml of acetone solution in 1000 ml HPLC grade water.
- Put channel A and B in HPLC grade water and Channel C and D in 0.25% v/v solution of acetone in water.
- Purge the all ports to rmove the air bubbles from all tubes.
- Set the flow rate at 1.0 ml/min. Keep 25% composition for each channels.
- Allow the system to stabilize for few minutes.
- Flush all the channels at flow rate of 1.0 ml/min for 20 minutes using 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
connecter |
|
Detector wavelength |
254 nm |
|
Run time |
30 minutes |
|
Injection Delay |
15 minutes |
- Injet 0 µL or minimum volume of HPLC grade water andrecord the gradient profile.
Run the gradient using channel 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 changein absorbance.
- The delay volume of the sysytem can be calculated in terms of ml by subtracting 5 minutes from the actual time in minutes taken for changes in absorbance.
Delay Time=5.95.0 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 NMT 1.0 ml
5. Injection Volume Accuracy:
- Materials requird are HPLC grade water, analyical weighing balance, Vials.
- Purge the instrumnet 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 |
20µL |
- Fill the HPLC vial wit HPLC grade water. Weigh the initial weight of vials in gram as W1.
- The density of water is 0.99982 g/ml at 20⁰ C and 0.9970 g/ml at 25⁰ C. So volume of water is equivalent to mass of water.
- Inject 20µL in 10 replicates of injections from the same HPLC vials.
- After completion remove the vial and weighthe final weight as W2.
- Calculate the average volume by using formula: (W1-W2)*1000/10
Acceptance criteria: The average volume of injection should be 20µL±0.4 µL
6. Injection Volume Precision:
- For injection volume presicion use same material, chromatographic system as described in Flow rate consistency.
- Prepare 10 ppm solution of caffeine by using same procedure.
- Inject 6 replicate of 10 ppm caffeine standard and calulate % 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 RSDfor peak area NMT 1.0%
7. Injection Volume Linearity:
- For injection volum linearity also materials are same, 10 ppm solution. Use same chromatographic system used in injection volume precision and flow rate consistency.
- Inject blank solution at first.
- In this process 10 ppm standard solution is injected by varying injection volumes such as 5µL, 10µL, 20µL, 50µL, 100µL. Concentration of caffeine standard is constant and injection volume is changed.
- Plot the linearity graph and calculate the value of R-square.
Acceptance criteria: R-square NLT 0.999
8. Autosampler tempeature accuracy:
- Manage one calibrated digital thermometer.
- Set the sample compartment temperature at 40⁰ C. Allow the system to stabilize for 10 minutes.
- After 10 minutes record the observed temperature using a calibrated probe with digital thermometer.
- Repeat the same procedure and record temprature observed at 40⁰C, 30⁰C, 15⁰C, 10⁰C, 5⁰C.
Acceptance critria: The observed temperature should be within ±2⁰C of the set temperature.
9. Calibration of column oven:
- Take digital thermometer.
- Set the oven temperature at 60⁰C. Allow the system to stabilise for 10 miutes. Record the temperaure observed.
- Repeat the same procedure and record the temperature at 60⁰C,50⁰C,30⁰C,20⁰C,10⁰C
Acceptance critria: The observed temperature should be within ±2⁰C of the set temperature.
10. Detector Linearity:
- Materials required are HPLC grade water and methanol, C-18 or C-8 Column, weighing balance,voumetric flask, 50 ml volumetric flask, 3 volumetric flask of 100 ml, beaker, Caffeine IPRS.
- Prepare mobile phase by mixing 1:1 ratio of HPLC grade water and HPLC grade methanol. Filter and sonicate to remove air bubble.
- Set the chromatographic condition 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 50 ml volumetric flask. Add 10 ml of methanol to dissolve and make up volume upto mark which is 1000 ppm solution.
- To prepare 1.0 ppm (0.001 mg/ml) Caffeine Standard solution:Diute 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:Diute 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:Diute 10.0 ml of 1000 ppm stock solution in 100 ml V.F. with mobile phase.
- Fill the mobel phase in vial as blank and 1 ppm,10 ppm,100 ppm solution in different vials and label.
- Allow the systm for saturation.
- Inject blank solution and then inject replicate 10 µL of each standard soluiton.
- Record the chromatogram and plot the graph.
Acceptance criteria: R-square value NLT 0.999
11. (A) Wavelngth Accuracy for Phooto diode array Detectors (PDA):
- Use same chromatographic system as used in detecto linearity.
- Prepar 10 ppm caffeine standard solution.
- Set the PDA detector wavelength at 200 nm to 400 nm.
- First inject blank solution and then inject 20 µL of 10 ppm standard solution for all 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) Wave length accuracy for variable wavelength detectors (VWD):
- Here also 10 ppm caffeine standard solution is used. Here test are performed at different standard i.e. 205 nm, 245 nm, 273 nm.
- Chromatograpic condition is same as detector linearity.
- Create 32 acquistion programs with same parameters but changing wavelngth at the interval of 1 nm each.
- Inject 20 µL of10 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 is same as above
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