1.0 what is the HPLC full form?
Answer: HPLC full form is High performance/ pressure liquid chromatography.
2.0 How many parameter of HPLC Calibration?
Answer: These parameter use in HPLC Calibration given below:
1.0 Calibration of pump
1.1 Flow rate Accuracy
1.2 Flow rate Precision
1.3 Gradient Composition
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2.0 Calibration of Auto sampler /Auto injector
2.1 Accuracy of Auto injector
2.2 Precision and linearity of Auto injector
2.3 Accurate position of vials in Auto Sampler
2.4 Carry over Test
2.5 Auto sampler Temperature calibration
3.0 Calibration of column oven compartment
3.1 Column oven calibration
4.0 Calibration of Detector
4.1 Noise and Drift test
4.2 Linearity of detector response
4.3 Wavelength Accuracy
3.0 What is the formula to determined flow rate accuracy and its acceptance criteria?
Answer: Determine the flow rate using the formula
Flow rate formula = Weight of water(mg/gm.) X Density Correction Factor of water / Time (in minutes/sec)
And its Acceptance criteria are :
Flow 0.5 millilitre/minutes (± 2 % of Set Flow) flow limit 0.49 to 0.51 ml.
Flow 1.0 millilitre/minutes (± 2 % of Set Flow) flow limit 0.98 to 1.02 ml.
Flow 2.0 millilitre/minutes (± 2 % of Set Flow) flow limit 1.96 to 2.04 ml.
4.0 How to determined flow rate precision and its acceptance criteria?
Answer: Flow rate precision determination and its acceptance criteria :
Flow rate precision shall be checked while precision and linearity of auto injector test parameter is performed. Upon completion of the precision and linearity of auto injector test, calculate the RSD of the Retention Time of Caffeine (25 ppm) from five replicate injections of 5µl and 100µl.
Acceptance criteria: RSD of retention time of caffeine from the five replicate injections of the 5µl and 100 µl should be NMT 1.0%
5.0 what is the full for of GPV?
Answer: GPV full form is Gradient propositioning valve.
6.0 How to calibrate GPV parameter in HPLC ant what is the acceptance criteria?
Answer: Determined GPV calibration parameter:
1.0 Gradient composition: Carry out the Gradient composition test as per below mentioned procedure
1.1 Fill the reservoir for A and B line with 100 % Water.
1.2 Fill the reservoir(Bottle) for the Line C and Line D with 0.5% acetone in water.
1.3 Purge all the lines vis. A, B and C, D with water and 0.5% Acetone for 20 minutes (each line 5minutes).
1.4 To prepare for GPV test, connect a tubing of an appropriate length and Restriction capillary in place of column.
1.5 Set flow rate at 2.0mililitre/minute and flush the pump (A 25%, B 25 %, C 25% and D 25%) with water for 15 minutes.
1.6 Set chromatographic conditions as:
| Mobile Phase | : | Channel A & B- Methanol Channel C & D 0.5% acetone in water |
| Flow Rate | : | 2.0ml/minute |
| Column Details | : | Restriction capillary |
| Injection Volume | : | 0.0µl |
| Run Time | : | 18.0 minutes |
Gradient profile shall be as
| Time | Flow rate (ml/min) | %A | %B | %C | %D | Curve |
| Initial | 2.000 | 50 | 50 | 0 | 0 | 11 |
| 2.00 | 2.000 | 0 | 0 | 50 | 50 | 11 |
| 6.00 | 2.000 | 50 | 50 | 0 | 0 | 11 |
| 10.00 | 2.000 | 45 | 45 | 10 | 0 | 11 |
| 12.00 | 2.000 | 50 | 50 | 0 | 0 | 11 |
| 14.00 | 2.000 | 45 | 45 | 0 | 10 | 11 |
| 16.00 | 2.000 | 50 | 50 | 0 | 0 | 11 |
| 18.00 | 2.000 | 50 | 50 | 0 | 0 | 11 |
1.7 Prepare sequence and save, run the sequence for gradient calibration.
1.8 After completion of run, calculate the gradient composition, name the peak as full scale, A/B/C and A/B/D in order.
Calculation Gradient propositioning valve (GPV) % for A/B/C and A/B/D in order using following formula:
1.9 Gradient propositioning valve (GPV) (For A/B/C) %= Height/Full scale (Height) X 100=……………. %
1.10 Gradient propositioning valve (GPV) (For A/B/D) % = Height/Full scale (Height) X 100=…………… %
1.11 Acceptance Criteria for GPV is 9.0 % to 11.0 %
7.0 How to calibrate auto sampler / Auto injector parameter in HPLC ant what is the acceptance criteria?
Answer: Determined auto sampler / Auto injector calibration parameter:
1.0 Calibration of auto sampler / Auto injector
Accuracy of Auto injector: Carry out the accuracy of auto injector test as per below mentioned procedure:
1.1 Set the temperature of the sample compartment 25°C.
1.2 Place four vial with cap and septa, filled with water in auto sampler, to check the accuracy of auto injector by injecting different injection volume from each vial.
1.3 Set the chromatographic condition of the HPLC system for determination as given below.
Chromatographic conditions:
| Column | : | C18 50 x 4.6mm, 3.5µ or Equivalent |
| Mobile phase | : | Methanol: Filtered and degassed water from Ultra-Pure water system / purified water (30: 70 v/v) |
| Flow rate | : | 1.0ml/min |
| Wave length | : | 273nm |
| Column oven temperature | : | 35°C |
| Auto-sampler Temp | : | 25°C |
| Run Time | : | 1.0 min |
Inject varying the injection volume from the vial as per table-1 below and weigh the vial before injecting (W1 g) and after injecting (W2 g) at every step. Table -1
Step Injection Volume (µl) No. of injection Acceptable Criteria
| Step | Injection Volume (µl) | No. of injection | Acceptable Criteria |
| 1 | 10 | 10 | 10 ± 1 µl |
| 2 | 20 | 10 | 20 ± 1 µl |
| 3 | 50 | 6 | 50 ± 1 µl |
| 4 | 100 | 5 | 100 ± 2 µl |
Injection volume in µl shall be calculated as
= (W1g – W2g) x 1000 / (Number of injection) x Density of water at 25°C
Note: Density of water at 25°C is 0.99704.
Acceptance criteria as per table-1 above.
8.0 How to perform calibration of auto Precision and Linearity of Auto Injector in HPLC and what is the acceptance criteria?
Answer: Determined of Precision and Linearity of Auto Injector calibration parameter in HPLC:-
1.0 Precision and Linearity of Auto Injector: This test shall be carried out as below mentioned procedure
1.1 Caffeine standard solution i.e. solution 1, solution 2, solution 3, solution 4 and solution 5 shall be prepared as per below mention procedure, Caffeine std solution 3 shall be used for Precision and linearity of Auto injector test however remaining shall be used for determination of “Linearity of detector response ‘and carry over test.
1.2 Preparation of standard solution (1000ppm): Weigh accurately about 25mg of Caffeine in 25ml volumetric flask dissolve and dilute to volume with mobile phase.
1.3 Preparation of solution 1(100 ppm): Dilute 10.0ml of standard solution to 100.0 millilitre with mobile phase.
1.4 Preparation of solution 2(50 ppm): Dilute 25.0millilitre of solution 1 to 50.0 millilitre with mobile phase.
1.5 Preparation of solution 3(25 ppm): Dilute 25ml of solution 2 to 50 ml with mobile phase.
1.6 Preparation of solution 4(10 ppm): Dilute 10ml of solution 1 to 100 millilitre with mobile phase.
1.7 Preparation of solution 5(5ppm): Dilute 25millilitre of solution 4 to 50 millilitre with mobile phase.
1.8 Preparation of solution 6(250 ppm): Dilute 5mililitre of standard solution to 20mililitre with mobile phase.
1.9 Prepare the mp. as Methanol and water (Ultra-pure water / purified water/water for injection) (30: 70 v/v). Filter and Degas. Purge the tubing properly.
1.10 Set the chromatographic conditions as mentioned below table.
Chromatographic conditions:
| Column | : | C18 50 x 4.6mm, 3.5µ or Equivalent |
| Flow rate | : | 1.0ml/min |
| Wave length | : | 273nm |
| Auto-sampler Temp | : | 15°C |
| Column oven temperature | : | 35°C |
| Run Time | : | 5.0 min |
1.11 Stabilize the system for 10-15 mins, purge the auto sampler for 5 minutes and inject the 5µl (five times), 10µl, 20µl, 30µl and 50µl (duplicate) and 100µl (five times) of the standard solution 3, inject blank solution for each level.
1.12 Calculate the average peak area of caffeine from the all the injections at each level in HPLC
1.13 Plot a linearity graph between injection volume on the x- axis and average area of Caffeine at each level on the y-axis.
Acceptance criteria:
• RSD of peak areas of Caffeine from the five replicate injections of the 5µl and 100µl should be Not more than 2.0%.
• The linearity coefficient ‘r’ obtained from the linearity graph of caffeine for different levels should be NLT 0.999.
8.0 How to perform calibration of Accurate position of vials in Auto sampler in HPLC?
Answer: Accurate position of vials in Auto sampler
1.0 Accurate position of vials in Auto sampler: Carry out the Accurate position vial in Auto sampler as per below mentioned procedure:
1.1 Use ultra-pure water as a sample and run the sample for one minute.
1.2 Prepare a new sequence for the above test, Place the vials at different positions in auto sampler as mentioned below table as per availability of number of vials in trays.
1.3 Always use new septa for above test.
For vial RACK A
| Position of Vial | 1 | 10 | 15 | 24 |
For vial RACK B
| Position of Vial | 25 | 35 | 40 | 48 |
For vial RACK C
| Position of Vial | 50 | 60 | 65 | 72 |
For vial RACK D
| Position of Vial | 75 | 85 | 90 | 96 |
For vial RACK E
| Position of Vial | 100 | 110 | 115 | 120 |
Set the chromatographic conditions as mentioned below:
Chromatographic condition:
| Column | : | C18 50 x 4.6mm, 3.5µ or Equivalent |
| Mobile phase | : | Methanol: Filtered and degassed water (Ultra-Pure water / purified water) (30: 70 v/v) |
| Flow rate | : | 1.0ml/min |
| Wave length | : | 273nm |
| Auto-sampler Temp | : | 25°C |
| Column oven temperature | : | 25°C |
| Run Time | : | 1.0 min |
| Injection Volume | : | 10µl |
1.4 Cross checks vial position from the chromatogram reports & sequence report and the septa must be penetrate by needle of Auto Injector.
Acceptance criteria
• The auto injector should be inject the sample from the given position of Vial from each Rack as mentioned in above table.
• The septa of each vial must be penetrating by the needle of auto injector in HPLC.