HPLC Calibration- A complete Guide – Part 2 of 3

HPLC Calibration Continued from …. A Complete Guide on HPLC Calibration -Part 1 of 3

Part 2 – HPLC Calibration on different Parameter starts from Energy check and completed on wavelength accuracy of Detector

In the previous article on HPLC Calibration we have covered following parameters.

  • Pressure Test.
  • Drift and Noise
  • Column oven and sample cooler
  • Pump by flow rate accuracy measurement.
  • Pump by gradient flow measurement.

A Complete Guide on HPLC Calibration – Part 2 of 3 (Continued…)

6.  HPLC Calibration : UV-Vis / PDA detector by reference energy check.

  • Calibration requirements: None
  • Mobile phase preparation: HPLC grade water or Methanol
  • System requirements: NA
  • Sample/standard preparation: NA
  • Calibration procedure:
  • Flush the system with mobile phase either HPLC grade water or HPLC grade methanol.
  • Check the reference energy for lamp in UV-Visible /PDA detector lamp at 225 nm.
Sr No Detector Observed Value Limit
1 UV-Visible NLT 15 nA
2 PDA NLT 10000 counts
  • General Note:
  • Lamp shall be replace, when reference energy of UV-Visible/ PDA detector lamp at 225 nm is less 15 nA/10000 counts respectively or fails to ignite or when the reference energy level of the lamp cause a decrease in sensitivity to the point where the baseline is too noisy and/or replacements of the lamp after one year interval, which ever is earlier.

*Reset the lamp hours counter to zero (0) at the time of replacement of lamp.

7.  HPLC Calibration : UV-VIS/PDA detector by linearity measurement.

  • Calibration requirements:
    • Caffeine
    • Balance
    • Water HPLC grade or equivalent
    • Acetonitrile HPLC grade.
  • Mobile phase preparation: Water and Acetonitrile (85 : 15)
  • System requirements:
Testing conditions Applied conditions
Column: C18 (25 cm x 4.6 mm), 5m Make-Hichrom or Merck or equivalent Column No.:
Flow rate: 1 ml/min
Wave length: 272 nm
Injection volume: 20ml
Run time: 10 min (Retention time: About 6.0 min)
Column/Sample cooler temperature
  • Sample/standard preparation:
    • Prepare five concentration solution of caffeine, which are 25 ppm, 50 ppm, 100 ppm, 150 ppm, and 250 ppm.
    • Preparation of stock solution : Take 100.0 mg weight of caffeine in 100 ml volumetric flask, dissolve it and dilute up to mark with HPLC grade water. Further dilute this solution to prepare the other standard solution as following (Section 4.3 to 4.7). (1000 ppm)
    • 25 ppm solution : Take 5 ml from stock solution with pipette in to 200 ml volumetric flask and dilute up to the mark with HPLC grade water.
    • 50 ppm solution : Take 5 ml from stock solution with pipette in to 100 ml volumetric flask and dilute up to the mark with HPLC grade water.
    • 100 ppm solution : Take 10 ml from stock solution with pipette in to 100 ml volumetric flask and dilute up to the mark with HPLC grade water.
    • 150 ppm solution : Take 15 ml from stock solution with pipette in to 100 ml volumetric flask and dilute up to the mark with HPLC grade water.
    • 250 ppm solution : Take 25 ml from stock solution with pipette in to 100 ml volumetric flask and dilute up to the mar with HPLC grade water.
  • Calibration procedure.
    • Set up the instrument as per the testing conditions.
    • Ensure that, the column is conditioned before injecting the sample.
    • Inject the all five linearity standard solution in duplicate and record the area of principal peak in observation table.
    • Plot a linearity curve of concentrations Vs corresponding mean area, using least square method. Calculate the squared correlation coefficient (r2) , and record the observations in observation table.
Observational table:
Sr. no Caffeine std

Conc.

Actual

Conc.

Area Mean Area Squared correlation coefficient (r2 = NLT 0.99)
1 25 ppm
2 50 ppm
3 100 ppm Intercept :NMT 3.0 % of 100 ppm area
4 150 ppm
5 250 ppm

Acceptance criteria :

  • Linearity – squared correlation coefficient (r2) = NLT0.99
  • Y intercept should be NMT 3.0% of 100 ppm area.

8.  HPLC Calibration : Auto sampler by carry over check.

  • Calibration requirements:
    • Caffeine
    • Balance
    • HPLC grade water.
    • HPLC grade Acetonitrile (ACN).
  • Mobile phase preparation:
    • Water: ACN (85:15) Mix 850 ml of HPLC grade water and 150 ml of acetonitrile and filter through 0.45 micron nylon filter, degas for 10 min by sonicator.
  • System requirements:

            Note: Needle should not be rinsed in this study.

Testing conditions Applied conditions
Column: C18 (25 cm x 4.6 mm), 5m Make – Hichrom or equivalent Column No.:
Flow rate : 1.0 ml/min
Wavelength: 272 nm
Injection volume: 20 ml
Run time: 10 min (Retention time: About 6.0 min)
Column/Sample cooler temperature
  • Sample/standard preparation:
    • Sample preparation(Conc. 1000 ppm):Weigh accurately  100 mg of caffeine and transfer it in 100 ml clean and dried volumetric flask, dissolve in and dilute up to the mark with water.
  • Calibration procedure :
    • Setup the HPLC system with the above conditions and keep the flow for 30 minutes to saturate the column with the mobile phase and inject 20 ml water as blank.
    • After getting the baseline from the blank inject the sample in duplicate and record the chromatogram.
    • Again inject 20 ml water as blank, if any peak observed at the principal peak retention time.
    • Calculate the % carryover using formula specified for carryover check and record the observation in observation table.
    • Compare the results obtained for compliance with the limits, as given in observation table.
  • Observation table :
No. of injection RT and area of caffeine in standard Area at RT of caffeine in blank (B) % Carry over Limits
RT Area NMT 0.2%
1
2
Mean area

Calculation: Calculate the % Carryover using following formula:

%Carryover = {Area at RT of Caffeine in Blank ( B)/ Mean Area of sample preparation (A) } *100

Acceptance criteria : % Carryover should be NMT 0.2%.

9.  HPLC Calibration : Auto sampler by linearity check.

  • Calibration requirements:
    • Caffeine
    • Balance
    • HPLC grade water or equivalent.
    • HPLC grade acetonitrile (ACN).
  • Mobile phase preparation (85 :15): Mix 850 ml of HPLC grade water and 150 ml of acetonitrile and filter through 0.45 micron nylon filter. Degas it for 10 min by sonicator.
  • System requirements:
Testing conditions Applied conditions
Column: C18 (25 cm x 4.6 mm), 5m Make – Hichrom or Equivalent Column No.:
Flow rate : 1.0 ml/min
Injection volume: As per Table – 1
Wavelength: 272 nm
Run time: 10 min (Retention time: About 6.0 min)
Column/Sample cooler temperature NA
  • Sample/standard preparation:
    • Sample preparation (Concentration 15 ppm): Weighed accurately 30 mg of caffeine and transferred it to 100 ml clean and dried volumetric flask, dissolved in and dilute up to the mark with water. Further diluted 5 ml of above solution to 100 ml with water and mixed well.
  • Calibration procedure :
    • Setup the HPLC system as per above Cinematographic conditions and saturate the column with the mobile phase for about 30 minutes.
    • Fill 5 vials with sample solutions respectively and place into vial positions 1, 29, 58, 87, 116 for Waters Alliance system

Note: Exact vial position depends on the shape of sample tray. The ideal selections are given    as following, which should be maintained. If total injection volume is more than the vial capacity then place the vial in next position or previous position.

  • 50 ml capacity loop: Start by injecting 5 ml and proceed with10 ml, 20 ml, 40 ml, and 50 ml from the vials from different locations in five replicate injection.
  • 100 ml capacity loop: Also for 100 ml loop capacity same injection pattern will follow additionally, 100 ml from the vials from different locations in five replicate injection.
  • 200 ml capacity loop: Inject 20 ml, 80 ml, 120 ml, 160 ml, and 200 ml from the vials from different locations in five replicate injection.
  • 500 ml/2000 µl capacity loop: Inject 20 ml, 80 ml, 200 ml, 400 ml, and 500 ml from the vials from different locations in five replicate injection.
  • Record the area due to principal peak. Take the mean area and calculate the % RSD for five replicate injection area and record the results in given the observation table.
  • Plot a linearity curve of concentrations Vs corresponding mean area, using least square method. Calculate the squared correlation coefficient r2, and record the observations.
Observation table
*Loop Capacity Area of Caffeine Squared correlation coefficient

(r2 = NLT 0.99 )

50 µl 5 µl 10 µl 20 µl 40 µl 50 µl
100 µl 5 µl 20 µl 50 µl 80 µl 100 µl
200 µl 20 µl 80 µl 120 µl 160 µl 200 µl
500 µl 20 µl 80 µl 200 µl 400 µl 500 µl
2000 µl 20 µl 80 µl 200 µl 400 µl 500 µl
Run – 1
Run – 2
Run – 3
Run – 4
Run – 5
Mean
%RSD

(NMT 2.0%)

  • based on the capacity of loop size study shall be designed so as to cover minimum and maximum capacity of the loop).

Acceptance criteria : Linearity – squared correlation coefficient (r2) = NLT0.99

10.  HPLC Calibration : UV-VIS/PDA detector by wavelength accuracy measurement.

  • Calibration requirements:
    • Caffeine Code no
    • Balance Code
    • Water HPLC grade or equivalent and Acetonitrile HPLC grade.
  • Mobile phase preparation:
    • Use filtered and degassed HPLC grade methanol as mobile phase.
  • System requirements:
Testing conditions Applied conditions
Column : Hypersil BDS C18 or equivalent (25 cm x 4.6 mm),5m, Column No.:
Flow Rate :1.0 ml/min
Wavelength : 201 nm to 209nm, 240 nm to 248nm 268 nm to 276 nm, for UV and PDA
Injection volume : 20 ml
Run time: 5 min. (10 min for PDA detector)
Column/Sample cooler temperature NA
  • Sample/standard preparation:
  • Prepare the sample for checking of wavelength accuracy by taking cleaned dried 250 ml volumetric flask and take about 50 mg of caffeine, dissolve and dilute up to the mark with HPLC grade methanol, take 10 ml of this solution in 100 ml volumetric flask and dilute up to the mark with HPLC grade methanol.(Conc. about 20 ppm).

                Note: Before calibration clean the system with isopropyl alcohol (IPA) or with the required solvent.

  • Calibration procedure.
    • Set up the instrument as per the given testing conditions (section 3).
    • Ensure that, the column is conditioned before injecting the sample.
    • Run the system for 201 nm to 209 nm, 240 nm to 248 nm and 268 nm to 276 nm by the increment of 1-nm wavelength and record the chromatograms .i.e. prepare the instrument methods with different wavelength if the detector is UV.

Note: In case of PDA detector select the range 201 nm to 209 nm, 240 nm to 248 nm and 268 nm to 276 nm and carryout only one sample single injection. Extract the data of 201 nm to 209 nm, 240 nm to 248 nm and 268 nm to 276 nm by the increment of 1-nm wavelength.

  • Record the observe area of each chromatogram in observation table.
  • Find the highest area for 201 nm to 209 nm, 268 nm to 276 nm and minimum area for 240 nm to 248 nm from the observation table and consider its respective wavelength as actual wavelength.
  • Observation table : Refer SOP for HPLC Calibration

               * Acceptance criteria : Maximum area should be at 205± 2 nm.

              **Acceptance criteria :Minimum area should be at 245 ± 2 nm.

              ***Acceptance criteria : Maximum area should be at 272 ± 2 nm.

Continue to next article : .. A complete Guide on HPLC Calibration -Part 3 of 3

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