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

In the HPLC Calibration – A complete guide article series, we have discussed about Monthly & Quarterly Calibration parameters, rest of the parameters are described below, in the end of the article all the relevant links has mentioned, in this article there are total 3 blogs, for complete Calibration process must read out all three blogs

11.  HPLC Calibration : Auto sampler for RI detector by linearity measurement.

• Calibration requirements:

  • Column : Symmetry C18 , 3.5 µm , (4.6 mm x 7.5 cm) column.
  • Caffeine
  • Balance
  • HPLC grade water or equivalent.
  • HPLC grade Methanol or equivalent.

• Mobile phase preparation:

  • Water : Methanol (75:25) Mix 750 ml of HPLC grade water and 250 ml of methanol and filter through 0.45 micron nylon filter, degas for 10 min by sonicator.

Note: This Auto sampler calibration parameter shall be performed when HPLC system consist only with RI detector.

• System requirements:      

Testing conditions	Applied conditions
Column: Symmetry C18, 3.5 µm, (4.6 mm x 7.5 cm) or equivalent.	Column No.:
Flow rate : 1.0 ml/min
Column oven temperature : 35°C
Flow cell temperature: 35°C
Injection volume : As per table -1
Run time: 5 min (Retention time: About 2.8 min)
Sensitivity for Waters make HPLC
Polarity + (positive)
*Sensitivity :64
*Response: 5
Rinsing solvent: HPLC grade water : methanol (20 :80)

• Diluent preparation:

  • Mix 80 ml of HPLC grade water and 20 ml of methanol and filter through 0.45 micron nylon filter, degas for 10 min by sonicator.

• Sample/standard preparation:

• • Preparation of stock solution (Conc. 2000 ppm):

    • Weigh accurately about 200 mg of caffeine in a clean and dried 10 ml volumetric flask, dissolve in and dilute up to the mark with Diluent. Take 1.0 ml of above solution in 10 ml volumetric flask and dilute up to the mark with diluent.

•     Calibration procedure :

  • Inject the sample preparations in duplicate and record the area of the principal peak in the given table.
  • Plot a linearity curve of Injection volume Vs corresponding mean area, using least square method. Calculate the squared correlation coefficient (r²), and record the observations in given table.

• Observation table:

Sr. no	Injection volume.	Area	Mean Area
1	5 ml
2	10 ml
3	20 ml
4	50 ml
5	100 ml

Note:

Location shall decide the calibration range for loop capacity and injection volume based on there working range for the calibration of Auto sampler.

Acceptance criteria :

Linearity – squared correlation coefficient (r²) = NLT 0.99

12.  HPLC Calibration : RI Detector by linearity measurement.

• Calibration requirements:

  • Column : Symmetry C18 , 3.5 µm , (4.6 mm x 7.5 cm) column.
  • Caffeine
  • Balance
  • HPLC grade water or equivalent.
  • HPLC grade Methanol or equivalent.

• Mobile phase preparation:

  • Water : Methanol (75:25) : Mix 750 ml of HPLC grade water and 250 ml of methanol and filter through 0.45 micron nylon filter, degas for 10 min by sonicator.

Note:

This Auto sampler calibration parameter shall be performed when HPLC system consist only with RI detector.

• System requirements:

Testing conditions	Applied conditions
Column: Symmetry C18, 3.5 µm, (4.6 mm x 7.5 cm) or equivalent.	Column No.:
Flow rate : 1.0 ml/min
Column oven temperature : 35°C
Flow cell temperature: 35°C

Testing conditions	Applied conditions
Injection volume : 20 µl
Run time: 5 min (Retention time: About 2.8 min)
Sensitivity for Waters make HPLC
Polarity + (positive)
*Sensitivity :64
*Response: 5
Rinsing solvent: HPLC grade water : methanol (20 :80)

• Diluent preparation:

  • Mix 80 ml of HPLC grade water and 20 ml of methanol and filter through 0.45m nylon filter, degas for 10 min.

• Sample/standard preparation:

  • Preparation of stock solution (Conc. 1000 ppm) : Weigh accurately about 100.0 mg of caffeine in a clean and dried 100 ml volumetric flask, dissolve in and dilute up to the mark with diluent.

• • 100 ppm-Linearity Solution 1: Dilute 1 ml of stock solution to 10 ml with diluent and mix well.

• • 200 ppm-Linearity Solution 2: Dilute 2 ml of stock solution to 10 ml with diluent and mix well.

• • 400 ppm-Linearity Solution 3: Dilute 4 ml of stock solution to 10 ml with diluent and mix well.

• • 500 ppm-Linearity Solution 4: Dilute 5 ml of stock solution to 10 ml with diluent and mix well.

• • 1000 ppm-Linearity Solution 5: Stock solution of 1000 ppm.

• Calibration procedure :

  • After stabilization of the system and column inject the diluent as blank.
  • Inject the all five linearity preparations in duplicate and record the areas of the principal peak in 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 in given table.
• Observation table: Refer SOP for HPLC Calibration

• Acceptance criteria :

  • Linearity – squared correlation coefficient (r²) = NLT 0.99

13.  HPLC Calibration :  fluorescence detector by linearity measurement .

• Calibration requirements:

  • HPLC grade water or equivalent.
  • Anthracene
  • Column
  • HPLC grade acetonitrile & HPLC grade water or equivalent.
  • Balance

• Mobile phase preparation:

  • Water: ACN ( 30:70): Mix 300 ml of HPLC grade water and 700 ml of acetonitrile and filter through 0.45 micron filters and degas about 10 min by Sonicator.

• System requirements:

Testing conditions	Applied conditions
Column : C18 (3.5 mm,4.6 mm x 75 mm) , Symmetry column or equivalent.	Column No.:
Flow rate : 1.0 ml/min.
Injection volume : 20 ml
Run time :10 min
Wavelength : Ex=249 nm,Em=402 nm
Polarity :+

Testing conditions	Applied conditions
Attenuation : 64
Response : STD
Gain :1, Unit: mv
Bandwidth :18
Column temperature : 30°C

•       Sample/standard preparation:

  • Preparation of stock solution (Conc. 100 ppb):

    • Prepare the standard by weighing, accurately about 50 mg of anthracene and transfer into clean dried 50 ml volumetric flask, dissolve in and dilute up to the mark with acetonitrile (stock solution). Take 1.0 ml of the stock solution in 100 ml volumetric flask and dilute up to the mark with acetonitrile. Further Take 5.0 ml the solution in 500 ml volumetric flask and dilute up to the mark with mobile phase.

  • Linearity Solution Preparation

    • 20 ppb-Linearity Solution 1: Dilute 2 ml of stock solution to 10 ml with mobile phase and mix it well.
    • 40 ppb-Linearity Solution 2: Dilute 4 ml of stock solution to 10 ml with mobile phase and mix it well.
    • 60 ppb-Linearity Solution 3: Dilute 6 ml of stock solution to 10 ml with mobile phase and mix it well.

• • • 80 ppb-Linearity Solution 4: Dilute 8 ml of stock solution to 10 ml with mobile phase and mix it well.
    • 100 ppb-Linearity Solution 5: Stock solution of 100 ppb .

• Calibration procedure

  • Ensure that, the column is conditioned before injecting the solution.
  • Set the instrument as per system requirement.
  • Inject the all five sample preparation in duplicate.
  • Record the area due to anthracene peak. Calculate mean area and record the results in 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 in observation table.

• Acceptance criteria

  • Linearity – squared correlation coefficient (r²) = NLT 0.99

14.  HPLC Calibration : fluorescence detector by wavelength accuracy measurement .

• Calibration requirements:

  • HPLC grade water or equivalent.
  • Column: NA

• Mobile phase preparation: NA

• System requirements:

Table

Parameters for emission wavelength calibration.	Parameter’s Setting for emission wavelength calibration.
Type	Sample scan
Grating to scan	Excitation scan
Scan wavelength range	340 to 360
Emission wavelength	397
Gain	1
EUFS	100000
Pace	100
Mark each nm	Off

 

Table 

Parameters for excitation wavelength calibration.	Parameter’s Setting for excitation wavelength calibration.
Type	Sample scan
Grating to scan	Emission scan
Scan wavelength range	375 to 430
Excitation wavelength	350
Gain	1
EUFS	100000
Pace	100
Mark each nm	Off

• Calibration Procedure:

  • Connect the restricted capillary to Fluorescence detector in place of column.
  • Keep the HPLC grade water in reservoir.
  • Purge the measuring cell of Fluorescence detector with water and allow to run the water through cell about 10 min.
  • Fill the water in measuring cell of Fluorescence detector.
  • Scan the sample (water fill in flow cell) from 340 nm to 360 nm and measure the emission wavelength (for emission wavelength 397 nm ). Record the observation in table.
  • For excitation wavelength calibration scan the sample (filled water in flow cell) from 375 nm to 430 nm and measure the highest peak listed first (for excitation wavelength 350 nm) peak. Record the observation in table.

Calibration Parameter	Acceptance Criteria	Observed value
Emission wavelength	397 ± 3 nm
Excitation wavelength	350 ± 3 nm

Note:   If the excitation wavelength and emission wavelength test fails, then there may be something other than pure water in flow cell or flow cell may be dirty . Therefore rinse the flow cell with water again and repeat the test.

Acceptance criteria :

Emission wavelength 397 ± 3 nm.

Excitation wavelength 350 ± 3 nm.

—————————————————–Completed Part 3 of 3———————————————————

Kindly visit below articles for Complete HPLC Calibration Procedure.

1. First Part
2. Second Part
3. Third Part

In the First article  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.

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

• UV-Vis / PDA detector by reference energy check.
• 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.
• Auto sampler for RI detector by linearity measurement.