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Standard Operating Procedure (SOP) and Format/Protocol for Calibration of HPLC system (Parameters are Pressure test, Drift and Noise, Flow Rate Accuracy, Autosampler by carryover check,  detector by linearity measurement, etc.)

Calibration of HPLC System

1.0   PURPOSE:

• • The purpose of this SOP is to describe the Procedure for Calibration of high-performance liquid Chromatography in Quality Control.

2.0   SCOPE:

• • This SOP is applicable to the High-Performance Liquid Chromatography system equipped with the following relevant detectors.

• • • UV-Visible Detector

• • • PDA Detector

• • • Refractive Index Detector (RID)

• • • Fluorescence Detector

3.0   REFERENCES:

• • Validation Template of the HPLC system.

• • SOP for  Maintenance of Laboratory Instrument. <Download>

• • SOP for Handling of out of Calibration results.<Download>
  • Operational manuals of Waters HPLC system.<Download>
  • SOP for Preparation of internal and external (Third Party) Calibration schedule and calibration practices. <Download>

4.0   RESPONSIBILITY: (SOP FOR CALIBRATION OF HPLC)

• • The analyst shall be responsible for:

• • Calibration of the HPLC System as per SOP.

• • To perform documentation of the Calibration of the HPLC Systems per the SOP.

• • Quality Control Head or Designee shall be responsible for:

• • To review and approve the SOP for Calibration of HPLC System.

• • Impart training to all concerned persons before the implementation of the HPLC Calibration SOP.

• • To ensure calibration of the instrument as per the SOP.

• • Ensure documentation as per the SOP.

• • To initiate the calibration activity immediately after the maintenance.

• • Quality Assurance shall be responsible for:

• • Check the SOP.

• • To ensure the implementation of the system as per SOP.

• • Quality Head shall be responsible for :

• • To review and approve the SOP.

5.0   ABBREVIATIONS USED IN THE SOP FOR CALIBRATION OF HPLC:

• • AU: Absorption Unit

• • ACN: Acetonitrile

• • EUFS: Emission/Excitation Unit Full Scale

• • GPV: Gradient Proportionality Valve

• • HPLC: High-performance Liquid Chromatography

• • NA: Not Applicable.

• • NLT: Not Less Than

• • nm: Nano Meter

• • NMT: Not More Than

• • PDA Detector: Photo Diode Array Detector

• • ppb: Parts Per Billion

• • ppm: Parts Per Million

• • RI Detector: Refractive Index Detector

• • RSD: Relative Standard Deviation

• • RT: Retention Time

• • UV-VIS Detector: Ultra Violet – Visible Detector

6.0   PROCEDURE FOR CALIBRATION OF HPLC SYSTEM:

• • General Instruction during Calibration of HPLC system :

• • Calibration, operation, cleaning, and precautions shall be done as per the respective instrument operation SOP

• • Each individual module shall be calibrated as per the defined calibration frequency.

• • At the time of calibration enter the details in the respective calibration template and compare the obtained results to the specified limits given in the respective template.

• • If observed any noncompliance, intimate the same to Head QC.

• • After completion of the calibration of the HPLC system submit the report for checking.

• • Update the calibration status of HPLC on the instrument label and fill the respective logs after completion of calibration.

• • Frequency – Calibration of HPLC System :
  • Following calibration parameters of HPLC shall be performed Quarterly ( ±7days)

• • • Pressure Test calibration of HPLC.

• • • Drift and Noise calibration of HPLC.

• • • Column oven and sample cooler calibration of HPLC.

• • • Calibration of Pump by flow rate accuracy measurement in HPLC.

• • • Calibration of the pump by gradient flow measurement in HPLC.

• • • UV-Vis / PDA detector by reference energy check calibration of HPLC.

• • Following calibration parameters of HPLC shall be performed Six-monthly ( ± 15 days ).

• • • UV-VIS/PDA detector by linearity measurement- Calibration of HPLC.

• • • Autosampler by carryover check – Calibration of HPLC.

• • • Autosampler by linearity check – Calibration of HPLC.

• • • UV-VIS/PDA detector by wavelength accuracy measurement – Calibration of HPLC.

• • • Autosampler for RI detector by linearity measurement – Calibration of HPLC.

• • Following calibration parameters of HPLC shall be performed Yearly(±30 days).

• • • RI Detector by linearity measurement – Calibration of HPLC.

• • • Fluorescence detector by linearity measurement – Calibration of HPLC.

• • • Fluorescence detector by wavelength accuracy measurement – Calibration of HPLC.

• • Also, HPLC calibrations parameters should be carried out immediately after the following maintenance.

Sr. No.	Name of the module	Type of Maintenance	Calibration required
1	Pump	Change of plunger assembly/pump seal / Check valve	Flow rate accuracy measurement.
2	Detector	Change of lamp / optical lens	The linearity of the response check and wavelength accuracy check.
3	Autosampler	Change of syringe/sample loop / Rotor	Carryover check and Linearity.
4	Column oven	Replacement of sensor	Calibration of column oven.
5	Sample cooler	Replacement of sensor	Calibration of sample cooler.

Note: Calibration requirement in case of other than above maintenance shall be evaluated case-by-case.

• Calibration Procedure & Acceptance Criteria of HPLC System:

• • Calibration procedure and acceptance criteria are given in respective calibration templates.

Annexure 1: Pressure test Calibration of HPLC:

• • Calibration requirements: None

• • Mobile phase preparation: HPLC grade water or methanol.

• • System requirements: NA

• • Sample/standard preparation: NA

• • Calibration procedure of HPLC System :

• • • The pressure test shall be carried out before pump calibration as per the procedure given below.

• • • Purge the system for each flow line with the mobile phase and use any of the flow lines for the pressure test.

• • • Ensure that the tubing from the reservoir to the column inlet shall be removed.

• • • Plug the outlet of the pump using a dead nut.

• • • Set the flow rate of the pump to 0.1 ml/min.

• • • Observe the pressure until it reaches the maximum psi/kgf.

• • • Repeat the same procedure for further two times and observe the same.

• • • In case the pressure does not reach the maximum and fluctuate at some psi/kgf, implies poor check valve performance or leaks within the pumping system.

• • • After satisfactory completion of the pressure test, proceed for further calibration of other parameters.

• • Verify the results against the acceptance criteria and in case of any non-compliance, inform Head-QC or designee.

Observation table

Sr. No.	Set Flow rate (ml/min)	Pressure observed (Maximum pressure should be observed)	Remarks
1	0.1
2	0.1
3	0.1

Annexure 2: Calibration of HPLC for Drift and Noise

• Calibration requirements:

• • Water HPLC grade

• • Methanol HPLC grade

• Mobile phase preparation:

• • Water: Methanol (70:30)

• • Mix ________ (700 ml) of HPLC grade water and _________ (300 ml) of methanol and filter through _________ (0.45m) nylon filter, degas for _______(10min) by sonication.

• System requirements:

Testing conditions	Applied conditions
Column: C18 (25 cm x 4.6 mm), 5m or equivalent.	Column No.:
Flow rate: 1.0 ml/min
Wavelength For UV: 254 nm
Wavelength For PDA: 254 nm (2D data collection at 3.6 nm bandwidth)
Injection volume: 0.0 ml
Run time: 15 min
Column/Sample cooler temperature	NA

•  Sample/standard preparation : NA
• Calibration procedure :

• • Flush the system with hot water for 15 minutes.

• • Ensure that, the column is conditioned before injecting the sample.

• • After conditioning, the column equilibrates the system for 15 min and then run the blank injection as per test condition for 15 min.

• • After blank injection run completion, process the data using the processing method.

• • Record the results in the observation table.

• Observation table :

Detector type	Peak Noise _µAU (micro AU)	Acceptance criteria of Noise (micro AU)	Drift_mAUHR (milli AU/Hr)	Acceptance criteria of Drift (milli AU/Hr)
For UV Detector		Less than or equal to 60 micro AU		Less than 10 milli AU/Hr
For PDA Detector		Less than or equal to 80 micro AU		Less than 10 milli AU/Hr

 

Acceptance criteria: Noise should be less than or equal to 60 micro AU for UV detector and less than or equal to 80 micro AU for PDA detector. Drift should be less than 10 milli AU/Hr for UV detector and less than 10 milli AU/Hr for PDA detector.

 Annexure 3: Calibration of column oven and sample cooler in HPLC. 

• Calibration requirements:

• • Digital temperature indicator

• Mobile phase preparation: NA
• System requirements:

Testing conditions	Applied conditions
Column temperature: As per the observation table.
Sample cooler temperature: As per the observation table.

• Sample/standard preparation: NA

• Calibration procedure of Drift & Noise in HPLC System:

• • Carry out the calibration for the temperature point at 30°C, 60°C and 15°C using digital temperature indicator.

• • Set the temperature of the column oven at 30°C, keep the temperature probe in the center of the column oven, and allow to stabilize the set temperature for at least 15 to 20 min or more requirement.

• • Check the displayed temperature of the column oven and temperature indicator device and record the reading in the observation table.

• • Similarly, carry out the calibration for 60°C temperature calibration in the same manner and note all the reading in observation table-1.

• • If column oven is available with cooler, set the temperature of column oven at 15°C, keep the temperature probe in the center of column oven, and allow to stabilize the set temperature for at least 15 to 20 min and note the reading in observation table-1.

• • Similarly set the sample cooler temperature at 4.0°C and 25.0°C and repeat the procedure above and record the results in observation table-2.

Note: QC shall decide the calibration range for the column oven and sample cooler temperature based on there working range. Sample cooler temperature calibration shall be applicable for only those system which has sample cooler facility.

• Observation Table :

• Table-1: For Column oven

Sr. No.	Set Temperature	Observed temperature after stabilized			Limits
		5 min	10 min	15 min
		Display of Temp. Indicator
1	30.0 °C				28.0 – 32.0 °C
2	60.0 °C				58.0 – 62.0 °C
3	15.0 °C				13.0 – 17.0 °C
Note: Thermometer used should be calibrated to at least 2 °C of the set temperature

• Table 2: For sample cooler

Sr. No.	Set Temperature	Observed temperature after stabilization			Limits (± 2°C)
		5 min	10 min	15 min
		Display of Temperature indicator
1	4.0°C				2.0 – 6.0 °C
2	25.0°C				23.0 – 27.0 °C
Note: The thermometer used should be calibrated to at least 2°C or better.

Calibration of Pump by flow rate accuracy measurement in the HPLC System.

• • Calibration requirements:

• • Where four inlet lines (A, B, C & D) are mixed into GPV assembly, before the pump (system operated with a single pump).

• • The mixing accuracy of lines is decided by GPV functionality, this is normally measured by GPV test in such case only one-time flow rate accuracy shall be performed.

• • In the case of a binary system where two pumps exist for each individual line then individual pump flow rate calibration shall be done.

Note: QC shall decide the calibration range based on there working range for the calibration of instruments.

• • Calibrated Stopwatch

• • 10 ml volumetric flask

• • Restricted capillary tube (5-meter Length x 0.17 mm ID)

• Mobile phase preparation :

• • HPLC grade water or equivalent (Which is filtered with 0.45-micron filter and degassed).

• System requirements :

Testing conditions	Applied conditions
Flow Rate: 0.5, 1.0, 3.0 ml/min.

• Sample/ standard preparation: NA

• Calibration procedure:

• • Connect the stainless steel tubing, or other restriction devices, in place of column connections with the instrument.

• • Set the vacuum degasser (if installed) to run continuously, or set the sparge rate to 100%.

• • Keep the mobile phase in the respective reservoir.

• • Purge the system for each flow line with filtered HPLC grade water and allow to run the water about 15 min prior to check the flow.

• • Ensure that the tubing from the reservoir to column inlet shall be free from the air bubble and system pressure should be constant.

• • Take 10 ml clean and dried volumetric flask and keep at the restricted capillary’s outlet in such a way that when the pump is on the water drops fall into the volumetric flask

• • Set the pump for 0.5 ml/minute flow-rate, start the pump and allow to run about 10 min.

• • Then keep the flask at restricted capillary’s outlet and then simultaneously start the stopwatch.

• • Stop the Stopwatch when the volume of water reached the mark in the volumetric flask.

• • Derive the time taken and record the observation in the observation table.

• • Repeat the same procedure for the flow rate of 1.0 ml/min and 3.0 ml/min in 10 ml volumetric flask and record the observations in the given table

• • Flowline used for pump calibration A/B/C/D

Sr. No.	Set Flow rate (ml/min)	The volume delivered by the pump (ml)	Theoretical Time (In seconds)	Limits (± 2% of the theoretical limit, seconds)	Actual time (seconds)
1	0.5	10	1200	1176-1224
2	1.0	10	600	588-612
3	3	10	200	196-204

 

Acceptance criteria : The actual time observed should be within ± 2.0% of the theoretical time.

 Annexure 5: Calibration of Pump by gradient flow measurement in HPLC.

• Calibration requirements:

• • Acetone

• • HPLC grade water or equivalent

• • Restricted capillary (5-meter Length x 0.17 mm ID)

• Mobile phase preparation

• • Mobile phase A (A=B if the pump is quaternary):

• • Take HPLC grade water 1-liter filter with 0.45-micron nylon filter is cleaned and dried mobile phase bottle and sonicate it for about 10 min.

• • Note: If the pump is quaternary, mobile phase A is to be kept in reservoir A and B both.

• • Mobile phase B (C=D if the pump is quaternary):

• • Take acetone 5 ml in a clean and dried 1-liter volumetric flask, dilute up to mark with HPLC grade water.

• • Filter it with 0.45-micron nylon filter paper and degas by sonication for about 10 min.

• • Note: If the pump is quaternary, mobile phase B is to be kept in reservoir C and D both.

• System requirements:

Testing conditions	Applied conditions
Column: Restricted capillary	ID No.:
Flow Rate: 2.0 ml/min.
Wavelength: 265 nm
Injection volume: 10 ml
Runtime: 30 minutes for binary (20 min if pump is quaternary)
Column oven/Sample cooler temperature	NA

• Sample/standard preparation:

• • Take HPLC grade water or equivalent as a standard/sample

• Calibration procedure for Gradient Flow Measurement :

  • If the column is attached with the instrument then remove it and connect with the restricted capillary tube.

• • Purge the system with HPLC grade water, which is previously filtered through 0.45m

• • Take the HPLC grade water previously filtered through 0.45m nylon filter and use as mobile phase for Pump-A ( Use for Pump A and Pump-B if the pump is quaternary)

• • Prepare 0.5% v/v solution of Acetone in HPLC Water and filter in 0.45m nylon filter and use as mobile phase for the Pump – B (Pump–C and Pump-D if the pump is quaternary).

• • Set the system gradient flow program as per table-1 for binary and table-2 for the quaternary pump.

• • Inject HPLC grade water or equivalent as a sample for five times and record peak height/area in respective tables 4 and 5.

• • Plot a linearity curve of peak height % (Theoretical) Vs observed peak height (µv) using the least square method.

• • Calculate the squared correlation coefficient (r²) and RSD of peak height and record the observations in the observation table for the binary pump.

• Flow program:

• • Table 1: Gradient flow programming table for the binary pump.

Flow	Time in minutes	% Channel –A	% Channel – B
2.0 ml	0	100	0
2.0 ml	5.0	100	0
2.0 ml	5.5	0	100
2.0 ml	6.0	100	0
2.0 ml	11.0	100	0
2.0 ml	11.5	30	70
2.0 ml	12.0	100	0
2.0 ml	17.0	100	0
2.0 ml	17.5	60	40
2.0 ml	18.0	100	0
2.0 ml	23.0	100	0
2.0 ml	23.5	90	10
2.0 ml	24.0	100	0
2.0 ml	29.0	100	0

• Table 2: Gradient flow programming table for the quaternary pump.

Time	Flow	%A	%B	%C	%D	Curve
0	2.00	50	50	0	0	11
2.00	2.00	0	0	50	50	11
6.00	2.00	50	50	0	0	11
10.00	2.00	45	45	10	0	11
12.00	2.00	50	50	0	0	11
14.00	2.00	45	45	0	10	11
16.00	2.00	50	50	0	0	11
18.00	2.00	50	50	0	0	11

• Table 4: Observation table for the binary pump.

• • Inject three standard trials before starting the actual sequence for system equilibration and also observe the pressure graph.

• • If it is found satisfactory then start the sequence.

Sr. No.	Peak Height (100%) Between 5.0 to 6.0 minutes	Peak Height (70%) Between 11.0 to 12.0 minutes	Peak Height (40%) Between 17.0 to 18.0 minutes	Peak Height (10%) Between 23.0 to 24.0 minutes	Linearity the squared correlation coefficient (r2) ( r2 = NLT0.99)
1.
2.
3.
4.
5.
Mean
%RSD (NMT2.0%)

• Table 5 Observation table for the quaternary pump.

• • Inject three standard trials before starting the actual sequence for system equilibration and also observed pressure graph. if it is found satisfactory than start the sequence.

Sr. No.	Peak Height (A + B) (100%) at Approx. 6 minutes	Peak Height (C) (10%) at Approx. 12 minutes	Peak Height (D) (10%) at Approx. 16 minutes
1.
2.
3.
4.
5.
Mean Þ
%RSD Þ (NMT 2.0%)

• Calculation :

• (Mean of second peak height/Mean of first peak height) X 100 =

____________ X 100 = _________% (Limit: Between 9.5% and 10.5%)

• (Mean of third peak height/Mean of first peak height) X 100 =

____________ X 100 = _________% (Limit: Between 9.5% and 10.5%)

Acceptance criteria Linearity – squared correlation coefficient (r2 ) = NLT 0.99 for binary. %RSD of peak height obtained for five replicate injections of the sample should be NMT 2.0% for quaternary.

Annexure 6: Calibration of UV-Visible / PDA detector by reference energy of the HPLC lamp.

• • Calibration requirements: None

• • Mobile phase preparation: HPLC grade water or Methanol

• • System requirements: NA

• • Sample/standard preparation: NA

• • Calibration procedure:

• • Flush the system with the mobile phase either HPLC grade water or HPLC grade methanol.

• • Check the reference energy for the lamp in the 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:

• • The lamp shall be replaced,
    • 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 causes a decrease in sensitivity to the point where the baseline is too noisy and/or replacements of the lamp after the one-year interval, whichever is earlier.

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

Acceptance criteria: Observed reading for reference energy of lamp for UV -Visible detector should be NLT 15 nA and for PDA detector NLT 10000 counts.

Annexure 7: Calibration of Autosampler by carryover check for HPLC System.

• • Calibration requirements:

• • • Column

• • • Caffeine

• • • Analytical Balance

• • • HPLC grade water.

• • • HPLC grade Acetonitrile (ACN).

• • Mobile phase preparation for Calibration of HPLC for Carryover Check:

• • Water: ACN (85:15): Mix  850 ml of HPLC grade water and 150 ml of acetonitrile and filter through 0.45m 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: 10min (Retention time: About 6.0 min)
Column/Sample cooler temperature	NA

• • 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 for carryover in the HPLC system:

• • 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 µl water as blank.

• • After getting the baseline from the blank inject the sample in duplicate and record the chromatogram.

• • Again inject 20 µl water as blank, if any peak observed at the principal peak retention time.

• • Calculate the % carryover using a formula specified for carryover check and record the observation in the observation table.

• • Compare the results obtained for compliance with the limits, as given in the observation table.

• • Observation table :

No. of injection	RT and area of caffeine in a standard		The area at RT of caffeine in the blank (B)	% Carryover	Limits
	RT	Area			NMT 0.2%
1
2
Mean area

• • Calculation: Calculate the % Carryover using the following formula: 

%Carryover = Area at RT of Caffeine in Blank( B) *100

                         Mean Area of sample preparation (A)

Acceptance criteria : % Carryover should be NMT- 0.2%.

Annexure 8: Calibration of UV-VIS/ PDA detector by linearity measurement.

• Calibration requirements:

• • Caffeine

• • Analytical Balance

• • Water HPLC grade or equivalent and Acetonitrile HPLC grade.

• Mobile phase preparation:

• • Water and Acetonitrile (85: 15): Take 850 ml  HPLC grade water or equivalent and 150 ml HPLC grade acetonitrile, filter it through 0.45-micron filter paper and sonicate it for about 10 min.

• 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: 1ml/min
Wavelength: 272 nm
Injection volume: 20ml
Run time: 10 min (Retention time: About 6.0 min)
Column/Sample cooler temperature	NA

• 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 follows. (1000 ppm)

• Preparation of 25 ppm solution :

• • Take 5ml from stock solution with a pipette into 200 ml volumetric flask and dilute up to the mark with HPLC grade water.

• Preparation of 50 ppm solution :

• • Take 5 ml from the stock solution with a pipette into a 100 ml volumetric flask and dilute up to the mark with HPLC grade water.

• Preparation of 100 ppm solution :

• • Take 10 ml from the stock solution with a pipette into a 100 ml volumetric flask and dilute up to the mark with HPLC grade water.

• Preparation of 150 ppm solution :

• • Take 15 ml from stock solution with help of pipette/s into 100 ml volumetric flask and dilute up to the mark with HPLC grade water.

• Preparation of 250 ppm solution :

• • Take 25 ml from stock solution with pipette into 100 ml volumetric flask and dilute up to the mar with HPLC grade water.

• Calibration procedure for Linearity Measurement in HPLC System.

• • Set up the instrument as per the testing conditions.

• • Ensure that, the column is conditioned before injecting the sample.

• • Inject the all five linearities standard solution in duplicate and record the area of the principal peak in the observation table.

• • Plot a linearity curve of concentrations Vs corresponding mean area, using the least square method.

• • Calculate the squared correlation coefficient (r²) , and record the observations in the 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 the 100 ppm area.

 

Annexure 9: Calibration of UV-VIS/ PDA detector by wavelength accuracy measurement in HPLC System

• Calibration requirements:

• • Caffeine

• • Analytical Balance

• • HPLC grade water and Acetonitrile.

• 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 10ml of the above solution in 100ml volumetric flask and dilute up to the mark with HPLC grade methanol.

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

• Calibration procedure of WL accuracy in the HPLC System.

• • Set up the instrument as per the given testing conditions.

• • 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.

• • Record the chromatograms .i.e. prepare the instrument methods with different wavelength if the detector is UV.

Note: In the 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 observed area of each chromatogram in the 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 : 

Wavelength	Area	The wavelength for maximum area	Acceptance criteria
201 nm			The maximum area should be at 205 ± 2 nm
202 nm
203 nm
204 nm
205 nm
206 nm
207 nm
208 nm
209 nm

 

Acceptance criteria : The maximum area should be at 205± 2 nm.

Annexure 10: Calibration of Autosampler by linearity measurement – HPLC System.

• Calibration requirements:

• • Caffeine

• • Analytical 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 a 0.45µ 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 the above solution to 100 ml with water and mixed well.

• Calibration procedure of Autosampler Linearity in HPLC System:

  • Setup the HPLC system as per the above Chromatographic 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 the 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 the next position or previous position.

• For 50 ml capacity loop:

• • Inject 5 ml, 10 ml, 20 ml, 40 ml, and 50 ml from the vials from different locations in five replicate injections.

• For 100 ml capacity loop:

• • Inject 5 ml, 20 ml, 50 ml, 80 ml, and 100 ml from the vials from different locations in five replicate injections.

• For 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 injections.

• For 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 injections.

• • Record the area due to the 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 the least square method.

• • Calculate the squared correlation coefficient r², and record the observations.

Note: Location shall decide the calibration range for loop capacity and injection volume based on there working range for the calibration of Autosampler.

• Observation table: Table-1

*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
2
3
4
5
Mean
%RSD (NMT 2.0%)

 

(* Tick √ whichever is applicable, based on the capacity of loop size study shall be designed so as to cover minimum and the maximum capacity of the loop)

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

Annexure 11: Calibration of RI Detector by linearity measurement (HPLC)

• Calibration requirements:

• • Column : Symmetry C18 , 3.5 µm , (4.6 mm x 7.5 cm)

• • Caffeine

• • Analytical 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 250ml of methanol and filter through 0.45µ nylon filter, degas for 10min by sonicator.

Note: This Autosampler calibration parameter shall be performed when the HPLC system consists only with an 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 20ml of methanol and filter through 0.45m nylon filter, degas for 10min.

• 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.

• Linearity preparation 1 (100 ppm):

• • Dilute______(1 ml) of stock solution to_____(10 ml) with diluent and mix well.

• Linearity preparation 2 (200 ppm):

• • Dilute______(2 ml) of stock solution to_____(10 ml) with diluent and mix well.

• Linearity preparation 3 (400 ppm):

• • Dilute______(4 ml) of stock solution to_____(10 ml) with diluent and mix well.

• Linearity preparation 4 (500 ppm):

• • Dilute______ (5 ml) of stock solution to_____(10 ml) with diluent and mix well.

• Calibration procedure of Linearity of RI Detector – HPLC:

• • Linearity preparation 5 (1000 ppm): Stock solution of 1000 ppm is already given above.

• • After stabilization of the system and column inject the diluent as blank.

• • Inject 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 the least square method.

• • Calculate the squared correlation coefficient (r²) and record the observations in the given table.

• Observation table:

Sr. no	Caffeine std Conc.	Actual conc.	Area	Mean Area	Squared correlation coefficient ( r2 = NLT 0.99)
1	100 ppm
2	200 ppm
3	400 ppm
4	500 ppm
5	1000 ppm

 

Acceptance criteria : Linearity – squared correlation coefficient (r2) = NLT 0.99

Annexure 12: Calibration of fluorescence detector by linearity measurement.

• Calibration requirements:

• • HPLC grade water or equivalent.

• • Anthracene

• • HPLC Column

• • HPLC grade acetonitrile & HPLC grade water or equivalent.

• • Analytical Balance

• Mobile phase preparation:

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

• System requirements:

Testing conditions	Applied conditions
Column : C18 (3.5mm,4.6mm x 75mm) , Symmetry column or equivalent.	Column No.:
Flow rate : 1.0 ml/min.
Injection volume: 20ml
Run time:10min
Wavelength : Ex=249nm,Em=402nm
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 50ml volumetric flask, dissolve in and dilute up to the mark with acetonitrile (stock solution).

• • Take 1.0ml of the stock solution in a 100 ml volumetric flask and dilute up to the mark with acetonitrile.

• • Further, Take 5.0ml of the solution in a 500 ml volumetric flask and dilute up to the mark with the mobile phase.
• Linearity preparation 1 (20 ppb):

• • Dilute 2 ml of stock solution to 10 ml with the mobile phase and mix it well.

• Linearity preparation 2 (40 ppb):

• • Dilute 4 ml of stock solution to 10 ml with the mobile phase and mix it well.

• Linearity preparation 3 (60 ppb):

• • Dilute 6 ml of stock solution to 10 ml with the mobile phase and mix it well.

• Linearity preparation 4 (80 ppb):

• • Dilute 8 ml of stock solution to 10 ml with the mobile phase and mix it well.

• Linearity preparation 5 (100 ppb):

• • The stock solution

• Calibration procedure

• • Ensure that, the column is conditioned before injecting the solution.

• • Set the instrument as per system requirement (Section no. 3).

• • Inject the all five sample preparation in duplicate.

• • Record the area due to the anthracene peak.

• • Calculate the mean area and record the results in the observation table.

• • Plot a linearity curve of concentrations Vs corresponding mean area, using the least square method.

• • Calculate the squared correlation coefficient (r²), and record the observations in the observation table.

• Observation table:

Sr. no	Anthracene std Conc.	Actual conc.	Area	Mean Area	The squared correlation coefficient (r2) (NLT 0.99)
1	20 ppb
2	40 ppb
3	60 ppb
4	80 ppb
5	100 ppb

 

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

Annexure 13: Calibration of fluorescence detector by wavelength accuracy measurement (HPLC).

• Calibration requirements:

• • HPLC grade water or equivalent.

• • Column: NA

• • Mobile phase preparation: NA

• • System requirements:

Table-1

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 – 2

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 the Fluorescence detector in place of the column.

• • Keep the HPLC grade water in a 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 the cell of the Fluorescence detector.

• • Scan the sample (water fill in the flow cell) from 340 nm to 360 nm and measure the emission wavelength (for emission wavelength 397 nm ). Record the observation in the table.

• • For excitation wavelength calibration scan the sample (filled water inflow cell) from 375 nm to 430 nm and measure the highest peak listed first (for excitation wavelength 350 nm) peak. Record the observation in the 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 inflow cell or flow cell may be dirty. Therefore rinse the flow cell with water again and repeat the test.

Acceptance criteria : The emission wavelength of 397 ± 3 nm. The excitation wavelength of 350 ± 3 nm.

 

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