Instrument Calibration Protocol of High Performance Liquid Chromatography

Instrument	High Performance Liquid Chromatography
Instrument Code Number
Reference SOP No.
Model No.
Make
Calibration Date
Next Due Date

1. HPLC System Cleaning :

Chromatographic Condition:

Column : Union

Detector : OFF

Flow Rate : 1.0 mL / minute

Injection Volume: 100 µL

a. Solution 1 Preparation: Prepare a solvent mixture for system cleaning (Water: Acetonitrile: Methanol: IPA) (125:125:125:125).

Total Quantity Prepared for Solution 1: _______________ mL.

Solution 1: Water : _____________ mL

Acetonitrile : _____________ mL

Methanol : _____________ mL

Isopropyl Alcohol: _____________ mL

b. Solution 2 Preparation: Take 495 ml of above solvent (Solution – 1) and add 5 ml Orthophosphoric acid. Shake it to mix.

Total Quantity Prepared for Solution 2: _______________ mL.

Solution 2: Solution 1: _________ mL

Orthophosphoric acid. : _____________ mL

Wash the system with Acetonitrile: Water (50: 50) for 30 minutes at 1.0 ml/ minute flow rate.

Acetonitrile: ____________ mL

Water : _______________ mL

Done By: Checked By:

2. Calibration :

Mobile Phase: Methanol HPLC grade.

Chromatographic Condition:

Column : Symmetry C₁₈ 150 x 4.6 mm, 5µ

Detector : UV Detector at 254 nm

Flow Rate : 1.0 mL / minute

Injection Volume : 20 µL

Column Temperature: Ambient (about 25°C)

Sampler Temperature: Ambient

Run Time : 5 minutes

a. Peak Area Reproducibility :

Note: Perform the Peak area reproducibility by using injection volume 5µL.

Standard Preparation: [Analytical Balance ID: _________________ ]

Weight of Uracil Standard: _______ mg

(Lot No. of Uracil __________________)

Solution A: Weigh accurately about 50 mg of Uracil Standard into 250 mL calibrated volumetric Flask. Add 100 mL of HPLC grade Methanol, sonicate to dissolve and make up the Volume with HPLC grade Methanol.

Solution B: Dilute 10 mL of Solution A to 100 mL with HPLC grade Methanol.

Inject Solution B, repeatedly 5 times and find the Tailing Factor and % RSD of

Uracil Standard Area.

Injection No.	Uracil Standard Area	Tailing Factor
1
2
3
4
5
Mean
% RSD		Not Applicable
Limit	NMT 1.0 %	NMT 2.0

b. Retention Time Reproducibility :

Note down the Retention Time of 5 Uracil Standard Injections injected in Peak Area

Reproducibility test and Calculate % RSD for Retention Time.

Injection No.	Retention Time
1
2
3
4
5
Mean
% RSD
Limit	NMT 1.0 %

c. Calibration of Detector:

i. Linearity Test: (For UV and PDA Detector):

Standard Preparation: [Analytical Balance ID: _________________]

Weight of Uracil Standard: _______ mg

(Lot No. of Uracil __________________)

Solution A: Weigh accurately about 25 mg of Uracil Standard into 50 mL calibrated volumetric flask. Add 20 mL of HPLC grade Methanol, sonicate to dissolve and make up the volume with HPLC grade Methanol.

Prepare the 5 solution of different concentration from Solution A;

Solution A	Final Volume	Concentration (in mcg / mL, ppm)
1 ml	100 ml with Methanol HPLC grade	5
2 ml	100 ml with Methanol HPLC grade	10
3 ml	100 ml with Methanol HPLC grade	15
4 ml	100 ml with Methanol HPLC grade	20
5 ml	100 ml with Methanol HPLC grade	25

Inject the above 5 ppm, 10 ppm, 15 ppm, 20 ppm, 25 ppm solution, separately. Find out area of the resulting chromatogram. Plot the Graph Area Vs Concentration.

Concentration (ppm)	Uracil Standard Area (UV)	Uracil Standard Area (PDA)
5
10
15
20
25

Acceptance Criteria: Graph of Area Vs Concentration should be Linear.

Correlation Co – Efficient: UV _____, PDA _______.

[Limit: Not less than 0.999]

Done By: Checked By:

ii. Wavelength Accuracy Test : (For UV Detector):

Standard Preparation: [Analytical Balance ID : _________________ ]

Chromatographic Condition:

Column : Symmetry C₁₈, 150 x 4.6 mm, 5µ

Detector : UV Detector

Flow Rate : 1.0 mL / minute

Injection Volume : 20 µL

Column Temperature: Ambient (about 25°C)

Mobile Phase : Water: Acetonitrile (50: 50)

Sampler Temperature : Ambient

Run Time : 5 Minutes

Note: Perform the UV detector wavelength accuracy test by using injection volume 10µL.

Weight of Caffeine Standard: _______ mg

(Lot No. of Caffeine __________________)

Sample Preparation:

Weigh accurately about 50 mg of Caffeine and dilute to 50 mL with Mobile Phase. Dilute 1 mL of this solution to 100 mL with Mobile Phase (10 ppm) (Solution B).

Inject the Solution B at different wavelength from 200 nm to 210 nm and from 268 nm to 277 nm each.

Report the area at each Wavelength and report the Wavelength having Maximum Area.

Wavelength from 200 nm to 210 nm:

Wavelength (nm)	Caffeine Standard Area
200
201
202
203
204
205
206
207
208
209
210

Observation: Area is Maximum at Wavelength ___________

Acceptance Criteria: Area should be Maximum at a Wavelength of 205 nm ± 2nm.

Wavelength from 268 nm to 277 nm:

Wavelength (nm)	Caffeine Standard Area
268
269
270
271
272
273
274
275
276
277

Observation: Area is Maximum at Wavelength ___________

Acceptance Criteria: Area should be Maximum at a Wavelength of 273 nm ± 2 nm

Done By: Checked By:

iii. Wavelength Accuracy Test : (For PDA Detector):

Chromatographic Condition:

Column : Symmetry C₁₈, 150 x 4.6 mm, 5µ

Detector : PDA Detector (200 nm – 400 nm)

Flow Rate : 1.0 mL / minute

Injection Volume : 20 µL

Column Temperature: Ambient

Sampler Temperature : Ambient

Mobile Phase : Water: Acetonitrile (50: 50)

Run time : 10 minutes

Weight of Caffeine Standard: _______ mg

(Lot No. of Caffeine __________________)

Sample Preparation: [Analytical Balance ID: _________________]

Weigh accurately about 50 mg of Caffeine and dilute to 50 mL with Mobile Phase. Dilute 1 mL of this solution to 100 mL with Mobile Phase (10 ppm) (Solution B).

Make one injection of diluent and one injection of Solution B at Wavelength ranging from 200 nm to 400 nm. Report the Wavelength corresponding to Maximum Area.

Observation: Area is Maximum at Wavelength ___________

Acceptance Criteria:

Area should be Maximum at a Wavelength of 205 nm ± 2nm.

Area should be Maximum at a Wavelength of 273 nm ± 2nm.

Done By: Checked By:

iv. Lamp Energy and Performance Check :

UseMobile Phase as 100 % Methanol.

Observation:

Reference Energy: ____________

Acceptance Criteria: Not Less than 15 nA at 225 nm.

Done By: Checked By:

d. Calibration of Gradient Valve:

[Analytical Balance ID: _________________ ]

Install Union in place of Column and Flush Solvent Lines (A, B, C and D) with

Respective solvents at flow rate of 2.0 mL / min.

Sample Preparation:

Prepare 5.6 mg / L of Propyl Paraben standard in HPLC grade Methanol.

Reservoir A and B: Methanol HPLC grade

Reservoir C and D: 5.6 mg / L of Propyl Paraben standard in HPLC grade

Methanol.

Chromatographic System:

Detector : UV detector at 254 nm

Flow rate : 2.0 mL / minute

Injection Volume : 20 µL

Column Temperature : Ambient (about 25°C)

Sampler Temperature : Ambient

Enter the following Time Programme;

Time	Flow (ml)	% A	% B	% C	% D	Curve
Initial	2.0	50	50	0	0	–
2	2.0	0	0	50	50	11
6	2.0	50	50	0	0	11
10	2.0	45	45	10	0	11
12	2.0	50	50	0	0	11
14	2.0	45	45	0	10	11
16	2.0	50	50	0	0	11
18	0.0	50	50	0	0	11

Observation:

Peak	% GPV
A / B / C
A / B / D

Acceptance Criteria: % GPV must be 10.0 % ± 0.5.

Done By: Checked By:

e. Calibration of Injection Volume [Injector linearity]:

Analytical Balance ID: _________________]

Solution A:

Weigh accurately about 50 mg of Uracil standard into 250 mL calibrated volumetric flask. Add 100 mL of Methanol HPLC, sonicate to dissolve and make up the volume with Methanol HPLC grade.

Solution B:

Dilute 10 ml of Solution [A] to 100 mL with Methanol HPLC grade.

Inject the above solution [B] 2µl, 5µL, 10µL, 20µL, 30µL, 40µL, 50µL, 80µl.

Inject the above solution [B] 5µL, 10µL, 20µL, 30µL, 40µL, 50µL, 80µL, 100µL and 200µL, separately. (For loop volume 200µl)

Inject the above solution [B] 5µL, 10µL, 20µL, 30µL, 40µL, 50µL, 80µl (For loop volume 100µL)

Note: Perform the Injector linearity by Inject the above solution [B] 2 µL, 3µL, 5µL, 8µl, 10µL, 12µL and 15µL separately.

Find out area of the resulting chromatogram and plot the graph area Vs injection volume and find out the regression coefficient.

Injection Number	Injection Volume	Area
1
2
3
4
5
6
7
8
9

Acceptance Criteria: Graph of Area Vs Injection Volume should be Linear.

Correlation Co – efficient: _________

Limit: Not less than 0.999

Done By: Checked By:

f. Calibration of Flow Rate:

[Analytical Balance ID: _________________ ]

First run the instrument 15 minutes by taking water as mobile phase.

Weigh the 50 ml beaker. Select one of the four Solvent lines A, B, C or D. Collect water in beaker up to five minutes for every flow rate 0.3mL / minute. Again weigh the beaker with water. Find out weight of water collected in five minutes in the beaker by subtracting Empty beaker weight. From weight of beaker with mobile phase (water). Convert wt. of water into volume by dividing its density (0.997043) at temperature 25 ± 2°C.

Note: Collect water in beaker up to five minutes for every flow rate 0.3, 0.5, 1.0, 2.0 and 3.0 mL / minute.

Calculate average flow rate:

Repeat the Procedure for flow rate 0.5 ml, 1.0, 2.0 and 3.0 mL / minute.

Observation:

For Solvent Line: _______

For 0.3 ml / minute

Sr. No.	1	2
Wt. of Beaker
Wt. of Beaker + Wt. of water collected in 5 min.
Wt. of water collected in 5 min. (A)
Volume of water collected in 5 min. (A / 0.997043)
Flow Rate / minute

Acceptance Criteria

0.3 ± 0.015 ml / min. (0.285 – 0.315 ml / min)

For 0.5 ml / minute

Sr. No.	1	2
Wt. of Beaker
Wt. of Beaker + Wt. of water collected in 5 min.
Wt. of water collected in 5 min. (A)
Volume of water collected in 5 min. (A / 0.997043)
Flow Rate / minute

Acceptance Criteria

0.5 ± 0.025 ml / min.(0.475 – 0.525 ml / min)

For 1.0 ml / minute

Sr. No.	1	2
Wt. of Beaker
Wt. of Beaker + Wt. of water collected in 5 min.
Wt. of water collected in 5 min. (A)
Volume of water collected in 5 min. (A / 0.997043)
Flow Rate / minute

Acceptance Criteria

1.0 ± 0.05 ml / min.(0.95 – 1.05 ml / min)

For 2.0 ml / minute

Sr. No.	1	2
Wt. of Beaker
Wt. of Beaker + Wt. of water collected in 5 min.
Wt. of water collected in 5 min. (A)
Volume of water collected in 5 min. (A / 0.997043)
Flow Rate / minute

Acceptance Criteria

± 0.10 ml / min. (1.90 – 2.10 ml / min)

For 3.0 ml / minute

Sr. No.	1	2
Wt. of Beaker
Wt. of Beaker + Wt. of water collected in 5 min.
Wt. of water collected in 5 min. (A)
Volume of water collected in 5 min. (A / 0.997043)
Flow Rate / minute

Acceptance Criteria

± 0.15 ml / min. (2.85 – 3.15 ml / min)

Done By: Checked By:

g. Calibration of Oven:

Keep the calibrated temperature sensor in the column oven & close the door.

Set the different temperature 25°C, 30°C, 40°C, 50°C, 60°C and observe the temperature on temperature sensor and LCD screen.

Calibrated Temperature Sensor ID: __________________

Sr. No.	Set Temperature	Observed Temperature on Calibrated Temperature Sensor	Observed Temperature on LCD Screen	Difference
1	25°C
2	30°C
3	40°C
4	50°C
5	60°C

Acceptance Criteria:

Difference should not be more than ± 2 °C of set temperature between calibrated temperature sensor and on LCD screen.

Done By: Checked By:

h. Calibration of Sample Cooler:

Keep the calibrated temperature sensor in the sample compartment and close the door. Set the different temperature 4°C, 15°C and 25°C and observe the temperature on temperature sensor and LCD screen. Wait for some time to achieve the set temperature.

Sr. No.	Set Temperature	Observed Temperature on Calibrated Temperature Sensor	Observed Temperature on LCD Screen	Difference
1	4°C
2	15°C
3	25°C

Acceptance Criteria: Difference should not be more than ± 2 °C of set temperature between calibrated temperature sensor and on LCD screen.

Done By: Checked By:

i. Calibration of Auto Sampler by Carry Over Check:

Chromatographic Condition:

Column : X bridge C₁₈, 50 x 4.6 mm, 3.5µ.

Flow rate : 1.0 mL / minute

Injection Volume : 10 µL

Column Temperature: 40 ºC

Blank : Mobile Phase

Note: The Needle wash time is set to Normal in instrument method where Enhanced kit installed.

[Analytical Balance ID: _________________]

Weight of Caffeine Standard: _______ mg

(Lot No. of Caffeine __________________)

Sample Preparation:

Solution A:

Dilute 1 mL of Solution B in to 100 mL volumetric flask. Dilute up to the volume with mobile phase. Further dilute 1 mL to 100 mL with mobile phase (0.4 ppm) (0.01 % of Solution B).

Solution B:

Weigh accurately 40 mg of Caffeine and transfer in a 10 mL clean and dried volumetric flask dissolve in and dilute up to the mark with mobile phase (4000 ppm).

Procedure:

Prepare two vials with Blank solution and place in position 1 and position 4.

Place vial containing Solution A in position 2 and Solution B in position 3.

Inject 2 replicates of Blank from Vial – 1 (Pre Blank)

Inject 3 replicates of Solution – A from Vial 2.

Make 1 injection of Solution – B from Vial 3.

Inject 3 replicates of Blank from Vial – 4 (Post Blank).

Injection	RT	Area
Pre Blank
Pre Blank
Sample – 1 Solution A
Sample – 2 Solution A
Sample – 3 Solution A

Average of Sample 1 to 3, Solution A

Solution B
Post Blank – 1
Post Blank – 2
Post Blank – 3

Criteria:

Area of peak obtained due to Caffeine in the post blank – 1 should not be more than the average area of Caffeine obtained in the injection of solution A.

Done By: Checked By:

j. Calibration of Fluorescence Detector :

Standard Preparation: [Analytical Balance ID: _________________]

Weight of Anthracene Standard: _______ mg

1. Peak Area Reproducibility:

Injection No.	Anthracene Standard Area
1
2
3
4
5
6
Mean
% RSD
Limit	Not More Than 1.0 %

Done By: Checked By:

2. Raman Signal to Noise Ratio Test :

Observation: ___________

Acceptance Criteria: Not Less Than 800.

Done By: Checked By:

k. Calibration of Refractive Index Detector:

i. Peak Area Reproducibility:

Chromatographic Condition:

Column : Symmetry C₁₈, 75 x 4.6 mm, 3.5 µ

Detector : RI Detector

Flow Rate : 1.0 mL / minute

Injection Volume : 20 µL

Column Temperature: (About 35 °C)

Mobile Phase : Methanol: Water (250: 750)

Sampler Temperature : Ambient

Sensitivity: 64

Run Time: 10 min

Weight of Caffeine Standard: _______ mg

(Lot No. of Caffeine __________________)

Sample Preparation: [Analytical Balance ID: _________________ ]

Solution A:

Weigh accurately about 100 mg of Caffeine Standard into 100 mL calibrated volumetric flask. Add 80 mL of Mobile Phase sonicate to dissolve and make up the volume with mobile phase.

Inject the above solution (Six replicate) separately. Find out Area Sensitivity and calculate the Relative Standard Deviation of the resulting chromatogram.

Injection No.	Caffeine Standard Area
1
2
3
4
5
6
Mean
% RSD
Limit	NMT 1.0 %

Done By: Checked By:

ii. Injection Linearity:

Inject the above solution 5 µL, 10 µL, 20 µL ,50 µL, 80 µL, 100 µL, 200 µL separately. Find out area of the resulting chromatogram. Plot the Graph Area Vs Injection Volume.

Injection Volume	Caffeine Standard Area
5
10
20
50
80
100
200

Acceptance Criteria: Graph of Area Vs Injection Volume should be Linear.

Correlation co-efficient: _______.

[Limit: Not less than 0.999]

Done By: Checked By:

iii. Peak Area Sensitivity % RSD:

Inject the 20 µL Solution A at sensitivity 16, 32, 64, 128 and 256. Calculate the % RSD of Area Sensitivity.

Sample Name	Area	Area Sensitivity	% RSD
Sensitivity 16
Sensitivity 32
Sensitivity 64
Sensitivity 128
Sensitivity 256

Acceptance Criteria: Not More Than 2.0 %.

Done By: Checked By:

1. UV Detector Noise and Drift Test :

Mobile Phase:

Instrument	Solvent line	—
e2695	A, B	100 % HPLC grade Water
—	C, D	100 % HPLC grade methanol
—	Seal Wash	70:30 HPLC grade Water/ Methanol
—	Needle wash	100 % HPLC grade Methanol

Chromatographic Condition:

Column :	XBridge C18 4.6 X 50 mm, 3.5 µm
Detector	UV Detector at 254 nm
Flow Rate	(1.0 mL / minute)
Injection Volume	(0.0 µL )
Column Temperature	(35°C ±2°C)
Run time	(15.0 min)
Sampler Temperature	(Ambient )

Isocratic Gradient program:

Time	Flow (mL/min)	%A	%C	Curve
	1	70	30	–
10.00	1	70	30	6
120.00	1	70	30	6
121.00	0	70	30	6

Event:

—	Time(min)	Event	Action	Channel
1	120.00	Lamp	Off	Channel A

Sample set: Create the sample set as mentioned below:

Sr. No.	Inj. Vol.(µl)	Number of Inj.	Sample Name	Function	Run Time (min)
Equilibration	—	—	—		10.00
Purge Injector	—	—	—		6.50
Equilibration	—	—	—		15.00
1	0.0	1	Blank_ Noise_ Drift Test	Inject immediate standard	15.00

Run the sequence with created sample set.

Processing method:

Noise and drift parameter settings in processing method as follows:

Parameters	Settings
Calculate Noise and Drift	Selected
Start Time (min)	3.00
Stop time (min)	15.00
Segment Width (sec)	30

Confirm the integration parameters by integrating all the channels and save the processing method. Process and print the result from preview publisher in ‘Detector Noise Drift Report’ report method

Process and print the result from preview publisher in ‘Detector Noise Drift Report’ report method

Sr. No.	Test	Observation	Limit	Remark (Pass/Fail)
1.	Detector noise (µAU)		NMT 60µAU
2.	Drift (mAU/Hr)		NMT 10mAU/Hr

Acceptance Criteria: Detector Noise : NMT 60 µAU

Drift : NMT 10 mAU/Hr

Done By: Checked By:

m. PDA Detector Noise and Drift Test:

Chromatographic Condition:

Inject mobile phase as blank with following chromatographic condition:

Column: Hypersil BDS C18, 100 mm X 4.6 mm, 3µm.

Mobile phase : Milli-Q Water: Acetonitrile (85:15 %v/v)

To create an Instrument method: Create instrument method as per following parameters:

Flow rate: 1.0 mL/min

Wavelength: 254 nm

Sampling rate: 2 point/sec

Column oven temperature: 30°C ±2°C

For 2998 detector and other advanced version of PDA detector select and tick (√) on Interpolate 656 nm and 370 nm line regions.

Sr. No.	Test	Observation	Limit	Remark (Pass/Fail)
1.	Detector noise (µAU)		<80µAU
2.	Drift (mAU/Hr)		<10mAU/Hr

Acceptance Criteria:

Detector noise : <80 µAU

Drift : <10 mAU/Hr

Done By: Checked By:

m. Calibration of ELSD Detector :

i. Reproducibility Test:

Acetaminophen standard stock solution:

Weighed ____ (100.0mg) Acetaminophen (Lot. No/B.No ) and transferred accurately into a 100 mL volumetric flask, dissolved in 20 ml of diluent and diluted up to the mark with diluent.

Acetaminophen standard Solution-1 (100ppm):

Transferred (5.0 mL) of Acetaminophen standard stock solution to 50 mL Volumetric flask and dilute to volume with diluent. (Concentration: 100 ppm)

Chromatographic Conditions:

Analytical column : Xterra MSC18, 100x 3.0mm, 5µm or equivalent

Mobile phase : Water: Methanol (90:10 v/v)

Flow rate : 1.0 mL/min

Injection volume : 10µl

Detector gain : 50

Data rate : 5

Nebulizer heating : 59 %

Time constant : 1.000 Sec

Drift tube : 50°C

Gas pressure : 25 psi

Column Temperature : 35°C

Sample temperature : 5°C

Run time : 5.0 min

Diluent : Milli-Q water or equivalent

Nebulizer : High flow or low flow

Injection No.	RT of Acetaminophen	Peak area of Acetaminophen
1
2
3
4
5
6
Mean
% RSD
Limit	NMT 3.0%	NMT 5.0%

Remarks: Results Complies/Does not Complies.

Done By: Checked By:

ii. Detector Linearity Test:

Acetaminophen standard stock solution:

Weighed ____ (100.0mg) Acetaminophen (Lot. No/B.No ) and transferred accurately into a 100 mL volumetric flask, dissolved in 20 ml of diluent and diluted up to the mark with diluent.

Acetaminophen standard Solution-1(100ppm):

Transferred (5.0 mL) of Acetaminophen standard stock solution to 50 mL

Volumetric flask and dilute to volume with diluent. (Concentration: 100 ppm)

Chromatographic Conditions:

Analytical column : Xterra MSC18, 100x 3.0mm, 5µm or equivalent

Mobile phase : Water: Methanol (90:10 v/v)

Flow rate : 1.0 mL/min

Injection volume : 10µl

Detector gain : 50, 100, 200, 500, 800, 1000

Data rate : 5

Nebulizer heating : 59 %

Time constant : 1.000 Sec

Drift tube : 50°C

Gas pressure : 25 psi

Column Temperature : 35°C

Sample temperature : 5°C

Run time : 5.0 min

Diluent : Milli-Q water or equivalent

Nebulizer : High flow or low flow

Detector Gain	Peak area of Acetaminophen	Gain value
50
100
200
500
800
1000

Acceptance Criteria: Graph of Peak area of Acetaminophen Vs Gain value Should be Linear.

Correlation co-efficient: _______. [Limit: Not less than 0.99]

Remarks: Calibration Satisfactory / Not Satisfactory

—	Prepared By	Checked By	Approved By
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