Instrument Calibration Protocol Ultra Performance Liquid Chromatography (UPLC)

INSTRUMENT	Ultra Performance Liquid Chromatography
Instrument ID. No.
Ref. SOP No.
Model No.
Calibration frequency	Half Yearly “-15 days
Date of calibration
Next due date

UPLC System Cleaning :

Chromatographic Condition :

Column : Union

: OFF

Flow Rate : 1.0 ml/minute

Injection Volume : 10 µL

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

Solution 2 Preparation:

Take 450 ml of above solvent (Solution – 1) and add 50 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

UPLC System Cleaning done: Satisfactory / Not Satisfactory.

UPLC Calibration :

1. Peak Area Reproducibility :

Chromatographic System :

Column: Acquity UPLC HSS T 100 x 2.1 mm, 1.8µ [Column No. : __ ]

Detector : UV detector at 273 nm

Flow rate : 0.4 ml/minute

Injection Volume : 3µl

Column Temperature : 40°C

Mobile phase : Acetonitrile: Water (70:30) HPLC grade.

Sample Preparation : [Analytical Balance ID : _________________ ]

Weigh accurately about 100 mg of Caffeine Standard into 100 ml Calibrated volumetric flask. Add 50 ml of Mobile phase, sonicate to dissolve and make up the volume with Mobile phase. – Solution [A]

Weight of Caffeine Standard: _______ mg

(Lot No. of Caffeine __________________)

Dilute 1 ml of above Solution [A] to 100 ml with Mobile phase. Solution [B] (10 ppm

Inject solution [B], 5 times repeatedly and find out the tailing factor and % RSD of area.

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

2. Retention Time Reproducibility:

Note down the retention time of injections injected in Peak Area Reproducibility and calculate % RSD.

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

3. Calibration of Detector : [ Analytical Balance ID : _________________ ]

Detector Linearity : (For TUV Detector)

Chromatographic System :

Column: Acquity UPLC HSS T 100 x 2.1 mm, 1.8µ [Column No.: ______]

Detector : UV detector at 273 nm

Flow rate : 0.4 ml/minute

Injection Volume : 3µl

Column Temperature : 40°C

Mobile phase : Acetonitrile: Water (70:30) HPLC grade.

Weight of Caffeine Standard: _______ mg Lot No. of Caffeine__________________)

Weigh accurately about 25 mg of Caffeine Standard into 50 ml calibrated volumetric flask. Add 20 ml of mobile phase, sonicate to dissolve and make up the volume with Mobile phase. – Solution [A].

Volume of Solution [A]	Final Volume	Conc. (ppm)

Inject the above 5 ppm, 10 ppm, 15 ppm, 20 ppm, 25 ppm solution separately. Find out area of the

area of the resulting chromatogram. Plot the graph Area Vs Concentration.

Concentration (ppm)	Area (TUV)
5
10
15
20
25

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

Correlation Co – efficient : TUV _____ [Limit : Not less than 0.999]

4. Wavelength Accuracy Test : [Analytical Balance ID : ___________ ]

For TUV Detector :

Chromatographic Condition :

Column: Acquity UPLC HSS T 100 x 2.1 mm, 1.8µ [Column No. :_____]

Detector : UV detector at 273 nm

Flow rate : 0.4 ml/minute

Injection Volume : 3µl

Column Temperature : 40°C

Mobile phase : Acetonitrile: Water (70: 30) HPLC grade.

Weight of Caffeine Standard : _______ mg (Lot No. of Caffeine : ________________)

Sample Preparation :

Weigh accurately about 100 mg of Caffeine Standard into 100 ml Calibrated volumetric flask. Add 50 ml of Mobile phase, sonicate to dissolve and make up the volume with Mobile phase. – Solution [A].

Dilute 1 ml of above Solution [A] to 100 ml with Mobile phase Solution [B] (10 ppm)

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

277 nm each. Find out the area at each wavelength and report the wavelength having maximum area.

a. 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 ± 2 nm.

b. 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 ± 2nm.

5. Lamp Energy And Performance Check :

Reference energy : _______ nA

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

6. Calibration of Gradient Valve : [Analytical Balance ID : _____________ ]

Install union in place of Column and flush solvent lines (A, B, C & D) with respective solvents at flow rate of 1 ml/min.

Prepare 5.6 mg / l of Propylparaben standard in Methanol HPLC grade.

Mobile phase :

Reservoir A and B : Methanol HPLC Grade

Reservoir C and D : 5.6 mg / lt. of Propyl Paraben standard in Methanol HPLC Grade

Chromatographic System :

Detector : UV detector at 254 nm

Flow Rate : 1.0 ml/minute

Injection Volume : 3µl

Column Temperature : Ambient (about 25°C)

Time	Flow (ml)	% A	% B	% C	% D	Curve
Initial	1.0	50	50	0	0	–
2	1.0	0	0	50	50	11
6	1.0	50	50	0	0	11
10	1.0	45	45	10	0	11
12	1.0	50	50	0	0	11
14	1.0	45	45	0	10	11
16	1.0	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 % ± 0.5.

7. Calibration of Injection volume :

Sample Preparation : [Analytical Balance ID : _________________ ]

Weigh accurately about 100 mg of Caffeine Standard into 100 ml Calibrated volumetric flask. Add 50 ml of Mobile phase, sonicate to dissolve and make up the volume with Mobile phase. – Solution [A]

Dilute 1 ml of above Solution [A] to 100 ml with Mobile phase Solution [B] (10 ppm)

Inject the above solution [B] 1µl, 2µl, 4µl, 6µl, 8µl separately. Find out area of the resulting chromatogram & plot the graph area vs injection volume and find out the regression coefficient.

Injection Number	Injection Volume	Area
1	1 µl
2	2 µl
3	4 µl
4	6 µl
5	8 µl

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

Correlation co-efficient : ______________ [Limit : Not less than 0.999]

8. Calibration of Flow Rate :

First run the instrument 15 minutes by taking water as mobile phase.Take 2 ml Volumetric Flask for 0.1ml flow and 5ml Volumetric Flask for 0.4 ml and 0.6 ml flow. Select one of the four Solvent lines A, B, C or D.Start stopwatch & same time collect water in 2 ml Volumetric Flask up to the meniscus of the flask for flow rate 0.1 ml/minute.Collect water in 5 ml Volumetric Flask for flow rate 0.4 & 0.6 ml/minute.Collect water in 5 ml Volumetric Flask for flow rate 0.4 & 0.6 ml/minute.Calculated Flow rate = Volume of Volumetric Flask / (Measured Time in Second / 60)

Observation:

For Solvent line _________

For 0.1 ml / minute :

Sr. No.	1	2
Calculated Flow

Acceptance Criteria

0.1 ± ml/min (± 2 %). (0.098 – 0.102ml/min)

For 0.4 ml / minute :

Sr. No.	1	2
Calculated Flow

Acceptance Criteria

0.4 ± ml/min (± 2 %). (0.392 – 0.408 ml/min)

For 0.6 ml / minute :

Sr. No.	1	2
Calculated Flow

Acceptance Criteria

0.6 ± ml/min (± 2 %). – 0.612 ml/min)

9. Calibration of Oven :

Keep the calibrated temperature sensor probe in the Column oven.Set the oven with different temperature as per table given below and observe the temperature on calibrated temperature sensor and on LCD screen.

Calibrated temperature sensor ID : __________________

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

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

10. Calibration of Sampler Cooler :Keep the calibrated temperature sensor probe in the Sampler cooler.Set the oven with different temperature as per table given below and observe the temperature on calibrated temperature sensor and on LCD screen

Calibrated temperature sensor ID : __________________

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

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

11. Calibration of Sampler Cooler :Keep the calibrated temperature sensor probe in the Sampler cooler.Set the oven with different temperature as per table given below and observe the temperature on calibrated temperature sensor and on LCD screen

Calibrated temperature sensor ID : __________________

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

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

12. Calibration of Auto Sampler by Carryover Check :

[Balance ID:_____________]

Mobile Phase : Solvent Line A and C: 100 % Water.

Solvent Line B and D: 100 % Acetonitrile.

Rinsing Solvent and Needle Wash : Water : Acetonitrile (90 : 10)

Chromatographic Condition :

Column: Acquity UPLC BEH C18, 50 x 2.1 mm, 1.7 µm [Column No. : ____ ]

Detector : UV detector

Injection Volume : 5.0 µL

Column Temperature: 40°C

Run Time : 1 minute and for challenge injection 11 minutes.

Detector Wavelength : 273 nm

Gradient Programme :

Time (minutes)	Flow Rate (mL/min)	% A	% B	% C	% D	Curve
0.00	0.6	90.0	10.0	0.0	0.0	—
30.00	0.6	90.0	10.0	0.0	0.0	11
30.50	0.0	90.0	10.0	0.0	0.0	11

Gradient Programme (For Challenge Injection – 4000 ppm) :

Time (minutes)	Flow Rate (mL/min)	% A	% B	% C	% D	Curve
0.00	0.6	90.0	10.0	0.0	0.0	—
1.00	0.6	90.0	10.0	0.0	0.0	6
1.40	0.6	0.0	100.0	0.0	0.0	6
5.00	0.6	0.0	100.0	0.0	0.0	6
5.50	0.6	90.0	10.0	0.0	0.0	6
40.00	0.0	90.0	10.0	0.0	0.0	11

Sample Preparation :

Weight of Standard: _______ mg (Lot No. of Caffeine : _________________)

Solution A :

Dilute 1 mL of Solution B in to 100 mL volumetric flask. Dilute up to the volume with diluent. Further dilute 1 mL to 200 mL with diluent (0.2 ppm) (0.005 % 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 diluent (4000 ppm).

Setup the UPLC system as per above-mentioned condition and keep the flow for 30 minutes to saturate the column with the mobile phase and 5 µL mobile phase as blank.

Inject two pre blank injections, solution A (0.2 ppm) (Three injection), Challenge injection

(4000 ppm) (One injection), followed by three post blank injection.

Record the result as mentioned in the table.

Injection	RT	Area
Pre Blank – 1
Pre Blank – 2
Solution A (0.2 ppm) – Inj – 1
Solution A (0.2 ppm) – Inj – 2
Solution A (0.2 ppm) – Inj – 3
Mean area of solution A	—
Challenge Injection (4000 ppm)
Post Blank – 1
Post Blank – 2
Post Blank – 3

% Carry Over: ___________

Limits: Not more than 0.005 %

Calculate the % carryover using the formula :

% Carryover

% Carryover = [(Area of Caffeine in Post Blank – 1) x (Standard Concentration) x 100]/[(area of Caffeine peak with Solution (A)) x (Challenge Concentration)]

Remarks : Calibration satisfactory / Not satisfactory

Calibrated By (Sign/ Date)

Checked By (Sign/ Date)