Capillary Electrophoresis Calibration Protocol

INSTRUMENT	:	Capillary Electrophoresis
Refer SOP No.	:
Model No.	:	7100
Instrument ID. No.	:
Date of Calibration	:
Make	:	Agilent
Frequency	:	Half yearly – 15 Days
Next Due Date	:

1. Injection Linearity Test (Set I ):

a. Inject 4-Hydroxy acetophenone solution 1 mM/L single injection and check the area.

b. Method Parameters (DAD):

Wave length: 325 nm

Band width: 10

Peak Width: >0.025 min (0.5 s response time 10Hz)

Stop time: As CE

Post Time: Off

Advanced:

Spectrum: None

Auto analog output:

Zero Off Set: 5%

Attenuation: 1000 mAU

Indirect UV margin for negative absorbance: 100 mAU

Auto balance: Pre run

Lamps on required for acquisition: UV lamp

c. Method Parameters (CE):

Vials:

Inlet Home: 04 Buffer

Outlet home: 05 Buffer

Cassette temperature: 20°C

High voltage system (Enable high voltage)

Voltage: 0.0 KV (positive polarity)

Current: 300 µA

Power: 6.0w

Low current alarm limit: off

Stop time: 4.0 min

Post time: Off

Replenishment: parallel

Preconditioning:

Function: Flush

Parameter: 60s (inlet: 6 outlet: 11)

Injection:

Function: Apply pressure

Parameter: 25 mbar for 3s (inlet – injection vial , outlet : outlet home vial)

Time table:

Time: 0.2

Function: change voltage

Parameter: 30 kV

Post Conditioning: Not applicable

d. Instrument curves:

Leak current: enable

2. Injection Linearity Test (Set II):

a. Inject 4-Hydroxy acetophenone solution 1 mM/L single injection and check the area.

b. Method Parameters (DAD):

Wave length: 325 nm

Band width: 10

Peak Width: >0.025 min (0.5 s response time 10Hz)

Stop time: As CE

Post Time: Off

Advanced:

Spectrum: None

Analog output

Zero Offset: 5%

Attenuation: 1000 mAU

Indirect UV margin for negative absorbance: 100 mAU

Auto balance: Pre run

Lamps on required for acquisition: UV lamp

c. Method Parameters (CE):

Vials:

Inlet Home: 04 buffer

Outlet home: 05 buffer

Cassette temperature: 20°C

High voltage system (Enable high voltage)

Voltage: 0.0 KV (positive polarity)

Current: 300 µA

Power: 6.0w

Low current alarm limit – off

Stop time: 4.0 min

Post time: 0.0 min

Replenishment: parallel

Preconditioning:

Function: Flush

Parameter: 60s (inlet: 6 outlet: 11)

Injection

Function: Apply pressure

Parameter: 50 mbar for 3s (inlet – injection vial, outlet: outlet home vial)

Time table

Time: 0.2

Function: change voltage

Parameter: 30 kV

Post Conditioning: Not Applicable

Instrument curves:

Leak current: enable

3. Injection Linearity Test (Set III):

a. Inject 4-Hydroxy acetophenone solution 1 mM/L single injection and check the area.

b. Method Parameters (DAD):

Wave length: 325 nm

Band width: 10

Peak Width: >0.025 min (0.5 s response time 10Hz)

Stop time: As CE

Post Time: Off

Advanced:

Spectrum: None

Analog output:

Zero Offset: 5%

Attenuation: 1000 mAU

Indirect UV margin for negative absorbance: 100 mAU

Auto balance: Pre run

Lamps on required for acquisition: UV lamp

c. Method Parameters (CE):

Vials:

Inlet Home – 04 buffer

Outlet home – 05 buffer

Cassette temperature – 20°C

High voltage system (Enable high voltage)

Voltage: 0.0 KV (positive polarity)

Current: 300 µA

Power: 6.0w

Low current alarm limit: off

Stop time: 4.0 min

Post time: Off

Replenishment: parallel

Preconditioning:

Function: Flush

Parameter: 60s (inlet: 6 outlet: 11)

Injection

Function: Apply pressure

Parameter: 50 mbar for 5s (inlet – injection vial, outlet: outlet home vial)

Time table

Time: 0.2

Function: change voltage

Parameter: 30 kV

Post Conditioning: Not Applicable

d. Instrument curves

Leak current: enable

4. Injection Linearity Test (Set IV):

a. Inject 4-Hydroxy acetophenone solution 1 mM/L single injection and check the area.

b. Method Parameters (DAD):

Wave length: 325 nm

Band width: 10

Peak Width: >0.025 min (0.5 s response time 10Hz)

Stop time: As CE

Post Time: Off

Advanced:

Spectrum: None

Analog output

Zero Offset: 5%

Attenuation: 1000 mAU

Indirect UV margin for negative absorbance: 100 mAU

Auto balance: Pre run

Lamps on required for acquisition: UV lamp

c. Method Parameters (CE):

Vials:

Inlet Home: 04 Buffer

Outlet home: 05 buffer

Cassette temperature: 20°C

High voltage system (Enable high voltage)

Voltage: 0.0 Kv (positive polarity)

Current: 300 µA

Power: 6.0w

Low current alarm limit: off

Stop time: 4.0 min

Post time: Off

Replenishment: parallel

Preconditioning:

Function: Flush

Parameter: 60s (inlet: 6 outlet: 11)

Injection

Function: Apply pressure

Parameter: 100 mbar for 5s (inlet – injection vial , outlet : outlet home vial)

Time table

Time: 0.2

Function: change voltage

Parameter: 30 kV

Post conditioning: Not Applicable

d. Instrument curves:

Leak current: enable

Origin	Migration Time Minutes	Area mAU*s	Actual Injection pressure mbar*s	Corrected Area mAU
Injection Linearity Set I
Injection Linearity Set II
Injection Linearity Set III
Injection Linearity Set IV

Result of Correlation coefficient

(Correlation coefficient: NLT 0.9900)

Note: Calculate the Correlation coefficient for injection pressure Vs corrected area

Remarks: Results found satisfactory / not satisfactory.

Analyzed By: Checked By:

5. Injection precision test:

a. Use 4-hydroxy acetophenone standard solution shall be used for calibration of injection precision test.

b. Inject 1 mM/L 4-hydroxy acetophenone standard solution 8 injections (first two injections used for system equilibration) and record the data.

c. Method parameters (DAD):

Wavelength: 325 nm

Band width: 10.0

Peak width: >0.10 min (2.0 s response time 2.5 Hz)

Stop time: As CE

Post Time: Off

Advanced (Spectrum)

Store: All in peak

Range: 190.0 to 450.0 nm

Step: 1.0 nm

Threshold: 1.0mAU

Analog out put

Zero off set: 5 %

Attenuation: 1000 mAU

Indirect UV margin for negative absorbance: 100 mAU

Auto balance: Pre run

Lamps on required for acquisition: UV lamp

d. Method Parameters (CE):

Vials:

Inlet Home: 04 buffer

Outlet home: 05 buffer

Cassette temperature: 20°C

High voltage system (Enable high voltage)

Voltage: 0.0 KV (positive polarity)

Current: 300 µA

Power: 6.0w

Low current alarm limit: off

Stop time: 4.0 min

Post time: Off

Replenishment: Serial

Preconditioning: Not applicable

Injection

Function: Apply pressure

Parameter: 50 mbar for 5s (inlet – injection vial, outlet: outlet home vial)

Time table

Time: 0.2

Function: change voltage

Parameter: 30 kV

Post conditioning: Not applicable

e. Instrument curves

Leak current – enable

Injections	Migration Time Minutes	Area mAU*s	Corrected Area mAU
Injection 1
Injection 2
Injection 3
Injection 4
Injection 5
Injection 6
Result
Acceptance Criteria	(%RSD: NMT 2.0%)	(%RSD: NMT 3.0%)

Remarks: Results found satisfactory / not satisfactory.

Analyzed By: Checked By:

6. Noise and Drift Test:

a. Inject the blank vial (empty vial) one injection as per the procedure.

b. Method parameters (DAD):

Wavelength: 254 nm

Band width: 4.0

Peak width: >0.05 min (1.0 s response time 5 Hz)

Stop time: As CE

Post Time: Off

Advanced (Spectrum)

Store: None

Analog out put

Zero off set: 5 %

Attenuation: 1000 mAU

Indirect UV margin for negative absorbance: 100 mAU

Auto balance: Pre run

Lamps on required for acquisition: UV lamp

c. Method Parameters (CE):

Vials:

Inlet Home: 04 buffer

Outlet home: 05 buffer

Cassette temperature – 20°C

High voltage system (Enable high voltage)

Voltage: 0.0 KV (positive polarity)

Current: 300 µA

Power: 6.0w

Low current alarm limit: off

Stop time: 11.00 min

Post time: Off

Replenishment: Parallel

Preconditioning: Not applicable

Injection: Not Applicable

Time table: Not Applicable

Post conditioning: Not Applicable

d. Instrument curves:

Leak current – Enable

Cassette temperature – Enable

Noise mAU	Drift mAU/Hr
Result:	Result:
Acceptance Criteria: NMT 0.05 mAU	Acceptance Criteria: NMT 2.00 mAU/Hr

Remarks: Results found satisfactory / not satisfactory.

Analyzed By: Checked By:

7. Response Linearity:

a. Use 4-hydroxy acetophenone standard solution shall be used for calibration of Response Linearity test.

b. Inject 0.1, 0.5, 1.0, 5.0 mM/L 4-hydroxy acetophenone standard solution, single injection each and record the data.

c. Method parameters (DAD):

Wavelength: 325 nm

Band width: 10.0

Peak width: >0.025 min (0.5 s response time 10 Hz)

Stop time: As CE

Post Time: Off

Advanced (Spectrum)

Store: None

Analog out put

Zero off set: 5 %

Attenuation: 1000 mAU

Indirect UV margin for negative absorbance: 100 mAU

Auto balance: Pre run

Lamps on required for acquisition: UV lamp

d. Method Parameters (CE):

Vials:

Inlet Home: 04 buffer

Outlet home: 05 buffer

Cassette temperature: 20°C

High voltage system (Enable high voltage)

Voltage: 0.0 KV (positive polarity)

Current: 300 µA

Power: 6.0w

Low current alarm limit: off

Stop time: 4.0 min

Post time: Off

Replenishment: Parallel

Preconditioning

Function: Flush

Parameter: 60s (Inlet: 6 outlet: 11)

Injection

Function: Apply pressure

Parameter: 50 mbar for 5s (inlet – injection vial, outlet: outlet home vial)

Time table

Time: 0.2

Function: change voltage

Parameter: 30 kV

Post conditioning: Not Applicable

e. Instrument curves:

Leak current: enable

Origin	Migration Time minutes	*Area mAUs**	Actual Concentration(mM)	Corrected Area
Run 1			0.1
Run 2			0.5
Run 3			1.0
Run 4			5.0
Result
Acceptance Criteria	Correlation coefficient NLT 0.9900

Note: Calculate the Correlation coefficient for concentration Vs corrected area

Remarks: Results found satisfactory / not satisfactory.

Analyzed By: Checked By:

8. Voltage Stability Test(Negative Test):

a. Inject blank empty vial single injection as per the procedure.

b. Method Parameters(DAD):

Wavelength: 325 nm

Band width: 10.0

Peak width: >0.025 min (0.5 s response time 10 Hz)

Stop time: As CE

Post Time: Off

Advanced (Spectrum)

Store: None

Analog out put

Zero off set: 5 %

Attenuation: 1000 mAU

Indirect UV margin for negative absorbance: 100 mAU

Auto balance: Pre run

Lamps on required for acquisition: UV lamp

c. Method Parameters (CE):

Vials:

Inlet Home: 04 buffer

Outlet home: 05 buffer

Cassette temperature: 20°C

High voltage system (Enable high voltage)

Voltage: 0.0 KV (positive polarity)

Current: 300 µA

Power: 6.0w

Low current alarm limit: off

Stop time: 10.0 min

Post time: Off

Replenishment: Parallel

Preconditioning: Not Applicable

Injection: Not Applicable

Time table

Time: 0

Function: change voltage

Parameter: “- 25 kV”

Post conditioning: Not Applicable

d. Instrument curves:

Leak current – enable

Result:

Acceptance Criteria: -25 KV ±1% (- 25.25 to -24.75 KV)

Remarks: Results found satisfactory / not satisfactory.

Analyzed By: Checked By:

9. Voltage Stability Test(Positive Test):

a. Inject blank empty vial single injection as per the procedure.

b. Method Parameters(DAD):

Wavelength – 325 nm

Band width – 10.0

Peak width – >0.025 min (0.5 s response time 10 Hz)

Stop time -As CE

Post Time – Off

Advanced (Spectrum)

Store – None

Analog out put

Zero off set – 5 %

Attenuation – 1000 mAU

Indirect UV margin for negative absorbance – 100 mAU

Auto balance – Pre run

Lamps on required for acquisition – UV lamp

c. Method Parameters (CE):

Vials:

Inlet Home – 04 buffer

Outlet home – 05 buffer

Cassette temperature – 20°C

High voltage system (Enable high voltage)

Voltage – 0.0 KV (positive polarity)

Current – 300 µA

Power – 6.0w

Low current alarm limit – off

Stop time – 10.00 min

Post time – Off

Replenishment – Parallel

Preconditioning: Not Applicable

Injection: Not Applicable

Time table

Time: 0

Function: change voltage

Parameter: “25 kV”

Post conditioning: Not applicable

d. Instrument curves:

Leak current – enable

Result:

Acceptance Criteria: 25 KV ±1% (24.75 to 25.25KV)

Remarks: Results found satisfactory / not satisfactory.

Analyzed By: Checked By:

10. Wave Length Accuracy Test:

a. Use 4-hydroxy acetophenone 1 mM/L for calibration of Wave Length Accuracy Test.

b. Inject single injection of 4-hydroxy acetophenone 1 mM/L standard solution as per the procedure.

c. Calculate the maxima and minima at 236, 258,325 wavelengths.

d. Method Parameters(DAD):

Wavelength: 325 nm

Band width: 10.0

Peak width: >0.025 min (0.5 s response time 10 Hz)

Stop time: As CE

Post Time: Off

Advanced (Spectrum)

Store: All in peak

Range: 190.0 to 450.0 nm

Step: 1.0 nm

Threshold: 1.0mAU

Analog out put

Zero off set: 5 %

Attenuation: 1000 mAU

Indirect UV margin for negative absorbance: 100 mAU

Auto balance: Pre run

Lamps on required for acquisition: UV lamp

e. Method Parameters (CE):

Vials:

Inlet Home – 04 buffer

Outlet home – 05 buffer

Cassette temperature: 20°C

High voltage system (Enable high voltage)

Voltage: 0.0 KV (positive polarity)

Current: 300 µA

Power: 6.0w

Low current alarm limit – off

Stop time: 4.0 min

Post time: Off

Replenishment: Parallel

Preconditioning: Not Applicable

Injection

Function: Apply Pressure

Parameter: 50 mbar for 5s (inlet – injection vial, outlet: outlet home vial)

Time table

Time – 0

Function – change voltage

Parameter – 30 kV

Post conditioning: Not Applicable

f. Instrument curves :

Leak current – enable

Wavelength 1:

Wavelength nm	Area mAU
232
233
234
235
236
237
238
239
240

Result: Maxima Observed at ___________ Wavelength (Acceptance Criteria- 236 ± 2 nm)

Remarks: Results found satisfactory / not satisfactory.

Analyzed By: Checked By:

Wavelength 2:

Wavelength nm	Area mAU
254
255
256
257
258
259
260
261
262

Result: Minima Observed at ___________ Wavelength (Acceptance Criteria- 258 ± 2 nm)

Remarks: Results found satisfactory / not satisfactory.

Analyzed By: Checked By:

Wavelength 3:

Wavelength nm	Area mAU
320
321
322
323
324
325
326
327
328

Result: Maxima Observed at ___________ Wavelength (Acceptance Criteria- 325 ± 2 nm)

Remarks: Results found satisfactory / not satisfactory.

Analyzed By: Checked By:

Remarks: Calibration satisfactory / Not satisfactory

Calibrated By

Signature/Date

Checked By

Signature/Date

Approved By

Signature/Date