Calibration Of Volumetric Glassware Biology Essay

To calibrate the provided volumetric flask, one mark pipette, graduated pipette and burette by using standard operating procedure.

Procedure:

1. Calibration of volumetric flasks:

Weigh a clean and dry volumetric flask.  Fill the volumes of the volumetric flask upto the mark with recently boiled and cooled distilled water at room temperature and weigh the flask.  Take the dry and clean beaker and collect the water from the volumetric flask. Check the weight of the empty volumetric flask and substracting the weight of the empty volumetric flask from the weight of the volumetric flask with water. Determine the volume in ml by the formula

Acceptance criteria:

Capacity in ml

5

10

25

50

100

200

250

500

1000

Tolerance

(±ml)

Class A

0.02

0.02

0.03

0.04

0.06

0.08

0.1

0.15

0.2

Class B

0.04

0.04

0.06

0.08

0.15

0.2

0.2

0.3

0.8

2. Calibration of one mark pipette or graduated pipette

Clean and dry the one mark or graduated pipette. Fill the volumes of the pipette upto the mark with recently boiled and cooled distilled water at room temperature. Take the dry and clean previously weighed beaker and collect the water from the pipette. Take the weight of beaker with water. Check the weight of water by subsracting the weight of the empty beaker from the weight of the beaker with water.

Determine the volume in ml by the formula

Acceptance criteria:

One mark pipette or graduated pipette

Capacity in ml

1

2

5

10

15

20

25

50

100

Tolerance

(±ml)

Class A

0.006

0.006

0.01

0.02

0.02

0.03

0.03

0.04

0.06

Class B

0.02

0.02

0.03

0.04

0.04

0.05

0.06

0.08

0.12

3. Calibration of burette

Clean and dry the burette. Fill the volumes of the burette upto the mark with recently boiled and cooled distilled water at room temperature. Take the dry and clean previously weighed beaker and collect the water from the pipette. Take the weight of beaker with water. Check the weight of water by subsracting the weight of the empty beaker from the weight of the beaker with water.

Determine the volume in ml by the formula

Acceptance criteria:

Burettes

Capacity in ml

10

25

50

100

Subdivision in ml

0.05

0.05

0.1

0.1

Tolerance

(±ml)

Class A

0.01

0.03

0.05

0.1

Class B

0.02

0.06

0.1

0.2

Calibration of UV/Visible Spectroscopy

Aim: 

To calibrate the provided UV/Visible spectrophotometer by using standard operating procedure.

Procedure:

Ensure that the connections of the instrument are proper.

1. Control of Absorbances:

Potassium dichromate Solution

Dry a quantity of potassium dichromate at 130°C. Weigh accurately about 60 mg of potassium dichromate and transfer into 1000 ml volumetric flask and dissolve in it with minimum quantity of 0.005M H2SO4. Volumes of the flask is make upto the mark with 0.005M H2SO4 to produce 1000 ml. Measure the absorbance of resulting solution at the wavelengths given below the anexture 1. Calculate value of A (1%,1cm) for each wavelength.

Acceptance criteria (anexture 1)

S. No

Wavelengths (nm)

A (1%, 1cm)

Limit

Minimum

Maximum

1

235.0

124.5

122.9 - 126.2

2

257.0

144.0

142.8 - 145.7

3

313.0

48.6

47.0 - 50.3

4

350.0

106.6

105.6 - 108.2

Stringent limits are adopted from IP & BP for acceptance criteria

2. Resolution power:

Prepare the 0.02% v/v solution os toluene in hexane and record the spectrum of a 0.02% v/v solution in the range of 260 nm to 420 nm (check the hexane for transmittance between 260 nm to 420 nm, using water as a blank and use only if transmittance is not less than 97%).

Acceptance criteria

The ratio of the absorbance at the maximum at about 269nm to that at the minimum at about 266nm is not less than 1.5.

3. Limit of Stray Light

Weigh accurately about 1.2 gm of potassium chloride previously dried at 105° for 2 hours, transfer into a 100 ml volumetric flask and dissolve in it with minimum quantity of distilled water. Volumes of the flask make upto the mark with distilled water. Measure absorbance of the above solution at  198, 199, 200, 201, 202 nm using water as blank.

Acceptance criteria

Absorbance is greater than 2.

4. Wavelength accuracy, resolution and baseline flatness (inbuilt test)

Attach printer directly to the instrument (instead of computer).

Go to Þ MODE press F3 key i.e. Maintenance.

Press ‘1’.

Press Start/Stop key.

After screen changes, ensure that nothing is kept in the optical path and press the Start/Stop key again.

Prints will come after all the three tests are over.

Acceptance criteria

Wave length accuracy : at 656.1 ± 0.3 nm and at 486.0 ± 0.3 nm.

Resolution : 1.0 nm or less.

Baseline flatness : ± 0.002Abs.

Calibration of digital polarimeter

Aim: 

To calibrate the provided digital polarimeter by using standard operating procedure.

Procedure:

Ensure that all the connections of the instrument are proper.

Turn ON the main power supply of instrument.

Prepare the different concentrations sucrose solution by using previously dried analytical grade sucrose at 105°C for 1 hour as follows:

S.NO

Concentration of Sucrose

(in %)

Weight taken (in g)

Dilution volume with water (ml)

1

10

10

100

2

20

20

100

3

30

30

100

4

40

40

100

5

50

50

100

Enter the actual concentration with respect to the weight taken in Mode: 1.

Measure the angular rotation for each concentration and calculate percentage relative standard deviation (% RSD) for 5 readings obtained in each concentration.

calculate the correlation coefficient for different concentration and checks reading obtained with annexure for acceptance criteria.

Precautions

Check the temperature of the solution.

Care should be taken to avoid air bubble in the path of light source, While filling polarimeter tube due.

While using tubes with removable end plates fitted with gasket tighten end plate only enough to ensure a leak proof seal between the end plate and the body of the tube.

Liquids and solutions of solid must be clear.

ANNEXURE

S. No

Concentration of Sucrose (in %)

Optical Rotation (µ)

at 25°C

Tolerance limit

Minimum

Maximum

1.

10.0

13.33°

13.18°

13.4°

2.

20.0

26.61°

26.36°

26.80°

3.

30.0

39.86°

39.54°

40.20°

4.

40.0

53.06°

52.72°

53.6°

5.

50.0

66.23°

65.9°

67.00°

  Acceptance criteria for % RSD : Not more than 1.0%

  Acceptance criteria for correlation coefficient: Not less than 0.995

Calibration of automatic potentiometric titrator

Aim:

To calibrate the provided automatic potentiometric titrator by using the standard operating procedure

Procedure:

Ensure that all the connections of the instrument are proper.

Turn ON the instrument and printer.

Calibration of Burette.

Steps:

Clean the inter changeable burette, fill distilled water in reservoir and fill the burette with distilled water from reservoir.

Go to AUX functions and set Eject volume to 0.10, 1.0, 10.0, 20.0 ml.

Quit and then press Eject.

Collect the volume in clean, dry and previously weighed beaker for each ejection. Weigh the beaker and find out the weight of ejected volume of distilled water, further find out the volume. Volume of the ejected distilled water is calculated by the following

Accepatance criteria:

S. No

Volume of ejection (in ml)

Accepatable volume (in ml)

Minimum

Maximum

1

0.10

0.0990

0.1010

2

1.0

0.99

1.01

3

10.0

9.98

10.02

4

20.0

19.97

20.03

Precautions:

Clean the inter changeable burette with acetone after calibration of burette is over.

Calibration of pH meter

Aim:

To calibrate the provided pH meter by using the standard operating procedure

Reagents:

Buffer solution pH 4.0

Buffer solution pH 7.0

Buffer solution pH 9.0

Distilled water

Preparation of buffer solution:

Dissolve the one buffer tablets or powder pack of pH 4.0, 7.0, 9.0 in 100 ml distilled water in a 100 ml volumetric flask.

Procedure:

Ensure that all the connections of the instrument are proper.

Rince the electrode thoroughly with distilled water to remove all traces of storage solution or previous solution and clean the electrode on a soft tissue paper to remove the excess of rinse distilled water.

Setup the electrode stand and fit the pH electrode into it.

Turn ON the instrument.

Set the temperature at 25°C by using temperature adjustable knob.

Dip the electrode in the beaker containing distilled water.

Allow 30 sec for the pH electrode to reach the thermal equilibrium with distilled water.

Adjust the pH meter at pH 7.0 by using calibration knob.

Withdrawn the electrode from beaker containing distilled water and measure the pH of the prepared buffer solution one by one and record it.

Accepatance criteria:

The measured pH should be within ±0.1 pH unit of the specified value for the buffers solution.

S. No

pH of buffer solution

Limits

Minimum

Maximum

1

4

3.996

4.004

2

7

6.993

7.007

3

9

8.991

9.009

Precausion:

After pH measurement wash the electrode with distilled water and clean the electrode with tissue paper.

Calibration of conductometer

Aim:

To calibrate the provided conductometer by using the standard operating procedure

Reagents:

Preparation of 0.01M KCl:

Weigh accurately about 0.7456 gm of potassium chloride, transfer into 100 ml volumetric flask and dissolve in it with minimum quantity of distilled water. Volumes of the flask made upto the marke with distilled water.

Procedure:

Ensure that all the connections of the instrument are proper.

Rince the electrode thoroughly with distilled water to remove all traces of storage solution or previous solution and clean the electrode on a soft tissue paper to remove the excess of rinse distilled water.

Setup the electrode stand and fit the electrode into it.

Turn ON the instrument.

Set the temperature at 25°C, cell constant 1and conductivity range at 2 millisec.

Dip the electrode in the beaker containing 0.01M KCl solution.

Allow 30 sec for the conductivity electrode to reach the thermal equilibrium with 0.01M KCl solution.

Adjust the conductivity meter at conductance 1.412mS/cm by using calibration knob.

Withdrawn the electrode from beaker containing 0.01M KCl solution.

Calibration of refractometer

Objective:                                                                                                                              

To provide a procedure for the calibration of refractometer.

         

PROCEDURE:

Calibrate the apparatus using following solvents;

Name of the liquid

Refractive Index

Temperature

Distilled water

1.3325

20°C ± 0.5°C

Toluene

1.4969

20°C ± 0.5°C

Carbon Tetrachloride

1.4603

20°C ± 0.5°C

Requirement: Tolerance: ± 0.0005

Maintenance / Repairs:

When the instrument do not comply with the requirement / tolerance range specified above, the instrument should be labeled "OUT OF CALIBRATION" and should be repaired / serviced.

Calibration of Gas Chromatography

Aim:

To calibrate the provided Gas Chromatography by standard operating procedure.

Reagent:

Preparation of 1.0% v/v toluene solution in n-Hexane:

Weigh accurately about 1 gm of toluene and transfer in to 100 ml volumetric flask. The volumes of the flask is make upto the mark with n-Hexane.

Procedure:

1. Calibration of carrier Gas flow setting

Select on "tool" at right hand corner of screen on instrument.

Select option utilities.

Touch on column flow calculation, in that set column length, diameter, flow and column oven temperature.

Set the carrier gas pressure by using carrier pressure knob.

2. Calibration of column oven temperature

Insert the calibrated digital thermometer from topside by piercing glass wool.

Set the column oven temperature on instrument to 50°C, 100°C, 200°C and 250°C. Allow to stabilize the set temperature for 5min and record temperature on digital thermometer at respective temperatures and match the recorded temperature with anexture 1.

Acceptance criteria (anexture 1)

S. No

Set temperature on GC

Limits

Minimum

Maximum

1

50°C

48°C

52°C

2

100°C

98°C

102°C

3

200°C

198°C

202°C

4

250°C

248°C

252°C

3. Calibration of detector

Attach the stainless steel packed column ( length 2 M X diameter 1/8 inch) with stationary phase [10 % C-20 M (Ch-W/HP) 80/100].

Set the chromatographic conditions as the followings

Injector temperature : 180°C

Detector temperature : 220°C

Column flow : 2.7 ml/min

Oven temperature : 110°C (Isothermal)

Run Time  : 5 minutes

Inject the blank and then six injections of 1.0% v/v toluene in n-Hexane solution in system and record chromatogram for all injections. Calculate the % RSD for the area of six 1.0 % v/v toluene in n-Hexane solution injections.

Acceptance criteria: The % RSD should not be more than 15.0%

Calibration of HPLC

Objective:

To provide a procedure for the calibration of HPLC.

Procedure:

1. Check point : leakage test (by pressure drop)

1. Ensure that, the instrument is ready for calibration and Start-up procedure is followed.

2. Place inlet tubing of the Pump in to the Water HPLC grade through suction filter.

3. Allow mobile phase to flow for about 5 min.

4. Block Pump outlet with the block screw.

5. The pressure rises and on crossing the 300 bar, "ERROR P-MAX" appears on the display window. Note the time. Press "CE" key and observe the pressure drop for 5 min.

6. After 5 min., record the pressure in calibration Log.

7. Make entry of the column usage in the Column Usage Log Register.

8. Make entry of the usage in to the Instrument Usage Log Register.

9. Compare the result for its compliance against limit given in the Calibration Log and

put the remark regarding the Calibration Status.

10. In case of non-compliance, follow the Maintenance Program.

2. Flow rate calibration

1. Ensure that, the instrument is ready for calibration and Start-up procedure is followed.

2. Ensure that, the Pump is passing the "Leakage Test (By Pressure Drop)".

3. Keep the Drain tube in such a way that the mobile phase (Water) drops falls in to 10 ml

clean, dry volumetric flask without touching the walls of the flask and start

immediately the stopwatch when first drop falls in to the flask.

4. Wait till the collected mobile phase reaches 10 ml mark of the volumetric flask and

Stop the stopwatch. .

5. Record the time required to collect the 10 ml mobile phase in calibration Log.

6. Repeat the procedure for 1.0 ml, 1.5 ml and 2.0 ml/ min. flow rates.

7. Repeat the step 3 to 6 but using Methanol HPLC grade as mobile phase instead of water.

8.  Compare the results for its compliance against limits given in the Calibration Log

and put the remark regarding the Calibration Status.

9. Make entry of the usage of the instrument and column in the Instrument Usage Log

Register and Column Usage Log Register respectively.

10. Prepare Calibration Status Label and display on the instrument at the designated place.

11. In case of non-compliance, follow the Maintenance Program.

Reproducibility and lineraity of injection volume

Solution Mixer Preparation :

1. Take clean and dry 50 ml volumetric flask.

2. Pipette out 1.0 ml of Benzene and Toluene in to the clean and dry 50 ml volumetric flask.

3. Make up the volume to 50 ml with Methanol and mix well.

        Chromatographic Condition:

Column                  :           ODS C18, (25 cm x 4.6 mm ID, 5 µm) 

Mobile Phase         :           Methanol : Water (70 : 30)

Flow Rate              :           1.0 ml/min.

Wavelength           :           254 nm

Injection Volume    :           20 µl

Calibration:

1. Ensure that, the instrument is ready for calibration and Start-up procedure is followed.

2. Ensure that, the instrument is set according to the Chromatographic conditions.

3. Follow the Instrument Operating procedure, Inject 10 µl in triplicate and record the

chromatograms.

4. Repeat the injection of above solution by injecting 15, 20, 25, 30 µl in triplicate.

5. Take the print out of the chromatograms and attach to the Calibration Log.

6. Record the Area and Retention times of the of the Benzene and Toluene peaks in the

Calibration Log.

7. Make entry of the usage of the instrument and column in the Instrument Usage Log

Register and Column Usage Log Register respectively.

8. Plot  the curve for area corresponding to Benzene to Toluene peaks v/s injection

volume, Find out the RSD (reproducibility) and record in the Calibration Log.

9. Find out the Correlation coefficient "r2" for each peak  at five levels and record in the

Calibration Log.

10. Compare the result for its compliance against limit given in the Calibration Log and

put the remark regarding the Calibration Status.

11. Prepare Calibration Status Label and display on the instrument at the designated place.

12. In case of non-compliance, follow the Maintenance Program.

Check point: D2 lamp energy check (detector)

1. Ensure that, the instrument is ready for calibration and Start-up procedure is followed.

2. On the Detector’s display window, some values at the functions "l(nm)", "abs(AU)",

"range (AUFS)" and "lamp" appears.

3. On the display, previously set value blinks at "l (nm)" function, enter the Wave length

to 254 nm by pressing numerical keys.

4. Press "Func Back" key and select "lamp" functions, enter 1 to select D2 lamp.

5. Further press "Func Back" key till "REF EN" appears.

6. Record the Reference Energy of the D2 Lamp at 254 nm in the Calibration Log.

7. Compare the result for its compliance against limit given in the Calibration Log and

put the remark regarding the Calibration Status.

8. In case of non-compliance, follow the Maintenance Program.

3. Linearity of detector response  

Solution Mixer Preparation:

1. Take three clean and dry 50 ml volumetric flasks.

2. Pipette out ml of Benzene and Toluene as per the following table in to the clean,

dry 50 ml volumetric flask separately.

3. Make up the volume to 50 ml with Methanol and mix well.

Solution (Level)

ml of Benzene to be taken

Ml of Toluene to be taken

To be Diluted with Methanol to

1

0.5

1.0

50 ml

2

1.0

1.0

50 ml

3

1.5

1.0

50 ml

Chromatographic Condition :

1. Set each of the equipment to the following parameters as per the Equipment’s

Parameter Set Up Procedure.

Column                  :           ODS C18, (25 cm x 4.6 mm ID, 5 m)

Mobile Phase         :           Methanol : Water (70 : 30)

Flow Rate              :           1.0 ml/min.

Wavelength            :           254 nm

Injection Volume    :           20 µl

Calibration  :

1. Ensure that, the instrument is ready for calibration and Start-up procedure is followed.

2. Ensure that, the instrument is set according to the Chromatographic conditions.

3. Follow the Instrument Operating procedure, Inject each of the Solutions in triplicate

and record the chromatograms.

4. Take the print out of the chromatograms and attach to the Calibration Log.

5. Record the Area and Retention times of the of the Benzene and Toluene peaks in the

Calibration Log.

6. Make entry of the usage of the instrument and column in the Instrument Usage Log

Register and Column Usage Log Register respectively.

7. Calculate the Area Ratio of Benzene to Toluene, Find out the Mean value of the ratios

and record in the Calibration Log.

8. Find out the Correlation coefficient "r2" from the Mean area ratio values of the three

levels.and record in the Calibration Log.

8. Plot the curve for area ratio corresponding to Benzene to Toluene peaks v/s

concentration, Find out the RSD (reproducibility) and record in the Calibration Log.

9. Compare the result for its compliance against limit given in the Calibration Log and

put the remark regarding the Calibration Status.

10. Prepare Calibration Status Label and display on the instrument at the designated

place.

11. In case of non-compliance, follow the Maintenance Program.