A CRITICAL REVIEW ON CALIBRATION OF ANALYTICAL INSTRUMENTS

About Authors:
Manoranjan Thunuguntla*, B.Krishnamoorthy, M.Muthukumaran, Amreen Nishat, Vasu Naik
Montessori Siva Sivani Institute Of Science & Technology College Of Pharmacy,
Mylavaram, Krishna District,
Andhra Pradesh
* manozph@gmail.com

Abstract¹:
Analytical instruments are used for specific analysis of drugs and pharmaceuticals. So, regular performance verification is made to ensure that instrument used in the analytical purpose should be properly calibrated and validated “to demonstrate that it is suitable for its intended purpose”. This article is prepared in the sense to get all the information on calibration of basic analytical instruments in laboratory and industrial scale of scientific institutions. The scope of this article is to get the procedures of calibration of analytical instruments at here. Calibration of instruments with their procedure and precautions of Thermometer, Ph Meter, Karl Fisher, Polarimeter, Conductivity Meter, Tablet Fraibilator, Hardness Tester, Disintegration Test Apparatus, Dissolution Test Apparatus, Potentiometer and U. V. Spectrophotometer are mentioned below.

REFERENCE ID: PHARMATUTOR-ART-1684

Introduction:
Calibration is the process by which ensure that an instrument readings are accurate the reference to establish standard. Calibration is performed by using primary standard. It is done to check the zero error deflection by using standard reference.

Need for calibration:
Calibration can be called for
* With a new instruments
* When a specified time period is elapsed
* When a specified usage (operating hours) has elapsed
* When an instrument has had a shock or vibration which potentially may have put it out of calibration
* Sudden change in weather
* Whenever observation appears questionable

These are the analytical instruments which are responsible to get the reports of the analyte in laboratory and industry scale in accurate by using the instruments having the status of READY TO USE.

The status of the instruments is identified by checking the calibration tag of the instruments. Tag consists of information regarding instruments like name of instrument, date of procurement, date of calibration, next calibration date and signature of calibrated person with date. Calibration of instruments is done in two ways internal calibration and external calibration. Internal calibration is done the in-house officials whom have sound knowledge on it. External calibration is done according to the instructions of the manufacturer and should be done in the government approved individual or institution.

Table No.: 1

Procedures for calibration of Analytical instruments:

I.Thermometer²:
Ice Point Method
• Fill an insulated container, such as a wide mouth “thermos” bottle with a mixture of potable crushed ice and water.
• The container must have crushed ice throughout to provide an environment of 32°F, so you may have to pack more ice into the container during the process.
• When the mixture of the water has stabilized after four or five minutes, insert the thermometer to be calibrated to the appropriate immersion depth.
• Be sure to hold the stem of the instrument away from the bottom and sides of the container (preferably one inch) to avoid error.
• If your thermometer is not accurate within +/- 2°F of 32°F., adjust the thermometer accordingly.
The ice point method permits calibration to within 0.1°F

Boiling Point Method
• After the water in the container has reached a complete “rolling” boil, insert the instrument to the appropriate immersion depth. The boiling point in Wisconsin is 212°F.
• Be sure there is at least a two-inch clearance between the stem or sensing element and the bottom and sides of the container.
• If your thermometer is not accurate within +/- 2°F of 212°F., adjust thermometer accordingly.
The boiling point method permits calibration to within 1.0°F.

II.    Digital Balance³:
· Place 500mg standard calibrated weight on the pan.
· Record the display of the weight.
· Remove the weight & again place the weight on the pan & record the display once again.
· Repeat the above operation to get ten readings Calculate the standard deviation of ten readings.
· The measurement uncertainty shall be considered satisfactory if three times of standard deviation of not less than 10 replicate weighing divided by amount weight does not exceed 0.001.
· Affix a ‘Calibration label’ on the instrument.
· Report any discrepancy noted at the time of calibration to Q.C Manager & notify the defect to rectify the instrument. & affix an ‘UNDER MAINTENANCE ‘ label on the instrument.

III. pH Meter Calibration⁴:
Use the squirt bottle to rinse and the plastic beaker to collect the water. Two buffer calibrations should be done before any measurements are taken. One buffer calibration should be done every two hours.

1. ALWAYS start with the yellow pH 7 buffers as the first calibration buffer.

2. Use fresh (< 1 week), room temperature buffers. They are stored in the refrigerator and should be taken out in time to reach room temperature. The pH meter does display temperature, which should be taken into account when using the buffers.

3. The buffers should be stirred using a stir plate and magnetic stirrer while they are being measured for calibration.

4. Use the two buffers that bracket the expected range of measurements. For example, if your samples are expected to be acidic, use the pH 7 and pH 4 buffers to calibrate. If they are expected to be basic use the pH 7 and pH 10 buffers.

5. Rinse the electrode and place into the first buffer.

6. Press the 2^nd then the cal keys. CALIBRATION is displayed above the main field and the time and date of the last calibration are displayed. After a few seconds, P1 is displayed in lower field. P1 indicates that the meter is ready for the first buffer and a value has not yet been entered. When READY is displayed, indicating electrode stability, the reading begins to flash, press the yes key. The display will remain frozen for two seconds. Then P2 will be displayed in the lower field indicating that the meter is ready for the second buffer.

7. Rinse the electrode and place into the second buffer. When READY is displayed, press the yes key. Press the measure key to end calibration. SLP appears in the lower field with the actual electrode slope, in percent, in the field.

8. The meter automatically advances to the measure mode. MEASURE is displayed above the main field.

9. Rinse the electrode and place into sample. Record pH directly from the meter display and temperature from the lower field.

ONE Buffer Calibration

1. Select one buffer that closely approximates the expected sample pH.

2. Repeat steps 5 and 6 from above.

3. Press the measure key. SLP will be displayed in the lower field and the electrode slope in memory in the main field. If necessary, enter the correct electrode slope determined by the two point calibration and press the yes key. If slope value is unknown, enter 100.0 or perform a two buffer calibration.

4. Rinse the electrodes and place into sample. Read the pH directly from the main display and temperature from the lower field.

Storage
When not in use, the pH electrode should always be stored in a storage solution of 1 gram of KCl per 200ml of either the Buffer 4 or 7.

Cleaning

1. Soak electrode in 0.1 M HCl for 15-20 minutes.
2. Rinse with DI H2O
3. Recondition by allowing soaking in either a 4 or 7 buffers for 30 minutes

NOTE: ALWAYS RINSE THE PROBE WITH DI WATER IN BETWEEN EACH MEASUREMENT.

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IV.Karl fischer Apparatus⁵:
Volume calibration
· Press CAL key, display shows ‘ARE YOU READY FOR CALIBRATION ENTER TO CONTINUE, STOP TO EXIT’, press ENTER key, display shows ‘FILL LIQUID, REMOVE AIR BUBBLE, ENTER TO CONTINUE’.
· Fill sufficient quantity of water in reservoir & connect to the INLET of the DISPENSOR. Flush sufficient quantity of water to remove the Air bubble. Keep an empty pre-weighed beaker under the out let tube. Press ENTER key.
· Display shows ENTER REQUIRED VOLUME, ENTER TO CONTINUE, enter the volume for dispensing. Press ENTER key.
· Display shows volume. Press ENTER key.
· Display shows ‘CALIBRATION IN PROGRESS’ the dispenser will dispense the set volume as per program.
·  After dispensing the volume weigh the beaker. Difference shows the weight of water.
·  Repeat this procedure for 3 to 4 times & determine the average volume dispensed. The Tolerance limits ± 0.1 ml.
· Record the results in KF apparatus calibration Record- F-1.
· If the dispenser does not give correct volume call service person from the company.

Precautions:
· Keep the instrument clean by using Methanol from the reservoir & circulating to the delivery tube.
· If fresh KF reagent is added to the old stock in KF reservoir, mix the solution in reservoir properly and recalibrate the reagent for new factor.

V.    Polarimeter⁶
1.      Prepare the standard solutions of sucrose in distilled water of different concentrations from 10 % To 50 % and sonicate if necessary . Use distilled water as a blank and operate the instrument.
2.      Clean the sample cell with distilled water and check the reading . Press ZERO to get the zero of the distilled water blank. Place the sucrose solution one after another from 10 % to 50 % and measure the optical rotation for each solution which should be comparable with values given below:-

Table No. 2

3. Check that instrument is clean and free from dust.
4. Following are the keys present on the instrument:
5. Switch on the instrument power supply, and wait for few minutes.
6. As soon as the power is switched on ,the monitor shows the company’s name and go on initializing For 5-13 minutes.                                                      
7. After that the Instrument is in the "READY" mode for automatic operation indicated by READY. Display
8. Lift the door of the sample compartment and place the sample cell filled with media in which sample is prepared (ensure that no air bubbles are there ). Push ZERO keys for zero clearing. 
9. Push  EDIT key and enter  identification and concentration of sample .
10. Place the sample cell filled with sample solution ( ensure that no air bubbles are there ) and press START key. Take the reading displayed on the digital screen.

VI. Conductivity meter
1.      After receiving a new conductivity meter remove the electrode cap and soak the meter for a few minutes in alcohol to remove oils.  Do not submerge the meter below the cap line.  Rinse with water and gently blot dry with a paper towel.
2.      Always calibrate the conductivity meter with the Sodium Chloride Standard Solution prior to each sampling event.  It should be calibrated to read 1000 µS/cm ± 10 µS/cm (preferably within 12 hours).  Remember that µS/cm and µmhos/cm are interchangeable.  The old data sheets still have µmhos/cm as the increment for reporting conductivity, but the new sheets, provided in this notebook, have been updated to reflect that the meters we now use report conductivity in µS/cm.
3.      To calibrate, use the following instructions which are also located inside the conductivity meter case. Open the battery compartment lid (end with lanyard loop).  The two white buttons are Increment (INC) and Decrement (DEC) calibration keys.
4.      Rinse the electrode in deionized water, then rinse it in calibration standard (solution), then dip it in a container of calibration standard.  Switch unit on (ON/OFF) key).  Wait several minutes for display  to stabilize Press the INC (increase) or DEC (decrease) keys to adjust reading to match the calibration standard value. After 3 seconds without a key press, the display flashes 3 times, then shows “ENT”.  The tester accepts calibration value; returns to measurement mode.  The meter is now calibrated. Replace battery cap.Be sure the probes are dry when storing the meter. 

VII. Friability⁷
· Switch on the power supply.
· Set the RPM to 25 and start the machine simultaneously with the stop watch. Count the actual rotations and not the time required for the same.  
· Similarly set the RPM to 100 and note the time required and actual rotations.
· Apparatus is in proper working condition if,Time required for 25 rotations is 1 min ± 05 sec.
· Time required for 100 rotations is 4 min ± 20 sec. 

VIII . Hardness tester
The calibration was realized by two alternating approximately equally skilled appraisers (A, B). The measurement points were along the diameter (rim to rim) of the CRM in equidistant intervals. Appraiser A performed a calibration (5 indentations) followed by appraiser B. The indentations of both appraisers were evenly distributed around the center of the filed of view complying with the standard’s requirement for the minimal spacing between the adjacent indentations (3´ the average indentation diagonal). The force application time was 10 seconds. The values of average hardness and standard deviation sH for individual calibrations

The test force/load F = 98.07 N (10 kg). According the direct calibration (VI/08)  the deviation for test force 98.07 N is -0.2 % and the deviation of the measurement device is -0.2 % for 0.1 mm and -0.2 % for 0.2 mm. The certified reference material (CRM) in form of hardness reference block with specified hardness Hc = 472.4 HV 10 and expanded uncertainty  UCRM = ± 9.448 HV 10 (coverage factor k = 2) was used as a standard. The ambient temperature was 20°C, relative humidity 48 %.

IX . Disintegration test apparatus⁹:
· Ensure the apparatus is properly connected to the power supply.
· When the power is switched ON the TIMER and TEMP. shows digital value.
· Set the temperature 37°C by using SET, TEMP. , D  , Ñ, DISP.SEL and  ENTER  keys.
· Set the timer 15.00 by using  SET, TIME. , D  , Ñ, DISP.SEL and  ENTER  keys.
· Start the temperature by push  the ENTER key.
· Fill the beaker of DT apparatus with  Purified water and adjust the temperature between 37 ± 1 ^OC.
· Fix the beaker in its position and adjust the level of water so that when the basket is in upper most position, wire mesh of the basket is 25 mm below the water level, and when it is at   lower most position the wire mesh is at least 25 mm above the bottom of the beaker.
· Start the basket – rack assembly and  Timer  simultaneously.
· Place the standardized calibrated thermometer  to record the actual temperature against the temperature displayed.
· Note the  temperature of water in the beaker and Oscillation per minute of the basket-rack assembly.
·  Switch OFF the apparatus and record the observations in the calibration
· ACCEPTANCE CRITERIA: The temperature shall be between 37 ±  1 ^OC.The frequency of basket rack shall be between 29 to 32 per minute.

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X. Dissolution test apparatus¹⁰:

PART ‘A’
The instrument shall be calibrated for RPM and Temperature.

For Temperature Calibration:
Measure the temperature of the water bath and of each jar with a calibrated thermometer and compare the result against the digital display on the apparatus.

Acceptance Criteria: 37°C ± 0.5°C

For RPM Calibration.
Calibrate the apparatus at 50 and 100 RPM. Compare the RPM shown on the digital display of the apparatus with the RPM measured with a stopwatch or Taco meter.

Acceptable criteria:  ± 1 RPM – for 50 RPM
                              ± 2 RPM – for 100 RPM

PART ‘B’ Apparatus Suitability Test Disintegrating Type
1. Use USP dissolution calibrator disintegrating type 50 mg prednisone tablets.
2. This USP Dissolution Calibrator is provided for the Apparatus Suitability Test in the general chapter of USP 24 or as per the method specified in the documents received along with the respective lot of the tablet.
3. Do not expose the tablets to excessive humidity. Store in dry, cool place.
4. Dissolution Media: Distilled water 500 ml.
5. Heat the medium with gentle stirring, to about 45° C, immediately filter under vaccum using a membrane filter, with stirring for about 5 minutes.
6. Weigh accurately about 10 mg of prednisone reference standard (already dried on 105°C  for 3 houre into a 100 ml volumetric flask and dissolve in 5 ml of ethanol. Make up to volume with distilled water.
7. Dilute 10 ml of the solution to 50 ml with distilled water.
8. Conduct the suitability test at conditions mentioned in the certificate of tablets using apparatus I and II.
9. After completion of the dissolution time withdraw filter and aliquot of the solution.
10. Discard the first 2 ml of solution and measure the concentration of prednisone at 242 nm against the absorbance of prednisone USP reference standard solution.
11.  The apparatus is suitable if each of the individual calculated values for each apparatus at all indicated speeds are within the specified ranges.

Apparatus Suitability Test Non-disintegration Type
a. Apparatus suitability test for non-disintegrating type Salicylic Acid 30 mg tablets.
b. This USP Dissolution Calibrator is provided for the Apparatus Suitability Test in the general chapter of USP 24 or as per the method specified in the documents received along with the respective lot of the tablet.
c. These tablets are pure salicylic acid with no binders or fillers, because of the physical properties of such tablets, some sticking may occur during storage. Gentle tapping of the bottle may be used to separate the tablets.
d. Do not expose the tablets to excessive humidity. Store in a dry, cool place.
e. Dissolution Medium – 0.05 M phosphate buffer pH 7.4 ± 0.05, 900 ml.
f. Weigh accurately about 27.22 g of monobasic potassium phosphate and dilute to 1000 ml with water.  (Solution A)
g. Weigh accurately about 8 g sodium hydroxide and dilute to 1000 ml with water 0.2M Solution (Solution B).
h. Place 500 ml of solution A and 391 ml of solution B and dilute to 2000 ml with water and adjust the pH to 7.4 ± 0.05 with either of the solution A/B.
i. Dry a portion of salicylic acid working standard over silica gel for 3 hours before use.
j. Weigh accurately about 33 mg of salicylic acid working standard into a 100 ml volumetric flask, add 1 ml methanol and dissolve the powder, Dilute to volume with phosphate buffer. Dilute 5 ml to 50 ml with buffer.
k. Place one Salicylic acid Non-disintegrating type tablet in each of the 6 containers and operate the apparatus at each of the speeds indicated in the certificates. Withdraw and filter an aliquot of the solution
l. Discard the first 2 ml of solution. Dilute 5ml of the filtrate to 50 ml with dissolution medium (phosphate buffer) and measure the concentration of Salicylic acid at 296 nm against the absorbance of Salicylic acid reference standard solution.
m. The apparatus is suitable if each of the individual calculated values for each apparatus at all indicated speed is within the specified ranges.

XI. U.V. Spectrophoto Meter¹¹:
Switch ON the instrument and allow it to warm for 10 minutes.

Preparation of Potassium dichromate solution-UV :
Dry a quantity of Potassium dichromate by heating to constant weigh at 130ºC. Weigh accurately 60.0 mg (57-63mg) and dissolve in 0.005M Sulphuric acid to produce 1000ml.

Control of absorbance:
Scan the user base line from 200nm-400nm with blank solution (0.005M Sulphuric acid) ands press Auto zero. Check the absorbance by using Potassium dichromate solution UV at various wavelengths indicated in the following table using 0.005M Sulphuric acid as blank.

The above table gives for each wavelength the exact value of  E1% (1%  1cm) and permitted limits.

The E1% (1% 1cm) is calculated as follows:
E1% (1% 1cm) =  Absorbance % 1000
                          Concentration % 100           

Control of wave length:
1.    Verify the wavelength scale using the absorption maxima of Holmium oxide filters.
2.    Switch ON the instrument and allow it to warm up.
3.    Bring the instrument in wavelength scanning position.
4.    Set the Wavelength limit from 200-600nm.
5.    Clean all the four windows of reference and sample compartment.
6.    Make auto zero.
7.    Put Holmium oxide film in the sample compartment of the instrument.
8.    Start scanning and take the print of wavelength scan.
9.    For checking wavelength accuracy narrow absorption bands should only used.
10.    The band at 360.8nm is particularly suitable.
11.    Other suitable bands are at 279.3nm and 536.4nm.
12.     Verify the wavelength scale using the absorption maxima.
13.    The permitted tolerances are, (a)1nm for the range 200-400nm.(b)3nm for the range 400-600nm.

Limit Of Stray Light:
Preparation of Potassium chloride solution:
Weigh accurately 1.2 g of Potassium chloride (dried at 120°) in 100ml volumetric flask. Dissolve the content in water and make up the volume.
Measure the absorbance at 200 nm at path length 1.0 Cm.
Absorbance should be greater than 2.0 .
Resolution:-Prepare a0.02% w/v solution of Toluene in Hexane .Record the spectrum

References:
1. jbsr.pharmainfo.in/documents/vol2issue2/2010020204.pdf
2. datcp.wi.gov/uploads/Food/pdf/ThermometerCalibration-7.pdf
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14. Practice and Theory of pH Measurement; Ingold: Urdorf, Switzerland, 1989.
15. pharmaguideline.com/2011/01/karl-fisher-apparatus-calibration.html
16. Y.Anjaneyulu, R.Marayya, Quality Assurance and Quality Management in Pharmaceutical industry,  harmabook Syndicate, 2005

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