US Environmental Protection Agency
Office of Pesticide Programs
Office of Pesticide Programs
Microbiology Laboratory
Environmental Science Center, Ft. Meade, MD
Standard Operating Procedure for
Calibration and Maintenance of pH Meters
SOP Number: EQ-01-08
Date Revised: 05-03-17
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SOP No. EQ-01-08
Date Revised 05-03-17
Page 1 of 12
SOP Number
EQ-01-08
Title
Calibration and Maintenance of pH Meters
Scope
This protocol describes the method for the operation, two-point
calibration, and maintenance of the pH meters used in the laboratory.
Application
pH meters are used to determine the pH of samples, such as media
and reagents used in the laboratory. The pH of media and reagents is
a critical attribute of such solutions for the proper performance of
media and reagents.
Approval Date
SOP Developer:
Print Name:
SOP Reviewer
Print Name:
Quality Assurance Unit
Print Name:
Branch Chief
Print Name:
Date SOP issued:
Controlled copy
number:
Date SOP withdrawn:
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SOP No. EQ-01-08
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TABLE OF CONTENTS
Contents Page Number
1.
DEFINITIONS
3
2.
HEALTH AND SAFETY
3
3.
PERSONNEL QUALIFICATIONS AND TRAINING
3
4.
INSTRUMENT CALIBRATION
3
5.
SAMPLE HANDLING AND STORAGE
3
6.
QUALITY CONTROL
3
7.
INTERFERENCES
3
8. NON-CONFORMING DATA
3
9.
DATA MANAGEMENT
4
10.
CAUTIONS
4
11.
SPECIAL APPARATUS AND MATERIALS
4
12.
PROCEDURE AND ANALYSIS
4
13.
DATA ANALYSIS/CALCULATIONS
12
14.
FORMS AND DATA SHEETS
12
15.
REFERENCES
12
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1. Definitions
Abbreviations/definitions are provided in the text.
2. Health and Safety
Follow procedures specified in SOP MB-01, Laboratory Biosafety.
The Study Director and/or lead analyst should consult the Safety Data
Sheet for specific hazards associated with media and reagents.
3. Personnel
Qualifications and
Training
Refer to SOP ADM-04, OPP Microbiology Laboratory Training.
4. Instrument
Calibration
As noted in section 12.
5. Sample Handling
and Storage
1. Measure the pH of media and reagents at room temperature (20°C
to 25°C) unless otherwise specified on the Media/Reagent
preparation sheet. For agar based media, the pH is to be taken on
a solidified sample (see section 12.5).
2. The buffers used for calibration and reagents (e.g., IN HC1, IN
NaOH, KC1 solution) are stored at room temperature in a
secondary containment unit.
3. Store electrodes for up to one week (and extended storage) in 25
mL of pH 7 or pH 4 buffer. Never store the electrodes in distilled
water.
4. For longer storage periods of electrodes used for pH meter units
530 and 530-P, remove the bulb protector, fill the wetting cap
with the 3M KC1 fill solution and push the wetting cap onto the
tip of the electrode.
5. The ExStik pH meter electrode has a sponge in the electrode
protective cap, when longer storage of the ExStik pH meter is
required, keep the sponge soaked in pH 4 solution to preserve
electrode.
6. Quality Control
For quality control purposes, the required information is documented
on the appropriate form (see section 14).
7. Interferences
1. When calibrating the pH meters and taking the pH of a sample,
rinse the electrodes thoroughly with de-ionized water and blot dry
with Kimwipes before proceeding to the next solution.
8. Non-conforming
Data
1. Management of non-conforming data is consistent with SOP
ADM-07, Non-Conformance Reports.
2. To pass calibration, for the 530 and 530-P pH meters, the slope
must be 100 ± 5%. If slope is out of range, several corrective
actions may be necessary to ensure pH meters are working
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properly. See section 12.6 for troubleshooting and corrective
actions.
3.
If a pH meter is not functioning as required, consult the technical
manual to determine the problem and perform the appropriate
corrective action.
9. Data Management
Data will be archived consistent with SOP ADM-03, Records and
Archives.
10. Cautions
1.
Do not stir the sample while the instrument is reading as this may
provide inaccurate readings.
2.
Discard used buffer solutions daily. Do not reuse.
3.
For 530 and 530-P pH meters:
a. Do not allow the 3M KC1 fill solution to run dry. Add 3M
KC1 fill solution whenever the level falls more than 25 mm
below the fill hole. Replace the 3M KC1 fill solution at least
once a month; record this activity on the pH Meter
Calibration Log Form (see section 14).
b. Always remove the wetting cap and the fill hole plug during
calibration and measurements. Replace the fill hole plug in
between use.
4.
Do not leave the electrode in organic solvents (for all pH meters)
as the tip and body may be damaged.
11. Special Apparatus
and Materials
1.
The list of pH meters and electrodes is maintained as an
attachment to the SOP. See section 14.
2.
Calibration Buffers: pH 4, pH 7 and pH 10.
3.
Fill solution: 3M KC1 solution.
4.
Storage Solutions: 3M KC1 solution for capping or pH 4, pH 7
and pH 10 for buffer immersion.
5.
pH Electrode Cleaning Solution.
6.
Ceramic replacement junction.
7.
8.
IN Sodium Hydroxide Solution (NaOH).
IN Hydrochloric acid (HC1).
12. Procedure and
Analysis
1.
Calibrate the pH meters at least once on the day of use as outlined
in sections 12.1 through 12.3. A two-point calibration is required,
using two standard pH buffers.
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2. The standard buffers used are pH 4, pH 7, and pH 10. Dispense a
small amount of the required buffers into a smaller container or
tube for calibration.
3. Use pH buffers for calibration one time only. After calibration,
discard the buffers as per manufacturer's recommendations.
4. Use two standard pH buffers to determine the pH of the medium
or reagent.
a. If the final pH of the sample falls between pH 7.0 and 10.0,
use the pH 7.0 and 10.0 buffers for calibration.
b. If the final pH of the sample falls between pH 4.0 and 7.0,
use the pH 4.0 and 7.0 buffers for calibration.
5. Record the expiration dates of the buffers on the pH Meter
Calibration Record Form (see section 14). Record the date the
buffers are changed under Notes on the form.
6. For pH meter unit 530 and 530-P, check the level of the 3M KC1
fill solution every day when in use to ensure that it is within 25
mm of the filling opening. Bring to level if needed. Record under
Notes any adjustments made with the fill solution on the pH
Meter Calibration Check Record Form (see section 14).
12.1 Two-point
calibration
pH Meter Model
M 530 - (Used for
liquid samples)
a. Perform a two-point calibration with calibration buffers 7
plus 4 or 10 (whichever is nearest to the expected sample
value)
b. Prior to use, verify that the pH meter is set at Auto endpoint
as indicated by a letter "A" in the upper left hand corner of
the display. If the letter "A" is not indicated, press the
"Auto" button until it is displayed. After the endpoint is
reached, brackets will appear around the A (e.g. [A]). The
temperature and slope will appear at the bottom of the
display. When selecting the buffer for calibration, the
selected buffer pH will also appear at the bottom of the
display, but once the calibration begins, the pH of the buffer
will disappear.
c. To calibrate, rinse the electrode tip with de-ionized water
and blot dry with Kimwipes, then place the tip of the
electrode into the pH 7 buffer and press "Cal" one time so
that "pH 7" and "Cal 1" appear.
d. The pH meter will automatically read the endpoint when the
reading is stable. The appropriate buffer symbol will appear
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on the display. Record the pH value on the pH Meter
Calibration Check Record Form.
e. Rinse the tip of the electrode with de-ionized water and blot
dry with Kimwipes.
f. Place the tip of the electrode in the second calibration buffer.
If pH 4 buffer is used, press "Cal" one time so that "pH 4"
and "Cal 3" appear. If pH 10 buffer is used, press "Cal" two
times so that "pH 10" and "Cal 3" appear.
g. The pH meter will automatically read the endpoint when the
reading is stable and the appropriate buffer symbol will
appear on the display. The display will show the electrode
slope value. Record the pH value and slope on the pH
Meter Calibration Check Record Form.
h. If the slope is less than 95% or greater than 105%, refer to
the troubleshooting section (see section 12.6) of the Corning
pH meter 530 and instructions manual (see section 15).
i. Rinse the electrode with de-ionized water and blot dry with
Kimwipes. Confirm the calibration by rechecking the pH of
the pH 7 buffer a second time (see 12.4 c). Record the pH
value and temperature on the pH Meter Calibration Check
Record Form.
12.2 Two-point
Automatic
calibration
'Autocar
pH meter
M 530-P (Used for
liquid samples)
a. Perform a two-point calibration with calibration buffers 7
plus 4 or 10 (whichever is nearest to the expected sample
value). For this pH meter, the specified Buffer set is 9.
b. Ensure the pH meter MODE is set to display the nominal pH
value for the recognized buffer and not the electrode voltage
(mV).
c. Start the calibration with . The number of the
specified buffer set is displayed (Buffer set 9)
d. The first buffer (Buffer 1) of the buffer set is requested.
e. Immerse the electrode tip in the first buffer solution (pH 7).
f. Press the key. The AR display indicator flashes. The
nominal pH value for the recognized buffer is displayed.
g. As soon as a stable value is recognized, the next buffer is
requested (Buffer 2).
h. Continue the two-point calibration by thoroughly rinsing the
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electrode with deionized water.
i. Immerse the pH electrode in the second buffer solution
(either pH 4 or pH 10).
j. Press the key. The AR display indicator flashes. The
nominal pH value for the recognized buffer is displayed.
k. As soon as a stable value is recognized, the value of the zero
point (ASY) is displayed for 10 seconds, then the value of
the slope (SLO) is displayed for 10 seconds.
1. After this, the meter automatically switches to the measuring
mode.
m. Record the pH value and slope on the pH Meter Calibration
Check Record Form.
n. If the slope is less than 95% or greater than 105%, refer to
the troubleshooting section (see section 12.6) of the Corning
pH meter 530-P and instructions manual (see section 15).
o. Rinse the electrode with de-ionized water and blot dry with
Kimwipes. Confirm the calibration by reading the pH of the
pH 7 buffer a second time (see 12.4 c). Record the pH value
and temperature on the pH Meter Calibration Check Record
Form.
12.3 Two-Point
calibration of
ExStik pH meter
(Used for solid
samples)
a. Perform a two-point calibration with calibration buffers 7
plus 4 or 10 (whichever is nearest to the expected sample
value).
b. To calibrate, rinse the electrode tip with de-ionized water
and blot dry with Kimwipes, then place the tip of the
electrode into the pH 7 buffer solution and momentarily
press the CAL key. Calibrate the pH 7 buffer first.
c. The ExStik automatically recognizes the solution and
calibrates itself to that value. Record the pH value on the pH
Meter Calibration Check Record Form.
d. Rinse the tip of the electrode with de-ionized water and blot
dry with Kimwipes.
e. Proceed to the second calibration buffer (e.g., pH 4 or 10) as
necessary.
f. During calibration, the pH reading flashes on the main
display.
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g. When calibration is complete, the ExStik automatically
displays END and returns to normal operation mode.
Record the pH value on the pH Meter Calibration Check
Record Form.
h. The appropriate circled indicator 4, 7, or 10 will appear on
the ExStik's LCD when a calibration has been completed.
The calibration data is stored until a new calibration is
performed.
Note: if the solution is off more than one pH unit from the pH
standards of 4, 7, or 10, the ExStik will assume an error and abort
the calibration. CAL and END will be displayed.
12.4 Liquid Sample pH
reading
(pH meter Models
530 and 530-P)
a. For all media and reagents other than those listed in
Attachment 2, measure the pH of the liquid sample prior to
autoclaving and verify after autoclaving.
See Attachment 2 (section 14) for the list of media and
reagents that do not require a pH verification for each batch.
b. Measure the pH of the liquid sample at room temperature
unless otherwise specified on the Media/Reagent preparation
sheet.
c. Place the tip of the rinsed and dried electrode in the sample
and press "read" (530) or "OK" (530-P) to start the
measurement. The decimal point will flash while the
electrode is reading.
Note: Do not stir the sample while the instrument is
reading. Add acid or base if any pH adjustments are
necessary and mix sample thoroughly by stirring. Once
sample is mixed, stop stirring the sample prior to re-reading
the pH. Repeat this process until the desired pH has been
reached.
d. When the pH reading is stable, record the initial pH of the
solution on the Media/Reagent preparation sheet (see SOP
MB-10, Media and Reagents Used in Efficacy Testing).
e. If the pH of the liquid falls outside of the desired range,
adjust the pH of the tempered sample using the specified
acid and base solutions and record the amount used on the
Media/Reagent preparation sheet.
f. Record the adjusted pH of the solution on the
Media/Reagent preparation sheet.
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g. Measure the pH of the sample after autoclaving. Let sample
adjust to room temperature prior to pH verification. Follow
steps 12.4 c-d.
h. The temperature (in °C) will display at the same time as the
pH. Record the temperature at which the pH of the solution
was measured on the Media/Reagent preparation sheet.
i. Note: Several liquid media do not require a pH verification
prior to and post autoclaving for each prepared batch. This
information is provided on the media and reagents
preparation sheets and in Attachment 2. Verify the pH of
each new lot of dehydrated media after autoclaving once per
lot.
12.5 Solid media pH
reading with
ExStik meter
a. For all solid media (e.g. agars), other than those listed in
Attachment 2, measure the pH of the agar prior to
autoclaving and verify after autoclaving.
Note: Refer to Attachment 2 (section 14) for list of media
that do not require pH verification for each batch.
b. Place the electrode on solidified medium, the main display
and bargraph indicate the pH reading while the lower
display reads temperature. Readings flash until they have
stabilized.
c. The bargraph is center zero, i.e. at pH 7 there is no display.
As the pH rises, the bar moves from the center to the right.
d. If the pH drops, the bar moves from the center to the left.
e. When the pH reading is stable, record the initial pH of the
solution on the Media/Reagent preparation sheet (see SOP
MB-10, Media and Reagents Used in Efficacy Testing).
f. If the pH of the liquid falls outside of the desired range,
adjust the pH of the tempered sample using the specified
acid and base solutions and record the amount used on the
Media/Reagent preparation sheet.
g. Record the adjusted pH of the solution on the
Media/Reagent preparation sheet.
h. Measure the pH of the sample after autoclaving. Let sample
adjust to room temperature prior to pH verification. Follow
steps 12.5 c-e.
12.6 Troubleshooting
a. If slope is out of range (< 95% or > 105%), conduct the
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and corrective
actions for pH
meters 530 and
530-P
following corrective actions in sequential order. If the first
corrective action resolves the problem, it is not necessary to
continue with subsequent corrective actions.
b. First corrective action: change the standard pH buffers and
recalibrate the instrument.
c. Second corrective action: change the 3M KC1 fill solution
in the electrode or bring up to volume and re-calibrate the
instrument.
d. Third corrective action: clean the electrode with the pH
Electrode Cleaning Solution (follow manufacturer's
instructions). See section 12.8 for Electrode Cleaning and
Maintenance. Record all troubleshooting activities under
the notes section of the pH Meter Calibration Record Form
(see section 14).
e. If the problem persists or cannot be determined or corrected,
call the manufacturer.
12.7 Troubleshooting
and corrective
actions for ExStik
pH meter
a. If the batteries are weak, the "BAT" indicator appears on the
LCD screen of the unit. Press the ON/OFF key to turn the
ExStik on or off. The auto power off feature shuts the
ExStik off automatically after 10 minutes of inactivity to
preserve battery life.
b. If the meter will not calibrate of displays a -1, reset the
meter and attempt to re-calibrate as follows:
i. Turn off the meter.
ii. Remove the battery cartridge from the top of the
meter.
iii. Press the ON/OFF button for 10 seconds to bleed off
all charges within the meter.
iv. Re-insert the batteries and power ON the meter.
v. Attempt to re-calibrate the meter.
c. If the unit appears to be locked (display frozen) it is possible
that the DATA HOLD mode has been inadvertently
accessed by pressing the MODE button. Simply press the
Mode button again or turn the meter off and restart if the
display appears frozen.
d. If the meter does not latch up and no button presses revive
it, remove the batteries, push the ON button for 10 seconds
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and then reinsert the batteries,
e. If needed, consult the user guide.
12.8 Electrode
Cleaning and
Maintenance for
pH meter units
530 and 530-P
a. Clean the electrode when the slope falls below 95% or is
greater than 105% for 530 and 530-P pH meters
b. Test the flow of the junction by letting the electrode hang in
the air for 1-2 hr. A proper flowing junction will have KC1
salt crystals forming on it. If none appear, review the
following suggestions for cleaning.
c. Soak the tip of electrode in the pH Electrode Cleaning
Solution for 1-2 hr. Rinse the electrode with de-ionized
water and soak in pH 7 buffer until stable.
d. For oil contamination: Carefully clean the tip of the
electrode using a cotton swab soaked with alcohol or
acetone. Rinse the electrode with de-ionized water and soak
in pH 7 buffer until stable.
e. If cleaning does not improve junction flow, replace the
junction. To replace, pull out the clogged junction, rinse the
electrode barrel with de-ionized water, and replace with a
clean junction. Then refill the electrode with 3M KC1 fill
solution up to the fill hole plug (see section 15).
f. After cleaning and maintenance activities, check the
calibration of the pH meter and record on the pH Meter
Calibration Form.
g. Record cleaning and maintenance activities under notes on
the pH Meter Calibration Record Form (see section 14).
12.9 Electrode
Replacement for
ExStik pH meter
a. For EXStik pH meter, the electrode has limited life based on
frequency of use and care among other factors.
b. To change the electrode, unscrew and completely remove
the electrode retaining collar.
c. Gently rock the electrode from side to side, pulling it away
from the meter, until it disconnects.
d. To attach an electrode, carefully plug the electrode into the
meter socket (note that the electrode connector is keyed,
ensuring proper connection).
e. Secure the electrode in place by tightly turning the collar in
place (a rubber gasket seals the electrode with the meter).
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13. Data Analysis/
Calculations
Not Applicable
14. Forms and Data
Sheets
Test Sheets. Test sheets are stored separately from the SOP under the
following file names:
Attachment 1: Inventory of pH meters and electrodes
Attachment 2: List of media and reagents for pH
verification per lot of dehydrated media
pH Meter Calibration Record .
£, EQ-01-08 Fl.docx
Form -
15. References
1. Corning Electrodes Information sheet for pH combination 3-in-l
electrodes.
2. Corning Labware & Equipment- Electrodes: How to Maintain
the Premium Performance of Your Corning Electrodes.
3. Instruction manual for Corning pH meter M 530.
4. Instruction manual for Corning pH meter M 530-P
5. ExStik waterproof pH meter user guide:
http://www.globalw.com/downloadsAVQ/phlOO.pdf
6. Corning Electrodes Information pamphlet for flat surface
electrodes.
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