US Environmental Protection Agency
Office of Pesticide Programs
Office of Pesticide Programs
Microbiology Laboratory
Environmental Science Center, Ft. Meade, MD
Standard Operating Procedure for
Verification of Digital Pipettes
SOP Number: QC-19-06
Date Revised: 01-23-14
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SOP No. QC-19-06
Date Revised 01-23-14
Page 1 of 13
SOP Number
QC-19-06
Title
Verification of Digital Pipettes
Scope
Describes process for verification of digital pipettes and the
calibration of the Artel PCS 2 Pipette Calibration System.
Application
The Artel PCS 2 pipette calibration system is used to verify volumes
of one milliliter or less. The gravimetric procedure is used to verify
volumes greater than one milliliter.
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Date SOP withdrawn: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. QC-19-06
Date Revised 01-23-14
Page 2 of 13
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
4
7.
INTERFERENCES
4
8. NON-CONFORMING DATA
4
9.
DATA MANAGEMENT
4
10.
CAUTIONS
4
11.
SPECIAL APPARATUS AND MATERIALS
5
12.
PROCEDURE AND ANALYSIS
5
13.
DATA ANALYSIS/CALCULATIONS
9
14.
FORMS AND DATA SHEETS
9
15.
REFERENCES
9
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SOP No. QC-19-06
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1. Definitions
Abbreviations/definitions are provided in the text.
1. Verification failure = Verification of pipette exceeds ±5% of the target
volume.
2. Ideal volume = Target volume; actual volume being measured, corrected
for temperature using the density of water at 21.0°C (0.997995 g/mL, see
section 15.1).
2. Health and
Safety
1. Follow procedures specified in SOP MB-01, Laboratory Biosafety. The
Study Director and/or lead analyst should consult the Material Safety Data
Sheet for specific hazards associated with products.
2. All kit reagents are non-hazardous, aqueous based and may be disposed of
by flushing down a drain.
3. Personnel
Qualifications
and Training
Refer to SOP ADM-04, OPP Microbiology Laboratory Training.
4. Instrument
Calibration
1. Refer to SOP EQ-03 (weigh balances) for details on method and
frequency of calibration.
2. Check the PCS instrument calibration twice a year using an instrument
calibrator kit.
3. Photocopy the PCS instrument calibration printout (it is printed on thermal
paper which fades over time) and store in the Pipette Verification and
Calibration Log Book.
4. Store certificates of calibration for PCS calibrator kit and PCS Reagent Kit
in the Pipette Verification and Calibration Log Book.
5. Acceptable limits for PCS Instrument Calibration are listed in the PCS 2
Pipette Calibration System Procedure Guide (refer to reference 15.2). If a
failure occurs, clean the calibration vials and perform the instrument
calibration check again. If the instrument fails again, purchase another
calibration set and recheck the calibration. If failures persist, schedule
service for the instrument and do not use the instrument for calibration
until it has been recertified.
6. Refer to Chapter 5 of the PCS Procedure Guide for detailed instructions
and pass/fail specifications.
5. Sample
Handling and
Storage
1. Store the calibration kit and the reagent kit in darkness at room
temperature (e.g., 20-25°C) in the same laboratory as the instrument.
2. Handle each blank reagent vial with care. The vial contains a
predetermined amount of liquid and any spillage will lead to inaccurate
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SOP No. QC-19-06
Date Revised 01-23-14
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results.
6. Quality Control
1. For quality control purposes, the required information is documented on
the appropriate form(s) (see section 14).
2. To provide maximum life and dependable system performance, periodic
cleaning should be performed. Refer to Chapter 7, Section 7.3, of the PCS
Procedure Guide for instructions on cleaning the cabinet, the vial holder,
and the filer wheel assembly.
7. Interferences
1. To ensure that accurate and precise results are obtained during pipette
verifications and instrument calibration checks, all vials should be free of
fingerprints or smudges prior to placement into the vial block. THUS,
AVOID HANDLING THE LOWER PORTION OF THE VIALS.
Fingerprints or smudges on the vials that fall into the optical path will
introduce errors that cause inaccurate readings. If the lower portion of the
vial is inadvertently touched, it should be properly cleaned prior to use
(refer to Chapter 2, Section 2.2 of the PCS Procedure Guide for cleaning
procedures).
2. Never remove the cap of a calibration standard.
3. Blank reagent vials should be moved immediately from the vial block into
the vial holder. Once the blank reagent vial has been inserted into the
instrument it must not be turned, moved, or removed until the entire
procedure has been completed, or the vial has been consumed.
4. Always take a fresh aliquot of sample solution and lightly cap. Always
use the aliquot of sample solution within 30 min to avoid evaporation.
8. Non-
conforming
Data
1. Management of non-conforming data will be specified in the study
protocol; procedures will be consistent with SOP ADM-07, Non-
Conformance Reports.
2. Do not use pipettes if the inaccuracy exceeds ±5% of the target volume.
3. See Chapter 7, Troubleshooting, of the PCS Procedure Guide for specific
system performance checks. Section 7.2 provides a troubleshooting guide
for symptoms, causes, and corrective actions.
9. Data
Management
1. Data will be archived consistent with SOP ADM-03, Records and
Archives.
2. Maintain an inventory of pipettes electronically using a Microsoft Excel
spreadsheet (refer to section 14). After each addition to or deletion from
the inventory, file a hard copy of the pipette inventory in the Pipette
Verification and Calibration Record Book.
10. Cautions
1. Keep the PCS Instrument out of direct sunlight and away from anything
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SOP No. QC-19-06
Date Revised 01-23-14
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hot or cold.
2. To ensure proper sample mixing, place the PCS Instrument on a sturdy
table.
3. Do not open the PCS Vial Holder Cover until you are prompted with
"Pipette Sample #" or "Remove Cap and Insert Blank."
4. If a pipette is dropped or damaged, it must be successfully verified or
recalibrated prior to use.
5. If a pipette fails the in-house verification assessment, it will not be used in
the laboratory and will be recalibrated prior to use.
11. Special
Apparatus and
Materials
1. ARTEL PCS 2 Pipette Calibration System Instrument, serial no. 6250. For
verifying pipettes with volumes 1 mL or less.
2. Calibrated balance. Capable of measuring 0.01 g. For verifying pipettes
with volumes greater than 1 mL.
3. Pipettes.
a. Rainin Adjustable Volume Pipettes
b. Gilson Microman Positive Displacement Pipettes
c. Gilson Distriman Continuously Adjustable Volume Repetitive
Pipette
12. Procedure and
Analysis
12.1 Pipette
Verification
Requirements
a. Twice a year, verify the performance of each pipette listed in
Attachment A, once using an in-house procedure (see 12.1, i-ii) and
once by a vendor who will also calibrate and service each pipette.
i. For pipettes dispensing volumes of 1 mL or less, use the Artel
PCS 2 pipette calibration system (refer to section 15.3).
ii. For pipettes dispensing volumes greater than 1 mL, use the
gravimetric verification procedure.
b. If a pipette is out of compliance prior to the biannual verification
(e.g., the pipette was dropped), verify the pipette using the Artel PCS
2 pipette calibration system or the gravimetric verification procedure.
c. Record the results on the Pipette Verification Record Sheet (refer to
section 14). The Pipette Verification Record Sheet is based on the
inventory of pipettes and may change over time. Note: after the
annual vendor calibration, complete the Verification Status and
Corrective Action Taken columns on the Pipette Verification Record
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SOP No. QC-19-06
Date Revised 01-23-14
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Sheet for the half of the year in which the pipettes are calibrated by a
vendor. Fill in the Percent Inaccuracy column with "N/A".
12.2 Using the Artel
PCS
a. Turn on the PCS instrument and the printer.
b. Load blank vials into the vial block at least 15 minutes prior to use.
Four vials can be loaded into the vial block at this time. Inspect for
smudges and clean them if necessary (refer to section 7.1).
c. Allow the instrument to self calibrate and follow the on-screen
instructions. Enter a pre-assigned Operator ID number.
d. When prompted, enter the four or five digit lot code shown on the
reagent kit insert and on the exterior label affixed to the top of the
reagent kit. If using a new lot, enter the lot information when
prompted.
e. After self calibration, the instrument will prompt the operator to
insert the CAL A Vial. Insert the CAL A calibration standard into
the vial holder with the CAL A label facing toward the front of the
PCS.
f. After closing the cover on the vial holder, the PCS will perform a
zero check. Once the zero check is complete, remove the CAL A
vial and place it back in the Reagent Kit.
g. Remove a blank vial from the block. Invert it several times to
thoroughly mix the contents.
h. Insert the blank reagent vial into the vial holder located under the vial
holder cover. Close the cover. The instrument will take a reading of
the blank.
i. Enter the pipette serial number (numerical digits only) and the
volume to be checked. Once entered, the instrument will indicate
which range solution should be used for that verification.
i. Refer to "Verification Volume" column in Sample Pipette
Verification Sheet for the pipette volumes to be verified.
j. Mix the range solution by inverting the bottle several times before
removing the cap. Recap the bottle as soon as possible to avoid
evaporation.
i. Each kit contains bottles of sample solution corresponding to
four different pipette volume ranges:
Range 1: For pipette volumes 200 to 5000 |iL.
Range 2: For pipette volumes 50 to 199 |iL.
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SOP No. QC-19-06
Date Revised 01-23-14
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Range 3: For pipette volumes 10 to 49 |iL.
Range 4: For pipette volumes 2 to 9 |iL.
ii. Use the proper range solution for the pipette volume being
verified.
k. Transfer an aliquot of sample solution, using a disposable transfer
pipette, into a sample aliquot container. Transfer pipettes and sample
aliquot containers are found in the reagent kit. Place the container
into the aliquot container holder to avoid spillage. Place the cap
lightly on the container to minimize evaporation.
1. Using the pipette to be verified, aspirate an aliquot of sample solution
from the sample aliquot container.
i. Use standard laboratory pipetting technique and follow
specific pipette manufacturer's instructions.
m. Lift the instrument vial holder cover and dispense the aliquot of
sample solution into the blank reagent vial directly into the liquid or
against the inside wall of the vial. If dispensing against the inside
wall, place the pipette tip above the meniscus but below the top of
the vial holder. The mixing action of the PCS will wash the solution
off the inside wall of the vial.
n. Once the aliquot is dispensed, carefully withdraw the pipette and
close the cover. The PCS will display the results of the previous
reading and will prompt for the addition of the next sample. Follow
the same above procedure for sample #2 and for each subsequent
sample addition. After each new sample addition, the instrument will
mix, read, and print the delivered volume.
o. After dispensing the desired number of samples for the verification
run (5 samples), press the "End of Run" button. The group statistics
will be computed and the results printed.
p. Within a blank vial's allowed capacity, additional verification runs at
any volume may be performed.
q. When verifying an adjustable volume pipette at multiple volumes,
start anywhere along its volume range. After dispensing the desired
number of samples at the first volume, press "Enter" (do not press
"End of Run"). The group statistics for the first volume will be
computed and the results printed. Follow the on screen instructions
and indicate the next volume to be verified when prompted. If
desired, continue to verify the same pipette at different volumes by
pressing the "Enter" key at the conclusion of each volume. After
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SOP No. QC-19-06
Date Revised 01-23-14
Page 8 of 13
pipetting the final sample at the last volume for the pipette press the
"End of Run" button. The group statistics and results for the last
volume and the footer will be printed.
12.3 Gravimetric
Verification
Procedure
a. Record all pertinent information for the gravimetric verification
procedure on the Pipette Verification - Gravimetric Analysis Form
(refer to section 14).
b. In advance of testing, fill a container with de-ionized water and allow
it to equilibrate to room temperature in the same laboratory with the
balance. List the balance that will be used on the appropriate form.
c. Place a small Erlenmeyer flask on the balance and record its mass on
the appropriate form.
d. Using the pipette to be verified, aspirate an aliquot of DI water from
the sample aliquot container and dispense into the Erlenmeyer flask.
e. Record the mass on the appropriate form.
f. Follow the procedure in 12.3d-e for each subsequent sample
addition; measure at least 5 samples. Do not tare between samples.
After each new sample addition, record the mass on the appropriate
form.
g. Input the measurements and other appropriate information into the
Pipette Verification - Gravimetric Analysis Spreadsheet.
12.4 Pipette
Verification
Record Sheet
a. From the data on the PCS printout for each pipette or the Pipette
Verification - Gravimetric Analysis Spreadsheet, record the
following on the Pipette Verification Record Sheet:
i. Percent Inaccuracy, record the value from the PCS printout
or the Pipette Verification - Gravimetric Analysis
Spreadsheet for each pipette.
(x — ideal volume) x 100
Percent Inaccuracy = -—
ideal volume
ii. Verification Status: record as "pass" or "fail". Record status
as "pass" if the percent inaccuracy is within ±5%, otherwise
record as "fail".
iii. Corrective Action: record as "yes" or "no". Record as "yes"
if the verification status is "fail" and describe the action taken
at the bottom of the page. If the verification status is "pass,"
record as "no."
b. Generate one Pipette Verification Record Sheet biannually and file in
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SOP No. QC-19-06
Date Revised 01-23-14
Page 9 of 13
the Pipette Verification and Calibration Log Book.
c. The Pipette Verification Record Sheet may be completed
electronically or by hand.
d. Photocopy the PCS printout for each pipette and store in the Pipette
Verification and Calibration Log Book.
13. Data Analysis/
Calculations
1. None
14. Forms and Data
Sheets
1. Sample Pipette Verification Record Sheet
2. Sample PCS Printout
3. Sample Pipette Inventory
4. Test Sheets. Test sheets are stored separately from the SOP under the
following file names:
MLB Pipette Inventory QC-19-06 Fl.xlsx
Pipette Verification Record Sheet QC-19-06_F2.xlsx
Pipette Verification - Gravimetric Analysis Form QC-19-06 F3.xlsx
Pipette Verification - Gravimetric Analysis , „ „ , . ,
c , , A QC-19-06 F4.xlsx
Spreadsheet -
15. References
1. CRC Handbook of Chemistry and Physics. 93rd ed. CRC Press: Boca
Raton, FL, 2012; p 6-8.
2. Artel, Inc. 1997. PCS 2 Pipette Calibration System Procedure Guide,
Doc #: 15A2135 Ver: 5.1, 03/28/97.
3. Artel, Inc. 2003. Artel Lab Report - Calibration Frequency for Pipettes,
Issue 6.
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SOP No. QC-19-06
Date Revised 01-23-14
Page 10 of 13
Sample Pipette Verification Record Sheet
OPP Microbiology Laboratory
Verification Date(s): Initials:
Manufacturer
Model No.
Serial No(s).
Volume
Range
Verification
Volume(s)
Percent
Inaccuracy1
Verification
Status2
Corrective
Action (Y/N)
X12523D
Gilson
M10
X12649D
1-10 nL
10 nL
BH15232
Gilson
M100
GG05125
10-100 nL
100 nL
GG05127
1-12.5 nL
10 nL
AE10020
10-125 nL
100 nL
100-1250 nL
900 nL
1-12.5 nL
10 nL
BD10010
10-125 nL
100 nL
100-1250 nL
900 nL
1-12.5 nL
10 nL
U10048H
10-125 nL
100 nL
100-1250 nL
900 nL
A0504243A
L0508039A
C0823986A
J0753884A
C0825980A
Rainin
L-1000
C0823596A
100-1000 nL
1000 nL
H0101474A
H0100977A
J0902334A
J0908624A
D0303509A
E0301364A
A0510192A
Rainin
L-200
L0509218A
20-200 nL
100 nL
C0821962A
J0750805A
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SOP No. QC-19-06
Date Revised 01-23-14
Page 11 of 13
Manufacturer
Model No.
Serial No(s).
Volume
Range
Verification
Volume(s)
Percent
Inaccuracy1
Verification
Status2
Corrective
Action (Y/N)
C0820542A
C0820661A
G0101809A
G0102379A
J0902612A
C0401654A
Rainin
L-100
C0825210A
10-100 nL
100 nL
C0825238A
A0507382A
C0822081A
J0724932A
C0822312A
Rainin
L-20
C0825315A
2-20 nL
10 nL
F0100492A
F0100448A
J0903005A
C0400398A
Rainin
L-2
H0100116A
0.1-2 nL
2 nL
HO100003A
Rainin
L-5000
D1080497A
0.5-5 mL
5 mL
Rainin
L-10000
A1058487A
1-10 mL
10 mL
Rainin
L-20000
L0931886A
2-20 mL
20 mL
Percent inaccuracy is recorded from the PCS printout for each pipette. Verification status = PASS if percent
inaccuracy is within ±5%. If verification status = FAIL, record "yes" in Corrective Action column and fill in Action
Taken below.
Corrective Actions:
Pipette Serial Number Action Taken
1)
2.)
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SOP No. QC-19-06
Date Revised 01-23-14
Page 12 of 13
Sample PCS Printout
OPP Microbiology Laboratory
PI . n 0i<
(Jl>ER«TOI? ID: t
RE«?EMI LOT «: 3^55?
TEMPERATURE CC>: 20 !
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SOP No. QC-19-06
Date Revised 01-23-14
Page 13 of 13
Sample Pipette Inventory
OPP Microbiology Laboratory
MLB Pipette Inrentory
Manufacturer
Mo-del Xe?
Serial Xoi'zL
X12523D
Gils on
Ml®
X326-10
BH15232
{Ms on
M100
GG05I2T
GGC5125
AE 30323
Gils on
Distriman
BD103I3
OOC45H
AG53-243A
LD53S33SA
C0S239S6A
JCT53SS-A
C3S25980A
Raimn
L-1000
C3S2359SA
K310U"4A
EC1305~"'A
709C233-A
JQS0S624A
D3303539A
E030135-A
Manufacturer
Model Xo
Sirtal Xot z s
A0510192A
L050S21SA
C3S219S2A
J075CS05A
Rainin
L-200
C0S235-2A
C3820S61A
G0101S59A
G31023m
J3902S32A
CG4Q1654A
Rainin
L-100
C3S25213A
C382523SA
A0'53"3S2A
CGS220SIA
J0'T2-9'32A
C0S22312A
Ramm
L-20
C0S253I5A
FOIOC-2SA
FGI0044SA
70933035A
CG40G3SSA
Rainin
L-2
KG133116A
KC103033A
Rainin
L-50'30
D13S049M
Rainin
L-10DM
A205S4S""A
Rainin
L-2000'0
LG931S5SA
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