US Environmental Protection Agency
Office of Pesticide Programs

Office of Pesticide Programs

Microbiology Laboratory

Environmental Science Center, Ft. Meade, MD

SOP for Verification of Digital Pipettes Using the
PCS 2 Pipette Calibration System

SOP Number: QC-19-05

Date Revised: 03-29-11


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SOP No. QC-19-05
Date Revised 03/29/11
Page 1 of 15

EPA/OPP MICROBIOLOGY LABORATORY
ESC, Ft. Meade, MD

Standard Operating Procedure
for

Verification of Digital Pipettes Using the PCS 2 Pipette Calibration System

SOP Number: QC-19-05
Date Revised: 03/29/11

Initiated By:		Date: / /

Print Name:	

Technical Review:	Date: / /

Print Name:

Technical Staff

QA Review:		Date: / /

Print Name:	

QA Officer

Approved By:		Date: / /

Print Name:	

Branch Chief

Effective Date: 	/	/	

Controlled Copy No.:	

Withdrawn By:

Date: / /


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SOP No. QC-19-05
Date Revised 03/29/11
Page 2 of 15

TABLE OF CONTENTS
Contents	Page Number

1.0	SCOPE AM) APPLICATION	3

2.0	DEFINITIONS	3

3.0	HEALTH AND SAFETY	 3

4.0	CAUTIONS	3

5.0	INTERFERENCES	3

6.0	PERSONNEL QUALIFICATIONS	4

7.0	SPECIAL APPARATUS AND MATERIALS	5

8.0	INSTRUMENT OR METHOD CALIBRATION	5

9.0	SAMPLE HANDLING AM) STORAGE	5

10.0	PROCEDURE AM) ANALYSIS	5

11.0	DATA ANALYSIS/CALCULATIONS	 10

12.0	DATA MANAGEMENT/RECORDS MANAGEMENT	10

13.0	QUALITY CONTROL	10

14.0	NONCONFORMANCE AND CORRECTIVE ACTION	10

15.0	REFERENCES	 11

16.0	FORMS AND DATA SHEETS	11


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SOP No. QC-19-05
Date Revised 03/29/11
Page 3 of 15

1.0 SCOPE AND APPLICATION:

1.1 The Artel PCS 2 pipette calibration system quickly and accurately measures the
actual volume dispensed by any pipette and will be used by the laboratory to
verify pipette performance. Results are traceable to NIST.

2.0 DEFINITIONS:

2.1	NIST = National Institute of Standards and Technology

2.2	Verification failure = Verification of pipette exceeds 5% of the target volume.

3.0 HEALTH AND SAFETY:

3.1 All kit reagents are non-hazardous, aqueous based and may be disposed of by
flushing down a drain.

4.0 CAUTIONS:

4.1	Keep the PCS Instrument out of direct sunlight and away from anything hot or
cold. Store the pipettes, the instrument, and the reagent kit at room temperature.

4.2	To ensure proper sample mixing, place the PCS Instrument on a sturdy table.

4.3	Do not open the PCS Vial Holder Cover until you are prompted with "Pipette
Sample #" or "Remove Cap and Insert Blank."

4.4	If a pipette is dropped or damaged, it must be successfully verified or recalibrated
prior to use.

4.5	If a pipette fails the in-house verification assessment, it will not be used in the
laboratory and will be recalibrated prior to use.

5.0 INTERFEREDCES: Issues discussed in this section must be recognized to avoid

interference during the verification process. Interference could lead to erroneous

readings.

5.1	Ensure that the reagent kit is at the same ambient temperature as the instrument by
storing them in close proximity to each other (in the same laboratory at room
temperature).

5.2	Once the blank reagent vial has been inserted into the instrument it must not be


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SOP No. QC-19-05
Date Revised 03/29/11
Page 4 of 15

turned, moved, or removed until the entire procedure has been completed, or the
vial has been consumed. If the vial is removed and sample solution has been
pipetted into the vial, this vial is no longer usable.

5.3	Blank reagent vials should be moved immediately from the vial block into the vial
holder, giving them no chance to warm or cool during the verification process.

5.4	Always take a fresh aliquot of sample solution if the solution has been in the
sample aliquot container for over one-half hour.

5.5	Always place the cap (lightly) on the aliquot containers between pipette sampling
to minimize reagent evaporation.

5.6	The PCS reagent kit should be stored at room temperature. Avoid storage
temperatures above 32C (90F). Kit components should be kept in the dark.

5.7	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. In the case of instrument calibration checks, false failures
may occur. All vials are cleaned and inspected prior to shipping. If the lower
portion of the vial is inadvertently touched, it should be properly cleaned prior to
use. The following procedure is recommended for cleaning all PCS vials:

5.7.1	Pour enough rubbing alcohol (isopropyl) into a small container to
cover approximately 1 inch of the vial when immersed in the alcohol.

5.7.2	Hold the vial by the cap and immerse (approximately 1 inch) in the
alcohol.

5.7.3	Remove the vial from the alcohol and, using a lint free wipe (e.g.,
Kimwipe), wipe the vial dry by rotating the vial in the lint free wipe
several times.

5.8	The power line to which the instrument is connected should be free of voltage
variations caused by other equipment such as water baths or refrigerators.

PERSONNEL QUALIFICATIONS:

6.1 Personnel are required to be knowledgeable of the procedures in this SOP.


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SOP No. QC-19-05
Date Revised 03/29/11
Page 5 of 15

Documentation of training and familiarization with this SOP can be found in the
training file for each employee.

7.0 SPECIAL APPARATUS AND MATERIALS:

7.1	ARTEL PCS 2 Pipette Calibration System Instrument, serial no. 6250

7.2	Rainin Adjustable Volume Pipettes

7.3	Gilson Microman Positive Displacement Pipettes

7.4	Gilson Distriman Continuously Adjustable Volume Repetitive Pipette
8.0 INSTRUMENT OR METHOD CALIBRATION:

8.1	Check the PCS instrument calibration quarterly. To check the instrument
calibration, use an instrument calibrator kit. It contains four vials of different
concentrations of mixed sample and blank reagents and an insert with calibrator
values, lot code, expiration date and instructions. The calibrator values contain
information about these concentrations derived from a reference instrument at the
factory which itself has been calibrated using standards traceable to NIST.

8.2	Photocopy the PCS instrument calibration check printout (it is printed on thermal
paper which fades over time) and store in the Pipette Verification and Calibration
Log Book.

8.3	Store certificates of calibration for PCS calibrator kit and PCS Reagent Kit in the
Pipette Verification and Calibration Log Book.

8.4	Refer to Chapter 5 of the PCS Procedure Guide for detailed instructions and
pass/fail specifications.

9.0 SAMPLE HANDLING AND STORAGE: Between uses, store the calibration kit in
darkness at room temperature (18-28C) to allow for a six month shelf life.

10.0 PROCEDURE AND ANALYSIS:

10.1.1 Perform pipette verification every six months (biannually) on the
micropipettes listed in Attachment A using the Artel PCS 2 pipette
calibration system (refer to reference 15.2).

10.1.1.1 If a pipette is suspected of being out of compliance prior to


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SOP No. QC-19-05
Date Revised 03/29/11
Page 6 of 15

the biannual verification, verify the pipette using the Artel
PCS 2 pipette calibration system.

10.1.2	Record the results on the Pipette Verification Record Sheet (refer to
section 16.0). The Pipette Verification Record Sheet is based on the
inventory of pipettes and may change over time.

10.1.3	All pipettes will be calibrated annually by a vendor.

NOTE: The annual vendor calibration will serve as pipette verification
for one half of the year. Fill in the Verification Status and Corrective
Action Taken columns on the Pipette Verification Record 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".

10.2	Turn on the PCS instrument and the printer.

10.3	Load blank vials into the vial block at least 15 minutes prior to their use. Four
vials can be loaded into the vial block at this time. Inspect for smudges and clean
them if necessary (refer to section 5.7).

10.4	Allow the instrument to self calibrate and follow the on-screen instructions. Enter
a pre-assigned Operator ID number.

10.5	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. Enter the lot
information if it is a new lot of reagents.

10.6	After self calibration, the instrument will prompt the operator to insert the CAL A
Vial.

10.6.1 IMPORTANT: The CAL A calibration standard should always be
inserted into the vial holder with the CAL A label facing toward the
front of the PCS. Be careful not to smudge or get fingerprints on the
lower portion of the vial as this is a part of the optical path and any
smudges will decrease the accuracy of the results (refer to section 5.7).
Never remove the cap of a calibration standard.

10.7	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.


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SOP No. QC-19-05
Date Revised 03/29/11
Page 7 of 15

10.8	Remove a blank vial from the block. Invert it several times to mix the contents
thoroughly, remove the cap and set it aside.

10.8.1 IMPORTANT: Each blank reagent vial must be handled with care.

The vial contains a predetermined amount of liquid and any spillage
will lead to inaccurate results. The lower portion of the vial is a part of
the optical path and smudges will decrease the accuracy of the results.
Touching it should be avoided (refer to section 5.7).

10.9	Open the vial holder cover and 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.

10.9.1 IMPORTANT: Once the blank reagent vial has been inserted into the
instrument it must not be turned, moved or removed until the
verification procedure is complete for all pipettes to be verified using
that vial.

10.10	Enter the pipette serial number. Enter the volume to be checked. Once entered,
the instrument will indicate which range solution should be used for that
verification.

10.10.1 Refer to "Verification Volume" column in Attachment A for the
pipette volumes to be verified.

10.11	Locate the appropriate range solution in the reagent kit. Mix the contents by
inverting the bottle several times before removing the cap. Recap the bottle as
soon as possible to avoid evaporation.

10.11.1 IMPORTANT: 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.

Range 3: For pipette volumes 10 to 49 |iL.

Range 4: For pipette volumes 2 to 9 |iL.

USE THE PROPER RANGE SOLUTION FOR THE PIPETTE VOLUME
BEING VERIFIED. If the improper sample solution has been pipetted into a
blank reagent vial, that vial is no longer usable and should be discarded.

10.12 Transfer an aliquot of sample solution, using a disposable transfer pipette, into a


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SOP No. QC-19-05
Date Revised 03/29/11
Page 8 of 15

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 any possibility of spillage. Place the cap lightly on the container to
minimize evaporation.

10.12.1 NOTE: Transfer only enough reagent to be completely used in half-
hour increments. The total volume of sample solution required
depends on the pipette volume, the total number of pipettes tested and
the total number of discrete data points being generated. The volume
graduations on the pipette as well as the 1 mL marks on the sample
aliquot containers can be used as rough guide.

10.13	Using the pipette to be verified, aspirate an aliquot of sample solution from the
sample aliquot container. Use your standard pipetting technique. Place the cap
lightly back on the sample aliquot container between sampling to avoid errors due
to reagent evaporation.

10.13.1 NOTE: When verifying the accuracy of a pipette there is an inevitable
linkage between the operator and pipette. For meaningful results, it is
recommended that you use your standard laboratory pipetting
technique and follow your specific pipette manufacturer's instructions.

10.14	Lift the instrument vial holder cover and dispense the aliquot of sample solution
into the blank reagent vial.

10.14.1 IMPORTANT: The sample solution may be dispensed directly into the
liquid or against the inside wall of the vial. If dispensing against the
inside wall, the pipette tip should be placed 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.

10.15	Once the aliquot is dispensed, carefully withdraw your pipette and close the
cover. The PCS will display the results of the previous reading and will prompt
you to add 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.

10.16	After dispensing the desired number of samples for the verification run (5
samples, refer to Attachment A), press the "End of Run" button. The group
statistics will be computed and the results printed.

10.17 Within a blank vial's allowed capacity, you may continue with additional


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SOP No. QC-19-05
Date Revised 03/29/11
Page 9 of 15

verification runs. You may perform verifications all at the same sample volume,
at lower or higher sample volumes but within the same range solution, or at lower
or higher sample volumes and with different range solutions.

10.17.1 IMPORTANT: In verifying pipettes from more than one range in a
given vial, always use the range of sample solution which is
appropriate to the volume of pipette being verified.

10.18	In verifying an adjustable volume pipette at multiple volumes, you may start
anywhere along its volume range. When you have dispensed 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. The instrument knows that you are verifying the same pipette
but at a different volume. You may continue to verify the same pipette at
different volumes by pressing the "Enter" key at the conclusion of each volume.
Be sure to pay strict attention to the range solution to be used for a particular
volume. After 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.

10.19	From the data on the PCS printout for each pipette, record the following on the
Pipette Verification Record Sheet:

10.19.1	Percent Inaccuracy, record the value from the PC S printout for each
pipette.

10.19.2	Verification Status: record as "pass" or "fail". Record status as "pass"
if the percent inaccuracy is within 5%, otherwise record as "fail".

10.19.3	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".

10.20	Generate one Pipette Verification Record Sheet biannually and file in the Pipette
Verification and Calibration Log Book.

10.21	The Pipette Verification Record Sheet may be completed electronically (using
Microsoft Excel) or by hand.

10.22	Photocopy the PCS printout for each pipette and store in the Pipette Verification
and Calibration Log Book.


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SOP No. QC-19-05
Date Revised 03/29/11
Page 10 of 15

11.0 DATA ANALYSIS/CALCULATIONS: None.

12.0 DATA MANAGEMENT/RECORDS MANAGEMENT:

12.1	Data will be recorded promptly, legibly, and in indelible ink on the appropriate
forms. Completed forms are archived in notebooks kept in secured file cabinets
in file room D217. Only authorized personnel have access to the secured files.
Archived data is subject to OPP's official retention schedule contained in SOP
ADM-03, Records and Archives.

12.2	Maintain an inventory of pipettes electronically using a Microsoft Excel
spreadsheet (refer to 16.3). 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.

13.0 QUALITY CONTROL:

13.1	For quality control purposes, the required information is documented on the
appropriate forms (refer to section 16.0).

13.2	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.

14.0 NONCONFORMANCE AND CORRECTIVE ACTION:

14.1	Acceptable limits for PCS Instrument Calibration are listed in the PCS 2 Pipette
Calibration System Procedure Guide (refer to reference 15.1). If Noise,

Accuracy, Inaccuracy Due to Noise, Overall System Inaccuracy, or Overall
Instrument Performance failure, 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. Do not use the instrument for calibration until it has been
recertified.

14.2	Do not use pipettes if the inaccuracy exceeds 5% of the target volume.

14.3	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.


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SOP No. QC-19-05
Date Revised 03/29/11
Page 11 of 15

15.0 REFERENCES:

15.1	Artel, Inc. 1997. PCS 2 Pipette Calibration System Procedure Guide, Doc #:
15A2135 Ver: 5.1, 03/28/97.

15.2	Artel, Inc. 2003. Artel Lab Report - Calibration Frequency for Pipettes, Issue 6.
16.0 FORMS AND DATA SHEETS:

16.1	Sample Pipette Verification Record Sheet

16.2	Sample PCS Printout

16.3	Sample Pipette Inventory

MS Excel spreadsheet: MLB Pipette Inventory


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SOP No. QC-19-05
Date Revised 03/29/11
Page 12 of 15

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

















1-12.5 nL

10 nL











AE10020

10-125 nL

100 nL













100-1250 nL

900 nL













1-12.5 nL

10 nL







Gilson

Distriman

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











Rainin

L-1000

C0825980A

100-1000 nL

1000 nL







C0823596A











H0101474A















H0100977A















J0902334A















J0908624A















A0510192A















L0509218A















C0821962A











Rainin

L-200

J0750805A

20-200 nL

100 nL







C0820542A











C0820661A















G0101809A















G0102379A












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SOP No. QC-19-05
Date Revised 03/29/11
Page 13 of 15

Manufacturer

Model No.

Serial No(s).

Volume
Range

Verification
Volume(s)

Percent
Inaccuracy1

Verification
Status2

Corrective
Action (Y/N)





J0902612A











Rainin

L-100

C0825210A

10-100 nL

100 nL







C0825238A











A0507382A















C0822081A















J0724932A











Rainin

L-20

C0822312A

2-20 nL

10 nL







C0825315A











F0100492A















F0100448A















J0903005A











Rainin

L-2

H0100116A

0.1-2 nL

2 nL







HO100003A







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.)

3.)


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SOP No. QC-19-05
Date Revised 03/29/11
Page 14 of 15

Sample PCS Printout
OPP Microbiology Laboratory

US EPA ~ MARYLAND

FT. MEAD. MD 20755

PATE: 00/00/00
TIT1E: 00:00

PIPETTE CALIBRATION

PIPETTE SERIAL sr 152348
OPERATOR ID; 1
REAGENT LOT It: 14758
TEMPERATURE  : 22.1

PIPETTE SAMPLE VOLUME:

SAMPLE RANGE: 3

SAMPLE It

1

2

3

4

5

NQ.PTS: 5

MEAN: 9.9?

STO: 0.03?

CV: 0.37*

INftCG: -0.28%

LAST INST. CALIBRATION CHECK: 2? Oct 00
SOFTWARE VERSION: 6.013
INSTRUMENT SERIAL St: 6250

ARTEL PCS , PATENTS PENDING

VOLUME

9.94

9.97

9.34
10.03

9.98


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SOP No. QC-19-05
Date Revised 03/29/11
Page 15 of 15

Sample Pipette Inventory
OPP Microbiology Laboratory

MLB Pipette Inventory



Marnqfttgnursr

Model No,.

Srriul

Velum* Range.

I'm [rfc_rrizK

Rspiicalt

s

JV'x'i--in'srr

.7:'

Gilson

m:c

X12523B
X12649B
BHI5252

:-:c aL

1C uL

5

6 months

Gilson

Distrimaii

^.JE; C C 2 c
BBLCCLC

U1CC-SH

1-12.5 jL-
1C-125 jL"
LCC-125C uL:

1C uL;
ICC jL-

9CC jL:

5

6 months

Rimm

L-1CCC

AC 5 C - 2-13 A
LC50SCSSA
CC325S86A
JC~55SS-r.-L
CCS2-9SCA
CC525596A
HC1C L-i~-A
HC1CCS"A
JC9C235-A
JC3C5624A

-CC-.COC uL

I #0*0 (iL

5

6 months



Ratntn

L-2CC

AC 51C i 92A
LC5C92ISA
CCS21962A
JC5CSC5A
CC82C5-2A
CC82C66 LA

gc:c:scsa

GC1C23"'9A

JCSC2612A

2C-2CC jL

ICC jL

5

6 months

Rimm

L-ICC

CCS2521CA
CCS2523SA

1C-1CC dL

ICC iiJL

5

6 months

Raima

L-20

ACr C 3S2A
C0S22C8 lA
JC~24952A
C 3 S 2 2 312 A
CCS25515A
FC _oC-rS2A
FC1CC44SA
JC9C30C5A

2-20 ji.L

10 jiL

5

6 months

Rxtnra

L-2

HC1CC116A

"A - " 
fiv 1 V V J .-J,

O.I-2 mL

2|iL

5

6 months



1125 |iL syringe: 1-12.5 |iL volume range
21250 hL syringe: 10-125 |iL volume range.
312.5 mL syringe: 100-1250 volume range


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