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-19-10 Date Revised: 02-01-19 ------- SOP No. QC-19-10 Date Revised 02-01-19 Page 1 of 6 SOP Number QC-19-10 Title Verification of Digital Pipettes Scope Describes the process for verification of digital pipettes. Application Pipettes are calibrated by an ISO 17025 accredited vendor at a frequency determined by the laboratory and are verified in-house using the gravimetric procedure as necessary. 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Name: Quality Assurance Unit Print Name: Branch Chief Print Name: Date SOP issued: Controlled copy number: Date SOP withdrawn: ------- SOP No. QC-19-10 Date Revised 02-01-19 Page 2 of 6 TABLE OF CONTENTS Contents Page Number 1.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 3 10. CAUTIONS 3 11. SPECIAL APPARATUS AND MATERIALS 4 12. PROCEDURE AND ANALYSIS 4 13. DATA ANALYSIS/CALCULATIONS 6 14. FORMS AND DATA SHEETS 6 15. REFERENCES 6 ------- SOP No. QC-19-10 Date Revised 02-01-19 Page 3 of 6 1. Definitions Abbreviations/definitions are provided in the text. 1. Verification failure = Verification of pipette exceeds ±5% inaccuracy and/or <5% of the coefficient of variation (% CV). 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). 3. Percent inaccuracy = percentage by which measured volume differs from the target volume 2. Health and Safety Follow procedures specified in SOP MB-01, Laboratory Biosafety. 3. Personnel Qualifications and Training Refer to SOP ADM-04, OPP Microbiology Laboratory Training. 4. Instrument Calibration 1. The pipets are calibrated by an ISO accredited vendor or verified in-house (refer to section 12.2) on a predetermined frequency (see the Pipette Verification Record Sheet, section 14). 2. Refer to SOP EQ-03 (weigh balances) for details on method and frequency of calibration. 5. Sample Handling and Storage None. 6. Quality Control For quality control purposes, the required information is documented on the appropriate form(s) (see section 14). 7. Interferences 1. Pipettes with inaccuracies that exceed the specifications listed in this SOP (see section 8.2). 8. Non- conforming Data 1. Manage non-conforming data consistent with SOP ADM-07, Non- Conformance Reports. 2. Do not use pipettes if the inaccuracy exceeds ±5% of the target volume and/or <5% CV (see 13.1). For corrective actions, see section 12.3. 9. Data Management 1. Archive data 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. If a pipette fails an in-house verification assessment, do not use it until it has been repaired (if necessary) and recalibrated by an ISO 17025 ------- SOP No. QC-19-10 Date Revised 02-01-19 Page 4 of 6 accredited vendor (see 12.1b). 11. Special Apparatus and Materials 1. 2. Calibrated balances. Capable of measuring 0.01 g for verifying pipettes with volumes greater than 1 mL and 0.0001 g for verifying pipettes with volumes less than or equal to 1 mL. a. For balances capable of measuring up to 0.00001 g, use the upper weight range of the balance in order to display a resolution of 0.0001 g prior to initiating gravimetric pipette verification procedure. Pipettes. b. Rainin Adjustable Volume Pipettes Gilson Microman Positive Displacement Pipettes c. d. Gilson Distriman Continuously Adjustable Volume Repetitive Pipettes e. Eppendorf Repeater Pipettes 12. Procedure and Analysis 12.1 Pipette Verification Requirements a. Verify and service pipettes using an ISO 17025 accredited vendor; refer to the Pipette Verification Record Sheet (see section 14) for the frequency of verification. b. If a pipette is dropped or broken, verify the pipette using the gravimetric verification procedure (see section 10). c. Record vendor or in-house verification results on the Pipette Verification Record Sheet (refer to section 14) and file in the Pipette Verification and Calibration Log Book. The Pipette Verification Record Sheet is based on the inventory of pipettes and may change over time. 12.2 Gravimetric Verification Procedure The gravimetric pipette verification procedure utilizes the relationship between the weight of a water sample (g) and its density (g/mL) to verify the volume being delivered at a specified temperature. a. Use the pipette tips that correspond to each pipette. b. Record all pertinent information for the gravimetric verification procedure on the Pipette Verification - Gravimetric Analysis Form (refer to section 14). c. In advance of testing, fill a container with de-ionized water and allow it to equilibrate to room temperature in the same laboratory as the balance. List the balance that will be used on the appropriate form. ------- SOP No. QC-19-10 Date Revised 02-01-19 Page 5 of 6 d. Place a small container on the balance (for example, a 50 mL Erlenmyer flask) and record its weight on the appropriate form. e. Using the pipette to be verified, aspirate an aliquot of DI water from the sample aliquot container and dispense into the small container. Refer to the Pipette Verification - Gravimetric Analysis Spreadsheet (see section 14) for the recommended verification volume for each pipette. f. Record the weight on the appropriate form. g. Follow the procedure in 12.2d-e for each subsequent sample addition; measure at least 5 samples. Do not tare between samples. After each new sample addition, record the weight on the appropriate form. h. Input the measurements and other appropriate information into the pipette-specific worksheet in the Pipette Verification - Gravimetric Analysis Spreadsheet (see section 14). i. Verify that the percent inaccuracy is within ±5% and the % CV is <5%. i. If either the percent inaccuracy or % CV are outside the required range, repeat the analysis. ii. If the ranges continue to be outside the required range, label the pipette as "Out of Service" and remove the pipette from use until it is repaired and calibrated by a vendor. 12.3 Pipette Verification Record Sheet a. From the data on the calibration certificate or Pipette Verification - Gravimetric Analysis Spreadsheet for each pipette, record the following on the Pipette Verification Record Sheet: i. Verification Status: record as "pass" or "fail." Record status as "pass" if the percent inaccuracy is within ±5% and the % CV is <5% from gravimetric analysis or if vendor calibration certificate indicates acceptable results, otherwise record as "fail." ii. Corrective Action: record as "yes" or "no." If the verification status is "pass," record as "no." Record as "yes" if the verification status is "fail," describe the action taken at the bottom of the page, and notify the Quality Assurance Officer (QAO). The QAO must determine if there was any impact on work conducted using the failed pipet. b. Complete the Pipette Verification Record Sheet electronically or by ------- SOP No. QC-19-10 Date Revised 02-01-19 Page 6 of 6 hand. 13. Data Analysis/ Calculations „ , . (x-ideal volume)x 100 1. Percent Inaccuracy = - ideal volume ° n/ CV fStandard deviation's w -^qq \ Mean J 14. Forms and Data Sheets 1. Test Sheets. Test sheets are stored separately from the SOP under the following file names: MLB Pipette Inventory QC-19-10 Fl.xlsx Pipette Verification Record Sheet QC-19-10_F2.xlsx Pipette Verification - Gravimetric Analysis Form QC-19-10 F3.xlsx Pipette Verification - Gravimetric Analysis , „ , „ . , c , , A QC-19-10 F4.xlsx Spreadsheet - 15. References 1. CRC Handbook of Chemistry and Physics. 93rd ed. CRC Press: Boca Raton, FL, 2012; p 6-8. ------- |