Proper Weighing, Reagent Handling, Volumetric Measurement and Pipetting Techniques Workshop

Transcription

1 Proper Weighing, Reagent Handling, Volumetric Measurement and Pipetting Techniques Workshop Reggie Morgan & Robin Parnell 07/24/2017- GLP Conference

2 Topics to be Covered Contamination and Cleaning Procedures Measuring Devices Proper Weighing Proper Pipetting Reagent Handling 2

3 Cleaning and Decontamination 3

4 Your data is only as good as your samples Sample Containers- Scrupulously Cleaned Deionized Water Rinse Solvent or Acid Rinse Preservatives added without introducing Contamination Microbiology- Sterilization After Addition of Chemicals Quality Control Checks Soap Residue ph indicator- Acid Rinse Sterility- Microbiology Sample Containers 4

5 Glassware Cleaning One of the Most Common Sources of Contamination is Improper Preparation of Glassware 5

6 Water Rinse with Deionized Water Rinse with High Purity Water if Needed Detergent Phosphate Free Microbiology- Inhibitory Testing per Lot Additional Rinsing Acid- Metals Solvents- Organics, Oil and Grease Glassware Cleaning 6

7 Measuring Devices 7

8 Measuring Devices Volumetric Flasks Volumetric Pipets Graduated Cylinders 8

9 Measuring Devices Class A Glassware Use Class A glassware to make volumetric measurements All Class A glassware is permanently marked Conforms to standards for measuring devices established by the American Society for Testing and Materials(ASTM) 9

10 Measuring Devices Use of Volumetric Measuring Devices Accurate Measurements of Samples Reagents Standards 10

11 Measuring Devices Optimizing Accuracy Choose a measuring device suitable for specific volume to be measured Choose a measuring device capable of measuring or delivering a desired level of accuracy Avoid the use of multiple measuring devices for a single measurement Allow liquids to come to room temperature before measuring 11

12 Measuring Devices Measurements are based on the Meniscus The curve in the upper surface of a liquid close to the surface of the container or another object, caused by surface tension. Depth must be measured with the meniscus at eye level and at the center of the meniscus, or the bottom of a concave meniscus. Manufacturers of glassware and other tools calibrate their measurement marks to account for the meniscus. 12

13 Measuring Devices Meniscus 13

14 Use of Volumetric Flasks Preparation of Standards Preparation of Sample Dilutions Preparation of Sample extracts and digestates Used in combination with Pipets Measuring Devices 14

15 Measuring Devices Use of graduated cylinders Not volumetric measuring devices Class A or B can be used when measuring volumes where solids prevent use of a pipet Use of Pipettes Major types of transfer pipets To Contain (TC) To Deliver (TD) Dual Purpose (TC/TD) Blow-Out 15

16 Measuring Devices To Contain (TC) Calibrated to hold or contain the exact amount specified by the calibration Must be completely emptied to provide the stated volume The letters TC are marked on the neck To achieve complete delivery, blow out the pipette with a bulb 16

17 Measuring Devices To Deliver (TD) Pipets designed to release the exact calibrated amount when the tip is held vertically against the receiving wall until draining stops Types: Mohr, serological, and volumetric The letters TD are marked on the neck 17

18 Measuring Devices Dual Purpose (TC/TD) Pipets which combines three calibrations Has a upper graduation mark which is the To Deliver and Blow-Out line Has a lower graduation which is the To Contain line and has a double band for Blow-Out 18

19 Measuring Devices Blow-Out Pipets intended for rapid use and emptied by forceful blow-out Don t deliver calibrated amount until completely emptied TD and TC/TD pipets can also be Blow-Out pipets Marked with a double band etching or fired-in marking 19

20 Proper Weighing 20

21 Location of the Balance Weighing Bench Stable (lab bench, lab table, stone bench) Antimagnetic (no steel plate) Protected against electrostatic charges (no plastic or glass) Wall or floor installation (not both) Space dedicated to the balance

22 Location of the Balance Work Room Vibration-free Free from drafts Temperature Keep the temperature of the room as constant as possible. Weighing results are influenced by temperature! Do not weigh near radiators or windows

23 Location of the Balance Light If possible, place the balance on a window-free wall. Direct sunlight will influence the weighing result. Place the balance a significant distance from lighting fixtures to avoid heat radiation.

24 Location of the Balance Air Do not place the balance in the air flow of air conditioners or devices with ventilators. Place the balance a sufficient distance from any potential strong currents of air. Do not place the balance next to a door. Avoid places with high traffic. Passersby will usually create a draft at the weighing location.

25 Operation of the Balance Switching On Do not disconnect the balance from the power supply unless moving to a new location. When you switch the balance off, use the display key. The balance is now in standby mode. The electronics are still energized and no warm-up period is necessary

26 Operation of the Balance Leveling Check to see if balance is level prior to use Align the balance. Make sure air bubble is in the center of the level indicator. Turn the leveling feet to make adjustments.

27 Operation of the Balance Adjustment Adjust the sensitivity of the balance regularly, especially: When you operate the balance for the first time When you change the location of the balance After leveling the balance After major changes in temperature, humidity or air pressure

28 Operation of the Balance Reading Check that the balance displays exactly zero at the start of each weighing. Tare, if needed, to avoid zeroing errors. Read the result only after the small round circle in the upper left on the balance display has faded.

29 Operation of the Balance Weighing Pan Always place what is being weighed in the middle of the weighing pan. This will prevent corner load errors. Draft Shield Open the draft shield only when necessary.

30 Operation of the Balance Weighing Vessel Use the smallest possible weighing vessel. Ensure that the weight of the weighing vessel plus the weight of what is being measured does not exceed the capacity of the balance.

31 Operation of the Balance Weighing Vessel The weighing vessel and the sample it contains should have the same temperature as the surroundings. If possible, do not use your hands to place the weighing vessel in the weighing chamber. You could alter the temperature or humidity of the weighing chamber.

32 Physical Influences Physical influences can cause unstable balance readings the result slowly drifts in one direction, unexpected value is displayed

33 Physical Influences The most frequent causes are: Influences of the weighing sample Influences on the balance from the surrounding area Moisture gain or loss by the weighing sample Electrostatically charged weighing samples or vessels Magnetic weighing samples or vessels

34 Physical Influences Temperature Problem The weight display of a weighing sample drifts in one direction Possible Reasons The balance has not been connected to the power supply long enough to allow sufficient warm-up There is a temperature gradient between the weighing sample and the surroundings that leads to air currents along the weighing vessel Cold objects appear heavier Warm object appear lighter

35 Physical Influences Corrective Measures Temperature Never weigh samples taken directly from a dryer or refrigerator Acclimate weighing sample to the temperature of the lab or weighing chamber Handle weighing vessels with tweezers or tongs Avoid putting your hand in the weighing chamber

36 Physical Influences Problem Moisture Gain/Evaporation The balance display continuously drifts while weighing a vessel Possible Reasons You are experiencing the weight loss of volatile substances or weight increase of hygroscopic weighing samples.

37 Physical Influences Corrective Measures Moisture Gain/Evaporation Use clean and dry weighing vessels and keep the weighing pan free from dirt and water drops Use vessels with narrow necks and mount covers or stoppers.

38 Physical Influences Problem Electrostatics Each weighing shows a different result. The weight display is unstable. The repeatability of the result is poor. Possible Reasons Your weighing vessel or the sample has become electrostatically charged. Materials such as glass, plastics or powder cannot dissipate electrostatic charges very well. Dry air increases the risk of this effect

39 Physical Influences Corrective Measures Electrostatics Increase the atmospheric moisture Screen electrostatic forces by placing the weighing vessel in a metal container Use other weighing vessels Use antistatic kits or guns

40 Physical Influences Problem Magnetism The weight of the sample depends on its position on the weighing pan. Display remains the same but the repeatability is poor. Possible Reasons You are weighing magnetic material and the additional forces that arise are wrongly interpreted as a load.

41 Physical Influences Magnetism Corrective Measures Screen magnetic forces with special vessel Non-magnetic support Reference for Weighing Section Mettler Toledo website

42 Measuring Devices Measurement Exercise #1 42

43 Pipetting 43

44 Pipetting Pipette types Piston-driven Air Displacement Vacuum assisted pipettes Volumetric pipettes Graduated pipettes Positive Displacement Pasteur pipette Pipetting Syringe

45 Pipetting Piston-driven Air Displacement Vacuum is generated by the vertical travel of a metal or ceramic piston within an airtight sleeve. Capable of being very precise and accurate Subject to inaccuracies caused by changes in the environment (mostly temperature, but also air pressure) Subject to inaccuracies in consistent technique

46 Pipetting Types: Piston-driven Air Displacement Adjustable or fixed Single-channel, multi-channel or repeater Conical tips or cylindrical tips Standard or locking Manual or electronic Manufacturer

47 Pipetting Vacuum Assisted Hollow narrow cylinders Require the use of an additional suction device Commonly made of borosilicate glass which is tough and chemically resistant Disposable and single use pipettes are often made of polystyrene

48 Pipetting Vacuum Assisted Volumetric Calibrated for one volume Used to measure extremely accurately measurements Used to make base stock, calibration standards and solutions for titrations Graduated Uses a series of marked lines to indicate different calibrated volumes Accuracy isn t as great as that of a volumetric pipette

49 Pipetting Positive Displacement Composed of a plunger which directly displaces the liquid Used to avoid contamination Most commonly used for: Volatile Viscous substances DNA

50 Pipetting Pasteur Not graduated or calibrated for any particular volume Used to transfer small amounts of liquids Pipetting syringe Combines functions of volumetric pipettes, graduated pipettes and burettes Basically a syringe on the end of a pipettor

51 Pipetting Technique Suggestions Prewet the pipette tip at least two times Work at temperature equilibrium Examine the tip before dispensing sample Keep pipette straight up while pipetting Pause consistently after aspiration (1 sec) Pull the pipette straight out

52 Pipetting Technique Suggestions cont. Minimize handling of the pipette and tip Immerse the tip to the proper depth 2-5mm below the meniscus Don t touch pipettes or tips to walls and bottom of containers Use proper pipette tip Use consistent plunger pressure and speed

53 Pipetting Pitfalls Aspirating too fast forcing liquid into the body of the pipette Pipetting at a slant causing an incorrect volume to be measured Bubbles in the pipette tip Not allowing the full volume to be pulled up by removing pipette tip too soon Rapid or jerky movement losing volume from tip Inconsistency in pipetting technique Proximity of solutions

54 Pipetting Devices Measurement Exercise #2 54

55 Reagent Handling 55

56 Reagent Handling Chemical Storage Chemical container labeling is the primary initial source of warning for the employee All original chemical containers must be labeled accurately and clearly with the following information: Identity of the chemical Date the chemical was received Date the chemical was opened and the expiration date Appropriate hazard warning such as corrosive, flammable, carcinogen, or oxidizer

57 Reagent Handling Chemical Storage Containers for prepared chemicals must contain the following information: Identity of the chemical The ID of the analyst that prepared the chemical and the date it was prepared Appropriate hazard warning such as corrosive, flammable, carcinogen, or oxidizer

58 Reagent Handling Chemical Storage Never place beakers or open containers containing chemicals in a refrigerator Volatile reagents must be stored in a designated refrigerator, freezer or cabinet according to protocol

59 Reagent Handling Chemical Storage Store bulk acids and bases separately in ventilated areas away from volatile organics and oxidizable materials Do not store strong acids or caustics above eye level

60 Reagent Handling Chemical Storage Store individual reagent containers in specifically labeled vented cabinets Make sure that all containers of prepared reagents, or containers that hold small amounts of reagents for short periods of time are clearly labeled

61 Reagent Handling Chemical Storage Ensure that flammable materials are stored in cabinets specifically designed and marked for flammable materials If the NFPA (National Fire Protection Association) fire hazard rating listed is 2, 3, or 4, the chemical MUST be stored in a flammable cabinet

62 Reagent Handling Chemical Storage NEVER store flammable chemical with acids, bases or strong oxidizers Flammable cabinets are not be used for storage of anything other than flammable chemicals

63 Reagent Handling Reagent Preparation Minimizing contamination of stock by: Never pipetting directly from stock reagent bottle Never pour unused reagent back into a stock reagent bottle

64 Documentation 64

65 It is better to record too much information than not enough Documentation If it isn t recorded there is no objective evidence it happened Enough detail must be recorded to reconstruct everything that happened as long as data records are maintained 65

66 Document Control System Document Control Purpose is to ensure only the most recent revisions are available to appropriate personnel, revisions are timely and receive required approvals Includes the following documents Quality Assurance Manual SOPs Operational Memos Policies

67 Document Control Development, Revision and Approval Technical Managers QA Manager Document Control Number Include effective date Revision Code

68 Document Control Document Distribution Electronic Read Only System PDF of final revision centrally located No printing or printing with Unofficial Copy watermark Paper System Documented distribution list Numbered copies Collection and disposal of previously distributed copies

69 Document Control Master List of All Current Documents Document type Title Document control number Effective date

70 Document Control Archived Listing Title Effective date Document control number Date taken out of service Date of disposal

71 Document Control Archival Filed with secure records and kept at least as long as data is maintained Recommend keeping SOPs longer to track historical changes to procedures and instrumentation Designate documents as OBSOLETE

72 Document Control Data Reports The laboratory must define what are considered official data records Example: Hard copies of instrumentation printout and bench sheets are official data records Data validation documentation Calculations Qualifying notes

73 Document Control All records maintained in organized file system in secured area to ensure regulatory requirements are met Secured, limited access Maintained for five years Document records disposal policy Document disposal of records

74 Electronic File naming convention (run number including date) Specific network directory Electronic back-up Document Control Paper Run or batch naming convention (number and date) Logbooks with numbered pages

75 Traceability The ability to verify the history, location, or application of an item by means of documented recorded identification. The capability (and implementation) of keeping track of a given set or type of information to a given degree, or the ability to chronologically interrelate uniquely identifiable entities in a way that is verifiable. 75

76 Traceability Reagent Log Lot Numbers Number of Containers and When Received Shelf Life When Put into Use When Taken out of Use Reagent Preparation Log Volume Prepared Lot Numbers of all Reagents Used Analyst Preparing Holding Time (Prep and expiration dates) 76

77 Traceability Sample Preparation Logs Sample Date and Time (if holding time is less than 48 hrs.) Volume Sample Used Reagents and Quantities Added Digestion/Distillation Start and End Temperature Unusual observations Identification of Analyst(s) 77

78 Traceability Temperature recordings Refrigerators, incubators, Ovens, ambient, etc. Ensure Thermometers Have had Annual Verification Ensure Thermometer is Sensitive Enough and in Proper Range (tenths place graduations) Celsius or Farenheit 78

79 Traceability Equipment Maintenance Serial Number Received Put into Service All Maintenance and Verifications Parts replaced and serial numbers if applicable Removed from Service and Disposal 79

80 Traceability Why Do I need to Write all of this stuff down? How will this help me 80

81 Troubleshooting 81

82 Definition A form of problem solving, often applied to repair failed products or processes. It is a logical, systematic search for the source of a problem so that it can be solved, and so the product or process can be made operational again. Troubleshooting is needed to develop and maintain complex systems where the symptoms of a problem can have many possible causes. 82

83 Troubleshooting Start with the simplest thing that could have gone wrong and work toward more complex problems 83

84 Troubleshooting Preparation Sample Preparation Correct volumes used to prepare aliquot for analysis Matrix matching Holding time of prepared aliquot Sample Preparation Prep logs reviewed against SOPs- correct sample volume, matrix, extract solvent Observations recorded that could impact results Instrument preparation Conditions Environment concerns 84

85 Troubleshooting Analysis/ Data Review Complete SOP review and objective evidence all steps were followed Comparison of bench sheet with other typical bench sheets Responses QC recoveries Unusual performance that did not result in failed QC QC trending and acceptability evaluation Bias or unusual recoveries Review of Calibration Standards preparation and responses Correlation Coefficient 85

86 Troubleshooting Instrumentation Review Analytical Conditions Internal Standard and/or surrogate recoveries Maintenance records Was there maintenance performed before PT analysis? Was there maintenance that was overdue? Does there need to be a defined maintenance schedule? 86

87 Troubleshooting Corrective and Preventive Action are the Results of Effective Troubleshooting Root Cause Analysis Description of what needs to be corrected Troubleshooting Records reviewed Systems evaluated Corrective Action Preventive Action

88 Troubleshooting Implementing Corrective and Preventive Action: Communicate changes Update SOPs Track completion and implementation using a database or spreadsheet Follow-up

89 Questions 89