Thank you for registering for this Best Practices for Volumetric Measurement on-demand course. This course is designed to provide refresher training to those who regularly perform volumetric measurements or to provide initial information for new laboratory personnel. We hope you find this course useful. This course like all of the courses currently offered in our program is made possible through a cooperative agreement between RTI International and the National Institute of Justice. We appreciate the funding from NIJ.
This course presents an overview of the volumetric measurements of liquids, proper techniques, evaluation of equipment, documentation, QA/QC and more.
For this module we will discuss the types of volumetric equipment and best selection criteria based on the needed measurement. Each of these equipment types will be discussed including uses and operation, troubleshooting, general maintenance, proper documentation quality assurance and control measures and safety.
Results of the graduate students indicated that the % inaccuracy, as indicated by the y-axis, measured as the mean of 10 readings for a student ranged from -10% to almost 30%, the imprecision measured as the %CV indicated by the x-axis, ranged from 1% to 37%, and only one of ten subjects’ measurements fell within the manufacturer’s specifications for inaccuracy and imprecision for this pipette, noted by the light blue area. Similarly, results for experienced laboratory technicians was even higher with % inaccuracy measured from -15% to greater than 35% and their imprecision ranged from less than 1% to almost 70%. These experienced technicians with years of pipetting experience tended to be more imprecise than inaccurate. An additional study by this group to investigate the effect of training and practice on pipetting technique demonstrated that a short training course given to the same 10 graduate students consisting of brief oral instruction in the theory of proper pipetting technique followed by 10 minutes of practice dramatically improved their pipetting technique and continued practice led to even better and acceptable accuracy and precision in pipetting technique of less than 5%.
Volumetric simply refers to the amount of space a substance occupies which can be accurately measured. This course will present an overview of the most commonly used volumetric glassware for forensic scientists including flasks, pipettes, burettes and graduated cylinders. Liquid handling equipment including dispenser pipettes and bottle dispensers are covered as well. There are other glassware types in the laboratory, such as Erlenmeyer flasks and beakers which should not be used for volumetric measurements rather only to estimate volumes. These are the major six categories of volumetric laboratory equipment we will discuss within the time limitation of this course.
Volumetric glassware will be discussed first. Here is a list of the topics we will cover including composition and classification, markings, meniscus readings, and specifics about each type of volumetric glassware.
For pipettes, waiting and delivery times are also tied to the classification scheme. The Delivery time is the time it takes for the meniscus of the liquid to flow from the top marking to the tip of the pipette or lower marking on the pipette. The waiting time follows the delivery time and it starts when the meniscus stops on it own. The waiting period allows the residual liquid to flow down the walls of the pipette causing the meniscus to rise again. At the end of the waiting period the meniscus should be reset and the liquid on the tip of the pipette should contact the inside wall of the receiving vessel and a 'pipette tip' should be rolled inside the vessel to allow the final liquid drop to exit pipette into vessel. Blow out pipettes do not require a waiting period, but generally blow-out pipettes are not recommend in forensic applications as they do not conform to accuracy requirements. Fortunately, blowout pipettes are generally marked on outside of glassware for easy recognition. Volumetric pipettes are classified based on their accuracy and delivery times. Class A pipettes are the highest grade pipettes produced with highly accurate instrumentation and batch verified for conformity of accuracy and precision. Class A pipettes are officially certified for their degree of accuracy, error limit and coefficient of variation which are defined by regulating bodies such as ISO and ASTM. Class A and Class AS have the same error limits but differ in their waiting and delivery times as 'AS' indicates a more swift delivery. Class A glassware such as cylinders usually have ring marks at major increments whereas Class B glassware has short marks for graduation. Class A and AS pipettes can be supplied with an individual serial number or not. Serialized pipettes are provided with a certificate of analysis and are usually sandblasted with a number to match the certificate. They are certified using calibrated instruments and following standardized procedures. Class B pipettes typically have twice the limits for accuracy, error limit and coefficient of variation in comparison to Class A glassware.
Here is a bulb pipette depicting many common markings. On the top right, you can see the conformity and standardization symbols followed by the reference temperature for standardization of 20 degrees Celsius, a commonly used temperature. Next, the classification of this pipette is designated as AS and the EX designation tells us it is a ‘To Deliver’ pipette. The last marking on the right is the country of origin. The manufacturer and the specific brand trademark are on the top left of the pipette. Finally, volumetric glassware may also have a marking detailing its tolerance and the nominal volume. For example, this pipette was validated to measure 25 mL plus or minus three-hundredths of a milliliter. which means it can measure between 24.97 and 25.03 mL. If this accuracy is sufficient, then it is safe to use the nominal value. On the other hand, if one wishes to obtain the highest possible accuracy it would be necessary to calibrate the pipette in one's own laboratory setting with the appropriate liquid to be measured.
Another term in the proper use of glass ware is the term meniscus. A meniscus describes the curvature in the surface of a liquid. When reading a meniscus to determine if you have reached the fill volume this should be done at eye level to prevent parallax error. It is easier to read the meniscus if a dark back ground is placed behind the glassware if the liquid is clear. Alternatively, a burette card which is usually a white card marked with a dark line can be held behind the glassware to contrast the line between glassware markings and the liquid level. The curvature is caused by the surface tension of the liquid to be attracted to the walls of the volumetric equipment which causes a concave or downward meniscus of adhesion. While a liquid’s attraction towards itself causes a convex or upward curvature meniscus of cohesion. Water has adhesive properties demonstrating a concave meniscus and mercury has cohesive properties demonstrating a convex meniscus.
The first category of glassware we will discuss is volumetric flasks. A volumetric flask is a flat-bottomed flask with a narrow neck. The top of the neck is specially ground-glass which accommodates a tight-fitting glass stopper or plastic cap. The calibration mark is a single gradation line on the neck indicates the exact volume the flask will contain at a specified temperature marked on the flask. This temperature is usually 20°C. Volumetric flasks are used to make solutions of known concentration by the dissolution of a known mass of solid or the dilution of a more concentrated solution. Volumetric flasks can be glass or a chemically resistant plastic such as polypropylene. Volumetric flasks come in two different standards with the higher standard flasks being Class A. Class A flasks are made with a more accurately placed graduation mark and can have a unique serial number for traceability and should be used when making important solutions such as calibrators or standards. Class B or equivalent flask are used to prepare solvents or solution for which a lower standard is permitted. Most of you have heard the term Q.S. which for all purposes means 'quantity sufficient' which indicates the volume needed to reach the predetermined and calibrated volumetric amount. Frequently used single volumes for flasks range from 10.00 to 1000 mL with significant figures of tolerance between a tenth to a hundredth of a decimal place beginning at the lowest volume. These zeros may be omitted in common use but they should be considered in calculations, as they indicate the accuracy of the volume measurement. Volumetric flasks less than 25 mL should be Class A accuracy if being used to prepare standards or solutions that require high accuracy.
Let’s look in more detail of the capacity of a set of typical graduated cylinders ranging from 10 milliliters to 1000 milliliters. In this table the graduation interval is the range of milliliters a specified graduated cylinder can measure. A primary marking is indicated by an extended or pronounced line that goes completely around the graduated cylinder in a designated increment, which is usually the first number of the graduation interval. So, a 250 mL graduated cylinder can measure 10 to 250 mL and primary markings every 10 milliliters, but that is not to say it can do so precisely over its entire graduation interval. A simple rule to keep in mind is that a narrower graduated cylinder permits a more precise measurement than a broader one because the same error in the height of the liquid leads to a smaller error in measured volume. The next column gives the Sub-Interval which is usually indicated by a less pronounced secondary mark that may not go completely around the graduated cylinder. The last column gives the error of measuring the liquid between two subintervals. So, for a 10 mL graduated cylinder a sub-interval is designated every 0.1 milliliters with a tolerance of plus or minus one-hundredth of a milliliter and your best estimate 9.1 mL mark would be between 9.01 and 9.19 mL. However, it should be kept in mind that graduated cylinders are not as precise as a volumetric flask, especially over its entire graduation interval. Graduation cylinders can have as much as a 10% error rate at a lower volume which can make it unacceptable in many instances for volumetric measurements. It is essential to evaluate which volumetric glassware is most appropriate for the job at hand.
When filling a volumetric flask with a desired liquid, transfer assistance can be achieved with the use of glass or disposable Pasteur or transfer pipettes, any of which should be investigated for contribution of contaminants or interferents for any assay the solution will be used in. In addition, funnels can be used to help transfer a liquid into a volumetric flask, taking precautionary steps to use the proper size to prevent spills or tipping of glassware. If a long stem funnel such as this is used it should not go below the fill lines. Sometimes a tight seal will cause backup and leakage at the funnel-flask seal and this can be prevented by using fingers to slightly loosen the seal and pouring more slowly. Again, plastic and glass funnels are available or should be checked for possibility of contaminant or interferent contributions. Finally, a wash bottle with the primary filling liquid can be used to rinse the transferring glassware, such as a beaker or graduated cylinder, and the funnel several times to insure that all weighed material is entirely transferred to the volumetric flask.
In today’s forensic laboratories pipettes, no matter the type, are probably the most utilized and essential volumetric instrument. Their accuracy and precision are vital to the defensibility of forensic results. We will begin this section by discussing glass pipettes and then move to positive displacement, dispenser and automatic pipettes. Their forensic applications are primarily to prepare analytical standards are calibrators. When possible, most laboratories use faster pipettes with disposable parts that can minimize sample contamination. We will discuss these pipetting aids in a few moments. There are all types of glass pipettes including bulb pipettes which measures a single volume in increments of 1 to 50 mL. It is known as a bulb pipette because of its characteristic bulging filling chamber in the glass about half way up its length. Alternatively, a straight-walled, graduated pipettes for which the calibration marks are confined along the length of the stem, as in Mohr pipette, or extended to the tip, as in serologic pipette can be used. The arrows indicate that the meniscus must be precisely on a calibration mark both at the beginning and at the end of a transfer for Mohr and a larger volume remains after delivery than with a serological pipette for which the measured liquid in the pipette exits all the way down to the tip. Graduated pipettes are commonly manufactured for 1 to 10 mL volumes with 0.1 to 0.5 mL increments, respectively. Bulb pipettes are usually more accurate, with an error of ± 0.1 or 0.2 mL. There are capillary pipettes which can measure volumes well below 200 microliters and glass pipettes which have been invented for specific uses for handling blood such as Sahli or blood dilution pipettes. As detailed earlier, glass pipettes have many markings, are classified by calibrated accuracy, speed and delivery temperature.
The two types of volumetric equipment we will discuss today are dispenser pipettes and bottles dispensers. Because both offer speed and facilitate repetitive measurements, they are very common in forensic laboratories.
A brief description of pipetting techniques is warranted for discussion of dispenser pipettes. For electronic and manual dispensing pipettes, forward pipetting techniques is the most common method and the most accurate and precise. Liquid is aspirated up into the tip of the pipette. Then, with one full movement of the piston with a controlled and steady removal of your finger or pushing the necessary button for an automatic, electronic pipette, all the liquid in the tip is discharged. This technique ensures that no liquid remains in the tip after pipetting and touching the side of the vessel with the pipettor tip helps to ensure no liquid is left on the tip. Reverse pipetting technique can be employed as well. The first steps of aspirating the liquid are the same as forward pipetting, but only a portion of the liquid is dispersed upon delivery. After delivery, the remaining liquid can be returned to stock or discarded prior to removal of the tip. Reverse pipetting is used for high viscosity, biological or foaming liquids, or very small volumes, this technique allows a selected volume plus an excess to be aspirated into the tip. Dilutions can be achieved by the simultaneous dispensing of two different liquids in different volumes. The first volume is aspirated, followed by an air gap, then the second volume. The two are then dispensed in one action. Generally, an electronic pipettor is used for this technique to improve throughput and reduces fatigue. For the same benefits, dispensing multiple aliquots of a single fluid using an electronic pipettor can be used for preparing serial dilutions. Some electronic models allow sequenced dispensing involving programming a sequence of different volumes to dispense. This is particularly useful for serology and related applications. Finally, the mixing of fluids is often undertaken during dilution procedures. Either a manual pipettor or various electronic models can perform mixing techniques.
Here are a few best practices for use of dispenser pipettes. The density of air is affected by temperature. Pipetting is no exception to this rule. The volume delivered by a pipette varies with air pressure, relative humidity, and vapor pressure of the sample; all of which are temperature dependent. It is also important to properly immerse the pipette tip 2 to 5 mm below the meniscus and clear of the container wall and bottom during sample aspiration to ensure proper volume. Try to use a consistent plunger pressure and speed with your pipetting technique by using a consistent and controlled motion. Lastly, minimize handling of the pipette and tip to prevent body heat transfer temperature effects. This can be done by simply setting the pipette down between prolonged deliveries. However, always use a pipette cradle and do not place flat.
When adjusting the volume it is important to remember not to adjust beyond the upper and lower range capacities. Take steps to handle all pipettors with care to prevent falls. Pipette cradles are the place one should place an unused pipette. Contamination can be an issue with dispenser pipettes. The pipette shaft assembly should be protected by not operating the pipette without a tip, not laying a filled pipette on its side, preventing immersion of the barrel and by not allowing the plunger to snap up when liquid is being aspirated. Excessive heat can damage a pipette and they should not be used to close to a flame.
Not only do pipettes have the common parts we see on the outside including the plunger, tip release button, digital display window for the measured volume, shaft or barrel with discharging end and the disposable pipette tips, but there are many internal parts as well. All dispenser pipettes have various moving parts including a springs, a piston and its seal, and piston housing or outer shell. There are many points of contact which must be checked regularly and maintained in good working order to prevent failure. The spring mechanism can be protected by setting the pipettor to its highest volume between uses to release pressure. Many forensic laboratories choose to have scheduled maintenance and calibration checks either internally or opting for an external vendor to ensure pipettors are routinely tested for compliance. Each dispenser pipette can and will be different in some way. New forensic scientists, even trained scientists changing laboratories or laboratory duties should have the competency of their pipetting skills verified. In addition, some type of in-service training should be provided before implementation of a new dispenser pipette, including competency verification. Forensic laboratories are set-up with standard operating procedures which in large part are routinely performed in the same environment by the same analyst. This type of maintained operational environment is optimal because it promotes constant evaluation of pipetting technique and external factors, observation of the pipette’s state of repair, and accountability. Assign pipette unique and traceable identification numbers to assist with documentation of maintenance and calibration and troubleshooting failed procedures. This will expedite resolution of a problem. With many repetitive motions required with pipetting it is important for safety and to prevent injury by evaluating the ergonomics and design of a dispenser pipette. Fatigue-free operation reduces or prevents muscular aliments of the hand, neck, arms and shoulders many times associated with repetitive motions such as pipetting.
Use of bottle-top dispensers are ideal for serial work of repetitive nature Which require rapid and consistent delivery of a liquid. Unlike glass pipettes, bottle dispensers do not have waiting times and their delivery time is less as well. Similarly, bottle dispensers have replaces graduated cylinder pours in many instances for forensic lab work for the aforementioned reasons. Bottle dispensers easily deliver 0.1 to 100 ml aliquots as stated earlier there are even dispenser burettes available commercially.
Another type of volumetric equipment is a bottle dispenser. Bottle dispensers are great for repeated discharge of identical quantities of liquid, usually reagents, at a measured amount for delivery. Major components of a bottle dispenser include five major parts: First, there is either a bottle-top lid that is part of a bottle dispenser unit complete with the bottle or direct dispenser for reagent bottles that can be affixed to a specific reagent bottle volume such as a 4 Liter bottle. Second there is a measuring cylinder with its accompanying piston. Types of pistons include floating piston and spring-loaded ‘wiping-seal’ piston. Floating piston are less resistant to corrosive reagents than spring-loaded pistons. There is some type of valve system necessary to move the liquid out of the dispensing bottle and into the receiving vessel. For glass measuring cylinder, a protection sleeve is another important component. Lastly, a discharge tube works with the valve system in dispensing the liquid.
Accuracy is how close to the intended target a measurement is. PRECISION is the reproducibility and repeatability of achieving the same or similar measurement many times using different operators and equipment or the same operator and equipment, respectively. If you take this graphical representation and move in a counter clockwise rotation first observing the upper left bull’s eye, you see that it has high precision but low accuracy. The upper right bull’s eye is the best case scenario have both highly precise and accurate measurements. The lower right bull’s eye has high accuracy but low precision and finally the lower left bull’s eye is both imprecise and inaccurate. You can not correct for low precision but you can correct for bias or the difference between the mean of measurements and the reference or target value. Inaccuracy or a high bias can be corrected as part of the calibration process. For volumetric equipment, accuracy and precision are implicitly measured by the significant figures of their tolerance or error rate.
We have discussed many types of volumetric glassware and equipment. Now it is time to discuss some key points for proper volumetric measurements. This section covers calibration, accuracy and precision, quality assurance and cleaning.
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RTI - Volumetrics
<ul><li>Agenda Why the Hype Overview </li></ul>INTRODUCTION
<ul><li>Volumetric Equipment </li></ul><ul><li>Best Practices for Laboratory Equipment Selection </li></ul><ul><li>Uses and Operation </li></ul><ul><li>Troubleshooting and General Maintenance </li></ul><ul><li>Proper Documentation </li></ul><ul><li>Quality Assurance </li></ul><ul><li>Safety </li></ul>Agenda INTRODUCTION
<ul><li>Volumetric Flasks </li></ul><ul><li>Glass Pipettes </li></ul><ul><li>Graduated Cylinders </li></ul><ul><li>Dispenser Pipettes </li></ul><ul><li>Bottle Dispensers </li></ul><ul><li>Burettes </li></ul>Overview INTRODUCTION Erlenmeyer and beakers should not be used to measure only to estimate. The amount of space a substance occupies.
<ul><li>Classification </li></ul><ul><ul><li>Type (Class A, AS, and B) </li></ul></ul><ul><ul><li>Determination </li></ul></ul><ul><ul><ul><li>suitability for official certification </li></ul></ul></ul><ul><ul><ul><li>degree of accuracy and error </li></ul></ul></ul><ul><ul><li>Graduation </li></ul></ul><ul><ul><li>Serial Number </li></ul></ul>Classification <ul><li>Waiting & Delivery Times (pipettes) </li></ul><ul><ul><li>Guided by application and equipment design </li></ul></ul><ul><ul><li>Classification (A vs. B) </li></ul></ul>VOLUMETRIC GLASSWARE
Markings VOLUMETRIC GLASSWARE BRAND trademark for the highest quality grade volumetric instruments Manufacturer Nominal volume Tolerance (error limit) DIN Certification mark Reference temperature (20 o C) calibration (TD, Ex = to deliver), waiting time(15 sec.) Class 'A', the highest quality grade, 'S' for swift delivery Country of origin The symbol for the conformity certification by a certifying organization
Reading the Meniscus <ul><li>CONVEX upward (cohesion) </li></ul><ul><ul><li>high point reading </li></ul></ul><ul><ul><li>mercuric </li></ul></ul><ul><li>CONCAVE downward (adhesion) </li></ul><ul><ul><li>low point reading </li></ul></ul><ul><ul><li>aqueous </li></ul></ul>VOLUMETRIC GLASSWARE Remember! - eye level reading - contrasting background
<ul><li>Types </li></ul><ul><ul><li>Glass </li></ul></ul><ul><ul><li>Plastic </li></ul></ul><ul><ul><li>Class A & B </li></ul></ul><ul><li>Use </li></ul><ul><ul><li>Single volume (10-1000 mL) </li></ul></ul><ul><ul><li>Q.S. ('quantity sufficient') means 'bring to volume' </li></ul></ul><ul><ul><li>High accuracy volume </li></ul></ul><ul><ul><li>TC at 20 degrees Celsius </li></ul></ul>Flasks VOLUMETRIC GLASSWARE
Graduated Cylinders *Source: KIMAX® Class A, Serialized and Certified Kimax VOLUMETRIC GLASSWARE 0.25 1 3 to 50 50 0.4 1 5 to 100 100 0.8 2 10 to 250 250 10 5 0.2 0.1 Sub-Interval ( s econdary mark; mL) 2.5 50 to 1000 1000 1.3 25 to 500 500 0.01 1 to 10 10 0.17 2 to 25 25 Tolerance ( mL) Graduation Interval (primary mark; mL) Capacity (mL)
Pipettes VOLUMETRIC GLASSWARE <ul><li>Check temperature </li></ul><ul><li>Fill with liquid </li></ul><ul><li>Read meniscus </li></ul><ul><li>Wipe off </li></ul><ul><li>Deliver to wall of the vessel </li></ul><ul><li>'Rolling' wipe of pipette tip on wall </li></ul><ul><li>Strict observance of waiting time </li></ul>Best Practices - Steps of use
<ul><li>Bulb </li></ul><ul><li>Graduated </li></ul><ul><ul><li>Mohr </li></ul></ul><ul><ul><li>Serological/Tissue </li></ul></ul><ul><li>Capillary (to contain up to 200 µL) </li></ul><ul><li>Blood </li></ul><ul><ul><li>Sahli </li></ul></ul><ul><ul><li>Blood dilution </li></ul></ul>Pipettes VOLUMETRIC GLASSWARE
<ul><li>Allow sample to come to ambient temperature </li></ul><ul><li>Pipette immersion below sample meniscus </li></ul><ul><li>Maintain controlled plunger pressure and speed </li></ul>Dispenser Pipettes Best Practices - Steps of use VOLUMETRIC EQUIPMENT Minimize pipette and tip handling.
<ul><li>Do not rotate the volume adjuster beyond the upper/lower range capacity </li></ul><ul><li>Do not allow contamination of the shaft assembly of pipette by: </li></ul><ul><ul><li>operating without a tip </li></ul></ul><ul><ul><li>laying a filled pipettor on its side </li></ul></ul><ul><ul><li>immersing the barrel </li></ul></ul><ul><ul><li>allowing the plunger to snap up when liquid is being aspirated </li></ul></ul>Dispenser Pipettes Best Practices - Troubleshooting VOLUMETRIC EQUIPMENT Remember! Do not heat pipettes or pass through a flame. Handle with care to prevent falls.
<ul><li>Store pipettors set to their highest volume to releases pressure on the spring mechanism </li></ul><ul><li>Routine maintenance and calibration checks </li></ul><ul><li>Competency training </li></ul><ul><li>Maintain the environment </li></ul><ul><li>Assign pipette unique and traceable identification numbers </li></ul><ul><li>Evaluate ergonomics and design </li></ul>Dispenser Pipettes Best Practices - Troubleshooting VOLUMETRIC EQUIPMENT Plastic tip Discharge end Digital display window Plunger To discard tip
<ul><li>Serial work </li></ul><ul><li>Rapid and consistent </li></ul><ul><li>Replaces graduated cylinder pours </li></ul><ul><li>Capability </li></ul><ul><ul><li>0.1 to 100 ml </li></ul></ul><ul><ul><li>Dispenser burettes </li></ul></ul>Bottle Dispensers Best Practices - Use VOLUMETRIC EQUIPMENT Waiting time not required. Meniscus setting not required.
Bottle Dispensers <ul><li>Repeated discharge of identical quantities of liquid, usually reagents, at a measured amount for delivery </li></ul><ul><li>Major components </li></ul><ul><ul><li>bottle-top lid or direct dispenser bottle </li></ul></ul><ul><ul><li>measuring cylinder/piston (e.g., floating piston, spring- loaded “wiping-seal” piston) </li></ul></ul><ul><ul><li>valve system </li></ul></ul><ul><ul><li>protection sleeve for glass cylinder </li></ul></ul><ul><ul><li>discharge tube </li></ul></ul>VOLUMETRIC EQUIPMENT
Accuracy and Precision VOLUMETRIC EQUIPMENT ACCURACY LOW HIGH PRECISION LOW HIGH
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