Department of Biochemistry Faculty of Science Chulalongkorn University 2310 221 Measurement of Weights and Volumes Dr. Somporn Kamolsiripichaiporn, 081-802-8766 ************************************************************* IMPORTANT GROUND RULES Students should have the following items reachable during the laboratory work: • Lab coat – wear at all times, name tag is appreciated • Eye protection – wear lab glasses (safety goggles) at all times to protect against impacts and splashes • Wear well covered shoes for own safety • Tissue paper for general use • Hand towel – to dry hand after washing • Experiment towel – for general use • Recording notebook and pen • Labeling pen Students should take care of their personal belongings: handbag, mobile phone, books, etc., and make sure they are safe from chemical spill and contamination. SAFETY POLICY 1.
If you should get a chemical into your eyes, wash with flowing water from the eyewash for approximately 15-20 minutes, and notify staff. 2. Never use an open flame when working with organic solvents. 3. In case of fire or accident, notify your teacher at once. (Note location of fire extinguisher and safety shower now so that you can use them if needed. Wet towels are very effective for smothering fires.) 4. Take special care when working with strong acids or strong bases. Contact with these materials can cause severe chemical burns. 5. Do not touch hot glassware or hot hardware. Think before you act. 6. Do not taste anything in the laboratory. (This applies to food as well as chemicals. Do not use the laboratory as an eating place and do not eat or drink from laboratory glassware.) 7. Exercise great care in noting the odor of fumes and, whenever possible, avoid breathing fumes of any kind. 8. When working with electrical equipment, observe caution in handling loose wires and make sure that all equipment is electrically grounded before touching it. 9. Avoid working alone in the laboratory. 10. To minimize hazard, confine long hair securely when in the laboratory.
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GLASSWARE AND EQUIPMENT FOR MEASUREMENT OF WEIGHTS AND VOLUMES I. Laboratory Wares, Glass Wares and Plastic Wares
Beakers Erlenmyer flasks Volumetric flasks Boiling flask Crucibles (porcelain, aluminum oxide, silica, platinum) Bottle Petri-dish Measuring cylinder Tubes Centrifuge tube
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Micro-tube Tubing Funnel Homogenizer Weighing bottle Dessicator Pipet Burette Cuvette etc.
Choice of use depends on • • • • •
Volume Purpose of use: transfer, storage, reaction, heating Property of substance Type and property of plastic (polyallomer, polyprolylene, polycarbonate) Disposable
Cleaning • • • •
Wash with detergent solution. Rinse with large amount of tap water. Final rinse with several small portions of deionized water (properly cleaned glassware will be coated with a uniform and unbroken film of water). Dry in the oven if necessary.
II. Volumetric Equipment • • •
•
•
Pipet, buret, volumetric flask, measuring cylinder. They are marked by the manufacturer to indicate the manner of calibration. TD (Ex) for “to deliver” - When the graduation line indicates the volume delivered from the vessel, the ware is marked “TD”. TC (In) for “to contain” at certain temperature - When the graduation line denotes the volume contained in the calibrated vessel, the ware is marked “TC”. The standard temperature for glass volumetric apparatus is 20oC and all apparatus is calibrated by manufacturers to contain or deliver the indicated capacity at this temperature.
Pipet
A pipet (also called a pipette or a pipettor) is a laboratory instrument used to transport a measured volume of liquid. There are many types of pipets. 2
Two types of markings on the pipet: TC or TD - A pipet with TC marking has been calibrated to contain a specified volume of liquid. - A pipet marked TD (the more common type of pipet) has been calibrated to deliver a specified volume of liquid.
Volumetric (Transfer) Pipets – fixed volume Volumetric pipets are designed to transfer a fixed amount of liquid when filled to the mark (e.g. 10 mL and only 10 mL). There is generally only one "fill-line" on a volumetric pipet.
Pipet stand
Measuring (graduated) Pipets
Mohr pipets are graduated but the graduations stop at a baseline before the pipet begins to narrow (see arrow on left). To accurately transfer fluid with this type of pipet, the meniscus must be precisely on a calibration mark both at the beginning and at the end of a transfer. Serological pipets are the type mostly used. Serological pipets have no base mark (the graduations continue onto the tip) and are graduated to deliver. Blow-out and not blow-out (gravity feed system) ???
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Examples - KIMBLE pipet: Calibrated to deliver to the tip, with two rings near the top, denoting that the small amount remaining in the tip after free delivery must be blown out and added to the main volume delivered. ASTM Specification E1380 for color coding All pipets are graduated to the tips and have permanently marked bands near the top end (unless otherwise specified) to indicate that the small amount remaining in the tip after free delivery has ceased must be blown out to obtain the total rated capacity.
Proper method of delivering contents 1. Draw liquid above top graduation using bulb or equivalent device. Always use a suction bulb when filling a pipet. Never do the suction by mouth! 2. Use forefinger (not thumb) for level control. 3. Wipe any drops off outside surface. 4. Zero meniscus at top graduation. Your eyes must be at the level of the liquid surface to avoid error due to parallax. 5. Dispense in vertical position with unrestricted outflow. 6. Touch off last remaining drop to surface of receiver (2 seconds after free-flow ceases).
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7. Only serological type pipets require last drops to be “blown out”. Do not “blow-out” volumetric (transfer) or meaasuring (Mohr) pipets as they are calibrated for after “touch-off” to remain. 8. Avoid contamination of the pipet tip if repeated use is required 9. Protect yourself from chemical around the pipet tip. Sources of error in pipetting 1. Failure of properly align the meniscus with the volume mark. 2. Parallax error: Your eye must be level with the volume mark and the pipet vertical. If you are looking up at the pipet, the meniscus will be too high when it appears to align with the mark. If you are looking down at the pipet, the meniscus will be too low when it appears to align with the mark. 3. Forcing the solution out of the pipet causes too much to be delivered. 4. Using a dirty pipet causes too little or contaminated solution to be delivered. Leaving little droplets behind on the walls (except for the small amount in the tip) causes too little solution to be delivered. 5. Forcing the pipet into the bulb usually causes liquid to be sucked into the bulb. This contaminates the bulb which must then be thoroughly rinsed and dried. 6. Allowing the tip of the pipet to rise above the liquid in the container usually causes the liquid to be sucked into the pipet bulb. 7. A broken or chipped pipet can reduce the amount of liquid held after transfer. This causes too much liquid to be delivered. Cleaning pipet • • •
Use a rubber bulb to draw detergent solution to a level 2 to 3 cm above the calibration mark. Drain the detergent solution and rinse well with tap water. Final rinse with de-ionized water thoroughly by filling in water and rotate the pipet to wet the entire interior surface.
For cleaning a number of pipets at the same time, rinse with tap water and dip in a container containing mild detergent with the point tip up and make sure the tips are covered with detergent liquid. Wash well with water and a final rinse of de-ionized water.
Automatic Siphon Pipet Washer - Rinser •
Constructed of high-density polyethylene which resists corrosive action of common cleaning solutions, reduces scratching and breakage of glassware.
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Inlet connector is near base for stability; water enters chamber at top to prevent back flow.
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Pipet Baskets • Fit washing chambers of corresponding size. • Perforated bottom plate is recessed for complete drainage.
Autopipet • • •
Pipetman, Eppendorf pipettor, Finpipet, Integrapette. Polypropylene tips – different color for different size. Different brands have different preference and advantage. Before using or purchasing, check if the pipet can be used with concentrated acid or certain solvents.
Single Channel Autopipet Plunger
Volume adjustment wheel
Volume indicator
Polypropylene tip
Tip ejector
Tip ejector
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How to use a Pipetman 1. Adjust the digital volume indicator by turning the volume adjustment wheel. For example, to use the P200 transfer 58 microliters, the digits should read 058. One should always dial down to the desired volume. Be gentle in order to avoid damaging the pipettor. Do not force the Pipetman to a setting that is higher than it is designed for. 2. Attach a disposable tip to the Pipetman. Do not touch the end of the tip with your fingers! 3. The plunger has two stops. To draw liquid into the tip, first depress the plunger to the first stop. Immerse the disposable tip into the sample solution and release the pressure on the plunger, allowing the plunger to return to the initial up position. Do not let the plunger pop or snap up. If you do, sample will be drawn into the Pipetman, contaminating the sample and Pipetman.
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4. Withdraw the tip from the sample solution and dispense the sample from the Pipetman. To dispense into an empty vessel, touch the end of the disposable pipette tip on the side wall of the vessel and depress the plunger to the first stop. Wait 1-2 seconds (especially for viscous solutions like glycerol) and then continue to depress the plunger to the second stop. If you are transferring a sample into another solution, place the pipette tip into the solution and then dispense. 5. Without removing pressure from the plunger, withdraw the tip from the tube or solution, and then allow the plunger to return to the up position. If you release the pressure on the plunger while the tip is in the solution, you will pull solution into the tip again. 6. Never invert or lay Pipetman down if liquid is in the tip. Do not use Pipetman to pipet concentrated acid! 7. Calibrate by weighing water several times and calculate mean and SD. Check the manual given by the manufacturer. 8. Clean tips by detergent solution, tap water and de-ionized water. Sonicator bath is useful or stir in detergent with magnetic bar. Different sizes of Pipetman Pipetman model
Adjustable range (μl)
Recommended range (μl)
Smallest increment (μl)
P-2
0–2
0.1 – 2
0.002
P-10
0 – 10
0.5 – 10
0.02
P-20
0 – 20
2 – 20
0.02
P-100
0 – 100
10 – 100
0.2
P-200
0 – 200
50 – 200
0.2
P-1000
0 – 1000
100 – 1000
2.0
P-5000
0 – 5000
500 – 5000
2.0
Reading the volume setting The volume indicator consists of three number dials and is read from top to bottom. For P2, P-10, P-20, P-100 and P-200, the black digits indicate microliters, while the red digits indicate tenths and hundredths of microliters. For P-1000, the red digits indicate milliliters and the black digits indicate microliters. Checking for accuracy Periodically test your Pipetman by measuring the weight of water that it delivers. For example, for a P-1000, ensure that the 1 ml setting delivers 1 g of water and the 50 μl setting delivers 50 mg.
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Safety issue The P-5000 uses a safety filter in the end of the shaft to help prevent liquid entering the shaft and contacting the piston, should the plunger be allowed to snap up during aspiration. This is particularly important using large volumes. If the filter gets wet, replace it.
Multichannel Autopipet
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Glass Syringe
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Hamilton syringe Used for accurate dispensing of volumes as small as 1 microliter Tight fitting metal plunger in a glass tube
How to use glass syringe properly 1. Draw solution into the syringe and expel it several times to wet the inside of the glass and the needle. 2. Slowly draw solution up until the plunger is at the line you want. 3. Watch carefully for bubbles forming. If you get bubbles between the solution and the plunger, expel the solution quickly to shoot the bubble out. 4. Repeat until you have no bubbles in the syringe. 5. Wash the syringe out repeatedly with water. Never leave salts or organics in the syringe or the plunger will become permanently locked. 6. Be careful not to bend the plungers. They bend easily and once bent are useless. Pasteur Pipet •
Non-calibrated disposable glass pipet
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Transfer liquid when volume is not important
These unbreakable all-in-one pipets eliminate the hazard of broken glass and exposure to infectious materials. Unlike glass Pasteur pipets, there is no need to attach a rubber bulb because the bulb is integrally molded into the product. Molded from see-through low density polyethylene, these pipets are inert to biological fluids and most acids. The low affinity surface reduces the loss of cells and valuable proteins due to binding. They work well wherever there is a need for quick, safe transfer of fluids.
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Dispenser
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To transfer aliquots of solution
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An aliquot is a measured fraction of the volume of a liquid sample
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Adjustable volume pipet dispensers for repetitive dispensing of precise liquid aliquots
One excess drop of liquid makes big error!!!!!
III. Selection and Handling of Reagents and Chemicals Choice of chemicals – – –
reagent grade primary standard grade (reference standard) special purpose reagent, e.g. spectrophotometric solvent
Rules for handling reagents and solutions: 1. Select the best grade for analytical work. Pick the smallest bottle that will supply the desired quantity. 2. Replace the top of every container immediately after removal of the reagent. 3. Make sure the stoppers of reagent bottles are kept clean, not lying around on the desktop. 4. Never return any excess reagent back to a bottle unless specifically directed. 5. Never insert spatulas, spoons or knives into a bottle that contains a solid chemical, shake vigorously or tap gently against a wooden table to break up any encrustation.
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6. Keep the reagent shelf and the laboratory balance clean and neat. Clean up any spills immediately. 7. Check how to dispose unwanted reagents and solutions. 8. Consult the Material Safety Data Sheet (MSDS) for safety and precaution in handling chemicals.
Centrifuge Tubes
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Reference: Beckman centrifuge manual IV. Weighing Balance – a delicate instrument that you must handle with care! • • •
Precision balance – up to 8000 g capacity, readability 0.1 – 0.001 g (100-1 mg) Analytical balance – up to 300 g capacity, readability 0.1 – 0.01 mg Micro and ultramicro balance – to 3 g capacity, readability 0.001– 0.0001 mg
Electronic Balances • •
Most are equipped with a built in standard weight, thus making periodic re-calibration simple and convenient. With some, periodic re-calibration is performed automatically. Automatic taring control: causes the display to read zero with a weighing container on the pan. Most balances permit taring to 100% of capacity.
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Analytical balance
Precision balance
Microbalance
Precautions • • •
Center the load on the pan as well as possible. Protect the balance from corrosion. Keep the balance and its case scrupulously clean.
Weighing chemicals normally kept in the freezer • • •
Allow the chemical to adjust to room temperature before weighing. Weigh the chemical as quickly as possible. Return the chemical to freezer as soon as finish weighing.
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A guide to proper use of the balance (NB: different brand and type of balance may vary) Using an Analytical Balance: Modified from http://www.dartmouth.edu/~chemlab/techniques/a_balance.html Do check the level indicator bubble before weighing. The two rear balance feet serve as leveling screws. Clean the balance pan with brush. Turn the balance on by pressing the control bar. The display lights up for several seconds, then resets to 0.0000.
Place creased, small weighing paper on the balance pan.
Close the sliding glass doors. Wait for the green dot on the left to go out. This is the stability indicator light, indicating that the weight is stable. (Some balance may not have this indicator light.)
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Press the control bar to cancel out the weight of the container or paper. The display will again read 0.0000.
Carefully add the substance to be weighed up to the desired mass. Do not attempt to reach a particular mass exactly.
Before recording the mass, close the glass doors and wait until the stability detector lamp goes out. Record mass of solid. TIPS: Don't lean on the bench while weighing. Make sure that the balance is placed on sturdy table or bench. Do record the mass of your container, if you will need it later. Clean up Use the brush provided to clean spills in the weighing chamber. Discard any disposable tare containers, weighing paper in the nearest wastebasket.
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V. Preparing Solution For simple solution: 1. Calculate the amount of chemical from required concentration and volume. If too small amount of chemical is needed, increase the volume or concentration to the level that can be weighed accurately (e.g. make ten times more concentrated and diluted later to the required concentration before use). 2. Weigh chemicals. 3. Transfer all chemical to appropriate glassware (beaker, Erlenmeyer flask or volumetric flask with powder funnel). 4. Dissolve well with water (de-ionized or distilled depending on use) or other solvent. Magnetic stirrer is useful for this step.
5. Transfer to volumetric flask or measuring cylinder and adjust to the final volume required. Mix well and transfer to appropriate container for storage.
Be careful when using distilled water bottle - difficult to control the flow and can get overshoot easily!
Use pasteur pipet with its tip touching the wall of the container and let water flow slowly
For solution with required pH: 1. 2. 3. 4.
Calculate the amount of chemical from required concentration and volume. Weigh chemicals. Transfer all chemical to beaker of appropriate size. Dissolve with water (de-ionized or distilled depending on use) or other solvent to 80% of required volume. Stir with magnetic stirrer. 5. Measure pH with pH meter. Be careful not to place electrode too close to the magnetic bar. 17
pH meter
6. Adjust pH with acid or base using pasteur pipet (or burette) until achieving the required pH with gentle stirring. Transfer to volumetric flask or measuring cylinder and adjust to the final volume required. 6. Mix well and transfer to appropriate container for storage. Mixing the solution in small tube • • • •
Finger tapping the tube for small volume. Parafilm wrapping and invert tube with the thumb pressing on the parafilm. Vortex – use proper tube. Stirring/heating plate with magnetic bar.
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Measuring pH - PH Meter
Portable pH meter
Bench top digital pH meter • • •
Some systems have a direct sensor electrode and a separate calomel reference electrode. Others have both electrodes built into a single electrode stem. Follow the instruction given for each pH meter. In addition to the pH meter of analog type, modern pH meters are digital and computerized. Portable pH meter is convenient for field work.
How to use pH meter in general 1. Remove the electrode(s) from the storage solution and rinse them off thoroughly with a squirt bottle. 2. Immerse the electrode(s) into a reference solution of known pH that is near the desired pH to calibrate the instrument. 3. With the electrode(s) immersed in the reference solution, switch the pH meter from standby mode to pH mode. 4. Correct the pH reading to match the pH of the reference solution. 5. Put the pH meter on standby and remove the electrode(s) from the reference solution. 6. Rinse the electrode(s) thoroughly with distilled water and place it in the sample solution. Swirl slightly and allow a few seconds for the pH value to stabilize before recording the pH. Adjusting pH of a solution Mix the solution well before measuring the pH. Add small amounts of acid or base, as appropriate and mix the solution until a stable reading is obtained. • Continue the operation until you achieve the desired pH. • If only slight adjustment is needed, use diluted acid or base so that you do not overshoot the desired value. • Be careful not to break the electrode during the adjustment. • •
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Be careful with the electrode
Storage and labeling of solution • • • • • • • •
Do not store solution in the glassware used for preparing or measuring solution, e.g. beaker, volumetric flask, Erlenmyer flask. Choose suitable container: type, materials and volume. Avoid using breakable materials. Consider the chemical property of the solution. NB: Alkaline solution has to be kept in plastic bottle while acid is kept in glass bottle. For solution of small volume, store in small tubes or bottles and place them in paper or plastic boxes. Store solution at appropriate temperature – room temperature, 4oC, -20oC or –70oC For samples to be analyzed, store in appropriate aliquots and use one at a time. Freeze and thaw effect – denature protein or enzyme. Check if chemical is light sensitive. Protect by aluminum foil wrap or store in brown/red bottle. Label clearly on the sticker in ink – your real name, name and concentration of solution, chemical identity, date prepared, warning if any (corrosive, toxic, carcinogen, etc.)
VI. Errors Chemical analyses are affected by two types of errors: •
Random or indeterminate error – causes data to be scattered more or less symmetrically around a mean value. Caused by many uncontrollable variables that are inevitable parts of every physical and chemical measurement. Example: in the calibration of a pipet - visual judgement of meniscus, drainage time and angle of the pipet, viscosity of the liquid
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•
Systematic or determinate error – causes the mean of a set of data to differ from the accepted value. It causes the results in a series of replicate measurements to be all high or low. Definite value, assignable cause and same magnitude for replicate measurements made in the same way. Example: Instrument errors – corrected by calibration (periodic calibration of equipment is always desirable because the response of most instruments change with time as a result of wear, corrosion and mistreatment). Method errors – instability of some species, side reaction, incompleteness of reaction, contamination. Personal errors – minimized by care and self-discipline, create habit to check instrument readings, notebook entries and calculations systematically.
Gross error – occur occasionally and are often large and may cause a result to be either high or low. It leads to outliers – differ markedly from all other data in a set of replicate measurements. (Gross errors are undetected mistakes that cause a measurement to be very much farther from the mean measurement than other measurements, http://www.definitionseek.com/chemistry/gross_error.html) VII. Validation Criteria Precision •
Describes the closeness of a series of measurements or agreement among several results that have been obtained under the prescribed conditions.
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Provides an indication of random errors.
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High precision – measurements are closely grouped, i.e. random errors are small.
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Precision of a set of replicate data – described by standard deviation, variance and coefficient of variation.
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Repeatability – precision under same conditions: analyst, apparatus, reagents, time interval, etc.
NB: Reproducibility – using same method but under different conditions, e.g. different laboratories, analysts, reagents, apparatus, etc. Accuracy •
Indicates the closeness (conformity) of a measurement to its true or accepted value (agreement between a result and its true value).
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•
Accounts for all types of error, particularly useful for systematic errors.
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Expressed by the absolute or relative error.
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Absolute error = Xi – Xt
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Relative error = ( Xi - Xt ) / Xt
(t = true or accepted value)
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x 100 %
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Accuracy and Precision
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Undesirable – Potential quality is high due to low intrinsic noise but the actual quality is poor because the value is biased
Accuracy and precision reflect how well the test method performs day to day in a laboratory Example: Determination of nitrogen in two pure compounds
Absolute error in the micro-Kjeldahl determination of nitrogen. Each dot represents the error associated with a single determination. Each vertical line labeled (xi – xt) is the absolute average deviation of the set from the true value. (Data from C.O. Willits and C. L.Ogg, J. Assoc. Anal.Chem., 1949, 32, 561)
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Sensitivity •
Capacity of the test procedure to record small variations in concentration Example: Protein determination - Biuret method VS Lowry’s method
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The smallest change in the measured quantity which consistently causes the output of the measuring instrument to change (responsiveness)
Resolution •
The smallest increment of change in the measured value that can be determined from the readout or recording instrument
Reliability Sensitivity to gross errors or a measure of the ease with which gross errors can be detected •
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Highly reliable result – small outliers can be noticed
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Unreliable result – large outliers will go unnoticed
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Reliability of a clinical lab test is determined by 4 indicators
NB: Sensitivity and specificity, dealing with how well the test is able to distinguish presence of disease from absence of disease •
Sensitivity is the proportion of true positives that are correctly identified by the test, i.e. ability of a test to correctly identify individuals who have a given disease or condition. - A test is 90% sensitive means the test can correctly identify the disease of 90 cases out of 100 people who are known to have that disease.
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Specificity is the proportion of true negatives that are correctly identified by the test i.e. ability of a test to correctly exclude individuals who do not have the disease or condition. - If 100 healthy individuals are tested and only 90 of those are found normal, the test is proven to be 90% specific.
References: 1. Analytical Biochemistry, Second Edition, by David J. Holme and Hazel Peck 2. Analytical Chemistry – An Introduction, Sixth International Edition, by Skoog/West/Holler 3. www.mt.com 4. www.dartmouth.edu 5. http://home.clara.net/rod.beavon/lab_book.htm 6. http://www.labtestsinline.org/understanding/features/realibility-2.html 7. http:/www.gmat.unsw.edu.au/anap/gps/gps_survey/chap2/241relb.htm
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