CHM250 Calibration and Measurement Lab Green Profile

Balance Calibration

Introduction: Balances that are properly operated, calibrated and maintained are crucial for laboratory operations. The accuracy of all quantitative results is based on the ability of the balances to produce accurate and reproducible measurements within the strictest of quality control limits. For balances to work properly, they must be placed in a suitable environment. The work area should be relatively free from drafts and vibrations. Work surfaces must be level and rigid. The ideal balance location is free of significant temperature fluctuations. The balance must be cleaned and leveled properly before use. Any unclean balance surface could be a source of contamination. Balances must be clean, zeroed, leveled and free of all debris. Balance lids and doors should be closed and hoods replaced after use. Avoid dropping anything directly onto the balance pan as the shock may affect or damage the balance.

The weight of a tarred vessel affects the accuracy and precision of a balance. The combined weights of the tarred vessel and material must be less than the maximum operating range of the balance. For example, if the analyst is weighing into a tarred 50 g beaker, the balance will behave as if it were loaded to greater than 50 g. Good laboratory procedure must be employed when weighing small amounts into large tarred amounts. Specific analytical methods may address this issue of choosing the appropriate balance and weighing techniques. A balance should not be used outside of its operating range or its limited usage range.

When weighing, place the object directly in the center of the pan. The potential inaccuracy of off-center weighing will be magnified if the balance is not leveled properly. Use extreme care when handling the “certified” weights. If a weight is dropped, inform your instructor. If a weight is inadvertently touched with a non gloved hand, see the manufacturers recommended cleaning procedures.

Balance Calibration Procedure: 1. Clean the balance with a suitable brush (camel hair or equivalent). Level the balance, if necessary. Please wear gloves to handle the weights and use the tongs provided in the boxes. 2. Perform the calibration using the designated weights. Record the mass of the weight, observed mass in grams, and the difference between the two numbers. a. Perform linearity and reproducibility tests. The linear range will be defined by seven weights. The difference values should be between +/-0.00003 for weights less than 1 grams, +/-0.0005 for weights less than 25 grams, and +/-0.002 for heaver weights b. The reproducibility test will consist of at least 10 weighings of a mass at approximately 10% of the balance’s maximum capacity. Suggested weights for the analytical balances, 50.0g for the upper range and 200 mg for the lower range. Make sure you calculate the mean, standard deviation, and relative standard deviation (%RSD). 3. Record all of your work in your laboratory notebook. Before you leave lab, make sure someone record checks your values. If you want to use excel to calculate the values you may, and you can use excel to directly record your values (your primary data).

Automatic (Eppendorf) Pipettes Introduction: Eppendorf pipettes are used to deliver small (μL), adjustable volumes of solutions. These pipettes have disposable plastic tips that fit onto a reusable hand-held pipette. The tip is filled and the solution dispensed with a push-button on the top of the pipette. Automatic pipets are designed to deliver aqueous solutions with an accuracy of within a few percentage points. The amount of liquid actually dispensed varies, however, depending on the viscosity, surface tension, and vapor pressure of the liquid. The typical automatic pipet is very accurate with aqueous solutions but is not always as accurate with other liquids. Pipettes should be used in their calibrated range, typically 10-100% of delivery volume.

Automatic pipettes must never be dipped directly into the liquid sample without a plastic tip. The pipette is designed so that the liquid is drawn only into the tip. The liquids are never allowed to come in contact with the internal parts of the pipette. The plunger has two detent, or “stop,” positions used to control the filling and dispensing steps. Most automatic pipettes have a stiffer spring that controls the movement of the plunger from the first to the second detent positions. The plungers of the pipettes should move smoothly and freely. The pipettes should be clean and free of visible residue. Some pipettes need the tip wetted before use. If this is needed for accurate delivery, the tip is wetted by drawing and expelling the liquid at least three times. Air displacement pipettes may be used in two modes, forward and reverse. In the forward mode the plunger is depressed to the stop, the liquid is pulled into the pipette tip and the plunger is completely depressed to expel the liquid. To reverse pipette, depress the plunger completely, pull up the liquid into the pipette tip, then expel the liquid by depressing the plunger to the stop.

Review the owners manual to make sure that the pipette is being used in the correct mode. Use the following technique when transferring volumes with an Eppendorf pipette: 1. Select the volume to be transferred by turning the knob near the top of the pipette. 2. Place a disposable tip on the end of the pipette 3. Push the button down to the first stop 4. Place the tip into the solution to be transferred and release the button 5. Push the button to the second stop to completely empty the tip.

Solutions which contain volatile organics are difficult to pipette. The vapor pressure generated by the organic material will force the solution out the pipette tip (if possible switch to glass pipettes or glass syringes). Positive displacement pipettes rely on plunger settings and are used for viscous samples. For accurate delivery of viscous solutions always use a positive displacement pipette.

Automatic (Eppendorf) Pipettes Procedure 1. Room temperature DI water will be used for all calibrations. No temperature correction is required if water temperature is between 13 C and 27 C. Measure and record water temperature and thermometer identification number (if applicable). 2. Variable Volume - For variable volume air displacement pipettes (single delivery), calibrate at a low- (~10% of total volume), a mid- (~50% of total volume) and a high- (100% of total volume) volume. For variable volume repipettors with various sizes of tips, calibrate using one tip at the lowest, middle and highest setting of the pipette. a. Pipette six aliquots of DI water at the lowest volume recording the weight of each aliquot. Calculate the average weight of the aliquots. Divide the resulting average by the specific gravity of water (0.998 g/mL). b. The difference between the pipette’s set volume and the delivered observed volume must be within 2% for the pipette to be calibrated (% recovery between 98.0 – 102%). The % RSD should be within 2%. % recovery: Avg (g) x 100 0.998 g/mL x expected volume (mL)

3. Repeat at the mid and high volume. 4. Calibrate at least two pipettes (i.e. a 10 mL pipette and a 1 mL pipette). Make sure you also record the Pipette Model and Serial Number as well as which balance you used. 5. Make sure your worksheet is record checked in your research notebook.

Calibration of Volumetric Glassware1 An important trait of a good analyst is the ability to extract the best possible data from his or her equipment. For this purpose, it is desirable to calibrate your own volumetric glassware (burets, pipets, flasks, etc.) to measure the exact volumes delivered or contained. This experiment also promotes improved technique in handling volumetric glassware.

Calibrating a 50-mL Buret - This procedure tells how to construct a calibration graph such as the one below to convert the measured volume delivered by a buret to the true volume delivered at 20°C.

1. Fill the buret with distilled water and force any air bubbles out the tip. See whether the buret drains without leaving drops on its walls. If drops are left, clean the buret with soap and water or soak it with cleaning solution.2 Adjust the meniscus to be at or slightly below 0.00 mL, and touch the buret tip to a beaker to remove the suspended drop of water. Allow the buret to stand for 5 min while you weigh a 125-mL flask fitted with a rubber stopper. (Hold the flask with a paper towel to prevent fingerprints from changing its mass.) If the level of the liquid in the buret has changed, tighten the stopcock and repeat the procedure. Record the level of the liquid. 2. Drain approximately 10 mL of water at a rate < 20 mL/min into the weighed flask, and cap it tightly to prevent evaporation. Allow 30 s for the film of liquid on the walls to descend before you read the buret. Estimate all readings to the nearest 0.01 mL. Weigh the flask again to determine the mass of water delivered. 3. Drain the buret from 10 to 20 mL, and measure the mass of water delivered. Repeat the procedure for 30, 40, and 50 mL. Then do the entire procedure (10, 20, 30, 40, 50 mL) a second time. 4. Use the table of water density at the end of this experiment to convert the mass of water to the volume delivered. Repeat any set of duplicate buret corrections that do not agree to within 0.04 mL. Prepare a calibration graph like the one above, showing the correction factor at each 10-mL interval.

EXAMPLE, Buret Calibration, When draining the buret at 24°C, you observe the following values: Final reading 10.01 10.08 mL Initial reading 0.03 0.04 Difference 9.98 10.04 mL Mass 9.984 10.056 g Actual volume delivered 10.02 10.09 mL Correction +0.04 +0.05 mL Average correction +0.045 mL

To calculate the actual volume delivered when 9.984 g of water is delivered at 24°C, use the conversion factor 1.003 8 mL/g in the table of water density (see below). We find that 9.984 g occupies (9.984 g)(1.003 8 mL/g) = 10.02 mL. The average correction for both sets of data is +0.045 mL.

To obtain the correction for a volume greater than 10 mL, add successive masses of water collected in the flask. Suppose that the following masses were measured:

Volume interval (mL) Mass delivered (g) 0.03–10.01 9.984 10.01–19.90 9.835 19.90–30.06 10.071 Sum 30.03 mL 29.890 g The total volume of water delivered is (29.890 g)(1.003 8 mL/g) = 30.00 mL. Because the indicated volume is 30.03 mL, the buret correction at 30 mL is –0.03 mL. What does this mean? Suppose that the calibration graph shown above applies to your buret. If you begin a titration at 0.04 mL and end at 29.00 mL, you would deliver 28.96 mL if the buret were perfect. The calibration graph tells you that the buret delivers 0.03 mL less than the indicated amount; so only 28.93 mL was actually delivered. To use the calibration curve, either begin all titrations near 0.00 mL or correct both the initial and the final readings. Use the calibration curve whenever you use your buret.

Density of water

a. Corrected for buoyancy with Equation 2-1 in the textbook b. Corrected for buoyancy and expansion of borosilicate glass (0.001 0% K-1)

Calibrating Other Glassware Other volumetric glassware can also be calibrated by measuring the mass of water they contain or deliver. Glass transfer pipets and plastic micropipets can be calibrated by weighing the water delivered from them. A volumetric flask can be calibrated by weighing it empty and then weighing it filled to the mark with distilled water. Perform each procedure at least twice. Compare your results with the tolerances listed in tables in Chapter 2 of the textbook. Graduated or Volumetric Pipettes Introduction: A less-expensive means of delivering known quantities of liquid is to use a graduated pipette. Because they are made of glass, they are inert to most organic solvents and reagents. Disposable serological pipettes may be an attractive alternative to standard graduated pipettes. Never draw liquids into the pipettes using mouth suction. A pipette bulb or a pipette pump must be used to fill pipettes. The calibrations printed on graduated pipettes are reasonably accurate, but you should practice suing the pipettes in order to achieve this accuracy. When accurate quantities of liquids are required, the best technique is to weigh the reagent that has been delivered from the pipette. Procedure 1. Automatic Pipette: Accurately weigh a HPLC vial, with screw cap and Teflon insert, on a balance or a 20mL scientilation vial. Determine its weight to the nearest milligram (0.001g). Using the automatic pipette, dispense 0.500 mL of water into the vial, replace the cap assembly (with the insert arranged Teflon side down), and weigh the vial a second time. Determine the weight of water dispensed. Calculate the density of water from your results. Repeat this process three times and record the average numbers. Repeat the experiment with 0.500mL of hexane. Dispose of any excess hexane in a designated waste container. Calculate the density of hexane from your data. Repeat this process three times and record the average numbers. Record the results in your notebook, along with your comments on any deviations from literature values that you may have noticed. At room temperature, the density of water is 0.997 g/mL, and the density of hexane is 0.660 g/mL. 2. Dispensing Pump: Accurately weigh a 20 dram vial, with screw cap, on a balance. Determine its weight to the nearest milligram (0.001g). Using a dispensing pump that has been adjusted to deliver 2.5 mL, dispense 2.5 mL of water into the vial, replace the cap assembly, and weigh the vial a second time. Determine the weight of water dispensed. Repeat this process three times. Calculate the density of water from your results. Record the results in your notebook, along with your comments on any deviations from the literature value that you may have noticed. 3. Graduated Pipette: Accurately weigh a HPLC vial, with screw cap and Teflon insert, on a balance. Determine its weight to the nearest milligram (0.001g). Using a 1.0mL graduated pipette, dispense 0.500 mL of hexane into the vial, replace the cap assembly (with the insert arranged Teflon side down), and weigh the vial a second time. Determine the weight of water dispensed. Repeat this process three times. Calculate the density of hexane from your results. Record the results in your notebook, along with your comments on any deviations from literature values that you may have noticed. 4. Volumetric Pipette: Accurately weigh a HPLC vial, with screw cap and Teflon insert, on a balance or a 20mL scientilation vial. Determine its weight to the nearest milligram (0.001g). Using a 1.0mL volumetric pipette, dispense 1.0 mL of hexane into the vial, replace the cap assembly (with the insert arranged Teflon side down), and weigh the vial a second time. Determine the weight of hexane dispensed. Repeat this process three times. Calculate the density of hexane from your results.

Final Questions: Answer the following questions in your lab notebook. 1. Criticize the following techniques a. A 100 mL graduated cylinder is used to measure accurately a volume of 2.8 mL. b. A one-piece polyethylene transfer pipette is used to transfer precisely 0.75 mL of a liquid that is being used as the limiting reactant. c. The volume markings on a 100 mL beaker are used to transfer accurately 5 mL of a liquid. d. A 5 mL automatic pipette is used to transfer a total of 10 mL of a liquid. 2. What measuring device would you use to measure the volume under each of the conditions described below? a. 5 mL of a solvent needed for a crystallization b. 0.76 mL of a liquid sample to be added to a known volume of solvent c. 1 mL of a solvent needed to dilute a sample No lab report is required for this lab. Show your laboratory notebook to your instructor to receive credit for this lab.

References 1 Adapted from Experiments To Accompany Quantitative Chemical Analysis, 8th Edition (2010) Daniel C. Harris

2 Prepare cleaning solution by dissolving 36 g of ammonium peroxydisulfate, (NH4)2S2O8, in a loosely stoppered 2.2-L (“one gallon”) bottle of 98 wt% sulfuric acid. Add ammonium peroxydisulfate every few weeks to maintain the oxidizing strength. EOSULF is an alternative cleaning solution for removing proteins and other residues from glassware in a biochemistry lab. EOSULF contains the metal binder EDTA and a sulfonate detergent. It can be safely poured down the drain. [P. L. Manske, T. M. Stimpfel, and E. L. Gershey, J. Chem. Ed. 1990, 67, A280.]