Chapter 4 Measurements, Solutions and Calibration
Similar to the production of high quality goods by master craftsmen, the analyst must use measurement tools of the highest quality, in order to prove an answer to a laboratory test that is indicative of the actual amount of the analyte in the sample. The tools the analyst uses are volumetric glassware, analytical balances and analytical instruments. To a large extent the accuracy of these tools is directly related to the initial cost and the time spent on maintenance and calibration. Each of these categories of tools and their care are the topics of this chapter.
Reading scales and displays generally involve seeing and recording numbers. Please, please remember that 'numbers by themselves mean nothing'. The engineering units are vital to knowing what the numbers mean. Always, always append the engineering unit [inches, kilos, kps]. Use appropriate notations [standard, fixed or scientific] and calculate according to significant numbers.
Erlenmeyer flasks and beakers are not volumetric measuring tools. They are, in many cases, marked with approximate volume indication. However, even in the most reliable flasks and beakers, there is a ±5 % uncertainty in where the volume line actually belongs. There are only five volumetric measuring devices recognized as suitable for accurate and precise analytical work. They are: burets, volumetric flasks, volumetric (transfer) pipets, glass measuring pipets and graduated cylinders. Produced and calibrated in accordance with ASTM standards.1 They are available in either of two grades, Class A and Class B. Class B is sometimes called student or economy grade, although in certain cases these designators indicate containers of even lower quality than Class B.
The ASTM standards define the tolerances within which the markings are placed on the glass, with Class A glassware having the smallest tolerances. The Class B tolerances are, in general, twice the acceptance range of the Class A values (see Tables 4-1 through 4-4). All plastic (Teflon®, polypropylene, polymethylpentene and polyethylene) volumetric containers are Class B. Class A volumetric glassware will always have a large “A” prominent near the label on the piece. Glassware without an obvious “A” is Class B or of lesser accuracy.
Some of the glassware manufacturers have been offering two types of Class A calibrated volumetric ware. The first is a generic Class A line of glassware. The second type is a certified/serialized Class A and comes complete with documentation and traceability to NIST, which is required for certification to ISO 9000 standards.
|
Table
4-1. Comparison of Class A and Class B tolerances for
Volumetric Flasks. Tolerances
are in mL. |
Table
4-2. Comparison of Class A and Class B tolerances for
Pipets. Tolerances are in mL. |
Table 4-3. Comparison of Class A and Class B tolerances for Graduated Cylinders. Tolerances are in mL. |
Table
4-4. Comparison of Class A and Class B tolerances for
Burets. Tolerances are in mL. |
||||||||
| Nominal Volume mL | Class A tolerance | Class B tolerance | Nominal Volume mL | Class A tolerance | Class B tolerance | Nominal Volume mL | Class A tolerance | Class B tolerance | Nominal Volume mL | Class A tolerance | Class B tolerance |
|
10 |
±0.02 |
±0.04 |
1 |
±0.006 |
±0.012 |
10 |
±0.08 |
±0.1 |
10 |
±0.02 |
±0.04 |
|
25 |
±0.03 |
±0.06 |
5 |
±0.01 |
±0.02 |
25 |
±0.14 |
±0.3 |
25 |
±0.03 |
±0.06 |
|
50 |
±0.05 |
±0.10 |
10 |
±0.02 |
±0.04 |
50 |
±0.20 |
±0.4 |
50 |
±0.05 |
±0.10 |
|
100 |
±0.08 |
±0.16 |
25 |
±0.03 |
±0.06 |
100 |
±0.35 |
±0.6 |
100 |
±0.10 |
±0.20 |
|
250 |
±0.12 |
±0.24 |
50 |
±0.05 |
±0.10 |
250 |
±0.65 |
±1.4 |
|||
|
500 |
±0.20 |
±0.40 |
100 |
±0.08 |
±0.16 |
500 |
±1.10 |
±2.6 |
|||
|
1000 |
±0.30 |
±0.60 |
1000 |
±2.00 |
±5.0 |
||||||
|
2000 |
±0.50 |
±1.00 |
2000 |
---- |
±10.0 |
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Pipets are used for accurate volume measurements and transfer. There are three types of pipets commonly used in the laboratory: volumetric pipets, graduated or Mohr pipets, and serological pipets. Volumetric pipets are used to deliver a single volume. Measuring pipets (graduated) will deliver fractions of the total volume.
Pipets come in a variety of shapes and sizes. Measuring pipets can be differentiated from volumetric pipets by the presence of a graduated volume scale on the side of the measuring pipet. There are two types of graduated pipets commonly found in the laboratory. The measuring pipet (Mohr) is graduated from a zero mark near the top of the pipet to a baseline near the tip of the pipet. Mohr pipets are intended to indicate the delivered volume of liquid by the difference between the initial and final liquid position, with delivery of the maximum calibrated volume leaving the tip of the pipet full of liquid. Mohr pipets come in calibrations with Class A and Class B tolerances, which are the same as those of the volumetric pipets. A serological pipet is graduated from a zero mark near the top of the pipet to the very tip of the pipet. It can be used to indicate the difference between the initial and final liquid levels similar to the Mohr pipet, however to deliver the whole calibrated amount, the pipet is blown out with a pipet bulb such that no liquid remains in the tip. The best serological pipets are available only with Class B tolerances. Pipets with wide tips for measuring sludges and other viscous liquids are serological pipets. Disposable serological pipets made of glass or plastic are available and calibrated to Class B or lesser accuracy.
Volumetric pipets have a bubble in the shaft of the pipet and, in general, a single calibration mark. Volumetric pipets come in “To Contain” (TC) and “To Deliver” (TD) calibrations. There is a special volumetric pipet called a Dual Purpose pipet which has calibration marks with Class A tolerances for both TC and TD use. The recommended procedure for emptying volumetric pipets is: held in the vertical position; unrestricted outflow; tip touched to wet surface or receiving vessel and kept in contact until the emptying is complete; Under no circumstances should the small amount remaining in the tip be blown out.
Proper use of volumetric glassware
The first consideration in the use of volumetric glassware is the temperature. Glass more closely resembles a very viscous solution than it does a crystalline solid. The properties of glass can be varied greatly by varying the additives. For example, addition of B2O3 produces a glass (called borosilicate glass) that expands and contracts little under large temperature changes. Thus it is useful for labware and cooking utensils. Glass is an amorphous solid containing a good deal of disorder. Not only do different types of glass have different rates of expansion but different areas of the same piece of glass have different rates of expansion, so, there is no universal correction factor for temperature variation. Instead, the temperature at the time of calibration is normally etched on the piece. When none is etched, 20º C is assumed.
Furthermore, the solutions change volumes, most expanding when raising the temperature. Water is significantly unique in its behavior, shrinking in volume from 0 to 4º C, thereafter expanding with temperature rise. This volume variation affects the reagent concentration. Diluting most acids and bases in water is exothermic, whereas dissolving ammonium chloride is endothermic. Therefore, final volume adjustments should always be delayed until the solution’s temperature has stabilized at the initial temperature.
‘Dilute to the mark’ and ‘read the meniscus’ are common instructions for preparation of solutions or titrating. Most calibration marks on volumetric glassware extend all the way around, aiding orientation of the eye and the container in order to see a single straight line just intersecting with the bottom curve of the liquid.
If the container is a TC device, the entire contents are the correct volume. Then transferring the contents to another container, a quantitative transfer must be made. This entails blowing out the pipet with a pipet bulb and rinsing the inside of the pipet with additional solvent from a squeeze bottle, or rinsing the flask or cylinder with additional solvent.
For TD containers, a flowtime must be observed.
Proper dispensing of solution from a TD pipet requires touching the pipet tip to the inside wall of the
receiving container and maintaining contact for at least the time determined by Table 4-5.
The reason is to allow all of the water film on the inside of the pipet to drain off, so you get the full
accuracy the pipet is capable of. If you watch, you can see that
takes a while. Really accurate pipets, like class A, should be designed
with narrow enough holes in the tip to make them drain so slowly that the film draining would keep up with the
bulk draining, and the timing could take care of itself.
Table 4-5.
Flowtimes for TD pipets
Nominal Volume mL
Class A Flowtime (sec)
Class B Flowtime (sec)
1
10
3
10
15
8
25
25
15
50
25
15
100
30
30
For solutions which have a different density or viscosity than pure water, the flowtimes are not correct, and the analyst should consider using a TC pipet because the error associated with the retained solution in the tip of the TD pipet generally exceeds the manufacturing tolerance of the TC pipet. Similar consideration apply to the use of pipet pumps and pipet bulbs. Certainly the analyst should never pipet by mouth, especially in a wastewater laboratory, therefore pipetting aids are necessary. However the calibration of the pipet is performed assuming only gravity is affecting the liquid flow and it is not being pushed out of the pipet rapidly by air pressure.
A solution consists of a dissolved substance, the solute, and a dissolving medium, the solvent. A solution is a homogeneous mixture and has a constant composition throughout. A solute need not be a solid. If the solution contains two liquids, the liquid that is in excess is conventionally called the solvent. The most common solvent is water.
Titration is an experimental procedure in which the unknown concentration of a known volume of solution is determined by measuring the volume of a solution of known concentration required to react completely with it.
The three types of titration reactions are:
The four general steps used during a chemical titration are:
When using a buret for dispensing solutions, such as in a titration, the correct procedure is to fill the buret to the very top, then open the stopcock and allow liquid to drain out until the air bubble in the tip of the buret is flushed. Refill the buret, drain until the meniscus matches the zero mark and record the volume. Perform the titration by adding drops from the buret to the rapidly stirred solution until the permanent endpoint is close (very near), indicated by the color change first occurring on addition of the drop, then rapidly fading back to the initial state. Very accurate work can be performed by opening the stopcock a little until a partial drop appears, closing the stopcock and the using a squeeze bottle to air wash the liquid off the tip of the buret and into the titration solution. Remember to wash the sides of the titration flask down with a little water to insure that all the reagent delivered to the flask has reacted with the solution. Before pouring the titrant into the buret, be sure mix it so that any condensed water vapor on the upper surfaces of the inside of the bottle is recombined with the rest of the solution. Otherwise the titrant will be too strong. this is important when pipetting standard solutions out of bottles. Record the end volume off the buret onto the benchsheet (lab data book). The fastest work with a buret is achieved by performing a rough titration on an aliquot of sample to give an idea of the volume required, then taking a second aliquot of sample, rapidly adding a little less than the volume needed from the buret, then carefully finishing the titration in a dropwise fashion. The most accurate results are obtained by performing careful titrations in duplicate, then averaging the values.
Documentation: Running all these tests will be effective only when properly documented. Neat, legible and complete information written (or recorded) will communicate to others, less is unacceptable. Document the raw data as soon as possible. Verify. The information is useful only if accurate and complete. Schedule time for transferring data to files for legal requirements and process control analysis. The lab technician will have completed the task ONLY when the results of the sample analysis are properly documented.
There is no standard laboratory form (Benchsheets). Most operators usually develop their own data sheets for recording test results and other important data. These data sheets should be prepared in a manner that makes it easy for you to record results, review them, and recover these results when it is necessary. Each facility will have different needs for collecting and recording data and may require several different data or worksheets or benchsheets.
Volumetric glassware should never be placed in an oven. The etched markings on the glassware weaken the piece at that point and the stresses from the heating are sufficient to cause a crack. Sterilization in an autoclave is permissible but allow a long slow cool down on the order of one to two hours. Likewise, graduated cylinders should never be used for mixing solutions. The exothermic reactions are sufficient to crack the container where etching identifies volumes.
Calibration of nonstandard volumetric measuring devices
There are numerous tools used in the laboratory for fluid measuring and transfer which one would like to calibrate to the maximum accuracy possible. Examples include BOD bottles, colorimetric tubes, syringes, diluters, bottle-top dispensers and disposable tip adjustable and fixed volume pipetters. These can easily be calibrated. All one needs is an analytical balance, a source of reagent water, a standardized thermometer, a calibration logbook and a table of water densities (or specific gravity) such as that found in the Handbook of Chemistry and Physics.
First, let everything equalize to room temperature and place the thermometer in the container of reagent water. Dispense (measure) a volume of water from the device to be calibrated onto the analytical balance. Record the weight in grams of water dispensed. Repeat this at least two more times, then determine the average weight. Record the temperature. Look up the density of water in the table and divide the average weight of water dispensed by the density (g/mL). The resulting number is the actual volume dispensed in mL.
Calibration of bottles and other containers is performed by calculating the weight of water at the ambient temperature corresponding to the desired volume, then adding water to the container while on the balance until the proper weight is obtained. The meniscus position is permanently marked by etching a line on the bottle with a diamond point pen or file. Once a glass container is etched with a volume marking it should never be subjected to thermal stress.
For devices which have moving parts and require preventative maintenance such as the diluters, dispensers and pipetters, this procedure should be repeated at regular intervals, such as weekly. The reason for this is that it serves as a check on the state of the seals and lubricants inside the devices. For example, one lab checks the calibrations on their disposable tip pipetters weekly and calculates the relative variation on the mean of three determinations from the true volume, and the relative standard deviations of the three determinations. For a 1,000 mL adjustable pipetter the first week gave 1.0141 mL (±1.59% variation) and 0.15% RSD, the second week gave 1.0170 mL (±1.86%) and 0.65 RSD and the third week gave 1.0454 mL (±4.8%) and 5.1% RSD indicating the seals/lubricant were failing. After cleaning/repair and relubrication, the calibration of the pipetter gave 1.0066 mL (±0.9%) and 0.28% RSD. Other than checking, the calibration on a regular basis, there was no indication that the device was in a failing mode. For adjustable volume pipetters, at least two volumes, one high and one low, should be checked. Some have found that these types of devices are irregular in their failures, but generally require major cleaning or replacement every one to three months.
Analytical balances
The weighing of solids in the laboratory is essential for preparing reagents and it is the final determinative instrument for solids analyses. It is quite rare to see the old chain and arm balances in the wide glass and wood cabinets still in use in analytical labs, however these are just as usable as modern electronic balances. In fact, some new instruments work on the same principle. Most analytical balances are reproducibly sensitive to the ten thousandth of a gram (0.00001 g)(1 mg = .001 g, 0.01 mg = 0.00001 g) if situated on a vibration free platform in an area not subject to stray air currents or temperature variation. Analytical balances have limited working ranges, often only to 100 or 200 grams. The more sensitive the balance, the smaller the range. Less sensitive balances such as top-loaders and mechanical triple beam scales should be used in situations where greater range is needed.
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