Contaminated glassware will virtually ensure failure. Glassware Many contaminants, including organics and metal ions, adhere to the inner walls of glass containers. Washing glassware, including pipets, with dilute detergent 0.
The final rinse should be with distilled or deionized water. Metal ion contamination can be greatly reduced from glassware by rinsing with concentrated nitric acid followed by extensive rinsing with purified water.
Dry equipment is required for most processes carried out in biochemistry laboratory. When you needed dry glassware in organic laboratory, you probably rinsed the piece of equipment with acetone, which rapidly evaporated, leaving a dry surface. Unfortunately, that surface is coated with an organic residue consisting of nonvolatile contaminants in the acetone. Glassware and plasticware should be rinsed well with purified water and dried in an oven designated for glassware, not one used for drying chemicals.
Quartz and Glass Cuvettes. Never clean cuvettes or any optically polished glassware with ethanolic KOH or other strong base, as this will cause etching. All cuvettes should be cleaned carefully with 0. The most common solvent for solutions used in the biochemical laboratory is water. Ordinary tap water contains a variety of impurities including particulate matter sand, silt, etc.
In addition, the natural degradation of microorganisms leads to the presence of by-products called pyrogens. Tap water should never be used for the preparation of any reagent solutions. For most laboratory procedures, it is recommended that some type of purified water be used.
There are five basic water purification technologies-distillation, ion exchange, carbon adsorption, reverse osmosis, and membrane filtration. Most academic laboratories are equipped with "in-house" purified water, which typically is produced by a combination of the above purifying technologies.
For most procedures carried out in a biochemistry teaching laboratory, water purified by deionization, reverse osmosis, or distillation usually is acceptable. For special procedures such as buffer standardization, liquid chromatography, and tissue culture, ultrapure water should be used. The water quality necessary will depend on the solutions to be prepared and on the biochemical procedures to be investigated. Water that is purified only by ion exchange will be low in metal ion concentration, but may contain certain organics that are washed from the ion-exchange resin.
These contaminants will increase the ultraviolet absorbance properties of water. If sensitive ultraviolet absorbance measurements are to be made, distilled water is better than deionized. Solution Preparation. The concentrations for solutions used in biochemistry may be expressed in many different units. In your biochemistry laboratory, the most common units will be: Molarity M ; concentration per liter of solution. Many solutions you use will be based on molarity. For practice, assume you require 1 liter of solution that is 0.
Add about mL of purified water and swirl to dissolve. Then add water so that the bottom of the meniscus is at the etched line on the flask. Stopper and mix well. The flask must be labeled with solution contents 0. In general, solid solutes should be weighed on weighing paper or plastic weighing boats, using an analytical or top-loading balance. Liquids are more conveniently dispensed by volumetric techniques; however, this assumes that the density is known. If a small amount of a liquid is to be weighed, it should be added to a tared flask by means of a disposable Pasteur pipet with a latex bulb.
The hazardous properties of all materials should be known before use and the proper safety precautions obeyed. The storage conditions of reagents and solutions are especially critical. Although some will remain stable indefinitely at room temperature, it is good practice to store all solutions in a closed container. This inhibits bacterial growth and slows decomposition of the reagents. If these are aqueous solutions or others that will freeze, be sure there is room for expansion inside the container.
Stored solutions must always have a label containing the name and concentration of the solution, the date prepared, and the name of the preparer.
Volumetric flasks, of course, have glass stoppers, but test tubes, Erlenmeyer flasks, bottles, and. Remember that hydrocarbon foil, a wax, is dissolved by solutions containing nonpolar organic solvents. Bottles of pure chemicals and reagents should also be properly stored. Many manufacturers now include the best storage conditions for a reagent on the label. Many biochemical reagents form hydrates by taking up moisture from the air. If the water content of a reagent increases, the molecular weight and purity of the reagent change.
However, if this reagent is stored in a moist refrigerator or freezer outside a desiccator, the moisture content may change to an unknown value. Practical biochemistry is highly reliant on analytical methods. Many analytical techniques must be mastered, but few are as important as the quantitative transfer of solutions.
Some type of pipet will almost always be used in liquid transfer. Since students may not be familiar with the many types of pipets and the proper techniques in pipetting, this instruction is included here.
Filling a Pipet. The use of any pipet requires some means of drawing reagent into the pipet. Liquids should never be drawn into a pipet by mouth suction on the end of the pipet. Small latex bulbs are available for use with disposable pipets Figure 1AA. For volumetric and graduated pipets, two types of bulbs are available. One type Figure lAB features a special conical fitting that accommodates common sizes of pipets. To use these, first place the pipet tip below the surface of the liquid.
Squeeze the bulb with your left hand if you are a right-handed pipettor and then hold it tightly to the end of the pipet. Slowly release the pressure on the bulb to allow liquid to rise to 2 or 3 ern above the top graduated mark. Then, remove the bulb and quickly grasp the pipet with your index finger over the top end of the pipet. The level of solution in the pipet will fall slightly, but not below the top graduated mark.
If it does fall too low, use the bulb to refill. Mechanical pipet fillers sometimes called safety pipet fillers, propipets, or pi-fillers are more convenient than latex bulbs Figure 1. Equipped with a system of hand-operated valves, these fillers can be used for the complete transfer of a liquid. The use of a safety pipet filler is outlined in Figure 1.
Never allow any solvent or solution to enter thepipet bulb. To avoid this, two things must be kept in mind: 1 always maintain careful control while using valve S to fill the pipet, and 2 never use valve S unless the pipet tip is. Examples of pipets and pipet fillers. If the tip moves above the surface of the liquid, air will be sucked into the pipet and solution will be flushed into the bulb. Other pipet fillers are used in a similar fashion. Disposable Pasteur Pipets.
Often it is necessary to perform a semiquantitative transfer of a small volume 1 to 10 mL ofliquid from one vessel to another. Since pouring is not efficient, a Pasteur pipet with a small latex bulb may be used Figure I.
Pasteur pipets are available in two lengths 15 em and 23 em and hold about 2 mL of solution. These are especially convenient for the transfer of nongraduated amounts to and from test tubes. If dilution is not a problem, rinsing the original vessel with a solvent will increase the transfer yield.
Used disposable pipets should be discarded in special containers for broken glass. Calibrated Pipets. Although most quantitative transfers are now done with automatic pipetting devices, which are described later in the chapter, instructions will be. How to use a Spectroline safety pipet filler. Using thumb and forefinger, press on valve A and squeeze bulb with other fingers to produce a vacuum for aspiration.
Release valve A leaving bulb compressed. To deliver the last drop, maintain pressure on valve E, cover E inlet with middle finger, and squeeze the small bulb. If a quantitative transfer of a specific and accurate volume of liquid is required, some form of calibrated pipet must be used.
Volumetric pipets Figure 1AF are used for the delivery of liquids required in whole milliliter amounts 1, 2, 3, 4, 5, 10, 15,20,25, 50, and mL. To use these pipets, draw liquid with a latex bulb or mechanical pipet filler to a level 2 to 3 em above the "fill line. Release liquid from the pipet until the bottom of the meniscus is directly on the fill line.
Transfer the pipet to the inside of the second container and release the liquid. Hold the pipet vertically, allow the solution to drain until the flow stops, and then wait an additional 5 to 10 seconds. Touch the tip of the pipet to the inside of the container to release the last drop from the outside of the tip. Remove the pipet from the container.
Some liquid may still remain in the tip. Most volumetric pipets are calibrated as "TD" to deliver , which means the intended volume is transferred without final blow-out, i. Fractional volumes of liquid are transferred with graduated pipets, which are available in two types-Mohr and serological.
Mohr pipets Figure lAG are available in long- or short-tip styles. Long-tip pipets are especially attractive for transfer to and from vessels with small openings. Virtually all Mohr pipets are TD and are available in many sizes 0. The marked subdivisions are usually 0. Selection of the proper size of pipet is especially important. For instance, do not try to transfer 0. Use the smallest pipet that is practical. The use of a Mohr pipet is similar to that of a volumetric pipet.
Draw the liquid into the pipet with a pipet filler to a level about 2 em above the "0" mark. Lower the liquid level to the 0 mark. Remove the last drop from the tip by touching it to the inside of the glass container. Transfer the pipet to the receiving container and release the desired amount of solution. The solution should not be allowed to move below the last graduated mark on the pipet.
Touch off the last drop. Serological pipets Figure 1. Their use is identical to that of a Mohr pipet except that the last bit of solution remaining in the tip must be forced out into the receiving container with a rubber bulb. This final blow-out should be done after 15 to 20 seconds of draining. Automatic Pipetting Systems. For most quantitative transfers, including many identical small-volume transfers, a mechanical microliter pipettor Eppendorf type is ideal.
This allows accurate, precise, and rapid dispensing of fixed volumes from 1 to u. The pipet's push-button system can be operated with one hand, and it is fitted with detachable polypropylene tips Figure 1. Other useful information about pipetting is available at the Web site www. The advantage of polypropylene tips is that the reagent film remaining in the pipet after delivery is much less than for glass tips. Mechanical pipettors are available in up to 25 different sizes.
Newer models offer continuous volume adjustment, so a single model can be used for delivery of specific volumes within a certain range. To use the pipettor, choose the proper size and place a polypropylene pipet tip firmly onto the cone as shown in Figure 1.
Tips for pipets are. A How to use an adjustable pipetting device. B Set the digital micrometer to the desired volume using the adjustment knob. Attach a new disposable tip to the shaft of the pipet.
Press on firmly with a slight twisting motion. C Depress the plunger to the first positive stop, immerse the disposable tip into the sample liquid to a depth of 2 to 4 mm, and allow the pushbutton to return slowly to the up position and wait I to 2 seconds.
D To dispense sample, place the tip end against the side wall of the receiving vessel and depress the plunger slowly to the first stop. Wait 2 to 3 seconds, and then depress the plunger to the second stop to achieve final blow-out. Withdraw the device from the vessel carefully with the tip sliding along the inside wall of the vessel. Allow the plunger to return to the up position. Discard the tip by depressing the tip ejector button.
Photos courtesy of Rainin Instrument Company, Inc. Pipetman is a registered trademark of Gilson Medical Electronics. Exclusive license to Rainin Instrument Company, Inc. Details of the operation of an adjustable pipet are.
For rapid and accurate transfer of volumes greater than 5 mL, automatic repetitive dispensers are commercially available. These are particularly useful for the transfer of corrosive materials. The dispensers, which are available in several sizes, are simple to use. The volume of liquid to be dispensed is mechanically set; the syringe plunger is lifted for filling and pressed downward for dispensing. Hold the receiving container under the spout while depressing the plunger.
Touch off the last drop on the inside wall of the receiving container. Special procedures are required for cleaning glass pipets. Immediately after use, every pipet should be placed, tip up, in a vertical cylinder containing a dilute detergent solution less than 0. The pipet must be completely covered with solution. This ensures that any reagent remaining in the pipet is forced out through the tip.
If reagents are allowed to dry inside a pipet, the tips can easily become clogged and are very difficult to open. After several pipets have accumulated in the detergent solution, the pipets should be transferred to a pipet rinser. Pipet rinsers continually cycle fresh water through the pipets. Immediately after detergent wash, tap water may be used to rinse the pipets, but distilled water should be used for the final rinse.
Pipets may then be dried in an oven. The purpose of each laboratory exercise in this book is to observe and measure characteristics of a biomolecule or a biological system. The characteristic is often quantitative, a single number or a group of numbers. These measured characteristics may be the molecular weight of a protein, the pH of a buffer solution, the absorbance of a colored solution, the rate of an enzyme-catalyzed reaction, or the radioactivity associated with a molecule.
If you measure a quantitative characteristic many times under identical conditions, a slightly different result will most likely be obtained each time. If the absorbance of a solution is determined several times at a specific wavelength, the value of each measurement will surely vary from the others. Which measurements, if any, are correct? Before this question can be answered, you must understand the source and treatment of numerical variations in experimental measurements.
Analysis of Experimental Data. An error in an experimental measurement is defined as a deviation of an observed value from the true value. There are two types of errors, determinate and indeterminate, Determinate errors are those that can be controlled by the experimenter and are associated with malfunctioning equipment, improperly designed experiments, and variations in experimental conditions. These are sometimes called human errors because they can be corrected or at least partially alleviated by careful design and performance of the experiment.
Indeterminate errors are those that are random and cannot be controlled by the experimenter. Specific examples of indeterminate errors are variations in radioactive counting and small differences in the successive measurements of glucose in a serum sample. Two statistical terms involving error analysis that are often used and misused are accuracy and precision. Precision refers to the extent of agreement among repeated measurements of an experimental value.
Accuracy is defined as the difference between the experimental value and the true value for the quantity. Since the true value is seldom known, accuracy is better defined as the difference between the experimental value and the accepted true value. Several experimental measurements may be precise that is, in close agreement with each other without being accurate. If an infinite number of identical, quantitative measurements could be made on a biosystem, this series of numerical values would constitute a statistical population.
The average of all of these numbers would be the true value of the measurement. It is obviously not possible to achieve this in practice. The alternative is to obtain a relatively small sample of data, which is a subset of the infinite population data.
The significance and precision of these data are then determined by statistical analysis. This section explores the mathematical basis for the statistical treatment of experimental data. Most measurements required for the completion of the experiments can be made in duplicate, triplicate, or even quadruplicate, but it would be impractical and probably a waste of time and materials to make numerous determinations of the same measurement.
Rather, when you perform an experimental measurement in the laboratory, you will collect a small sample of data from the population of infinite values for that measurement. To illustrate, imagine that an infinite number of experimental measurements of the pH of a buffer solution are made, and the results are written on slips of paper and placed in a container.
It is not feasible to. By doing this, you have collected a sample of data. By proper statistical manipulation of this small sample, it is possible to determine whether it is representative of the total population and the amount of confidence you should have in these numbers. The data analysis will be illustrated here primarily with the counting of radioactive materials, although it is not limited to such applications. Any replicate measurements made in the biochemistry laboratory can be analyzed by these methods.
Radioactive decay with emission of particles is a random process. It is impossible to predict with certainty when a radioactive event will occur. Therefore, a series of measurements made on a radioactive sample will result in a series of different count rates, but they will be centered around an average or mean value of counts per minute.
Table 1. A similar table could be prepared for other biochemical measurements, including the rate of an enzyme-catalyzed reaction or the protein concentration of a solution as determined by the Bradford method. The arithmetic average or mean of the numbers is calculated by totaling all the experimental values observed for a sample the counting rates, the velocity of the reaction, or protein concentration and dividing the total by the number of times the measurement was made.
The mean is defined by Equation 1. The mean counting rate for the data in Table 1. If the same radioactive sample were again counted for a series of ten observations, that series of counts would most likely be different from those listed in the table, and a different mean would be obtained.
If we were able to make an infinite number of counts on the radioactive sample, then a true mean could be calculated. The true mean would be the actual amount of radioactivity in the sample.
Although it would be desirable, it is not possible experimentally to measure the true mean. Therefore, it is necessary to use the average of the. Since it is not usually practical to observe and record a measurement many times as in Table 1. Please read our short guide how to send a book to Kindle. The file will be sent to your email address. It may take up to minutes before you receive it.
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Utopia book 2 pdf. Process systems analysis and control coughanowr solutions pdf. Microelectronic circuits and devices pdf. Walter rudin principles of mathematical analysis 3rd edition pdf. If you only knew book. The book offers the latest safety and environmental precautions in each experiment to inform students and instructors of potential hazards and proper disposal of materials. For anyone interested in science.
Biology and other natural sciences. ISBN Your tags:. Send-to-Kindle or Email Please login to your account first Need help? Please read our short guide how to send a book to Kindle. Photoinduced Proton Transport through Chloroplast Membranes. Activity and Thermal Stability of Gel-immobilized Peroxidase. About the Author s. Previous editions. Modern Experimental Biochemistry, 2nd Edition.
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