Laboratory Equipment: A Comprehensive Guide, Study Guides, Projects, Research of Biotechnology

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LABORATORY INSTRUMENTATION AND TECHNIQUES Dr. Mathew Folaranmi OLANIYAN Associate Professor Department of Medical Laboratory Science Achievers University, Owo-Nigeria i DEDICATION This book is dedicated to Almighty God and my children(Olamide, Ajibola and Oluwatobi) ii CHAPTER TEN ION SELECTIVE ELECTRODES AND POTENTIOMETRY…………………. 58 CHAPTER ELEVEN HOT AIR/BOX OVEN…………………………………………………………………… 79 CHAPTER TWELVE ELIZA READER…………………………………………………………………………… 83 CHAPTER THIRTEEN REFRIGERATOR…………………………………………………………………………… 88 CHAPTER FOURTEEN LABORATORY MIXER…………………………………………………….…………… 100 CHAPTER FIFTEEN POLYMERASE CHAIN REACTION (PCR ) MACHINE………………..………. 101 CHAPTER SIXTEEN LABORATORY INCUBATOR…………………………………………………………. 105 CHAPTER SEVENTEEN MICROTOMES…………………………………………………………………………. ….. 112 CHAPTER EIGHTEEN ENZYME-LINKED IMMUNOSORBENT ASSAYS (ELISAs) TECHNIQUE. 120 CHAPTER NINETEEN MICROSCOPY TECHNIQUE………………………………………………………….. 123 CHAPTER TWENTY HISTOLOGICAL TECHNIQUES – MICROTOMY………………………………. 138 CHAPTER TWENTY ONE SPECTROPHOTOMETRY AND COLORIMETRY………………………………. 164 CHAPTER TWENTY TWO ELECTROPHORESIS……………………………………………………………………. 174 CHAPTER TWENTY THREE POLYMERASE CHAIN REACTION (PCR)………………………………………. 182 CHAPTER TWENTY FOUR FLOROMETRY /SPECTROFLOROMETRY……………………………………… 187 CHAPTER TWENTY FIVE LYOPHILISATION (FREEZE-DRYING)……………………………………….. … 201 CHAPTER TWENTY SIX OSMOMETRY……………………………………………………………………………… 205 CHAPTER TWENTY SEVEN TURBIDIMETRY AND NEPHELOMETRY…………………………………………. 208 CHAPTER TWENTY EIGHT CONDUCTOMETRY, POLAROGRAPHY AND POLAROGRAPHY…………. 210 CHAPTER TWENTY NINE RADIOIMMUNOASSAY (RIA)………………………………………………………………. 215 CHAPTER THIRTY AUTOANALYZERS/ AUTOMATED ANALYSER………………………………. 218 CHAPTER THIRTY ONE SOLVENT EXTRACTION…………………………………………………………………….. 225 CHAPTER THIRTY TWO CHROMATOGRAPHY………………………………………………………………………….. 228 CHAPTER THIRTY THREE FLOW CYTOMETRY……………………………………………………………………………. 240 LIST OF REFERENCES…………………………………………………………………………. 244 iv Laboratory apparatus is a set of equipment or tools or a machine that is used for a particular purpose. Laboratory apparatus is the individual instruments or pieces of equipment, or the entire set of equipment to conduct projects and experiments. The most common utensils and appliances that you need while performing hands on activities in a laboratory. The laboratory apparatus depends upon the type of laboratory you are in and the experiment you are going to perform. Laboratory tool is any physical item that can be used to achieve a goal, especially if the item is not consumed in the process. Tools that are used in particular fields or activities may have different designations such as "instrument", "utensil", "implement", "machine", "device," or "apparatus". The set of tools needed to achieve a goal is "equipment". The knowledge of constructing, obtaining and using tools is technology. 2 CHAPTER TWO AUTOCLAVE An autoclave is essentially just a large steel vessel through which steam or another gas is circulated to sterilize things, perform scientific experiments, or carry out industrial processes. Typically the chambers in autoclaves are cylindrical, because cylinders are better able to withstand extreme pressures than boxes, whose edges become points of weakness that can break. The high-pressure makes them self-sealing (the words "auto" and "clave" mean automatic locking), though for safety reasons most are also sealed manually from outside. Just like on a pressure cooker, a safety valve ensures that the steam pressure cannot build up to a dangerous level. A medical autoclave is a device that uses steam to sterilize equipment and other objects. This means that all bacteria, viruses, fungi, and spores are inactivated. However, prions, such as those associated with Creutzfeldt- Jakob disease, may not be destroyed by autoclaving at the typical 134 °C for three minutes or 121 °C for 15 minutes[citation needed]. Although that a wide range species of archaea, including Geogemma barosii, can survive at temperatures above 121 °C, no archaea are known to be infectious or pose a health risk to humans; in fact their biochemistry is so vastly different from our own and their multiplication rate is far too slow for microbiologists to worry about them. Autoclaves are found in many medical settings, laboratories, and other places that need to ensure the sterility of an object. Many procedures today employ single-use items rather than sterilizable, reusable items. This first happened with hypodermic needles, but today many surgical instruments (such as forceps, needle holders, and scalpel handles) are commonly single- use rather than reusable items (see waste autoclave). Autoclaves are of particular importance in poorer countries due to the much greater amount of equipment that is re-used. Providing stove-top or solar autoclaves to rural medical centres has been the subject of several proposed medical aid missions. 3 Because damp heat is used, heat-labile products (such as some plastics) cannot be sterilized this way or they will melt. Paper and other products that may be damaged by steam must also be sterilized another way. In all autoclaves, items should always be separated to allow the steam to penetrate the load evenly. Autoclaving is often used to sterilize medical waste prior to disposal in the standard municipal solid waste stream. This application has become more common as an alternative to incineration due to environmental and health concerns raised because of the combustion by-products emitted by incinerators, especially from the small units which were commonly operated at individual hospitals. Incineration or a similar thermal oxidation process is still generally mandated for pathological waste and other very toxic and/or infectious medical waste. In dentistry, autoclaves provide sterilization of dental instruments according to health technical memorandum 01-05 (HTM01-05). According to HTM01-05, instruments can be kept, once sterilized using a vacuum autoclave for up to 12 months using sealed pouches Working Principle Why is an autoclave such an effective sterilizer? An autoclave is a large pressure cooker; it operates by using steam under pressure as the sterilizing agent. High pressures enable steam to reach high temperatures, thus increasing its heat content and killing power. Most of the heating power of steam comes from its latent heat of vaporization. This is the amount of heat required to convert boiling water to steam. This amount of heat is large compared to that required to make water hot. For example, it takes 80 calories to make 1 liter of water boil, but 540 calories to convert that boiling water to steam. Therefore, steam at 100… C has almost seven times more heat than boiling water. Steam is able to penetrate objects with cooler temperatures because once the steam contacts a cooler surface, it immediately condenses to water, producing a concomitant 1,870 fold decrease in steam volume. This creates negative pressure at the point of condensation and draws more steam to the area. Condensations continue so long as the temperature of the condensing surface is less than that of steam; once temperatures equilibrate, a saturated steam environment is formed. 4 Next, steam is pumped through the chamber at a higher pressure than normal atmospheric pressure so it reaches a temperature of about 121– 140°C (250–284°F). Once the required temperature is reached, a thermostat kicks in and starts a timer. The steam pumping continues for a minimum of about 3 minutes and a maximum of about 15–20 minutes (higher temperatures mean shorter times)—generally long enough to kill most microorganisms. The exact sterilizing time depends on a variety of factors, including the likely contamination level of the items being autoclaved (dirty items known to be contaminated will take longer to sterilize because they contain more microbes) and how the autoclave is loaded up (if steam can circulate more freely, autoclaving will be quicker and more effective). Autoclaving is a bit like cooking, but as well as keeping an eye on the temperature and the time, the pressure matters too! Safety is all-important. Since you're using high-pressure, high-temperature steam, you have to be especially careful when you open an autoclave that there is no sudden release of pressure that could cause a dangerous steam explosion. To be effective against spore forming bacteria and viruses, autoclaves need to: ◾Have steam in direct contact with the material being sterilized (i.e. loading of items is very important). ◾Create vacuum in order to displace all the air initially present in the autoclave and replacing it with steam. ◾Implement a well designed control scheme for steam evacuation and cooling so that the load does not perish. The efficiency of the sterilization process depends on two major factors. One of them is the thermal death time, i.e. the time microbes must be exposed to at a particular temperature before they are all dead. The second factor is the thermal death point or temperature at which all microbes in a sample are killed.The steam and pressure ensure sufficient heat is transferred into the organism to kill them. A series of negative pressure pulses are used to vacuum all possible air pockets, while steam penetration is maximized by application of a succession of positive pulses 7 Test for the efficacy of an Autoclave(quality assurance) There are physical, chemical, and biological indicators that can be used to ensure that an autoclave reaches the correct temperature for the correct amount of time. If a non-treated or improperly treated item can be confused for a treated item, then there is the risk that they will become mixed up, which, in some areas such as surgery, is critical. Chemical indicators on medical packaging and autoclave tape change color once the correct conditions have been met, indicating that the object inside the package, or under the tape, has been appropriately processed. Autoclave tape is only a marker that steam and heat have activated the dye. The marker on the tape does not indicate complete sterility. A more difficult challenge device, named the Bowie-Dick device after its inventors, is also used to verify a full cycle. This contains a full sheet of chemical indicator placed in the center of a stack of paper. It is designed specifically to prove that the process achieved full temperature and time required for a normal minimum cycle of 274 degrees F for 3.5–4 minutes To prove sterility, biological indicators are used. Biological indicators contain spores of a heat-resistant bacterium, Geobacillus stearothermophilus. If the autoclave does not reach the right temperature, the spores will germinate when incubated and their metabolism will change the color of a pH-sensitive chemical. Some physical indicators consist of an alloy designed to melt only after being subjected to a given temperature for the relevant holding time. If the alloy melts, the change will be visible Some computer-controlled autoclaves use an F0 (F-nought) value to control the sterilization cycle. F0 values are set for the number of minutes of sterilization equivalent to 121 °C (250 °F) at 100 kPa (15 psi) above atmospheric pressure for 15 minutes . Since exact temperature control is difficult, the temperature is monitored, and the sterilization time adjusted accordingly. Application of autoclave Sterilization autoclaves are widely used in microbiology, medicine, podiatry, tattooing, body piercing, veterinary science, mycology, funeral homes, dentistry, and prosthetics fabrication. They vary in size and function depending on the media to be sterilized. 8 Typical loads include laboratory glassware, other equipment and waste, surgical instruments, and medical waste.A notable recent and increasingly popular application of autoclaves is the pre-disposal treatment and sterilization of waste material, such as pathogenic hospital waste. Machines in this category largely operate under the same principles as conventional autoclaves in that they are able to neutralize potentially infectious agents by utilizing pressurized steam and superheated water. A new generation of waste converters is capable of achieving the same effect without a pressure vessel to sterilize culture media, rubber material, gowns, dressing, gloves, etc. It is particularly useful for materials which cannot withstand the higher temperature of a hot air oven. Autoclaves are also widely used to cure composites and in the vulcanization of rubber. The high heat and pressure that autoclaves allow help to ensure that the best possible physical properties are repeatably attainable. The aerospace industry and sparmakers (for sailboats in particular) have autoclaves well over 50 feet (15 m) long, some over 10 feet (3.0 m) wide. Other types of autoclave are used to grow crystals under high temperatures and pressures. Synthetic quartz crystals used in the electronic industry are grown in autoclaves. Packing of parachutes for specialist applications may be performed under vacuum in an autoclave which allows the parachute to be warmed and inserted into the minimum volume. 9 Care and Maintenance of centrifuges Mechanical stress Always ensure that loads are evenly balanced before a run. „Always observe the manufacturers maximum speed and sample density ratings. „Always observe speed reductions when running high density solutions, plastic adapters, or stainless steel tubes. Many rotors are made from titanium or aluminum alloy, chosen for their advantageous mechanical properties. While titanium alloys are quite corrosion-resistant, aluminum alloys are not. When corrosion occurs, the metal is weakened and less able to bear the stress from the centrifugal force exerted during operation. The combination of stress and corrosion causes the rotor to fail more quickly and at lower stress levels than an uncorroded rotor. 12 1- A tabletop micro laboratory centrifuge 2- Laboratory macro/bench centrifuge 4- An Eppendorf laboratory centrifuge 13 CHAPTER FOUR WEIGHING BALANCE Balances are designed to meet the specific weighing requirement in the laboratory working environment. These balances come in precision designs and operating characteristics that allows making quick and accurate measurements. Further, these balances can also be tubes to transfer data to computer for further analysis as well as can have piece count functions and hopper functions. With end usage of these scales in precision weighing applications in laboratories, these also offer excellent value of money invested. Here, our expertise also lies in making these available in both standard and custom tuned specifications. The range offered includes Analytical Balances, General Purpose Electronic Balance, Laboratory Balances and Precision Weighing Balances. The history of balances and scales dates back to Ancient Egypt. A simplistic equal-arm balance on a fulcrum that compared two masses was the standard. Today, scales are much more complicated and have a multitude of uses. Applications range from laboratory weighing of chemicals to weighing of packages for shipping purposes. To fully understand how balances and scales operate, there must be an understanding of the difference between mass and weight. Mass is a constant unit of the amount of matter an object possesses. It stays the same no matter where the measurement is taken. The most common units for mass are the kilogram and gram. Weight is the heaviness of an item. It is dependent on the gravity on the item multiplied by the mass, which is constant. The weight of an object on the top of a mountain will be less than the weight of the same object at the bottom due to gravity variations. A unit of measurement for weight is the newton. A newton takes into account the mass of an object and the relative gravity and gives the total force, which is weight. Although mass and weight are two different entities, the process of determining both weight and mass is called weighing. 14 measurements to be made quickly thus making it a more convenient choice when exact measurements are not needed. Top-loaders are also more economical than analytical balances. Modern top-loading balances are electric and give a digital readout in seconds. Torsion Balance Measurements are based on the amount of twisting of a wire or fiber. Many microbalances and ultra-microbalances, that weigh fractional gram values, are torsion balances. A common fiber type is quartz crystal. Triple-Beam Balance This type of balance is less sensitive than a top- loading balance. They are often used in a classroom situation because of ease of use, durability and cost. They are called triple-beam balances because they have three decades of weights that slide along individually calibrated scales. The three decades are usually in graduations of 100g, 10g and 1g. These scales offer much less readability but are adequate for many weighing applications. Precision Weighing Balances are laboratory standard high precision balances that are based on latest process technology and features best displayed increment of 0.001g (1mg) with maximum capacity available. These perfectly match up the applications demanding more than a standard balance and assist in simplifying complex laboratory measurements including in determining difference between initial & residual weights. Here, the calculation of the density of solids & liquids also eliminates need for time consuming manual calculation and data logging. The standard features include protective in-use cover and security bracket, working capacities from 0.1 mg to 230 gm, pan size of 90 mm, ACC of 0.1 mg, internal calibration, display using LCD with back light, standard RS-232 C interface and hanger for below balance weighing. Balance and Scale Care and Use A balance has special use and care procedures just like other measuring equipment. Items to be measured should be at room temperature before weighing. A hot item will give a reading less than the actual weight due to convection currents that make the item more buoyant. And, if your balance is enclosed, warm air in the case weighs less than air of the same volume at room temperature. 17 Another important part of using a balance is cleaning. Scales are exposed to many chemicals that can react with the metal in the pan and corrode the surface. This will affect the accuracy of the scale. Also, keep in mind that a potentially dangerous situation could occur if a dusting of chemicals is left on the balance pan. In many lab and classroom situations, more than one person uses a single scale for weighing. It would be impossible for each person to know what everyone else has been weighing. There is a chance that incompatible chemicals could be brought into contact if left standing or that someone could be exposed to a dangerous chemical that has not been cleaned from the balance. To avoid damaging the scale or putting others in danger, the balance should be kept extremely clean. A camel's hair brush can be used to remove any dust that can spill over during weighing. Calibration is another care issue when it comes to scales. A scale cannot be accurate indefinitely; they must be rechecked for accuracy. There are weight sets available that allow users to calibrate the scale themselves or the scales can be calibrated by hiring a professional to calibrate them on site. The correct weight set needs to be chosen when calibrating a scale. The classes of weight sets start from a Class One which provides the greatest precision, then to Class Two, Three, Four and F and finally go down to a Class M, which is for weights of average precision. Weight sets have class tolerance factors, and as a general rule, the tolerance factor should be greater than the readability of the scale. A scale should be calibrated at least once a year or per manufacturer’s guidelines. It can be done using calibration weight sets or can be calibrated by a professional. The readability of the scale will determine which weight set will be appropriate for calibrating the scale 18 What is the difference between accuracy and precision? Accuracy tells how close a scale gets to the real value. An inaccurate scale is giving a reading not close to the real value. Precision and accuracy are unrelated terms. A precise scale will give the same reading multiple times after weighing the same item. A precise scale can be inaccurate by repeatedly giving values that are far away from the actual value. For instance a scale that reads 5.2g three times in a row for the same item is very precise but if the item actually weighs 6.0g the scale is not accurate. 19 Construction and Dimensions: Laboratory water baths usually have stainless steel interiors and either chemically resistant plastic or epoxy coated steel exteriors. Controllers are either analogue or digital. Bath dimensions can be a bit misleading when litre capacity is quoted because it depends how high you measure (water baths are never filled to the top). To compare different bath volumes it is best to compare the internal tank dimensions. Laboratory Water Bath Accessories: Lift-off or hinged plastic (depending on bath temperature) or stainless steel lids are available as well as different racks to hold tubes, etc. Lids with holes with concentric rings are available for boiling water baths to hold different size flasks. Care and maintenance It is not recommended to use water bath with moisture sensitive or pyrophoric reactions. Do not heat a bath fluid above its flash point. Water level should be regularly monitored, and filled with distilled water only. This is required to prevent salts from depositing on the heater. Disinfectants can be added to prevent growth of organisms. Raise the temperature to 90 °C or higher to once a week for half an hour for the purpose of decontamination. Markers tend to come off easily in water baths. Use water resistant ones. If application involves liquids that give off fumes, it is recommended to operate water bath in fume hood or in a well-ventilated area. The cover is closed to prevent evaporation and to help reaching high temperatures. Set up on a steady surface away from flammable materials. 22 CHAPTER SIX ANAEROBIC JARS Lid with Clamp with Palladium O-ring gasket clamp screw catalyst pellets Envelope containing sodium bicarbonate and sodium borohydride Anaerobic indicator (methylene blue) Copyright © 2004 Pearson Education, ne. ubishing as Berjamin Cummings 23  Method of use 1a.The culture: The culture media are placed inside the jar, stacked up one on the other, and 24 The chemical indicator generally used for this purpose is "chemical methylene blue solution" that since synthesis has never been exposed to elemental oxygen. It is colored deep blue on oxidation in presence of atmospheric oxygen in the jar, but will become colorless when oxygen is gone, and anaerobic conditions are achieved. 27 Compound Light Microscope Arm: supports upper parts and provides carrying handle Stage clips: hold slide CHAPTER SEVEN MICROSCOPE in place on the stage Mechanical stage: movable stage that aids in accurate positioning of] |: = slide : Coarse-adjustment knob: used to bring specimen into ~ focus; used only with low-power objective Fine-adjustment knob: used to f bring specimen into final focus Scanning power objective (4x): shortest objective lens used to scan the whole slide ‘@PGregory Eyepieces (ocular lenses) - magnifying power of 10x Body tube: holds nosepiece at one end and eyepiece at other end; conducts light rays Nosepiece: Revolving device that holds Objectives | objective lenses: see info at bottom ofillustration Stage: holds and supports L microscope slides Diaphragm control lever: controls amt. of illumination used to view specimen Condensor: lens system below stage used to focus the beam of light on the specimen Light source: attached lamp that directs beam of light up through specimen Low-power objective (10x): lens next in length (longer than scanning) used to view specimen in greater detail High-power objective (40x): lens next in length (longer than low-power) used to view specimen in even greater detail Oil immersion objective (100%): used in conjunction with immersion oil to view specimen in greatest detail 28 Principles of Electron Microscopy Uses electromagnetic lenses, electrons and fluorescent screen to produce image Resolution increased 1,000 fold over brightfield microscope 0 To about 0.3 nm (1x10°) Magnification increased to 100,000x Two types of electron microscopes o Transmission o Scanning jul-ta 13 Principles of Light Microscopy: Magnification Compound Microscope : microscope has two magnifying lenses = Lenses include ocular lens and objective lens Lenses combine to enlarge objects = Magnification is equal to the product of the ocular lens x the objective lens: 10x X 100x = 1,000x jul-ta 29 10. Mechanical Stage (Slide Mover): Mechanical stage consists of two knobs with rack and pinion mechanism. The slide containing the object is clipped to it and moved on the stage in two dimensions by rotating the knobs, so as to focus the required portion of the object. 11. Revolving Nosepiece: It is a rotatable disc at the bottom of the body tube with three or four objectives screwed to it. The objectives have different magnifying powers. Based on the required magnification, the nosepiece is rotated, so that only the objective specified for the required magnification remains in line with the light path. (ii) Optical Parts: These parts are involved in passing the light through the object and magnifying its size. The components of optical parts include the following: 1. Light Source: Modern microscopes have in-built electric light source in the base. The source is connected to the mains through a regulator, which controls the brightness of the field. But in old models, a mirror is used as the light source. It is fixed to the base by a binnacle, through which it can be rotated, so as to converge light on the object. The mirror is plane on one side and concave on the other. It should be used in the following manner: (a) Condenser Present: Only plane side of the mirror should be used, as the condenser converges the light rays. (b) Condenser Absent: (i) Daylight: 32 Plane or concave (plane is easier) (ii) Small artificial light: High power objective: Plane side Low power objective: Concave side 2. Diaphragm: If light coming from the light source is brilliant and all the light is allowed to pass to the object through the condenser, the object gets brilliantly illuminated and cannot be visualized properly. Therefore, an iris diaphragm is fixed below the condenser to control the amount of light entering into the condenser. 3. Condenser: The condenser or sub-stage condenser is located between the light source and the stage. It has a series of lenses to converge on the object, light rays coming from the light source. After passing through the object, the light rays enter into the objective. The ‘light condensing’, ‘light converging’ or ‘light gathering’ capacity of a condenser is called ‘numerical aperture of the condenser’. Similarly, the ‘light gathering’ capacity of an objective is called ‘numerical aperture of the objective’. If the condenser converges light in a wide angle, its numerical aperture is greater and vice versa. If the condenser has such numerical aperture that it sends light through the object with an angle sufficiently large to fill the aperture back lens of the objective, the objective shows its highest numerical aperture. Most common condensers have numerical aperture. If the numerical aperture of the condenser is smaller than that of the objective, the peripheral portion of the back lens of the objective is not illuminated and the image has poor visibility. On the other hand, if the numerical aperture of condenser is greater than that of the objective, the back lens may receive too much light resulting in a decrease in contrast. 33 There are three types of condensers as follows: (a) Abbe condenser (Numerical aperture=1.25): It is extensively used. (b) Variable focus condenser (Numerical aperture =1.25) (c) Achromatic condenser (Numerical aperture =1.40): It has been corrected for both spherical and chromatic aberration and is used in research microscopes and photomicrographs. 4. Objective: It is the most important lens in a microscope. Usually three objectives with different magnifying powers are screwed to the revolving nosepiece. The objectives are: (a) Low power objective (X 10): It produces ten times magnification of the object. (b) High dry objective (X 40): It gives a magnification of forty times. (c) Oil-immersion objective (X100): It gives a magnification of hundred times, when immersion oil fills the space between the object and the objective The scanning objective (X4) is optional. The primary magnification (X4, X10, X40 or X100) provided by each objective is engraved on its barrel. The oil-immersion objective has a ring engraved on it towards the tip of the barrel. Resolving Power of Objective: It is the ability of the objective to resolve each point on the minute object into widely spaced points, so that the points in the image can be seen as distinct and separate from one another, so as to get a clear un-blurred image. 34 The limit of resolution of an objective (d) is the distance between any two closest points on the microscopic object, which can be resolved into two separate and distinct points on the enlarged image. Points with their in-between distance less than ‘d’ or objects smaller than ‘d’ cannot be resolved into separate points on the image. If the resolving power is high, points very close to each other can be seen as clear and distinct. Thus, the limit of resolution (the distance between the two resolvable points) is smaller. Therefore, smaller objects or finer details can be seen, when’d’ is smaller. Smaller ‘d’ is obtained by increasing the resolving power, which in turn is obtained by using shorter wavelength of light (λ) and greater numerical aperture. Limit of resolution = d = λ/2 n.a. Where, λ = Wave length of light and n.a. = Numerical aperture of the objective. If λ green = 0.55 p and n.a. = 1.30, then d = λ/2 n.a. = 0.55/2 X 1.30 = 0.21 µ. Therefore, the smallest details that can be seen by a typical light microscope is having the dimension of approximately 0.2 µ. Smaller objects or finer details than this cannot be resolved in a compound microscope. 5. Eyepiece: The eyepiece is a drum, which fits loosely into the draw tube. It magnifies the magnified real image formed by the objective to a still greatly magnified virtual image to be seen by the eye. Usually, each microscope is provided with two types of eyepieces with different magnifying powers (X10 and X25). Depending upon the required magnification, one of the two eyepieces is inserted into the draw tube before viewing. Three varieties of eyepieces are usually available. 37 They are the Huygenian, the hyper plane and the compensating. Among them, the Huygenian is very widely used and efficient for low magnification. In this eyepiece, two simple Plano-convex lenses are fixed, one above and the other below the image plane of the real image formed by the objective. The convex surfaces of both the lenses face downward. The lens towards the objective is called ‘field lens’ and that towards eye, ‘eye lens’. The rays after passing through the eye lens come out through a small circular area known as Rams-den disc or eye point, where the image is viewed by the eye. Total magnification: The total magnification obtained in a compound microscope is the product of objective magnification and ocular magnification. Mt = Mob X Moc Where, Mt = Total magnification, Mob = Objective magnification and Moc = Ocular magnification If the magnification obtained by the objective (Mob) is 100 and that by the ocular (Moc) is 10, then total magnification (Mt) = Mob X Moc =100 X 10 =1000. Thus, an object of lq will appear as 1000 µ. Useful magnification: It is the magnification that makes visible the smallest resolvable particle. The useful magnification in a light microscope is between X1000 and X2000. Any magnification beyond X2000 makes the image blurred. 38 CHAPTER EIGHT SPECTROPHOTOMETER AND COLORIMETER A spectrophotometer is an instrument which is used to measure the intensity of electromagnetic radiation at different wavelengths. Important features of spectrophotometers are spectral bandwidth and the range of absorption or reflectance measurement. Spectrophotometers are generally used for the measurement of transmittance or reflectance of solutions, transparent or opaque solids such as polished gases or glass. They can also be designed to measure the diffusivity on any of the listed light ranges in electromagnetic radiation spectrum that usually covers around 200 nm- 2500 nm using different controls and calibrations. Spectrophotometry is a quantitative measurement of the reflection or transmission properties of a material as a function of wavelength. It is more specific than the common term electromagnetic spectroscopy which deals with visible light near ultraviolet and near infra-red. It does not cover the time resolved spectroscopic techniques which means that "anything that allows to measure temporal dynamics and kinetics of photo physical processes". Spectrophotometry involves the use of spectrophotometer. The basic measurement principle used by a spectrophotometer is relatively simple and easy to understand. 39 There is a proportional relationship between the absorbance and concentration, and this forms the basis of quantitative analysis. Here, ε is the sample’s absorption coefficient and L is the cell’s optical path length. The measurement method shown in eliminates the influence of reflection from the cell surface and absorption by the solvent, and ensures that only the absorption due to the sample is measured. Monochromatic light is light that consists of a single wavelength. To be precise, it has a spectral bandwidth (slit width). For example, monochromatic light with a wavelength of 500 nm and a spectral bandwidth of 2 nm is light that covers a wavelength interval (full width at half maximum) spanning 499 and 501 nm. 2.The Configuration of a Spectrophotometer The indispensable elements of a spectrophotometer consist, as shown in of a light source, a spectrometer, a sample compartment, and a detector. Although I said in the previous section that the sample is exposed to monochromatic light, there are instruments in which white light is passed through the sample before being passed into the spectrometer. This method is employed in high-speed photometry instruments that use array detectors. 3.Light Source The desirable properties of a light source are as follows: a) Brightness across a wide wavelength range b) Stability over time c) A long service life d) Low cost Although there are no light sources that have all of these properties, the most commonly used light sources at the moment are the halogen lamps used for the visible and near-infrared regions and the deuterium lamps used for the ultraviolet region. Apart from these, xenon flash lamps are sometimes used. (1) Halogen Lamp 42 Emission Intensity Distribution of Halogen Lamp (3,000K) The principle for light emission is the same as that for a standard incandescent bulb. Electric current is supplied to a filament, the filament becomes hot, and light is emitted. The bulb in a halogen lamp is filled with inert gas and a small amount of a halogen. While the tungsten used as the filament evaporates due to the high temperature, the halide causes the tungsten to return to the filament. This helps create a bright light source with a long service life. The emission intensity distribution of a halogen lamp can be approximated using Planck’s law of radiation. It has relatively high levels of each of the properties a) to d) mentioned above. (2)Deuterium Lamp Emission Intensity Distribution of Deuterium Lamp1) A deuterium lamp is a discharge light source in which the bulb is filled with deuterium (D2) at a pressure of several hundred pascals. Although 400 nm is, in general, an approximate usage limit at the long wavelength end, because the degree of attenuation at this end is quite low, light of wavelengths greater than 400 nm is used. In the region beyond 400 nm, there are also large numbers of bright line spectra. Among these, the bright line spectra at 486.0 nm and 656.1 nm are particularly intense, and can be used for the wavelength calibration of spectrophotometers. 43 The usage limit at the short wavelength end is determined by the transmittance of the window material. 4.Monochrometer Cross Section of Diffraction Grating Spectroscopy is the technique of splitting light that consists of various wavelengths into components that correspond to those wavelengths. The element that splits this light is called a dispersive element. Prisms and diffraction gratings are typical dispersive elements. Prisms used to be commonly used as the dispersive elements in spectrometers, but recently, diffraction gratings have become the most commonly used type of dispersive element. The diffraction gratings used in spectrophotometers have from several hundred to approximately 2,000 parallel grooves per millimeter cut into them at equal intervals. If this diffraction grating is exposed to white light, because of interference, the white light is dispersed in a direction perpendicular to the grooves, and light components of specific wavelengths are reflected only in specific directions. λ1 to λ3 represent wavelengths. The wavelengths change continuously and so if a diffraction grating is exposed towhite light, it appears iridescent. The way that the clear side of a CD appearsto glitter with iridescence when it is exposed to light is based on the same mechanism as the spectroscopy performed with a diffraction grating. 5.Sample Compartment Two light beams pass through the compartment, and that this is therefore the sample compartment of a “double-beam spectrophotometer”. The monochromatic light that leaves the spectrometer is split into two beams before it enters the sample compartment. A spectrophotometer in which only one beam passes through the sample compartment is called a “single- beam spectrophotometer”. 44 Spectral Sensitivity Characteristics of a Silicon COLORIMETER A colorimeter is a device used for measuring colours, or colorimetry. It measures the absorbance of different wavelengths of light in a solution. It can be used to measure the concentration of a known solute. A colorimeter is an instrument that compares the amount of light getting through a solution with the amount that can get through a sample of pure solvent. A colorimeter contains a photocell is able to detect the amount of light which passes through the solution under investigation. A colorimeter is a light- sensitive instrument that measures how much color is absorbed by an object or substance. It determines color based on the red, blue, and green components of light absorbed by the object or sample, much as the human eye does. When light passes through a medium, part of the light is absorbed, and as a result, there is a decrease in how much of the light reflected by the medium. 47 A colorimeter measures that change so users can analyze the concentration of a particular substance in that medium. The device works on the basis of Beer-Lambert's law, which states that the absorption of light transmitted through a medium is directly proportional to the concentration of the medium. Types of colorimeters There are many different types of colorimeters, including the color densitometer, which measures the density of primary colors, and the color photometer, which measures the reflection and transmission of color. Styles include digital, also called laboratory, and portable. Digital versions are most often used in a lab setting for sampling or in the classroom for educational purposes. Portable versions can be carried anywhere, regardless of environmental conditions, to test things like water and soil samples on site. The spectrophotometer, a type of photometer that measures light intensity, is often grouped together with colorimeters, but it is technically a different device. Both rely on Beer-Lambert's law to calculate the concentration of a substance in a solution, but they do so in different ways. A colorimeter measures only red, green, and blue colors of light, while a spectrophotometer can measure the intensity of any wavelength of visible light. In general, spectrophotometers are more complicated and less rugged than most colorimeters; they should be handled with utmost care and require regular recalibration. A colorimeter is a light-sensitive device used for measuring the transmittance and absorbance of light passing through a liquid sample. The device measures the intensity or concentration of the color that develops upon introducing a specific reagent into a solution. There are two types of colorimeters — color densitometers, which measure the density of primary colors, and color photometers, which measure the color reflection and transmission. Design of Colorimeter The three main components of a colorimeter are a light source, a cuvette containing the sample solution, and a photocell for detecting the light passed through the solution. The instrument is also equipped with either colored filters or specific LEDs to generate color. The output from a colorimeter may be displayed by an analog or digital meter in terms of transmittance or absorbance. 48 In addition, a colorimeter may contain a voltage regulator for protecting the instrument from fluctuations in mains voltage. Some colorimeters are portable and useful for on-site tests, while others are larger, bench-top instruments useful for laboratory testing. Working Principle The colorimeter is based on Beer-Lambert's law, according to which the absorption of light transmitted through the medium is directly proportional to the medium concentration. In a colorimeter, a beam of light with a specific wavelength is passed through a solution via a series of lenses, which navigate the colored light to the measuring device. This analyses the color compared to an existing standard. A microprocessor then calculates the absorbance or percent transmittance. If the concentration of the solution is greater, more light will be absorbed, which can be identified by measuring the difference between the amount of light at its origin and that after passing the solution. In order to determine the concentration of an unknown sample, several sample solutions of a known concentration are first prepared and tested. The concentrations are then plotted on a graph against absorbance, thereby generating a calibration curve. The results of the unknown sample are compared to that of the known sample on the curve to measure the concentration. Applications Colorimeters are widely used to monitor the growth of a bacterial or yeast culture. They provide reliable and highly accurate results when used for the assessment of color in bird plumage. They are used to measure and monitor the color in various foods and beverages, including vegetable products and sugar. Certain colorimeters can measure the colors that are used in copy machines, fax machines and printers. 49 In the printing industry, a colorimeter is a basic element in a color management system. Other printing industry applications include checking the electronic components and quality of pulp paper and measuring the quality of printing ink. Diamond merchants use colorimeters to measure the optical properties of precious stones. In cosmetology, the device is used to measure the sun protection factor of products applied to the skin. Colorimeters can analyze skin tones and tooth color to help diagnose certain diseases, and hospitals even use some types of this device to test the concentration of hemoglobin in blood. 52 CHAPTER NINE FLAME PHOTOMETERS FLAME PHOTOMETERS In flame photometry, a branch of atomic spectroscopy also called “flame atomic emission spectrometry,” atoms are examined in the spectrometer. This technique is suited to the quantitative and qualitative determination of a variety of cations—particularly for alkali and alkaline earth metals— since they are excited to higher levels of energy at low flame temperatures Schematic Representation of the Flame Photometer Flame Pholoactector Filter Major Components: 1. Sample Delivery Fuel od mee ource Monochromator Detector Read out device Nebuliser awn » Components of a Flame Photometer: Flame containing fhe ae. 4. CeO (oi in monochromator pooner Yead- out to be analysed (lacs a waveleagtt) ful dir 5, Wet The last three components of a flame photometer are similar to those of a spectrophotometer. The flame however takes place of the light source and sample compartment 53 Monochromators: Filters and monochromators are needed to isolate the light of specific wavelength from remaining light of the flame. For this simple filters are sufficient as we study only few elements like Ca, Na, K and Li. So a filter wheel with filter for each element is taken. When a particular element is analyzed, the particular filter is used so that it filters all other wavelengths. Detector: Flame photometric detector is similar to that used in spectrophotometry. The emitted radiation is in the visible region i.e. 400nm to 700nm. Further the radiation is specific for each element so simple detectors are sufficient for the purpose like photo voltaic cells, photo tubes etc. Recorders and display: These are the devices to read out the recording from detectors. Flame photometer uses/ Applications 1. For qualitative analysis of samples by comparison of spectrum emission wavelengths with that of standards. 2. For quantitative analysis to determine the concentration of group IA and IIA elements. For example a) Concentration of calcium in hard water. b) Concentration of Sodium, potassium in Urine c) Concentration of calcium and other elements in bio-glass and ceramic materials. Flame photometry limitations: Unlike other spectroscopy methods, flame photometry finds little use in research and analysis. This is due to 1. Limited number of elements that can be analyzed. 56 2. The sample requires to be introduced as solution into fine droplets. Many metallic salts, soil, plant and other compounds are insoluble in common solvents. Hence, they can’t be analyzed by this method. 3. Since sample is volatilized, if small amount of sample is present, it is tough to analyze by this method. As some of it gets wasted by vaporization. 4. Further during solubilisation with solvents, other impurities might mix up with sample and may lead to errors in the spectra observed. How Flame Photometers Work • These instruments are fairly simple, consisting of four basic components: a flame or “burner,” a nebulizer (or aspirator) and mixing chamber, color filters, and a photo detector. Flame photometers are also very cost effective and easy to use. • In a flame photometer, the solution is aspirated through a nebulizer (or aspirator) into the flame. After the sample matrix evaporates, the sample is atomized. Atoms then reach an excited state by absorbing heat from the flame. When these excited atoms return to their lowest-energy state, they give off radiation in certain wavelengths, leading to the creation of a line spectrum. • A filter pre-selected based on the atom being analyzed is used in flame photometry. The emission line’s intensity is then practically measured and is related to the solution’s original concentration. 57 CHAPTER TEN ION SELECTIVE ELECTRODES AND POTENTIOMETRY The pictures below show all the equipment needed for an Ion Analyser measurement:  One Ion-Selective electrode and one Reference electrode are inserted into a dual electrode head.  The head, one temperature sensor and one pH electrode are connected to a 2-channel electrode-computer interface.  The interface is connected to a serial or USB port of a computer running the 2-channel measurement software.  The computer screen shows the calibration graph for a potentiometric ammonium measurement, with sample results plotted on it. 58 Education and Research: Wide range of applications. b) Advantages. 1) When compared to many other analytical techniques, Ion-Selective Electrodes are relatively inexpensive and simple to use and have an extremely wide range of applications and wide concentration range. 2) The most recent plastic-bodied all-solid-state or gel-filled models are very robust and durable and ideal for use in either field or laboratory environments. 3) Under the most favourable conditions, when measuring ions in relatively dilute aqueous solutions and where interfering ions are not a problem, they can be used very rapidly and easily (e.g. simply dipping in lakes or rivers, dangling from a bridge or dragging behind a boat). 4) They are particularly useful in applications where only an order of magnitude concentration is required, or it is only necessary to know that a particular ion is below a certain concentration level. 5) They are invaluable for the continuous monitoring of changes in concentration: e.g. in potentiometric titrations or monitoring the uptake of nutrients, or the consumption of reagents. 6) They are particularly useful in biological/medical applications because they measure the activity of the ion directly, rather than the concentration. 7) In applications where interfering ions, pH levels, or high concentrations are a problem, then many manufacturers can supply a library of specialised experimental methods and special reagents to overcome many of these difficulties. 8) With careful use, frequent calibration, and an awareness of the limitations, they can achieve accuracy and precision levels of ± 2 or 3% for some ions and thus compare favourably with analytical techniques which require far more complex and expensive instrumentation. 61 9) ISEs are one of the few techniques which can measure both positive and negative ions. 10) They are unaffected by sample colour or turbidity. 11) ISEs can be used in aqueous solutions over a wide temperature range. Crystal membranes can operate in the range 0°C to 80°C and plastic membranes from 0°C to 50°C. Ion-Selective Electrodes are part of a group of relatively simple and inexpensive analytical tools which are commonly referred to as Sensors. The pH electrode is the most well-known and simplest member of this group and can be used to illustrate the basic principles of ISEs. a) The pH Electrode This is a device for measuring the concentration of hydrogen ions and hence the degree of acidity of a solution - since pH is defined as the negative logarithm of the hydrogen ion concentration; i.e. pH=7 means a concentration of 1x10-7 moles per litre. (To be more precise, the term ‘concentration’ should really be replaced by ‘activity’ or ‘effective concentration’. This is an important factor in ISE measurements. The difference between activity and concentration is explained in more detail later, but it may be noted here that in dilute solutions they are essentially the same. The most essential component of a pH electrode is a special, sensitive glass membrane which permits the passage of hydrogen ions, but no other ionic species. When the electrode is immersed in a test solution containing hydrogen ions the external ions diffuse through the membrane until an equilibrium is reached between the external and internal concentrations. Thus there is a build up of charge on the inside of the membrane which is proportional to the number of hydrogen ions in the external solution. Because of the need for equilibrium conditions there is very little current flow and so this potential difference can only be measured relative to a separate and stable reference system which is also in contact with the test solution, but is unaffected by it. 62 A sensitive, high impedance millivolt meter or digital measuring system must be used to measure this potential difference accurately. The potential difference developed across the membrane is in fact directly proportional to the Logarithm of the ionic concentration in the external solution. Thus, in order to determine the pH of an unknown solution, it is only necessary to measure the potential difference in two standard solutions of known pH, construct a straight line calibration graph by plotting millivolts versus pH (= - Log [H+]) then read off the unknown pH from the measured voltage. In order to measure the electrode potential developed at the ion-selective membrane the ISE/pH electrode must be immersed in the test solution together with a separate reference system and the two must be connected via a millivolt measuring system. At equilibrium, the electrons added or removed from the solution by the ISE membrane (depending on whether it is cation or anion sensitive) are balanced by an equal and opposite charge at the reference interface. This causes a positive or negative deviation from the original stable reference voltage which is registered on the external measuring system. The relationship between the ionic concentration (activity) and the electrode potential is given by the Nernst equation: E = E0 + (2.303RT/ nF) x Log(A) Where E = the total potential (in mV) developed between the sensing and reference electrodes. E0 = is a constant which is characteristic of the particular ISE/reference pair. (It is the sum of all the liquid junction potentials in the electrochemical cell, see later) 2.303 = the conversion factor from natural to base10 logarithm. R = the Gas Constant (8.314 joules/degree/mole). 63 v) It is more usual to plot a calibration graph using the ionic concentration with a logarithmic scale on the X-axis rather than the pX factor (analogous to pH) on a linear axis. vi) Some ISEs will only work effectively over a narrow pH range. Types of Ion Selective Electrodes a) General Discussion Ion selective electrodes come in various shapes and sizes. Each manufacturer has it’s own distinctive features, but very few give details of the internal construction of the electrode or composition of the ion- selective membranes. These are the most important factors which control the performance of the electrode, and are often kept as closely guarded trade secrets. Nevertheless, there are certain features that are common to all. All consist of a cylindrical tube, generally made of a plastic material, between 5 and 15 mm in diameter and 5 to 10 cm long. An ion-selective membrane is fixed at one end so that the external solution can only come into contact with the outer surface, and the other end is fitted with a low noise cable or gold plated pin for connection to the millivolt measuring device. In some cases the internal connections are completed by a liquid or gel electrolyte, in others by an all-solid-state system. Ion-selective membranes are currently only available for a limited number of commonly occurring ionic species. CATIONS: Ammonium (NH4+), Barium (Ba++), Calcium (Ca++), Cadmium (Cd++), Copper (Cu++), Lead (Pb++), Mercury (Hg++), Potassium (K+), Sodium (Na+), Silver (Ag+). 66 ANIONS: Bromide (Br-), Chloride (Cl-), Cyanide (CN-), Fluoride (F-), Iodide (I-), Nitrate (NO3-), Nitrite (NO2-), Perchlorate (ClO4-), Sulphide (S-), Thiocyanate (SCN-). The manner in which these different membranes select and transport the particular ions is highly variable and in many cases highly complex. It is far beyond the scope of this work to explain in detail the exact mechanism for each ion. Moreover, it is not necessary for the analyst to understand these mechanisms in order to use the electrodes satisfactorily. Nevertheless, it may be of interest to the general reader to give some indication of these processes. There are two main types of membrane material, one based on a solid crystal matrix, either a single crystal or a polycrystalline compressed pellet, and one based on a plastic or rubber film impregnated with a complex organic molecule which acts as an ion-carrier. The development of these organic membranes was based on biological research which revealed that some antibiotics and vitamins can induce cationic permeation through cell membranes. One example of each membrane type is described below as an illustration of the range of technologies employed. b) Crystal-Membrane Electrodes e.g. Fluoride. The Fluoride electrode is a typical example of the first type. Here the membrane consists of a single lanthanum fluoride crystal which has been doped with europium fluoride to reduce the bulk resistivity of the crystal. It is 100% selective for F- ions and is only interfered with by OH- which reacts with the lanthanum to form lanthanum hydroxide, with the consequent release of extra F- ions. This interference can be eliminated by adding a pH buffer to the samples to keep the pH in the range 4 to 8 and hence ensure a low OH- concentration in the solutions. c) Impregnated-PVC-Membrane Electrodes e.g. Potassium. The Potassium electrode was one of the earliest developed and simplest examples of the second type. The membrane is usually in the form of a thin disc of PVC impregnated with the macrocyclic antibiotic valinomycin. This compound has a hexagonal ring structure with an internal cavity which is almost exactly the same size as the diameter of the K+ ion. Thus it can form complexes with this ion and preferentially conducts it across the membrane. Unfortunately it is not 100% selective and can also conduct small numbers of sodium and ammonium ions. Thus these can cause errors in the potassium determination if they are present in high concentrations. The majority of other ISEs suffer from similar limitations (see later section on ‘interference’). 67 d) Care and Maintenance of ISEs. When handling ISEs, care should be taken to avoid damaging the membrane surface. If the electrodes are in frequent use then they can simply be left hanging in the electrode holder with the membrane surface open to the air but protected by a clean dry beaker. For prolonged storage in a cupboard or drawer, the membrane should be protected by covering with the rubber or plastic cap which is normally provided with the electrode. After extensive use the membranes may become coated with a deposit or scoured with fine scratches which may cause a slow or reduced response (low slope) or unstable readings. Crystal membranes can be regenerated by washing with alcohol and/or gently polishing with fine emery paper to remove any deposit or discoloration, then thoroughly washing with de-ionised water to remove any debris. After this, they may require soaking in the concentrated standard solution for several hours before a stable reading can be re- established. It must be noted, however, that prolonged immersion of crystal membranes in aqueous solutions will eventually cause a build up of oxidation products on the membrane surface and thus inhibit performance and shorten the active life. Conversely, PVC membranes should not even be touched, let alone polished, and can be often be regenerated by prolonged (several days) soaking in the standard solution, after removing any deposit with a fine jet of water, or rinsing in alcohol. REFERENCE ELECTRODES In order to measure the change in potential difference across the ion-selective membrane as the ionic concentration changes, it is necessary to include in the circuit a stable reference voltage which acts as a half-cell from which to measure the relative deviations. a) The Silver / Silver Chloride Single Junction Reference Electrode. The most common and simplest reference system is the silver / silver chloride single junction reference electrode. This generally consists of a cylindrical glass tube containing a 4 Molar solution of KCl saturated with AgCl. The lower end is sealed with a porous ceramic frit which allows the slow passage of the internal filling solution and forms the liquid junction with the external test solution. Dipping into the filling solution is a silver wire coated with a layer of silver chloride (it is chloridised) which is joined to a low- noise cable which connects to the measuring system. 68 The main disadvantage of this arrangement is the fact that it is the reference element which is the most likely to cause problems or fail, long before the ISE head does, but the whole unit has to be replaced when failure does occur. In contrast to pH electrodes, some ISEs are produced as mono- electrodes for use with separate reference systems. One reason for this is because ISE membranes have a far lower impedance than pH sensors and are less susceptible to stray electrostatic fields. Thus it is not necessary to screen the sensor head by surrounding it with the reference system. More importantly, the membranes and internal construction of ISEs are generally far more expensive than pH sensors and it is much more cost-effective to have separate units in which the reference system can be replaced independently from the ISE. e) Multiple Electrode Heads: Separable Combinations. A new concept for combination electrodes has recently been introduced. Both the ISEs and the reference electrodes are made in the form of 8mm diameter tubes fitted with a gold plated plug-in connector. These can be inserted separately into special multiple electrode heads which are fitted with the cables and connectors for attaching to the measuring system. The rigid plastic head ensures that the ISE and reference system remain firmly linked together at a regular distance apart during operation, but either one can easily be replaced in the event of failure or need to change the analysis. Moreover, the replacement electrodes are relatively inexpensive compared to conventional electrodes because they do not incorporate the expensive low-noise cables. The ELIT Electrode Head System A practical and cost effective way to combine Ion Selective and Reference Electrodes ELIT Electrode Heads are manufactured from a robust plastic material and fitted with low noise cables and connectors which are compatible with any standard mV/pH/ion meter. The standard version, for use with an ELIT Ion Analyser / Computer Interface, has a BNC plug, but DIN, US or S7 versions are available if required. The sockets on the head and the pins on the plug-in electrodes are gold plated to assure good contact. 71 Advantages of this 'electrode combination' over conventional combination electrodes:  Use of one reference electrode for several ion-selective electrodes.  Replacement of a defective reference system without sacrificing the more expensive ISE.  Expensive low-noise cable and connector are attached to the re-usable head and do not need to be replaced if the ISE becomes defective.  ISE is less expensive than conventional types with cable & connectors permanently attached.  ISE can be stored dry and the RE wet.  Increased distance between the ISE and the reference system reduces electrical interference and increases the precision of measurement. Five types of ELIT Heads are currently available:  Mono Head for inserting one ELIT ISE or Redox electrode, for use with a conventional reference electrode (or a combination pH electrode).  Dual Head for use as an electrode combination: one ISE or Redox and one ELIT reference electrode.  Triple, Four or Seven- electrode head for connecting up to six sensors with one reference, for use in simultaneous multi-component analysis using an ELIT four or eight-channel computer interface. 72 MEASURING PROCEDURES a) Adding ISAB As noted above (Chap. 6) ISAB (Ionic Strength Adjustment Buffer) is normally added to samples and standards in order to ensure that all measured solutions have the same Ionic Strength and avoid errors due to differences between the measured activity and the actual concentration. Indeed, many ISE manufacturers advocate always adding ISAB to samples and standards, irrespective of whether the samples have high ionic strength or not, because this can help to stabilise the liquid junction potential of the reference electrode and hence reduce errors in measurement, and reduce the time taken to reach a stable reading - but this may not be necessary if the reference electrode has an "equi-transferrent" filling solution (i.e. both ions have the same, or nearly the same, mobility - e.g. Lithium Acetate, or Potassium Nitrate). Nevertheless, it must be noted that most electrode systems will give faster stabilisation with ISAB when measuring low concentration samples in the non-linear range. For many applications, however, it may be unnecessary (for samples with IS below 0.01M for monovalent ions and 0.001M for divalent ions), or ineffective (for samples with IS greater than about 0.1M), or inconvenient, to add ISAB to all the samples and standards. One simple way to avoid adding ISAB is to dilute the samples to a level where the activity effect is insignificant. But this requires a knowledge of the Ionic Strength of the samples, and care must be taken to avoid diluting so much that the measurements would fall within the non-linear range of the electrode. In some applications, where only the approximate concentration of the samples are required, or the differences between samples are more important than the actual concentrations, the effect of the ionic strength can often be ignored. Alternatively, if the highest possible precision and accuracy is required then using the Sample Addition or Standard Addition methods may be a better solution than adding ISAB. If it is decided that ISAB should be added then the most important factor is that it should be added equally to standards and samples. 73 A big advantage of this method is that it can be used to measure large batches of samples covering a wide range of concentrations very rapidly without having to change range, recalibrate or make any complicated calculations. Moreover, if ISAB is not being used, it is not necessary to measure the volume of the samples or standards. Quite acceptable results can be obtained for some elements by simply dangling the electrodes in a river or pond or effluent outflow without the need to take samples in small beakers. b) Incremental Methods There are three main types of incremental methods in general use: •Standard (or Known) Addition, •Sample Addition, •Sample Subtraction. Standard Addition and Sample Addition Methods. These methods involve measuring the voltage in a relatively large, accurately measured, volume of sample (for standard addition) or standard (for sample addition) then adding a much smaller volume of standard (or sample) and taking a second reading after the voltage has stabilised in the mixture. For full details see: www.nico2000.net/datasheets/staddl.html Summary of Advantages over Direct Potentiometry •The electrodes remain immersed throughout the process so that there is little change in the liquid junction potential of the reference electrode (which can often be changed by several millivolts when the electrodes are removed from one solution and placed in another) between calibration and sample measurement - and therefore this source of measurement error is virtually eliminated. •Calibration and sample measurement are both made essentially at the same time and in the same solution so that ionic strength and temperature differences between standard and sample are not significant and ISAB is not normally required. 76 •Once the approximate concentration for the samples is known, the calibration (slope) can be "fine tuned" by analysing a standard with a concentration that lies within the range of the samples (and is at the same temperature) and then adjusting the slope and re-calculating the results until the standard gives the correct answer. This "fine tune" procedure is very quick and easy using the ELIT ISE/pH Ion Analyser Software. •Measuring the slope at or very near to the sample concentration means that these methods can be used with old or worn electrodes which may not be completely linear over their whole range, as long as the slope is stable and reproducible over the limited range of the samples. Sample Subtraction method. This involves adding a small amount of sample solution to a standard solution of an ion with which it will react stochiometrically to form a complex or precipitate, thus reducing the concentration of both ions. The ISE used will be sensitive to the reactive ion in the standard, not the sample. The big advantage of this method is that it can extend the range of ions measurable by ISEs to others for which no ion-sensitive membranes are available. For example, there is currently no ISE capable of detecting the sulphate ion. However, sulphate can be removed from solution by precipitating as barium sulphate, and there is an ISE which is sensitive to barium. Therefore, sulphate can be measured by first measuring the voltage in a pure barium chloride standard. Then adding a known volume of a sample containing sulphate, wait for precipitation to be completed, and measure the voltage on the barium electrode again. The amount of barium used can then be calculated using a similar equation to that used for Sample Addition and the sulphate content in the sample will be the same as this - since each sulphate ion will combine with one barium ion. c) Potentiometric Titrations Potentiometry is generally valuable as a technique for detecting the end- point of titrations where there is often a drastic change in the concentrations of the reactants and thus a big shift in the electrode potential. These end point determinations can often be made more precisely than other ISE methods because they depend on the accuracy of the volumetric measurements rather than the measurement of the electrode potential. 77 For example, when a calcium solution is titrated against the complexing reagent EDTA there is a gradual decrease in the Ca concentration as more EDTA is added until the end point when all the Ca disappears from solution. The progress of this titration can be monitored using a calcium electrode. This method can also be used to extend the range of ions measurable by ISEs. For example aluminium cannot be measured by direct potentiometry but it can be titrated by reacting with sodium fluoride and monitoring the reaction using a fluoride electrode. It can also be used for elements for which it is difficult to maintain stable standard solutions or which are toxic and it is undesirable to handle concentrated standard solutions. For example, cyanide solutions can be titrated against a hypochlorite solution which forms a complex with the cyanide ions and effectively removes them from solution. The amount of cyanide in the original solution is proportional to the amount of hypochlorite used from the start of the titration until the end-point when there is no further change in the cyanide electrode potential. 78  To prevent slipping and this protects the bench surface the hot air oven is mounted on four rubber foots. The scale is calibrated in 5degC steps. WORKING PRINCIPLE The working of the hot air oven is based on the hot air inside the chamber of oven by the forced circulation. As it is a universal scientific fact that in any chamber hot air rises above, So by utilizing this principle when the hot air reaches the top of chamber it is circulated back to bottom by a fan installed inside the chamber and hence optimum amount of heat is achieved gradually inside the hot air oven. After heating the content of the oven for two hours at 160 @c, the articles are allowed to remain there, till the temperature comes down to 40 @c. then the sterilized materials is then removed from the oven. APPLICATIONS 1. It is mainly used for the sterilization of glasswares such as pestle and motar, petridishes, flasks, pipettes, bottles, test tubes etc. 2. It is used for the sterilization of powders such as sulphacetamides, sulphadiazenes, kaolin, zinc oxide, starch etc. 3. Injections where fixedoils is used as the vehicle are sterilised by the dry heat method 4. Example: injections of progestrone, injection of testosterone propionate and injections of oestradiols dipropionate. 5. It is also used for sterilisation of scalpels, scissors, spatula, blades and glass syringes. 6. The chemicals, glassware in laboratories, research institutions, industries, hospitals use hot air ovens are suitable for temperature upto 250degC. 7. Hot air ovens suits to various applications like heating, drying, sterilizing & baking. ADVANTAGES 1. It is used for the sterilisation of those substances which gets spolied during moist heat sterilisation. Eg: oily materials and powders. 2. The method is suitable for sterilisation of assembled equipments such as all glas syringes due to expose to high temeprature for a long time. 3. It is not so damaging to glass and metals equipments as moist heat. 4. Dry heat will not corrode or rust instruments or needles. 5. Dry heat will sterilize instruments containing many parts that can not be disassembled 81 DISADVANTAGES 1. This method is not suitable for the surgical dressings. 2. This method is not suitable for the most of the medicaments, rubber and plastic good because the articles are exposed to a very high temperature for a long period. 3. Dry heat penetrates slowly and unevenly. 4. Dry heat requires long exposure times to effectively achieve sterility. 5. Dry heat requires higher temperatures that many items cannot be safely exposed to. 6. Dry heat requires specialized packaging materials that can sustain integrity under high heat conditions. 7. Dry heat may require different temperature and exposure times, depending on the type of item being sterilized. PRECAUTIONS 1. Glass apparatus must be wrapped with the clean cloth or filter paper and containers must be plugged with non assorbants cotton wool. 2. The article and substances which are to be sterilised should not be placed at the floor of the oven as it receieves direct heat and becomes much hotter. 3. The oven should not be over loaded with the materials meant for sterilisation. 4. There should be sufficient space in between the artilcles, so that there is uniform distribution of heat. 5. 82 CHAPTER TWELVE ELIZA READER Elisa Reader Principle The basic principle in Elisa readers are the special filters for only 5-6 standard wavelengths for all Elisa kits (which depends from substrate type). Always check your kit’s instructions with the reader filters (or the substrate electronic absorbance spectrum). For instance, you can measure the maximum to reach the highest sensitivity of your elisa photometer by putting your colored substrate in the plate reader for the absorbance spectra. The Elisa photometers have these filters which fit to almost all substrates commonly used. Elisa Reader vs. Spectrophotometer The major difference between the Elisa plate reader and the spectrophotometer is that the Elisa readers are commonly used for intensity measurements on a large number of samples where you can also use a very small volume of sample. The spectrophotometer appears to be more sophisticated and much detailed as discussed below: Spectrophotometer •The instrument is more accurate than the Elisa microplate reader •It can measure at any wavelength •It can record a spectrum •It can measure the kinetic continuously •Moreover, spectrophotometer offers full monochromator and more sensitive detection, anisotropy measurements. The only disadvantage with this instrument being that you can easily pick up artifact spectra (based on unclean samples or misaligned optics) therefore there’s the need to know your instrument well and use the recommended correction factor files for the instrument and do an many controls as possible of all the components of the solution you may be having. Elisa Reader •The elisa reader commonly referred to as the microplate reader is much faster compared to spectrophotometer •This instrument used multiple sample at the same time •Smaller volumes can be used such as 500-200 ul for 96-well plates •It can be equipped with robot for screening too 83 Test samples are located in specially designed plates with a specific number of wells where the procedure or test is carried out. Plates of 8 columns by 12 rows with a total of 96 wells are common. There are also plates with a greater number of wells. For specialized applications, the current trend is to increase the number of wells (384-well plates) to reduce the amount of reagents and samples used and a greaterthroughput.Thelocationoftheopticalsensorsofthe microplate reader varies depending on the manufacturers: these can be located above the sample plate, or directly underneath the plate’s wells. Nowadays microplate readers have controls regulated by microprocessors; connection interfaces to information systems; quality and process control programs, which by means of a computer, allow complete test automation. ELIZA MICROPLATE READER Equipment required for ELISA testing In order to perform the ELISA technique, the following equipment is required: 1. Microplate reader. 2. Microplate washer . 3. Liquid dispensing system (multi-channel pipettes may be used). 4. Incubator to incubate the plates. Figure 1 illustrates how this equipment is interrelated. Mechanical phases of the ELISA technique Using the equipment When an ELISA test is conducted, it typically follows these steps: 1. A first washing of the plate may be done using the microplate washer. 2. Using a liquid dispenser or the multi-channel pipettes, wells are filled with the solution prepared to be used in the test. 3. Theplateisplacedintheincubatorwhereatacontrolled temperature, a series of reactions take place. Stages 1, 2 and 3 can be repeated several times depending on the test, until the reagents added have completed their reactions. Finally, when all the incubation steps have been completed, the plate is transferred to the microplate reader.The reading of the plate is done and a diagnosis can be deduced. Biochemical phases of the ELISA technique1 The ELISA technique from a biochemical point of view: 1. The plate wells are coated with antibodies or antigens. 2. Samples, controls and standards are added to the wells and incubated at temperatures ranging between room temperature and 37 °C for a determined period of time, according to the test’s characteristics. During the incubation, the sample’s antigen binds to the antibody coated to the plate; or the antibody in the sample binds to the antigen coated on the plate, according to their presence and quantity in the sample analyzed. 3. After incubation, the unbound antigen or antibodies are washed and removed from the plate by the microplate washer using an appropriate washing buffer. 86 4. Next, a secondary antibody, called the conjugate, is added. This harbours an enzyme which will react with a substrate to produce a change of colour at a later step. 5. Then begins a second period of incubation during which this conjugate will bind to the antigen-antibody complex in the wells. 6. After the incubation, a new washing cycle is done to remove unbound conjugate from the wells. 7. A substrate is added. The enzyme reacts with the substrate and causes the solution to change in colour. This will indicate how much antigen-antibody complex is present at the end of the test. 8. Once the incubation time is completed, a reagent is added to stop the enzyme-substrate reaction and to prevent further changes in colour. This reagent is generally a diluted acid. 9. Finally, the plate in is read by the microplate. The resulting values are used to determine the specific amounts or the presence of antigens or antibodies in the sample. Note: Some of the wells are used for standards and controls. Standards allow the cut-off points to be defined. The standards and controls are of known quantities and are used for measuring the success of the test, evaluating data against known concentrations for each control. The process described above is common, although there are many ELISA tests with test-specific variants. 87 CHAPTER THIRTEEN REFRIGERATOR How Does a Refrigerator Work? In the refrigeration cycle, there are five basic components: fluid refrigerant; a compressor, which controls the flow of refrigerant; the condenser coils (on the outside of the fridge); the evaporator coils (on the inside of the fridge); and something called an expansion device. Here’s how they interact to cool your food. 1. The compressor constricts the refrigerant vapor, raising its pressure, and pushes it into the coils on the outside of the refrigerator. 2. When the hot gas in the coils meets the cooler air temperature of the kitchen, it becomes a liquid. 88