Instrumentation PDF

INSTRUMENTATION CCLAB INTRODUCTION In addition to this, they are also used in the medical laboratory to preserve some reagents Medical labor...

INSTRUMENTATION CCLAB INTRODUCTION In addition to this, they are also used in the medical laboratory to preserve some reagents Medical laboratories perform several chemical such as Pregnancy test kits and biological tests on a daily basis. In order to Rapid plasma regain (RPR) test kits. generate accurate and precise results, the labs Blood grouping anti sera and others which are require a wide range of effective and kept in the refrigerators to prevent their sophisticated equipment. deterioration which may happen if they at The complexity and range of equipment room temperature. required by the lab varies with the applications or tests they need to perform. HOT AIR OVEN Hot air ovens are laboratory testing equipment that are MICROSCOPE used to sterilize materials such as glassware, Microscopes are the most commonly found lab chemicals, and sealed containers. They are also used instruments. They are used to view micro-sized for drying, baking, curing, and heat-treating various objects which cannot be seen by naked eye. substances. WATER BATH A water bath is a laboratory equipment that is used to incubate samples at a constant temperature over a long period of time. Water bath is a preferred heat source for heating flammable chemicals instead of an ANALYTICAL BALANCES open flame to prevent ignition. Nowadays analytical and electronic are the most popularly used balances in medical laboratory to provide a precision and accuracy for reagent and standard preparation. INCUBATOR An incubator is a device used to grow and maintain microbiological cultures or cell cultures. The incubator maintains optimal temperature & humidity An incubator is required where as dry heat block or a water bath may be used. REFRIGERATORS AUTOCLAVE Refrigerators are physical means of preserving An autoclave is used to sterilize samples and various laboratory specimens. lab instruments, such as plastic tubes, pipette The suppress the growth of bacteria and tips, glassware and surgical instruments in a maintain the specimens with little alteration. research laboratory. It uses temperature, INSTRUMENTATION CCLAB pressure and steam for the decontamination of A Colorimeter is a light and sensitive device used to materials. measure the transmittance and absorbance of light The working principle of an autoclave is based that passes through a liquid sample. on the downward displacement of air through gravity. Usually, an autoclave functions on standard settings Pressure: 15 psi, Steam Temperature: 121°C for 15 minutes. pH meter pH meter is an instrument used to measure the pH or hydrogen ion concentration of the solution by the potential difference between two electrodes. Used for Water Purification Equipment used for water purification and distillation includes deionized (DI) water systems, water distillers, reagent-grade water systems, and laboratory filters. DESSICATORS The primary purpose of a desiccator is to protect hygroscopic materials, chemicals, and samples from Blood Gas Analyzers moisture, ensuring they remain stable and maintain Blood gas analyzers use a person's blood to their intended properties measure the pH and partial pressure of oxygen, as well as carbon dioxide. It can also evaluate bicarbonate concentration in the blood. This test can be performed very quickly, and results are generally available within minutes after the test. HAEMOGLOBINOMETER As the name suggests, a haemoglobinometer is used colorimeter/photometer for estimating hemoglobin in the blood. It uses INSTRUMENTATION CCLAB spectrophotometry for calculating hemoglobin B) a head : fixed to the shaft , with buckets for concentration. It is a portable device, therefore can be holding the centrifuge tubes. easily carried. The result is expressed in gram C) tubes : containing the liquid to be hemoglobin per 100 ml of the blood sample. centrifuged Centrifugation mechanism: When the spindle rotates the tubes are subjected to centrifugal force. They swing out to the horizontal and the particles in suspension in the liquids in the tubes are thrown to the bottom of the tubes. FUNDAMENTALS OF CENTRIFUGES CENTRIFUGE Centrifuges are commonly found in lab instruments that are used to separate fluids based on their density. They sediment particles (cell, bacteria, casts, parasites, blood etc.) suspended in fluid by centrifugal force. DEFINITION A centrifuge is a laboratory device that is used for separation of fluids based on density. Centrifugation targets These particles form the centrifuge deposit which can be separated from the supernatant fluid and examined. The deposit may contain, for example: blood cells; parasite eggs (in diluted stools); cells from the urinary tract ( in urine). PRINCIPLE A body is rotated in a circular movement at speed. This creates a force that drives the body away from the center of the circular movement. Components of a Centrifuge A) central shaft (spindle) : that rotate at high speed TYPES OF CENTRIFUGE INSTRUMENTATION CCLAB Hand operated centrifuge: CENTRIFUGE CATEGORIES This is operated manually by turning Benchtop centrifuges a handle,It takes two or four tubes. Are broad class of centrifuges with The hand-operated centrifuge can be maximum speed in RCFs can range used : from 100-50.000 x g. To examine urinary deposits Tubes volumes can range from 1ml To concentrate certain to a few liters. parasites in stools The speed is insufficient for Refrigerated benchtop centrifuges satisfactory separation of Ideal for centrifugation of samples erythrocytes from plasma in blood. that may be temperature sensitive , such as live cells or proteins. Electric centrifuge: Sample volumes from under 1 ml Electric centrifuges are more accurate than hand- to few liters. operated centrifuges and should be used whenever Speed can reach up to 60.000 x g possible. Electric centrifuges are used with two types Clinical benchtop centrifuges of head — the “swing-out” head and the “angle” head. Low speed centrifuges ideal for the separation of whole blood components, such as red blood cells as well as other body fluids. Their speed range between 200- 6.000 rpm. Microcentrifuge Used for samples of small volume 1ml( PCR tube), 2 ml ,1.5ml, 0.5 ml. “Swing-out” head: Their speeds up to 16.000 x g. The head is designed to swing the tubes out to a horizontal position during centrifuging. This is the type most frequently needed. Vacuum centrifuge / concentrators Use vacuum centrifugal force , temperature and or gas to remove liquid solvent for the concentration or desiccation of samples systems. This centrifuge is ideal for purification of preparation of samples such as : nucleic acids and proteins. For evaporation of solvents vacuum centrifuges typically utilize built in heating systems. “Angle” head: The “angle” head holds the tubes at an angle of about 45° during centrifuging. It is useful for certain techniques, e.g. agglutination tests in blood-grouping by the test tube method. Instruction for use: You should always follow the manufacturer's instructions when using the centrifuge. Installing the centrifuge: The centrifuge must be placed on rubber pads or a mat on a flat level surface. Balancing the tubes INSTRUMENTATION CCLAB Balance the tubes that are opposite each other by Always balance the centrifuge buckets before starting weighing them in their buckets on the open two pan the centrifuge. Failure to do this can cause excessive balance. wear or the centrifuge may move. Ensure that the lid is closed before starting the centrifuge. When starting the centrifuge, gradually increase the speed, turning the knob slowly, until the desired speed is reached. Stop the centrifuge gradually. Never try to slow the centrifuge down with your hand. Never open the centrifuge until it has come to a complete stop. Remove the tubes slowly and carefully. FUNDAMENTALS OF GLUCOMETER Glucometer: “A blood glucose meter is often called a glucometer or glucose meter or sugar meter. Glucometer is fundamentally a medical device which measures the concentration of glucose in human blood.” GLUCOMETER - USES It is measured by obtaining a small drop of blood via pricking the skin and placed on a test strip so that the meter can read it. GLUCOMETER IS USED BY PEOPLE WHO HAVE DIABETES TO: Control the taxes of glucose. 70mg/dL hypoglycemia 180mg/dL hyperglycemia Recognize when they need to use INSULINE. Glucometer - Equipment Lancet Meter with test strip inserted Test strips Lancet pen top Lancet pen with lancet inserted SAFETY PRECAUTIONS Check that the tubes are the correct size for the centrifuge. Tubes that are too long or too small may break. Fill the tubes to no more than three-quarters full to prevent spillage in the bowl. INSTRUMENTATION CCLAB 2. Isoelectric Focusing 3. Imunoelectrophoresis EFFECTS A better lifestyle for diabetics. Control of glucose. Reduction the rates of diabetics emergency. Lower expenses on hospitals care. Paper Electrophoresis * It is the form of electrophoresis that is carried out on FUNDAMENTALS OF ELECTROPHORESIS filter paper. ELECTROPHORESIS * This technique is useful for separation of small DEFINITION: It describes migration of charged charged molecules such as amino acids and small particles or molecules under the influence of electric proteins. field. * The serum proteins are separated into 5 distinct PURPOSE FOR CARRYING OUT bands- albumin, α-, α2-, β- and γ-globulins. ELEECTROPHORESIS 1. To determine the number, amount and mobility of components in given sample or to separate them. 2. Determination of molecular weight of proteins and DNA sequencing. 3. To obtain information about the electrical double layers surrounding the particles. FACTORS AFFECTNG ELECTROPHORETIC MOBILITY 1. CHARGE Gel Electrophoresis 2. SIZE * It is a technique used for the separation of DNA, 3. SHAPE RNA, or protein molecules according to their size and TYPES OF ELECTOPHORESIS charge using an electric current applied to the gel 1. Zone Electrophoresis matrix. a) Paper Electrophoresis * The serum proteins can be separated to about 15 b) Gel Electrophoresis distinct bands. INSTRUMENTATION CCLAB * The antibodies when come in contact with antigens, Types of gel: precipitation occurs, resulting in the formation of 1. Agarose gel precipitin bands. 2. Polyacrylamide gel 3. Sodium dodecyl sulphate (SDS) POINT OF CARE TESTING (POCT) IN CLINICAL CHEMISTRY Point-of-Care Testing (POCT) is clinical laboratory testing conducted close to the site of patient care where care or treatment is provided; Point-of-care testing (POCT), also known as near-patient testing, refers to any analytical test performed outside the laboratory and may be located either within a hospital as an adjunct to the main laboratory or for primary healthcare outside the hospital setting. ADVANTAGES OF POCT IN CLIN. CHEM LABORATORY Immediate Results - POCT enables immediate patient results, allowing for timely treatment decisions, Reduced Turnaround Time - POCT reduces the time taken to obtain results, leading to faster patient diagnoses, Polyacrylamide is employed for the determination of Enhanced Patient Care - POCT contributes molecular weights of proteins in a popularly known to enhanced patient care by providing real time electrophoresis technique known as SDS-PAGE. data for treatment, ISOELECTRIC FOCUSING POCT DEVICES USED IN CLIN. CHEM * This technique is based on the immobilization of the LABORATORY Blood Glucose Meters - These devices are molecules at isoelectric pH during electrophoresis. * It is ideal for separation of amphoteric substances. widely used for monitoring blood glucose levels in diabetic patients * It’s gels contain synthetic buffers called ampholytes that smooth the pH gradients. Portable Coagulometers - Portable * The serum proteins can be separated to as many as coagulometers help In monitoring blood coagulation parameters, essential for 40 bands. IMMUNOELECTROPHORESIS anticoagulant therapy. * It is a two stage process, Electrophoresis is Handheld Chemistry Analyzers - These conducted in first stage and immunoprecipitation compact analyzers provide rapid results for using antibodies against specific proteins in the second various clinical biochemistry tests. stage. INSTRUMENTATION CCLAB APPLICATIONS IN CLINICAL CHEMISTRY 1. Emergency Departments - POCT Is essential in FUNDAMENTALS OF SPECTOPHOTMETER emergency departments for quick assessment and triage of patients Biochemical analyzers can be used in hospital 2. Outpatient Clinics - POCT facilitates on-the-spot laboratories to perform various tests like albumin tests, diagnosis and management for outpatient care and sugar level tests, or to detect levels of enzymes and monitoring creatinine in the blood 3. Home Care Settings - A POCT devices enable It is a fully automated analyzers. accurate testing and monitoring for patients receiving Spectrophotometer - An instrument that measures care at home the amount of photons (the intensity of light) absorbed CHALLENGES AND LIMITATIONS OF POCT IN after it passes through sample at specific wavelength. CLIN. * Almost all of the energy available at Earth's CHEM LABORATORY surface comes from the sun. The sun gets its Quality Control - Maintaining high-quality energy from the process of nuclear fusion. standards in POCT requires strict adherence * This energy eventually makes its way to the to quality control protocols outer regions of the sun and is radiated or Operator Training - Proper Training of emitted away in the form of energy, known as healthcare professionals is crucial to ensure electromagnetic radiation. accurate and reliable test results. * A particle of electromagnetic radiation is Regulatory Compliance - Adhering lo known as a photon, regulatory requirements is imperative to * Electromagnetic radiation, also known as ensure the safety and reliability of POCT radiant energy (or radiation), is spread in the devices form of electromagnetic waves. QUALITY CONTROL AND ASSURANCE IN POCT * Electromagnetic waves are waves that can 1 Internal Quality Control - Regular internal quality cause charged particles (such as electrons) to control checks are performed to monitor the precision move up and down. These waves have both of POCT devices electrical and magnetic properties and can 2 External Quality Assessment - Participation In travel through gases, liquids, solids, and external quality assessment programs ensures the through empty space (or a vacuum) at nearly reliability of POCT results. 300,000 kilometers per second (the speed of 3 Proficiency Testing - Regular proficiency testing Is light). conducted to evaluate the performance of POCT * Electromagnetic waves are characterized by operators and devices wavelength and frequency. Future Developments and Trends in POCT * The wavelength is the distance between 1 Technological Advancements - Ongoing two wave crests or troughs. The highest point technological innovations will lead to more of a wave is called the crest, and the lowest sophisticated and accurate POCT devices point of a wave is called the trough. 2 Point-of-Care Biomarkers – Advancements in * Frequency is expressed in hertz (Hz) and biomarker detection will expand the applications of refers to the number of wavelengths that pass POCT in clinical biochemistry, a fixed point in 1 second. The shorter the 3 Connectivity and Integration – Enhanced wavelength is, the higher its frequency will be. connectivity will enable seamless integration of POCT The reverse is also true. For example, radio data with electronic health records waves have the longest wavelength and the lowest frequency. TAKEAWAYS * The electromagnetic spectrum is the term used by scientists to describe the entire range of light that exists. From radio waves to gamma rays, most of the light in the universe is, infact, invisible to us! Light is a wave of alternating electric and magnetic fields INSTRUMENTATION CCLAB Properties of Light 1.) Particles and Waves Light waves consist of perpendicular, oscillating electric and magnetic fields Parameters used to describe light - amplitude (A): height of wave's electric vector - Wavelength (λ): distance (nm, cm, m) from peak to peak - Frequency (v): number of complete oscillations that the waves makes each second Hertz (Hz): unit of frequency / second (s") 1 megahertz (MHz) = 10°Hz Parameters used to describe light t- Energy (E): the energy of one particle of light (photon) is proportional to its frequency where: E = photon energy (Joules) v = frequency (sec-1) h = Planck's constant (6.626x10-34J-s) As frequency (v) increases, energy (E) of light increases Relationship between Frequency and Wavelength where: c = speed of light (3.0x1010 cm/s in vacuum)) v = frequency (sec-1) A = wavelength (cm) Relationship between Energy and Wavelength As wavelength (λ) decreases, energy (E) of light increases INSTRUMENTATION CCLAB Absorption of Light 1.) Colors of Visible Light Many Types of Chemicals Absorb Various Forms of Light Light is made up of wavelengths of light, and each wavelength is a particular color. The color we see is a result of which wavelengths are reflected back to our eyes. o White light is actually made of all of the colors of the rainbow because it contains all wavelengths, and it is described as polychromatic light. Light from a torch or the Sun is a good example of this, o Light from a laser is monochromatic, which means it only produces one color. 3.) Beer's Lambert's Law Beer's law states that the concentration of a substance is directly proportional to the amount of light absorbed or inversely proportional to the logarithm of the transmitted light. Lambert's law stated that absorbance of a 2.) Colors of object material sample is directly proportional to its Objects appear different colors because they thickness (path length) absorb some colors (wavelengths) and Light of a particular wavelength enters the reflected or transmit other colors. The colors ‘sample’. we see are the wavelengths that are reflected Light scatters from particles in solution, or transmitted. reducing light transmission For example, a red shirt looks red because the Light is absorbed by molecules/particles dye molecules in the fabric have absorbed the reducing light transmission wavelengths of light from the violet/blue end » The relative amount of light absorbed (A) through a of the spectrum. sample is dependent on: - distance the light must pass through the sample (cell length path - b) - amount of absorbing chemicals in the sample (analyte concentration — c) - ability of the sample to absorb light (molar absorptivity - α) INSTRUMENTATION CCLAB Absorbance is sometimes called optical density (OD) Absorbance is useful since it is directly related to the analyte concentration, cell pathlength and molar absorptivity. This relationship is known as Beer's Law where: A = absorbance (no units) α = molar absorptivity (L/mole-cm) b = cell pathlength (cm) c = concentration of analyte (mol/L) Beer’s Law Beer's Law allows compounds to be quantified > The relative amount of light passing through the by their ability to absorb light, Relates directly sample is known as the transmittance (T) to concentration (c) Absorptivity depends on molecular structure and the way in which the absorbing molecules react with different energies. For any particular molecular type, absorptivity changes as wavelength of radiation changes. The amount of light absorbed at a particular wavelength depends on the molecular and ion types present and may vary with concentration, pH, or temperature. b is the length of light path (1 cm) through the solution. Because the path length and molar absorptivity are constant for a given wavelength - Absorbance (A) is the relative amount of light absorbed by the sample and is related to transmittance (T) > It cannot be measured directly by a spectrophotometer but rather is mathematically derived from % T INSTRUMENTATION CCLAB * LED Lamp - Produce a single wavelength of light, thus, LED lamp does not require a monochromator. Its life is very long. LED light source has little variation in bandwidth, and it's stable. LED lamp is a low-cost light source. * Deuterium Lamp - A continuous spectrum in the ultraviolet region is produced by electrical excitation of deuterium at low pressure. It is Also known as D2 lamp, its wavelength range is from of 190nm - 370nm. Because of its high temperature behavior, the normal glass housing is not suitable, but requires quartz, or other materials. Life time of a typical deuterium lamp is about 1000 hours. An UV / Vis spectrophotometer will design a deuterium lamp with a halogen lamps, in order to cover the entire UV and visible light wavelength. * Xenon Lamp - Xenon lamp provides high energy light source, and it Spectrophotometer can reach a steady state in a short time period. Its light 1.) Basic Design covers the entire UV and visible wavelength range, - An instrument used to make absorbance or from 190nm to 1100nm. An xenon lamp flashes in a transmittance measurements is known as a frequency of 80Hz, so that the life time is longer than spectrophotometer deuterium lamp or halogen lamp. However, the cost of a xenon lamp is higher. * Hydrogen Gas Lamp and Mercury Lamp are commonly used in UV absorption measurements as well as visible light. * Globar (silicon carbide rod): Infra-Red Radiation at wavelengths: 1200 - 40000 nm NiChrome wire (750 nm to 20000 nm) for IR Region. Laser Sources: Light Source: - Acronym for: light amplification by stimulated - The light source typically yields a high output of emission of radiation polychromatic light over a wide range of the spectrum - These devices transform light of various frequencies into an extremely intense, focused, and nearly non- divergent beam of monochromatic light - Used when high intensity line source is required - Cost as varied as possible wavelength range: from two-dollar pointers to million-dollar devices * Properties of laser sources include Spatial coherence: Wave fronts of all the photons are “launched in unison,” all moving in step with the others Types of light sources used in spectrophotometers (coherent). include: - Strong, concentrated, tight beam (directional). * Incandescent lamps and lasers - Laser light is typically monochromatic — emitting Incandescent Lamps: only one wavelength * Halogen Lamp - Also known as tungsten or quartz lamp, and the wavelength range of halogen lamp is in the visible light region, which is in the range of 320nm to 700 nm. If the instrument is equipped with a halogen lamp only, it means the instrument can only measure visible light. General halogen lamp life is about 2000 hours, or more INSTRUMENTATION CCLAB compounds absorb some wavelengths of light while transmitting others. * Filters ranges: A) Long-Pass Filter: A long-pass configuration transmits longer wavelengths above a specified range while attenuating shorter wavelengths. B) Short-Pass Filter: A short-pass configuration Wavelength Selector (monochromator) transmits shorter wavelengths over an active range Spectral Isolation while attenuating longer wavelengths. - system for isolating radiant energy of a C) Bandpass Filter: Short-pass and long-pass filters desired wavelength and excluding that of other can be combined to form a bandpass filter, which wavelength is called spectral isolation and it is features lower transmittance values and rejects any achvied with the help of Monochromator. wavelengths outside a predetermined interval. The Monochromator consists of these parts: size of the interval depends on the number of filter ~ Entrance slit — layers used in the device. ~ Collimating lens or mirror — ~ Dispersion element: A special plate with hundreds of parallel grooved lines. The grooved lines act to separate the white light into the visible light spectrum Thin-Film Optical Filters - Although colored-glass filters can be configured in a wide variety of forms, their transmission curves normally cannot be customized to fit a specific * Filters: application. Thin-film optical filters, however, can be - In applications requiring control of light designed to meet nearly any type of transmission transmissions, optical filters can serve as an effective curve, making them more effective for certain means of selectively blocking or transmitting light applications. In addition to standard light control based on certain properties, such as wavelengths or systems, thin-film filters can be used to transmit or intensity block laser light, which may contain extra wavelengths * Colored Optical Filters than those in the main laser line. - An interference filter or dichroic filter is an Neutral-Density Filters optical filter that reflects one or more spectral bands - A neutral-density filter is typically used to attenuate or lines and transmits others, while maintaining a light at all wavelengths equally. nearly zero coefficient of absorption for all wavelengths of interest. - Absorption filters, commonly manufactured from dyed glass or pigmented gelatin resins. These INSTRUMENTATION CCLAB Sample Cell: - A cuvette (French: cuvette = "little vessel") is a small tube of circular or square cross section, sealed at one end, and designed to hold samples for spectroscopic experiments. Prism - The prism is another type of monochromator. A narrow beam of light focused on a prism is refracted as it enters the denser glass. Short wavelengths are refracted more than long wavelengths, resulting in dispersion of white light into a continuous spectrum. The prism can be rotated, allowing only the desired wavelength to pass through an exit slit Photo Detector: ~ Measures the amount of light passing through the sample. ~ Usually works by converting light signal into electrical signal ~The least expensive of the devices is known as a Diffraction gratings barrier-layer cell, or photocell. - A diffraction grating consists of many parallel grooves ~The photocell is composed of a film of light-sensitive (15,000 or 30,000 per inch) etched onto a polished material, frequently selenium, on a plate of iron. Over surface (Thin layer of aluminum-cupper alloy on flat the light-sensitive material is a thin, transparent layer glass surface).. of silver. When exposed to light, electrons in the light- - Diffraction, the separation of light into component sensitive material are excited and released to flow to wavelengths, is based on the principle that the highly conductive silver in comparison with the wavelengths bend as they pass a sharp corner. silver, a moderate resistance opposes the electron flow - The degree of bending depends on the wavelength. toward the iron, forming a hypothetical barrier to flow As the wavelengths move past the corners, wave fronts in that direction. Consequently, this cell generates its are formed. own electromotive force, which can be measured. The produced current is proportional to incident radiation. Sample Cell: sample container of fixed length (b). - Usually a square cuvet - Made of material that does not absorb light in the wavelength range of interest 1. Glass - visible region 2. Quartz - ultraviolet 3. NaCl, KBr — Infrared region INSTRUMENTATION CCLAB ~The third major type of light detector is the photomultiplier (PM) tube, which detects and amplifies radiant energy. ~incident light strikes the coated cathode, emitting electrons. The electrons are attracted to a series of anodes, known as dynodes, each having a successively higher positive voltage These dynodes are of a material that gives off many secondary electrons when hit by single electrons. Initial electron emission at the cathode triggers a multiple cascade of electrons within the PM tube itself. Because of this amplification, the PM tube is 200 times more sensitive than the Phototube: phototube. ~A phototube is similar to a barrier-layer cell in that it has photosensitive material that gives off electrons when light energy strikes it. ~ it differs in that an outside voltage is required for operation. ~ Phototubes contain a negatively charged cathode and a positively charged anode enclosed in a glass case. ~The cathode is composed of a material (e.g., rubidium or lithium) that acts as a resistor in the dark but emits electrons when exposed to light. ~The emitted electrons jump over to the positively charged anode, where they are collected and return through an external, measurable circuit. The cathode usually has a large surface area. ~PM tubes are used in instruments designed to be extremely sensitive to very low light levels and light flashes of very short duration. ~The accumulation of electrons striking the anode produces a current signal, measured in amperes, that is proportional to the initial intensity of the light. The analog signal is converted first to a voltage and then to a digital signal through the use of an analog to- digital (A/D) converter. Digital signals are processed electronically to produce absorbance readings Readout device. - In the past nearly all spectrophotometer used ammeters or galvanometers. Newer digital devices and printers have now replaced these, and many instruments relay their electrical output directly to INSTRUMENTATION CCLAB computer circuits where calculations are performed, Single Beam Spectrophotometer allowing direct reporting of sample concentration. Advantages Microprocessor and recorders - Single beam instruments are less expensive - High energy throughput due to non-splitting of source beam results in high sensitivity of detection Disadvantages - Instability due to lack of compensation for disturbances like electronic circuit fluctuations, voltage fluctuations, mechanical component's instability or drift in energy of light sources. Such drifts result in abnormal fluctuations in the results. Double Beam Spectrophotometer 2.) Types of Spectrophotometers Advantages of Double Beam Single-Beam Instrument: sample and blank are - Modern improvements in optics permit high level of alternatively measured in same sample chamber. automation and offer the same or even better level of detection as compared to earlier single beam systems. - Instability factors due to lamp drift, stray light, voltage fluctuations do not affect the measurement in real- time. Disadvantages - The cost factor is more than offset by the advantages offered by modern double beam systems Difference between Colorimeter and Spectrophotometers Colorimeters Double-Beam Instrument - Continuously compares sample and blank - A colorimeter is generally any tool that characterizes - Automatically corrects for changes in electronic color samples to provide an objective measure of color characteristics. signal or light intensity of source - Colorimeters use a colored light beam to measure sample concentration. - A colorimeter uses a tristimulus absorption filter to isolate a broad band of wavelengths. It is generally rugged and less complex than a spectrophotometer. - In colorimetry, colored light passes through an optical filter to produce a single band of wavelengths. - Both of these techniques use the Beer-Lambert Law to determine concentration. The difference is that colorimeters measure the absorbency of light in a sample. Spectrophotometers - A spectrophotometer is a photometer that can measure intensity as a function of wavelength of light. - Spectrophotometers use a white light that is passed through a slit and filter to analyze samples. - A spectrophotometer uses an interference filter or a grating and prism to isolate a narrow band of wavelengths. It is a more complex instrument than a colorimeter. - In spectrophotometers, the white light is passed through a special filter that disperses the light into many bands of wavelengths. - Spectrophotometers measure the amount of light that passes through it. INSTRUMENTATION CCLAB