This document provides information about using a spectrophotometer to analyze compounds. It discusses key concepts like:
- Absorbance increases linearly with concentration according to Beer's Law (A = εcl)
- Transmittance and absorbance can be converted between each other using mathematical relationships
- Common absorbing biochemicals like nucleic acid bases and amino acids each have characteristic absorption spectra
- Obtaining a spectrum of an unknown dye allows identification by comparing peaks to sample spectra
- A standard curve relating absorbance to concentration can be used to determine the concentration of unknown samples
The document then provides guidance on experiments students will perform to obtain a spectrum of an unknown dye, calculate its extinction coefficient, generate a standard
This document provides an overview of fluorometry, including basic concepts, instrumentation, and applications. It discusses how fluorescence occurs when a molecule absorbs light at one wavelength and reemits light at a longer wavelength. Factors that affect fluorescence such as temperature, pH, and dissolved oxygen are also covered. The relationship between fluorescence intensity and concentration is explained. Additionally, the document defines fluorescence polarization and describes various types of quenching including self-quenching, chemical quenching, and collisional quenching.
Beer's law and Lambert's law describe the absorption of light in materials. Beer's law states that absorbance is directly proportional to concentration, while Lambert's law states absorbance is directly proportional to path length. Beer and Lambert combined their laws into the Beer-Lambert law, which states absorbance is equal to the molar absorptivity (a constant for a given substance and wavelength) multiplied by the path length and concentration. The Beer-Lambert law is commonly used for quantitative analysis but has limitations at very high concentrations due to interactions between molecules.
This document discusses Beer's law and Lambert's law, which describe how the intensity of light passing through an absorbing medium decreases exponentially with increasing thickness and concentration of the medium. It states that Beer's law relates the decrease in intensity to both the thickness and concentration, while Lambert's law relates it only to thickness. The document also describes deviations from the linear relationship predicted by these laws that can occur, including positive deviations where concentration changes have a greater than expected effect, and negative deviations where changes have a smaller effect. Possible causes of deviations, both instrumental and physicochemical, are outlined.
This document provides an introduction to spectrometric methods and the Beer-Lambert law. It defines key terms like absorbance, transmittance, molar absorptivity, and wavelength. The Beer-Lambert law states that absorbance is directly proportional to concentration, path length, and molar absorptivity. It also explains that absorbance follows a linear relationship with concentration at a given path length and wavelength for a single analyte. Deviations from Beer's law can occur under certain circumstances.
This document discusses photometry and spectrophotometry. It defines photometers as instruments that use filters to select wavelengths of light, while spectrophotometers use monochromators like prisms or gratings to select wavelengths. Beer's law and Lambert's law relating the absorption of light to properties of the absorbing material are also described. The key components of a spectrophotometer including its light source, wavelength selector, sample cell, and detector are summarized. Double beam and multichannel spectrophotometers are mentioned as are applications in chemistry, biology, and quality control testing of spectrophotometers.
Spectrophotometry: basic concepts, instrumentation and applicationBasil "Lexi" Bruno
This document provides an overview of spectrophotometry, including basic concepts, instrumentation, and applications. It describes how spectrophotometers work by isolating specific wavelengths of light and measuring their absorption by a sample. The key relationship discussed is Beer's Law, which states that absorbance is directly proportional to concentration. Instrumentation components are also outlined, including light sources, monochromators for selecting wavelengths, and various methods for spectral isolation like filters, prisms and diffraction gratings.
1. Three common calibration techniques are described: calibration curve method, standard additions method, and internal standard method.
2. The calibration curve method involves preparing standard solutions of a known analyte concentration and measuring the analytical signal. A calibration curve of signal vs. concentration is made to determine unknown concentrations.
3. The standard additions method is useful when sample matrix effects are present. Known amounts of standard are added to samples and the signal response is measured. The intercept of the standard additions plot indicates the original analyte concentration in the sample.
4. The internal standard method corrects for variations in sample volume, position, and matrix. A known amount of a second element is added to standards and samples. Concent
This document provides an overview of fluorometry, including basic concepts, instrumentation, and applications. It discusses how fluorescence occurs when a molecule absorbs light at one wavelength and reemits light at a longer wavelength. Factors that affect fluorescence such as temperature, pH, and dissolved oxygen are also covered. The relationship between fluorescence intensity and concentration is explained. Additionally, the document defines fluorescence polarization and describes various types of quenching including self-quenching, chemical quenching, and collisional quenching.
Beer's law and Lambert's law describe the absorption of light in materials. Beer's law states that absorbance is directly proportional to concentration, while Lambert's law states absorbance is directly proportional to path length. Beer and Lambert combined their laws into the Beer-Lambert law, which states absorbance is equal to the molar absorptivity (a constant for a given substance and wavelength) multiplied by the path length and concentration. The Beer-Lambert law is commonly used for quantitative analysis but has limitations at very high concentrations due to interactions between molecules.
This document discusses Beer's law and Lambert's law, which describe how the intensity of light passing through an absorbing medium decreases exponentially with increasing thickness and concentration of the medium. It states that Beer's law relates the decrease in intensity to both the thickness and concentration, while Lambert's law relates it only to thickness. The document also describes deviations from the linear relationship predicted by these laws that can occur, including positive deviations where concentration changes have a greater than expected effect, and negative deviations where changes have a smaller effect. Possible causes of deviations, both instrumental and physicochemical, are outlined.
This document provides an introduction to spectrometric methods and the Beer-Lambert law. It defines key terms like absorbance, transmittance, molar absorptivity, and wavelength. The Beer-Lambert law states that absorbance is directly proportional to concentration, path length, and molar absorptivity. It also explains that absorbance follows a linear relationship with concentration at a given path length and wavelength for a single analyte. Deviations from Beer's law can occur under certain circumstances.
This document discusses photometry and spectrophotometry. It defines photometers as instruments that use filters to select wavelengths of light, while spectrophotometers use monochromators like prisms or gratings to select wavelengths. Beer's law and Lambert's law relating the absorption of light to properties of the absorbing material are also described. The key components of a spectrophotometer including its light source, wavelength selector, sample cell, and detector are summarized. Double beam and multichannel spectrophotometers are mentioned as are applications in chemistry, biology, and quality control testing of spectrophotometers.
Spectrophotometry: basic concepts, instrumentation and applicationBasil "Lexi" Bruno
This document provides an overview of spectrophotometry, including basic concepts, instrumentation, and applications. It describes how spectrophotometers work by isolating specific wavelengths of light and measuring their absorption by a sample. The key relationship discussed is Beer's Law, which states that absorbance is directly proportional to concentration. Instrumentation components are also outlined, including light sources, monochromators for selecting wavelengths, and various methods for spectral isolation like filters, prisms and diffraction gratings.
1. Three common calibration techniques are described: calibration curve method, standard additions method, and internal standard method.
2. The calibration curve method involves preparing standard solutions of a known analyte concentration and measuring the analytical signal. A calibration curve of signal vs. concentration is made to determine unknown concentrations.
3. The standard additions method is useful when sample matrix effects are present. Known amounts of standard are added to samples and the signal response is measured. The intercept of the standard additions plot indicates the original analyte concentration in the sample.
4. The internal standard method corrects for variations in sample volume, position, and matrix. A known amount of a second element is added to standards and samples. Concent
UV-visible spectroscopy is a technique that uses light in the visible and adjacent ranges. It works by measuring how much light is absorbed by a sample at each wavelength.
The document discusses the basic principles of spectroscopy including the laws of absorption. It describes the instrumentation used in UV-visible spectroscopy including light sources, wavelength selectors, sample holders and detection devices.
The document also covers electronic transitions that can occur, different types of spectrometers, and applications of UV-visible spectroscopy in chemistry, physics and other fields.
This document describes spectrophotometry and Beer's law. It discusses how spectrophotometers work by measuring the absorption of light by chemical substances over various wavelengths. It then describes two experiments conducted - the first determined the maximum absorbance wavelengths of different colored solutions, the second used Beer's law to calculate the concentrations of green solutions from their measured absorbances. The results showed that maximum absorbance correlated with complementary colors and absorbance increased with concentration as predicted by Beer's law.
This document provides information on atomic absorption spectroscopy (AAS), including:
1) AAS is a technique used to determine the concentration of chemical elements in samples by measuring light absorption by free atoms. It can analyze over 62 elements and is commonly used in pharmaceutical, food, and environmental applications.
2) The basic components of an AAS instrument are a hollow cathode lamp, monochromator, atomizer, detector, and nebulizer. Samples are atomized in a flame or graphite furnace then irradiated to cause absorption of specific wavelengths that are measured.
3) AAS is based on the principle that free atoms generated from a sample can absorb radiation at specific frequencies, allowing quantification of elemental
A short lecture about Atomic Spectroscopy: Flame Photometry, Atomic Absorption, and Atomic Emission with Coupled Plasma (FP, AA and ICP-AES). Presented at 28.03.2011, Faculty of Agriculture, Hebrew University of Jerusalem, by Vasiliy Rosen, M.Sc.
Atomic absorption spectroscopy is a technique that uses the absorption of light to measure the concentration of atomic absorption in a sample. It works by vaporizing the sample into atoms, then measuring how much light is absorbed by the atoms at a specific wavelength. The amount of absorption is directly related to the concentration of the element being measured. The key components of an atomic absorption spectroscopy system are the lamp sources, which emit specific wavelengths of light corresponding to the element being analyzed, the atomization process which turns solid or liquid samples into gaseous atoms, detectors that convert light signals into electrical signals, and monochromators that isolate the desired wavelength of light. Potential sources of interference must also be considered and addressed. Common applications of atomic absorption spectroscopy
1) Spectrophotometry is a technique used to measure how much light of varying wavelengths is absorbed by a substance.
2) The document outlines procedures for using a spectrophotometer to determine the wavelength of maximum absorbance (lambda max) of a basic fuchsin dye solution.
3) It then discusses how to use the Beer-Lambert law to determine the relationship between absorbance and concentration by measuring absorbance values of serial dilutions of the dye solution.
This document discusses turbidimetry and nephelometry. Turbidimetry measures cloudiness or haziness in a fluid sample by detecting scattered light, while nephelometry specifically measures light scattering. Both operate on the principles of light absorption and scattering when passed through a sample. A turbidimeter/nephelometer contains a light source, sample holder, and detector and can be used to measure water and air pollution, detect contaminants, and determine endpoints in titrations. Turbidimetry has applications in water treatment plants and the food industry and provides advantages of low cost and not requiring zero adjustments, though it cannot determine particle size and bubbles can interfere with readings.
1. UV-visible spectroscopy is used to detect functional groups, impurities, and perform qualitative and quantitative analysis of compounds.
2. It works by measuring how much light is absorbed by a sample at different wavelengths, providing information on functional groups and molecular structure.
3. Key applications include detection of impurities, structure elucidation, and determination of concentration through Beer's law.
Sample preparation is an essential part of HPLC analysis to provide a reproducible and homogenous solution suitable for injection onto the column. The goal of sample preparation is to remove interferences and ensure the sample is compatible with the HPLC method without damaging the column. Sample matrices can be organic or inorganic solids, semisolids, liquids or gases, with liquids being easiest to prepare. Solid and semisolid samples require reducing particle size through processes like blending or grinding. Filtration is also important to remove particles that could damage the column. Common pretreatment methods for liquid samples include liquid-liquid extraction and solid phase extraction, while newer techniques are used for solid samples like supercritical fluid extraction. Derivatization can improve
An amino acid analyzer uses ion-exchange chromatography and post-column reaction with ninhydrin to detect and quantify amino acids in solutions. The system works by automatically injecting samples, separating amino acids using buffers of varying pH in a column, reacting the column eluent with ninhydrin in a coil to produce colored compounds, and using a photometer and recorder to identify and measure the amino acids based on their retention times and absorption peaks. The amino acid analyzer can analyze over 40 amino acids and is used to detect amino acids in tissues, fluids, foods and more.
It give detail information on the measurement of the intensity of scattered light at right angles to the direction of the incident light as a function of the concentration of the dispersed phase
UV -Vis Spectrophotometry- Principle, Theory, Instrumentation and Application...Dr. Amsavel A
UV -Vis Spectrophotometry- Principle, Theory, Instrumentation and Application in Pharmaceutical Industry Dr. A. Amsavel.
UV &Visible Spectroscopy-Absorption Theory
Electronic Transitions
Beer- Lambert Law
Chromophores & Auxochrome
Factors Influence the Absorption
UV-Vis Spectrophotometer-Instrumentation
Operation of the Spectrophotometer
Qualification & Calibration
Application
(MS) is an analytical technique that produces spectra (singular spectrum) of the masses of the atoms or molecules comprising a sample of material. The spectra are used to determine the elemental or isotopic signature of a sample, the masses of particles and of molecules, and to elucidate the chemical structures of molecules, such as peptides and other chemical compounds,so it is considered one f the very important diagnostic analytical techniques .
UV-VIS spectroscopy analyzes the absorption of light in the ultraviolet-visible spectral region by molecules. White light is composed of a range of wavelengths, which can be separated by a prism into the visible colors from violet to red. Different functional groups and conjugated systems in molecules absorb at characteristic wavelengths. The absorbance of a solution is proportional to the concentration of the absorbing species, as described by Beer's Law. UV-VIS spectroscopy is used to determine structural features and study reactions.
This document discusses spectrophotometry, including the Beer-Lambert law, instruments used, and applications. Spectrophotometry measures light intensity as a function of wavelength by diffracting light into a spectrum and detecting intensities. Instruments include a light source, monochromator to produce monochromatic light, cuvettes to hold samples, and detectors to convert light to electrical signals. Applications include concentration measurement using standard solutions, detecting impurities, structure elucidation of organic compounds, studying chemical kinetics, and determining functional groups and molecular weights.
- The document is a presentation on ultraviolet spectroscopy submitted by Moriyom Akhter and Md Shah Alam from the Department of Pharmacy at World University of Bangladesh.
- It defines ultraviolet spectroscopy and discusses key concepts like absorption spectra, types of electronic transitions that can occur, Beer's and Lambert's absorption laws, instrumentation components, and applications in qualitative and quantitative analysis.
- The presentation also examines effects of chromophores and auxochromes on absorption spectra and maximum wavelengths, and how solvents can shift absorption peaks.
UV-VIS reflectance spectroscopy is a technique that measures the diffuse reflectance of a sample across UV and visible wavelengths. It works by directing light at a sample inside an integrating sphere, which captures reflected light and directs it to a detector. The ratio of reflected to incident light at each wavelength is the reflectance spectrum. Reflectance is affected by factors like particle size, homogeneity, and packing density. It finds applications in pharmaceutical analysis and other industries to qualitatively and quantitatively analyze samples like drugs, proteins, and chemicals.
This document describes how to use a spectrophotometer to measure the wavelengths of light absorbed by different colored solutions. It provides steps for taking absorbance readings of solutions and recording the results. Absorbance data is presented showing that green solution absorbed most light while yellow absorbed least. Beer's law is explained, relating absorbance to concentration and path length of light through a solution.
UV-visible spectroscopy is a technique that uses light in the visible and adjacent ranges. It works by measuring how much light is absorbed by a sample at each wavelength.
The document discusses the basic principles of spectroscopy including the laws of absorption. It describes the instrumentation used in UV-visible spectroscopy including light sources, wavelength selectors, sample holders and detection devices.
The document also covers electronic transitions that can occur, different types of spectrometers, and applications of UV-visible spectroscopy in chemistry, physics and other fields.
This document describes spectrophotometry and Beer's law. It discusses how spectrophotometers work by measuring the absorption of light by chemical substances over various wavelengths. It then describes two experiments conducted - the first determined the maximum absorbance wavelengths of different colored solutions, the second used Beer's law to calculate the concentrations of green solutions from their measured absorbances. The results showed that maximum absorbance correlated with complementary colors and absorbance increased with concentration as predicted by Beer's law.
This document provides information on atomic absorption spectroscopy (AAS), including:
1) AAS is a technique used to determine the concentration of chemical elements in samples by measuring light absorption by free atoms. It can analyze over 62 elements and is commonly used in pharmaceutical, food, and environmental applications.
2) The basic components of an AAS instrument are a hollow cathode lamp, monochromator, atomizer, detector, and nebulizer. Samples are atomized in a flame or graphite furnace then irradiated to cause absorption of specific wavelengths that are measured.
3) AAS is based on the principle that free atoms generated from a sample can absorb radiation at specific frequencies, allowing quantification of elemental
A short lecture about Atomic Spectroscopy: Flame Photometry, Atomic Absorption, and Atomic Emission with Coupled Plasma (FP, AA and ICP-AES). Presented at 28.03.2011, Faculty of Agriculture, Hebrew University of Jerusalem, by Vasiliy Rosen, M.Sc.
Atomic absorption spectroscopy is a technique that uses the absorption of light to measure the concentration of atomic absorption in a sample. It works by vaporizing the sample into atoms, then measuring how much light is absorbed by the atoms at a specific wavelength. The amount of absorption is directly related to the concentration of the element being measured. The key components of an atomic absorption spectroscopy system are the lamp sources, which emit specific wavelengths of light corresponding to the element being analyzed, the atomization process which turns solid or liquid samples into gaseous atoms, detectors that convert light signals into electrical signals, and monochromators that isolate the desired wavelength of light. Potential sources of interference must also be considered and addressed. Common applications of atomic absorption spectroscopy
1) Spectrophotometry is a technique used to measure how much light of varying wavelengths is absorbed by a substance.
2) The document outlines procedures for using a spectrophotometer to determine the wavelength of maximum absorbance (lambda max) of a basic fuchsin dye solution.
3) It then discusses how to use the Beer-Lambert law to determine the relationship between absorbance and concentration by measuring absorbance values of serial dilutions of the dye solution.
This document discusses turbidimetry and nephelometry. Turbidimetry measures cloudiness or haziness in a fluid sample by detecting scattered light, while nephelometry specifically measures light scattering. Both operate on the principles of light absorption and scattering when passed through a sample. A turbidimeter/nephelometer contains a light source, sample holder, and detector and can be used to measure water and air pollution, detect contaminants, and determine endpoints in titrations. Turbidimetry has applications in water treatment plants and the food industry and provides advantages of low cost and not requiring zero adjustments, though it cannot determine particle size and bubbles can interfere with readings.
1. UV-visible spectroscopy is used to detect functional groups, impurities, and perform qualitative and quantitative analysis of compounds.
2. It works by measuring how much light is absorbed by a sample at different wavelengths, providing information on functional groups and molecular structure.
3. Key applications include detection of impurities, structure elucidation, and determination of concentration through Beer's law.
Sample preparation is an essential part of HPLC analysis to provide a reproducible and homogenous solution suitable for injection onto the column. The goal of sample preparation is to remove interferences and ensure the sample is compatible with the HPLC method without damaging the column. Sample matrices can be organic or inorganic solids, semisolids, liquids or gases, with liquids being easiest to prepare. Solid and semisolid samples require reducing particle size through processes like blending or grinding. Filtration is also important to remove particles that could damage the column. Common pretreatment methods for liquid samples include liquid-liquid extraction and solid phase extraction, while newer techniques are used for solid samples like supercritical fluid extraction. Derivatization can improve
An amino acid analyzer uses ion-exchange chromatography and post-column reaction with ninhydrin to detect and quantify amino acids in solutions. The system works by automatically injecting samples, separating amino acids using buffers of varying pH in a column, reacting the column eluent with ninhydrin in a coil to produce colored compounds, and using a photometer and recorder to identify and measure the amino acids based on their retention times and absorption peaks. The amino acid analyzer can analyze over 40 amino acids and is used to detect amino acids in tissues, fluids, foods and more.
It give detail information on the measurement of the intensity of scattered light at right angles to the direction of the incident light as a function of the concentration of the dispersed phase
UV -Vis Spectrophotometry- Principle, Theory, Instrumentation and Application...Dr. Amsavel A
UV -Vis Spectrophotometry- Principle, Theory, Instrumentation and Application in Pharmaceutical Industry Dr. A. Amsavel.
UV &Visible Spectroscopy-Absorption Theory
Electronic Transitions
Beer- Lambert Law
Chromophores & Auxochrome
Factors Influence the Absorption
UV-Vis Spectrophotometer-Instrumentation
Operation of the Spectrophotometer
Qualification & Calibration
Application
(MS) is an analytical technique that produces spectra (singular spectrum) of the masses of the atoms or molecules comprising a sample of material. The spectra are used to determine the elemental or isotopic signature of a sample, the masses of particles and of molecules, and to elucidate the chemical structures of molecules, such as peptides and other chemical compounds,so it is considered one f the very important diagnostic analytical techniques .
UV-VIS spectroscopy analyzes the absorption of light in the ultraviolet-visible spectral region by molecules. White light is composed of a range of wavelengths, which can be separated by a prism into the visible colors from violet to red. Different functional groups and conjugated systems in molecules absorb at characteristic wavelengths. The absorbance of a solution is proportional to the concentration of the absorbing species, as described by Beer's Law. UV-VIS spectroscopy is used to determine structural features and study reactions.
This document discusses spectrophotometry, including the Beer-Lambert law, instruments used, and applications. Spectrophotometry measures light intensity as a function of wavelength by diffracting light into a spectrum and detecting intensities. Instruments include a light source, monochromator to produce monochromatic light, cuvettes to hold samples, and detectors to convert light to electrical signals. Applications include concentration measurement using standard solutions, detecting impurities, structure elucidation of organic compounds, studying chemical kinetics, and determining functional groups and molecular weights.
- The document is a presentation on ultraviolet spectroscopy submitted by Moriyom Akhter and Md Shah Alam from the Department of Pharmacy at World University of Bangladesh.
- It defines ultraviolet spectroscopy and discusses key concepts like absorption spectra, types of electronic transitions that can occur, Beer's and Lambert's absorption laws, instrumentation components, and applications in qualitative and quantitative analysis.
- The presentation also examines effects of chromophores and auxochromes on absorption spectra and maximum wavelengths, and how solvents can shift absorption peaks.
UV-VIS reflectance spectroscopy is a technique that measures the diffuse reflectance of a sample across UV and visible wavelengths. It works by directing light at a sample inside an integrating sphere, which captures reflected light and directs it to a detector. The ratio of reflected to incident light at each wavelength is the reflectance spectrum. Reflectance is affected by factors like particle size, homogeneity, and packing density. It finds applications in pharmaceutical analysis and other industries to qualitatively and quantitatively analyze samples like drugs, proteins, and chemicals.
This document describes how to use a spectrophotometer to measure the wavelengths of light absorbed by different colored solutions. It provides steps for taking absorbance readings of solutions and recording the results. Absorbance data is presented showing that green solution absorbed most light while yellow absorbed least. Beer's law is explained, relating absorbance to concentration and path length of light through a solution.
Spectrophotometry uses the principle that molecules absorb specific wavelengths of light. A spectrophotometer directs a beam of light through a sample and measures the amount of light absorbed. It contains a light source, wavelength selector like a prism or grating to produce monochromatic light, sample holders, a detector to measure transmitted light intensity, and a readout device. It works based on Beer's law, where absorbance is directly proportional to concentration, molar absorptivity, and path length. This allows spectrophotometry to quantify the concentration of an analyte by its optical properties.
A spectrophotometer uses light to measure the concentration of solutes in solution. It works by passing light through a sample in a cuvette and measuring the amount of light absorbed. The main components are a light source, monochromator to separate wavelengths, sample cuvette, detector, and display. Common light sources are tungsten halogen lamps and xenon flash lamps. Monochromators use dispersion devices like prisms, filters, or diffraction gratings. Detectors convert light to electrical signals and displays show output. Measurements rely on Beer's Law relating absorption to concentration.
A spectrophotometer measures the amount of light absorbed by a sample. Early models took weeks for results and were only 25% accurate. In 1940, Arnold Beckman invented the first modern spectrophotometer, the Beckman DU, which provided results within minutes that were 99.99% accurate. A spectrophotometer uses a light source, dispersion devices like prisms or filters, sample cells, detectors, and a display. It is used to identify compounds and determine absorbance and transmission of light in chemistry.
A spectrophotometer is a device that measures light intensities at different wavelengths. It works by splitting light into a spectrum and detecting intensities with a charged couple device. Key components include an illuminant, optics to direct light, a monochromator to split light, and a photodetector. There are single and dual beam types, with dual beam minimizing errors by comparing sample and reference beams. Calibration ensures repeatability, and spectrophotometers are used to measure color and help with tasks like quality control and color matching.
UV/visible spectroscopy involves the interaction of electromagnetic radiation with matter. Absorption spectroscopy measures the absorption of UV or visible light, while emission spectroscopy measures light emitted from a sample. The wavelength and frequency of electromagnetic radiation are inversely related by the equation c=λν. Electronic transitions in molecules, such as σ→σ*, π→π*, n→σ*, and n→π* can be detected using UV/visible spectroscopy. Beer's law states that absorbance is directly proportional to concentration and path length. Chromophores are functional groups in molecules that absorb UV or visible light.
Atomic Absorption Spectroscopy uses the principle that free atoms generated from a sample can absorb radiation at specific frequencies, allowing the technique to quantify the concentration of various metals and metalloids present. The sample is atomized using a flame or graphite furnace and exposed to light from a hollow cathode lamp, with absorption measured to generate calibration curves and determine unknown concentrations. AAS is a common analytical technique used across various fields like environmental analysis, food testing, and pharmaceutical applications.
A flame photometer is a device used to determine the concentration of certain metal ions in a solution by measuring the intensity of light emitted from a flame. It works by nebulizing the sample solution into a flame, where the metal ions are atomized and excited to emit light of characteristic wavelengths. The intensity of the emitted light is directly proportional to the concentration of the metal ion in the original solution. Common metal ions that can be analyzed using flame photometry include sodium, potassium, lithium, and calcium.
Flame photometry (more accurately called Flame Atomic Emission Spectrometry)is a branch of spectroscopy in which the species examined in the spectrometer are in the form of atoms
A photoelectric flame photometer is an instrument used in inorganic chemical analysis to determine the concentration of certain metal ions among them sodium, potassium, calcium and lithium.
Flame Photometry is based on measurement of intensity of the light emitted when a metal is introduced into flame.
The wavelength of colour tells what the element is (qualitative)
The colour's intensity tells us how much of the element present (quantitative)
A spectrophotometer is an instrument that measures the amount of light transmitted through a sample. It uses light energy in the ultraviolet (UV) or visible light spectrum to detect molecules in a solution. The spectrophotometer shines a beam of light on a sample, and measures how the sample interacts with the light through absorption, reflection, or transmission. By comparing the transmittance or absorbance values of an unknown sample to standards of known concentration, the spectrophotometer can determine the concentration of the unknown sample.
A colorimeter is an instrument that measures the intensity of light to determine the concentration of colored compounds in solution. It works by passing light through sample solutions and measuring how much light is absorbed, based on Beer's Law. Key components include a light source, wavelength selector, sample holder, light detector, and readout device. Colorimeters are widely used in clinical and industrial settings to analyze biochemical, food, soil, and water samples colorimetrically.
Photometry is used to measure light intensity and is applied in techniques like colorimetry, spectrophotometry, and turbidometry. Colorimetry determines concentrations of colored compounds by measuring light absorbed at visible wavelengths. It follows Beer's and Lambert's laws - the amount of light absorbed increases exponentially with increasing concentration and path length. A colorimeter consists of a light source, monochromator/filters, sample holder, detector, and readout. It is used to estimate biochemical compounds in body fluids through color reactions.
It is the most common analytical technique used in biochemical estimation in clinical laboratory.
It involves the quantitative estimation of color.
A substance to be estimated colorimetrically, must be colored or it should be capable of forming chromogens (colored complexes) through the addition of reagents.
The document discusses measurement, calibration, and units of measurement. Some key points:
- Measurement is the first step to control and improvement. If you can't measure something, you can't understand or control it.
- The International System of Units (SI) defines seven base units including the meter, kilogram, second, ampere, kelvin, mole, and candela. Other units are derived from these base units.
- Calibration establishes the relationship between measurement instruments and reference standards under specific conditions. Regular calibration helps ensure accuracy and traceability to national standards.
- Factors like instrument specifications, use, environment, and measurement accuracy needed should be considered when determining calibration frequency.
Colorimetry is a common analytical technique used in clinical laboratories to estimate biochemical substances. It involves measuring the color intensity of a colored solution, which is proportional to the concentration of the colored substance. The solution is passed through a colorimeter, which measures the amount of light absorbed. Beer's law and Lambert's law describe how the absorption is related to concentration and path length. The colorimeter contains a light source, filter to select the wavelength, cuvette to hold the sample, and detector to convert light intensity to an electrical signal. Standards and blanks are used for calibration. Colorimetry is used to estimate many substances in blood, urine, and cerebrospinal fluid.
This document describes an experiment using Beer's Law to determine the concentration of an unknown nickel sulfate (NiSO4) solution. Students will create standard NiSO4 solutions of known concentrations and measure their absorbance. A Beer's Law plot of absorbance vs. concentration will be constructed and used to determine the concentration of the unknown solution by interpolation. Care must be taken to use the same cuvette and orientation for all trials to ensure accurate measurements. Absorbance and % transmittance have an inverse logarithmic relationship described by the Beer-Lambert law.
The Beer-Lambert Law describes how the intensity (I) of monochromatic light is attenuated when passing through a solution due to absorption. I decreases exponentially with increasing concentration (c) of the absorbing material, according to Beer's Law, and with increasing path length (l) through the material, according to Lambert's Law. The relationship can be expressed as: A (or extinction, E) = kcl, where k is the material's molar extinction coefficient and c and l have the above definitions. A plot of E versus c at constant l will yield a straight line passing through the origin.
Spectrophotometry is a method to measure how much a chemical substance absorbs light by measuring the intensity of light as a beam of light passes through sample solution. The basic principle is that each compound absorbs or transmits light over a certain range of wavelength
This document discusses the principles of colorimetry, specifically Beer's Law and Lambert's Law. It describes how colorimetry can be used to quantitatively estimate the concentration of a colored substance or solution. The amount of light absorbed by a solution is directly proportional to its concentration and path length. This relationship can be expressed by the formula A=ɛ x C x L, where A is absorbance, ɛ is the molar extinction coefficient, C is concentration, and L is path length. The document also provides details on the hardware components of a colorimeter and describes procedures to generate a calibration curve and use it to determine the concentration of an unknown sample.
Photometry techniques like colorimetry, spectrophotometry, and turbidometry measure the intensity of light absorbed or transmitted by a solution. Colorimeters contain a light source, monochromators/filters to select wavelengths, a sample holder (cuvette), photodetectors, and readout devices. The amount of light absorbed follows Beer's and Lambert's laws - absorption increases exponentially with concentration and path length. A colorimeter is used to quantify compounds in biological samples like blood and urine by measuring absorbance and relating it to a standard curve using the Beer-Lambert law. Colorimeters provide a simple and inexpensive way to perform quantitative analysis of colored compounds.
Application of centrifugation and SpectrophotometryAmany Elsayed
Centrifugation techniques such as velocity sedimentation centrifugation and fractional centrifugation are used to separate cellular components by size and weight. Velocity sedimentation centrifugation involves spinning a sample at low or high speeds to separate heavier precipitates from lighter organelles. Fractional centrifugation uses successive centrifugations at increasing speeds to separate cellular organelles into pellet and supernatant fractions. Spectrophotometry measures the absorption or emission of light by molecules. Beer's law states that absorbance is directly proportional to concentration, with absorbance measured using spectrophotometers and concentration determined using calibration curves.
This document discusses spectrophotometry techniques for measuring light absorption by molecules. It covers the electromagnetic spectrum, Beer-Lambert law, applications of UV-vis spectroscopy like determining cell density and protein concentration, and methods for measuring absorbance of molecules like DNA, RNA, proteins, and other biological compounds. Key concepts explained include the relationship between absorbance, molar extinction coefficient, concentration, and path length in the Beer-Lambert law.
Determination the Calibration Curve of Cobalt Nitrate by SpectrophotometerHaydar Mohammad Salim
This document describes an experiment to determine the calibration curve of cobalt nitrate using a spectrophotometer. Key steps include preparing cobalt standard solutions of known concentrations, measuring their absorbance, and using the results to construct a calibration curve relating absorbance to concentration. The calibration curve will then allow determination of the concentration of an unknown cobalt solution based on its measured absorbance. The document provides background on spectrophotometry and Beer's Law, which states that absorbance is directly proportional to concentration and path length.
Spectroscopy deals with the interaction of electromagnetic radiation with matter. There are two main types of spectra - absorption and emission. Absorption spectra occur when molecules absorb energy and are excited to higher energy levels, while emission spectra occur when molecules fall to lower energy levels and emit energy. Spectroscopy can provide information about the electronic, vibrational, and rotational energy levels of atoms and molecules. Beer-Lambert's law describes the quantitative relationship between absorbance of a solution and its concentration, with limitations at high concentrations or for fluorescent samples. Infrared spectroscopy specifically involves transitions between vibrational energy levels, detected in the mid-infrared region.
Optical techniques like photometry, spectrophotometry, and colorimetry are used in clinical laboratories. They are based on Beer's law and Lambert's law. Spectrophotometry measures light intensity at selected wavelengths using a light source, monochromator, sample cuvettes, detector, and display. It provides more sensitivity than colorimetry which determines color intensity based on light absorption. Both techniques rely on the principle that absorbed light is inversely proportional to concentration according to Beer-Lambert's law.
UV-visible spectroscopy involves using ultraviolet or visible light to analyze molecular structure and dynamics through light absorption, emission, or scattering. A spectrophotometer measures the amount of light transmitted through a sample. Absorption causes the sample's color, as molecules absorb all wavelengths except the color observed. UV-VIS spectrophotometers use light from 200-350 nm or 350-700 nm. Beer's Law states absorbance is proportional to concentration and path length. A standard curve relating absorbance and concentration of known standards is used to determine unknown concentrations. Accuracy requires testing standards and controls repeatedly.
This document discusses spectrophotometry techniques for measuring light absorption by molecules. It covers the electromagnetic spectrum, Beer-Lambert law, applications of UV-Vis spectroscopy like determining cell density and protein concentration, and methods for protein concentration measurements including Bradford assay and measuring absorbance at 280nm. Key concepts explained are molar extinction coefficient, absorbance spectra of biomolecules, and dilutions for preparation of solutions.
Colorimetry uses the human eye to determine the concentration of colored species, while spectrophotometry uses instruments to make quantitative measurements beyond the visible range. This experiment demonstrates both techniques on dyes. Colorimetry involves visual observations of solutions and mixtures, while spectrophotometry uses a Spec-20 spectrophotometer to measure transmittance and absorbance according to Beer's law. The results will be plotted and analyzed.
* Mb = 0.05 mol/L (molarity of initial solution)
* Vb = 25.0 mL (volume of initial solution)
* Va = 75.0 mL (total volume of diluted solution)
* Using the dilution equation:
MbVb = MaVa
(0.05 mol/L)(25.0 mL) = Ma(75.0 mL)
1.25 mmol = Ma(75.0 mL)
Ma = 1.25 mmol/75.0 mL = 0.0167 mol/L = 0.0167 M
The molarity of the diluted solution is 0.0167 M.
This document discusses spectrophotometry and the Beer-Lambert law. It provides:
1) An overview of how spectrophotometers work by measuring the absorption of light by chemical compounds and relating absorption to concentration according to the Beer-Lambert law.
2) A description of the basic components of a spectrophotometer including a light source, wavelength selector, sample cuvette, detector, and readout device.
3) Explanations of how to prepare and measure standards to generate a calibration curve to determine unknown concentrations.
This document provides information about using a spectrophotometer for quantitative analysis. It discusses how spectrophotometers work based on the Beer-Lambert law relating absorbance of light to analyte concentration. The key components of a spectrophotometer are described including the light source, wavelength selector, sample cuvette, detector, and readout device. General procedures are outlined for preparing standard solutions to generate a calibration curve and determining concentrations of unknown samples.
This document provides information about using a spectrophotometer for quantitative analysis. It discusses how spectrophotometers work based on the Beer-Lambert law relating absorbance of light to analyte concentration. The key components of a spectrophotometer are described including the light source, wavelength selector, sample cuvette, detector, and readout device. General procedures are outlined for preparing standard solutions to generate a calibration curve and determining concentrations of unknown samples.
UV-VISIBLE SPECTROPHOTOMETRY AND INORGANIC PHOSPHATE DETERMINATION.pdfTatendaMageja
This document discusses UV-visible spectrophotometry and its application to determining inorganic phosphate concentration. It begins by explaining the basic principles of spectrophotometry, how it works, and Beer's Law which states that absorbance is directly proportional to concentration. It then discusses inorganic phosphate determination specifically, describing how phosphate reacts with molybdate and is reduced to form molybdenum blue, which absorbs at specific wavelengths. The procedure for determining an unknown phosphate concentration using standards to generate a calibration curve is outlined. Applications to nutrient analysis and clinical correlations are also briefly mentioned.
1. The document discusses various topics in spectroscopy including: the study of how compounds interact with electromagnetic radiation and the measurement of absorbance spectra; the absorption of energy in different spectral regions; and the relationship between wavelength, frequency, and energy of electromagnetic radiation.
2. Key concepts explained are Beer's law and how absorbance is directly proportional to concentration, molar absorptivity, and path length; electronic transitions involved in UV/visible absorption; and applications of spectroscopy techniques like UV-Vis and fluorescence spectroscopy.
3. Examples are provided to illustrate calculating molar absorptivity from absorbance data and determining concentration from Beer's law.
UV-Visible Spectrometry is a technique used to analyze how molecules interact with light in the UV-Visible region. It works based on Beer's Law, where the absorbance of a solution is directly proportional to concentration and path length. The key components of a UV-Vis spectrometer are a radiation source, monochromator, sample cells, and detectors. It can be used for structure elucidation, quantitative analysis, detection of impurities, and studying chemical kinetics. Larger conjugated systems absorb at longer wavelengths with higher intensities. Solvents and functional groups can also impact absorption spectra.
UV-Visible Spectrometry is a technique used to analyze how substances interact with electromagnetic radiation in the UV-Visible region. It works based on Beer's Law, where the absorbance of a substance is directly proportional to its concentration and path length. The key components of a UV-Vis spectrometer are a radiation source, monochromator, sample cells, and detectors. It has various applications including structure elucidation, quantitative analysis, detection of impurities, and studying chemical kinetics.
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2. Before the prac class begins
experimental work….
• The spectrophotometer actually measures
transmittance: %T = (IT/I0)*100.
• This is the intensity of light transmitted
through the solution (IT) divided by the
intensity of light entering the solution, the
incident light (I0). The problem with this
measurement is that as the solution becomes more
concentrated the %T decreases. The relationship is also
not linear.
• Absorbance is actually log10I0/IT
3. Relationship between %Transmittance and
light path length and concentration
100
80
60
40
20
0 length
%T
100
80
60
40
20
0
%T
concentration
Transmittance = IT/I0 = e-αcl
where α is an extinction constant, c is
concentration and l is light path length
4. Absorbance increases linearly
with concentration
From IT/I0 = e-αcl
I have used α here to describe a constant that is proportional to the extinction
coefficient. As you can see from the maths it is not the mMolar extinction
coefficient
taking logs of both sides and inverting
Ln (I0/IT) = αcl
Converting to Log10
Log10(I0/IT) = εcl
Hence the Beer-Lambert Law
A = εcl
5. Before the prac class begins
experimental work….
• Absorbance increases linearly with
concentration as predicted by the Beer-
Lambert Law
A = εcl
• Explain why the working range of a
spectrophotometer is 0.1 – 1.0. Remember
Abs is a log scale. An absorbance of 1.6 is 2%
light transmitted while an absorbance of 2 is 1%
light transmitted. The class specs can not
accurately distinguish 1% from 2%. An
absorbance of 1.0 is 10% transmitted light.
6. Before the prac class begins
experimental work….
• For the mathematically minded:
• Transmittance = IT/I0*100
• Absorbance = log10(I0/IT)
Converting Transmittance to Absorbance
• %T/100 = IT/I0 100/%T = I0/IT
Taking logs on both sides
• Log 100 – log %T = log I0/IT
• 2 – log%T = Absorbance
7. Before the prac class begins
experimental work….
• Going back the other way
• Transmittance = IT/I0*100
• Absorbance = log10(I0/IT)
Converting Absorbance to Transmittance
• Abs = log(I0/IT)
• 10Abs
= (I0/IT), inverting
• 10-Abs
= (IT/I0),
• 100* 10-Abs
= 100*(IT/I0),
• 102-Abs
= %T
8. Experiment 1: Identifying a
compound by spectrophotometry
• If a compound absorbs light its absorption
spectrum is a unique property of that
compound.
• The molecular structure is responsible for
the absorption properties
• The most common feature of absorbing
compounds are conjugated double bonds,
often as an aromatic ring
9. Experiment 1: Identifying a
compound by spectrophotometry
• Conjugated double bonds result in pi
electrons above and below the ring or
chain and these electrons can be “moved”
to higher levels by photons of light.
• As the electrons are promoted to higher
levels “allowed” by the molecular structure
they absorb light of a specific wavelength,
based on the energy required for the
transition (∆E).
10. Experiment 1: Identifying a
compound by spectrophotometry
• This amount of absorbed energy (∆E) will
determine the λ of light absorbed.
• The ∆E is inversely proportional to the
wavelength of light absorbed ie. ∆E = hc/λ,
where h is Planck’s constant and c is the
velocity of light. (Remember this from
physics!??)
11. Common Absorbing Biochemicals
• The bases of nucleic
acids
NH
NN
H
N
O
NH2
Guanine
N
N
H
NH2
O
Cytosine
NH
N
H
O
O
H3C
Thymine
N
NN
H
N
NH2
Adenine
14. Amino Acid absorption Properties
H2N CH C
CH2
OH
O
HN
Tryptophan
H2N CH C
CH2
OH
O
OH
Tyrosine
H2N CH C
CH2
OH
O
Phenylanine
H2N CH C
CH2
OH
O
N
NH
Histidine
H2N CH C
CH2
OH
O
SH
Cysteine
15. The Dyes
• The dyes chosen for this experiment are
different colours (A – F)
• Each pair of students will be assigned a
dye by the demonstrator. They use this
dye for the whole practical.
• Students should take note of the colour of
their dye and record the concentration
(mM) on the bottle
16. Obtaining a Spectrum for the dye.
• Using the Shimadzu spectrophotometers
in spectrum mode (mode 2 on main menu)
place a 1 mL plastic cuvette full of H2O in
the holder and obtain a baseline correction
(F1). This will take some time.
• Then, using the same cuvette, fill it with
the dye solution and obtain a spectrum.
Find the peaks. If you are unclear how to
do this practise beforehand.
17. Obtaining a Spectrum for the dye.
• The reason for doing this is to find the
absorbing region of the dye. It takes a long
time to obtain a spectrum from 600 nm to
350 nm. A quick narrowing of the range is
to be encouraged.
• Get students to consider the colour of the
solution and how this might give clues to
the absorption minima and maxima
18. The relationship between colour
and absorption
• A compound will be yellow if it reflects light
in the yellow wavelengths and absorbs
light of other wavelengths.
• Yellow compounds (often red crystals)
usually absorb in the blue range ~450 –
350 nm and have an absorption minimum
>550 nm
19. Absorption Spectrum: Dye C
0.0
0.2
0.4
0.6
0.8
1.0
1.2
350 400 450 500 550 600
w avelength (nm)
Absorbance
Dye C is yellow -
red and has an
absorption
minimum in the
yellow/red region
Dye C: Riboflavin
20. Obtaining the spectrum
• Once the baseline is corrected and the
absorbing range determined, find the
absorbance of the dye every 10 nm within
the absorbing range. Every 50 nm will do
outside the absorbing range.
• If the baseline correction is done you don’t
have to zero every time you change
wavelengths. This saves heaps of time.
21. Obtaining the spectrum
• Students must, by the next lab, plot the
spectrum. It would be a good idea to get
them to this now if there are free
computers. Otherwise get them to identify
the dye by the peaks, comparing to the
sample spectra at the back of this section
of the lab manual.
• From the concentration on the bottle
estimate the extinction coefficient.
22. Obtaining the spectrum
• Make sure you are very familiar with the
Excel chart drawing process as you will
need to help the students here. Practise
with the spectro.xls spreadsheet provided.
It has the raw data obtained for riboflavin
and the worked solution.
• Check out what is expected graph-wise in
the worked solution. Students should have
practised much of this with the Excel task
in the last practical.
23. Calculating the Extinction
Coefficient
• This comes directly from the relationship
A = εcl,
Where ε is the extinction coefficient
expressed in the units of c, the
concentration. In this experiment the conc
units will be mM so ε will have the units
mM-1
cm-1
. Round the value off to 1 dec pl.
24. Discussion
• Predict which of the following parameters would
change with dilution? How would they change?
• The number of peaks
• The λ max
• Aλ1/Aλ2
• Absorbance at λ max
• Extinction Coefficient
• Transmittance at λmax
• Can you predict what would happen to the
absorption spectrum if you diluted your dye with
another dye?
25. Experiment 2: The Standard Curve
• Although identifying a compound by
spectro is a useful property,
spectrophometry is used more often to
measure the concentration of a
compound.
• Sometimes the extinction coefficient can
be used directly. This occurs when the
compound of interest has an intrinsic
absorbance.
26. Experiment 2: The Standard Curve
• If the compound of interest does not have its
own intrinsic absorbance then a coloured
derivative must be made by reacting it with
reagents. Then a standard curve must be
produced.
• In today’s practical students will gain experience
at producing and using a standard curve, even
though in this situation you would normally use
the extinction coefficient.
27. Experiment 2: The Standard Curve
• Using the same solution as the one used
to obtain the spectrum, get the students to
dilute it so that there are at least 5 points
for the line. My suggestion is 200, 400,
600, 800, 1000 uL of dye, then make each
up to 1 mL with water.
• Mix well and obtain the absorbances at
the λmax.
28. The standard curve
Standard Curve: Riboflavin
y = 0.0125x
R2
= 0.9999
0.0
0.2
0.4
0.6
0.8
1.0
1.2
0 20 40 60 80 100
[Riboflavin] (nmol/mL)
Absorbance@445nm
29. The standard curve
Standard Curve: Riboflavin
y = 0.0125x
R2
= 0.9999
0.0
0.2
0.4
0.6
0.8
1.0
1.2
0 20 40 60 80 100
[Riboflavin] (nmol/mL)
Absorbance@445nm
Plot the
concentration in
nmol/tube or
nmol/mL
30. The standard curve
• The main point of confusion in this task is how to
plot the concentration. The purists would plot it
in mM or uM and this would be correct BUT
confusing for the students when they come to
back calculate with it.
• Instead plot it in nmoles per tube which in this
case is nmol/mL. Note that the riboflavin
concentration range is ~0 – 80 nmol/mL. It is not
“neat” due to the concentration of the starting dye
solution 0f 0.0836 mM.
31. How to use the standard curve
• The standard curve is used to find the
concentration of an unknown solution of
riboflavin.
• This practical session has 2 different
unknowns the students must determine
the concentration of; one which is in the
working range of the spectrophotometer or
standard curve i.e. 0.1 – 1.0, the other is
outside the range.
32. Unknown 3a
• This unknown can be directly determined
by measuring its absorbance without
dilution.
• However it is always good practise to do
at least one dilution when estimating a
concentration.
• The obvious dilution is a 1 in 2 dilution.
This is your chance to introduce dilutions
to the students.
33. Dilutions
• A 1 in 2 dilution is 1 part riboflavin
unknown C1 and 1 part H2O.
• If you wanted to make up a 1 in 2 dilution
of unknown C1 which could be easily read
off the standard curve you would make it
up to 1 mL.
• This would mean 500 uL of unknown C1
and 500 uL H2O.
35. Slope 0.01245 Intercept 0.000571
Unknown C1
Dilution factor 1 2
A445 0.829 0.417
[Riboflavin]
(nmol/tube=mL) 66.5 33.4
Original Conc.
(nmol/mL) 66.5 66.9
Average
(nmol/mL) 66.7
From standard curve
or using SLOPE
function in Excel
From standard curve
or using
INTERCEPT
function in Excel
[Riboflavin]
(nmol/mL) =
(A445-
intercept)/slope
[original] =
[riboflavin]*
dilution
factor
Average of 2
values
36. Quick tips
• You can use the extinction coefficient
obtained in the first experiment or the
standard curve. Get the students to try
both methods.
• To get from the Absorbance to the
concentration you solve the equation of
the standard curve for x; you know the y
value (Abs) and you want to find out the x
value (conc.)
37. Why we express the
concentration in nmol/tube
• Provided you make the unknown dilutions
to the same volume as the standards you
can directly work out how much there is in
the tube straight from the graph.
• In the next exercise, unknown C2 or C3, it
will be a real advantage
38. Discussion Q from this section
• Where does the extinction coefficient fit in to the
std curve?
– It is the gradient, but the units of the ext. coefficient
are in the conc. Units on the x-axis
• What would happen to the absorbance
response and the equation of the line if:
– you measured the absorbance at a wavelength other
than the λmax?
– It would be linear but the gradient i.e. the ext.
coefficient would be lower See the varyQ worksheet
in the spectro.xls
39. What would happen to the absorbance
response and the equation of the line if:
• you expressed the concentration in different units (try M
and % (w/v), obtaining the molecular weight of the dye
from your demonstrator)?
– The equation changes, in particular the gradient. The
easiest way is to try this. Use the data given in the
spectro spreadsheet.
– you made the dye solutions up in 3 mL instead of 1
mL?
– No difference, the absorbance is dependent on the
concentration, which is volume independent. The only
problem is if the cuvette is not full enough to cover
light source
– you used cuvettes with a 2 cm light path instead of 1
cm? The slope would be double as the absorbance
would double for each tube
40. Experiment 3b: Unknown outside
the working range of the
spectrophotometer
• What concentration of riboflavin gives an
absorbance of 0.5? (always aim for the middle of
the range)
Using A = εcl……0.5 = 12.5*c*1
Conc = 0.04 mM 40 uM 40 nmol/mL
• So we need 40 nmoles of riboflavin in 1
mL to get an absorbance of 0.5
41. Experiment 3b:
• Now how much of our unknown do we
need to add to give an absorbance of 0.5?
• Our unknown, C2, lies between 0.5 and 1
mM. Undiluted this unknown would give
an absorbance between 6 and 12…way
too high!
• So we need to dilute our unknown…but by
how much
42. Experiment 3b:
• Let’s consider the upper end of the range,
1 mM.
• If our unknown is 1 mM, which is
1 umol/mL or 1 nmol/uL then…..
• as we need 40 nmol/mL to give an
absorbance of 0.5 so we would need to
add 40 uL (40/1).
43. Experiment 3b:
• Then let’s consider the lower end of the
range, 0.5 mM.
• If our unknown is 0.5 mM, which is
0.5 umol/mL or 0.5 nmol/uL then…..
• as we need 40 nmol/mL to give an
absorbance of 0.5 so we would need to
add 80 uL (40/0.5).
44. Experiment 3b:
• So we need to add between 40 and 80 uL
of our unknown and make these up to
1 mL mix well …….
• Then measure the absorbances of our
samples.
0.396 0.490 0.594 0.681 0.774
46. The back calculations
• The volume of the unknown (uL) and the
A445 are entered in directly as data.
• To calculate the [Riboflavin] (nmol/mL) you
solve the standard curve equation for x. This
gives the #nmoles of riboflavin in each cuvette.
31.76 39.31 47.66 54.65 62.12
47. The back calculations
From the [Riboflavin] (nmol/mL) we need to know
the concentration in the original unknown C2
31.76 39.31 47.66 54.65 62.12
?
48. The back calculations
Each cuvette has a known volume of the
original unknown added and we know
#nmoles of riboflavin in each cuvette
40 uL 50 uL 60 uL 70 uL 80 uL
31.76
nmoles
39.31
nmoles
47.66
nmoles
54.65
nmoles
62.12
nmoles
49. If we simply divide the #nmoles by the volume
added in uL we have the original concentration
of the unknown riboflavin in nmol/uL which is
mM. After all we only added two solutions to each cuvette; water
and riboflavin. We hope the nmoles came from the dye not the
water.
40 uL 50 uL 60 uL 70 uL 80 uL
0.79
mM
0.79
mM
0.79
mM
0.78
mM
0.78
mM
Average = 0.79 mM
50. For unknown C3: range 1 – 2 mM
• The upper range:
2 mM 2 umol/mL 2 nmol/uL
• So we need to add 40/2 = 20 uL
• The lower range:
• 1 mM 1 nmol/uL
• So we need 40/1 = 40 uL