Presentation on theme: "Introduction to Instrumental Analysis - Spectrophotometry"— Presentation transcript:
1 Introduction to Instrumental Analysis - Spectrophotometry Clinical Analytical ChemistryCLS 231Introduction to Instrumental Analysis - SpectrophotometryLecture 11Done by Lecturer : Amal Abu-Mostafa
2 Session Objectives: Introduction to Spectrophotometry Properties of LightColors & WavelengthsWhat are Spectroscopy and SpectrophotometryInstruments of MeasurementAbsorption of LightThe SpectrophotometerDefinitions & SymbolsBeer’s LawSpectrophotometric techniquesApplications of a spectrophotometerOverview of Quantitative Spectrophotometry
3 Introduction to Spectrophotometry Properties of Light:Electromagnetic radiation moves in wavesLight (called electromagnetic radiation) moves in waves.Wavelength = different types of light have different wavelengths. Some are longer than others. For instance, in the visible light spectrum, red light waves are longer than blue light waves.Wavelengths are commonly given in ????Lambda λ
6 What are Spectroscopy and Spectrophotometry?? Light can either be transmitted or absorbed by dissolved substances.Presence & concentration of dissolved substances is analyzed by passing light through the sample.Spectroscopes measure electromagnetic emissionSpectrophotometers measure electromagnetic absorption
7 Instruments of Measurement Two most common:Visible SpectrophotometerAtomic-Absorption Spectrophotometer
8 Instruments of Measurement What do visible spectrophotometers measure?Amount of light absorbed by the dissolved substanceQualitativeQuantitativeThe absorption of light indicates the presence of the substance. This is a qualitative measurement.The amount of light absorbed measures the concentration of the dissolved substance. This is a quantitative measurement.
9 Absorption of Light White light All colors Polychromatic light When white (polychromatic) light passes through a coloured solution some of the light is absorbed by the substances in the solution, and the rest passes through.For Example: Green solution absorbs light other than green.
10 Absorption of Light Monochromatic light Light of one color For example: If white light is made to pass through a red filter, all light except red is filtered out and absorbed. Therefore, only red light hits the solution.Red light is absorbedby the green solution
12 The Spectrophotometer a) Single-beamb) Double-beam
13 The Spectrophotometer Contains:Light source (Lamp)Optical filters or prismTube or cuvettephotoelectric cell, or detector, or Photomultiplier tube.
14 The Spectrophotometer Light source (Lamp)UV light from 200 to below 380 nm = deuterium or hydrogen lamp.Visible region from 380 nm to 700 nm = tungsten or tungsten-halogen.
15 The Spectrophotometer Optical filters/prisms:To limit light to a certain wavelengthMonochromator can isolate a specific wavelength of white light and allow it to pass through the solution being analyzed.Tubes or cuvettes:Visible range = glass cuvetteUV range = quartz cuvettePhotocell: To detect transmitted light,Or Detector: Convert radiant energy (photons) into an electrical signal.Or Photomultiplier tube: very sensitive detector
16 Definitions & Symbols: SpectrophotometryDefinitions & Symbols:Radiation Intensity (I)It : is the radiation transmitted by the solution.Io : is the radiation transmitted by the pure solvent (blank).Transmittance (T)It’s also referred to as %T or T x 100%T = It x 100IoIo It
17 Definitions & Symbols: SpectrophotometryDefinitions & Symbols:ABSORBANCE (A)A = log(1/T) = -log(T)A = log Io = log Io – log ItItAbsorbance is what is generally recorded from a spectrophotometer.
18 Beer’s LawMore dissolved substance = more absorption and less transmittance.Beer-Lambert’s Law is:A = l CLog Io = l CItA= Absorbance (no units)Io = intensity of incident lightIt = intensity of transmitted light = molar extinction coefficient, molar absorptivityc = concentration of the absorbing species (mol/L)l = path length of the light-absorbing sample (cm)
19 Sample ProblemCytosine has a molar extinction coefficient of 6 x 103 mol-1 cm-1 at 270 nm at pH 7. Calculate absorbance of 1 x 10-3 M cytosine solution in 1mm cell at 270 nm.Solution:A = Log Io = l CIt = 6 x 103 mol-1 .cm-1l = 1mm = 0.1 cmC = 1 x 10-3 MA = l c = (6 x 103)x (0.1) x (1 x 10-3)= 6 x 10-1= 0.6
20 Spectrophotometric techniques Are used to measure the concentration of solutes in solution by measuring the amount of light that is absorbed by the solution in a cuvette placed in the spectrophotometer.Spectrophotometry: Measures light absorbed by solution at a specific wavelength.One of the simplest and most widely used methods to determine the amount of protein or nucleic acid present in a given solution
21 Applications of a spectrophotometer Determines the presence and concentrations of samples.Determines the purity of a sample.Look at the change of samples over time.
22 Overview of Quantitative Spectrophotometry A. Measure the absorbance of standards containing known concentrations of the analyteB. Plot a standard curve with absorbance on the Y axis and analyte concentration on the X axisC. Measure the absorbance of the unknown(s)D. Determine the concentration of material of interest in the unknowns based on the standard curve