Introduction to Instrumental Analysis - Spectrophotometry Clinical Analytical Chemistry CLS 231 Introduction to Instrumental Analysis - Spectrophotometry Lecture 11 Done by Lecturer : Amal Abu-Mostafa

Session Objectives: Introduction to Spectrophotometry Properties of Light Colors & Wavelengths What are Spectroscopy and Spectrophotometry Instruments of Measurement Absorption of Light The Spectrophotometer Definitions & Symbols Beer’s Law Spectrophotometric techniques Applications of a spectrophotometer Overview of Quantitative Spectrophotometry

Introduction to Spectrophotometry Properties of Light: Electromagnetic radiation moves in waves Light (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

Electromagnetic Spectrum

Colors & Wavelengths Visible Light COLOR WAVELENGTH (λ in nm) Ultraviolet < 380, UV region (200-380 nm) Violet 380 – 435 Blue 436 – 480 Greenish-blue 481 – 490 Bluish-green 491 – 500 Green 501 – 560 Yellowish-green 561 – 580 Yellow 581 – 595 Orange 596 – 650 Red 651 – 780 Near Infrared > 780 Visible Light

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 emission Spectrophotometers measure electromagnetic absorption

Instruments of Measurement Two most common: Visible Spectrophotometer Atomic-Absorption Spectrophotometer

Instruments of Measurement What do visible spectrophotometers measure? Amount of light absorbed by the dissolved substance Qualitative Quantitative The 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.

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.

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 absorbed by the green solution

The Spectrophotometer

The Spectrophotometer a) Single-beam b) Double-beam

The Spectrophotometer Contains: Light source (Lamp) Optical filters or prism Tube or cuvette photoelectric cell, or detector, or Photomultiplier tube.

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.

The Spectrophotometer Optical filters/prisms: To limit light to a certain wavelength Monochromator can isolate a specific wavelength of white light and allow it to pass through the solution being analyzed. Tubes or cuvettes: Visible range = glass cuvette UV range = quartz cuvette Photocell: To detect transmitted light, Or Detector: Convert radiant energy (photons) into an electrical signal. Or Photomultiplier tube: very sensitive detector

Definitions & Symbols: Spectrophotometry Definitions & 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 100 Io Io It

Definitions & Symbols: Spectrophotometry Definitions & Symbols: ABSORBANCE (A) A = log(1/T) = -log(T) A = log Io = log Io – log It It Absorbance is what is generally recorded from a spectrophotometer.

Beer’s Law More dissolved substance = more absorption and less transmittance. Beer-Lambert’s Law is: A =  l C Log Io =  l C It A= Absorbance (no units) Io = intensity of incident light It = intensity of transmitted light  = molar extinction coefficient, molar absorptivity c = concentration of the absorbing species (mol/L) l = path length of the light-absorbing sample (cm)

Sample Problem Cytosine 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 C It  = 6 x 103 mol-1 .cm-1 l = 1mm = 0.1 cm C = 1 x 10-3 M A =  l c = (6 x 103)x (0.1) x (1 x 10-3) = 6 x 10-1 = 0.6

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

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.

Overview of Quantitative Spectrophotometry A. Measure the absorbance of standards containing known concentrations of the analyte B. Plot a standard curve with absorbance on the Y axis and analyte concentration on the X axis C. Measure the absorbance of the unknown(s) D. Determine the concentration of material of interest in the unknowns based on the standard curve

LINEAR RANGE: If there is too much or too little analyte, spectrophotometer cannot read the absorbance accurately.

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