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Introduction to Instrumental Analysis

- Spectrophotometry

Lecture 11

Done by Lecturer : Amal Abu-Mostafa

Lecture 11

Done by Lecturer : Amal Abu-Mostafa

Clinical Analytical ChemistryCLS 231

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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

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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

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Electromagnetic Spectrum

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Colors & WavelengthsCOLOR 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

Vis

ible

Lig

ht

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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

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Instruments of Measurement

Two most common:

1. Visible Spectrophotometer

2. Atomic-Absorption Spectrophotometer

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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.

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Absorption of Light

White lightAll 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.

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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

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The Spectrophotometer

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The Spectrophotometer

a) Single-beam

b) Double-beam

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The Spectrophotometer

Contains:Light source (Lamp)

Optical filters or prism

Tube or cuvette

photoelectric cell, or detector, or Photomultiplier tube.

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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.

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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

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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

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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.

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Beer’s LawMore 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)

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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 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

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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

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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.

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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

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

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Thank you