S P E C T R O P H O TO M E T R Y

Judith Maye G. Bolico, RMT, ASCPi

 Instrumentation
Judith Maye G. Bolico, RMT, ASCPi
Judith Maye G. Bolico, RMT, ASCPi

Four Basic Discipline

COLORIMETRY
LUMINESCENCE ELECTROANALYTICAL CHROMATOGRAPHY
METHODS
Judith Maye G. Bolico, RMT, ASCPi

Colorimetry

• Radiant energy EMITTED, TRANSMITTED ABSORBED, or REFLECTED under

controlled condition

a) SPECTROPHOTOMETRY

b) FLAME EMISSION SPECTROPHOTOMETRY

c) ATOMIC ABSORPTION SPECTROPHOTOMETRY

d) MASS SPECTROMETRY

e) REFLECTANCE SPECTROMETRY
Judith Maye G. Bolico, RMT, ASCPi

Luminescence
• Measurement of light when electrons return from an EXCITED STATE to a
LOWER ENERGY level

a) FLUORESCENCE

b) CHEMILUMINESCENCE

c) NEPHELOMETRY and TURBIDIMETRY

Judith Maye G. Bolico, RMT, ASCPi

Electroanalytical Methods
• Measurement of ELECTRICAL, POTENTIAL, CURRENT or RESISTANCE to quantify analytes.

a) Electrophoresis

b) Potentiometry

c) Ion-selective electrode

d) Osmometry

e) Amperometry

f) Coulometry

g) Flow Cytometry

h) Conductometry and Impedance

Judith Maye G. Bolico, RMT, ASCPi

Chromatography
• SEPARATION of analytes.

a) Gas

b) Liquid

c) Thin Layer

d) Adsorption

e) Partition

f) Affinity

g) HPLC
Judith Maye G. Bolico, RMT, ASCPi

Terminologies

• Wavelength
- Distance between two successive peaks
- Expressed in terms of nanometer (nm)\

• Regions of Spectrum:
- 400-700nm = Visible Spectrum
- <400nm = UV
- >700 = IR
Judith Maye G. Bolico, RMT, ASCPi

Terminologies

• Frequency
- Number of vibrations of wave per second

- Lower wave frequency = Longer wavelength

- Higher wave frequency = Shorter wavelength

Judith Maye G. Bolico, RMT, ASCPi

I. Colorimetry
• A. Spectrophotometric measurement
- Measurement of light intensity in a narrower wavelength

- Specific Wavelength

- Use of prisms, and grating

• B. Photometric measurement
- Without consideration of wavelength

- Uses filter

- Measurement of light intensity

Judith Maye G. Bolico, RMT, ASCPi

I. Colorimetry
• A. Spectrophotometry
- Involves the measurement of LIGHT TRANSMITTED by a solution to determine the
concentration of the LIGHT-ABSORBING substances in the solution.

- Beer Lambert’s Law

- Relationship between abs (at a given wavelength) and concentration.

- Conc of substance is = amount of light absorbed

- Conc of substance is ≠ logarithm of transmitted light.

Judith Maye G. Bolico, RMT, ASCPi

I. Colorimetry
• A. Spectrophotometry
- Increased concentration, increased intensity of color, increased light absorbed,
decreased light transmitted.
Wavelength (nm) Color absorbed Complementary Color
(color observed)

350-430 Violet Yellow-Blue

430-475 Blue Yellow
475-495 Green-blue Orange
495-505 Blue-green Red
505-555 Green Purple
555-575 Yellow-green Violet
575-600 Yellow Blue
600-650 Orange Green-blue
650-700 Red Blue-Green
Judith Maye G. Bolico, RMT, ASCPi

I. Colorimetry
• A. Spectrophotometry
Judith Maye G. Bolico, RMT, ASCPi

Components of Spectrophotometer

1 2 3 4 5 6
1. Light Source 2. Slits 3. 4. Sample Cell/ 5. 6. Read-out
Monochromator Cuvette Photodetector device
Judith Maye G. Bolico, RMT, ASCPi

Light Source

• Provides polychromatic light and

must generate sufficient radiant
energy or power to measure the
analyte of interest.
Judith Maye G. Bolico, RMT, ASCPi

Light Source
• 2 TYPES

• A. Continuum Source

- Emits radiation that changes in intensity

- Widely used in the laboratory

- Example:

1. Tungsten Iodide Lamp/ Incandescent Tungsten

▪ MOST common light source in the visible and near infrared region

2. Deuterium Lamp

▪ Routinely used to provide UV radiation in analytic spectrometers.

3. Xenon Discharge Lamp

▪ Produces a continuous source of radiation

▪ Covers both UV and Visible range.

Judith Maye G. Bolico, RMT, ASCPi

Light Source
• 2 TYPES

• B. Line Source

- Emits limited radiation and wavelength.

- Example:

1. Mercury and Sodium Vapor Lamps

• UV and Visible Region

2. Hollow Cathode Lamp

• Used in AAS
Judith Maye G. Bolico, RMT, ASCPi

Light Source
• Factors for choosing a Light Source

1. Range

2. Spectral Distribution within the range

3. Source of radiant production

4. Stability of the radiant energy

5. Temperature
Judith Maye G. Bolico, RMT, ASCPi

Light Source
• Alternative for Tungsten bulb
UV Visible and UV IR
Deuterium Lamp Mercury Arc Merst glower
Hydrogen Lamp Globar
Xenon Lamp
Judith Maye G. Bolico, RMT, ASCPi

Entrance Slit

• Minimizes unwanted or stray light

• Prevents the entry of scattered light into the monochromator system

• Stray Light

- Any wavelengths outside the band transmitted by the monochromator.

- It doesn’t originate from the polychromatic source.

- Causes absorbance error.

- Limits max. absorbance that a spectrophotometer can achieve

- Most common cause of loss of linearity at high-analyte concentration.

Judith Maye G. Bolico, RMT, ASCPi

Monochromator

• Isolates specific or individual

wavelength of light.
Judith Maye G. Bolico, RMT, ASCPi

Monochromator
• Kinds of Monochromator

A. Prisms
✓ Wedge-shaped piece of glass, quartz or NaCl

✓ It can be rotated, allowing only the desired wavelength to pass through an exit slit

B. Diffraction Gratings
✓ Most commonly used

✓ Better resolution than prism

✓ Wavelengths are bent as they pass a sharp corner

C. Filters
✓ Simple, least expensive

✓ Produce monochromatic light based on the principle of constructive interference of waves.

D. Holographic gratings
Judith Maye G. Bolico, RMT, ASCPi

Exit Slit

• Controls the width of light beam (bandpass)

• Allows only a fraction of the spectrum to reach the sample

cuvet

• Bandpass
- Total Range of Wavelengths transmitted.
Judith Maye G. Bolico, RMT, ASCPi

Cuvet

• Also known as:

Absorption Cell/Analytical Cell/Sample Cell.

• Holds the solution whose concentration is to

be measured.
Judith Maye G. Bolico, RMT, ASCPi

Cuvette
• Kinds of Cuvette

A. Alumina Silica Glass

✓ Most commonly used. (350-2000nm)

B. Quartz/Plastic
✓ Used for measurement of solution requiring visible and uv spectra

C. Borosilicate Glass
✓ Visible region; alkaline solution

D. Soft Glass
✓Visible region; for acidic solution
Judith Maye G. Bolico, RMT, ASCPi

Photodetector

• Detect and convert transmitted light into

photoelectric energy

• Detects the amount of light that passes

through the sample in the cuvette.

• Light energy into electrical energy

Judith Maye G. Bolico, RMT, ASCPi

Photodetector
• Kinds of photodetector

A. Photomultiplier
✓ Most commonly used detector –measures visible and UV regions

✓ Detect very low level of lights

✓ Most sensitive. Should never exposed to light because it will burn out.

B. Barrier layer cell/ Photocell/ Photovoltaic Cell

✓ Simplest detector; least expensive

✓ Film of selenium on iron plate coated with silver

C. Phototube
✓ Contains cathode and anode enclosed in a glass case.

✓ Second widely used; needs outside voltage to operate.

Judith Maye G. Bolico, RMT, ASCPi

Meter or Read-
out Device
• Displays output of the detection system.

• Examples:
Galvanometer

Ammeter

LED Display