METHODS

SPECTROPHOTOMETRIC

Descriptors
1.Introduction and generalities
2. Molecular spectrophotometry
4.Instrumentation and analytical applications
Spectroscopic optical methods
 This is the generic name for a very large number of instrumental
methods that use instrumental techniques in which an optical signal
is generated whose foundation is based on the interaction of
electromagnetic radiation with the analyte.
When electromagnetic radiation interacts with the sample containing
the analyte, different phenomena can arise, among which the
absorption and emission of light are the most relevant and give rise to
spectrophotometric methods of absorption and (or) emission.
The methods can be molecular or atomic absorption, depending on
whether the analyte is in a molecular or atomic state.
There are also fluorescent methods, in which there is absorption
preceded by emission
All of these methods are also called spectroscopic, because they are
based on the absorption or emission spectra of the analyte.
 The electromagnetic spectrum
Given the width of lines and spectral frequencies it contains, the
electromagnetic spectrum, the spectrum that results from the
interaction of matter-radiant energy, can be very complex and cover wide
areas: Vis-Uv, IR, Microwaves...etc.

Characteristics of electromagnetic radiation

Photon energy = hv
v=c
C- light speed

When l increases , the energy and frequency of the photon decreases

 Types of interaction

guy wavelength interaction

AND <10nm core emission
x-roy <10nm atomic ionization

uv 10-380nm electronic transactions

Vis 380-800nm electronic transactions
GO 800nm-100um link interactions
Radio meters nuclear absorption

Absorption: light is absorbed by an atom, ion or molecule that

acquires a higher energy state.
Emission: energy is released from an atom, ion or molecule that
acquires a lower energy state.

We will start by considering just some absorption methods!!!

 RADIATION ABSORPTION
ELECTROMAGNETIC
*
M+hn=M*
PROCESS BY WHICH A SPECIES IN A TRANSPARENT
MEDIUM, SELECTIVELY CAPTURES CERTAIN RADIATION
FREQUENCIES

The ABSORPTION of light by a substance takes place when the light

has the necessary energy to cause the necessary changes. These
changes (always discreet) can be:

•Electronic (atoms and (or) molecules)

•Vibrational (molecules only)
•Rotational (molecules only)
 Absorption spectroscopy

Atomic Molecular
Absorption Absorption

The spectrum is made up of lines, In absorption by molecules, vibrational and

due to the large number of rotational transactions between electronic
possible transactions, which sublevels come into play.
include the electronic sublevels The final result is translated into a
within each level. spectrum of bands.
energy ENERGY TRANSITIONS

uv
visibl
e

G
O

state state state

vibrational electronic rotationa
l
 TYPES OF SPECTRUM

l CONTINUOUS
SPECTRUM
LINE SPECTRUM
 Molecular absorption spectrophotometry
It is useful for both qualitative and quantitative analysis
Electronic
transactions Interactions
link
UV A NA Visible nearby N |R | Yo

UJ\W“NJ Spectrum
By varying the wavelength within each area of the electromagnetic spectrum, the
absorption spectrum of a substance in each region can be obtained.

Qualitative information:
It is obtained by comparing the spectrum of the sample with that of a
reference material.
This information is more relevant in IR spectroscopy and less in the case of
Vis-UV spectroscopy.

Vis-UV spectra can be achieved using the same equipment.

IR spectroscopy (more information) requires another type of instrument
 LAMBERT-BEER'S LAW

absorbance
total At =
measureme T, l
nt
to the

Q
= Transmittance
P
0
(T)
 MOLAR ABSORPTIVENESS

DEPENDS ON:

FEATURES OF THE
SOLVENT
ABSORBENT SPECIES
WORKING WAVELENGTH

UNITS

L /mol.cm

ADDITIVE PROPERTY OF ABSORBANCE

THE TOTAL ABSORBANCE OF A SAMPLE AT A
DETERMINED WAVELENGTH IS THE SUM OF THE
ABSORBANCES OF EACH OF ITS CONSTITUENTS AT SAID
WAVELENGTH.

This requires selecting the l of the analyte very well!

LIMITATIONS OF THE BEER LAW

REAL

$ variation in absorptivity due to solvent

 $ Concentration

APPARENTS

^ instrumental: polychromatic radiation

^ chemicals: chemical changes of species

absorbents by interaction with the solvent
 Deviations from Beer's law

Most species comply with the law in a certain concentration range. Outside of it,
they experience positive or negative deviations. This is well observed in the
calibration:

absorbance response due to self-

absorption or low light
passing through the
cuvette
target response,
interference or poor
sensitivity
concentration

It is necessary to ensure that you are working in the

linear interval!!
 WAVELENGTH SELECTION
OF WORK

Efforts should be made to measure absorbances in the environment closest

to l max . absorption in the spectrum (errors are minimized) and maximum
sensitivities are achieved.

Measuring away from that point of maximum absorption:

Small variations in measurement translate into large errors.
 BASIC COMPONENTS OF INSTRUMENTS
FOR MOLECULAR ABSORPTION MEASUREMENTS
IN
THE UV-VISIBLE
RADIANT ENERGY SOURCE
WAVELENGTH SELECTOR
CUVETTES (Sample)
DETECTOR
READING DEVICE
Comparative basic instrumentation in molecular spectroscopic methods

Vis-UV Spectroscopy:

IR Spectroscopy:

Molecular fluorescence:
(issue)

The three components go

together
as source and sample unit

detector reader
RADIANT ENERGY SOURCE
KEEP GOING
STABLE

Luminous intensity INTENSE

Lamp of

300 500 700 900

1100
wavelength (nm)
 Energy sources

In molecular absorption, the sample must be irradiated with light energy from a
source. Depending on the work area, the properties of the source are different.
The sources must be uniform in intensity.

IR sources are produced by the

passage of current through
incandescent materials:
ZrO2-yttrium oxides (400-20,000nm)
Cr-Ni (750-20,000 nm), SiC..etc

deuterium lamp
(UV)
tungsten source (W)
D2 * electrical energy
Used in VIS and near IR
produces light in ranges of 350-2200 nm D,* —.D,hv
160-380nm
 VISIBLE SPECTRUM AND COLORS
COMPLEMENTARY
display complementary
l (nm) color l (nm) color
380-420 violet 520 -550 yellow green
420 -440 blue violet 550 -580 yellow
440 -470 blue 580 -620 orange
470 - 500 green Blue 620 -680 red
500 -520 green 680 -780 purple
520 -550 yellow green 380 -420 violet
550 -580 yellow 420 -440 blue violet
580 -620 orange 440 -470 blue
620 -680 red 470 - 500 green Blue
680 -780 purple 500 -520 green
A SOLUTION APPEARS BLUE IN COLOR WHEN IT IS
ILLUMINATED WITH POLYCHROMATIC LIGHT BECAUSE IT
ABSORBS ALL COLORS EXCEPT BLUE, WHICH IS THE
COLOR IT ALLOWS TO PASS
 WAVELENGTH SELECTOR

CHARACTERISTICS
MAXIMUM TRANSMISSION WAVELENGTH
PERCENTAGE TRANSMITANCE AT MAXIMUM
EFFECTIVE BANDWIDTH

DESIGNS
CUT FILTERS
ABSORPTION FILTERS
INTERFERENCE FILTERS
* PRISM MONOCHROMATORS
* NETWORK MONOCHROMATORS
 WAVELENGTH SELECTION
WITH MONOCHROMATORS

BASIC COMPONENTS

ENTRY AND EXIT SLOTS

COLLIMATING MIRROR

DISPERSANT ELEMENT (PRISM OR NET)

MIRROR OR FOCUSING LENS

ENTRY AND EXIT WINDOWS

 MONOCHROMATORS

with
diffraction
grating

with prism
 BUCKETS

NORMAL FACES TO THE BEAM DIRECTION

G
O

MINIMUM ABSORPTION AT THE WORKING WAVELENGTH

1. Ordinary glass or silica (VIS) .
2. Fused silica or quartz (UV) .
3. NaCl, NaI, etc (IR) .
 DETECTORS

In the end the selected light has to be detected and quantified .

*This is achieved with the use of detectors whose purpose is to convert the
instrument's response into a measurable signal.
*Depending on the type of light you are working with, there are different
types of detectors:

guy X interval property use

detector
extent typical
nm

Phototube 150-1000 current uv

Photomultiplier 150-1000 current UVIVis
Solid state 350 - 3000 variable varied
Torque/thermal 600-20,000 current AI

Thermistors 600-20,000 IR resistance

PHOTOTUB
In IR spectroscopy, a Fourier transform
processor (FTIR) can be coupled to the detector. E
 DETECTOR REQUIREMENTS

THEY SHOULD RESPOND TO A WIDE RANGE OF

WAVELENGTHS

THEY MUST GIVE A FAST RESPONSE

THEY SHOULD BE SENSITIVE TO LOW LEVELS OF

RADIATION

THEY SHOULD PRODUCE AN EASILY AMPLIFIABLE

ELECTRICAL SIGNAL

THEY SHOULD HAVE LOW NOISE LEVEL

THE SIGNAL MUST BE PROPORTIONAL TO THE RADIANT

POWER
 PHOTOTUBES AND PHOTOMULTIPLIERS

They are based on the photoelectric effect, in which the incidence of a photonic
beam on a metal is capable of generating electrical energy.

The photon hits the cathode coated with

a photoemitter. cathode
A current is obtained that is
proportional to the photonics.
The tubes produce "dark
anode
PHOTOTUB
currents" due to thermal E
effects.

In the case of
photomultipliers, the signal is amplified through the use of dynodes in series.
 DETECTOR

PHOTOMULTIPLIER TUBE

Each dynode is connected to an

external 90 V source, generating in
the collector an avalanche of 10 6
to 10 7 electrons for each incident
photon in the case of a
photomultiplier tube with 9
dynodes. manifol
d

array photodiode
Made up of a series of photodiodes spaced at regular
intervals within an integrated circuit chip
 It can measure multiple wavelengths at the same time.

Currently, it is the most widely used spectrophotometric

detector coupled
to a liquid
chromatography
system.

Signal and readout processor

It is the last basic component of instrumentation

Every spectrophotometer must have:

1. Self-amplifying system that produces a measurable signal
2. A signal processing system, which allows data to be eliminated,
averaged, provide reading output, direct the signal output, etc.
3. A signal output: digital, recorder, etc.
4. Have some ability to process numbers/FTIR
 Spectrophotometers

They may be :
1 Single beam or simple beam

1 - light source 4-cell sample

2 - wave length selector 5 - detector
3 - shutter 6 - reading
 2 Double beam spectrophotometer

• The beam can be unfolded at regular intervals with the help of a chopper
• Half of the light passes through the sample and the other a “target”
• The measured absorbance takes into account the sample/blank absorption ratio and
allows correction of deviations in the detector and the output source.
• The chopper change speed is a limitation on what the instrument can select.

3 IR Spectrophotometers
They are of two types:
1) Dispersive (they rely on a monochromating and scanning system to produce
the spectrum).
2) Fourier transform (they use a combination of constructive and destructive
interference along with the transform to produce the spectrum).

Dispersive

fountain sample monochromator detector

They are generally double beam

the monochromator precedes the sample

Infrared with Fourier transform

Basis:
 At a fixed time many l are passed through the cell and the signal is recorded.
With each sweep, any l can traverse the cell many times. The interferogram is
obtained like this:

Analytical applications

Molecular absorption spectrophotometry can be applied to both qualitative and

quantitative analysis.

Qualitative analysis: the sample spectra must be compared with those

corresponding to standards in order to identify the coincident absorption bands.
This type of analysis is more frequent in UV and IR to identify organic compounds
and Vis is rarely used.
 Quantitative analysis: it is based on the Beer-Lambert law in the range of law
enforcement concentrations. The calibration curve is established using standards
and the absorbance of the sample of unknown concentration is referred to the curve
(sometimes a single standard is sufficient).
Procedure:
•In Vis. spectrophotometry, the analyte is usually incorporated into a complex
species that presents intense absorption bands.
•The selection of the l at which the absorbance is measured can contribute to
increasing the selectivity of the measurement (if it absorbs only the analyte)
•Whenever possible, the absorbance should be measured at l max

It is a routine technique and especially sensitive in fluorescence.

ANALYTICAL APPLICATIONS

WIDE FIELD OF APPLICATIONS

(organic and inorganic analysis)
HIGH SENSITIVITY
 GOOD SELECTIVITY

GOOD PRECISION

OPERATIONAL EASE

APPLICATION TO NON-ABSORBENT SPECIES

(FORMATION OF COMPLEXES)