ANALYTICAL TECHNIQUES Flashcards
-a method of studying and
measuring a specific spectrum, it is widely
used for the spectroscopic analysis of
sample materials.
Spectrometry
-Measures the light transmitted by a solution to
determine the concentration of the substance in the
solution
SPECTROPHOTOMETRY
measure how much a chemical substance absorbs
light by measuring the intensity of light as a beam of light
passes through sample solution.
.SPECTROPHOTOMETRY
-solution has an ability to absorb and transmit light,
and only ___ can be measured.
transmitted light
defined as the proportion of incident light
that is transmitted and is usually expressed as a percentage
Transmittance
Transmittance formula
%T = I/Io x 100
is the critical measure used in the
calculation of concentration.
absorbance
Absorbance is calculated as follows:
A= 2-log%T
Instruments that measure transmitted light energy of a
solution by reading its absorbance in a meter.
Optical Instruments
Spectrophotometry takes advantage of the property of ___
solutions to absorb light of specific wavelength
colored solutions
is a form of electromagnetic energy
Transmitted via electromagnetic waves
Light
Waves is measured in ____ between the peaks and
valleys *(wavelength).
nanometer
*The ____ the wavelength, the ____ the
electromagnetic energy.
shorter. higher
These are radiant energy from short wavelength gamma
rays to long wavelength radiowaves.
ELECTROMAGNETIC ENERGY
basic unit of ELECTROMAGNETIC ENERGY
photons
photons are also known as
light quantum
is a minute energy packet of
electromagnetic radiation ,discrete energy packets
photons
Types of Electromagnetic energies:
a. Cosmic rays
b. Gamma rays
c. X–rays
d. Visible rays
e. Ultra–Violet (UV)
f. Infrared
g. Radio, TV, Microwaves
THIS LAW STATES THAT: The concentration of a substance is
Directly proportional to the amount of light absorbed
Inversely proportional to the amount of transmitted light
Beer’s Law
states that a chemical solution's concentration is
directly proportional to its light absorption.
Beer’s Law
To convert a value from percent transmittance (%T)
to absorbance, use the following
equation:
Absorbance= 2 - log(%T).
wave like properties,
discrete packets of energy traveling in waves.
a.Photons / light particles
the linear distance between
successive wave peaks and measured in units of
nanometers (10 -9 )m)
Wavelenghts
number of wave peaks per
given unit of time
frequency
–height of the peak
Amplitude
shortest wavelength.
microwaves
interactions of light with matter occur
when a photon intercepts an atom,ion or molecule.
Excitation
Components of a spectrophotometer
1.)Light Source
2.)Entrance Slit
3.)Monochromator
4.)Exit Slit
5.)Sample cell
6.)Photodetector
7.) read out device
-Provide incident light /radiant energy for the system
Light Source
Light Source - typical source in most spectrophotometers.
-.For visible and near infrared region
spectrum (320 to 700nm)
incandescent Tungsten or Tungsten iodide lamp
Light Source -For UV spectrum (below 350nm)
Deuterium-discharge lamp
-exclude unwanted or stray light
Entrance Slit
Isolates specific wavelength of interest from the light source
Monochromator
-controls the width of the light beam
Exit Slit
-Also known as cuvette or analytical cell
Sample cell
-Converts transmitted radiant energy into an
equivalent amount of electrical energy.
Photodetector
it is the simplest types of absorption spectrometer, it is
designed to make one measurement at a time at one specified
wavelength
Single beam spectrophotomer
It is an instrument that splits the monochromatic light into two
components. One beam passes through the sample, and the
other through a reference black solution
Double-beam spectrometer
TYPES OF spectrophotomer
a. Single beam spectrophotomer
b. Double-beam spectrometer
Measures light emitted by excited atoms
used to measure sodium, potassium and lithium because
they are easy to excite
Flame Emission
is used in clinical chemistry for the
determination of electrolytes
Flame photometry
-It measures the light emitted by a single atom burned in a
flame
Flame photometry
The following electrolytes produces the following colors:
1. Sodium –
2. Potassium –
3. Lithium –
4. Magnesium –
5. Calcium
- Sodium – yellow
- Potassium – violet
- Lithium – red
- Magnesium – blue
- Calcium – red
Principle: excitation of electrons from lower to higher
energy state
Flame Emission
Flame Emission- Flame using ___ is used to excite the
atoms (higher energy state)
propane
Components of FES
A. Nebulizer (atomizer)
B. Burner
C. Monochromator system
D. Photosensitive detector (photomultiplier
tube)
Deliver a fine spray of sample
containing the metallic ion to the burner.
Nebulizer (atomizer)
A fuel gas (propane) with an oxidizing agent (compressed air) burned to produce the flame.
Burner
-Allow only emitted line spectrum of specific element
to strike the PMT.
Monochromator system
Measures light absorbed by ground state atoms
Atomic Absorption
Measures concentration through the detection of
absorbance of electromagnetic radiation by atoms
instead of molecules.
Atomic Absorption
**100 times more sensitive than FES
Atomic Absorption
Light Source AAS
Hallow cathode lamp
measure the concentrations of
solution that contain fluorescing
molecules
Fluorometer
Photometric measurement of light emitted
by a substance that has been previously
excited by a source of UV light,
FLOUROMETRY
*It is more sensitive than fluorometry
CHEMILUMINESCENCE
-In this method, no excitation radiation is required and no monochromator are needed because it
arises from one specie.
CHEMILUMINESCENCE
-For measuring abundant large particles and bacterial
suspensions
Turbidimetry
In coagulation
analyzers, turbidimetric measurements detect clot formation in the sample curvets.
Turbidimetry
Turbidimetric assays
have long been available in clinical chemistry to
quantify protein concentration in biologic fluids, such
as
urine and cerebrospinal fluid (CSF).
Measures the amount of light blocked (absorbance) by
suspension of particles
Turbidimetry
It is essentially a measurement of blocked light and the amount of
blocked light is directly proportional to the number of
particles in a solution.
Turbidimetry
determines the amount of light
scattered by the small particles or colloids in the sample cuvet.
-For measuring the amount of antigen-antibody complexes
Nephelometry
Principle: Light scattered by the small particles is measured at an
angle (forward or 90 degrees) to the incident light.
Nephelometry
Dependent on particle size and concentration
Turbidimetry
Dependent on wavelength and particle size.
Nephelometry
Migration of charge particles in an electric field. Separate proteins on the basis of their electric charge
densities
ELECTROPHORESIS
definition: The process of separating the charged
constituents of a sample by means of an electrical current.
ELECTROPHORESIS
it is a method used to separate, detect
and identify one or more proteins in a complex mixture.
Western Blotting –
-Migration of small ions, produced by electromigration causes the
movement of the charged ions due to low electric potential.
1.Iontophoresis
-Migration of charged macromolecules in a porous
support (paper. Cellulose acetate or agarose gel)
used for separating charged particles such as nucleic acids,
proteins, and biopolymers.
Zone electrophoresis
Result of electrophoresis consisting of separated strands
of a macromolecule
Electrophoretogram
gel - routinely used for protein analysis
Polyacrylamide gel
Separation is performed in narrow-bore fuse silica capillaries
Capillary electrophoresis
Techniques used to separate complex mixtures or
specimen compounds between two mobile and
stationary phase
CHROMATOGRAPHY
Chromatography Phase - carries the complex mixture
Mobile Phase
Chromatography Phase - through which mobile
phase flows
Stationary phase
holds the stationary phase
Column
separated components Chromatography Phase
Eluate
Modes of Separation Chromatography
- Adsorption
2.Partition - Steric Exclusion
- Ion Exchange
-Also known as liquid-solid chromatography
-Based on the competition between the sample and
the mobile phase for adsorptive sites on the solid
stationary phase.
Adsorption
-Stationary phase chromatography
Acidic polar (silica gel)
Basic polar (alumina)
Nonpolar (charcoal)
Also known as liquid-liquid chromatography
Partition
Also known as gel filtration, gel permeation, size-
exclusion, molecular exclusion or molecular sieve
chromatography
Steric Exclusion
-Stationary phase is coated on a sheet of paper or bound
to glass or plastic plate
Planar Chromatography
Chromatography -The stationary phase is packed into a tube or coated onto
the inner surface of the tube.
Column Chromatography
is the study of electron
movement in an oxidation or reduction reaction at a
polarized electrode surface.
Electrochemistry
Anode/cathode system , electrons spontaneously flow from an electrode of high electron affinity to an
electrode of low electron affinity, if the electrodes are connected via
SALT BRIDGE.
Measures current or voltage (potential) generated by
the activity of specific ions
ELECTROCHEMISTRY
Blood Gas Instruments ELECTROCHEMISTRY
pH Electrode
pCO 2 Electrode
pO 2 Electrode
Blood Gas Instruments Measure hydrogen ion activity
pH Electrode
Blood Gas Instruments pH electrode with a CO 2 -permeable membrane
and bicarbonate buffer.
Severinghaus electrode
pCO 2 Electrode
Blood Gas Instruments Measures current flow produced
from loss or gain of electrons.
pO 2 Electrode
Definitive identification and quantitation of samples
or compounds eluting from GC or HPLC columns.
MASS SPECTROMETRY
Measuring drugs of abuse in urine.
Measuring low-level and mixed-polarity analytes.
Vitamin D, testosterone and immunosuppressant drugs
MASS SPECTROMETRY
-measurement of voltage potentials is based on the
measurement of a potential (voltage) difference between two
electrodes immersed in solution under the condition of zero
current electrochemical measurements .
POTENTIOMETRIC METHODS
potentiometric apparatus used to
measure the concentration of hydrogen ions in solution. It
measures the potential difference between one half-cell and a
reference electrode.
pH meter
half-cell)-sensitive and responds to changes in concentration of a particular ion species in the solution in which the electrode is immersed.
- an electrochemical half-cell that interacts with the analyte of interest.
indicator electrode
measure a
potential across a membrane specific for a certain analyte.
ISE -ion-selective electrode
type of ISE
most commonly used for pH measurement.
Glass-membrane electrode
–measurement of the amount of
electricity passing between two electrodes in an
electrochemical cell.
1.Coulometry
1.Coulometry the unit of electric charge
coulomb (symbol: C)
measurement of the
current flowing through an electrical cell when a
potential is applied.
Amperometry
Measure of solute particles in a solution in terms of
their colligative properties (osmotic pressure, vapor
pressure, boiling point and freezing point).
OSMOMETRY
number of moles of particle per
kilogram of water and depends only on the number of particles
, not on what types of particles are present.
Osmolality
solution are related to the
number of solute particles per kilogram of solvent.
Colligative Properties
