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PRINCIPLE AND
INSTRUMENTATION
PRESENTED BY- MISS. SHIVANI JAGNNATH PATIL
M.PHARM-I YEAR (PHARMACEUTICAL QUALITY ASSURANCE)
1
FLAME PHOTOMETRY
2
FLAME PHOTOMETRY
Introduction
• Flame photometry is also known as Flame emission spectroscopy.
• Flame photometry is based on measurement of intensity of light emitted
when metal is introduced into flame.The wavelength of colour tells us
what the element is and the colour intensity tells us how much of element
is present.
• A photoelectric flame photometer is an instrument used in inorganic
chemical analysis to determine the concentration of certain metal ions
among them sodium,potassium,calcium and lithium.
3
PRINCIPLE
- The basic principle upon which Atomic Spectroscopy works is
based on the fact that "Matter absorbs light at the same wavelength at
which it emits light".
- The compounds of the alkali and alkaline earth metals (Group II)
dissociate into atoms when introduced into the flame. Some of these atoms
further get excited to even higher levels. But these atoms are not stable at
higher levels.Hence, these atoms emit radiations when returning back to the
ground state. These radiations generally lie in the visible region of the
spectrum. Each of the alkali and alkaline earth metals has a specific
wavelength.
4
Element Emitted wavelength Flame color
Sodium 589 nm Yellow
Potassium 766 nm Violet
Barium 554 nm Lime green
Calcium 622 nm Orange
Lithium 670 nm Red
For certain concentration ranges,
The intensity of the emission is directly proportional to the
number of atoms returning to the ground state. And the light
emitted is in turn proportional to the concentration of the
sample.
5
INSTRUMENTATION
6
Parts of flame photometer
A simple flame photometer consists of the following basic components:
Source of flame: A Burner in the flame photometer is the source of flame. It can be
maintained in at a constant temperature. The temperature of the flame is one of
critical factors in flame photometry.
Fuel-Oxidant mixture
Temperature (°C)
Natural gas-Air 1700
Propane-Air 1800
Hydrogen-Air 2000
Hydrogen-Oxygen 2650
Acetylene-Air 2300
Acetylene-Oxyen 3200
Acetylene-Nitrous oxide 2700
Cyanogen-Oxygen 4800
7
Nebuliser: Nebuliser is used to send homogeneous solution into the flame at a balanced
rate.
Optical system: The optical system consists of convex mirror and convex lens. The convex
mirror transmits the light emitted from the atoms. Convex mirror also helps to focus the
emissions to the lens. The lens helps to focus the light on a point or slit.
Simple colour filters: The reflections from the mirror pass through the slit and reach the
filters. Filters will isolate the wavelength to be measured from that of irrelevant emissions.
Photo-detector: The intensity of radiation emitted by the flame is measured by photo
detector. Here the emitted radiation is converted to an electrical signal with the help of
photo detector. These electrical signals are directly proportional to the intensity of light.
8
Events occur in Flame:
The oxidants in flame photometer are mainly air, oxygen or nitrous oxide. The temperature of the
flame depends on the ratio of fuel and oxidant.
The processes occurring during flame photometer analysis are summarized below:
Desolvation: Desolvation involves drying a sample in a solution. The metal particles in the solvent are
dehydrated by the flame and thus solvent is evaporated.
Vaporization: The metal particles in the sample are also dehydrated. This also led to the evaporation of the
the solvent.
Atomization: Atomization is the separation of all atoms in a chemical substance. The metal ions in the
sample are reduced to metal atoms by the flame.
Excitation: The electrostatic force of attraction between the electrons and nucleus of the atom helps them
to absorb a particular amount of energy. The atoms then jump to the higher energy state when excited.
Emission: Since the higher energy state is unstable the atoms jump back to the ground state or low energy
energy state to gain stability. This jumping of atoms emits radiation with characteristic wavelength. The
radiation is measured by the photo detector.
9
Applications of flame photometer
1.Flame photometer can be applied both for quantitative and qualitative analysis of elements.
The radiations emitted by the flame photometer are characteristic to particular metal. Hence
with the help of Flame photometer we can detect the presence of any specific element in the
given sample.
2.The presence of some group II elements is critical for soil health. We can determine the
presence of various alkali and alkaline earth metals in soil sample by conducting flame test
and then the soil can be supplied with specific fertiliser.
3.The concentrations of Na+ and K+ ions are very important in the human body for
conducting various metabolic functions. Their concentrations can be determined by diluting
and aspirating blood serum sample into the flame.
4.Soft drinks, fruit juices and alcoholic beverages can also be analysed by using flame
photometry to determine the concentrations of various metals and elements.
10
ULTRAVIOLET SPECTROSCOPY
11
ULTRAVIOLET SPECTROSCOPY
Spectroscopy
• It is the branch of science that deals with the study of interaction of matter with
light. OR
• It is the branch of science that deals with the study of interaction of
electromagnetic radiation with matter.
• The study of molecular or atomic structure of a substance by observation of its
interaction with electromagnetic radiation
• QUANTITATIVELY - For determining the amount of material in a sample
• QUALITATIVELY – For identifying the chemical structure of a sample
12
Principles of Spectroscopy
• The principle is based on the measurement of spectrum of a sample containing atoms /
molecules.
• Spectrum is a graph of intensity of absorbed or emitted radiation by sample verses
frequency (v) or wavelength (ʄ).
• Spectrometer is an instrument design to measure the spectrum of a compound.
1. Absorption Spectroscopy:
• An analytical technique which concerns with the measurement of absorption of
electromagnetic radiation.
• e.g. UV (185 - 400 nm) / Visible (400 - 800 nm) Spectroscopy, IR Spectroscopy (0.76 - 15
ʅm)
2. Emission Spectroscopy:
• An analytical technique in which emission (of a particle or radiation) is dispersed
according to some property of the emission & the amount of dispersion is measured.
• e.g. Mass Spectroscopy
13
THE LAWS OF SPECTROPHOTOMETRY
There are two very important basic laws and a third one which is a combination of the two.
• LAMBERTS LAW – ABSORBANCE (A) proportional to the PATHLENGTH (l) of the absorbing medium.
• BEERS LAW - ABSORBANCE (A) proportional to the CONCENTRATION (c) of the sample.
• BEER- LAMBERT LAW - ABSORBANCE (A) proportional to c x l
A α cl
A = Ecl (A is a ratio and therefore has no units)
The constant E is called the MOLAR EXTINCTION COEFFICIENT
14
IMPORTANCE OF THE BEER LAMBERT LAW
A = Ecl but if E and l are constant
ABSORBANCE α CONCENTRATION and should be linear
relationship Prepare standards of the analyte to be quantified at
known concentrations and measure absorbance at a specified
wavelength. Prepare calibration curve.
From measuring absorbance of sample
Concentration of analyte in sample can be obtained from the
calibration curve
E can be obtained from the slope of the calibration curve for a
given wavelength (l)
15
PRINCIPLE OF UV
• UV absorption spectra arise from transition of electron or electron within a molecule or an ion
from a lower to higher electronic energy level and UV emission spectra arise from reverse type
of transition.
• The UV radiation region extends from 10 nm to 400 nm and the visible radiation region extends
from 400 nm to 800 nm.
• Near UV Region: 200 nm to 400 nm
• Far UV Region: below 200 nm
• Far UV spectroscopy is studied under vacuum condition.
• The common solvent used for preparing sample to be analyzed is either ethyl alcohol or
hexane.
16
INSTRUMENT
ATION
• Components of UV-Visible
spectrophotometer
• Source
• Filters & Monochromator
• Sample compartment
• Detector
• Recorder
17
APPLICATION
• Detection of Conjugation
• Detection of functional group
• Detection of impurities
• Detection of geometrical isomers.
18
INFRARED SPECTROSCOPY
19
PRINCIPLE
• Infrared spectroscopy (IR) measures the bond vibration frequencies in a molecule and
is used to determine the functional group.
• Just below red in the visible region
• Wavelengths usually 2.5-25 mm
• More common units are wavenumbers, or cm-1 , the reciprocal of the wavelength in
centimeters (104/mm = 4000-400 cm-1 )
• Wavenumbers are proportional to frequency and energy The IR Region
• The IR region is divided into three regions: the near, mid, and far IR. The mid IR
region is of greatest practical use to the organic chemistry
20
INSTRUMENTATION
1. Radiation source
2. Monochromator
3. Solvents, sample
cells, samples
4. Readout / Recorder
21
APPLICATION
• IR is used for determination of impurities.
• It is used for analysis of organic compounds
• IR is used for determination of molecular structure.
22
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
23
CHROMATOGRAPHY
• It is define as, it is analytical method in which separation of active constituent in complex mixture, and
the mixture was distributed in two phases i.e. stationary phase and mobile phase is known as
chromatography.
• The technique is used for separation, purification, Identification and extraction of compound.
• It is method it can consist of two phases stationary phase and mobile phase.
• Stationary phase is constant phase or column packaging material. Mobile phase is moveable phase.
• The basic principle of chromatography is based on Adsorption and partition chromatography.
- Adsorption chromatography - The affinity of molecules towards stationary phase is known as
Adsorption chromatography.
- Partition chromatography - The molecule can moves in two phases of liquid is known as partition
chromatography.
24
HPLC
 HPLC is a High Performance liquid Chromatography or High Pressure Liquid Chromatography or High
Priced Liquid Chromatography.
 It is column chromatography.
 It is Liquid Chromatography.
 It is modified from of gas chromatography, it is applicable for both Volatile as well as Non volatile
compound.
 It can mainly divided by two types 1. Normal phase HPLC 2. Reversed Phase HPLC.
 It is having a high resolution and separation capacity. It is used as qualitative as well as quantitative
analysis.
 High performance liquid chromatography (HPLC) is a chromatographic technique used to separate a
mixture of compounds in analytical chemistry and biochemistry with the purpose of identifying,
quantifying or purifying the individual components of the mixture.
25
PRINCIPLE
High Performance Liquid Chromatography [HPLC] is based on adsorption
as well as partition chromatography is depending on the nature of
stationary phase, if stationary phase is solid principle is based on
adsorption chromatography and if stationary phase is liquid principle is
based on partition chromatography.
 It is important for determination of volatile and non volatile compounds.
It is important for determination of Retention Time (the time is required ,
after sample injection maximum angle peak reaches to detector)
26
TYPES OF HPLC SEPARATIONS
1. Normal Phase: Separation of polar analytes by partitioning onto a polar, bonded
stationary phase.
2. Reversed Phase: Separation of non-polar analytes by partitioning onto a non-polar,
bonded stationary phase.
3. Adsorption: In Between Normal and Reversed. Separation of moderately polar analytes
using adsorption onto a pure stationary phase (e.g. alumina or silica)
4. Ion Chromatography: Separation of organic and inorganic ions by their partitioning
onto ionic stationary phases bonded to a solid support.
5. Size Exclusion Chromatography: Separation of large molecules based in the paths they
take through a “maze” of tunnels in the stationary phase.
27
INSTRUMENTATION
o Solvent storage bottle
o Gradient controller and mixing unit
o De-gassing of solvents
o Pump
o Pressure gauge
o Pre-column
o Sample introduction system
o Column
o Detector
o Recorder
28
DETECTORS
• Absorbance (UV/Vis and PDA)
• Refractive index (detects the change in turbidity)
• Fluorescence (if the analyte is fluorescent)
• Electrochemical (measures current flowing through a pair
of electrodes, on which a potential difference is imposed,
due to oxidation or reduction of solute)
• Conductivity (for ions)
• Light scattering
• Mass spectrometry (HPLC-MS)
29
Types of pumps used in HPLC
• DISPLACEMENT PUMPS
• RECIPROCATING PUMPS
• PNEUMATIC PUMP
HPLC Columns Used in Analysis
• Normal Phase Columns.
• Reverse Phase Columns.
• Ion Exchange Columns.
• Size Exclusion Columns
APPLICATION
o Drug Discovery
o Clinical Analysis
o Forensic Chemistry
o Drug Metabolism study
o Environmental chemistry
o Diagnostic studies
o Determination of Green Florescent Protein
o Structural Determination
30
THANK YOU
31

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Principle and instrumentation

  • 1. PRINCIPLE AND INSTRUMENTATION PRESENTED BY- MISS. SHIVANI JAGNNATH PATIL M.PHARM-I YEAR (PHARMACEUTICAL QUALITY ASSURANCE) 1
  • 3. FLAME PHOTOMETRY Introduction • Flame photometry is also known as Flame emission spectroscopy. • Flame photometry is based on measurement of intensity of light emitted when metal is introduced into flame.The wavelength of colour tells us what the element is and the colour intensity tells us how much of element is present. • A photoelectric flame photometer is an instrument used in inorganic chemical analysis to determine the concentration of certain metal ions among them sodium,potassium,calcium and lithium. 3
  • 4. PRINCIPLE - The basic principle upon which Atomic Spectroscopy works is based on the fact that "Matter absorbs light at the same wavelength at which it emits light". - The compounds of the alkali and alkaline earth metals (Group II) dissociate into atoms when introduced into the flame. Some of these atoms further get excited to even higher levels. But these atoms are not stable at higher levels.Hence, these atoms emit radiations when returning back to the ground state. These radiations generally lie in the visible region of the spectrum. Each of the alkali and alkaline earth metals has a specific wavelength. 4
  • 5. Element Emitted wavelength Flame color Sodium 589 nm Yellow Potassium 766 nm Violet Barium 554 nm Lime green Calcium 622 nm Orange Lithium 670 nm Red For certain concentration ranges, The intensity of the emission is directly proportional to the number of atoms returning to the ground state. And the light emitted is in turn proportional to the concentration of the sample. 5
  • 7. Parts of flame photometer A simple flame photometer consists of the following basic components: Source of flame: A Burner in the flame photometer is the source of flame. It can be maintained in at a constant temperature. The temperature of the flame is one of critical factors in flame photometry. Fuel-Oxidant mixture Temperature (°C) Natural gas-Air 1700 Propane-Air 1800 Hydrogen-Air 2000 Hydrogen-Oxygen 2650 Acetylene-Air 2300 Acetylene-Oxyen 3200 Acetylene-Nitrous oxide 2700 Cyanogen-Oxygen 4800 7
  • 8. Nebuliser: Nebuliser is used to send homogeneous solution into the flame at a balanced rate. Optical system: The optical system consists of convex mirror and convex lens. The convex mirror transmits the light emitted from the atoms. Convex mirror also helps to focus the emissions to the lens. The lens helps to focus the light on a point or slit. Simple colour filters: The reflections from the mirror pass through the slit and reach the filters. Filters will isolate the wavelength to be measured from that of irrelevant emissions. Photo-detector: The intensity of radiation emitted by the flame is measured by photo detector. Here the emitted radiation is converted to an electrical signal with the help of photo detector. These electrical signals are directly proportional to the intensity of light. 8
  • 9. Events occur in Flame: The oxidants in flame photometer are mainly air, oxygen or nitrous oxide. The temperature of the flame depends on the ratio of fuel and oxidant. The processes occurring during flame photometer analysis are summarized below: Desolvation: Desolvation involves drying a sample in a solution. The metal particles in the solvent are dehydrated by the flame and thus solvent is evaporated. Vaporization: The metal particles in the sample are also dehydrated. This also led to the evaporation of the the solvent. Atomization: Atomization is the separation of all atoms in a chemical substance. The metal ions in the sample are reduced to metal atoms by the flame. Excitation: The electrostatic force of attraction between the electrons and nucleus of the atom helps them to absorb a particular amount of energy. The atoms then jump to the higher energy state when excited. Emission: Since the higher energy state is unstable the atoms jump back to the ground state or low energy energy state to gain stability. This jumping of atoms emits radiation with characteristic wavelength. The radiation is measured by the photo detector. 9
  • 10. Applications of flame photometer 1.Flame photometer can be applied both for quantitative and qualitative analysis of elements. The radiations emitted by the flame photometer are characteristic to particular metal. Hence with the help of Flame photometer we can detect the presence of any specific element in the given sample. 2.The presence of some group II elements is critical for soil health. We can determine the presence of various alkali and alkaline earth metals in soil sample by conducting flame test and then the soil can be supplied with specific fertiliser. 3.The concentrations of Na+ and K+ ions are very important in the human body for conducting various metabolic functions. Their concentrations can be determined by diluting and aspirating blood serum sample into the flame. 4.Soft drinks, fruit juices and alcoholic beverages can also be analysed by using flame photometry to determine the concentrations of various metals and elements. 10
  • 12. ULTRAVIOLET SPECTROSCOPY Spectroscopy • It is the branch of science that deals with the study of interaction of matter with light. OR • It is the branch of science that deals with the study of interaction of electromagnetic radiation with matter. • The study of molecular or atomic structure of a substance by observation of its interaction with electromagnetic radiation • QUANTITATIVELY - For determining the amount of material in a sample • QUALITATIVELY – For identifying the chemical structure of a sample 12
  • 13. Principles of Spectroscopy • The principle is based on the measurement of spectrum of a sample containing atoms / molecules. • Spectrum is a graph of intensity of absorbed or emitted radiation by sample verses frequency (v) or wavelength (ʄ). • Spectrometer is an instrument design to measure the spectrum of a compound. 1. Absorption Spectroscopy: • An analytical technique which concerns with the measurement of absorption of electromagnetic radiation. • e.g. UV (185 - 400 nm) / Visible (400 - 800 nm) Spectroscopy, IR Spectroscopy (0.76 - 15 ʅm) 2. Emission Spectroscopy: • An analytical technique in which emission (of a particle or radiation) is dispersed according to some property of the emission & the amount of dispersion is measured. • e.g. Mass Spectroscopy 13
  • 14. THE LAWS OF SPECTROPHOTOMETRY There are two very important basic laws and a third one which is a combination of the two. • LAMBERTS LAW – ABSORBANCE (A) proportional to the PATHLENGTH (l) of the absorbing medium. • BEERS LAW - ABSORBANCE (A) proportional to the CONCENTRATION (c) of the sample. • BEER- LAMBERT LAW - ABSORBANCE (A) proportional to c x l A α cl A = Ecl (A is a ratio and therefore has no units) The constant E is called the MOLAR EXTINCTION COEFFICIENT 14
  • 15. IMPORTANCE OF THE BEER LAMBERT LAW A = Ecl but if E and l are constant ABSORBANCE α CONCENTRATION and should be linear relationship Prepare standards of the analyte to be quantified at known concentrations and measure absorbance at a specified wavelength. Prepare calibration curve. From measuring absorbance of sample Concentration of analyte in sample can be obtained from the calibration curve E can be obtained from the slope of the calibration curve for a given wavelength (l) 15
  • 16. PRINCIPLE OF UV • UV absorption spectra arise from transition of electron or electron within a molecule or an ion from a lower to higher electronic energy level and UV emission spectra arise from reverse type of transition. • The UV radiation region extends from 10 nm to 400 nm and the visible radiation region extends from 400 nm to 800 nm. • Near UV Region: 200 nm to 400 nm • Far UV Region: below 200 nm • Far UV spectroscopy is studied under vacuum condition. • The common solvent used for preparing sample to be analyzed is either ethyl alcohol or hexane. 16
  • 17. INSTRUMENT ATION • Components of UV-Visible spectrophotometer • Source • Filters & Monochromator • Sample compartment • Detector • Recorder 17
  • 18. APPLICATION • Detection of Conjugation • Detection of functional group • Detection of impurities • Detection of geometrical isomers. 18
  • 20. PRINCIPLE • Infrared spectroscopy (IR) measures the bond vibration frequencies in a molecule and is used to determine the functional group. • Just below red in the visible region • Wavelengths usually 2.5-25 mm • More common units are wavenumbers, or cm-1 , the reciprocal of the wavelength in centimeters (104/mm = 4000-400 cm-1 ) • Wavenumbers are proportional to frequency and energy The IR Region • The IR region is divided into three regions: the near, mid, and far IR. The mid IR region is of greatest practical use to the organic chemistry 20
  • 21. INSTRUMENTATION 1. Radiation source 2. Monochromator 3. Solvents, sample cells, samples 4. Readout / Recorder 21
  • 22. APPLICATION • IR is used for determination of impurities. • It is used for analysis of organic compounds • IR is used for determination of molecular structure. 22
  • 23. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY 23
  • 24. CHROMATOGRAPHY • It is define as, it is analytical method in which separation of active constituent in complex mixture, and the mixture was distributed in two phases i.e. stationary phase and mobile phase is known as chromatography. • The technique is used for separation, purification, Identification and extraction of compound. • It is method it can consist of two phases stationary phase and mobile phase. • Stationary phase is constant phase or column packaging material. Mobile phase is moveable phase. • The basic principle of chromatography is based on Adsorption and partition chromatography. - Adsorption chromatography - The affinity of molecules towards stationary phase is known as Adsorption chromatography. - Partition chromatography - The molecule can moves in two phases of liquid is known as partition chromatography. 24
  • 25. HPLC  HPLC is a High Performance liquid Chromatography or High Pressure Liquid Chromatography or High Priced Liquid Chromatography.  It is column chromatography.  It is Liquid Chromatography.  It is modified from of gas chromatography, it is applicable for both Volatile as well as Non volatile compound.  It can mainly divided by two types 1. Normal phase HPLC 2. Reversed Phase HPLC.  It is having a high resolution and separation capacity. It is used as qualitative as well as quantitative analysis.  High performance liquid chromatography (HPLC) is a chromatographic technique used to separate a mixture of compounds in analytical chemistry and biochemistry with the purpose of identifying, quantifying or purifying the individual components of the mixture. 25
  • 26. PRINCIPLE High Performance Liquid Chromatography [HPLC] is based on adsorption as well as partition chromatography is depending on the nature of stationary phase, if stationary phase is solid principle is based on adsorption chromatography and if stationary phase is liquid principle is based on partition chromatography.  It is important for determination of volatile and non volatile compounds. It is important for determination of Retention Time (the time is required , after sample injection maximum angle peak reaches to detector) 26
  • 27. TYPES OF HPLC SEPARATIONS 1. Normal Phase: Separation of polar analytes by partitioning onto a polar, bonded stationary phase. 2. Reversed Phase: Separation of non-polar analytes by partitioning onto a non-polar, bonded stationary phase. 3. Adsorption: In Between Normal and Reversed. Separation of moderately polar analytes using adsorption onto a pure stationary phase (e.g. alumina or silica) 4. Ion Chromatography: Separation of organic and inorganic ions by their partitioning onto ionic stationary phases bonded to a solid support. 5. Size Exclusion Chromatography: Separation of large molecules based in the paths they take through a “maze” of tunnels in the stationary phase. 27
  • 28. INSTRUMENTATION o Solvent storage bottle o Gradient controller and mixing unit o De-gassing of solvents o Pump o Pressure gauge o Pre-column o Sample introduction system o Column o Detector o Recorder 28
  • 29. DETECTORS • Absorbance (UV/Vis and PDA) • Refractive index (detects the change in turbidity) • Fluorescence (if the analyte is fluorescent) • Electrochemical (measures current flowing through a pair of electrodes, on which a potential difference is imposed, due to oxidation or reduction of solute) • Conductivity (for ions) • Light scattering • Mass spectrometry (HPLC-MS) 29 Types of pumps used in HPLC • DISPLACEMENT PUMPS • RECIPROCATING PUMPS • PNEUMATIC PUMP HPLC Columns Used in Analysis • Normal Phase Columns. • Reverse Phase Columns. • Ion Exchange Columns. • Size Exclusion Columns
  • 30. APPLICATION o Drug Discovery o Clinical Analysis o Forensic Chemistry o Drug Metabolism study o Environmental chemistry o Diagnostic studies o Determination of Green Florescent Protein o Structural Determination 30