Flame Photometry and Spectroscopic Techniques Notes

Flame Photometry (FAES)

Flame Atomic Emission Spectroscopy (FAES) is an analytical technique for determining the concentration of specific chemical elements in a sample.

Components of Flame Photometry

1. Source of flame

2. Nebulizer

3. Optical System

4. Filter

5. Photon detector

6. Compressed fuel supply

Working Principle of Flame Photometry

A constant source of flame is required. Sources include gases and oxygen (air and fuel, respectively).

Types of Flame Sources

• Total consumption sources: The entire sample is consumed. Disadvantage: flame imbalance.

• Laminar flow (premixed): Uses a mixture of the sample, hydrogen, and oxygen. The sample is introduced into the flame and burns uniformly. Disadvantage: reflections from the convex mirror.

Nebulizer

Introduces a homogenous solution to the flame (though it is not an active component of the Flame Photometer).

• Laminar flow prevents a source of homogenous solution to the flame

Optical System

Consists of convex mirrors and lenses.

• The convex mirror transmits emitted radiation from the atom and focuses it on the lens.

• The convex lens focuses the emitted light on the filter.

Filter

Separates a particular element's atom to be measured at a specific wavelength, leaving metal salts and elements of interest respectively.

A simple prism or interference flame photometer is made up of a filter wheel into which filters are inserted to measure the intensity of radiation at a certain wavelength.

Photon Detector (LCD)

Liquid Crystal Detector measures the intensity of radiated light. This measured radiation lies within the visible region of the spectrum. Conventional Photodetector functions as a photovoltaic cell or phototube.

Operational Protocol of the Flame Photometer

1. Prepare standard working solutions (containing standard metal salts and elements of interest) and freshly prepare in distilled water.

2. Calibrate the flame photometer by ensuring the air and fuel supply are adequate and stable for 5 minutes

3. Switch on the instrument, lift the lid of the filter chamber, and insert the appropriate color filter.

4. Adjust the instrument by spraying the highest standard.

5. Adjust the flame photometer's sensitivity by spraying the highest standard working solution into the flame; record the maximum deflection.

6. Spray other standard solutions directly into the flame; record galvanometer readings.
   Note: After spraying each standard solution, wash the apparatus adequately before spraying the next standard solution.

7. After spraying the highest standard, spray the flame with deionized or distilled water and adjust it to zero.

8. Spray the flame with the unknown sample, take galvanometer readings, and wash the apparatus.

Calculate the mean of the galvanometer readings for both standard and specimen samples.

Plot a graph of the mean (from galvanometer readings) against concentration.

Extrapolate the concentration of the unknown sample from the graph.

Applications

1. Quantification and qualification of elements.

2. Detection of alkali and alkali earth metals in soil samples.

3. Detection of Sodium and Potassium concentration in blood samples.

4. Determination of the presence of elements in beverages and alcohol.

5. Calcium and magnesium concentration in cement.

6. Nickel and Lead concentration in petroleum products.

Advantages

1. Simple and economical method.

2. Quick, sensitive, and selective.

3. Allows for quantitative and qualitative analysis of smaller samples

Disadvantages

1. Analyses are sometimes inaccurate.

2. Only liquid samples can be analyzed.

3. Most elements cannot be analyzed.

Circular Dichroism (CD)

Circular Dichroism is an absorption spectroscopy method based on the differential absorption of Left-Circularly Polarized Light (L-CPL) and Right-Circularly Polarized Light (R-CPL).

In this method, absorption of light and other electromagnetic radiation by the substance varies depending on wavelength and polarization.

Brief History

The concept was first observed in the 19th century by French physicist Jean Baptiste Biot, known for his work on optical activity. Quantitative measurement became feasible with advancements in instrumentation in the 20th century.

Principles

Chirality and Optical Activity

Chiral molecules are not superimposable on their mirror images. They rotate the plane of polarized light.

Differential Absorption

$A = \varepsilon cl$

• $A$ = Absorbance

• $\varepsilon$ = Molar absorptivity (L/mol.cm)

• $c$ = Concentration of absorbing species (mol/L)

• $l$ = Path length (cm)

Circular Dichroism is measured using a Circular Dichroism Spectrophotometer.

The difference in molar absorptivity is given by:

$\Delta \varepsilon = \varepsilon<em>{L} - \varepsilon</em>{R}$

$\Delta A = A<em>{L} - A</em>{R}$

$\Delta A = (\varepsilon<em>{L} - \varepsilon</em>{R}) c l$

Circular Dichroism Spectrophotometer

Components and Working Principle

1. Light Source: A lamp produces a broad spectrum of light.

2. Monochromator: Selects a specific wavelength from the light source.

3. Polarizer: Converts the light into linearly polarized light.

4. Photoelastic Modulator (PEM): Rapidly switches the linearly polarized light to L-CPL and R-CPL, which passes through the sample.

5. Detector: Measures the intensity of the transmitted light, capturing differences in absorption.

The sample is placed in a quartz cuvette.

The information from the Detector is used to create a CD spectrum, which plots the difference in absorption ($\Delta \varepsilon$) against wavelength.

Applications

1. Determination and analysis of protein secondary structure and folding.

2. Determination of Nucleic Acid Conformation.

3. Study of interactions such as Protein-Ligand interactions and DNA-Drug interactions.

Advantages

1. Allows for rapid analysis.

2. Non-destructive and requires minimal sample.

3. Sensitive to international changes.

Disadvantages

1. Expensive instrumental requirement.

2. Requires relatively high concentration of sample.

3. Interpretation is usually complex.

Optical Rotatory Dispersion (ORD)

Optical Rotatory Dispersion is the variation in the plane of polarized light of a substance due to a change in wavelength.

The degree of rotation depends on the wavelengths of light used.

Specific Rotation

It is the observed angle of rotation ($\alpha$) of plane-polarized light at a specific wavelength ($\lambda$).

$[\alpha]_{\lambda} = \frac{\alpha}{l \cdot c}$

• $[\alpha]_{\lambda}$ = Specific rotation at wavelength $\lambda$

• $\alpha$ = Angle of rotation

• $l$ = Path length (dm)

• $c$ = Concentration (g/mL)

Principles

Linearly Polarized Light

Optical Activity

Specific Rotation

Circular Birefringence

This refers to the phenomenon where a chiral substance causes rotation of plane-polarized light.

ORD traces its origin back to the early 19th Century when Jean Baptiste Biot discovered Optical Activity.

Spectropolarimeter

Optical Rotatory Dispersion is measured using a Spectropolarimeter.

Working Principle

1. Unpolarized Light Source: Emits light of several wavelengths.

2. Monochromator: Isolates a particular wavelength and transmits it to the Nicol Prism

3. Nicol Prism: Turns light into linearly polarized light.

4. Sample: The linearly polarized light then passes through the sample (chiral molecules), which changes the plane of polarization to the right (dextrorotatory, +) or left (levorotatory, -).

5. Analyzer and Detector

The analyzer measures the angle of rotation, while the detector measures the intensity of light.

An ORD curve is plotted by plotting specific rotation ($[\alpha]_{\lambda}$) against wavelengths ($\lambda$).

Application

1. Determination of Enantiomeric Purity

2. Structural analysis of Chiral Molecules

3. Analysis of Biomolecules (Enantiomeric Purity and Composition)

Advantages

1. Non-Destructive

2. Quantitative analysis

3. Sensitivity to Chiral Centers

Disadvantages

1. Sensitivity to impurities

2. Limited structural detail

3. Cases Complexity of Interpretation

Polarimetry

Polarimetry is a method in biochemistry used to measure the rotatory forces of optically active molecules.

Principle

Optical rotation is dependent on:

1. Number of molecules on the path of the electromagnetic radiation

2. Concentration

3. Length of the containing vessel

4. Wavelength

5. Temperature

The wavelength at which the measurement is absorbed is specifically the Sodium D-line with a wavelength of 589 nm

The polarimeter measures the plane of polarized light when Optive electromagnetic radiation is passed through an optically active molecule.

Components of Polarimeters

1. Unpolarized Light Source: Includes sunlight or flames etc.
   In Polarimetry, a Sodium Lamp at a wavelength of 589 nm is used as a Source of Light.

2. Sample Cell: A scent for containing the Optically active molecule which rotates the plane of Polarized light.

3. Fixed Polarizer: Consists of a Nicol Prism made up of calcite. Converts the unpolarized light into a Linear Polarized signal.

4. Movable Polarizer (Analyzer): It is also a Nichol Prism made up of Calcite. It measures the rotatory perversity as an angle of rotation of the molecule of interest.

5. Detector (or Eye): Captures the signal of rotation of the Optically active molecule.

The polarimeter consists of 2 Nichel Prisms:

• One is the Polarizer

• The other is the Analyzer

There are 2 types of Rotation:

• Clockwise / Dextrorotatory rotation

• Anticlockwise / Levorotatory rotation.

Application

1. It is used to identify transformed Optically active molecules

2. It can determine an unknown molecule through the angle of rotation

3. It is used to analyze enzymes and antibiotics.

4. It is used to determine the Vanity, Purity and Concentration in Food, Beverages and Pharmaceuticals

Spectrophotometry

Spectrophotometry is an analytical technique used to measure the amount of light absorbed by a sample.

Principle

The principle is a very crucial technique in biochemistry in determining how matter behaves when exposed to light energy.

Components

1. Light Source: Tungsten or Halogen bulb

2. Collimator: Focuses the light on the Monochromator.

3. Monochromator: Disperses or isolates a particular wavelength.

4. Wavelength Selector

5. Cuvette or Sample Container: Made of Quartz, Plastic or glass

6. Photocell: Designed to pick up electron trays/signals and transmit them to the meter bron

7. Meter: Reader of the signals

Spectrophotometer Info

Spectrophotometer Tells how compound behaves

Nature of Compounds

Nature of a compound varies depending on the concentration of a solution and standard path length through which the light passes.

Electromagnetic Spectrum

• UVA = 320 - 400nm

• UVB = 280-320nm

• UVC = 100 - 280nm

• Visible Region = 400 nm - 700 nm

• Infrared $\lambda$ = 700 - 120 nm

Beer-Lambert Law

$A = \varepsilon cl$

$A = \log<em>{10} (I</em>0/I)$

• $A$ = Absorbance

• $\varepsilon$ = Molar absorptivity (L/mol·cm)

• $c$ = Concentration of absorbing species (mol/L)

• $l$ = Path length (cm)

• $I_{0}$ = Incident light

• $I$ = Transmitted light

The amount of Light absorbed by a Substance is directly proportional to its concentration.

Relationships

• If concentration C is in M, then $\alpha$ = molar absorption

• If concentration C is in g/L, then $\alpha$ = specific absorption coefficient

• Thickness/paths lengths of sample container or cuvette is always 1 cm.

• Ratio of Incident ray (I0) to Transmitted Ray (I) = Optical density & Absorbance

Example Calculations

A solution of Naphthalene contained in a 1cm cuvette transmits 63% of the incident light (I0 of 260 nm). Calculate:

1. The transmission ($I$) of

   • 3.2 g/L

   • 4.0 g/L

   • 1.8 g/L

2. If $a_{1\%}^{1 cm}$ is 1572, calculate the molecular weight of the compound

Solutions

Initial Steps

• Initial ray ($I_0$) for a starting solution = 100 %

• Percentage Volume (%V) = 15.72

• Thickness/paths lengths of sample container oscuvette is always 1 cm

Part A - Calculation of Absorbance for 3.2 g/L

$\log(I_0/I) = a c l$
$0.201 = 2.4 abs^{-1} g^{-1} L cm^{-1} \times 3.2 g/L \times 1 cm$
$I = 54 \%$

Part B - Calculation of Absorbance for 4.0 g/L

$\log(I_0/I) = a c l$
$\log(100/I) =0.084 abs^{-1} g^{-1} L cm^{-1} \times 4.0 g/L \times 1 cm$
$I = 46 \%$

Part C - Calculation of Absorbance for 1.8 g/L

$\log(I_0/I) = a c l$
$\log(100/I) =0.084 abs^{-1} g^{-1} L cm^{-1} \times 1.8 g/L \times 1 cm$
$I = 71 \%$

Part D - Molecular Weight Determination

$a<em>M = a</em>{1}^{1} \times molecular weight$
$MW = 187 g/mol$

Applications of Spectrophotometry

1. Qualitative analysis: Used for the identification of substances based on its absorption or transmission spectra.

2. Molecular weight determination

3. Control of purity: By assessing the purity of all substances by brand saying comparing the absorption rate of a sample with that of a standard

Fluorimetry (or Spectrofluorometry)

Fluorimetry is a type of electromagnetic spectroscopy that analyzes the fluorescence from a sample. Fluorescence is the ability of a substance to give out energy in the form of light due to the presence of a Fluorophore (fluorescent molecules).

Principle of Fluorimetry

Based on fluorescence, a molecule absorbs light at a certain wavelength, becomes excited, and moves from its ground state to a higher-energy state. Due to the instability of molecules at this new higher-energy state, then drops to lower - stable ground rotating / reading enery with the in forma of light

Spectrofluorometer Components & Principle

1. Light source: Xenon Bulb Provides the light needed for excitation

2. Excitation monochromator: It receives the light from Xenon bulb-supplies to source to excited of excite the sample by sending light

3. Slit By adjusting the slit width, the intensity of light reaching the sample can be controlled Narrower slits reduce its increased

4. Sample holder (cuvette) Contains the sample. Only two sides of the cuvette are transparent.

5. Emission monochromator: Receives the specific wavelength of emitted sends it to the Photocell

6. Photocell. Convert the light photon from receives the of the monochromator its electrical signal meter

Important Terms and Formulae

1. Quantum Yield ($\phi$): Also known as efficiency of fluorescence.

2. Limit of Detection (LOD).

3. Inner Filter effect

Formulae

• Quantum Yield ($\phi$):
  $\varphi = \frac{\text{Number of Emitted Photons}}{\text{Number of Absorbed Photons}}$

• Limit of Detection (LOD):

$\text{LOD} = \frac{3 \sigma}{S}$

where:
$\sigma$ = Standard deviation of the blank measurements
$S$ = Slope of the calibration curve (Signal versus Concentration)

Inner Filter Effect

Occurs when high concentrations of the fluorescent species absorb a significant portion of the excitation & emission light leading to deviations from Linearity

Corrected by formula for Inner Filter effect.

Fluorimetry Corrections and Intensity

• Quantum Yield

$I = I_0 c \epsilon$

• $I$ = Fluorescence Intensity

• $L_0$ = Intensity of excitation light

• $c$ = Concentration of fluorescent species (µm)

• $\epsilon$ = Molar absorptivity of fluorescent species at excitation wavelength (L·mol-1·cm-1)

• $l$ = Path length of the cuvette containing the sample (1 cm)

Inner Filter effect.

Applications of Fluorimetry

1. In the Study of structure and function of

2. Detection of pollutants

3. Chemical analysis

4. Clinical Diagnostics

PH Meter

Principle of PH Measurement

PH measurement is a potentiometric measurement ie is potential is directly proportional to pH and the reference electrode whose potential is independent of PH and the liquid to be measured.

Components of a PH meter

1. An indicating electrode references

2. Reference electrode

Working Principle

Before using the PH meter, it is calibrated with standard buffer solutions of known pH values.

The pH electrode and reference electrode are immersed in the solution whose pH is to be determined.

The pH meter measures the voltage difference between the two electrodes (pH electrode and the reference electrode)

The potential of the measuring electrode (pH or indicating electrode) depends only on the pH of the solution, while the potential of the reference electrode is unaffected by the pH, so it provides a stable reference voltage.

Electrical Potentials and Equations

The relationship between measured cell voltage (mV), pH, and temperature (C) is defined by the Nernst equation:

$E = E_0 - \frac{2.3RT}{nF} pH$
Where:

• $E$ = Cell voltage (potential)

• $E_0$ = Standard electrode potential

• $R$ = Universal gas constant (8.314 J/mol·K)

• $T$ = Temperature in Kelvin

• $n$ = Number of moles of electrons transferred in the cell reaction

• $F$ = Faraday constant (96485 C/mol)

$E = y_o \times\times$ (xaxis)

Assuming temperature remains constant, any change in cell voltage is caused directly by changes in the pH of the sample.

The slope factor at 25°C has a value of 59.15mV (Ideal slope). Electrical constant of the sample voltage.

The slope is determined through constant slope with the electric conductivity. The slope electrical conductivity of the pH of the sample

For a unit change in pH, an ideal measuring system will evidence a change of 51.96 mV

This is used in measurement. Of slope electrical constant performance of indicated in by

How does the PH meter measure p given solution.

measure pit of given solution and indication it

Working Principle Details

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Colometry and Heat transfer

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• Change in terms

Types if Heat transfer

Bomb calorimetry
Thermal intitation calorimetry
Adiabatic calorimetry

Calormetery is the

measurement of Phase transitions such as Glass transitions or for heat great in that sensitivity. the operation of such transition heat
heat.
Phase transfers more

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mass the the any the need counter

to diff the molecules and enthalpy the

$A = \log_{10} (I*0/I)$ - $A$ = Absorbance - $\varepsilon$ = Molar

wavelength of emitted sends it to the Photocell 6. **Photocell.** Convert the light photon from receives the of the monochromator its electrical signal meter ### Important Terms and Formulae 1. **Quantum Yield ($\phi$):** Also known as efficiency of fluorescence. 2. **Limit of Detection (LOD)**. 3. **Inner Filter effect** #### Formulae - **Quantum Yield ($\phi$):**
$\varphi = \frac{\text{Number of Emitted Photons}}{\text{Number of Absorbed Photons}}$$- **Limit of Detection (LOD):**$\text{LOD} = \frac{3 \sigma}{S}$where:<br>$\sigma$= Standard deviation of the blank measurements<br>$S = Slope of the calibration curve (Signal versus Concentration) #### Inner Filter Effect Occurs when high concentrations of the fluorescent species absorb a significant portion of the excitation & emission light leading to deviations from Linearity Corrected by formula for Inner Filter effect. ### Fluorimetry Corrections and Intensity - Quantum Yield I = I_0 c \epsilon$-$I$= Fluorescence Intensity -$L\_0$= Intensity of excitation light -$c$= Concentration of fluorescent species (µm) -$\epsilon$= Molar absorptivity of fluorescent species at excitation wavelength (L·mol-1·cm-1) -$l = Path length of the cuvette containing the sample (1 cm) Inner Filter effect. ### Applications of Fluorimetry 1. In the Study of structure and function of 2. Detection of pollutants 3. Chemical analysis 4. Clinical Diagnostics ## PH Meter ### Principle of PH Measurement PH measurement is a potentiometric measurement ie is potential is directly proportional to pH and the reference electrode whose potential is independent of PH and the liquid to be measured. ### Components of a PH meter 1. An indicating electrode references 2. Reference electrode ### Working Principle Before using the PH meter, it is calibrated with standard buffer solutions of known pH values. The pH electrode and reference electrode are immersed in the solution whose pH is to be determined. The pH meter measures the voltage difference between the two electrodes (pH electrode and the reference electrode) The potential of the measuring electrode (pH or indicating electrode) depends only on the pH of the solution, while the potential of the reference electrode is unaffected by the pH, so it provides a stable reference voltage. ### Electrical Potentials and Equations The relationship between measured cell voltage (mV), pH, and temperature (C) is defined by the Nernst equation: E = E_0 - \frac{2.3RT}{nF} pH$<br>Where: -$E$= Cell voltage (potential) -$E_0$= Standard electrode potential -$R$= Universal gas constant (8.314 J/mol·K) -$T$= Temperature in Kelvin -$n$= Number of moles of electrons transferred in the cell reaction -$F$= Faraday constant (96485 C/mol)$E = y_o \times\times$$ (xaxis) Assuming temperature remains constant, any change in cell voltage is caused directly by changes in the pH