MDM Flashcards
What is analytical spectroscopy?
Analytical spectroscopy is the science of determining how much of a substance is present by accurately measuring how much light is absorbed or emitted by atoms or molecules within it
What is an example of an instrument for analytical spectroscopy? What is its function?
Spectrophotometer - meausures the intensity of light absorbed by atoms or molecules
- can be double or single beam
What is needed for a spectrophotometer to function properly?
- A source that generates a broad band of electromagnetic radiation
- a dispersion device that selects from the broadband radiation of the source e.g a particular wavelength
- a sample area
- one or more detectors to measure the intensity of radiation
What are the 2 different light sources?
- Tungsten lamp 350 to 2000nm suitable for colourimetry - halogen light (VISIBLE LIGHT - ONES IN KITCHEN E.G)
- deuterium lamp 200 to 370nm, UV spectroscopy
both light sources need to provide a constant intensity at all wavelengths with low noise and long-term stability
What is a monochromator?
monochromator is either a prism (triagle light pass through gives u a rainbow) or diffraction gratings, most quantitative measurments light must be monochromatic
SNGLE WAVELENGTH E.G LAZER
monochromators contain
- an enterance slit
- collimating mirrow (lens)
- a dispersing device (prism or grating)
- a focusing mirror (or lens)
- an exit slit
What is diffraction grating?
It is where,
polychromatic radiation enters through enterance slit
the beam is collimated (makes radiation parallel before hitting dispersing element) and strikes dispersing element at an angle
beam is split into component wavelength by frating
by moving the dispersing element or exit slit, radiation of a particluar wavelength leaves
what is a detector
a detector converts a light signal into an electrical signal
it should give a linear response over a wide range with low noise and high sensitivity
several different types, but usually photomultiplier tube or a photodiode array
What is a photomultiplier tube?
Approx 1000V connected across 16 dynodes each more positive than the last
avalanche of electrons giving a measurable current for a few photons
How does it work?
Light enters the tube anstrikesthephoto emissivecathode, thisreleases electrons which are attracted to the dynode,
morectrons ae released which are then attracted to the next dynode which is held move positive than the last, process repeats and generates a cascade of electrons at the anode and amplifies the signal.
window allows light to pass you, dynodes are held as a positive charge. electrons stike dynode more electrons are generated when they are given off they are m0re attracted to the dynodes because there are more positively charge. at the end when they hit the anode, 1x10-7 electrons for every photon of light - causes an avalanche effect
What is a photodiode array?
Some modern spectrophotometers contain an array of photodiode detectors positioned side by side on a silicon crystal
- single beam instrument
- meausres whole spectrum simultaneously
useful for uv/vis absorption of sampes rapidly passing through a sample flow cell e.g HPLC or CE detector
- only reolution of 1nm
- need more detectors to improve resolution
Summary - examples of spectrophotometer, light source, monochromator, detector?
Spectrophotometer - measures the intesnity of light absorbed or emitted by atoms or molecules
Light source - Tungsten and deuterium
Monochromator - either prism or diffraction grating
detector - PMT or photodiode array detector
Which light sources are used in absorption spectroscopy and why?
- Hollow Cathode Lamp (HCL): These lamps are commonly used in atomic absorption spectroscopy (AAS). They contain a cathode made of the element of interest, and when an electric current passes through the lamp, it generates a characteristic emission spectrum for that element. HCLs are particularly useful for the analysis of trace metal ions.
- Flame Emission Source: Flame atomic emission spectroscopy (FAES) employs a flame, typically an air-acetylene flame or a nitrous oxide-acetylene flame, as the light source. The atoms in the sample are vaporized and excited in the flame, producing characteristic emission lines that are detected and used for qualitative and quantitative analysis.
- Inductively Coupled Plasma (ICP): ICP is a high-temperature ionization source used in inductively coupled plasma atomic emission spectroscopy (ICP-AES) and inductively coupled plasma mass spectrometry (ICP-MS). It produces a high-temperature, ionized argon plasma that excites and ionizes atoms, making them emit characteristic wavelengths of light.
- Continuum Sources: For techniques like atomic absorption spectroscopy (AAS) or atomic fluorescence spectroscopy (AFS), continuum sources such as deuterium lamps and xenon lamps are used to provide a broad spectrum of light. This light is then passed through a monochromator to select the desired wavelength for analysis.
- Laser Sources: Laser-induced breakdown spectroscopy (LIBS) and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) use lasers, often in the form of pulsed lasers, to ablate and atomize solid samples. The light emitted during the process contains information about the elemental composition.
- X-ray Sources: X-ray fluorescence spectroscopy (XRF) uses X-ray sources to excite inner-shell electrons of atoms, leading to characteristic X-ray emissions that are detected for elemental analysis.
Do you understand how a monochromator works?
A monochromator is an optical device used to isolate and select a single, narrow range of wavelengths (or colors) from a broader spectrum of light. It’s a crucial component in many analytical instruments, such as spectrophotometers and spectrometers, where the separation and analysis of light by its constituent wavelengths are required.
Entrance Slit: The monochromator starts with an entrance slit that allows the incoming light to enter. The light is typically a mixture of different wavelengths.
Collimating Optics: After passing through the entrance slit, the light is directed through collimating optics, which may include lenses or mirrors. The collimating optics serve to make the light rays parallel.
Dispersive Element: The collimated light is then directed toward a dispersive element, which is often a diffraction grating or a prism. The dispersive element separates the different wavelengths of light based on their angles of diffraction or dispersion.
Diffraction Grating: In a grating monochromator, a diffraction grating is used. The grating has closely spaced lines that act as a series of slits. When light passes through the grating, it is diffracted, and the different wavelengths are spread out at different angles.
Prism: In a prism monochromator, a prism is used to disperse the light. The different wavelengths of light are bent by different amounts as they pass through the prism, causing them to spread out.
Selection of Wavelength: To select a specific wavelength from the dispersed light, a movable slit or aperture is placed at the desired angle. By adjusting the position of this slit, only light of the chosen wavelength is allowed to pass through.
Exit Slit: The selected wavelength, now isolated, passes through an exit slit. The exit slit can be adjusted in width to control the bandwidth or resolution of the monochromator.
Detector: Finally, the isolated wavelength is directed towards a detector, which measures the intensity of the light. This detector is connected to the analytical instrument to record the data.
By adjusting the angle of the dispersive element or the position of the entrance and exit slits, a monochromator can select different wavelengths of light for analysis
Can you describe how a PMT works?
Approx 1000V connected across up to 16 dynodes, each diode is more positive than the last,
it is an avalanche of electrons giving a measurable current for a few photons
A Photomultiplier Tube (PMT) is a highly sensitive photon detector used to measure low levels of light in various applications, including scientific research, medical instruments, and analytical equipment. PMTs work on the principle of photoemission and electron multiplication. Here’s how a PMT works:
Photon Detection: When a photon of light enters the PMT, it strikes the photocathode, which is typically a photosensitive material (e.g., cesium-antimony compounds). The energy from the incoming photon is absorbed by the photocathode, causing the emission of an electron through the photoelectric effect. This process converts the photon’s energy into an electron with kinetic energy.
Electron Emission: The emitted electron is then accelerated and focused by a series of electrodes within the PMT. These electrodes are designed to provide a voltage gradient that causes the electron to move toward the next electrode, typically referred to as the “dynode.”
Electron Multiplication: As the accelerated electron strikes the first dynode, it releases additional secondary electrons through a process known as secondary emission. Each secondary electron is accelerated and strikes the next dynode, releasing even more electrons. This cascade effect continues as the electrons are multiplied at each dynode stage.
Sequential Dynodes: A typical PMT consists of multiple dynodes arranged in a sequence. The number of dynodes can vary depending on the PMT model, and each dynode stage provides electron multiplication. This cascade amplification results in a significant increase in the number of electrons produced.
Anode and Current Amplification: At the final dynode, a large number of electrons are collected and focused onto an anode. The anode generates an electric current that is directly proportional to the number of incident photons, making it a measure of the light intensity. This electric current can be read and processed by external electronics.
Output Signal: The amplified electric current is the output signal of the PMT, which can be further processed and analyzed by external equipment, such as amplifiers, data acquisition systems, or other detectors.
PMTs are highly efficient at converting photons into measurable electrical signals, making them valuable in applications that require sensitive detection of light, such as in fluorescence spectroscopy, scintillation counting, particle physics experiments, and more.
What are the differences between a diode array and a single beam spectrometer?
Design:
Single-Beam Spectrophotometer: This is the traditional type of spectrophotometer. It consists of a single optical path where light from the source passes through a sample, and the intensity of transmitted or reflected light is measured at a single fixed wavelength at a time.
Diode Array Spectrophotometer: Diode array spectrophotometers use an array of photodetectors, such as charge-coupled devices (CCDs) or complementary metal-oxide-semiconductor (CMOS) detectors, to simultaneously measure the entire spectrum of wavelengths. They capture multiple data points across a wide range of wavelengths simultaneously.
Speed:
Single-Beam Spectrophotometer: Single-beam instruments measure one wavelength at a time and require manual adjustments when scanning across different wavelengths. This can be time-consuming when obtaining a full spectrum.
Diode Array Spectrophotometer: Diode array spectrophotometers are much faster because they capture the entire spectrum in a single measurement, making them suitable for rapid scanning and kinetic studies.
Wavelength Range:
Single-Beam Spectrophotometer: Single-beam instruments are typically designed for specific wavelength ranges, and they require the manual insertion of different filters or gratings to change the wavelength.
Diode Array Spectrophotometer: Diode array spectrophotometers cover a wide range of wavelengths without the need for manual adjustments or changing components. They can be versatile for various applications.
Accuracy and Precision:
Single-Beam Spectrophotometer: Single-beam instruments can offer high accuracy and precision when used within their specified wavelength range.
Diode Array Spectrophotometer: Diode array spectrophotometers can also provide high accuracy and precision, and their ability to capture a full spectrum in a single measurement can reduce potential errors associated with wavelength changes.
Applications:
Single-Beam Spectrophotometer: These are suitable for routine measurements at specific wavelengths, such as absorbance at a fixed wavelength. They are commonly used in teaching laboratories and for simple applications.
Diode Array Spectrophotometer: Diode array instruments are versatile and suitable for a wide range of applications, including quantitative analysis, kinetics, and applications where a full UV-Visible spectrum is needed.
In summary, diode array spectrophotometers offer the advantage of speed and the ability to capture full spectra simultaneously, making them more versatile and efficient for many applications. Single-beam spectrophotometers are simpler in design and are suitable for basic measurements at a single wavelength or when a full spectrum is not required. The choice between the two depends on the specific needs of the analysis and the available budget.
Why does polychromatic light passing through a substance decrease?
- Reflection at phase boundaries - i.e. liquid/air, glass/liquid etc. This is caused by differences in the refractive index of the different materials through which the light is passing
- scattering of light caused by non-homogeneity of the sample
-absorbance by atoms or molecules in solution
What wavelength would you select?
Scan sample over region of interest and not wavelength at highest absorption
also use Brittish pharmacopeia to find max wavelength of a particluar drug
gives greatest sensitivity and peaks tend to be fairly flat depending on sample
What molecular orbitals are in single and double bonds?
Single and electrons
moelcular orbitals called sigma orbitals and sigma electrons
double bonds - 2 types
sigma orbital = pair of bonding electrons
and a pi
What are the energy levels? which transition will give an absorbance oeak between 200-750nm?
- bonding sigma
- bonding pi
- non bonding n
- antibonding pi star
5 antibonding sigma star
n to pi star and pi to pi star transitions will generate an absorption peak between 200-750nm
What is the equation for change in energy? what is the theory for energy and wavelength
chnage in energy = h x v
change in energy = h x c/wavelength
larger the energy the lower the waveelngth required
What is the A11 equation and what is A11?
£=A11 x Mr /10
A11 value is useful in pharmaceutical analysis where the molecular weight of the sample is unknown
- e.g when analysing a protein or DNA or
- where a mixture of several components is being analysed in the same sample ( ointment or suspension)
explain Beers law?
Beer’s Law, also known as the Beer-Lambert Law or Beer’s Law of Absorption, is a fundamental principle in analytical chemistry used to relate the concentration of a solute in a solution to the absorbance of light by that solution. It is a vital tool for quantitative analysis, particularly in spectrophotometry.
The basic equation for Beer’s Law is:
A=ε⋅b⋅c
Where:
A is the absorbance of the solution.
ε (epsilon) is the molar absorptivity or molar absorptivity coefficient (also called molar extinction coefficient), which is a constant specific to the substance being analyzed.
b is the path length of the sample through which the light passes (typically measured in centimeters).
c is the concentration of the solute in the solution, usually expressed in molarity (moles per liter).
Key points about Beer’s Law:
Direct Proportionality: Beer’s Law states that absorbance (A) is directly proportional to both the concentration of the solute (c) and the path length (b) of the sample. In other words, as the concentration or path length increases, so does the absorbance.
Molar Absorptivity (
ε): The molar absorptivity is a constant for a given substance at a specific wavelength of light. It depends on the nature of the solute, the solvent, and the wavelength of the incident light. It is a measure of how strongly the substance absorbs light at that wavelength.
Linearity: Beer’s Law holds true as long as the concentration range is within a linear region. Outside of this range, deviations may occur due to factors like non-linearity or the saturation of the absorbance.
Wavelength Specific: Beer’s Law is specific to a particular wavelength of light. The choice of wavelength depends on the absorption characteristics of the substance being analyzed.
Applications: Beer’s Law is widely used in spectrophotometry and colorimetry for quantitative analysis of various substances, including determining the concentration of analytes in solutions, such as in chemistry, biology, environmental science, and pharmaceuticals.
By measuring the absorbance of a sample and knowing the molar absorptivity and path length, you can use Beer’s Law to calculate the concentration of the solute in the solution. It’s a powerful tool for quantitative analysis in various scientific and industrial applications.
beers law with concentration
Low concentration directly proportional
high concentration curve
beers law describes the absorption behaviour of dilute solutions only
below 10mM conc effects not usually observed
above 10mM the close distance between absorbing molecules can alter ability to absorb a given wavelength
What can landa max show?
identification of drugs and unknown compounds
characteristic of a particular chromophore
should remain constant
be aware that when its ionised the position of landa max can change
What happens when the landa max becomes longer?
A shift in landa max towards longer wavelength is referred as a bathochromatic or RED shift
Occurs due to an action of an auxochrome
- this is a functional group attatched to a chromophore which does not absorb light energy itself, but influences the wavelengths of light absorbed by the chromophore
- examles include NH2, OH and SH groups
- possess long pairs of non-bonded electrons which can interact with the pi electrons of the chromophore and allow light of longer wavelength to eb absorbed.
What happens to aniline in acidic and alkaline conditions?
NH2 is an auxochrome
In pH>7 aniline is a base which is not ionised.
the landa max for benzene is 204nm compared to aniline 230nm. Due t the lone pair of electrons of NH2 interacting with the ring electrons it inc the electron density throughout the ring
In pH < 7 aniline in acidic solution reacts to form anilinium salt
lone pairs of electrons is now involved in bond formation to an H+ ion
no longer acts as a auxochroms
landa max returns to value of benzene
landa max =203nm
What is a hypsochromic effect?
also known as a blue shift is when a shift in landa max to a shorter wavelength.
Occurs when compounds with a basic auxochrome ionise and the lone pair is no longer able to interact with the electrons of the chromophore
- also been seen when different solvents or at elevated temperatures
- useful to identify amines, R_NH2
What is a bathochromic effect?
usually associated with increases in light absorption (hyperchromic effect)
What is a hypsochromic effect?
occurs with a decrease in absorbance (hypochromic effecect)
What do weak acids do to ionisation, give an example and what are the effects?
Example - Phenols - paracetamol (weak acids)
Ionisation causes;
an increase in the intensity of light absorption and
the position of landa max moves to longer wavelength
Effects
due to ionisationa nd loss of H atom as an H+ ion
- results in full negative charge on oxygen
- Interacts more effectively with ring than lone pair of electrons present in unionised form
What are the two methods of drug assay?
Comparative US
- a test/ a std
- advantage can be used if the drug undergoes a chemical reaction during assay
- disadv an authentic sampke of the drug in question must be available for comparison
Absolute EU
- absorbance meausred and BL equation used to calculate drug
- used in uk and in europe
How do you know if Beer’s law is obeyed?
when there is a colomn of numbers in an exam question - ALWAYS plot a graph - conc at bottom absorbance on the side
when absorbance is v concentration is plotted and a straight line is observed we can say beers law is obeyed for this drug at this particular wavelength.
How is A11 and E (molar absorbidity) calculated
Can be read of graph is axis are in %w/v or calculated using each data point and converting them into conc of g/ and taking average A=A11 x c x l
remember the concentration e.g if its 1%w (mg)/v you need to find conc 0.001 divided by 100 = 0.001%w/v
Molar absorbidity
A = E x c x l
c 1mg per 100ml = 0.01gL
What type of bonds will produce absorption in the UV and visible region?
Absorption of ultraviolet (UV) and visible light in molecules is primarily associated with electronic transitions within the molecule. The types of chemical bonds and electronic structures that can produce absorption in the UV and visible region are as follows:
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π-
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∗
π
∗
Transitions: These transitions involve the movement of electrons from a lower-energy
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π (pi) orbital to a higher-energy
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∗
π
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(pi-star) anti-bonding orbital. This type of transition is commonly associated with conjugated systems, such as those found in organic compounds like conjugated dienes and polyenes.
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π-
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∗
π
∗
transitions often result in absorption in the UV or visible region.
n-
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∗
π
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Transitions: These transitions involve the movement of electrons from a non-bonding (n) orbital to a higher-energy
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∗
π
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anti-bonding orbital. Molecules with lone pairs of electrons, like carbonyl compounds (e.g., ketones and aldehydes), can exhibit n-
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∗
π
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transitions that result in UV or visible absorption.
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σ-
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∗
σ
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Transitions:
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σ-
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∗
σ
∗
transitions involve the movement of electrons from a lower-energy
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σ (sigma) bonding orbital to a higher-energy
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∗
σ
∗
(sigma-star) anti-bonding orbital. These transitions are less common and typically occur at shorter wavelengths in the vacuum UV region.
Metal-Ligand Transitions: In coordination compounds, transition metals can absorb visible light through metal-ligand charge transfer (MLCT) or ligand-field transitions. The specific transitions depend on the metal, ligands, and the coordination complex’s structure.
Conjugation: Extended conjugation of double bonds or delocalized
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π electrons in a molecule can lead to absorption in the UV or visible region. Examples include aromatic compounds like benzene, which absorb in the UV region due to the resonance-stabilized
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π electrons in the ring.
Halogenation: Halogens, such as chlorine, bromine, and iodine, in organic compounds can lead to UV absorption due to their lone pair electrons participating in n-
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∗
π
∗
transitions. Halogenated organic compounds can show UV absorption bands.
Color Centers: In ionic solids, defects or impurities can create color centers. These arise from electron transitions associated with defects in the crystal lattice and can produce visible absorption.
What effect does have on different functional groups to its spectrum?
The pH of a solution can significantly affect the absorption spectrum of molecules with specific functional groups, particularly in the UV and visible region. Here are some examples of how pH can influence the absorption spectrum of different functional groups:
Amino Groups (NH2):
In a basic (high pH) solution, amino groups (-NH2) can become deprotonated, forming amine ions (-NH2⁻). This can shift the absorption spectrum to longer wavelengths, resulting in a bathochromic shift (redshift). This effect is observed in amino acids and amines.
In an acidic (low pH) solution, amino groups remain protonated, resulting in shorter-wavelength absorption and a hypsochromic shift (blueshift).
Carboxyl Groups (COOH):
In a basic solution, carboxyl groups (-COOH) can lose a proton, forming carboxylate ions (-COO⁻), leading to a bathochromic shift.
In an acidic solution, carboxyl groups remain protonated, resulting in a hypsochromic shift.
Phenolic Groups (ArOH):
In a basic solution, phenolic groups can lose a proton, forming phenolate ions (ArO⁻), which leads to a bathochromic shift.
In an acidic solution, phenolic groups remain protonated, resulting in a hypsochromic shift.
Imine Groups (C=N):
In a basic solution, imine groups can become deprotonated, forming imine ions (C=N⁻), leading to a bathochromic shift.
In an acidic solution, imine groups remain protonated, resulting in a hypsochromic shift.
Quinone Groups (C=O, C=O):
The absorption spectrum of quinone groups can be influenced by pH. Changes in pH can affect the equilibrium between the keto and enol tautomers, resulting in shifts in the absorption spectrum. These shifts can be bathochromic or hypsochromic.
Chromophores in Dyes and Indicators:
Many synthetic dyes and pH indicators contain specific functional groups that exhibit pH-dependent color changes due to changes in their electronic structure. For example, litmus paper changes color in response to pH changes.
