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Questions and Answers
What is the main focus of spectroscopy in biology?
What is the main focus of spectroscopy in biology?
Which of the following factors can cause alterations in the results of spectroscopy?
Which of the following factors can cause alterations in the results of spectroscopy?
The Lambert Beer Law is primarily used to relate which of the following?
The Lambert Beer Law is primarily used to relate which of the following?
What does spectroscopy help to determine about biomolecules?
What does spectroscopy help to determine about biomolecules?
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What aspect of light does spectroscopy primarily investigate?
What aspect of light does spectroscopy primarily investigate?
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What type of light is commonly used for measuring molecular presence using spectroscopy?
What type of light is commonly used for measuring molecular presence using spectroscopy?
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Which part of the spectroscopy process involves the measurement of light that is not absorbed by the sample?
Which part of the spectroscopy process involves the measurement of light that is not absorbed by the sample?
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In infrared spectroscopy, what is measured on the y-axis of the output graph?
In infrared spectroscopy, what is measured on the y-axis of the output graph?
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What does the presence of light photons that are not absorbed indicate in a spectroscopy experiment?
What does the presence of light photons that are not absorbed indicate in a spectroscopy experiment?
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What do the microwaves in spectroscopy primarily help to measure?
What do the microwaves in spectroscopy primarily help to measure?
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What is the relationship between frequency (f) and the period (T) of a wave?
What is the relationship between frequency (f) and the period (T) of a wave?
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What is the unit of frequency in the context of waves?
What is the unit of frequency in the context of waves?
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If a wave's speed is represented as 'v', how is it related to frequency and wavelength?
If a wave's speed is represented as 'v', how is it related to frequency and wavelength?
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What does the term 'wavelength' refer to in wave mechanics?
What does the term 'wavelength' refer to in wave mechanics?
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How is the period (T) of a wave defined in relation to wavelength and speed?
How is the period (T) of a wave defined in relation to wavelength and speed?
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What do the bands in a spectrum represent?
What do the bands in a spectrum represent?
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What is the primary role of a spectroscopist?
What is the primary role of a spectroscopist?
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Which physical property can NOT be inferred from a spectrum?
Which physical property can NOT be inferred from a spectrum?
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What describes the nature of electromagnetic radiation?
What describes the nature of electromagnetic radiation?
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What role do wave numbers (cm-1) play in infrared spectroscopy?
What role do wave numbers (cm-1) play in infrared spectroscopy?
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Study Notes
UV-Vis Spectroscopy of Biomolecules
- UV-Vis spectroscopy measures the interaction of electromagnetic waves with matter.
- Electromagnetic waves have different frequencies and wavelengths.
- Resonance occurs when the frequency of the light matches the vibrational frequency of the molecule, which causes absorption.
- The Lambert Beer Law describes the relationship between absorbance, concentration, and path length.
- High concentrations and stray photons can affect absorbance readings.
- Chromophores in biological samples absorb UV-Vis light, with different chromophores having different absorption patterns.
Spectroscopy
- Spectroscopy studies how light interacts with matter (atoms and molecules).
- The output of light is transformed by molecules in the sample.
- Spectroscopy determines the molecular organization (bond distance, bond strength, molecular geometry) of different molecules (gases, liquids, and solids).
- Biologists use spectroscopy to study large biological molecules, such as proteins and nucleic acids. This includes determining concentration, structural features, conformational changes, and molecular interactions.
Spectroscopy Processes
- Spectroscopy involves measuring how much light is transmitted or absorbed by a sample.
- The sample absorbs different types of light (microwaves, infrared, UV Visible, NMR).
- The detector compares the initial light (from a light source) with the output light that interacted with the sample.
- The output light can be measured by counting the photons.
- The y-axis can represent transmittance or absorbance.
Resonance
- Resonance is a phenomenon where a system oscillates or vibrates with maximum amplitude for a particular frequency.
- The frequency of a swing depends on its length.
- Resonance can occur when the frequency of an oscillation (light) matches the intrinsic frequency of a system or molecule, resulting in increased amplitude of oscillation or absorption of energy.
- This effect is crucial in spectroscopy for analyzing and identifying molecules in a sample.
Spectra
- Spectra are graphical representations of how a sample absorbs or transmits light over a range of wavelengths (energy).
- The x-axis typically represents wavelength, frequency, or energy.
- Different molecular features cause distinct absorption bands in the spectrum.
- An understanding of the spectrum allows deductions about molecular properties, such as size, bond lengths, mass, and charge.
The Lambert Beer Law
- The Lambert Beer law quantifies the relationship between the absorbance of a substance, concentration, and the path length.
- Absorbance depends on the concentration of a substance and the length of the light path through the sample.
- Variations in absorbance values reflect concentration changes.
- The law is used in determining the concentration of a substance from its absorption.
Stray Light
- Stray light in spectroscopy refers to light that does not pass through the sample but still reaches the detector.
- Stray light affects spectrophotometer readings at higher concentrations, particularly with inexpensive instruments.
- The detector will erroneously register higher light transmission than what actually passes through the sample.
- Correcting for stray light in instrumentation is essential for accurate UV-Vis measurements, especially at high concentrations.
Cuvettes
- Use high transmittance materials (e.g., quartz).
- Quartz is an expensive material.
- Quartz cuvettes typically have high transmittance, minimizing absorption of light by the cuvette itself.
- Plastic polymers can serve as good alternatives when cost is a primary factor.
- Use cuvettes appropriate for the wavelength of light being used, as some materials will have better transmittance than others.
Chromophores
- Chromophores are molecules that absorb light in specific wavelengths.
- Different types of biomolecules (like carbohydrates, lipids, proteins, nucleic acids) have different characteristic chromophores, resulting in different UV-Vis absorption bands.
- The presence of particular chromophores is essential for making certain types of molecular analysis possible in biological samples.
- Biological samples containing carbohydrates or lipids often do not absorb in common UV-Vis spectra regions.
Protein Analysis
- Proteins generally have no absorption in visible light; they are usually transparent.
- Proteins can absorb in the UV region (280 nm), if they contain aromatic amino acids (Trp, Tyr), or have disulfide bonds.
- The absorbance at 280 nm provides useful information about the protein concentration, based on the existence of aromatic amino acids.
Nucleic Acid Analysis
- Nucleic acids are colorless in visible light and strongly absorb in the UV region, at 260 nm.
- The absorbance at 260 nm, especially from DNA and RNA, correlates with their concentration.
- Nucleic acid contaminants can affect this measurement, sometimes requiring the ratio of the absorbance at 260 and 280 to be analyzed for contaminants.
Specific Absorption
- The maximum absorption of a molecule is wavelength-specific.
- This maximum is used for determining concentration.
- The wavelength at which a molecule absorbs light most strongly is a useful characteristic for determining that molecule's concentration within a sample.
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Description
Test your knowledge on UV-Vis spectroscopy and its application in analyzing biomolecules. This quiz covers fundamental concepts such as the interaction of light with matter, the Lambert Beer Law, and the role of chromophores in biological samples. Understand how these principles are crucial in the study of molecular structures.
