31 Questions
What is the primary purpose of fluorescence spectroscopy?
To excite electrons in a sample with light and measure lower energy light emission
Which characteristic is associated with fluorescent molecules?
Highly conjugated systems, many rings, and rigid and planar structure
What is the Stoke shift in fluorescence spectroscopy?
The difference between excitation and emission wavelengths
How do fluorescence spectrophotometers avoid interference?
By measuring emission at right angles to the light source
What does size exclusion chromatography use to separate molecules?
A column and a mobile and stationary phase
How does size exclusion chromatography separate molecules?
Based on size, with small molecules entering beads and large ones excluded
What is the primary principle behind dialysis?
Separating molecules based on size using a semi-permeable membrane
How does electrophoresis separate molecules?
Based on charge in an electric field, with agarose gels acting as a molecular sieve
What is the main purpose of UV/visible Spectroscopy?
All of the above
What happens when a compound absorbs UV energy?
Electrons are promoted to higher energy levels
How is transmittance calculated in UV spectroscopy?
As the percentage of light that passes through the substance
What does high absorbance indicate in UV spectroscopy?
A high concentration of absorbing molecules
Which law states that light absorption is proportional to the path length?
Lambert's law
At which wavelength does DNA absorb UV light?
$260nm$
Which amino acid side chains absorb UV light around $280/274nm$?
$Tryptophan and tyrosine$
Which prosthetic group absorbs UV light at $400nm$?
$Haem$
Fluorescence spectroscopy uses light to excite electrons, causing them to emit higher energy light.
False
Stoke shift is the term for the difference between excitation and emission wavelengths in fluorescence spectroscopy.
True
Fluorescence spectrophotometers measure emission at right angles to avoid interference.
True
Dialysis separates molecules based on charge in an electric field.
False
Agarose gels are used in size exclusion chromatography to separate molecules based on size.
False
Proteins can be separated using electrophoresis, with DNA running in a thinner gel.
False
SDS-PAGE separates proteins by denaturing them and running them in a thicker gel.
False
Size exclusion chromatography separates molecules based on their charge.
False
UV energy is similar to the bonding in organic molecules.
True
Absorbance is calculated as log10(I0/I)
True
Beer's law states that light absorption is proportional to the number of absorbing molecules (concentration)
True
Lambert's law states that light absorption is proportional to path length (width of sample the light is passed through)
True
DNA absorbs UV light at 260nm.
True
Peptide bond in proteins absorbs UV light at 190nm.
True
Tryptophan and tyrosine amino acid side chains absorb around 280/274nm.
True
Study Notes
-
Fluorescence spectroscopy involves exciting electrons in a sample with light, causing them to emit lower energy light
-
GFP, a naturally fluorescent protein, is widely used in biotechnology
-
Fluorescent molecules have highly conjugated systems, many rings, and are rigid and planar
-
Stoke shift is the difference between excitation and emission wavelengths, with larger shifts increasing sensitivity
-
Fluorescence spectrophotometers use a light source, prism, slits, and measure emission at right angles to avoid interference
-
Fluorescence offers enhanced sensitivity and specificity, but can be affected by energy transfer and not all compounds are fluorescent
-
Molecules can be separated using techniques like size exclusion chromatography, which uses a column and a mobile and stationary phase
-
Size exclusion chromatography separates molecules based on size, with small molecules entering beads and large ones excluded
-
Dialysis uses a semi-permeable membrane to separate molecules based on size, with small molecules passing through and large ones remaining in solution
-
Electrophoresis separates molecules based on charge in an electric field, with agarose gels acting as a molecular sieve
-
DNA and proteins can be separated using electrophoresis, with DNA negatively charged and separated using agarose gels in a tank.
-
Proteins can be separated using SDS-PAGE, which denatures them and runs them in a thinner gel.
-
Fluorescence spectroscopy involves exciting electrons in a sample with light, causing them to emit lower energy light
-
GFP, a naturally fluorescent protein, is widely used in biotechnology
-
Fluorescent molecules have highly conjugated systems, many rings, and are rigid and planar
-
Stoke shift is the difference between excitation and emission wavelengths, with larger shifts increasing sensitivity
-
Fluorescence spectrophotometers use a light source, prism, slits, and measure emission at right angles to avoid interference
-
Fluorescence offers enhanced sensitivity and specificity, but can be affected by energy transfer and not all compounds are fluorescent
-
Molecules can be separated using techniques like size exclusion chromatography, which uses a column and a mobile and stationary phase
-
Size exclusion chromatography separates molecules based on size, with small molecules entering beads and large ones excluded
-
Dialysis uses a semi-permeable membrane to separate molecules based on size, with small molecules passing through and large ones remaining in solution
-
Electrophoresis separates molecules based on charge in an electric field, with agarose gels acting as a molecular sieve
-
DNA and proteins can be separated using electrophoresis, with DNA negatively charged and separated using agarose gels in a tank.
-
Proteins can be separated using SDS-PAGE, which denatures them and runs them in a thinner gel.
Test your knowledge of UV/visible spectroscopy with this quiz. Learn about the principles and applications of spectroscopy, including compound identification, molecular structure determination, and substance quantification.
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