Chapter 18: Emission Spectroscopy | CHM 214 | 159

Jacob Stewart
16 Apr 202105:24
EducationalLearning
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TLDRThe video script delves into the principles of emission spectroscopy, emphasizing its analytical advantages over absorption spectroscopy. It highlights the sensitivity of fluorescence spectroscopy, which enables the detection of even single molecules, a feat not achievable with absorption methods. The script also discusses the limitations of fluorescence, such as the requirement for molecules to possess fluorescent properties, and the innovative use of fluorescent tags in biological systems, like GFP, to overcome these challenges.

Takeaways
  • 🌟 Emission spectroscopy involves the analysis of light emitted by molecules, as opposed to absorption where light is absorbed.
  • 🚀 Fluorescence is a type of emission spectroscopy that is particularly useful for analytical purposes, such as measuring concentrations of molecules.
  • 🚫 A limitation of fluorescence spectroscopy is that not all molecules fluoresce, which restricts its applicability to certain types of molecules.
  • 🔍 Fluorescent tags, like the green fluorescent protein (GFP), can be attached to non-fluorescent molecules to enable tracking and observation.
  • 🏆 The discovery of GFP led to a Nobel Prize due to its revolutionary impact on biological chemistry and research.
  • 💡 Fluorescence spectroscopy is more sensitive than absorption spectroscopy, allowing for the detection of smaller concentrations of material.
  • 📊 In absorption spectroscopy, a large amount of light is initially present and a small dip indicates absorption, whereas in fluorescence spectroscopy, the increase from zero signal is easier to detect.
  • 🌈 The background signal in fluorescence spectroscopy is effectively zero, which simplifies the detection process and enhances sensitivity.
  • 🔬 Fluorescence spectroscopy has been used to detect single molecules, a feat not possible with traditional absorption spectroscopy.
  • 🛠️ Future discussions will include the setup and use of instruments for measuring fluorescence or emission spectroscopy.
Q & A

  • What is the fundamental difference between absorption and emission spectroscopy?

    -In absorption spectroscopy, a molecule absorbs light and we measure the light that is missing. In emission spectroscopy, the molecule itself emits light upon excitation, and we observe and measure this emitted light.

  • Why is fluorescence spectroscopy particularly useful for analytical purposes?

    -Fluorescence spectroscopy is highly useful for analytical purposes because it allows for the measurement of concentrations and can be more sensitive than absorption spectroscopy, enabling the detection of smaller concentrations of material.

  • What is the main drawback of fluorescence spectroscopy?

    -The main drawback of fluorescence spectroscopy is that not all molecules fluoresce, which limits its usefulness as some molecules undergo internal system crossing and emit no light.

  • How does attaching a fluorescent tag to a molecule help in emission spectroscopy?

    -Attaching a fluorescent tag to a molecule allows us to track the molecule in biological systems, even if the molecule itself does not have fluorescence properties. This technique is powerful and has been used with green fluorescent protein (GFP), which led to a Nobel Prize for its discovery.

  • Why is fluorescence spectroscopy more sensitive than absorption spectroscopy?

    -Fluorescence spectroscopy is more sensitive because it starts from a zero background signal, making it easier to detect a change from zero to something. This zero-background technique allows for the detection of much smaller concentrations of material.

  • What is the significance of the zero-background technique in fluorescence spectroscopy?

    -The zero-background technique in fluorescence spectroscopy means that there is no background signal to worry about. This makes it easier to observe a change from zero to a measurable signal, which is crucial for detecting very low concentrations of a substance.

  • What is the single-molecule level detection capability of fluorescence spectroscopy?

    -Fluorescence spectroscopy has been demonstrated to detect the presence of a single molecule, which is a significant advantage over absorption spectroscopy that cannot achieve this level of sensitivity.

  • How does the process of inner system crossing in a molecule affect its ability to be observed in emission spectroscopy?

    -If a molecule undergoes inner system crossing, it may not emit any light, making it invisible to emission spectroscopy. This is why it's important to find molecules with fluorescence properties to observe them via emission spectroscopy.

  • What is the role of a monochromator in absorption spectroscopy?

    -In absorption spectroscopy, a monochromator is used to filter the light source, allowing only light of a specific wavelength to pass through the sample. This helps in accurately measuring the amount of light absorbed at different wavelengths.

  • How does the signal versus wavelength plot differ between absorption and fluorescence spectroscopy?

    -In absorption spectroscopy, the signal versus wavelength plot starts with a large amount of signal (light from the source) and shows a small dip where absorption occurs. In fluorescence spectroscopy, the background signal is zero, and the plot shows an increase in signal from nothing when the sample is present and emitting light.

  • What will be covered in the next video regarding fluorescence or emission spectroscopy?

    -The next video will discuss how to set up an instrument to measure fluorescence or emission spectroscopy, providing practical insights into the experimental setup and procedures.

Outlines
00:00
🌟 Understanding Emission Spectroscopy and Fluorescence

This paragraph introduces the concept of emission spectroscopy and its intricacies compared to absorption spectroscopy. It explains how molecules can emit light and the significance of processes like fluorescence and phosphorescence in analytical applications. The main drawback of fluorescence spectroscopy is highlighted, which is the limitation due to not all molecules fluorescing. However, the paragraph also discusses how attaching fluorescent tags, such as the green fluorescent protein (GFP), can overcome this limitation. The advantages of fluorescence spectroscopy, particularly its higher sensitivity and ability to measure lower concentrations of substances, are emphasized, explaining the zero-background technique that allows for easier detection against a null signal.

05:02
🔬 Advancements in Emission Spectroscopy: Single Molecule Detection

The second paragraph delves into the advancements in emission spectroscopy, particularly the ability to detect single molecules using fluorescence spectroscopy. This is a significant achievement as it is not possible with absorption spectroscopy. The paragraph sets the stage for the next video, where the setup and measurement techniques for fluorescence or emission spectroscopy will be discussed. The mention of single molecule detection showcases the high sensitivity and potential applications of emission spectroscopy in various fields.

Mindmap
Keywords
💡Emission Spectroscopy
Emission spectroscopy is an analytical technique used to study the interaction of light with matter by observing the light emitted from a sample. In the video, it is contrasted with absorption spectroscopy, highlighting that emission spectroscopy involves the detection of light produced by the sample itself after excitation, rather than measuring the decrease in light intensity as it passes through the sample. This technique is crucial for understanding molecular properties and behaviors, especially in the context of fluorescence and phosphorescence.
💡Fluorescence
Fluorescence is a type of photoluminescence and a specific process in emission spectroscopy where a molecule absorbs light at a certain wavelength and then re-emits light at a longer wavelength. The video explains that fluorescence is highly useful for analytical purposes, such as measuring concentrations, but its limitation is that not all molecules fluoresce. The example given is the attachment of fluorescent tags to molecules in biological systems, like the green fluorescent protein (GFP), to track and observe them.
💡Phosphorescence
Phosphorescence is another process related to emission spectroscopy where a molecule emits light after being excited to a higher energy state, similar to fluorescence, but the key difference is that phosphorescence involves a slower return to the ground state through the emission of light over a longer period. The video mentions phosphorescence as an alternative emission process that can be observed in certain molecules, expanding the scope of spectroscopic analysis.
💡Inner System Crossing
Inner system crossing refers to a non-radiative process where a molecule transitions from an excited state to a lower energy state without emitting light. In the context of the video, this process is mentioned as a reason why some molecules do not fluoresce, as they undergo inner system crossing instead of emitting light, thus limiting the applicability of fluorescence spectroscopy.
💡Green Fluorescent Protein (GFP)
Green Fluorescent Protein (GFP) is a protein that exhibits fluorescence, originally isolated from the jellyfish Aequorea victoria. The video highlights the significance of GFP in biological chemistry as a tagging tool, allowing scientists to track and observe specific molecules within living organisms. The discovery and application of GFP have been so impactful that it was recognized with a Nobel Prize.
💡Sensitivity
In the context of spectroscopy, sensitivity refers to the ability of a technique to detect and measure small quantities of a substance. The video explains that fluorescence spectroscopy is more sensitive than absorption spectroscopy because it can measure smaller concentrations of material. This is due to the zero-background nature of the technique, where the detection starts from no light and any increase in light is easily noticeable, unlike absorption spectroscopy where a decrease in light intensity from a high background needs to be measured.
💡Zero-Background Technique
A zero-background technique, as described in the video, is a method that allows for the detection of a signal without any background noise or interference. In the case of fluorescence spectroscopy, the background signal is effectively zero because no light is emitted from a sample that does not fluoresce. This makes it easier to detect and quantify even the smallest amounts of fluorescent material, as any increase in light is indicative of the presence of the target substance.
💡Monochromator
A monochromator is an instrument used in spectroscopy to isolate a single wavelength of light from a broad spectrum. In the video, it is mentioned as part of the setup in absorption spectroscopy, where it is used to shine light through the sample and measure the light that is absorbed. The monochromator plays a crucial role in determining the specific wavelengths that are being analyzed, which is essential for understanding the interaction between light and the sample.
💡Concentration Measurement
The measurement of concentration involves determining the amount of a particular substance within a given volume of a solution or mixture. In the video, it is discussed in the context of fluorescence spectroscopy, which is highlighted as a more sensitive method for measuring concentrations compared to absorption spectroscopy. The ability to measure smaller concentrations is particularly useful in analytical applications, such as tracking the presence and quantity of specific molecules in complex biological systems.
💡Single Molecule Detection
Single molecule detection refers to the ability to observe and analyze individual molecules, which is a significant achievement in the field of spectroscopy. The video mentions that fluorescence spectroscopy has been demonstrated down to the single molecule level, meaning that it is possible to detect and study the presence and behavior of just one molecule. This level of sensitivity and precision has revolutionized the study of molecular interactions and dynamics, allowing for a deeper understanding of biological processes at the most fundamental level.
Highlights

Understanding emission spectroscopy and its processes is crucial for analyzing light emission from molecules.

Emission spectroscopy involves observing the light emitted by molecules, as opposed to absorption where light is missing.

Fluorescence, a type of emission spectroscopy, is highly useful for analytical purposes and measuring concentrations.

The main drawback of fluorescence spectroscopy is that not all molecules fluoresce, limiting its applicability.

Molecules that do not fluoresce may undergo internal system crossing and emit no light, making them invisible to emission spectroscopy.

In biological systems, non-fluorescent molecules can be tagged with fluorescent markers to track them using emission spectroscopy.

The discovery of the green fluorescent protein (GFP), which is used in biological chemistry, led to a Nobel Prize.

Fluorescence spectroscopy is more sensitive than absorption spectroscopy due to its ability to measure smaller concentrations of material.

In absorption spectroscopy, a large amount of light is present, and any absorption results in a small dip, making it harder to detect changes.

Fluorescence spectroscopy starts from zero background signal, making it easier to detect an increase in signal from nothing.

The zero-background technique of fluorescence spectroscopy allows for the measurement of single molecules, which is not possible with absorption spectroscopy.

The sensitivity of fluorescence spectroscopy is due to its ability to detect a change from zero to something, as opposed to a large amount to less.

The upcoming video will cover the setup and measurement process for fluorescence or emission spectroscopy.

Emission spectroscopy is particularly powerful in biological chemistry and has significant applications in the field.

The limitations of emission spectroscopy can be overcome by attaching fluorescent tags to molecules of interest.

The process of emission spectroscopy involves exciting the sample with a light source and detecting the light it emits.

Transcripts

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Emission SpectroscopyMolecular AnalysisFluorescence DetectionPhosphorescence PropertiesAnalytical TechniquesBiological ChemistryGreen Fluorescent ProteinSensitivity AdvantageSingle Molecule