Beginner’s Guide to Fluorescence Spectroscopy, Study notes of Chemistry

Download Beginner’s Guide to Fluorescence Spectroscopy and more Study notes Chemistry in PDF only on Docsity! AROMETRIX  Beginner’s Guide to  Fluorescence Spectroscopy          Abstract    In 2017, Arometrix investigated and validated the use of fluorescence  spectroscopy as a powerful tool for real-time molecular monitoring during  short-path distillation. Our research paper, ​“In Situ Fluorescence Spectroscopy  for In-Line Distillation Process Monitoring”​, was peer-reviewed and published  by Cannabis Science and Technology a year later. Additionally, we had the  privilege of speaking and delivering our research presentation at the Cannabis  Science Conference in Maryland.    Since then, Arometrix has been a pioneer in the cannabis production industry,  helping customers to optimize cannabis process control, new staff training,  and batch quality. Now, ​this novel technology is not only available for  short-path distillation,​ but also for wiped film evaporation, ethanol extraction,  chromatography, conversion reaction, and soon even more.    Many of Arometrix’s customers are laboratory technicians, managers, and  consultants who are very knowledgeable about their processes; ​however, the  area of “fluorescence spectroscopy” is still a bit of a mystery to most.​ ​It was  for the purpose of advancing the knowledge in this specific field of  spectroscopy that this guide was made.            Table of Contents  Chapter 1: An Introduction to Fluorescence Spectroscopy 3  What is fluorescence spectroscopy? 3  Spectroscopy 3  Fluorescence 3  What does the “Spectra” in Spectroscopy mean? 3  What are waves, wavelengths, nanometers and peak structures? 4  Chapter 2: Spectroscopy Methods 6  Standard Light Detectors and Measurement Systems 6  Fluorometer 6  UV detector 6  Spectrometer 6  Spectrophotometers 7  HPLC/GC - Advantages & Disadvantages 8  Fluorometer - Advantages & Disadvantages 8  Spectrophotometer - Advantages & Disadvantages 8  The Human Eye - Advantages & Disadvantages 8  What is the difference between Fraction Finder and HPLC? 9  Chapter 3: Overview of Relevant Key Concepts 10  Electromagnetic Radiation 10  Spectrum 10  Waves & Electromagnetic Radiation 10  Light Scattering 10  Excitation 11  Photon 11  Transmission vs Reflection 11  Summary 12        If you notice on the Fraction Finder display’s Spectrum View (see below), the X-axis is  wavelength.    Nanometers​ are a unit of length measurement with a  magnitude of 10​-9​.(eg. 1 meter is 0.000000001 nanometers).  Wavelengths of light are generally represented in  nanometers.    However, not ​all​ light is measured in nanometers.    In fact, a lot of people refer to light with eV (energy  measure/photon), Hz, and even cm^(-1); the last two refer to  light frequency instead of wavelength.    Peak structure ​refers to the shape of the spectral signal. Not  all​ chemicals/molecules will have a unique Peak Structure,  but they can have different, or even slightly different, peak  structures which leads to them being resolvable.      Image from Arometrix, “Chemical Cheat Sheet”    The Fraction Finder measures the amount of light at different wavelengths. The collection of  these intensity values, with respect to the wavelengths, is what makes peak structures.    Putting these concepts into context:​ An example of these concepts can be seen in our  Fraction Finder ​Chemical Cheat Sheet​. Note how we refer to each molecule's wavelength  and waveform (waveform is a colloquial term that we use to describe peak structure shape  and intensity).    We’ve included two screenshots from the Cheat Sheet above.    ● The upper image is of our Reference/Excitation peak, which is simply the internal  reference peak that the system uses, showing a sharp peak structure at a wavelength  region of 360-370 nanometers  ● The lower image still includes the Reference/Excitation peak, but it also shows the  Delta-9 THC Indicator, which displays a short broad peak structure at a wavelength  region of about 450-470 nanometers.                    Chapter 2: Spectroscopy Methods    Standard Light Detectors and Measurement Systems  There are several different individual detectors and whole measurement systems used for  the purpose of measuring light. Below we will define the ones that are relevant to this field.    Fluorometer  ● Basic meaning​:​ A fluorometer is something that  measures fluorescence data, specifically, the intensity  of fluorescence  ● Technical measurement method​: ​Measures light at a  right angle (90 degree) from the excitation light        Image from Matthias M.  UV detector  ● Basic meaning​: ​A UV (ultra-violet) detector,  commonly referred to as UV-Vis, is a detector that  measures the amount of UV or visible light  absorbed by components of a mixture being eluted  (elution occurs during substance removal)  ● Technical measurement method​:​ Measures  average intensity over various wavelengths; you  can’t distinguish between wavelengths with a UV  detector.  ● What is UV?​ Ultraviolet light (UV) is a domain of  light, typically considered to be light with a  wavelength between 10 nm to 400 nm. Basic  Example: the light that you protect yourself from  with sunscreen.  Image from Ibsen Photonics  Spectrometer  ● Basic meaning​: ​A spectrometer  is a detector that measures  spectral data. It distinguishes the  wavelengths that were absorbed.  Most of the time a spectrometer  is a component of a  spectrophotometer.  ● Technical measurement  method​: Measures light absorption as a function of wavelength simultaneously.    Image from Edinst    Spectrophotometers  ● Basic meaning​: ​A spectrophotometer is also a measurement system that measures  spectral data. It focuses on the  relative intensities of the  wavelengths that were  absorbed ​or​ the wavelengths  that were reflected. It is a  complete system that  measures the absorption of  light. It measures intensity as a  function of wavelength one  after the other.  ● Technical measurement  method​: ​Similar to a  spectrometer, but measures  absorption as a function of  wavelength serially.  Image from Shimadzu    Furthermore,​ ​the detector’s geometry is what classifies it as a specific measurement system.        Image from Arometrix, Chris M.        Chapter 3: Overview of Relevant Key Concepts    Electromagnetic Radiation  Basic meaning:​ ​Spectroscopy uses spectra in the investigation of electromagnetic (EM)  radiation. It is the interaction of, and emission of, light.    Technical meaning​: ​This relates to the interrelation of electric currents or fields and  magnetic fields. Specifically, this is defined as a fundamental physical force that is responsible  for interactions between charged particles, which occur because of their charge and for the  emission and absorption of photons, that is about a hundredth the strength of the strong  force, and that extends over infinite  distances but is dominant over atomic  and molecular distances. Radiation  refers to the emission of energy as  electromagnetic waves or as moving  subatomic particles, especially  high-energy particles which cause  ionization. In other words, it refers to the  way that electrically charged particles  radiate, or scatter, upon interaction.        Image from E. Campostrini  Spectrum  The full range of wavelengths of electromagnetic radiation  Waves & Electromagnetic Radiation  The way in which charged particles interact and oscillate (or vary in magnitude or position) in  magnetic fields. More specifically, this is a variation of an electromagnetic field in the  propagation of light or other radiation through a medium or vacuum. This periodic  disturbance of the particles of a substance may be propagated without net movement of the  particles.  Light Scattering  Light that has been diffused through a media (i.e.  something in) and (at least partially) re-emitted  (i.e. something out).    A real-life example of this is a prism.        Image from Jochem Vreeman  Fluorescence​ is a light scattering event because light is being re-emitted. Furthermore, it is  an inelastic scattering phenomena where light is emitted from a sample due to electron  excitation and relaxation by a specific energy of light.    With ​inelastic scattering​, during a given particle collision, energy is absorbed by one or more  particles. Examples of this include Fluorescence and Raman scattering. Comparatively, with  elastic scattering​, during a given particle collision, energy is not absorbed by either particle.  An example of this is any type of diffraction, such as X-Ray. Even the formation of rainbows is  inherently an elastic process because the light doesn’t change its energy, just its direction.  Excitation  The application of energy to a particle, object, or physical system. In other words, this is  considered the addition of energy, so exciting something means applying/adding energy to  it. The state that a particle is in during excitation (when an atom or molecule has absorbed  energy) is referred to as the ​excited state.  Photon  A particle representing a quantum of light or other electromagnetic radiation  Transmission vs Reflection  Transmission​ refers to the light that has passed straight through a media. An example of this  is visible light passing straight through a clean window. ​Reflection​ refers to the light that  bounces off an interface. An example of this is visible light bouncing off a mirror.                                      Summary  Arometrix hopes that this guide has been educational to those who are beginners in the field  of fluorescence spectroscopy. Moving forward, Arometrix plans to continuously update this  document as we receive more questions and comments on the subject matter. With that  said, if you would like to discuss this paper, learn more about our technology, or get a  product quote, please do not hesitate to get in touch with our team. We love to hear from  you!    Get In Touch  ● Phone: (240) 492-6556  ● Email: ​sales@arometrix.com ● Website: ​www.arometrix.com  ● Instagram: ​@arometrix    FRACTION FINDER EXTRACTION FINDER    FRACTION FINDER ​ULTRA