Spectrophotometry Lecture 2 - New Mansoura University PDF

New Mansoura University Faculty of Pharmacy Pharm D Program ___________________________________________________________________________ Instrumental Analysis (PMC-205) Level II Students-Semester 3 Spectrophotometry...

New Mansoura University Faculty of Pharmacy Pharm D Program ___________________________________________________________________________ Instrumental Analysis (PMC-205) Level II Students-Semester 3 Spectrophotometry [Lecture 2] Professor Dr. Rania El-Shaheny  New Mansoura University Faculty of Pharmacy Pharm D Program ___________________________________________________________________________ After this lecture, you should know: ◼ What are the basic components of the spectrophotometer? ◼ What are the types and properties of each of these components?. ◼ What are the differences between UV- spectrophotometer and visible- spectrophotometer?  New Mansoura University Faculty of Pharmacy Pharm D Program ___________________________________________________________________________ Spectrophotometer  New Mansoura University Faculty of Pharmacy Pharm D Program ___________________________________________________________________________ Components of Spectrophotometer Spectrophotometer is the instrument used for spectrophotometric measurements. The basic components of the spectrophotometer includes the following: Entrance Exit Slit Slit 0.20 Readout Filter, Prism Light or Grating Source Wavelength Selector Detector Sample (Monochromator)  New Mansoura University Faculty of Pharmacy Pharm D Program ___________________________________________________________________________ Components of Spectrophotometer 1- Light Source. 2- Wavelength Selector (Monochromator). 3- Sample Compartment & Cuvette. 4- Detector. 5- Readout device.  New Mansoura University Faculty of Pharmacy Pharm D Program ___________________________________________________________________________ 1- Light Source.  New Mansoura University Faculty of Pharmacy Pharm D Program ___________________________________________________________________________ Components of Spectrophotometer 1- Light Source: It must provide a stable and intense light over a broad wavelength range. There are 2 main types of light sources: 1- Tungsten lamp 2- Deuterium lamp Used as a source Used as a source of Visible light. of UV light. 8  New Mansoura University Faculty of Pharmacy Pharm D Program ___________________________________________________________________________ 2- Monochromators Filter Prism Grating  New Mansoura University Faculty of Pharmacy Pharm D Program ___________________________________________________________________________ 2- Wavelength Selector (Monochromator): It isolates a narrow band of light of certain wavelength (monochromatic light) from the all wavelengths emitted by the light source (polychromatic light). Wavelength Light Polychromatic light Selector Monochromatic light Source (Monochromator) Filter Prism Grating  New Mansoura University Faculty of Pharmacy Pharm D Program ___________________________________________________________________________ 2- Wavelength Selector (Monochromator): …. Continued There are 3 types of monochromators: Filters – Prisms – Gratings (I) Filters: - It selects a certain range of wavelengths by filtering out undesirable ones. - There are 2 types of filters used in spectrophotometry: (A) Absorption filter: {used in the Visible region only} It absorbs the undesired part of the incident light and transmits the desired part (i.e. transmits the desired band of wavelengths). (B) Interference filter: {used in both Visible & UV regions} It reflects the undesired part of the incident light and transmits the desired part (i.e. transmits the desired band of wavelengths. - Advantages of filters: Simple, cheap, and stable. - Disadvantages of filters: One filter can isolate only one band of wavelengths (i.e. filters do not have variable wavelength settings) so they are used only when measurements are made at a fixed wavelength. 11  New Mansoura University Faculty of Pharmacy Pharm D Program ___________________________________________________________________________ (II) Prisms: - The prism splits light into beams of different wavelengths. - Its action is based on the refraction (bending ‫ )انكسار‬of the incident light by a degree that differs according to the wavelength. - There are 2 types of prisms used in spectrophotometry: (A) Glass prisms: ……… used in the Visible region only (Q. T or F?) {because glass is opaque (not transparent) to UV light}. (B) Quartz prisms: ……… used in both Visible & UV regions. - The main disadvantage of prisms is the non-linear dispersion of light so it needs expensive variable-width exit slits. 12 Violet red  New Mansoura University Faculty of Pharmacy Pharm D Program ___________________________________________________________________________ (III) Gratings: (the most commonly used) (Q. Give name?) - A Grating is a highly polished surface with a large number of parallel, equally-spaced grooves. - There are 2 types of gratings used in spectrophotometry: (A) Reflection Grating ……… the most commonly used type. (B) Transmission Grating  New Mansoura University Faculty of Pharmacy Pharm D Program ___________________________________________________________________________ (A) Reflection Grating In reflection grating, when the incident light strikes the grooved surface of the grating, the beams of different wavelengths are reflected at different angles.  New Mansoura University Faculty of Pharmacy Pharm D Program ___________________________________________________________________________ (B) Transmission Grating It is similar to the reflection grating but the dispersed beams are transmitted through the grating. (i.e. when the incident light strikes the grooved surface of the grating, the beams of different wavelengths are transmitted at different angles). -------------------------------------------------------------- - The major advantage of gratings over prisms is the linear dispersion of light which allows the use of fixed-width exit slits. ----------------------------------------------------------------------------------------------------------- 3- Sample Compartment and Cuvette Spectrophotometer  New Mansoura University Faculty of Pharmacy Pharm D Program ___________________________________________________________________________ 3- Sample Compartment and Cuvette: 0.20 3 1 2 Sample Cuvette There are 2 types of sample cuvettes (cell): (A) Glass or Plastic cuvette: used in the Visible region only. {because glass is opaque (not transparent) to UV light}. (B) Quartz cuvette: used in both Visible and UV regions.  New Mansoura University Faculty of Pharmacy Pharm D Program ___________________________________________________________________________ Question: Why do the sample cuvettes have two frosted or opaque sides ??? Answer: 1- To prevent light scattering out of the cuvette at the sides. The frosted or opaque sides help to prevent the scattered light signal from being lost and giving inaccurate readings. 2- To restrict the use of the cuvette in one orientation, so that you get more reproducible readings. This is very important especially with plastic cuvettes. 3- The frosted or opaque sides acts as a guide of where you should pick up or hold the cuvette, so that the clear optical sides do not become contaminated with fingerprints.  Sample Compartment = cuvette holder This compartment is a cell positioner that can hold up to six sample cuvettes cells at a time  New Mansoura University Faculty of Pharmacy Pharm D Program ___________________________________________________________________________ Remember Visible UV Spectrophotometry Spectrophotometry (Colorimetry) (1) Light source Tungsten lamp Deuterium lamp (2) Wavelength Selector (Monochromator): Absorption filter or Filters Interference filter Interference filter or Prisms Glass or Quartz Quartz or Gratings ------------------- (3) Sample cuvette Glass, Plastic or Quartz Quartz  New Mansoura University Faculty of Pharmacy Pharm D Program ___________________________________________________________________________ 4- Detector: 0.20 3 4 1 2 It is a device that detects (measures) the amount of light passing through the sample and converts it into an electrical signal which is then converted to a readable form by the readout device. The most commonly used types of detectors in UV/Visible spectrophotometer are: (I) Phototubes. (II) Photomultipliers.  (I) Phototubes. Light Cathode (‒) [photo-emissive material] Anode (+) [collecting electrode] Principle (Mechanism): A phototube is a light-sensitive electronic device that is designed to emit an electric current when exposed to light. It is composed of: a photo-emissive or photo-sensitive cathode and a collecting anode. When the beam of light reaches the cathode, it will cause the emission of equivalent number of electrons that are attracted to the anode.  New Mansoura University Faculty of Pharmacy Pharm D Program ___________________________________________________________________________ (II) Photomultiplier tubes (PMT) (the most commonly used detector) In order to obtain greater sensitivity to very weak light, multiplication of the initial photoelectrons (i.e. electrons emitted from the cathode after exposure to light) by employing secondary emission processes.  New Mansoura University Faculty of Pharmacy Pharm D Program ___________________________________________________________________________ (Principle (Mechanism): The photomultiplier contains several anodes of a gradual increasing positive potential called “Dynodes” present in one bulb. When electrons are emitted form the photocathode, they are attracted to dynode 1 and liberate more electrons then to dynode 2 (because it has higher potential than dynode 1) and the process continues until reaching the last anode. The final resulting current is 106-108 times greater than the primary current.  Principle of Photomultiplier Detector (Video) Animation on YouTube showing the principle of photomultiplier detector https://www.youtube.com/watch?v=f61eMq4Wg4w You can download the above animation file with subtitles from the following link: http://www.mediafire.com/download/tfw77m3ewhbte0s/Animation_Spectro_Ph otomultiplier.mp4  New Mansoura University Faculty of Pharmacy Pharm D Program ___________________________________________________________________________ 5- Readout device: 0.20 3 4 5 1 2 It convert the signal coming from the detector into a readable form displayed as Transmittance or Absorbance.  Types of Spectrophotometer 1- Single-beam Spectrophotometer: It needs to change the cuvette to adjust blank (reference) 0.20 reading so that it is slow and can not be used for automatic scanning of the full wavelength range. 2- Double-beam spectrophotometer: - No need to change the cuvette for blank. - Rapid automatic (Readout) scanning of different wavelengths (full (Blank) wavelength range).  New Mansoura University Faculty of Pharmacy Pharm D Program ___________________________________________________________________________ There are 2 possibilities for the double-beam spectrophotometer (B) After passing through the (A) After passing through the monochromator, the light beam is passed monochromator, the light beam is split by through a chopper which blocks one beam a beam splitter into two separate beams, at a time [i.e. at one time, it allows the beam one for the reference (blank) and the other to pass through the reference (blank) only for the sample and then the two beams and at the other time, it allows the beam to are measured by two separate detectors pass through the sample] and then the at the same time. beam is measured using one detector which alternates between measuring the sample beam and the reference beam in synchronism ‫ تزامن‬with the chopper.  Principle of double-beam spectrophotometer (Video) Video on YouTube showing the principle of double-beam spectrophotometer https://www.youtube.com/watch?v=O39avevqndU You can download the above animation file with subtitles from the following link: http://www.mediafire.com/download/tc92n5y58kri3iv/Animation_Spectro_Double- beam+Spectrophotometer+with+subtitles.mp4  New Mansoura University Faculty of Pharmacy Pharm D Program ___________________________________________________________________________ Summary Today we learnt the components of spectrophotometer: 1. Light source: Tungsten lamp & Deuterium lamp. 2. Wavelength selector (monochromator):  Filters: Absorption filter & Interference filter.  Prisms: Glass prism & Quartz prism.  Gratings: Reflection grating & Transmission grating. 3. Sample compartment & Cuvette: Glass, Plastic & Quartz. 4. Detector: I. Phototubes. II. Photomultipliers. 5. Readout device.  New Mansoura University Faculty of Pharmacy Pharm D Program ___________________________________________________________________________ I- Complete the following sentences: 1- The basic components of the spectrophotometer ………… , ………… , ………… , ………… and …………. 2- The source of light in visible spectrophotometry is ……… while in UV spectrophotmetry is ………. 3- Types of filters include …………. and ….......... 4- …...... is a type of monochromators depending on reflection of light striking a grooved surface at different reflection angles. 5- Linear dispersion of light is an advantage of …………… over …………….. monochromators. 6- …… are anodes of a gradual increasing positive potential present in photomultiplier detectors. 7- ………….. is a type of monochromators characterized by a large number of parallel equally-spaced grooves.  New Mansoura University Faculty of Pharmacy Pharm D Program ___________________________________________________________________________ III- Write () or (): 1- Tungsten lamp is used as a light source in colorimetry ( ). 2- Filters are used as a wavelength selector in scanning variable wavelengths ( ).  New Mansoura University Faculty of Pharmacy Pharm D Program ___________________________________________________________________________