this basic spectrophotometer training session should enable you to know: basics of how a spec’...
TRANSCRIPT
![Page 1: This basic spectrophotometer training session should enable you to know: Basics of how a Spec’ Works What the Individual Components Actually Do Commonly](https://reader030.vdocuments.us/reader030/viewer/2022013101/5697c01d1a28abf838cd0725/html5/thumbnails/1.jpg)
Basic Theory of Basic Theory of SpectrophotometersSpectrophotometers
![Page 2: This basic spectrophotometer training session should enable you to know: Basics of how a Spec’ Works What the Individual Components Actually Do Commonly](https://reader030.vdocuments.us/reader030/viewer/2022013101/5697c01d1a28abf838cd0725/html5/thumbnails/2.jpg)
This basic spectrophotometer training session should enable you to know:
• Basics of how a Spec’ Works• What the Individual Components Actually
Do• Commonly used Terminology
![Page 3: This basic spectrophotometer training session should enable you to know: Basics of how a Spec’ Works What the Individual Components Actually Do Commonly](https://reader030.vdocuments.us/reader030/viewer/2022013101/5697c01d1a28abf838cd0725/html5/thumbnails/3.jpg)
What’s a What’s a Spectrophotometer?Spectrophotometer?
![Page 4: This basic spectrophotometer training session should enable you to know: Basics of how a Spec’ Works What the Individual Components Actually Do Commonly](https://reader030.vdocuments.us/reader030/viewer/2022013101/5697c01d1a28abf838cd0725/html5/thumbnails/4.jpg)
Light SourcesLight Sources
Deuterium / D2
Tungsten Halogen
Xenon Lamp Covers Whole Range
![Page 5: This basic spectrophotometer training session should enable you to know: Basics of how a Spec’ Works What the Individual Components Actually Do Commonly](https://reader030.vdocuments.us/reader030/viewer/2022013101/5697c01d1a28abf838cd0725/html5/thumbnails/5.jpg)
Dispersing ElementsDispersing Elements
Filter
Prism
Grating
![Page 6: This basic spectrophotometer training session should enable you to know: Basics of how a Spec’ Works What the Individual Components Actually Do Commonly](https://reader030.vdocuments.us/reader030/viewer/2022013101/5697c01d1a28abf838cd0725/html5/thumbnails/6.jpg)
DetectorsDetectors
Photocell
Photodiode
Photomultiplier
![Page 7: This basic spectrophotometer training session should enable you to know: Basics of how a Spec’ Works What the Individual Components Actually Do Commonly](https://reader030.vdocuments.us/reader030/viewer/2022013101/5697c01d1a28abf838cd0725/html5/thumbnails/7.jpg)
Spectral Bandwidth Spectral Bandwidth
If SBW of instrument is 8nm
696 704
Eg. Set to 700nm
Also often referred to as:- Bandwidth, Bandpass, Slit Width, Resolution.
![Page 8: This basic spectrophotometer training session should enable you to know: Basics of how a Spec’ Works What the Individual Components Actually Do Commonly](https://reader030.vdocuments.us/reader030/viewer/2022013101/5697c01d1a28abf838cd0725/html5/thumbnails/8.jpg)
Spectral Bandwidth Spectral Bandwidth
Optimum SBW is 1/10th of the Natural Bandwidth of the peak
![Page 9: This basic spectrophotometer training session should enable you to know: Basics of how a Spec’ Works What the Individual Components Actually Do Commonly](https://reader030.vdocuments.us/reader030/viewer/2022013101/5697c01d1a28abf838cd0725/html5/thumbnails/9.jpg)
Spectral Bandwidth Spectral Bandwidth
The effect of SBW on the ability to resolve spectral peaks
![Page 10: This basic spectrophotometer training session should enable you to know: Basics of how a Spec’ Works What the Individual Components Actually Do Commonly](https://reader030.vdocuments.us/reader030/viewer/2022013101/5697c01d1a28abf838cd0725/html5/thumbnails/10.jpg)
SBW
Optics
Overall Instrument RecapOverall Instrument RecapSource Lamp
Entrance Slit
Grating
Exit Slit
Sample
Detector
![Page 11: This basic spectrophotometer training session should enable you to know: Basics of how a Spec’ Works What the Individual Components Actually Do Commonly](https://reader030.vdocuments.us/reader030/viewer/2022013101/5697c01d1a28abf838cd0725/html5/thumbnails/11.jpg)
Instrument TypesInstrument Types
• Simple Fixed Filter Instruments• Fixed Grating Instruments Scanning Grating Instruments - Fixed
SBW Scanning Grating Instruments - Variable
SBW
![Page 12: This basic spectrophotometer training session should enable you to know: Basics of how a Spec’ Works What the Individual Components Actually Do Commonly](https://reader030.vdocuments.us/reader030/viewer/2022013101/5697c01d1a28abf838cd0725/html5/thumbnails/12.jpg)
Simple Uses of a Simple Uses of a SpectrophotometerSpectrophotometer
• Multiple Absorbance• Fixed Absorbance• Fixed Concentration using Factor• Fixed Concentration using Standard(s) Scanning• Absorbance at Fixed v Time - Kinetics
![Page 13: This basic spectrophotometer training session should enable you to know: Basics of how a Spec’ Works What the Individual Components Actually Do Commonly](https://reader030.vdocuments.us/reader030/viewer/2022013101/5697c01d1a28abf838cd0725/html5/thumbnails/13.jpg)
Spectrum Regions - Typical Wavelengths*
Ultra Violet 180 nm to 400 nm1800 Å to 4000 Å.18 µm to .4 µm55,556 cm-1 to 25,000 cm-1( 5.5 * 106 Hz to 2.5 * 106 Hz )
Visible 400 nm to 800 nm4000 Å to 8000 Å.400 µm to .8 µm25,000 cm-1 to 12,500 cm-1( 2.5 * 106 Hz to 1.25 * 106 Hz )
Near Infra Red 800 nm to 2500 nm8000 Å to 25000 Å.8 µm to 2.5 µm12,500 cm-1 to 4000 cm-1 (1.25 * 106 Hz to 4 * 105 Hz )
Infra Red 2,500 nm to 15,000 nm25,000 Å to 150,000 Å2.5 µm to 15 µm4,000 cm-1 to 667 cm-1( 4 * 105 Hz to 6.67 * 104 Hz )
Far Infra Red 15,000 nm to 200,000 nm150,000 Å to 2,000,000 Å15 µm to 200 µm667 cm-1 to 50 cm-1( 6.67 * 104Hz to 5 * 103 Hz )
*Reference: *Dyer, John R. 1965. Applications of Absorption Spectroscopy of Organic Compounds (2nd printing). Englewood Cliffs, NJ Prentice-Hall: pp 1,22.
![Page 14: This basic spectrophotometer training session should enable you to know: Basics of how a Spec’ Works What the Individual Components Actually Do Commonly](https://reader030.vdocuments.us/reader030/viewer/2022013101/5697c01d1a28abf838cd0725/html5/thumbnails/14.jpg)
Conversions
Nanometer ( nm, millimicron ) 1 nm = 1 x 10-9 meters
Angstrom ( Å ) 1 Å = 1 x 10-10 meters
Micron ( µm, micrometer ) 1 µm = 1 x 10-6 meters
Wave Number ( cm-1 ) 1 cm-1 = # of waves per cm
(Inverse Centimeter)
To convert wave number to/from nm:
nm = 1 / (WaveNumber)(10-7)
WaveNumber = 1 / ( nm )(10-7)
![Page 15: This basic spectrophotometer training session should enable you to know: Basics of how a Spec’ Works What the Individual Components Actually Do Commonly](https://reader030.vdocuments.us/reader030/viewer/2022013101/5697c01d1a28abf838cd0725/html5/thumbnails/15.jpg)
Absorbance vs % Absorbance vs % TransmittanceTransmittance
%T Absorbance100 050 0.30125 0.602
12.5 0.9036.25 1.204
3.175 1.498
Abs = - [ log t ]t = %T / 100t = 10 (̂-Abs)
![Page 16: This basic spectrophotometer training session should enable you to know: Basics of how a Spec’ Works What the Individual Components Actually Do Commonly](https://reader030.vdocuments.us/reader030/viewer/2022013101/5697c01d1a28abf838cd0725/html5/thumbnails/16.jpg)
0
20
40
60
80
100
120
1 2 3 4 5 6
Concentration
% T
![Page 17: This basic spectrophotometer training session should enable you to know: Basics of how a Spec’ Works What the Individual Components Actually Do Commonly](https://reader030.vdocuments.us/reader030/viewer/2022013101/5697c01d1a28abf838cd0725/html5/thumbnails/17.jpg)
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1 2 3 4 5 6
Concentration
Ab
s
![Page 18: This basic spectrophotometer training session should enable you to know: Basics of how a Spec’ Works What the Individual Components Actually Do Commonly](https://reader030.vdocuments.us/reader030/viewer/2022013101/5697c01d1a28abf838cd0725/html5/thumbnails/18.jpg)
0
20
40
60
80
100
120
1 2 3 4 5 6
Concentration
% T
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1 2 3 4 5 6
ConcentrationAb
s
![Page 19: This basic spectrophotometer training session should enable you to know: Basics of how a Spec’ Works What the Individual Components Actually Do Commonly](https://reader030.vdocuments.us/reader030/viewer/2022013101/5697c01d1a28abf838cd0725/html5/thumbnails/19.jpg)
Sample Holders and Sample Holders and GlasswareGlassware
1cm Cell / CuvetteSingle Holder, Multi-Cell HolderLong Pathlength Cell - Up to 10cmTest TubesFlow Cells - Sipper System (Can link to Auto-
sampler)Solid Sample Holders eg. For Filters
![Page 20: This basic spectrophotometer training session should enable you to know: Basics of how a Spec’ Works What the Individual Components Actually Do Commonly](https://reader030.vdocuments.us/reader030/viewer/2022013101/5697c01d1a28abf838cd0725/html5/thumbnails/20.jpg)
SipperSipper
Easy Sampling - High Throughput
![Page 21: This basic spectrophotometer training session should enable you to know: Basics of how a Spec’ Works What the Individual Components Actually Do Commonly](https://reader030.vdocuments.us/reader030/viewer/2022013101/5697c01d1a28abf838cd0725/html5/thumbnails/21.jpg)
GENESYS 10 with sipper GENESYS 10 with sipper pumppump
Cell Holder
Flowcell
Pump
Sample inlet spout
![Page 22: This basic spectrophotometer training session should enable you to know: Basics of how a Spec’ Works What the Individual Components Actually Do Commonly](https://reader030.vdocuments.us/reader030/viewer/2022013101/5697c01d1a28abf838cd0725/html5/thumbnails/22.jpg)
What industries use What industries use SpectrophotometersSpectrophotometers
Academia & Teaching Biological & Biochemical Research Bio’ & Biochem’ Monitoring & Quality Control Chemical Research Chemical Process Monitoring & QC Pharmaceutical Research Pharmaceutical Monitoring & QC Environmental Laboratories Hospital Pharmacy & Clinical Laboratories Industrial Laboratories etc. etc. etc. Basically just about ANY laboratory!
![Page 23: This basic spectrophotometer training session should enable you to know: Basics of how a Spec’ Works What the Individual Components Actually Do Commonly](https://reader030.vdocuments.us/reader030/viewer/2022013101/5697c01d1a28abf838cd0725/html5/thumbnails/23.jpg)
But wait… there’s more . . .But wait… there’s more . . .
After a pH meter and a Balance, a Visible or UV-Vis Spectrophotometer is the next most common piece of
equipment in most laboratories!Anything that goes near to, into, or is in contact with your body MUST either have been tested by or have
had it’s components tested by a Visible or UV-Vis Spectrophotometer.
FoodsBeverages
PharmaceuticalsCosmeticsChemicals
FabricsDyes
Etc. etc. etc.
![Page 24: This basic spectrophotometer training session should enable you to know: Basics of how a Spec’ Works What the Individual Components Actually Do Commonly](https://reader030.vdocuments.us/reader030/viewer/2022013101/5697c01d1a28abf838cd0725/html5/thumbnails/24.jpg)
Customer QuestionsCustomer Questions What Wavelength(s) / Wavelength Range? Type of Work?
Simple Absorbance Concentration (Factor, Single / Multi Standard) Scanning Kinetics
Number of Methods Number of Samples Sample Volume Sample Presentation Needs
(Instrument screen, Printer, Computer)
![Page 25: This basic spectrophotometer training session should enable you to know: Basics of how a Spec’ Works What the Individual Components Actually Do Commonly](https://reader030.vdocuments.us/reader030/viewer/2022013101/5697c01d1a28abf838cd0725/html5/thumbnails/25.jpg)
Questions?Questions?