Basic Theory of Basic Theory of SpectrophotometersSpectrophotometers

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

What’s a What’s a Spectrophotometer?Spectrophotometer?

Light SourcesLight Sources

Deuterium / D2

Tungsten Halogen

Xenon Lamp Covers Whole Range

Dispersing ElementsDispersing Elements

Filter

Prism

Grating

DetectorsDetectors

Photocell

Photodiode

Photomultiplier

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.

Spectral Bandwidth Spectral Bandwidth

Optimum SBW is 1/10th of the Natural Bandwidth of the peak

Spectral Bandwidth Spectral Bandwidth

The effect of SBW on the ability to resolve spectral peaks

SBW

Optics

Overall Instrument RecapOverall Instrument RecapSource Lamp

Entrance Slit

Grating

Exit Slit

Sample

Detector

Instrument TypesInstrument Types

• Simple Fixed Filter Instruments• Fixed Grating Instruments Scanning Grating Instruments - Fixed

SBW Scanning Grating Instruments - Variable

SBW

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

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.

 

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)

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)

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

Concentration

Ab

s

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

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

SipperSipper

Easy Sampling - High Throughput

GENESYS 10 with sipper GENESYS 10 with sipper pumppump

Cell Holder

Flowcell

Pump

Sample inlet spout

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!

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.

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)

Questions?Questions?