SpectrophotometerSpectrophotometer

••Measures the light that passes through Measures the light that passes through a liquid samplea liquid sample

••Spectrophotometer gives readings in Spectrophotometer gives readings in Percent Transmittance (%T) and in Percent Transmittance (%T) and in Absorbance (A)Absorbance (A)

The Electromagnetic SpectrumThe Electromagnetic Spectrum

= c / E = h

Absorbance and Absorbance and Complementary ColorsComplementary Colors

Io I

Cell withPathlength, b,

containing solution

lightsource detector

blank where Io = I

concentration 2concentration 1

b

with sample I < Io

The process of light being absorbed by a solution

As concentration increased, less light was transmitted (more light absorbed).

The law states that the amount of light absorbed by a solution The law states that the amount of light absorbed by a solution

(colored) is proportional to the concentration of the absorbing (colored) is proportional to the concentration of the absorbing substance and to the thickness of the absorbing material substance and to the thickness of the absorbing material (path length). Absorbance is also called optical density (path length). Absorbance is also called optical density

A = abcA = abc

where a – molar absorptivity, b – where a – molar absorptivity, b – pathlength, and c – molar pathlength, and c – molar

concentrationconcentration

Some terminologySome terminology

I – I – intensityintensity where I where Ioo is initial intensity is initial intensity

T – T – transmissiontransmission or %T = 100 x T or %T = 100 x T(absorption: Abs = 1 – T or %Abs = 100 - %T)(absorption: Abs = 1 – T or %Abs = 100 - %T)

T = I/ IT = I/ Ioo

A – A – absorbanceabsorbance

A = - log T = -log I/ IA = - log T = -log I/ Ioo

The blank contains The blank contains allall substances substances expect the analyte.expect the analyte.

Is used to set the absorbance to zero:Is used to set the absorbance to zero:

AAblankblank = 0 = 0 This removes any absorption of light This removes any absorption of light

due to these substances and the cell.due to these substances and the cell. All measured absorbance is due to All measured absorbance is due to

analyte.analyte.

Conventional Conventional SpectrophotometerSpectrophotometer

1. A stable and cheap energy source.2. A monochromator to break the polychromatic radiation into component and wavelength/bands of wave length.3. Transparent vessels (cuvettes) to hold the sample.4. A photo sensitive detector and associated amplifier and recorder

Conventional Conventional SpectrophotometerSpectrophotometer

Optical system of a split-beam spectrophotometer

LIGHT SOURCES

 UV Spectrophotometer

1. Hydrogen Gas Lamp

2. Mercury Lamp

Visible Spectrophotometer

1. Tungsten Lamp

IR Spectrophotometer

1. Carborundum (SIC)

Light SourceLight Source

Deuterium Arc LampDeuterium Arc Lamp UV RegionUV RegionWavelength Range :Wavelength Range :190~420nm190~420nm

Tungsten LampTungsten Lamp Wavelength Range : Part of the UV and the whole of the Wavelength Range : Part of the UV and the whole of the VisibleVisiblerange (range ( 약약 350 ~ 2,500nm)350 ~ 2,500nm)

Xenon LampXenon Lamp Wavelength Range : 190~800nmWavelength Range : 190~800nm

MonochromatorMonochromator Accepts polychromatic input light from a lamp and Accepts polychromatic input light from a lamp and

outputs outputs monochromatic lightmonochromatic light ComponentsComponents : Entrance slit, Dispersion device, Exit slit. : Entrance slit, Dispersion device, Exit slit. The resolving element are of two kinds namely,The resolving element are of two kinds namely, prismsprisms and diffraction gratings. Simple glass prisms and diffraction gratings. Simple glass prisms

are used for visible range. For UV region silica, fused are used for visible range. For UV region silica, fused silica or quartz prism is used. Fluorite is used in vaccum silica or quartz prism is used. Fluorite is used in vaccum UV range. UV range.

Gratings Gratings are often used in the monochromators of are often used in the monochromators of spectrophotometers operating in UV, visible and infra red spectrophotometers operating in UV, visible and infra red regions. Their resolving power is far superior to that of regions. Their resolving power is far superior to that of prisms & they yield a linear resolution of spectrum.prisms & they yield a linear resolution of spectrum.

Dispersion DevicesDispersion Devices

• Non-linear dispersion• Temperature sensitive

• Linear Dispersion• Different orders

CELLS

UV Spectrophotometer

Quartz (crystalline silica)

 Visible Spectrophotometer

Glass

 IR Spectrophotometer

NaCl

Open-topped rectangular standard cell (a) and apertured cell (b) for limited sample volume

Cell Types ICell Types I

Cell Types IICell Types II

Micro cell (a) for very small volumes and flow-through cell (b) for automated applications

Detection DevicesDetection Devices

Most detectors depend on the photoelectric Most detectors depend on the photoelectric effect, where incident light photons) liberates effect, where incident light photons) liberates electrons from a metal or other material surface.electrons from a metal or other material surface.

Important requirements for a detector Important requirements for a detector (1)high sensitivity to allow the detection of low levels of radiant (1)high sensitivity to allow the detection of low levels of radiant

energy,energy, (2)short response time,(2)short response time, (3)long term stability, and (3)long term stability, and (4)an electronic signal which is easily amplified for typical read out (4)an electronic signal which is easily amplified for typical read out

apparatus, Ultraviolet and visible radiation detectors are photocells, apparatus, Ultraviolet and visible radiation detectors are photocells, phototubes and photo multiplier tubes.phototubes and photo multiplier tubes.

Photomultiplier Tube DetectorPhotomultiplier Tube Detector

Anode

• High sensitivity at low light levels• Cathode material determines spectral sensitivity• Good signal/noise• Shock sensitive

Amplification and ReadoutAmplification and Readout

Radiation detectors generate electronic Radiation detectors generate electronic signals which are proportional to the signals which are proportional to the transmitter light. transmitter light.

These signals need to be translated to a These signals need to be translated to a form that is easy to interpret. form that is easy to interpret.

This is accomplished by using amplifiers, This is accomplished by using amplifiers, ammeters, potentiometers, and ammeters, potentiometers, and potentiometric recorders.potentiometric recorders.

1. Qualitative Analysis1. Qualitative Analysis2. Quantitative Analysis2. Quantitative Analysis3. Molecular weight determination3. Molecular weight determination4. Study of cis-trans Isomerism4. Study of cis-trans Isomerism5. Other Physiochemical studies5. Other Physiochemical studies6. Control of Purification6. Control of Purification7. 7. Difference SpectroscopyDifference Spectroscopy8. Turbidimetry8. Turbidimetry

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