UV-VIS SpectroscopyUV-VIS Spectroscopy

Dr. AKM Shafiqul IslamDr. AKM Shafiqul Islam

Spectrometer ExperimentSpectrometer Experiment

Irradiance in Irradiance out

Pathlength

Irradiance, P, is measured in W·m-2

Transmittance, T, is the fraction of original light not absorbed by the sample, or

Where po = is power of incident light, p = power of transmitted light and b = path length

kb

P

PT 10

0

• A light source can be a lamp, laser, or even a light bulb. A monochromator (“one color”) is used to select a particular wavelength. This is typically a grating, but can be a prism or filter. The light then passes through the sample, containing the analyte. Afterwards, the light is detected.

Putting this in logarithmic formPutting this in logarithmic form

kbP

PT

0

loglog

In 1852, Beer and Bernard each In 1852, Beer and Bernard each stated that a similar law holds for the stated that a similar law holds for the dependence of concentration, cdependence of concentration, c

Where k’ is a new constantWhere k’ is a new constant

ck

P

PT 10

0

ckP

PT

0

loglog

Combining these two laws which describes Combining these two laws which describes the dependence of the dependence of TT on both the path on both the path length and concentrationlength and concentration

Where Where aa is a combined constant of is a combined constant of kk and and k’k’

abcP

PT

0

loglog

abc

P

PT 10

0

It is more convenient to remove negative sign on It is more convenient to remove negative sign on right hand side, we getright hand side, we get

The transmittance is given byThe transmittance is given by

or,or,

abcP

P

TTA 0log

1loglog

100%0

P

PT

100/%TT

Now, we can writeNow, we can write

TT

A %log100log%

100log

Absorbance and Beer’s LawAbsorbance and Beer’s Law AbsorbanceAbsorbance, A, the amount light absorbed by the , A, the amount light absorbed by the

sample is related to transmittance:sample is related to transmittance:

Beer’s law relates the absorbance of a chemical to Beer’s law relates the absorbance of a chemical to its concentration:its concentration:

bb is the pathlength, typically in cm, and is the pathlength, typically in cm, and cc is the is the concentration of the chemical species in Mconcentration of the chemical species in M

is the molar absorptivity, the unit that tells how is the molar absorptivity, the unit that tells how much light is absorbed for a given wavelength. much light is absorbed for a given wavelength. has units of Mhas units of M-1-1 cm cm-1-1

TP

PA loglog 0

bcA

Wavelengths and ColorWavelengths and Color

Beer’s Law AssumptionsBeer’s Law Assumptions

The light being shined on the sample must be The light being shined on the sample must be monochromatic (one color or wavelength)monochromatic (one color or wavelength)

The analyte must not be participate in a The analyte must not be participate in a concentration dependent equilibriumconcentration dependent equilibrium This isn’t a good technique for many weak acid This isn’t a good technique for many weak acid

systems, as dilution increases dissociation and systems, as dilution increases dissociation and HA and AHA and A-- probably don’t have the same probably don’t have the same absorbanceabsorbance

To do a Spectroscopic AnalysisTo do a Spectroscopic Analysis

You need:You need: A continuous light sourceA continuous light source A wavelength selectorA wavelength selector A sample cellA sample cell A detectorA detector

The sample cell is called The sample cell is called cuvetcuvet and can be made and can be made of many substancesof many substances Glass (good for visible)Glass (good for visible) Quartz (UV-vis)Quartz (UV-vis) NaCl/KBr (IR)NaCl/KBr (IR)

Spectroscopic ProcedureSpectroscopic Procedure You may have a single-beam or double beamYou may have a single-beam or double beam

Single-beam instrument has one sample holder, you Single-beam instrument has one sample holder, you must swap blank and samplemust swap blank and sample

Double-beam instrument splits light output between Double-beam instrument splits light output between two holders so you can measure blank and sampletwo holders so you can measure blank and sample

A A baselinebaseline spectrum is a spectrum of a reference spectrum is a spectrum of a reference solution (solvent or reagent blank)solution (solvent or reagent blank)

We try to do an analysis at the We try to do an analysis at the λλmaxmax if we can if we can Sensitivity is greatest at maximum absorbanceSensitivity is greatest at maximum absorbance Curve is relatively flat in case the monochromator Curve is relatively flat in case the monochromator

drifts and is off by a little in wavelengthdrifts and is off by a little in wavelength

The Single-Beam SpectrometerThe Single-Beam Spectrometer

How Do UV spectrometers work?How Do UV spectrometers work?

Two photomultiplier inputs, differential voltage drives amplifier.

Matched quartz cuvettes

Sample in solution at ca. 10-5 M.

System protects PM tube from stray light

D2 lamp-UV

Tungsten lamp-Vis

Double Beam makes it a difference technique

Rotates, to achieve scan

The polychromatic light is separated into The polychromatic light is separated into monochromatic wavelengths by diffraction. monochromatic wavelengths by diffraction.

nn = d(sin = d(sin + sin + sin ))

Optics of the Grating MonochromatorOptics of the Grating Monochromator

In the equation nIn the equation n = d(sin = d(sin + sin + sin ) n is the order of ) n is the order of the diffraction n = 1, 2, 3 etc, d is the number of the diffraction n = 1, 2, 3 etc, d is the number of lines etched on the grating, lines etched on the grating, is the angle of the is the angle of the incident beam and incident beam and is the angle of the emerging is the angle of the emerging beam.beam.

Optics of the Grating MonochromatorOptics of the Grating Monochromator

The photodiode array detectorThe photodiode array detector

The photodiode array detectorThe photodiode array detector

Key components:(1) Light Source(2) Monochromator(3) Sample/reference holder(4) Radiation detection(5) Readout device

UV Instrumentation

Spectroscopic ProcedureSpectroscopic Procedure

We should always try to keep the absorbance We should always try to keep the absorbance reading of our sample below 1.reading of our sample below 1. Because % transmittance is related Because % transmittance is related

logarithmically with concentration, it means that logarithmically with concentration, it means that from 1-99% transmittance we can detect ~ 2 from 1-99% transmittance we can detect ~ 2 orders of magnitude in analyte concentration.orders of magnitude in analyte concentration.

Any orders of magnitude greater than that will be Any orders of magnitude greater than that will be detected in the range of 0-1% T.detected in the range of 0-1% T.

In order to maximize accuracy, you should dilute In order to maximize accuracy, you should dilute the solution if you have to so that the transmittance the solution if you have to so that the transmittance reading is not maxed out in that region.reading is not maxed out in that region.

Spectroscopic ProcedureSpectroscopic Procedure You should always try to keep the absorbance You should always try to keep the absorbance

reading of your sample below 1.reading of your sample below 1. Because % transmittance is related Because % transmittance is related

logarithmically with concentration, it means that logarithmically with concentration, it means that from 1-99% transmittance you can detect ~ 2 from 1-99% transmittance you can detect ~ 2 orders of magnitude in analyte concentration.orders of magnitude in analyte concentration.

Any orders of magnitude greater than that will Any orders of magnitude greater than that will be detected in the range of 0-1% T.be detected in the range of 0-1% T.

In order to maximize accuracy, you should dilute In order to maximize accuracy, you should dilute the solution if you have to so that the the solution if you have to so that the transmittance reading is not maxed out in that transmittance reading is not maxed out in that region.region.

An Electronic SpectrumAn Electronic SpectrumA

bsor

banc

e

Wavelength, , generally in nanometers (nm)

0.0400 800

1.0

200

UV Visiblemaxwith certain extinction

Make solution of concentration low enough that A≤ 1

(Ensures Linear Beer’s law behavior)

Even though a dual beam goes through a solvent blank, choose solvents that are UV transparent.

Can extract the value if conc. (M) and b (cm) are known

UV bands are much broader than the photonic transition event. This is because vibration levels are superimposed on UV.

Ultraviolet SpectroscopyUltraviolet Spectroscopy

200-400 nm photons excite electrons from a 200-400 nm photons excite electrons from a bonding orbital to a bonding orbital to a * antibonding orbital.* antibonding orbital.

Conjugated dienes have MO’s that are closer in Conjugated dienes have MO’s that are closer in energy.energy.

A compound that has a longer chain of A compound that has a longer chain of conjugated double bonds absorbs light at a conjugated double bonds absorbs light at a longer wavelength. =>longer wavelength. =>

ChromophorChromophoree

ExampleExample SolventSolvent maxmax (nm) (nm) maxmax Type of Type of transitiotransitio

nn

AlkeneAlkene nn-Heptane-Heptane 177177 13,00013,000 **

AlkyneAlkyne nn-Heptane-Heptane 178178

196196

225225

10,00010,000

2,0002,000

160160

**

__

__

CarbonylCarbonyl nn-Hexane-Hexane

nn-Hexane-Hexane

186186

280280

180180

293293

1,0001,000

1616

LargeLarge

1212

nn**

nn**

nn**

nn**

CarboxylCarboxyl EthanolEthanol 204204 4141 nn**

AmidoAmido WaterWater 214214 6060 nn**

AzoAzo EthanolEthanol 339339 55 nn**

NitroNitro CHCH33NONO22IsooctaneIsooctane 280280 2222 nn**

NitrosoNitroso CC44HH99NONO Ethyl etherEthyl ether 300300

665665100100

2020__

nn**

NitrateNitrate CC22HH55ONOONO22DioxaneDioxane 270270 1212 nn**

C6H13HC CH2

C5H11C C CH3

CH3CCH3

O

CH3CH

O

CH3COH

O

CH3CNH2

O

H3CN NCH3

Absorption Characteristics of Some Common Chromophores

Solvents for UV (showing high Solvents for UV (showing high energy cut-offs)energy cut-offs)

Water 205

CH3CN 210

C6H12 210

Ether 210

EtOH 210

Hexane 210

MeOH 210

Dioxane 220

THF 220

CH2Cl2235

CHCl3 245

CCl4 265

benzene 280

Acetone 300

Various buffers for HPLC, check before using.

Deviation from Beer’s LawDeviation from Beer’s Law

Beer’s law is only valid for low concentration, up Beer’s law is only valid for low concentration, up to 10 mM;to 10 mM;

The intermolecular distances in a given solution The intermolecular distances in a given solution will decrease, eventually reach a point at which will decrease, eventually reach a point at which neighboring molecules mutually affect the neighboring molecules mutually affect the charge distribution of the othercharge distribution of the other affect affect

Chemical processes such as the reversible Chemical processes such as the reversible association-dissociation of analyte molecules, or association-dissociation of analyte molecules, or the ionization of a weak acid in an unbuffered the ionization of a weak acid in an unbuffered solvent.solvent.

Instrumentation limitation-incident beam may be Instrumentation limitation-incident beam may be polychromatic . polychromatic .

-Processes other than analyte absorption result in significant decrease in the power of the incident beam;- Reference cell is used to correct these processes;- Reference cell is often prepared by adding distilled water to an absorption cell; - The reference cell is then placed in the path of the light beam, and the power of the radiation exiting the reference cell is measured and taken as P0 for the sample cell.

Background Correction

• Linear calibration curve;• Nonlinear calibration

Calibration Curves

An Example--PulegoneAn Example--Pulegone

Frequently plotted as

log of molar

extinction

So at 240 nm, pulegone has

a molar extinction of

7.24 x 103

Antilog of 3.86

O

ExampleExample

A solution containing a compound of A solution containing a compound of formula weight 280 g/mol absorbed formula weight 280 g/mol absorbed 65.0% of the light with 450 nm 65.0% of the light with 450 nm wavelength in a 2.00 cm cell at a wavelength in a 2.00 cm cell at a concentration of 15.0 × 10concentration of 15.0 × 10-3-3 g/L. g/L. Calculate its molar absorptivity at 450 Calculate its molar absorptivity at 450 nm. nm.