UV Vis Principle

7/26/2019 UV Vis Principle

1/38

UV-VISIBLEUV-VISIBLE

SPECTROPHOTOMETRYSPECTROPHOTOMETRY


2/38

2

ObjectivesObjectives To understand the process of absorption of light by molecules To understand the relationships between Absorbance,

Transmittance, path length and analyte concentration (Beer-Lambert Law).

To be familiar with the quantitative applications of the Beer-LambertLaw in the multi-component analysis of mixtures, and in standard

addition. To be aware of the chemical and instrumental limitations of the

Beer-lambert Law.

To be aware of the major components of the instruments used for

measurement of absorbance. To understand the experimental requirements for

spectrophotometric measurements.

To be familiar with typical applications of spectrophotometric

analysis.


3/38

3

1. Introduction1. IntroductionSpectrophotometry - measurement ofSpectrophotometry - measurement of

light absorption to quantifylight absorption to quantifyconcentration ofconcentration of analyteanalyte

1.1 Light Absorption1.1 Light Absorption

Absorption of a photon by a molecule results in an excited stateexcited state

Different wavelengths of light can be absorbed, which cause

different forms of excitation


4/38

4

1. Introduction1. Introduction Absorption of

UV and visible

light by a

molecule

causes

electronic

excitation1020 1018 1016 1014 1012 108

Cosmic

rays -rays X-rays UV

Visible

Infrared MicrowaveR

adio

Electronicexcitation

Bond breakingand ionization Vibration Rotation

Visible Spectrum

400 500 600 700


5/38

5

1.1. IntroductionIntroduction

Bonding in organic molecules is based on overlap betweenand

atomic orbitals.Electrons inElectrons inandandorbitalsorbitalscan be excited to thecan be excited to theantibonding** andand**orbitalsorbitals- involves absorption of different energies/wavelengths.- involves absorption of different energies/wavelengths.


6/38

6

Electronic energy levels ofpolyatomic molecules

* (antibonding)

* (antibonding)

n (non-bonding)

(bonding) (bonding)

1.1. IntroductionIntroduction * and * transitions aremore likely than n * and n *

Stronger absorptions occur forthese transitions.

Wavelength and intensity ofabsorption is affected by:

molecular geometry ofmolecule

types of substituent groups

natureof solvent (polarity)

The part of the molecule whichabsorbs light is called thechromophorechromophore..


7/38

7

1. Introduction1. Introduction1.2 Complementary Colours1.2 Complementary Colours

When white light is absorbed by a chromophore, the eye detect

the colours that are notnot absorbed. This is called the

complementary colour to the colour absorbed.

Determination of concentration depends on detection of change

in colour intensity (absorption) at a particular wavelength.

ofmaximumabsorption

Colour Absorbed Colour Observed

380-440 violet-blue green-yellow440-500 blue-green orange-red

500-580 green-yellow violet-blue

580-680 orange-red blue-green

680-780 purple green


8/38

8

2. Colorimetric Analysis2. Colorimetric Analysis2.1 Photometric measurement2.1 Photometric measurement

(a)visual comparison using colour standards

P Po


9/38

9

2. Colorimetric Analysis2. Colorimetric Analysis(b) Colorimeter/Photometer

Filters used to select narrow wavelength

Detection with photosensing device

PPo

Filter

wheel

Photodetector


10/38

10

2. Colorimetric Analysis2. Colorimetric Analysis2.22.2 SpectrophotometricSpectrophotometricAnalysisAnalysis

Spectral bandwidth 1 nm, i.e very monochromatic light.

can operate in visible and UV ranges

Colorimetry and spectrophotometry provide sensitivesensitivemethods ofanalysis, i.e. ppm to ppb ranges.

PPo

PhotodetectorMonochromator


11/38

11

3. Quantifying Light Absorption3. Quantifying Light Absorption

PPo

b

Absorbing solution

of concentration,c.

Reflected

beam

Pr

Pa

3.1 Incident Light3.1 Incident Light

P0 = Pr + Pa + P (1)

Pr 4% for air-glass interface;I.e.use blank to zero P0= Pa+ P (2)


12/38

12

3. Quantifying Light Absorption3. Quantifying Light Absorption3.2 Lamberts Law3.2 Lamberts Law

Formonochromaticlight passing

though a transparent medium:

3.3 Beers Law3.3 Beers LawThe intensity of a monochromatic

light beam decreaseswithconcentrationincrease.

A = logP0

P

dPP

= - k' dc

Pa

P

-dPP

= k' 0

c

dc

lnP0

P = k'c or,

logP0P

= k"c

A = k"c

A = kb

-dP = - k P db

P=Po

P=P

-dP

P

=k

b=0

b=b

db

lnP0

P = kb

Absorbance, A, defined as:


13/38

13

3. Quantifying Light Absorption3. Quantifying Light Absorption3.4 Beer-Lambert Law3.4 Beer-Lambert Law

Combining these gives:

A = a b c Where: a is the absorptivity,absorptivity,b is thepath lengthpath length, and c is the

concentration.concentration.

When c is in mol/L, and b is in cm, A = b c

Where is the molar absorptivitymolar absorptivity.

TransmittanceTransmittancedefined as: T = P/Po Hence:

A = log (1/T) = log(100/%T) = 2 - log(%T)


14/38

14

4. Applications of the Beer-Lambert Law4. Applications of the Beer-Lambert Law4.14.1 Analysis of a singleAnalysis of a single analyteanalyte

At fixed and b, is constant for a given chromophore.

Add chromogenic reagentschromogenic reagents to sample and standards, and measureabsorbance.

Assumes that the chemical matrix of standard is the same as thesample.

Ax

Cx

4

3

2

1

0 2 3

A

A

A

A

A0

C C 1 C C C 4

Concentration


15/38

15

4. Applications of the Beer-Lambert Law4. Applications of the Beer-Lambert Law

6005004003000.0

0.1

0.2

0.3

0.4

0.5

0.6

MN

M+N

Wavelength (nm)

1111 2222

4.2 Analysis of mixtures4.2 Analysis of mixtures

Beer-Lambert Law applies to mixtures of of non-interacting components.

At1: A1 = 1,M bcM + 1,N bcN,at and 2: A2 = 2,M bcM + 2,N bcN

1,M, 1,N, 2,M, 2,N are found from separate calibrations at1and 2. b is aconstant.


16/38

16

4. Applications of the Beer-Lambert Law4. Applications of the Beer-Lambert Law Measure A1and A2 at 1and 2, and solve for cMand cN.

Most accurate when differences between values are the greatest,

i.e choose appropriate values.

Accuracy decreases as the number of components increases.

See Problem 5


17/38

17

4.34.3 Standard AdditionStandard Addition

-Multi Point Method -Multi Point Method Used for samples with complex

matrix chemicalinterference if

calibration method applied.

Measure A of sample+ chromogenicreagent.

Repeat with added increments of

standard to same vol. of sample.


Vol. Std added (mL)0 Vs


18/38

18


Sample Conc = Cx, Vol sample = Vx , Vol std added =Vs. Std Conc =Cs, Total vol = Vt.

Plot A vs Cs, gives straight line: A = + Vs

Where = bCxVx/Vt, and = bCs/ Vt Hence:

Cx= Cs / Vx

Before addition: A =b VxCx

Vt

After addition: A =b VsCs

Vt+

b VxCx

Vt


19/38

19

4. Applications of the Beer-Lambert Law4. Applications of the Beer-Lambert Law4.44.4 Standard Addition-Two Point MethodStandard Addition-Two Point Method

See problem 4

Before addition: A1=b VxCx

Vt

After addition: A2 =b VsCs

Vt +b VxCx

Vt

and hence: Cx =A1Cs Vs(A2- A1)Vx


20/38

20

5. Limitations of the Beer-Lambert Law5. Limitations of the Beer-Lambert Law5.1 Concentration effects5.1 Concentration effects

B-L law applies to dilute

solutions (negligible interactionbetween solute ions).

Higher concentrations ofanalyte (i.e. > 10-2M) or high

electrolyte concentrations,molecular/ionic interactions

reduced light absorption atsome's.

= f(n, refr. index of soln). Ifchanges in n large, deviation

from B-L observed.

Replace with n/(n2+ 2)2inB-L equation

Concentration

Deviation from B-L law

(loss of sensitivity)

Adherence to B-L law


21/38

21

5. Limitations of the Beer-Lambert Law5. Limitations of the Beer-Lambert Law5.2 Instrumental deviations5.2 Instrumental deviations

Occur whenpolychromaticpolychromatic rather than monochromatic light is

used.

For a light beam consisting of 'and , B-L Law becomes:

A = log(P0'+ P0") - log(P0'10-'bc + P0"10

-"bc)

Only when ' " is B-L law obeyed, i.e A = e'bc

Concentration

' "Band A

' < "Band B

'

Wavelength

Band B

Band A

' "

"


22/38

22

6. Experimental Considerations6. Experimental Considerations6.1 Wavelength selection6.1 Wavelength selection

Choose where A is large to obtain best sensitivity.

Choose where dA/d = 0 or is small.

Wavelength

?

Ab

sor

bance


23/38

23

6. Experimental Considerations6. Experimental Considerations6.2 Chromogenic reagents for colorimetric analysis6.2 Chromogenic reagents for colorimetric analysis

Should be stable and pure,

Should not absorb at of measurement, but.

Should react rapidly with analyte to give a stable chromophore.

Absorptivity,,should not be sensitive to minor pH, T, electrolytechanges, etc.

Should be selective.

For UV measurements, often measure particular chromophoredirectly, I.e. no reagent necessary (e.g. nitrate in GDR expt).


24/38

24

7. Instrumentation7. Instrumentation7.17.1 PrinciplePrinciple

7.2 Photometer/Colorimeter7.2 Photometer/ColorimeterSource: Generally visible light (Tungsten)

selector: Filters, simple monochromator

Sample blank: Single beam, no ref.

Detector: Photocell or photodiode.

Output: Microammeter/galvanometer/digital

Advantages / Disadvantages:

Lower cost

Light source fluctuations during measurement.

Light poorly monochromated.

Light

Source

Wavelength

selection

Sample/

Blank

Signal

detection

Data/

Output


25/38

25

7. Instrumentation7. Instrumentation

Single beam spectrometerSingle beam spectrometer


26/38

26



27/38

27


7.37.3 UV-visible SpectrophotometerUV-visible Spectrophotometer

Source: UV-deuterium,visible-quartz halogen

selector: Grating or Prism monochromator, scanning.

Sample/blank:Dual beam, can reference source fluctuations

Detector: Phototube, photodiode, photomultiplier.

Output: Digital, VDU,

Advantages: More sensitive,

UV and visible spectra, Scanning or diode array simultaneous spectral aquisition

better resolution (narrow spectral bandwidth)


28/38

28


Scanning, double beam UV-vis spectrometerScanning, double beam UV-vis spectrometer


29/38

29


Diode array spectrometerDiode array spectrometer


30/38

30


7.4 Probe photometers7.4 Probe photometers

To Detector

Optical fibre bundle

Optical filter

Mirror


31/38

31


7.5 Optical Sensing Devices (7.5 Optical Sensing Devices (OptrodesOptrodes,, OptodesOptodes)) Reflectance spectroscopy

Used for miniaturized in-situmeasurement of glucose, pH

To Detector

Optical fibre bundle


32/38

32

8.Applications8.Applications

Clinical,environmental, industrial and forensic Screening of drugs

e.g. UV spectral identification and quantitation of amphetamines

Determination of cyanide e.g. in Tylenol tablets

Use of UV-vis for detection after liquid, paper or thin-layerchromatographic separation

Collect,

extract,

analyseby UV-VisS

td

1

Std2

Sample

Elution

Flow-through

UV-vis spectro-

photometerPump

Time/Distance

Concentration


33/38

34


7.6 Flow Injection Analysis7.6 Flow Injection Analysis


34/38

35


35/38

36


36/38

37

8. Applications8. Applications

Rapid ScreeningRapid Screening White powder is seized in a police

raid. Is it heroin? UV spectrum of heroin shows maxat

278 nm gives tentative identification.

Allows elimination of other materials

(e.g. starch, sugar, which are commondiluents used in heroin).

Allows thousands of other substancesto be eliminated.

Need to do confirmatory test, eg gaschromatography, or mass spec.


37/38

38


Determination ofDetermination of

ethanol in expiredethanol in expired

breath (Thebreath (TheBreathalyserBreathalyser))

2K2Cr2O7+ 3C2H5OH 8H2SO4

2Cr2(SO4)3 + 2K2SO4+

3CH3COOH + 11 H2O

Measure K2Cr2O7 at 420nm


38/38

39


Screening (Fuel cell) breath testerScreening (Fuel cell) breath testerAbsorbed methanol reacts giving small current.

Approx 3000 tests,

Documents

UV Vis Principle