UV-VIS SpectroscopyUV-VIS Spectroscopy An obvious difference between certain compounds is

their color. The human eye is functioning as a spectrometer

analyzing the light reflected from the surface of a solid or passing through a liquid.

Although we see sunlight (or white light) as uniform or homogeneous in color, it is actually composed of a broad range of radiation wavelengths in the ultraviolet (UV), visible and infrared (IR) portions of the spectrum.

• The component colors of the visible portion can be separated by passing sunlight through a prism, which acts to bend the light in differing degrees according to wavelength.

• Visible wavelengths cover a range from approximately 400 to 800 nm. The longest visible wavelength is red and the shortest is violet.

• The wavelengths of what we perceive as particular colors in the visible portion of the spectrum are displayed

Violet:   400 - 420 nmViolet:   400 - 420 nm Indigo:   420 - 440 nmIndigo:   420 - 440 nm Blue:   440 - 490 nmBlue:   440 - 490 nm Green:   490 - 570 nmGreen:   490 - 570 nm Yellow:   570 - 585 nmYellow:   570 - 585 nm Orange:   585 - 620 nmOrange:   585 - 620 nm Red:   620 - 780 nmRed:   620 - 780 nm

Color wheel: complementary colors are diametrically opposite each other. Thus, absorption of 420-430 nm light renders a substance yellow, and absorption of 500-520 nm light makes it red. Green is unique in that it can be created by absoption close to 400 nm as well as absorption near 800 nm.

When white light passes through or is reflected by a colored substance, a characteristic portion of the mixed wavelengths is absorbed. The remaining light will then assume the complementary color to the wavelengthsabsorbed.

• Electromagnetic radiation such as visible light is commonly treated as a wave phenomenon, characterized by a wavelength or frequency.

• Wavelength is the distance between adjacent peaks, and may be designated in meters, centimeters or nanometers (10-9 meters).

• Frequency is the number of wave cycles that travel past a fixed point per unit of time, and is usually given in cycles per second, or hertz (Hz).

The visible spectrum constitutes but a small part of the total radiation spectrum.

This electromagnetic spectrum ranges from very short wavelengths (including gamma and x-rays) to very long wavelengths (including microwaves and broadcast radio waves). The following chart displays many of the important regions of this spectrum, and demonstrates the inverse relationship between wavelength and frequency.

The Electromagnetic SpectrumThe Electromagnetic Spectrum

The energy associated with a given segment of the spectrum is proportional to its frequency.

* = c

E = h = frequency

= wavelength

C = velocity of light ( 3 x 1010 cm / sec )

E = energy

h = Planck’s constant (6.626068 × 10-34 m2 kg / s )

Wavelength * frequency =velocity

Interaction between light and matter:Interaction between light and matter:When radiation interacts with matter a number of processes can occur including:

reflection scattering absorbance fluorescence/phosphorescence (absorption and re-

emission)

photochemical reaction

When measuring UV-Visible spectra we want only the absorbance process to occur. Any of the other processes will adversely affect the precision of our measurements.

Electronic SpectraElectronic Spectra

Absorption occurs due to the fact that all

molecules possess electrons which can be

exited or raised to higher energy level. Many

can be excited by radiation of UV or visible

wavelengths but others are only excited by

vacuum UV.

Electronic SpectraElectronic Spectra The amount of energy a molecule can have in each

form is not a continuum but a series of levels or states.

The photons of UV and Visible light have sufficient energy to cause transitions between the different electronic energy levels of bonds (double bonds) in molecules, whereas photons of infra-red radiation have sufficient energy to cause transitions between vibration energy levels.

The Energy difference between the electronic energy levels is given by the equation:

E = h The absorption of energy excites the electron from the ground state to an electronic excited state

Any change in electronic energy is accompanied by vibration and rotational energy levels.

Chemical Structure and UV Chemical Structure and UV absorbanceabsorbance

The absorbance energy depends on the nature of the bonds within a molecule. Electrons in organic molecules may be in :Strong bond ,weaker bond or non bonding n. When energy is absorbed all these types of electrons can be elevated to excited anti-bonding states , represented with an asterisk:

Ene

rgy

n

Some chromophores and the wavelengths of their Some chromophores and the wavelengths of their absorbance maxima.absorbance maxima.

Chromophore Formula Example (nm)Carbonyl (ketone) RR'C=O Acetone 271Carbonyl (aldehyde) RHC=O Acetaldehyde 293Carboxyl RCOOH Acetic Acid 204Amide RCONH2 Acetamide 208Ethylene RCH=CHR Ethylene 193Acetylene RC=CR Acetylene 173Nitrile RC=N Acetonitrile <160Nitro RNO2 Nitromethane 271

*The position of the absorbance maximum of a chromophore is not fixed, as it is to some extent dependent upon the molecular environment of the chromophore.

Spectroscopy is the study of the absorbance and emission of electromagnetic radiation (light) by matter.

The collection of frequencies absorbed by a sample is its absorption spectrum.

SpectroscopySpectroscopy

AbsorbanceAbsorbance

I0 Incident radiation I Transmitted radiation

Absorbance = log (I0 / I)

path-length

sample

Transmittance = 100* ( I/ I 0 )

Absorbance may be presented as

transmittance (T = I/I0) or

absorbance (A= log I0/I).

If the sample compound does not absorb light at a specific wavelength, I() = I0()

I=I0 T = 1 and A = 0

If the sample compound absorbed light, I()< I0 ()

I<I0 T< 1 and A > 0

Beer's law :Beer's law :

The amount of light absorbed is proportional to the number of absorbing molecules through which the light passes.

The result of plotting absorbance against concentration is shown below.

A= A= b c b c

The extinction coefficientThe extinction coefficientIs a characteristic of a given substance at a specific wavelength.

A= absorbance, C = sample concentration in moles/literb = length of light path through the cuvette in cm

A= A= b c b c==b Cb C

A solution of 0.249 mg of the unsaturated aldehyde in 95% ethanol (1.42 • 10-5 M) ;1 cm cuvette. Using the above formula, ε = 36,600 for the 395 nm peak, and 14,000 for the 255 nm peak. Note that the absorption extends into the visible region of the spectrum, so it is not surprising that this compound is orange colored.

                                                                                                 

            

Calibration of a single component Calibration of a single component /single wavelength/single wavelength Determine the optimum wavelength Measure a blank spectrum (I0) Measure the Absorbance of the component spanning the

entire concentration range. By linear regression determine the best line that goes

through the data.

Abs

orba

nce

Wavelength

NH3 Calibration data - spectra . OMA-300 , 60 cm cell, int 370, avg 16

0

0.2

0.4

0.6

0.8

1

1.2

195 200 205 210 215 220 225 230

Wavelength (nm)

Abs

orba

nce

(AU

)

NH3 Calibration data - spectra . OMA-300 , 60 cm cell, int 370, avg 16

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

210 210.5 211 211.5 212 212.5 213 213.5 214

Wavelength (nm)

Abs

orba

nce

(AU

)

NH3 calibration curve - OMA-300 60 cm flow cell int 370 avg 16

y = 854.19x

R2 = 0.9997

0.000

100.000

200.000

300.000

400.000

500.000

600.000

0 0.1 0.2 0.3 0.4 0.5 0.6

Absorbance (AU)

NH

3 C

on

cen

trat

ion

PP

M

Multi componentsMulti componentsPrinciple of Additivity: Principle of Additivity: The absorbance at any wavelength of a mixture is equal to the sum of the absorbance of each component in the mixture at that wavelength. Absorbance at Wavelength 1 =

Absorbance of component X at Wavelength 1 +Absorbance of component Y at Wavelength 1

0.0663

0.26900.3353

0

0.2

0.4

0.6

0.8

1

1.2

220 240 260 280 300 320

Wavelength

Ab

sorb

ance

SO2 1%

H2S 1%

H2S 1% +SO2 1%

Total Absorbance at 230nm = H2S abs at 230nm + SO2 abs at 230nm 0.3353 = 0.0663 + 0.2690

0

0.2

0.4

0.6

0.8

1

1.2

1.4

220 230 240 250 260 270 280 290 300 310 320

nm

AU

SO2

H2S

total

0

0.2

0.4

0.6

0.8

1

1.2

1.4

220 230 240 250 260 270 280 290 300 310 320

nm

AU

H2S 1% +SO2 1%

H2S 0.25% +SO2 1.25%

Least Squares FitLeast Squares Fit

A way to reduce sensitivity to random noise is to use more data points.

The least squares method uses data points over a wavelength range and is an "over-determined" calculation, i.e.. more data points are used than are necessary.

Concentrations of components are determined by minimizing the sum of the squares of absorbance differences between measured and calculated spectra.

A(1) = AH2S( +ASO2(1) =

H2S 1) CH2S b + SO21) CSO2 b+ error

A(2) = AH2S( +ASO2(2) =

H2S 2) CH2S b + SO22) CSO2 b+ error

A(n) =

AH2S(n +ASO2(n) =

H2S n) CH2S b + SO2n) CSO2 b+ error

A(1) = H2S 1) CH2S b + SO21) CSO2 b+ error

A(2) = H2S 2) CH2S b + SO22) CSO2 b+ error

A(n) = H2S n) CH2S b + SO2n) CSO2 b+ error

Instead of using two data points at 210 and 230 nm we will use data points at 1 nm intervals over the range 200 - 240 nm. Assuming random measurement error of ±10% as before, the actual measured spectrum could be as shown below.

Summary of Terms

Absorbance = log (I0 / I)

Transmittance = 100* ( I/ I 0 ) b c

Extinction coefficient =a characteristic of a given substance under a precisely defined set of conditions of wavelength, solvent, temperature and other parameters.

Least squares method uses data points over a wavelength range and is an "over-determined" calculation

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

230 240 250 260 270 280 290

nm

Ab

sorb

ance

Benzene

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

230 240 250 260 270 280 290

nm

Ab

sorb

ance

Toluene

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

230 240 250 260 270 280 290

nm

Ab

sorb

ance

Aromatics

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

190 200 210 220 230 240 250

nm

AU

NH4 50ppm

NH4 100ppm

NH4 500ppm

Formate500ppmFormate1000ppm

Formate1500ppm

Fig 1: shows the absorbance spectra of samples no 1-6 (see the above table)

0

0.01

0.02

0.03

0.04

0.05

0.06

200 210 220 230 240 250 260 270 280

nm

AU

H2S

THT

CS2

COS

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

1.80

500 600 700 800 900 1000 1100

Wavelength (nm)

Abs

orba

nce

(AU

)

Cu+2

-2

0

2

4

6

8

10

12

14

850 860 870 880 890 900 910 920 930 940 950

wavelength (nm)

abs

orba

nce

(A

U)

1_BHC

2_BHC

3_FAO_SCN

4_SCN-RAW

9-ALKYATE

12-FAWLEY XYLENE

11-POWERFORMERFEED

10-RAFFINATE

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

210 215 220 225 230 235 240 245 250

wavelength (nm)

Ab

sorb

ance

(A

U)

H2S

240 260 280 300 320 340 360 380 400Wavelength

Ab

sorb

ance

Dl

Cl2

0.00

0.02

0.04

0.06

0.08

0.10

0.12

190 200 210 220 230 240 250Wavelength (nm)

Abs

orba

nce

(AU

)NO 20.4 PPMNO 26 PPMNO 30.2 PPMNO 35 PPMNO 39.5 PPM

0.00

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

0.10

190 200 210 220 230 240 250Wavelength (nm)

Abs

orba

nce

(AU

)

NH3 46 PPMNH3 35 PPMNH3 21 PPMNH3 15.3 PPMNH3 8.2 PPM

0.000

0.005

0.010

0.015

0.020

0.025

0.030

190 290 390 490 590

Wavelength (nm)

Abs

orba

nce

(AU

)NO2 40.8 PPMNO2 36.2 PPMNO2 30.2 PPMNO2 26.9 PPm

0.000

0.001

0.002

0.003

190 200 210 220 230 240 250Wavelength (nm)

Abs

orba

nce

(AU

)

NH3 1 PPMNO 1 PPMNO2 1 PPM

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

190 210 230 250 270 290 310Wavelength (nm)

Abs

orba

nce

(AU

)SO2 500PPMSO2 375PPMSO2 250 PPMSO2 201 PPMSO2 112 PPm

SpecificationsSpecificationsThe use of microprocessors, computers, RAM and disk memory and the advent of diode-arrays make the modern spectrophotometer very different to its ancestors.

The classical specifications for optical performance of scanning spectrophotometers are not necessarily directly relevant to the problem-solving capability of the diode-array spectrophotometer but they are extensively used so it is important to understand them and their significance.

Wavelength AccuracyWavelength Accuracy

The maximum difference between the true wavelength and the wavelength set on the instrument.

Wavelength accuracy is important when results or methods obtained or generated on one instrument are to be used or set up on another instrument.

Usually calibration standards are run on the instrument and then unknowns are measured on the same instrument.

Wavelength accuracy is usually measured using standards which have narrow, well-defined absorbance bands.

Wavelength accuracy < +/- 0.5nm

Measuring Wavelength accuracyMeasuring Wavelength accuracy

Wavelength ReproducibilityWavelength Reproducibility

Indicates the maximum difference between the true measurement wavelength when the same wavelength is set on the instrument at different times.

When measurements are made at absorbance maxima, wavelength reproducibility has little effect on the precision of absorbance measurements, but on the side of a band, as shown , large errors can be created.

a. neither accurate nor preciseb. precise but not accuratec. accurate but not precised. precise and accurate

Photometric AccuracyPhotometric AccuracyIndicates the maximum difference between the true absorbance of a sample and that measured by the spectrophotometer.

The photometric accuracy should be determined at different absorbance levels (checks for linearity) and at all wavelengths.

In practice photometric accuracy is usually specified at about 1.0 A at one wavelength.

Photometric accuracy = +/- 0.005AU

Neutral density glass filters : give an approximately constant absorbance at all wavelengths but currently-available standards are calibrated at 440 nm Metal-on-quartz filters which are calibrated in the UV region are available, but there are often problems with inter-reflection errors.Potassium dichromate in dilute sulfuric acid: but not stable, so must be made fresh each time, and preparation errors can easily occur.

There are no ideal standards for photometric accuracy currently available.

Measuring photometric accuracyMeasuring photometric accuracy::

Noise Noise (Photometric Precision)(Photometric Precision)

Short term (seconds) variation in the measured absorbance.

It arises because of fluctuations in lamp intensity and noise in the detector and associated electronics. It is specified either as peak-to-peak or rms (root mean square) noise..

Noise becomes increasingly important as the measured absorbance decreases. It defines the limit of sensitivity of an instrument. For this reason noise is usually measured and specified at 0 A i.e.. with no sample.

Measuring noise levelsMeasuring noise levelsNoise level is measured by the following test: The absorbance spectra of air is measured with an integration time of 0.5 sec over 60 seconds. An RMS calculation of the noise at single wavelengths is used , in order to pass this test the following condition must be fulfilled:

whereR - measured absorbance valueR - average of the absorbance values to 5 points either side of R n - total number of absorbance values read

( )( ) .R Rn AU

2

1 0002

DriftDrift

Long term (hours) variations in measured absorbance.

For single beam instruments the drift is important because it is an indicator of how frequently the blank must be re-measured to ensure a desired precision.

Most instruments will drift significantly after switching on as the instrument warms up so drift is usually specified after an appropriate warm-up time (usually 1 hour).

Measuring DriftMeasuring Drift

To measure the change in the zero absorbance per time. The spectra of air is measured every 5 seconds during one hour. The stability is the difference between the maximum and minimum absorbance at a specific wavelength. In order to pass this test the following condition needs to be fulfilled:

(Environment temperature should remain ±1°C during this test).

A A AUmax min . 0001

Stray LightStray LightStray light is light which reaches the detector which has a wavelength other than that is wanted.

The term stray light is used to cover both true "stray" light and "false" light.

True stray light is random and varies from instrument to instrument and even from measurement to measurement. It arises primarily from imperfections in or dirt on the optical components.

Measuring stray lightMeasuring stray lightUsing a blocking filter that transmits light above a certain wavelength and blocks all light below that wavelength. Any measured absorbance at wavelengths below the blocking wavelength is due to stray light.

NaNO2 340nm <0.05NaI 220nm <0.07%KCl (1.2%) 200 nm < 1%

The transmittance at the specific wavelengths must be within the stray light limit for the instrument to pass the test.

ResolutionResolution

The ability of the spectrophotometer to resolve adjacent absorbance bands.

Resolution depends upon two factors 1. the instrumental bandwidth 2. sampling interval of the signal recording process as shown below.

For a conventional scanning spectrophotometer the resolution is usually the same as the instrumental bandwidth because the sampling interval is significantly smaller than instrumental bandwidth.

For a diode-array instrument the sampling interval is usually the same as the instrumental bandwidth

Resolution is important when insufficient the measured absorbance will be influence as below. .

The spectrum a, measured with 1 nm resolution, is the "true" spectrum. As the resolution is decreased the effect is to flatten the spectral features until, eventually, the two bands merge into one.

The instrumental band width should be 5 times smaller than the natural bandwidth of the absorbance band being measured to get a good approximation to the true absorbance values (<1% error).

Measuring the resolutionMeasuring the resolutionBy measuring the absorbance spectrum of a solution of 200ppm toluene in hexane. The ratio between the absorbance at 269nm and 266nm is used.

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

230 240 250 260 270 280 290

nm

Ab

so

rb

an

ce 266nm266nm 269mn269mn

AbsAbs

at nm

at nm

max

min

.269

266

15>

Caffeine with salicylic acid impurityCaffeine with salicylic acid impurity

Internal referencingInternal referencing

Is a technique that is used to reduce noise and drift. This may be due to sample problems or to instrumental drift.

If the shift is the same at all wavelengths then by subtracting a "reference" wavelength, as near as possible to the baseline, from the "analytical" wavelength the shift can be eliminated from the measurement as shown:

Internal referencingInternal referencing

High SensitivityHigh Sensitivity

High light throughput result in low noise Wavelength averaging : The signal-to-noise ratio improves as

more data points are averaged. Time averaging: ( conventional spectrophotometers acquire

data serially and diode-array spectrophotometers acquire data in parallel, equivalent scan times are 30.0 and 0.1 sec respectively). noise can be theoretically reduced by the square root of number of measurements With a diode-array spectrophotometer, longer scan times are practical (with a conventional spectrophotometer, they rapidly become impractical because they are so long that productivity is very low and drift errors may become significant.)

Measurement StatisticsMeasurement Statistics

Measurement times of 0.5 or 1.0 sec are used and 5 or 10 spectra are measured and averaged together.

In addition, the standard deviation for each data point is calculated; this is a measure of the reliability of the data point.

The statistical information on the reliability of data points is important for "chemometric" techniques such as least squares with maximum likelihood.