Modern methods in techniques of analytical

chemistry: Spectroscopic techniques.

1) Describe the absorption of radiation by molecules and its relationship to molecular structure. 2)Make quantitative calculations, relating the amount of radiation absorbed to the concentration of an absorbing analyte.

3)Describe the instrumentation required for making measurements.

Scope:

The Electromagnetic Radiation• Light is a form of electromagnetic radiation.

Electromagnetic radiation can be considered as a form of radiant energy that is propagated as a transverse wave.

It vibrates perpendicular to the direction of propagation, imparts a wave motion to the radiation

Wavelength, λ = c/ v

Wavenumber, ṽ = v/c =1/λ

Wavelength ,λ is the distance between the neighboring peaks of two wave.

Frequency, v (nu) the number of cycles passing a fixed point per unit time.

Frequency is measure in Hertz, 1 Hz= 1 s-1.

Wavenumber, ṽ (nu tilde) the number of complete wavelengths in a given length.

e.g.: A wave number of 5 cm-1 indicates there are 5 complete wavelength in 1 cm.

Where c = velocity of light (3 x 1010 cm/s)

Electromagnetic radiation possesses certain amount of energy

E = hν = hc/λ

E = energy of a photonh = Planck’s Constant (6.62 x 10-34 joule second (J-s))

It is apparent that the shorter the wavelength or the greater frequency, the greater energy.

In spectrometric methods, the sample solution absorbs electromagnetic radiation from appropriate source, and the amount absorbed is related to the concentration of analyte in the solution.

109 1

07 1

05 1

03 1

01 1

0-1

10-3

10-5

10-7

10-9

10-1

1

gamma

X-rays

Ultra Violet

Infra red

microwave

Radio waves

500

600

700

Violet, indigo, blue

Green, yellow

Orange, red

Electromagnetic Spectrum

How does matter absorb radiation?

There are 3 basic process:1)Rotational transition- molecules rotate, absorb radiation and raised to higher

rotational energy level

2) Vibrational transition- Atoms or group of atoms within molecule vibrate

relative to each other, absorb radiation and raised to higher vibrational energy level.

3) Electronic transition- Electrons of molecule raised to higher electron energy.

Wavelenghts and Color

Wavelength of Maximum

absorption (nm)

Color absorption Color Observed

380-420 Violet Green-yellow

420-440 Violet-blue Yellow

440-470 Blue Orange

470-500 Blue-green Red

500-520 Green Purple

520-550 Yellow-green Violet

550-580 Yellow Violet-blue

580-620 Orange Blue

620-680 Red Blue-green

680-780 Purple Green

Irradiance or radiant power, P (Wm-2), is the energy per second per unit area of the beam of light.

Schematic of a simple spectrometer.

Derivation of Beer’s LawDerivation of Beer’s Law

Irradiance In, P0Irradiance In, P0

Irradiance Out, P

Irradiance Out, P

PathlenghtPathlenghtReadout

Transmittance is T=Po/P =10-kbTransmittance is T=Po/P =10-kb

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

Po = power of incident lightP= power of transmitted light

Putting transmittance in logarithmic form:

log T = log P/Po = -kb

Similar law holds for the dependence of T on concentration, c

T=Po/P =10-k’c and log T = log P/Po = -k’c

Combining these two laws,

T = P/P0 =10-abc (Beer’s Law)

Where ε is the analyte’s molar absorptivity. ε = a x molecular weight

log T = log P/P0 = -abc

A = -log T = log 1/T = log P0/P = abc = log 100 – log %T

Continue…..

Where

A = absorbance, a = analyte’s absoptivity b = pathlenght through the material; c= concentration,

OR A = εbc

Spectroscopy Nomenclature Recommended Name Unit

Absorbance (A) -

Absorptivity (a) cm-1 g-1 L

Pathlength (b) cm

Transmittance (T) -

Wavelength (λ) nm

Concentration (c) moles per liter (moles L-1)

Example 1:

A sample in a 1.0-cm cell is determine with a spectrometer to transmit 80% light at certain wavelength. If the absorptivity of this substance at this wavelength is 2.0, What is the concentration of the substance?

Solution:

The percent of the transmittance is 80%, and so T = 0.80.

log 1/T =abc

log 1/0.80 = 2.0 cm-1g-1L x 1.0 cm x c

log 1.25 = 2.0 g-1L x c

c = 0.10/2.0 = 0.050 gL-1

Example 2:

A solution containing 1.00 mg ion (as thiocyanate complex) in 100 mL was observed to transmit 70.0% of the incident light compared to an appropriate blank. (a)What is the absorbance of the solution at this wavelength?(b) What fraction of light would be transmitted by a solution of iron four times as concentrated.

Solution:

(a)T = 0.700 A = log 1/0.700 = log 1.43 = 0. 155

(b) 0.155 = ab (0.0100 g/L) ab = 15.5 L/g From A= abc A = 15.5 L/g x (4x0.1000 g/L) = 0.620

log 1/T = 0.620 T = 0.240

The absorbance of the new solution could have been calculated more directly:

Beer’s Law Assumption/Limitation

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

• The analyte must not be participate in a concentration dependent equilibrium

This isn’t good technique for many weak acid systems, as dilution increases dissociation and HA and A- probably don’t have the same absorbance.

Beer’s Law Assumption/LimitationProblems: Calibration curves are found to be nonlinear because occuring of deviation. Deviation from linearity are divided into three categories:

1)Fundamental : Law is valid for low concentration analyte. At higher concentration, there will be interaction between particle of analyte that may change the value of ε.

2) Chemical: when the absorbing species is involved in an equilibrium reaction.

HA ↔ H+ + A-

HA will absorbs the wavelength and contribute to ε and C value. However, if the equilibrium shifts to right, less HA available for absorption and will result in non linearity of the curve.

3) Instrumentation: 2 principal limitation: i) Stricly valid for purely monochromatic instrumentation. ii) The effect of ‘leakage’ light from imperfections within wavelength selector. This phenomena is called stray radiation.

Deviation from beer’s law:continue…

From the beer’s law, the absorbance against the conc. A straight line passing through origin is obtained (linear graph)However, deviation might occurs. Deviation is due to the

following factors:- A foreign substance having colour particle may affect the

absorption & extinction coefficient. Deviation also occur if colored solute ionized or dissociates in

the solution; e.g.- benzyl alcohol in chloroform Due to the presence of impurities that fluoresce or absorb at

the absorption wave length. If monochromatic light is not used deviation may occurs. If width of the slit is not proper. If the solution species undergoes polymerization

Block of Diagram - Spectrometer

Source Monochromator Sample DetectorReadout (meter or recorder)

Sources : Tungsten lamp (visible), hydrogen or deuterium discharge tube (ultraviolet), hot wires, light bulb or glowing seramic (IR), laser Monochromator: prism, difraction grating, optical filters.Sampel cell: cuvets, KBr, UV/IR quartz.Detector: phototube, photomultiplier tube, spectrophotometer (UV) thermocouples, bolometers (IR)

Two types of Monochromator

Grating: • A fundamental property of gratings is that the angle of deviation of all but one of the diffracted beams depends on the wavelength of the incident light. • Therefore, a grating separates an incident polychromatic beam into its constituent wavelength components, i.e., it is dispersive. Because of their dispersive properties, gratings are commonly used in monochromators and spectrometers.

Two types of Monochromator

Prism: •Electromagnetic radiation is refracted because index of refraction of prism material is different from air.•Shorter wavelengths are refracted more than longer wavelength•The effects of refraction is to ‘spread’ the wavelength apart into different wavelength•By rotating the prism, different wavelength can be made to pass through an exit slid and to the sample

Types of Instruments

•Single–beam : instrument has one sample holder, you must swap blank and sample.• less expensive, sophisticated instrument and excellent result can be obtained.• Modern single beam instrument are smaller, faster, more sensitive and more economical then the older versions.

Spectroscopic – Single Beam

Spectroscopic – Double Beam

•Double-beam: instrument splits light output between two holders so you can measure blank and sample.•Normally use as a recording instrument

26

Introduction to UV-Visible Absorption Spectroscopy

Applications

Protein

Amino Acids (aromatic)

Pantothenic Acid

Glucose Determination

Enzyme Activity (Hexokinase)

Pyridoxine Vitamin B12 Niacin Metal Determination (Fe) Fat-quality Determination (TBA) Enzyme Activity (glucose oxidase)

UV-Vis Spectrophotometer

1) The spectrophotometer should be turned on at least 30 minutes before calibration. Adjust the wavelength to the required setting.

2) With the 'Press for Zero Set' button depressed adjust the 'Zero Adjustment' dial until the meter needle is aligned with 0 % transmission (lower scale).

3) Then place an appropriate blank in a cell in the cell holder and adjust the '100 % Transmission' dial until the meter needle is aligned with 100 % transmission (lower scale).

4) The blank can be replaced with the sample(s) of interest and the absorbance read from the upper scale.

5) This procedure should be made for each wavelength of interest. The spectrophotometer should remain on until all the required readings are made.

Calibration of the Spectrophotometer

Example :

ppm Zn Absorbance

0.00 0.000

1.00 0.193

2.00 0.344

3.00 0.565

4.00 0.727

unknown 0.270

1) Plot a graph of absorbance versus the concentration of standard Zn on a graph paper.2) Recognize a linear regression of the plot.3) Calculate the concentration of Zn in the unknown.

Regression line and equation

Regression line and equation

Calculate the concentration of Zn in the unknown.

Solution: y = 0.1816 x + 0.0006 x = 0.1816 (0.270) + 0.0006 = 0.0496 ≈ 0.05

Concentration Vs. Absorbance

absorbanceRegression line (absorbance)

Fourier Transform Infra Red-FTIR

FTIR

The IR source will split into 2 path

The IR source will split into 2 path

Instead of grating monochromators, FTIR uses interferometer to obtain a spectrum

Functional Groups Vibration mode Frequency (cm-1)Alkane

C-H strechching 3000-2850 -CH2

CH2 (long chain)bending 1465

700

-CH3 bending 1450 dan 1375 Alkene

C=C bending 1660-1600 =C-H bending

1000-650

Carboxylic AcidC-OH strechching 3400-2400 C=O strechching 1730-1700C-O strechching 1320-1210

AlcoholO-H

C-O-Hstrechching

bending3400-33001440-1220

C-O strechching 1260-1000Ester

strechching

strechching

1750-1735

1740-1715

EpoxideC-O strechching 1300-1000

Ring strechching

Between 880 and 750 Beyween 950 and 815

1280-1230

R OR

O

C CH

O

ORH

H

O

Correlation Table- FTIR

Example FTIR Spectrum

OH

3456 cm-1

CH

2925 cm-

1

CH

2854 cm-

1

CH2

1465 cm-

1

CH3

1374 cm-

1

CH2

722 cm-1

=C-H

1035 cm-

1

C=C

1641 cm-

1

C-O

1246 cm-

1

C=O

1736 cm-

1

Atomic Spectrometric Methods

Atomic absorption spectrophotometry (AAS)

-To measure metal content (refer to periodic table) e.g. copper, iron, natrium, magnesium, manganese, zinc, etc.- cannot detect non-metal compound

Hollow cathode lamp- Source of the analytical light line for the element of interest - Give a constant and intense beam of that analytical line

Nebulizer - Suck up liquid sample at a controlled rate - Create a fine aerosol spray for introduction into the flame - Separate large and fine aerosol- Mix the aerosol, fuel and oxidant thoroughly before combusted in slotted burner which provide a flame

• Flame - Create atoms of the element of interest, Feo, Cuo, Zno, etc.

• Monochromator - Isolate the analytical line photons passing through the flame - Remove scattered light of other wavelengths from the flame- In doing this, only a narrow spectral line impinges on the PMT.

Photomultiplier tube (PMT)- This is the detector. The PMT determines the intensity of photons of the analytical line exiting the monochromator. -The PMT is the most commonly used detector for atomic absorption spectroscopy. -However, solid state detectors are now replacing conventional vacuum-type photomultipliers. -High tech electronics amplify, filter, and process the electrical signal, using a series of chips and microprocessors, transmitting the result to an internal or external computer which handle all data-handling and display

Graphite Furnace AA-The graphite furnace is an electrothermal atomiser system that can produce temperatures as high as 3.000°C. -The heated graphite furnace provides the thermal energy to break chemical bonds within the sample held in a graphite tube, and produce free ground state atoms.- Ground-state atoms then are capable of absorbing energy, in the form of light, and are elevated to an excited state. -The amount of light energy absorbed increases as the concentration of the selected element increases. -Flame AA can only analyse solutions, but graphite furnace can accept very small absolute quantities of solution, slurry or solid samples.

How to run your sample?

Chapter 8&9 - 41

ANALYTICAL TECHNIQUES

• Beer's law, A = k×C, not always true making a calibration curve necessary.

• Effect from matrix:e.g. high viscosity, chemical reaction with analyte

• Standard addition method is used to minimize the effects from the matrix

Example of standard addition plot

Concentration of analyte in sample = intercept at y-axis

slope

AAS - ApplicationQualitative analysis

- to identify an element. - However, due to interferences, atomic emission is far more convenient to qualitative emission.

Quantitative analysis

-Quantitative analysis with spectroscopic methods is based on the Beer-Lambert Law.