instrumental chemical analysis - philadelphia university · fieser–woodward rules • empirical...

Instrumental Chemical Analysis

Dr. Ahmad Najjar Philadelphia University

Faculty of Pharmacy

Department of Pharmaceutical Sciences

2nd semester, 2016/2017

Ultraviolet and visible spectroscopy

L6 page 1


Spectrophotometry • Spectroscopy is a general term referring to the interactions (absorption, emission) of

various types of electromagnetic radiation with matter.

L6 page 2

• Spectrophotometry is a method to measure how much a chemical substance absorbs or emits light by measuring the intensity of light (electromagnetic radiation).

• Electromagnetic spectrum refers to the full range of all frequencies of electromagnetic radiation, which is refers to the waves of the electromagnetic field, propagating through space carrying electromagnetic energy.


Spectrophotometry • Electromagnetic radiation (EMR) has been described in terms

of a stream of photons that travel in a wave-like pattern. Each photon contains a certain amount of energy, and all electromagnetic radiation consists of these photons.

L6 page 3

• All electromagnetic radiations travels in a straight line at the speed of light (3 x 108 m/s). The only difference between the various types of electromagnetic radiations is the amount of energy found in the photons.

18

1

1

sm.103.00λνcvacuuminlight ofvelocity

)cm :unit ;cm per waves of number(wavenumberν

Hertz ,sec ,cycles/sec of units(frequencyν

)nm μm, cm,m,: units(wavelengthλ

J.s 6.62x10 constant splanck' ish

νchλ

chνh (E)Energy

34-

ν/νλνc

1/λν

Crest Crest

Trough


Spectrophotometry Electromagnetic radiation in the domain ranging between 180 and 780 nm, has been studied extensively. This portion of the electromagnetic spectrum, designated as the ‘UV/Visible’. Generally provide little structural information but is very useful for quantitative measurements.

L6 page 4


Problem 1: Calculate the wavenumber of a beam of IR radiation with a wavelength of 3μm.

Problem 2: The frequency of a radiation is 3x1012 s-1. Calculate the wavelength of the radiation.

Problem 3: Calculate the energy of 530-nm photon of visible radiation

L6 page 5 Answer: wavenumber = 1/ 𝝀 = 3,333 cm-1

Answer: 𝝀 = 𝒄/ = 10-4 m

Answer: E = h = h 𝒄/𝝀 = 3.75 x 10-19 J

Legend: γ = Gamma rays HX = Hard X-rays SX = Soft X-Rays EUV = Extreme-ultraviolet NUV = Near-ultraviolet Visible light (colored bands) NIR = Near-infrared MIR = Mid-infrared FIR = Far-infrared EHF = Extremely high frequency (microwaves) SHF = Super-high frequency (microwaves) UHF = Ultrahigh frequency (radio waves) VHF = Very high frequency (radio) HF = High frequency (radio) MF = Medium frequency (radio) LF = Low frequency (radio) VLF = Very low frequency (radio) VF = Voice frequency ULF = Ultra-low frequency (radio) SLF = Super-low frequency (radio) ELF = Extremely low frequency(radio)


Spectrophotometric methods

• A group of techniques that relies on the interaction of EMR and matter. There are many types of methods based on either molecular or atomic interactions:

Absorption (excitation) Emission (luminescence, relaxation or deactivation):

Non-radiative Relaxation (vibrational or internal conversion) Radiative photoluminescence (luminescence after absorption) :

Fluorescence: • Resonance fluorescence • Non-resonance fluorescence

Phosphorescence

L6 page 6



Molecular orbital energies:

• Electrons in atoms exist in atomic orbitals (consist of electronic levels only) while electrons in molecules exist in molecular orbitals (consist of electronic, vibrational and rotational levels).

L6 page 7

• Each molecular orbital has energy level represent electronic state S. between each electronic state S there lies several vibrational levels V, themselves also sub-divided into a collection of rotational levels R.



L6 page 8

L6 page 9





L7 page 1



• Each molecular energy state is comprised of an electronic, vibrational and rotational component.

• The energy captured during the course of photon absorption can be expressed as

Etot = Erot + Evib + Eelec

• Promotion of an electron from one occupied orbital (HO) to an unoccupied

orbital (LU) with the apparition of a singlet state giving rise rapidly to a more stable triplet state. This process corresponds to a return of an excited species to the ground state.

a- radiative process b- internal conversion c- inter system crossing

L7 page 2



L7 page 3


The UV/Vis spectrum • UV/Vis spectrometers collect the data (transmittance or absorbance) over the

required range of wavelengths and generate the spectrum of the compound under analysis as a graph.

• The spectrum exhibit peaks over

the investigated wavelengths range. The wavelength at which the top of the peak occurs is called lmax (lambda max). Some compounds show more than one lmax.

• Spectrum profile is affected by several conditions like : sample state, pH, solvent nature, presented metal ions, temperature and concentration.

L7 page 4


The UV/Vis spectrum • The recorded spectra of compounds in the

condensed phase, whether pure or in solution, generally present absorption bands that are both few and broad, while those spectra obtained from samples in the gas state yield spectra of detailed ‘fine structure’.

• Examples:

L7 page 5


Electronic transitions of organic compounds

• Organic compounds represent the majority of the studies made in the UV/Vis. The observed transitions involve electrons engaged in or or non-bonding n electron orbitals of light atoms such as H, C, N, O. The character of each absorption band will be indicated in relation to the molecular orbitals (MO) concerned and the molar absorption coefficient .

L7 page 6


Electronic transitions of organic compounds * Appears in saturated hydrocarbons. Hexane (gas state): lmax =135nm. All

solvents have this transition. It is strong transition and needs high energy.

n* mainly if n electron from an atom of O, N, S, Cl present in saturated hydrocarbons system. Examples: methanol: lmax= 183nm, ether: lmax= 190nm, ethylamine: lmax=210nm. Weak transition.

n* this transition is usually observed in molecules containing a hetero atom carrying lone electron pairs as part of an unsaturated system. Example: ethanal: lmax =293nm. Weak transition.

* for unsaturated systems. Example: ethylene: lmax=165nm. Strong transition.

dd inorganic salts containing electrons engaged in d orbitals are responsible for transitions of weak absorption located in the visible region. These transitions are generally responsible for their colours. That is why the solutions of copper salt [Cu(H2O)6] 2+ is blue, while potassium permanganate yields violet solutions.

L7 page 7


Electronic transitions of organic compounds

L7 page 8


Chromophore groups • Chromophore: unsaturated groups or any functional group that absorbs at near UV or

Vis region when it is attached to non absorbing saturated residue with no unshared pair of e.

L7 page 9


Chromophore groups • More chromophores in the same molecule cause

bathochromic effect (Red shift: shift to longer wavelength) and hyperchromic effect (increase in intensity). In contrast the shift to shorter wavelengths (Blue shift) is called Hypsochromic effect and the decrease in intensity is called Hypochromic effect.

• In the conjugated chromophores electrons are delocalized over larger number of atoms causing a decrease in the energy of to * transitions and an increase in due to an increase in probability for transition.

• They are groups that do not confer color but increase the coloring power of a chromophore, they called Auxochromes. They are functional groups that have non-bonded valence electrons and show no absorption at l> 220 nm; they absorb in the far UV. (e.g. -OH and -NH2 groups cause a red shift)

L7 page 10


Chromophore groups

L7 page 11


Chromophore groups

L7 page 12


Chromophore groups

L7 page 13


Fieser–Woodward rules • Empirical rules to set up a correlation

between structures and positions of the absorption maxima.

• Many system were studied and rules were established for these systems such as: Heteroannular Diene (Transoid and Cisoid), Polyene, and unsaturated carbonyl (enone).

L8 page 1

• In such systems, the chemical structure was fragmented to basic structure and substituents.

λmax = Base value + Σ Substituent Contributions + Σ Other Contributions

• For enones and dienones we could start with the following basic structures:


Fieser–Woodward rules

L8 page 2

• For enones and dienones we could start with the following basic structures:


Fieser–Woodward rules

Component Contribution

Base- cyclohexenone + 215 nm

Substituents at α-position: 0

Substituents at β-position: 1 alkyl group + 12 nm

Substituents at γ-position: 0

Substituents at δ-position: 0

Substituents at ε-position: 0

Substituents at ζ-position: 1 alkyl group + 18 nm

Other Effects: 2 Double bonds extending conjugation 2 x 30 = + 60 nm

Homoannular Diene system in ring B + 35 nm

1 Exocyclic double bond + 5 nm

Calculated λmax 345 nm

L8 page 3


Solvent effects: solvatochromism • Solvents decrease the sharpness and fine details in

the spectrum peaks due to the large interaction between molecules, the strong intermolecular forces cause the electronic peaks to blend, giving only a single smooth absorption band.

• Polar solvents stabilize both non-bonding electrons in the ground state and * electrons in the excited state. This will lowering the energy state for both n and * electrons, but n state will be affected strongly.

L8 page 4


Solvent effects: solvatochromism

L8 page 5


Solvent effects: solvatochromism

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Effect of pH • pH of the solution could affect the chemical structure of the molecule. Rings may

opened or closed, saturation and conjugation could be affected, also charges may appeared and this with affect the polarity and electrons delocalization.

• Actually this is what happens for acid/base indicator molecules, like phenolphthalein.

• In basic solution, the central carbon becomes part of a double bond becoming sp2 hybridized instead of sp3 hybridization and leaving a p orbital to overlap with the -bonding in the rings. This makes the three rings conjugate together to form an extended chromophore absorbing longer wavelength visible light to show a fuchsia color.

L8 page 7


Effect of pH

L8 page 8

An animation of the pH dependent reaction mechanism: H3In+ → H2In → In2− → In(OH)3−

Methyl orange is a different example. What's happened here?? See: http://www.chemguide.co.uk/analysis/uvvisible/theory.html


Effect of pH

L8 page 9


Effect of pH

L8 page 10


Instrumentation in the UV/Visible

• UV/Vis spectrometers main components are : Source, Wavelength selector (Dispersive system or Discriminator or Monochromator), Sample container and Radiation transducer (Detector)

• Two optical schemes are well-known in UV/Vis spectrometers design. In the first design on which the majority of instruments are based, the spectrum is obtained in a sequential manner as a function of time (one wavelength after another). In the second, the detector ‘sees’ all of the wavelengths simultaneously.

L9 page 1

• Light sources:

for the visible region of the spectrum, an incandescent lamp fitted with a tungsten filament;

for the UV region (<350nm) a deuterium arc lamp under a slight pressure;

alternatively, for the entire region 200 to 1100 nm, a xenon arc lamp can be used.

• Dispersive systems and monochromators

Sequential instruments: the light emitted by the source is dispersed through either a planar or concave grating which forms part of a



L9 page 2

monochromator assembly. This device permits the extraction of a narrow interval of the emission spectrum. The wavelength or more precisely the width of the spectral band, which is a function of the slit width, can be varied gradually by rotating the grating.

Simultaneous instruments: this category of instrument functions according to the spectrograph principle. The light beam is diffracted after travelling through the measuring cell.



L9 page 3

• Detectors: The detector converts the intensity of the light reaching it to an electrical signal.



Photoelectric effect: light incident on the surface of a metal causes electrons to be ejected.

Two types of detector are used, either a photomultiplier tube or a semiconductor (charge transfer devices or silicon photodiodes).

Photomultiplier tubes (PMTs) amplifies the number of photoelectrons through the use of a dynode chain. When a dynode struck by a single energetic electron, it will emit several electrons. If 6-8 dynodes are chained together, then a single photoelectron incident on the first can generate 106-108 electrons at the anode.

L9 page 4


Instrumentation in the UV/Visible • Optical Materials:

Lenses, mirrors, wavelength-selecting elements and sample containers, which are usually called cells or cuvettes, must transmit radiation in the wavelength region being investigated.

In UV/Visible spectrophotometers, cells were made of quarts, glass or plastic for visible radiations, while it should be only quartz when using UV radiations.

L9 page 5

• Block Diagrams: A- Sequential Spectrometer Single-Beam Instruments

Double-Beam Instruments



L9 page 6

• Block Diagrams: A- Sequential Spectrometer



L9 page 7

http://pharmacydocs.blogspot.com/2017/01/ultraviolet-visible-spectroscopy.html

• Block Diagrams: B- Simultaneous Spectrometer (also called multichannel)



L9 page 8


Quantitative analysis: laws of molecular absorption • Lambert–Beer law

Example Calculate the absorbance of a solution having a %T of 89 at 400 nm.

A = log (100/%T) = log(100/89) = 0.051

Example A solution of Co(H2O)2+ has an absorbance of 0.20 at 530 nm in a 1.00 cm cell. Is known to be 10 L mol-1 cm-1. What is its concentration?

A = bC C = A/(b) = 0.20/(1.00x10) = 0.020 M

L9 page 9



Example The Absorbance of an unknown MnO4

- solution is 0.500 at 525 nm. When measures under identical conditions, a 1.0x10-4 M MnO4

- is found to have an absorbance of 0.200. Determine the concentration of the unknown.

known

unknown

known

unknown

known

unknown

C

C

Cb

Cb

A

A

..

..

MCC

unknownunknown 4

4105.2

100.1200.0

500.0 -

-

In general, when the absorbance is to be measured at a single wavelength, the absorption maximum is chosen. This is the point of maximum response so better sensitivity and lower detection limits. We will also have reduced error in our measurement (Why!!)

L9 page 10



Conditions for applying Beer-Lambert law – The light used must be monochromatic – The concentrations must be low – The solution must be neither fluorescent or heterogeneous – The solute must not undergo to photochemical transformations – The solute must not undertake variable associations with the solvent

Deviations from linearity are divided into three categories:

– Fundamental – Chemical – Instrumental

Ideally, according to Beer's law, a calibration curve of absorbance versus the

concentration of analyte in a series of standard solutions should be a straight line

with an intercept of zero and a slope of ab or εb.

L9 page 11



At high concentrations the individual particles of analyte no longer behave independently of

one another. The resulting interaction between particles of analyte may change the value of a

or ε.

The absorptivity, a, and molar absorptivity, ε, depend on the sample's refractive index.

Since the refractive index varies with the analyte's concentration, the values of a and ε will change. For sufficiently low concentrations of analyte, the refractive index remains essentially constant, and the calibration curve is linear.

L9 page 12


Quantitative analysis: laws of molecular absorption • Additivity of absorbances

Example We need to measure a metal-reagent complex (MR) which absorbs at 522 nm ( = 1.18x104). The solution also contains 1.00x10-4M excess reagent (R) with an of 5.12x102 at 522 nm. If the total absorbance is 0.727 at 522 nm in a 1.00 cm cell, what is the concentration of MR?.

MC

C

bCbCAAA

MR

MR

RRMRMRRMRTotal

5

424

1072.5

)1000.1()00.1()1012.5()00.1()1018.1(727.0

-

-

L10 page 1


Quantitative analysis: laws of molecular absorption • Additivity of absorbances

At two different wavelength max

Example Two metal complexes (X & Y) demonstrate at least some absorption over the entire visible range. A mixture was measured at two using a 1 cm cell and the following data was obtained. A1 = 0.533 A2 = 0.590 Determine the concentration of each species.

X

Y

4

X

2

2

3

Y

3

X

Y

3

X

3

1

C ngsubstitutiby

)C(1.45x10)C(5.64x100.590 λAt

3.55x10

C2.96x100.533C

)C(2.96x10)C(3.55x100.533 λAt

-

1 2

X 3.55x103 5.64x102

Y 2.96x103 1.45x104

M1.20x10C

3.55x10

))(3.60x10(2.96x100.533C And

M3.60x10C

C1.45x103.55x10

C2.96x100.5335.64x100.590

4

X

3

53

X

5

Y

Y

4

3

Y

32

-

-

-

-

-

L10 page 2


Quantitative analysis: laws of molecular absorption • Isobestic point

An isosbestic point is the wavelength in which the absorbance of two or more species are the same.

Assume compound A, which is transformed by a reaction of first order to compound B. The separately recorded spectra of A and B are cross over at a point I when one is superimposed upon the other.

For the wavelength of point I, the absorbances of the two solutions are the same and by corollary the coefficients A and B are equal. A will always be of the same value at the isobestic point.

isosbestic point is observed when studying coloured indicators as a function of pH, or kinetic studies of particular reactions. The isobestic point is useful to measure the total concentration of two species in equilibrium, i.e. an isomerization reaction.

L10 page 3


Quantitative analysis: laws of molecular absorption • Spectrophotometric Titrations

useful for locating the equivalence points of titrations. This application of absorption measurements requires that one or more of the

reactants or products absorb radiation or that an absorbing indicator be added to the analyte solution.

A photometric titration curve is a plot of absorbance (corrected for volume change) as a function of titrant volume.

Typical photometric titration curves. Molar absorptivities of the substance titrated, the product, and the titrant are A, P, and T, respectively.

L10 page 4


Derivative spectrometry

• The principle of derivative spectrometry consists of calculating, by a mathematical procedure, derivative graphs of the spectra to improve the precision of certain measurements. This procedure is applied when the analyte spectrum does not appear clearly within the spectrum representing the whole mixture in which it is present.

• This can result when compounds with very similar spectra are mixed together.

• The traces of the successive derived spectral curves are much more uneven than the one of the original spectrum (called zeroth order spectrum). These derivative plots amplify the weak slope variations of the absorbance curve.

• The procedure of obtaining the first derivative graph, dA/d=(d/d)bC, can be

• extended to successive derivatives (nth derivatives).

L10 page 5

instrumental chemical analysis - philadelphia university · fieser–woodward rules • empirical...

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