pharmaceutical analytical chemistry phc 213 1€¦ · [2] increasing the vibration of constituent...

PHARMACEUTICAL ANALYTICAL CHEMISTRY PHC 213 1

Spectrophotometry

Spectrophotometry is one of the spectroscopic methods in which the study of interaction between light energy and matter is used for qualitative and quantitative determination of different substances.

Advantages of spectrophotometry: 1. Wide applications. 2. Ease and convenience. 3. High selectivity. 4. High sensitivity.


Electromagnetic Radiation (EMR)

• -rays

• X-rays

• UV (Ultraviolet)

• Visible light (Vis)

• IR (Infrared)

• Microwaves

• Radiowaves


Electromagnetic Spectrum

-ray x-ray Visible IR w Rw

10-9 10-7 10-5 10-3 10-1 102

Wavelength (, cm )

Frequency ( , Hz )

108 1012 1014 1016 1018 1020

UV

Wavelength (nm)

400 500 600 700 800


The UV- Visible Radiation

• The UV region of spectrum extends from about 100 - 200 nm (far or vacuum UV) and from about 200 - 400 nm (near UV).

• The visible region of spectrum extends from about 380 nm to about 780 nm. The eye can normally detect only the colors within this wavelength range, that is why it is called visible.

• The visible spectrum consists of seven colors which are; violet, indigo, blue, green, yellow, orange and red, each color has characteristic wave length region.


The UV- Visible Radiation

• When all the wavelengths or colors of the visible light are transmitted or reflected together, the light appears as white light.

• While if all wavelengths or colors of visible light are absorbed, it appears black.

• Colored substances appear colored because they selectively absorb some of wavelengths of visible light and transmitt or reflect other wavelengths or colors (apparent color).


The UV-Visible Region Wavelength nm Color Complementary

(apparent) color

200-400

400-435 Violet Yellow-green

435-480 Blue Yellow

480-490 Blue-green Orange

490-500 Green-blue Red

500-560 Green Purple

560-580 Yellow-green Violet

580-595 Yellow Blue

595-650 Orange Green-blue

650-750 Red Blue-green


Light and Radiation • Light can be described as a wave. This wave has an electric

component and a magnetic component which are perpendicular

to each other.

E = Energy in joules (J)

u= Frequency (Hz)

h = Plank’s constant (6.63x10-27 erg. s)

= Wavelength (m) = C/ u

C = Speed of light (3 x 108 m/s in vacuum) = u

E = h u

E = h C/


Properties of Electromagnetic Radiation (EMR)

• 1-Wave Properties:

• Wavelength (λ):

• It is distance between two successive maxima or minima of wave (nm).

• Frequency (ʋ):

It is the number of cycles (waves) per second

[Hertz (Hz)].

It is inversely proportional to the wave length

ʋ α 1 / λ

ʋ = C / λ


2-Particle properties: • EMR can be viewed as a stream of particles

known as photons. The energy of a photon depends upon the frequency of radiation and can be expressed by Max Plank relation;

E α ʋ

E = h ʋ

Where h is Plank's constant

h = 6.63 x 10-34 j/s or,

= 6.63 10-27 erg/s


Interaction of substance with EMR

• When a molecule interact with EMR, it will absorb energy and the molecule is said to be excited because electrons undergo transition from original energy level (ground state = Eg) to an excited state (Es).

• Transition energy is given by:

Et or ∆E = Es – Eg = h ʋ

M + E → M* (excitation)

• After a brief period (10-6-10-9 S) M* relaxes to its ground state.

M* → M + E (relaxation)


Interaction of photons with matter

A molecule may absorb light energy in three ways:

E total = E transitional + E vibrational + E rotational

[2] Increasing the vibration of constituent nuclei (vibrational)

when molecule absorb IR irradiation.

[3] Raising an electron to a higher energy level (transitional energy)

when molecule absorb visible and UV light.

[1] Increasing the rotation of molecule around its axis (rotational)

when molecule absorb F-IR irradiation.


Interaction of photons with matter

When a molecule absorbs photons in the UV-VIS region, the absorption

of energy results in displacing an outer electron (valence electron) in the

molecule. The molecule is said to undergo transition from the ground

state of energy level to an excited state of energy level.


Interaction of photons with matter An excited molecule loses energy and returns to the ground state.

The energy is released in the form of:

Heat: electrons return directly to ground state.

Light (fluorescence): electrons return via a second excited state.

Molecular collisions. PHARMACEUTICAL ANALYTICAL CHEMISTRY PHC 213 14

Types of Electronic Transition

Anti-bonding

Anti-bonding

n Non-bonding

Bonding

Bonding n -

-

n -

-

200 300

Wavelength, nm

150 250

-

-


Absorption Spectrum

Absorption spectrum: it is characteristic to substance, and the wavelength at

which the maximum absorption is recorded and used to trace the substance

strength to enhance the sensitivity.

Absorption band spectrum for some molecules

max

Amax

Is a plot of absorption intensity versus the wavelength of the absorbed light


Some Important Terms

Chromophores: (Chrom = color, phore = carrier)

They are functional groups which confer color on substances capable of absorbing

UV and/or visible light. They have unsaturated bonds.

Examples: C = C, - C = O, - N = N, and – C ≡ N ( electrons).

Auxochromes: They are functional groups which can not confer colors on substances

but have the ability to increase the coloring power of chromophores, they do not absorb

radiations longer than 200 nm, but when attached to a given chromophore, cause a

shift to a longer wavelength with increase in absorption intensity.

Examples: - OH, - NH2.



Hypsochromic Bathochromic

Hyp

erc

hro

mic

H

ypo

rch

rom

ic

Wavelength, nm

A

bso

rba

nce

APEX



• Bathochromic shift ( red shift ): it is the shift of max to a longer wavelength

due to substitution or solvent effects.

• Hypsochromic shift ( blue shift ): it is the shift of max to a shorter wavelength.

• Hyperchromic effect: enhancement of molecule absorptivity (or absorption

intensity).

• Hypochromic effect: decrease of molecule absorptivity (or absorption intensity).


Factors Affecting Absorption Spectrum

Affect what ?

Maximum wavelength ( max )

Intensity ( )

Effect of pH on absorption spectra

Effect of solvent on absorption spectra


Factors Affecting Absorption Spectrum

NH2 NH3

HCl

• • + Cl

Aniline

280 nm

1,430

Anilinium

254 nm

160

max

Aniline

Effect of pH on absorption spectra

The UV spectrum of aniline in acid medium shows: hypsochromic shift with hypochromic effect.

This shift is due to the protonation of the amino group, hence the pair of electrons is no longer

available and the spectrum becomes similar to that of benzene (thus called benzenoid spectrum).


Factors Affecting Absorption Spectrum Effect of pH on absorption spectra

Phenol

270 nm

1,450

Phenolate

290 nm

2,600

• •

• •

OH • • • •

• • Na

+ O O

OH-

H+

max

Phenol

The UV spectrum of phenol in acidic medium is completely different from its

spectrum in alkaline medium (using same concentration). The spectrum in alkaline

medium exhibits bathochromic shift with hyperchromic effect. The red shift is due

to the participation of the pair electrons in resonance with the -electrons of the

benzene ring, thus increasing the delocalization of the -electrons.


Applications of Spectrophotometry 1. Qualitative Analysis

Analysis of Morphine

since morphine is a phenolic compound,

the observation of bathochromic shift with

hyperchromic effect in KOH is consistent

with, but not definite proof of, the

presence of morphine in the sample.

HO O

HNH3C

OH

Since other phenolic compounds show similar behavior, this test is

a definite proof of the absence of morphine in the sample.

For definite proof for the presence of morphine, better method

(e.g. infrared spectroscopy) should be used. PHARMACEUTICAL ANALYTICAL CHEMISTRY PHC 213 23

Applications of Spectrophotometry Quantitative Analysis

1. Determination of proper max :

450 550 650 350

Wavelength, nm PHARMACEUTICAL ANALYTICAL CHEMISTRY PHC 213 24

Applications of Spectrophotometry 2. Generating the calibration curve at max :

Unknown Standard solutions

Concentration

Ab

sorb

an

ce

Linear equation: A = a + b C

Unknown conc. Is determined by:

Calibration curve: graphically


Laws of light absorption

It relates absorption capacity to the thickness of an absorbing solute

(path length of light).

Lambert’s Law:

Log Io / I = K b

Io Incident light

I Transmitted light

K Proportionality constant

b Light path length

It relates absorption capacity to the concentration of an absorbing

solute.

Beer’s Law:

Log Io / I = K C

Io Incident light

I Transmitted light

K Proportionality constant

C Concentration PHARMACEUTICAL ANALYTICAL CHEMISTRY PHC 213 26


Log Io / I = A (absorbance)

It relates absorption capacity to the thickness of an absorbing solute

(path length of light) and the concentration. It is a combination between

Beer’s law and Lambert's law.

Beer’s - Lambert’s Law:

Log Io / I = a b C

A = a b C Usually b = 1 cm A = a C

Io Incident light

I Transmitted light

a Absorptivity

b Light path length (in cm)

C Concentration (in g/L)



Beer’s - Lambert’s Law: A = a b C

Concentration

Ab

sorb

an

ce



ε (Epsilon), Molar absorptivity,

if concentration (c) expressed as molar solution.

A = ε b c

Beer’s - Lambert’s Law: A = a b C

Expressions of a

A one percent one centimeter

if c is expressed in g/100 mL

A1%

1cm b c A

1%

1cm A =

a absorptivity,

if concentration (c) expressed as gram / Liter.

A = a b c

ε at max it is called εmax. = ε x 10 / molecular weight A1%

1cm PHARMACEUTICAL ANALYTICAL CHEMISTRY PHC 213

29

• The amount of radiation absorbed may be measured in a number of ways:

• Transmittance, T = I / Io % Transmittance, %T = 100 T

• Absorbance,

• A = log10 Io / I A = log10 1 / T A = log10 100 / %T A = 2 - log10 %T



• The last equation, A = 2 - log10 %T , is worth remembering because it allows you to easily calculate absorbance from percentage transmittance data.

• The relationship between absorbance and transmittance is illustrated in the following diagram



• A drug with molar absorptivity ε = 1.6 x 102

L/mole.cm yields an absorbance of 0.73 when measured in a 1-cm cell. Calculate the concentration.

• A = ε b c c = A/ ε b

• C = 0.73/160 = 0.0046 M



• Cytosine has a molar absorptivity of 6x103 at 270 nm at pH 7. Calculate the absorbance and percent transmission of 1x10-5 and 1x10-4 M cytosine solution in a 1-cm cell.

• A = log(I0/I) = ε b c = 6x103 x 1 x 1x10-5 = 0.06 • in percent transmission, I/I0 x 100, • T% = 10-0.06 x 100 = 101.94 (%) = 87.10 % • Similarly, for 1x10-4M, the absorbance A is given as, • A = log(I0/I) = ε b c = 6x103 x 1x 10-4 = 0.6 • in percent transmission, I/I0 * 100, • T% = 10-0.6 x 100 = 101.4 (%) = 25.12 %



Spectrophotometer

• The instrument used for quantitative measurement of absorbance or transmittance of UV/Vis light.

• Must contain five basic components

1- Light source: required to emit the wavelength of interest.

2- Monochromator : used to split the light into the different wavelengths.

3-A sample compartment : holds the sample to be analysed.

4-A detector.

5-The readout


Essential parts of spectrophotometer


Types of spectrophotometers

Amplifier Meter Light

source

Sample

cuvette

Monochromator

Detector

Single-Beam Spectrophotometers:


Components of spectrophotometer Light source:

- UV measurement: hydrogen or deuterium discharge lamp

(190 – 375 nm)

- Visible measurement: Tungsten lamp

(350 – 1000 nm)

Monochromator:

Function: To select light beam of certain wavelength.

- Filter

- Prisms

- Grating


Components of spectrophotometer

Filters: function via selective absorption of unwanted wavelength and

transmitting the complementary color. It consists of colored glass, or

dye suspended in gelatin and sandwiched between two glass plates.

Monochromators:

Prisms: function via refraction of light.


Components of spectrophotometer Monochromators:

Gratings: Consist of large number of parallel ruled very close to each

other on a highly polished surface, e.g. aluminium, or aluminized glass

(600 groove/mm). Each ruled groove functions as a scattering center

for light falling on its edge and through diffraction and interference the

grating disperses the light beam into almost single wavelength.

Incident

light Diffracted

light



• Transparent

• Quartz for UV measurements

• Glass or Quartz cell for VIS measurements

• Pathlength: usually 0.5, 1 or 1 cm

Sample Cell (Cuvette) :

Sample cuvette



Receive light emerged from the sample,

which excite electrons and generate an

electric current that proportional to the

received light intensity.

Detectors :

Anode (iron)

Electrons

Cathode (selenium) Light

beam

Photocell Photomultiplier tube PHARMACEUTICAL ANALYTICAL CHEMISTRY PHC 213

41


The function of the meter to translate the received current (current

intensity proportional to light emerged from sample) to signals on

paper or computer (integration of data).

Recorders :


Types of spectrophotometers Double-Beam Spectrophotometers:

Light

source

Monochromator

Amplifier Meter

Blank cuvette

Sample

cuvette

Detector 1

Detector 2

Beam

splitter


pharmaceutical analytical chemistry phc 213 1€¦ · [2] increasing the vibration of constituent...

Documents