Pharmaceutical Analysis 2

Instrumental Methods of Analyses

2
3 Categories

1. Spectrometry
2. Chromatography
3. Electroanalytical Chemistry
4 Spectrometry

▹ Science that deals with light and its absorption and emission by
solutions and other material substances.
5 Spectrometry

▹ The intensity of transmitted radiant energy is a function of the

concentration of chemical species which absorbs the radiant
energy.
7
8 Internal Energy

1. Rotational transition
2. Vibrational transition
3. Electronic transition
9
10 2 Theories of Radiant Energy

1. Light is consist of moving particles, often called

photons or quanta.
2. Light is consist of moving electromagnetic
disturbances, referred to as electromagnetic waves.
11 Particle Theory of Light

▹ Equation which relates the energy of a photon to the frequency of

radiation.
𝐸 = ℎ𝑣
Where:
E = energy in ergs
v = frequency, Hz
h = Planck’s constant (6.62 x 10-27 ergs・s or 6.62 x 10-34 J sec)
12 Wave Theory of Light

▹ Mathematical expression of wave theory.

𝑐 = 𝜆𝑣
Where:
c = velocity of light (3.0 x 1010 cm/s)
𝜆 = wavelength in cm
13 Terminologies

▹ Radiant Energy – refers to energy in UV, visible, and IR regions of

the electromagnetic spectrum.
▹ Wavelength – length of an electromagnetic wave (Greek symbol, 𝜆)

▹ Frequency – the number of the moving electromagnetic waves that

pass a fixed point in 1 sec (Greek symbol, 𝑣)
▹ Wave number – reciprocal of wavelength.
14 Sample Problems

▹ What is the frequency of a light that has a wavelength of

537 nm?
𝜆 = 537 𝑛𝑚
𝑐𝑚
𝑐 = 3 𝑥 10!"
𝑠
𝑣 =?
15 Sample Problems

▹ What is the wavelength of light that has a frequency of

7.89 x 1014 sec-1? Express the answer in both centimeters
and nanometers.
𝑣 = 7.89 𝑥 10!# /𝑠
𝑐𝑚
𝑐 = 3 𝑥 10!"
𝑠
𝜆 =?
16 Sample Problem

▹ What is the energy associated with a frequency of 6.18 x

1012 sec-1?
ℎ = 6.62 𝑥 10$%& 𝐽 • 𝑠𝑒𝑐
𝑣 = 6.18 𝑥 10!% /𝑠𝑒𝑐
𝐸 =?
17 Sample Problem

▹ What is the energy associated with a wavelength of 497

nm?
𝑐𝑚
𝑐 3 𝑥 10!"
𝑣= = 𝑠 = 6.03 𝑥 10!# /𝑠
𝜆 0.0000497 𝑐𝑚

𝐸 = ℎ𝑣
𝐸 = 6.62 𝑥 10$%# 𝐽 • sec x (6.03 x 10!# /𝑠)
18 Seatwork
▹ What is the wavelength of light with an energy of 5.92 x 10-12 J.
Express your answer in both centimeters and nanometers.
19 Seatwork
▹ The wavelength for the detection of lithium by its atomic emission
spectra is 680.8 nm. What is the energy of the photon of radiation
that corresponds to this emission line for lithium in joules?
20 Seatwork
▹ Light with a frequency of 7.26 x 1014 Hz lies in the violet region of
the visible spectrum. What is the wavelength of this frequency of
light? Answer in units of nm.
21 Ranges of the λ of the radiant energy

Region Wavelength
Ultraviolet 220 – 380 nm

Visible 380 – 780 nm

Near Infrared 780 – 3000 nm

Medium Infrared 3.0 – 15 µm

Far Infrared 15 – 300 µm

22
23 λ in VIS
λ (nm) Color absorbed Color transmitted

380 – 450 Violet Yellow green

450 – 480 Blue Yellow

480 – 490 Green blue Orange

490 – 500 Blue green Red

500 – 570 Green Purple to red violet

570 – 590 Yellow Blue

590 – 620 Orange Green blue

620 – 780 Red Blue green

24 Other terminologies
▹ Chromophore - a functional group which absorbs radiant energy in
the UV or visible region of the spectrum.

▹ Colorimetry – branch of spectrophotometry in which the absorption

measurement is made in the visible region of the spectrum.

▹ Colorimeter and filter photometer – used to designate instruments

which have the necessary electronics to permit measurement of
radiant power buy which use a filter instead of a prism or diffraction
grating for the purpose of increasing the sensitivity of the
measurement.
25 Other terminologies
▹ Bathochromic or red shift (auxochromes)– introduction of NH2 or
OH to aromatic system, increases the wavelength of absorption.
▹ Hypsochromic shift or blue shift – addition of proton to amino
group can reduce conjugation, reduction of wavelength of
absorption.
▹ Hypochromic effect – decreases the intensity of absorption.
▹ Hyperchromic effect – increases the intensity of absorption.
26
27 Absorption Spectra

▹ Absorption spectrum – a plot of the amount of light

absorbed by a sample vs. the wavelength of the light.

▹ Absorbance – the amount of light absorbed.

▸ aka. Optical density, absorbancy, extinction
coefficient
▸ Symbolized as A.
28 Formula:

1
𝐴 = 𝑙𝑜𝑔 = −log(𝑇)
𝑇

𝑃!"#$%
𝐴 = 𝑙𝑜𝑔
𝑃&'"()*'$
30 Absorption Spectra

▹ Transmission spectrum – a plot of the amount of light

transmitted by a sample rather than the light absorbed
vs wavelength of the light.

▹ Transmittance – the amount of light transmitted.

▸ The ratio of the radiant power transmitted by the
solution to the radiant power transmitted by the
blank.
▸ Symbolized as T.
32 Formula:

𝑃&'"()*'$
𝑇=
𝑃!"#$%

𝑇 = 10+,
%𝑇 = 10-+,
33 Conversion

▹ Express the following absorbances in terms of percent

transmittance:
▸ 0.038
▸ 0.958
▸ 0.399
▹ Convert the following transmittance data to absorbances:
▸ 15.8%
▸ 0.492
34 Answer

▹ Absorbance to % Transmittance: %𝑇 = 10&$'

▸ %𝑇 = 10&$' = 10&$"."%) = 91.6220%
▸ %𝑇 = 10&$' = 10&$".*+) = 11.0154%
▸ %𝑇 = 10&$' = 10&$".%** = 39.9025%
▹ Transmittance to Absorbance: A=-log(T)
▸ 𝐴 = − log 𝑇 = − log 0.158 = 0.8013
▸ 𝐴 = − log 𝑇 = − log 0.492 = 0.3080
35 Fundamental Laws of Spectrophotometry
▹ Beer’s Law – states that the power of a transmitted radiant beam
decreases exponentially as the concentration of the solution
containing the absorbing chemical species increases
arithmetically.
▹ Lambert’s or Bouguer’s Law – states that the power of a
transmitted radiant beam decreases exponentially as the
thickness of the solution containing the absorbing chemical
species increases arithmetically
▹ Beer-Lambert or Beer-Bouguer’s Law – relates the power of the
incident and the transmitted radiant energy to the thickness and
concentration of the solution containing the absorbing chemical
species.
36
37 Formula:

𝐴 = 𝑎𝑏𝑐
Or
𝐴 = 𝜀𝑏𝑐
Where:
A = absorbance
a = absorptivity
b = path length in cm
c = concentration in g/L or M
𝜀 = Molar absorptivity
38 Sample Problems

▹ A solution of c= 2.0 x 10-5 moles/L of chlordiazepoxide

dissolves in 0.1N NaOH was placed in a fused silica cell
having an optical path of 1cm. The absorbance A was
found to be 0.648 at a wavelength of 260 nm.
▸ a. What is the molar absorptivity?
▸ b. If the solution has A of 0.298 in a 1-cm cell at 260
nm, what is c?
39 Solution

a.A = εbc
𝐴 0.648 !𝟏 𝑴!𝟏
𝜀= = = 𝟑𝟐𝟒𝟎𝟎𝒄𝒎
𝑏𝑐 10!"𝑚𝑜𝑙𝑒𝑠
(1𝑐𝑚)(2.0× 𝑜𝑟 𝑀)
𝐿

$ '.)*+
b. 𝑐 =
%&
= (-).''/0:; 1:; )(3/0) = 𝟗. 𝟏𝟗𝟕𝟓×𝟏𝟎!𝟔 𝑴
40 Problems

▹ Aminacrine is a highly conjugated acridine ring produces a complex

UV spectrum that includes absorption maxima at 260, 313, 326, 381,
400 and 422 nm. The molar absorptivities of the absorbances at 260
and 313 nm are 63900 and 1130 /mole/L cm respectively. What is
the min amount of amicrine that can be detected at each of these
two wavelength in 1 cm cell, when A = 0.002?
41 Solution

▹ For 260 nm
$ '.'') !𝟖 𝑴
▸ 𝑐= = = 𝟑. 𝟏𝟐𝟗𝟗×𝟏𝟎
%& (5-*''/0:; 1:; )(3/0)

▹ For 313 nm
$ '.'') !𝟔 𝑴
▸ 𝑐= = = 𝟏. 𝟕𝟔𝟗𝟗×𝟏𝟎
%& (33-'/0:; 1:; )(3/0)
42 Sample Problem

▹ Guanosine has a max absorbance of 275 nm. The molar absorptivity

is 8400 in a 1 cm path length, the absorbance is 0.7, what is the
concentration of guanosine?
43 Solution

$ '.7
▹ I
𝑐 = %& = (+.''/0:; 1:; )(3/0) = 𝟖. 𝟑𝟑𝟑×𝟏𝟎 !𝟓 𝑴
44 Beer’s Plot

▹ A plot of absorbance values against a series of known solute

concentrations called a Beer’s plot should yield a straight line.
From this plot, the unknown solute concentration is determined
merely by measuring the absorbance value of the solution and
finding the concentration value which corresponds to the
measured absorbance value.
47 Sample Problem

Solution
Concentration (µg/mL) Absorbance at 390 nm
number
1 25.2 0.210
2 35.3 0.295
3 45.4 0.375
4 55.4 0.465
5 Sample (unknown) 0.235
48 Sample Problem

1. Determination of the concentration of the sample taken in the final

dilution
2. Determine the amount of sulfisoxazole prior to dilution.
3. Determine the quantity of sulfisoxazole present per tablet
4. Determine the %labeled amount.
49 Solution

Step 1. Plot the standard curve. Input the data in the calculator in
linear regression mode where x=concentration and y=absorbance.
Input data for solution numbers 1-4.
Step 2. To get the concentration of the sample at final dilution,
extrapolate using the absorbance of unknown sample.
0.235𝑥I = 𝟐𝟖. 𝟐𝟓𝟖𝟐𝝁𝒈/𝒎𝑳
50 Spectrophotometer
 Ultraviolet and Visible Spectroscopy

53
54 UV-Vis Spectroscopy
▹ Radiation in the wavelength range 200 – 700 nm is passed through
a solution of a compound. The electrons in the bonds within the
molecule become excited so that they occupy a higher quantum
state and in the process absorb some of the energy passing
through the solution.
55 Applications
▹ Quantification of drugs in formulations
▹ Determination of the pKa values of some drugs
▹ Determination of partition coefficients and solubilities of drugs.
▹ Used to determine the release of drugs from formulations with
time.
▹ Can be used to monitor the reaction kinetics of drug degradation,
▹ ID test
56 Strengths
▹ Easy-to-use, cheap and robust method
▹ Routine method for determining some physico-chemical
properties of drugs
57 Limitations
▹ Only moderately selective.
▹ Not readily applicable to the analysis of mixtures
 Infrared Spectrophotometry

62
63 IR Spectrophotometry
▹ Electromagnetic radiation ranging between 400 cm-1 and 4000 cm-
1 (2500 and 20000 nm) is passed through a sample and is absorbed
by the bonds of the molecules in the sample causing them to
stretch or bend.
64 IR Spectrophotometry
65 IR Spectrophotometry
Fig. 3. Vibration modes of a methylene group.
66

C–H C≡C C=C Fingerprint Region

67 Applications
▹ Qualitative fingerprint check for identity of raw materials
▹ Preliminary check for compound ID
▹ Can be used to characterize samples in the solid and semi-solid
states
▹ Used to detect polymorphs of drugs.
68 Strengths
▹ Provides complex fingerprint which is unique to the compound
being examined.
▹ Computer control of instruments
69 Limitations
▹ Rarely used as a quantitative technique.
▹ Usually can only detect gross impurities in samples.
▹ Sample preparation requires a degree of skill.
▹ Lacks robustness
70 Preparation of Samples
 Atomic Emission Spectrophotometry
(AES)

73
74 AES
▹ Atoms are thermally excited so that they emit light and the
radiation emitted is measured.
75 Applications
▹ Quantification of alkali metals
▹ Determination of metallic impurities
78 AES
 Atomic Absorption
Spectrophotometry (AAS)

79
80 AAS
▹ Atoms of a metal are volatilized in a flame and their absorption of a
narrow band of radiation produced by a hollow cathode lamp,
coated with the particular being determined, is measured.
81 Applications
▹ Determination of metal residues remaining from the
manufacturing process in drugs.
82 Strengths
▹ More sensitive than AES.
83 Limitations
▹ Only applicable to metallic elements.
▹ Each element requires a different hollow cathode lamp for its
determination.
84 AAS
Fig. 7. Schematic Diagram of an AAS
 Fluorescence spectrophotometry

85
86 Fluorometry
▹ Certain molecules, particularly those with a chromophore and a
rigid structure, can be excited by UV /visible radiation, and will
then emit the radiation absorbed at a longer wavelength. The
radiation emitted can then be measured.

TERM DESCRIPTION

Luminescence EMISSION OF LIGHT

ABSORBS UV light à RE-REMITS
Fluorescence
LIGHT
Phosphorescence DELAYED emission of light
87 Applications
▹ Determination of fluorescent drugs in low-dose formulations.
▹ Limit tests for impurities that are fluorescent.
▹ Useful for studying the binding of drugs to components in complex
formulations.
▹ Used in bioanalysis.
 Turbidimetry and Nephelometry

92
93 Turbidimetry and Nephelometry
▹ transmitted or reflected light respectively is measured after
radiant energy passes through a turbid solution or suspension.
▹ Nephelometry is based on the measurement of the brightness of
light reflected by a cloud of finely divided particles suspended in
a liquid.
▹ Turbidimetric methods are used in the official assay of the
majority of antibiotics, calcium panthothenate, Vitamin B12, and
other medicinal agents.
94 Turbidimetry and Nephelometry
 Nuclear Magnetic Resonance
Spectroscopy

95
96 NMR Spectroscopy
▹ Radiation i n the radiofrequency region is used t o excite atoms,
like protons o r carbon-13 atoms, s o that their spins switch from
being aligned with to being aligned against an applied magnetic
field.
97 Applications
▹ Characterization of the exact structure of raw materials,
intermediates and finished products.
▹ Determine impurities
▹ Used for fingerprinting mixtures
▹ Good potential for non-destructive quantitative analysis of drugs.
98 Strengths
▹ More information about molecular structure
▹ Reproducible results
▹ Very stable system
99 Limitations
▹ Relatively insensitive technique for samples <1mg for proton NMR
and <5 mg for carbon-13 NMR.
▹ Expensive instrumentation.
100 NMR
Fig. 9. Alignment with applied magnetic field.
101

End of Lecture!