analysis method

1. Mohanad AlBayati
Mohanad AbdulSattar Ali Al-Bayati, BVM&S, MS. Physiology, PhD.
Assistant Professor of Pharmacology and Toxicology
Department of Physiology and Pharmacology
College of Veterinary Medicine
University of Baghdad
Al-Ameria, Baghdad
Phone: 0964 7700766550
E. Mail: aumnmumu@covm.uobaghdad.edu.iq
aumnmumu@yahoo.com
Pharmaceutical Analysis
Spectroscopy
General
Spectrophotometer visible and Ultraviolet

3. Spectra UV IR AE AA ME NMR MASS
Wave lenght Infrared
Ultraviolet
and visible
Atomic
absorption
Nuclear magnetic
resonance
Atomic
emission
Molecular
emission
Spectroscopy
Mass
spectrometry

4. Spectra UV IR AE AA ME NMR MASS
Wave length Infrared
Ultraviolet
and visible
Atomic
absorption
Nuclear magnetic
resonance
Atomic
emission
Molecular
emission
Spectroscopy
Mass
spectrometry
What type of spectra
Rang of wave length
Electron energy
Electron in bounds and excitation

5. Spectroscopy
Ultraviolet and visible

6. Spectroscopy

7. Spectroscopy
The interaction between radiation and matter is a fascinating area in its own right
Most drug molecules absorb radiation in UV. Region of the spectrum
Some are coloured and thus absorb radiation in the visible region
The interaction between radiation and matter is a fascinating area in its own right
The absorption of UV. and visible radiation
Excitation of electrons within the molecular structure to higher energy state
Electron transition
Transition occur from the bottom vibrational state in the electronic ground of the molecule
To any one a number vibrational levels in the electronic excited state
Width of UV spectra
Electronic energy : Vibrational : rational transition

8. Spectroscopy
Excitation of electron from the ground to the excited electronic state
Bottom
Vibration state

9. Spectroscopy
Factors dependent of absorption of radiation UV-Visible region
1. Atomic bonds
Strong bond eg. π detected by short wave length < 150nm
weak bond σ detected by long wave length > 200nm
2. Structure of bonds single or double bonds
3. System of double bonds “chromophores” most commone
4. Symmetry – A symmetry chromophores and polarisation, two daimentional concept

10. curve is prepared by measuring the
absorbance of samples with known
amounts

11. Spectroscopy
Beer lambert low
logIo/It = A = εbc
Io intensity of radiation
It intensity of transmitted radiation
A absorbance
ε constant , molar extension coefficient; 1 M of analyte
b pathlength of the cell in 1 cm
c concentration of the analyte in moles litre -1
Io It
Concentration of the analyte
𝑐 =
𝐴
𝐴(1%, 1𝑐𝑚)

12. Spectroscopy
- Light sourse
- Monochromator
- Optics

13. Spectroscopy
Diode array instruments
“Photodiode spectrophotometer”
Dissolution multicomponent formulation
Calibration of absorbance scale
0.006% w/v solution Potassium dicromate in 0.005% M H2SO4
235nm 122.9 - 126.2
257nm 124.4 - 145.7
313nm 47.0 - 50.3
350nm 104.9 - 108.2
Calibration of absorbance scale
5% w/v solution holmium prechlorate
241.15±1nm, 287.51±1 nm, 369.5±1nm
Determination of instrumental resolution
0.02%w/w toluene in hexane
269nm to 266nm
Determination of stray light; light which falls on the detector within a UV
instrument without having passed through the sample.
1.2% of KCl in water - water blank 200nm

14. Spectroscopy
Steroid enones; similar λ max. of enone chromophore strength “ A(1%,1cm)value is
based on the the absorption of 1% w/v solution” decrease molecular weight increase
steroidal absorption.
Ephedrine: benzoid chromophore; Like benzene ring chromophore, no polar group,
weak symmetry forbidden band 260nm λmax below 200, vibrational fine structure
preserved, chromophore dose not interact strongly with solvent.
Ketoprofen: extended benzene chromophore; symmetry of the benzene ring is altered
due to four double bonds give λmax 204nm reach to 262nm, Pathchromic shift.
Procaine: amino group auxochrome; benzen group –C=O group interact with amino
acid in alkaline media.
Phenylephrine: phenolic hydroxyl group auxochrome; in both alkaline bathochromic
and hypechromic and acidic media lone pair electron interact with benzene ring

15. Spectroscopy
Determination of pka values
1. Determine λmax. nm
2. Determine absorbance (1%, 1cm) at λmax. nm
3. Different pH dilution measured absorbance
4. pKa= 𝑝𝐻 + 𝑙𝑜𝑔 +
𝐴𝑖−𝐴
𝐴−𝐴𝑢
A: Absorbance in Known pH
Ai: Absorbance of the fully ionized
Au: Absorbance of the un-ionized
5. Select suitable pH value within ± 1 of pKa that mean measured a
number of closely spaced pH value.

16. Spectroscopy
Pharmaceutical Quantitative analysis
Rely heavily on simple analysis to determine active ingredients in formulation, based on
1. Standard A (1%, 1cm)
2. Calibrated instrument
3. No interference from excipients
4. Free suspended matter
5. Data need to calculate the % of state content in sample:
a. State content per dosage form
b. Weight of (n)dosage forms
c. Weight extract of assay
d. Absorbance of standared
e. Absorbance of extract solution
f. Expected content in dosage form =
𝑐
𝑏
×a×n
g. Dilution factor = extract filtrate 𝑚𝑙 ÷ 𝑡𝑜𝑡𝑎𝑙 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛
h. Concentration in diluted tablet extract =
𝑒
𝑑
I. Concentration in original dosage form = h × 𝑔
j. Volume of original extract = (Z) ml
k. amount of original extract= I × 𝐸𝑥𝑡𝑟𝑎𝑐𝑡 𝑓𝑖𝑙𝑡𝑟𝑎𝑡𝑒 𝑚𝑙
6. Percentage of Stated content =
𝑘
𝑓

17. Spectroscopy
weight of unknown=
𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑆𝑎𝑚𝑝𝑙𝑒
𝐴𝑏𝑠𝑜𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑆𝑡𝑎𝑛𝑑𝑎𝑟𝑑
× 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑

18. Spectroscopy
Application in Preformulation and Formulation
Partition coefficient of drugs in buffer
Solubility, turbidity
Release of a drug from a formulation