This document provides an overview of classical and bioanalytical methods for analyzing biomolecules. It discusses several classical methods like IR spectroscopy, UV-Vis spectroscopy, mass spectrometry, chromatography techniques and X-ray crystallography. It notes limitations of classical methods for complex biomolecules. The document then introduces bioanalytical methods like electrophoresis, ligand binding assays for improved analysis of proteins, DNA and other biomacromolecules. It provides details on the principles and procedures of electrophoresis as a key bioanalytical technique.

1. “Bio molecules analysis, a
comparison of Classical and
Bioanalytical Methods”
Presented by :Ayesha Abdul Ghafoor
Presented to :Professor Dr. Bushra Khan
MS-1 (Analytical Chemistry)
Roll Number :
Course Stream :Bioanalytical Methods
1

2. Table of Contents
• Biomolecules
• Biomolecules analysia
• Classical Methods of Analysis
 IR spectroscopy
 UV/Vis Spectroscopy
 Mass Spectrometry
 Chromatography
 Gas Chromatography
 Liquid Chromatography
 X-Ray Crystallography
• Limitations of Classical analytical tools
• Bioanlytical Methods
• Proteins Analysis
• Electrophoresis
• Conclusion
• References
2

3. Biomolecules
A biomolecule is any molecule that is produced by
a living organism, including large polymeric
molecules such as proteins, polysaccharides,
lipids, and nucleic acids as well as small
molecules such as primary metabolites, secondary
metabolites, and natural products. A more general
name for this class of molecules is a biogenic
substance.
3

4. Biomolecules analysis
There are two kinds of analytical methods employed to study
Biomolecules by analysts
– Classical Methods
– Bioanalytical Methods
4

5. Classical Methods
There are various method by which scientist try to study
life molecules but bio molecules large and complex
structures make results ambiguous to interpret.
Examples of Classical Methods are
IR Spectroscopy
UV/Vis Spectroscopy
Mass Spectrometry
Gas Chromatography
High Performance Liquid Chromatography
X-Ray Crystallography
5

6. IR Spectroscopy
• Infrared spectroscopy exploits the fact that
molecules have specific frequencies at which they
rotate or vibrate
• These absorptions are resonant frequencies, i.e.
the frequency of the absorbed radiation matches
the frequency of the bond or group that vibrates
• absorption of energy is according to Plank’s
equation
E=hv
6

7. There are two types of bond vibration which leads to IR Spectra
• Stretch – Vibration or oscillation along the line of the bond
H H
C C
H H
• Bend – Vibration or oscillation not along the line of the bond
symmetric asymmetric
H H H H
C C C C
H H H
H
scissor rock twist wag
in plane out of plane
7

8. IR spectroscopy setup
• IR source emits an IR beam which is split into 2
identical beams, one goes through the sample and the
other through a reference cell.
Reference cell typically consists of the solvent that the
sample is dissolved in.
IR used to measure the amount of energy absorbed
when the frequency of the infrared light is varied
• In pulsed Fourier transform IR, a single pulse is sent
through the sample. This
• pulse will contain many frequencies. This will allow
for a much faster test.
8

9. Fig: Instrumental layout and Functioning of IR
Spectrograph
9

10. UV-Vis Spectroscopy
• Ultraviolet–visible spectroscopy refers to absorption
spectroscopy or reflectance spectroscopy in the
ultraviolet-visible spectral region. The absorption or
reflectance in the visible range directly affects the
perceived color of the chemicals involved. In this
region of the electromagnetic spectrum, molecules
undergo electronic Transitions i.e.
• UV- organic molecules
– Outer electron bonding transitions
– conjugation
• Visible – metal/ligands in solution
– d-orbital transitions
10

11. Instrumentation
UV/Vis Spectrograph composed of following
components
– Source (Deutrium Lamp ,Tungsten lamp)
– Monochromator
– Sample holder
– Diode Array Detector
– Recorder
11

12. Fig: Uv/Vis Spectroscope Functioning Lay Out
12

13. Mass Spectrometry
• Different elements can be uniquely identified by
their mass to charge ratio
13

14. MS Principle
• Find a way to “charge” an atom or molecule
(ionization)
• Place charged atom or molecule in a magnetic
field or subject it to an electric field and
measure its speed or radius of curvature relative
to its mass-to-charge ratio (mass analyzer)
• Detect ions using microchannel plate or
photomultiplier tube
14

15. Mass Spec Equation
m 2Vt2
=
z L2
m = mass of ionL = drift tube length
z = charge of ion t = time of travel
V = voltage
15

16. How does a mass spectrometer work?
Create ions Separate ions Detect ions
• Ionization • Mass analyzer • Mass spectrum
– MALDI-TOF
method
• MW
• Database
– MALDI – Triple Quadrapole analysis
– Electrospray • AA seq
(Proteins must be – MALDI-QqTOF
• AA seq and MW
charged and dry)
– QqTOF
• AA seq and protein modif.
16

17. Functioning of Mass
Spectrometry (MS)
• Introduce sample to the instrument
• Generate ions in the gas phase
• Separate ions on the basis of differences in m/z
with a mass analyzer
• Detect ions
17

18. Chromatography
It can be defined as
“It involves passing a mixture dissolved in a mobile phase
through a stationary phase, which separates the analyte to
be measured from other molecules in the mixture based on
differential partitioning between the mobile and stationary
phases”
• By Chromatography we can do both qualitative as
well as Quantitative analysis
• There are two Chromatographic techniques falls in
Classical methods
– Gas Chromatography (GC)
– Liquid Chromatography (HPLC)
18

19. Theoretical Plate
An imaginary unit of the column where equilibrium has been
established between S.P & M.P
(length of the column)
(no of theoretical plates)
HETP is given by Van Deemter equation
HETP=
A = Eddy diffusion term or multiple path diffusion which arises
due to packing of the column
B = Molecular diffusion, depends on flow rate
C = Effect of mass transfer,depends on flow rate
u = Flow rate
19

20. Efficiency ( No. of Theoretical plates)
It can be determined by using the formula
n = 16 Rt2
w 2
N = no. of theoretical plates
Rt = retention time
W = peak width at base
The no. of theoretical plates is high, the column
is highly efficient
For G.C the value of 600/ meter
20

21. GAS LIQUID CHROMATOGRAPHY
Principle
Partition of molecules between gas (mobile
phase) and liquid (stationary phase).
21

22. Instrumental layout
• Carrier gas
• Flow regulators & Flow meters
• Injection devices
• Columns
• Temperature control devices
• Detectors
• Recorders & Integrators
22

23. Fig : Functioning and Instrument Set up for Gas
Chromatograhy
23

24. How a Gas Chromatography Machine
Works
– First, a vaporized sample is injected onto the
chromatographic column.
– Second, the sample moves through the column through the
flow of inert gas.
– Third, the components are recorded as a sequence of peaks
as they leave the column.
24

25. HPLC Chromatography
• HPLC stands for
– High performance Liquid Chromatography
– High pressure Liquid Chromatography
– Highly Priced Liquid Chromatography
25

26. HPLC chromatography
• Separation is based on the analyte’s relative
solubility between two liquid phases
Mobile Phase Stationary Phase
Solvent Bonded Phase
26

27. Instrumentation
Gradient
Controller
•
Pump Column
Detector
Injector
Mobile Phases
27

28. Working of HPLC
28

29. X-RAY CRYSTALLOGRAPHY
29

30. Principle of X-Ray Crystallography
• X-rays are diffracted by electrons
• Diffraction: constructive or destructive
interference of scattered waves
• Pattern of diffracted x-rays useful to obtain
orientation of atoms in space (molecular
structure)
30

31. Scattering from a molecule
• Molecule is composed of many electrons
• The electron starts vibrating with the same frequency as x-ray beam
hit them
• Each electron will scatter secondary radiation uppon exposure to x-
rays
• The scattered secondary beams will interact and cause interference
• The scattering from a molecule is dependent on number of and
distances between electrons i.e. on structure
• If we would know the amplitudes and phases of scattered molecule, we
could calculate the structure of molecule...
Primary beam
31

32. The electron density equation
1
(xyz) F(hkl) exp[ 2 i(hx ky lz) i hkl ]
V h k l
• h,k,l – indices of reflections
• xyz – coordinates
• F – amplitude of reflections
• – phase of reflections
• V- unit cell volume
32

33. Instrumentation and Working
Source of X-ray
mount crystal
measure intensity and position of diffraction spots
rotate crystal
repeat data collection
33

34. Sampling ,Working and Results
Collection
34

35. Limitations of Classical Analytical
Methods
• Classical methods
– MS produce lots of fragments of Biomoleculs which
lead us to false results
– IR vibrations are numerous and we cant account all of
them
– Same for UV/Vis
– Chromatography i.e. GC is only for volatile
compounds while most or biomolecules are thermally
stable and by volatilizing them they lose their living
caharacteristics
• These limitations force analyst to make Bio
analytical method for Biomolecules
35

36. Bio analytical Methods
• Bioanalysis is a sub-discipline of analytical chemistry
covering the quantitative measurement of biological
molecules xenobiotics (drugs and their metabolites and in
unnatural locations or concentrations) and biotics
(macromolecules , proteins, DNA, large molecule drugs,
metabolites) in biological systems.
• Examples of Advance Bioanlytical Methods are
– Electrophoresis
– Ligand binding assays
 Dual polarisation interferometry
 ELISA (Enzyme-linked immunosorbent assay)
 MIA (magnetic immunoassay)
 RIA (radioimmunoassay)
36

37. Proteiomics
• Proteins play crucial roles in nearly all biological processes.
These many functions of proteins are a result of the folding of
proteins into many distinct 3D structures.
• Protein analysis tries to explore how amino acid sequences
specify the structure of proteins and how these proteins bind
to substrates and other molecules to perform their functions.
• Protein analysis allows us to understand the function of the
protein based on its structure.
37

38. Electrophoresis
“Electrophoresis separates molecules on the basis
of their charge and size. The charged
macromolecules migrate across a span of gel
because they are placed in an electrical field. The
gel acts as a sieve to to retard the passage of
molecules according to their size and shape.”
Electrophoresis is one of very important Bioanalytical
method widely used in proteiomics ,cell biology and
genetics .
38

39. Electrophoresis Principle
• The most known and widely used equation of
electrophoresis was developed in 1903 by
Smoluchowski. He finds out Electrophoretic mobility
by following expression
where εr is the dielectric constant of the dispersion medium, ε0 is the
permittivity of free space (C² N−1 m−2), η is dynamic viscosity of the
dispersion medium (Pa s), and ζ is zeta potential (i.e., the electrokinetic
potential of the slipping plane in the double layer )
39

40. Procedure of Electrophoresis
• Remove comb and observe wells.
• Place carbon paper in each end of the tray.
• Cover with buffer, making sure the allow buffer
to overflow into each end of the tray.
• Load gels.
• Connect the electrodes.
• Turn on power supply.
• Allow gels to run – make sure you see bubbles
coming from the electrodes.
40

41. PROCEDURE (CONTINUED)
• It will take about 30 minutes for the gel to run.
• Turn off power supply and remove electrodes.
• Pour off buffer into the designated container.
• Carefully remove gel from gel box and place in
glad container and cover with stain.
• Store in appropriate location.
41

42. Fig ; Instrumentation and working of
Electrophoresis
42

43. Conclusion
Many scientific endeavours are dependent upon
accurate quantification of drugs and endogenous
substances in biological samples; the focus of bio
analysis in the pharmaceutical industry is to provide a
quantitative measure of the active drug and/or its
metabolite(s) for the purpose of
pharmacokinetics, toxicokinetics, bioequivalence and
exposure–response.Classical methods are fail to be so
accurate except Bioanalytical methods .Bioanalytical
methods also applies to drugs used for illicit
purposes, forensic investigations, anti-doping testing
in sports, and environmental concerns
43

44. References
1. Booth, Brian P (2009-04-03). "Welcome to Bioanalysis" (PDF).
Bioanalysis 1 (1): 1–2..
2. Hill, Howard (2009-04-03). "Development of bioanalysis: a short
history“ (PDF). Bioanalysis 1 (1): 3–7.
3. Dobson CM (2000). "The nature and significance of protein
folding". In Pain RH (ed.). Mechanisms of Protein Folding.
Oxford, Oxfordshire: Oxford University Press
4. Harris, Daniel C. (1999). "24. Gas Chromatography". Quantitative
chemical analysis (Chapter) (Fifth ed.). W. H. Freeman and
Company. pp. 675–712
5. Paula, Peter Atkins, Julio de (2009). Elements of physical chemistry
(5th ed. ed.). Oxford: Oxford U.P. pp. 459
6. Skoog, et al. Principles of Instrumental Analysis. 6th ed. Thomson
Brooks/Cole. 2007, 169-173.
7. "Ultraviolet Spectroscopy and UV Lasers", Prabhakar Misra and
Mark Dubinskii, Editors, Marcel Dekker, New York, 2002
44

45. Continued
8. Lindsay, S. ; Kealey, D. (1987). High performance liquid
chromatography Wiley.. from review Hung, L. B.; Parcher, J. F.; Shores,
J. C.; Ward, E. H. (1988).
9. "Theoretical and experimental foundation for surface-coverage
programming in gas-solid chromatography with an adsorbable carrier
gas". J. Am. Chem. Soc. 110 (11): 1090
10. KM Downard (2007). "William Aston – the man behind the mass
spectrograph". European Journal of Mass Spectrometry 13 (3): 177–190
11. Tanaka, K.; Waki, H.; Ido, Y.; Akita, S.; Yoshida, Y.; Yoshida, T.
(1988). "Protein and Polymer Analyses up to m/z 100 000 by Laser
Ionization Time-of flight Mass Spectrometry
12. Ealick SE "Advances in multiple wavelength anomalous diffraction
crystallography". Current Opinion in Chemical Biology 4 (5): 495–9
(2000).
13. Dukhin, S.S.; B.V. Derjaguin Electrokinetic Phenomena. J.
Willey and Sons (1974).
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