Upload
ayesha-ghafoor
View
558
Download
2
Embed Size (px)
DESCRIPTION
Citation preview
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
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
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.
4
Biomolecules analysis
There are two kinds of analytical methods employed to study Biomolecules by analysts
– Classical Methods– Bioanalytical Methods
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 SpectroscopyUV/Vis SpectroscopyMass SpectrometryGas ChromatographyHigh Performance Liquid ChromatographyX-Ray Crystallography
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
7
There are two types of bond vibration which leads to IR Spectra• Stretch – Vibration or oscillation along the line of the bond
• Bend – Vibration or oscillation not along the line of the bondH
H
C
H
H
C
scissor
asymmetric
H
H
CCH
H
CC
H
HCC
H
HCC
symmetric
rock twist wagin plane out of plane
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.
9
Fig: Instrumental layout and Functioning of IR Spectrograph
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
11
Instrumentation
UV/Vis Spectrograph composed of following components– Source (Deutrium Lamp ,Tungsten lamp)– Monochromator – Sample holder– Diode Array Detector– Recorder
12
Fig: Uv/Vis Spectroscope Functioning Lay Out
13
Mass Spectrometry
• Different elements can be uniquely identified by their mass to charge ratio
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
15
Mass Spec Equation
mz
2Vt2
=
m = mass of ionL = drift tube lengthz = charge of ion t = time of travelV = voltage
L2
16
How does a mass spectrometer work?
• Ionization method– MALDI– Electrospray(Proteins must be
charged and dry)
• Mass analyzer– MALDI-TOF
• MW – Triple Quadrapole
• AA seq
– MALDI-QqTOF• AA seq and MW
– QqTOF• AA seq and protein modif.
Create ions Separate ions Detect ions
• Mass spectrum• Database
analysis
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)
19
Theoretical PlateAn 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
20
Efficiency ( No. of Theoretical plates)
It can be determined by using the formula
n = 16 Rt2
w2
N = no. of theoretical plates
Rt = retention time
W = peak width at baseThe no. of theoretical plates is high, the column
is highly efficientFor G.C the value of 600/ meter
21
GAS LIQUID CHROMATOGRAPHY
Principle
Partition of molecules between gas (mobile phase) and liquid (stationary phase).
22
Instrumental layout
• Carrier gas• Flow regulators & Flow meters• Injection devices• Columns• Temperature control devices• Detectors• Recorders & Integrators
23
Fig : Functioning and Instrument Set up for Gas Chromatograhy
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.
25
HPLC Chromatography
• HPLC stands for – High performance Liquid Chromatography– High pressure Liquid Chromatography– Highly Priced Liquid Chromatography
26
HPLC chromatography
• Separation is based on the analyte’s relative solubility between two liquid phases
Stationary PhaseMobile Phase
Solvent Bonded Phase
27
Instrumentation
Pump
Injector
ColumnDetector
Mobile Phases
Gradient Controller
•
28
Working of HPLC
29
X-RAY CRYSTALLOGRAPHY
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)
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
32
The electron density equation
• h,k,l – indices of reflections• xyz – coordinates • F – amplitude of reflections• a – phase of reflections• V- unit cell volume
(xyz ) 1
VF(hkl)
l
k
h exp[ 2i(hx ky lz ) i hkl ]
33
Instrumentation and WorkingSource of X-raymount crystalmeasure intensity and position of diffraction spotsrotate crystal repeat data collection
34
Sampling ,Working and Results Collection
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
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)
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 .
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 )
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.
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.
42
Fig ; Instrumentation and working of Electrophoresis
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
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
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).