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“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

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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

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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.

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Biomolecules analysis

There are two kinds of analytical methods employed to study Biomolecules by analysts

– Classical Methods– Bioanalytical Methods

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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

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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

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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

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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.

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Fig: Instrumental layout and Functioning of IR Spectrograph

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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

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Instrumentation

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

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Fig: Uv/Vis Spectroscope Functioning Lay Out

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Mass Spectrometry

• Different elements can be uniquely identified by their mass to charge ratio

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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

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Mass Spec Equation

mz

2Vt2

=

m = mass of ionL = drift tube lengthz = charge of ion t = time of travelV = voltage

L2

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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

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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)

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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

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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

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GAS LIQUID CHROMATOGRAPHY

Principle

Partition of molecules between gas (mobile phase) and liquid (stationary phase).

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Instrumental layout

• Carrier gas• Flow regulators & Flow meters• Injection devices• Columns• Temperature control devices• Detectors• Recorders & Integrators

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Fig : Functioning and Instrument Set up for Gas Chromatograhy

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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.

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HPLC Chromatography

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

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HPLC chromatography

• Separation is based on the analyte’s relative solubility between two liquid phases

Stationary PhaseMobile Phase

Solvent Bonded Phase

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Instrumentation

Pump

Injector

ColumnDetector

Mobile Phases

Gradient Controller

•

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Working of HPLC

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X-RAY CRYSTALLOGRAPHY

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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)

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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

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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 ]

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Instrumentation and WorkingSource of X-raymount crystalmeasure intensity and position of diffraction spotsrotate crystal repeat data collection

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Sampling ,Working and Results Collection

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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

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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.

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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 .

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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 )

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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.

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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.

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Fig ; Instrumentation and working of Electrophoresis

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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

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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

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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).