Capillary electrophoresis - Mass spectrometry

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PRESENTED BY:
Vasantha T. S
2nd Sem M pharm
Dept. of Pharm.chemistry
SACCP. B G Nagara.
PRESENTED TO:
Dr. T. Yunus Pasha
Head of the department
Dept. of Pharm. Chemistry
and Pharm.Analysis.
SACCP. B G Nagara.
CAPILLARY ELECTROPHORESIS- MASS SPECTROMETRY (CE-MS)

2. CAPILLARY ELECTROPHORESIS - MASS
SPECTROMETRY (CE-MS)
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3. CONTENTS:-
• Introduction
• Principle
• Instrumentation
• Applications
• References
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4. CAPILLARY ELECTROPHORESIS- MASS SPECTROMETRY (CE-MS)
Capillary
electrophoresis
(high separation
efficiency in liquid
phase)
+ CE-MS
Mass
spectrometry
(high separation
Efficiency in gas
phase)
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6. INSTRUMENTATION:-
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7. CAPILLARY ELECTROPHORESIS
 Capillary electrophoresis is an analytical technique that separates
ions based on their electrophoretic mobility with the use of an
applied voltage, 1000volts/cm.
 A capillary is present by connecting anode and cathode together.
The movement of components along the capillary by 2 interactions.
1) Electrophoretic mobility
2) Electro osmotic flow
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8. Electrophoretic mobility (µep)
Migration of charged particles in a stationary
medium under the influence of an applied electric
field.
The positive components move towards the
negatively charged cathode.
Electrophoretic mobility is given by the equation:
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10. Electro osmotic flow:-
 The interior wall of capillary contains charged sites that are
created by the ionization of silanol groups on the fused silica.
 The positive component interact with the negatively
charged inert surface in the capillary.
 The EOF along with electrophoretic mobility results in
effective separation of components.
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11.  By definition, Movement of the separation buffer through the silica
capillary as a results of the existence of a zeta potential at the
solvent/ silica interface.
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12.  At very low pH, ionization of silanol groups are very poor, results in
slow EOF.
 If pH increases, no. of ionized sites increases results in increase of
EOF.
 At very high pH, maximum ionization sites and maximum EOF.
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14. INTERFACING CE WITH MS
ELECTROSPRAY IONIZATION (ESI)
1) Sheath flow interface
2)Sheath less interface
3) Liquid junction interface
CONTINUOUS FLOW FAST ATOM BOMBARDMENT (CF-FAB)
MATRIX ASSISTED LASER DESORPTION IONIZATION (MALDI)
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15. ELECTROSPRAY IONIZATION (ESI)
• It is an evaporative technique.
• Sample introduced through the capillary.
• At the tip of the capillary high voltage will be applied.
• Nitrogen is supplied as nebulizing gas which helps to spray the sample
analyte.
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16. • Desolvation gas is heated nitrogen gas which helps to vaporize the
sample.
• The high potential, droplets will be ionized.
• Heated desolvation gas will evaporate the solvent & it will produce
the molecular ion.
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18. STRATEGIES FOR COUPING CE TO MS VIA ESI:-
Sheath flow interface:-
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19. This consists of a Central tube (the CE capillary) surrounded by a
second stainless steel tube-the sheath liquid tube.
The sheath liquid flows between this tube and the inner CE capillary.
Between the sheath liquid tube and the third outer tube, or glass
tube, flows the nebulizing gas that helps in the nebulizing process.
For this type of interface, a sheath liquid is constantly injected inside
the nebulizer through a coaxial canal, external to the CE capillary.
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20. The background electrolyte (BGE) and the sheath liquid are forming a
junction at the extremity of the ESI nebulizer, and sprayed in a single
process.
Sheath liquid:
 Commonly used: 1:1 mixture of water-methanol with 0.1% acetic
acid or formic acid.
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21. Functions:-
 The sheath liquid is connected to the CE outlet electrode,
therefore the junction formed with the BGE enables to maintain
the electric field.
 The electrospray process is optimal at flow rates in the μL/min
range and because of the electroosmotic flow, EOF in CE is of the
order of 20-200nL/min, there is an obvious discrepancy between
the EOF and the requirements of electrospray. In order to match
the effluent flow to the requirements for electrospray, a make-up
liquid is provided by the sheath liquid.
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22.  In the CE-MS coupling there is a high voltage applied to the inlet side
of the capillary and also a high voltage potential between the sprayer
needle and the end- plates near the MS entrance capillary.
 The potential b/w the sprayer needle and the MS entrance is approx.
3-5 kV.
 If the potential is negative, then positive ions will enter the MS- this
is called positive ion mode.
 If the potential is positive, then negative ions will enter the MS and
this is called negative ion mode.
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23. Sheath less interface:-
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24.  CE capillary is coupled directly to an ESI source with a sheath less
interface system.
 The electric contact for ESI is realized by using capillary coated with
conductive metal.
 Because no sheath liquid is used, the system has high sensitivity, low
flow rates and minimum background.
 These interface designs, all have challenges including low
mechanical robustness, poor reproducibility.
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25.  The latest sheath less interface design features porous ESI emitter
through chemical etching.
 The design effectively provides robust interfacing with mass
spectrometry and addresses the reproducibility challenges associated
with previous designs.
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26. Liquid junction interface :-
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27.  This technique uses a stainless steel tee to mix separation electrolyte from
CE capillary with make up liquid.
 The CE capillary and ESI needle are inserted through opposite sides of the
tee and a narrow gap is maintained.
 The electrical contact is established by make up liquid surrounding the
junction between 2 capillaries.
 System easy to operate.
 The sensitivity is reduced and the mixing of 2 liquids could degrade
separate.
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28. CONTINUOUS-FLOW FAST ATOM BOMBARDMENT :-
CE can be coupled to FAB ionization using a continuous flow
interface.
 The interface must match the flow rate between the 2 systems.
 The CF-FAB requires a relatively high flow rate but CE need low flow
rate for better separation.
 A make-up flow can be used using a sheath flow or liquid junction.
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30.  Desorption ionization technique.
 Sample and a matrix mixed to form sample – matrix mixture.
 Gas like xenon or argon will be enter the chamber and become
radical.
 Radical ion react with Xe or Ar, already present in chamber.
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31.  Accelerated neutral atoms hit to the sample-matrix mixture.
 Free radical cations will be removed by electric field.
 Accelerated neutral atoms will be bombarded to the sample-matrix
mixture & ionize the sample.
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32. COUPLING CE WITH MALDI-MS :-
 Desorption technique.
 Sample is placed in a matrix.
 Matrix made up of 2,4-dihydroxybenzoic acid and cinnamic acid.
 Matrix liquified at beginning.
 Allow it for solidification.
 Now, sample is entrapped in the matrix.
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33.  Sample : matrix = 1:10000
 Laser hit onto the matrix.
 Transfer of laser energy from matrix to sample.
 Sample particles getting kicked out, i.e.; desorbed from matrix.
 The sample particles become charged now due to the proton transfer to
sample.
 Ionized sample-molecular ion.
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34.  Off-line coupling of CE to MALDI, the CE effluent could be sprayed or
added dropwise on MALDI target plate then dried and analyzed by MS.
 For online coupling, a moving target with continuous contact to CE
capillary end is required.
 The moving target takes analytes into MS where it is desorbed or
ionized.
 Musyimi et al. Developed a new technique where rotating ball was
used to transfer CE to MS.
 The sample from CE is mixed with matrix coming through another
capillary.
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35. As the ball rotates the sample is dried before it reaches ionization
region.
 This technique has high sensitivity since no make-up fluid is used.
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36. MASS SPECTROMETRY
PRINCIPLE:-
 MS is an instrumental technique in which sample is converted
to rapidly moving positive ions by electron bombardment and
charged particles are separated according to their masses.
 Organic molecules are bombarded with electrons.
 Converted into highly energetic positively charged ions –
molecular ions/parent.
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37.  Further break into smaller ions- fragment ions/daughter ions.
 The formed ions are separated by deflection in magnetic field
according to their mass and charge.
 Mass spectrum- relative abundance(%) vs mass/charge ratio.
 Loss of electron from a molecule leads to free radical cation.
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39. PRINCIPLE OF CE-MS :-
 In CE-MS combine the high efficiency and high speed of
CE with high selectivity and high sensitivity offered by MS
detection.
 Separation first on the basis of an analyte’s charge-to-
size ratio and then on the basis of its mass-to-charge
ratio.
 First separating the ionic components of a sample by
applying voltage to the sample.
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41.  The ions will move through the capillary at different rates due to
charge and frictional forces.
 The separated samples is then sprayed into the mass spectrometer
which produces a spectra.
 The spectra is used to identify the individual components of the
sample.
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42. APPLICATIONS:-
1) Drug analysis and bioanalysis.
 Suitable for analysis of drugs in various matrices.
 In impurity profiling.
 Chiral analysis.
 Determination of drugs.
Eg: Analysis of Tetrandrine and Fangchinoline which are components
of some Chinese medicines.
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44. 2. Analysis of intact proteins and peptides.
 Providing fragmentation data that then be compared against
databases to identify unknown peptide or protein.
 Biopharmaceutical characterization.
 Glycoprotein analysis and Top-down protein analysis.
 Assessment of protein-ligand interactions.
 Metalloprotein characterization.
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45. 3. Analysis of amino acids.
 Amino acids have also been analyzed by CE-MS and although the CE
separation was not fully resolved, this was remedied by the MS.
Eg; separation and quantitative analysis of amino acids in urine.
 A good separation of 27 amino acids , including the isomers L-
leucine, L-isoleucine and L- allo isoleucine, in less than 30 min.
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46. 4. Food analysis and foodomics.
 Application of CE-MS in food safety and quality, as well as
in other aspects related to food traceability and bioactivity
following classical food analysis as well as novel foodomics
approaches.
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47. 5. Metabolomics.
 Metabolomics is a rapidly emerging field of functional genomics
research whose aim is the comprehensive analysis of low molecular
weight metabolites in a biological sample.
 CE-ESI-MS offers a convenient format for the separation of complex
mixtures of cationic, anionic and/or zwitterionic metabolites, as well
as their isobaric /isomeric without complicated sample handling.
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48. 6. Separation of isomeric compounds:-
 Glucose-6-phosphate and Fructose-6-phosphate,
which have the same chemical formulae and
molecular weights, are not be resolved by LC-MS, but
can be and quantitated by CE-MS.
 Separation of Scopolamine and two stereoisomers of
Hyoscyamine
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50. 7. Forensic sciences with focus on forensic
toxicology.
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51. REFERENCE:-
1) Capillary Electrophoresis- Mass Spectrometry (CE-MS)
Principles and Applications. Edited by Gerhardus de Jong.
2) Capillary Electrophoresis- Mass Spectrometry (CE-MS)
Wikipedia.
3) https://www.slideshare.net/AnusreeAnu11/capillary-
electrophoresismass-spectrometry
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