Capillary Electrophoresis-Mass Spectrometry is the combination of the two techniques which involves an interface.

1. CAPILLARY ELECTROPHORESIS-
MASS SPECTROMETRY (CE-MS)
PREPARED BY
ANUSREE V
2ND SEMESTER M.PHARM
PHARMACEUTICAL CHEMISTRY
AL SHIFA COLLEGE OF PHARMACY

2. CAPILLARY ELECTROPHORESIS-MASS
SPECTROMETRY (CE-MS)
Capillary electrophoresis
(high separation efficiency
in liquid phase)
Mass spectrometry (high
separation efficiency in gas
phase)
CE-MS

4. INSTRUMENTATION

5. 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.

6. 1. Electrophoretic mobility
2. Electroosmotic flow
Electrophoretic mobility(Uep )
 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:

8. Electroosmotic 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 resutls in effective separation of
components.

9.  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.

10.  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.

12. INTERFACING CE WITH MS
 ELECTROSPRAY IONIZATION (ESI)
 Sheath flow interface
 Sheath less interface
 Liquid junction interface
 CONTINUOUS FLOW FAST ATOM BOMBARDMENT (CF-FAB)
 MATRIX ASSISTED LASER DESORPTION IONIZATION (MALDI)

13. 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.

14.  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.

16. STRATEGIES FOR COUPING CE TO MS
VIA ESI
Sheath flow interface

17.  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.

18.  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.

19.  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.
functions

20.  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.

21. Sheath less interface

22.  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.
 However, these interface designs, all have challenges including low mechanical
robustness, poor reproducibility.

23.  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.

24. Liquid junction interface

25.  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.
 This system easy to operate.
 However, the sensitivity is reduced and the mixing of 2 liquids could degrade separate.

26. 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.

28.  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.

29. 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.

30. 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.

31.  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.

32.  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.

33.  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.

34. 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

35.  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.

37. 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.

38.  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.

39. 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.

41. 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.

42. 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-alloisoleucine, in less than 30 min.

43. 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.

44. 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.

45. 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.

47. 7. Forensic sciences with focus on forensic toxicology.

48. REFERENCE
 Gordon A. Ross; Capillary Electrophoresis- Mass spectrometry: Practical
implementation and applications; LC.GC Europe- January 2001.
 Rodrigues KT, et al; CE-MS for the analysis of aminoacids; Methods Mol Biol. 2018
 www.biocompare.com
 www.humanmetabolome.com
 Julie Schappler, Victor Gonzalez-Ruiz, Serge Rudaz; CE-MS in drug analysis and
bioanalysis; 16 June 2016.

49.  Christian W. Klampfi, Markus Himmelsbach; Sheath liquids in CE-MS: Role, Parameters,
and Optimization; 16 June 2016.
 Rob haselberg, Govert W Somsen; CE-MS for the analysis of intact proteins; 16 June
2016.
 Tanize Acunha, Clara Ibanez, Virginia Garcia-Canas; CE-MS in food analysis and
foodomics; 16 June 2016.
 Nadia Porpiglia, Elena Giacomazzi, Rossella Gottardo, Franco Tagilaro; CE-MS in

50.  Akiyoshi Hirayama, Tomoyoshi Soga; CE-MS in Metabolomics; 16 June 2016.
 Venkateswarlu N; A Review on Capillary Electrophoresis- Mass spectrometry (CE-
MS);Research & Reviews: Journal of Pharmaceutical Analysis.
 Wikipedia.