CAPILLARY ELECTROPHORESIS-

MASS SPECTROMETRY (CE-MS)

CAPILLARY ELECTROPHORESIS-MASS
SPECTROMETRY (CE-MS)

Capillary electrophoresis
(high separation efficiency
in liquid phase)

CE-MS
Mass spectrometry (high
separation efficiency in gas
phase)
INSTRUMENTATION
 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. 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:

 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.
 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.
 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.

 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)

 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.
 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.
STRATEGIES FOR COUPING CE TO MS
VIA ESI

Sheath flow interface

 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.
 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.
 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.

 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.
Sheath less interface
 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.
 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.
Liquid junction interface
 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.
 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.

 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.

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.
 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.

 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.

 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.
 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.

 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

 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.

 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.
 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.

 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.
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.
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.
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.
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.
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.

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

 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.