Microchip capillary electrophoresis coupled with mass spectrometry (MCE-MS) provides advantages like shorter analysis times, lower sample volumes, and higher separation efficiencies compared to conventional capillary electrophoresis. MCE uses microfabricated chips with channels and reservoirs. Effective ionization interfaces like electrospray ionization are required to couple the microchip separation to MS detection. Applications of MCE-MS include analysis of amino acids, peptides, proteins, and other biomolecules. Further development is needed to establish universal ionization methods for both micro- and macroscale analyses.

1. MICROCHIP CAPILLARY
ELECTROPHORESIS–MASS
SPECTROMETRY
By
Shereen Mohamed Shehata
Supervised by
Dr. Sarah Al-Rashood
Pharmaceutical Chemistry Department
College of Pharmacy, King Saud University

2. 1- Introduction
• Microchip capillary electrophoresis (MCE) is a modification of
conventional capillary electrophoresis (CE) on a smaller scale
• MCE components are similar to those of conventional CE

3. Conventional capillary electrophoresis
Detection

4. Advantages of MCE:
MCE can provide:
 Separation of the analytes with nano-picoliters scale
sample volumes
 Parallel sample analysis
 Shorter analysis times
 Increased separation efficiencies
 Lower detection limits
 Portability and disposability of devices
 Reduced reagent consumption and waste generation
 Higher electric field strength compared to CE

5. Coupling mass spectrometric (MS) detection with MCE allows:
 Highly selective detection
 Structural information of the analytes

6. 2- Microchip design
 In MCE–MS, it is important to combine a separation
channel with an ionization interface on a single microchip
Classical microchip for the electrophoretic analyses consists
of:
• Four reservoirs
• A cross-type injector
• A separation channel

7. 2.1. Chip material
Substrate materials for MCE should:
(i) Support a stable electroosmotic flow (EOF)
(ii) Have good optical clarity
(iii) Be easily microfabricated
(iv) Be compatible with the running buffer
(v) Have good thermal/electrical properties to minimize
Joule heating or electrical breakdown

8. a) Quartz and glass
Advantages:
 Inert towards a variety of different solvents
 Give a faster EOF so, the separation time can be reduced
 A wide variety of the coating techniques is advantageous
for suppressing the adsorption of analytes onto the
microchannel

9. a) Quartz and glass
Disadvantages:
 The production is expensive and time-consuming
 The devices are fragile
So, cheap polymeric materials have been used as the chip
substrates.

10. b) Poly (dimethylsiloxane) (PDMS)
• Is the most popular polymer in microfabrication technology
• However, PDMS generates a reduced EOF relative to that of
glass, which can influence the efficiency of separation

11. c) Poly (methylmethacrylate) (PMMA)
Advantages:
• Has high thermal conductivity
• Low cost
• High dielectric constant
• Easily fabricated

12. d) SU-8
• Recently utilized as a chip material
• It is a chemically stable material with glass-like surface
properties with respect of the surface charge and EOF
• These polymeric materials are disposable

13. 2.2. Channel geometry
• Multiple channels can be combined without any connector
• Typically, MCE consists of injection channel that may be:
 Cross- injection channel layout
 T- injection channel layout
 Double-T injection channel layout
• Another channel, crossed to the injection channel, for the
separation

14. Typical channel designs for MCE: (a) cross injection, (b) T-injection, and
(c) double-T injection channel layouts

15. • To increase resolution in MCE we can use folded separation
channels (spiral-shaped) so, increasing the length of the
separation channel while maintaining small chip size
• Also, on-line sample preconcentration was found to
enhance both the plate numbers and sensitivities

16. 3. Ionization interface
Ranging from
small molecules
to large peptides
and proteins
For ionizing
analytes
dissolved in a
liquid phase
Electrospray
ionization (ESI) is
the most suitable

17. Electrospray is produced directly from the
microchannel outlet
On-chip
ionization
External emitter, such as fused silica capillary, a
nanospray needle, or a microsprayer attached to
the microchannel outlet
Off-chip
ionization
Chip-based ESI interfacing techniques can be divided into:

18. Considerations for coupling microchip to
MS
• Minimization of dead volume
• Establishing a steady flow rate for sample introduction
• Minimizing sample adsorption to the microfluidic device
• Solvent compatibility of the device
• Careful selection of the pH, as this will determine:
The analyte charge
The magnitude and direction of EOF

19. 3.1. ESI interface using tapered capillary tip
• The nanospray tip was attached to an opening drilled at the
end of a separation channel of a glass microchip
• When a five peptide mixture is analyzed by this MCE–MS,
three peptides are detected as a single peak due to serious
peak broadening

20. Modifications to improve the tapered capillary tip
efficiency and eliminate band broadening
a) Dead volume elimination:
 By removing the conical shape of the access hole
 Using a glass microchip having a monolithically integrated
tapered ESI emitter
The dead-volume-free MCE–MS device is very attractive
especially for the analysis of proteins and metabolites

21. b) Laminar flow induced by the negative pressure at the
tip can be controlled by:
 Developing a microchip to which the spray tip can be
attached using a PEEK screw without adhesive
 Using a microchip with a long separation channel
 The length of the inserted capillary tip should be kept to a
minimum
The microchip modified along these findings can realize the
MCE–MS analysis of drug components, peptides, tryptic
digestions, and proteins

22. c) Clogging in the fine ESI tip and/or narrow separation
channel can be solved by:
 The use of disposable chip substrates such as PDMS,
and cyclic olefin polymer (COP)
 By coating the channel surface with hydroxypropylmethyl
cellulose (HPMC), the efficiency can be improved from
numbers of 20,000 to 89,000

23. 3.2. Sheath-liquid and gas assisted
microsprayer ESI interface
• Using teflon/stainless-steel tubes and liquid junction based
microsprayer
• The liquid junction acts as a hydrodynamic and flowing
liquid bridge between the exit of the separation channel
and the inlet of the microsprayer

24. Schematic drawing of the MCE/MS apparatus and the expanded view of the liquid
junction

25. Applications of microsprayer
 Carnitine and three acetylcarnitines in human urine are
separated in less than 48 seconds
 Analysis of human plasma samples containing imipramine
and desipramine
 Glycoproteins and glycopeptides are successfully analyzed
with a limit of detection (LOD) at femtomole level

26. 3.3. Chamber-type ESI interface
• A sub-atmospheric chamber-type results in a stable flow
volume and ESI
• By using the developed ESI interface:
 Eight-angiotensin peptide mixture
 Four-protein mixture
 Bovine serum albumin (BSA) tryptic digests
Successfully
separated
and detected
within 8 min

27. 3.4. ESI interface using metal coated
capillary
• An ESI interface with a metalized nanospray, which allows
the direct application of the ESI voltage
 To improve the detection sensitivity, on-line sample
preconcentration has been carried out
 A conductive rubber coated nanospray was used to obtain
longer lifetime and robustness

28. 3.4. ESI interface using metal coated
capillary (cont.)
 To suppress band broadening, a hybrid nanospray ESI
interface in which PDMS nanospray structure is coated with
graphite powder to provide electric conductivity

29. 3.5. ESI interface monolithically integrating
sharp-pointed structure
• Recently, spray emitters with more simple structures and a
dynamic coating of the PDMS channel with a cationic
polymer, PolyE-323, has been developed
 Three peptide sample can be separated with acceptable
resolution
 However, the separation efficiencies and detection
sensitivities are insufficient
 This may be due to the formation of a large Taylor cone at
the ESI emitter tip

30. 3.6. LDI interface
• The high sensitivity of matrix assisted laser
desorption/ionization (MALDI) for large biomolecules
makes it ideal for interfacing with microdevices
• A combination of a gel electrophoresis chip with matrix free
infrared- LDI (IR-LDI) has been used for detection of
bradykinin and bovine insulin

31. 3.6. LDI interface (cont.)
 Microchip isoelectric focusing MCIEF–MALDI technique
using a resin tape cover and freeze-drying process has
been developed
 The band widths of separated proteins remain constant
before and after the fixation by freeze-drying
Thus, no band broadening during the transfer process from
MCIEF to MALDI resulting in good resolution of proteins

32. Schematic representation of experimental procedure in MCIEF–MALDI-MS.
Illustrations show cross-sectional views of chip during each process

33. 4. Coupling of MCE–MS with packed beds
• By combining a sample pretreatment site with an
electrophoretic separation channel
 To combine protein digestion with MCE–MS, a microchip
consisting of immobilized trypsin bead beds has been
fabricated
 A microchip format packed with C18 RP-packings has been
employed to the preconcentration of tryptic digests,
resulting in the LOD of 5 nM

34. 5. Applications
The advances in MCE–MS have been applied to the analysis
of:
 Amino acids
 Peptides
 Tryptic digests
 Proteins
 Amines
 Sugars
 Pharmaceutical compounds

35. 5. Applications (cont.)
• Through the advances in the development of novel
ionization interface and channel coating techniques, the
applications of MCE–MS will be extended to various areas
such as :
 Clinical
 Food
 Forensic
 Environmental researches

36. 6. Conclusion and future perspectives
MCE–MS has a great potential in bioanalysis
It can provide speed analyses in seconds and high efficiencies
For MS detection, two types of ionization interfaces, ESI and LDI
Multiple separation channels can be combined on microchips without any connector

37. 6. Conclusion and future perspectives (cont.)
 On the other hand, introduction of a universal ionization
method, both in macro- and microscales, remains another
great challenge for the future development
 As well, quite different types of solutions may be awaiting
widespread use. Just one example is a recently reported
carbon-nanotube nanomechanical resonator, which is
capable of measuring masses of individual atoms without
ionization