CEC HPLC CE

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Capillary Electrochromatography (CEC)

• CEC HPLC CE
• High separation efficiency of CE
• Outstanding separation selectivity of HPLC

• Pros1
• High efficiency
• High selectivity
• High sample loading capacity
• Capable to simultaneously separate
• both charged and neutral species
• Fast method development (from HPLC)
• Hyphenation with MS

• Cons1
• Column fabrication and robustness of
• the column
• Only a few stationary phases designed
• specifically for CEC
• Gradient elution requires specialized
• (home made) instrumentation

• Three modes of CEC
• Packed column
• Monolithic (in-situ)
• Open tubular (coatings)

1. Karlsson, et al, Anal. Chem., 2000, 72, 4394
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Two Major Solutions Developed in our Laboratory
for Chiral CE-MS
Chiral MEKC-MS Using Molecular Micelles
Chiral CEC-MS Using Internally Tapered Pack
Columns
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Major problem in coupling Chiral CEC to MS?

• Typically, CEC with UV-detection for chiral
  analysis requires two end frits and is generally
  fabricated using a standard square-tip untapered
  capillary of 50-100 mm i.d. (Figure a).

• Conventional untapered capillary is very
  problematic in CEC-MS, due to two
• Important reasons
• First, the short unpacked segment of 1 cm
  adjacent to the outlet frit that
• usually serves as the electrospray tip is prone
  to frequent breakage at the outlet
• end during installation in the MS nebulizer.

2. Second problem is the bubble formation at
the outlet end that results in irreproducible
retention times of the injected analytes.
A tapered column was fabricated by our research
group an externally tapered tip was prepared by
first heating a 3-4 cm long segment at the middle
of a 1.5 m long, 75 mm i.d. capillary using a
burner for 20 s. This was followed by pulling
the capillary quickly and cooling it to room
temperature. Then, this external tapered
capillary (Figure b) was slurry packed with a
CSP---?fragile outlet-end of the external taper
that tended to break during the voltage ramp or
due to arcing in the spray chamber.
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Discovery of a New Fabrication Procedure for the
Development of Internal Tapered Column An
Approach Towards a Rugged and a Reproducible
CEC-MS.
Microscopic images (a-e) showing the preparation
of internal taper for CEC-MS columns
(magnification 100 X) (a) before and (b-d)
after heating for about 15 s, 30 s, and 45 s
(magnification 100 X and 400 X), respectively
(e) showing that the CEC column packed with the
internal taper is flushed with mobile phase. A
schematic (f) of internal tapered CEC-MS column
showing dimension of packed and unpacked
segments.
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Manufacture of CEC-MS Internal Taper Tip
Fused silica capillary
Rotation of capillary in flame causes internal
channel to taper
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Simultaneous enantioseparation Chiral
separation Achiral separation
Critical achiral separation regions A
S-oxprenolol R-alprenolol B
R-pindolol R-metoprolol C S-pindolol,
R-propranolol S-metoprolol D R-carteolol
S-atenolol
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Conventional micelles versus molecular micelles
Unpolymerized micelle
Advantages of molecular micelles for MEKC-ESI-MS
A

• Fixed micellar structure (no fragment, less
  suppression )

ESI-MS

• High molecular weight produce no spectral clutter
  in low mass region

Surfactant monomers

• Zero critical micelle concentration (CMC)

60Co ?-radiation

• Lower surface activity

B
ESI-MS
Micelle polymer
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MEKC-MS comparison of monomeric and polymeric SUCL
1 Ate, 2 Met 3 Cart, 4 Pind 5 Oxp, 6
Tal 7 Alp 8 Prop
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Comparison of MEKC-UV and MEKC-MS separation of
eight beta blockers
Capillary length to UV detector 60 cm
MEKC-UV
Alp
Tal
S/Navg 7
20
Oxp
Pro
Met
UV absorbance (?200 nm)
Ate
Pin
Car
10
0
10
20
30
12000
Met
Capillary length to MS detector 120 cm
MEKC-ESI-MS
S/Navg 116
Oxp
Alp
Peak intensity (SIM, 8 ions)
Pro
8000
Ate
Pin
Tal
Car
10
20
30
40
50
60
Retention time (min)