Separation buffer

On-line sample-stacking techniques " and, more recently, the use of isotacho-phoresis have added to the potential benefits of CE by permitting the concentration of analyte in a large volume by exploiting the difference in the electric field between the dilute sample and system buffer. The electric field is much stronger in the dilute buffer-sample and hence analyte ions move faster until they reach the border with the separation buffer. At this point they slow down, causing the analyte to concentrate as a sharp sample band at the interface. 

Figure 10 Separation of ABEI-DSC-derivatized n-octylamine (2) and n-propylamine (3) with ECL detection. Remaining ABEI (1) and ABEI DSC (not shown) are also detected. Conditions 20% methanol in 5 mM sodium borate separation buffer, pH 10.9 5-s injection at 25 kV, 1.0 X 10 6 for each labeled amine 25-kV separation potential 10-mm platinum wire electrode. (From Ref. 85, with permission.)...

Figure 3.12 Metabolic profiling by capillary electrophoresis, (a) Comparative carbohydrate profiles of M. truncatula tissue obtained using 4-aminobenzonitrile derivatization, capillary electrophoresis with a 150 mM borate buffer, pH = 9, and on-column UV detection at 214 nm. (b) Anion profile from M. truncatula using capillary electrophoresis and indirect UV detection. The separation buffer was 5 mM K2C1O4, 1% Waters OFM-Anion BT, pH 8.0.

The next important milestone in CE was achieved in 1984, when Terabe et al. described the method of micellar electrokinetic capillary chromatography (MECC or MEKC). By simply adding a surfactant to the separation buffer electrolyte, it was possible to separate both charged and neutral compounds simultaneously in CE. Erom this point on, the technique developed rapidly with many applications resulting in a demand for identification information. Coupling of CE to mass spectrometry was a next challenge and the... 

The separation mechanism is quite different from other chromatographic techniques and a broader spectrum of possible impurities can be detected at the same system (e.g., inorganic small cations, anions by indirect detection, chiral separations by adding a chiral selector, proteins and peptides by adding a polymer to the separation buffer, etc.)... 

Based on the theory, the separation of enantiomers requires a chiral additive to the CE separation buffer, while diastereomers can also be separated without the chiral selector. The majority of chiral CE separations are based on simple or chemically modified cyclodextrins. However, also other additives such as chiral crown ethers, linear oligo- and polysaccharides, macrocyclic antibiotics, chiral calixarenes, chiral ion-pairing agents, and chiral surfactants can be used. Eew non-chiral separation examples for the separation of diastereomers can be found. 

Separation is performed using free-zone electrophoresis, where the capillary is filled with a separating buffer at a defined pH and molarity. This buffer is also called a BGE. During separation, the polarity is set to cathodic or anodic mode, also called normal and reverse mode, depending on the charge of the molecule cation or anion. For anions, the capillary is usually dynamically coated with an electroosmotic flow (EOF) modifier to reverse the EOF and separate the analytes in the co-electroosmotic mode. 

The effectiveness of the coating has been investigated by separating phenolic compounds in the nonaqueous media. The EOF was found to be anodic and dependent on the pH of the separation buffer. In anofher study [66], imidazole containing zwitterionic salt (N-3-(-triethoxysilylpropyl)-4,5-dihy-droimidazole) was attached to the silica capillary wall via the formation of a covalent bond (Figure 6.8). 

Figure 12.3. Typical electropherogram from an injection reproducibility test. CE conditions capillary, 50 qm ID x 50 cm (40 cm to detector) temperature, 20°C detection, 325 nm with 10-nm bandwidth injection, 3 s (5 kPa) 1 mM 4-hydroxyacetophenone applied voltage, +30 kV separation buffer, 20 mM borate at pH 9.2 conditioning, 2-min high-pressure rinse (100 kPa) with 20 mM borate buffer pH 9.2 between runs.

Acidic (-COOH, -SH) or basic (NR2) functional groups are indicators of the appropriate pH range for separating multiple analytes. Also, hydrophobic functionality is an indication that organic solvents should be added to the separation buffer. This addition can enhance the solubility of the analytes in the separation buffer, decreasing analyte-wall interactions and enhancing resolution of such components (85). 

AA and IAA Lemon juice Juice expressed and Capillary zone Separation buffer = 0.1 M... 

AA and IAA Orange juice IAA added as inter- Capillary zone Separation buffer =... 

Metaphosphoric Micellar electro- Separation buffer = UV absorbance Internal standardization... 

Column rinsed with separation buffer for 2 min between runs. 

Column flushed with separation buffer between... 

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