Chiral Separations Using Proteins and Peptides as Chiral Selectors - Chiral Separations by Capillary Electrophoresis - page 144

wash the capillaries between runs by sodium hydroxide or sodium dodecylsulfate
(SDS) in order to remove the adsorbed proteins completely [5]. Two approaches to
avoid the adsorption of proteins or peptides to a capillary wall are the use of chemi-
cally coated capillaries and the use of additives to minimize their interactions with
the capillary wall. The permanent-coatings by linear polyacrylamide are most fre-
quently used [32]. In addition, polyethylene glycol [33]-, methylcellulose [5]-, linear
poly(dimethylacrylamide) [34]-, poly(acrylamide- co -allyl- -d-glucopyranoside- co -

allylglycidyl ether) [34]-, poly(dimethylacrylamide- co -allylglycidyl ether) [34]-, and
pullulan [23]-coated capillaries are used. The additives to minimize the protein-wall
interactions include hydroxypropylcellulose (HPC), dextran, o -phosphoryletha-
nolamine (PEA), 2-(cyclohexylamino)ethanesulfonic acid (CHES), and 3-[(3-chloram-
idopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO) [5].

β

6.4.1.2 Interference of UV Detection
When a protein or peptide is added in the running buffer, the background signal due
to the protein or peptide interferes with detection of an analyte. To overcome this
problem, a partial i lling technique was i rst introduced by Valtcheva et al. [20] for the
separation of -blockers using CBH I (Cel7A) as a chiral selector. The technique was

run automatically using a commercial CE instrument [25] with a slight modii cation.
In the technique, the capillary was partially i lled with a solution containing a protein
and the protein was not in the detector cell when the analyte reached the cell. Figure
6.3 schematically illustrates the operating principle of the partial i lling technique.

β

(A) (B)

[+] [-] [+] [-]

(C) (D)

1 2 3

FIGURE 6.3 Schematic illustration of the partial i lling technique. 1 = separation zone;
2 = running buffer; 3 = sample solution; arrows indicate detection window. (A) The separation
zone is introduced from the injection end to a point short of the detector cell, (B) the sample
solution is introduced into the capillary; (C) a high voltage is applied between both ends of
the capillary after both ends are dipped into the running buffer and the analytes migrate
toward the detector; (D) a separated zone reaches the detector cell but the separation zone
does not reach this cell. (From Tanaka, Y. and Terabe, S., J. Chromatogr. A , 694, 279, 1995.
With permission.)