Page 134 - Multidimensional Chromatography

126 Multidimensional Chromatography

As enunciated above, a high-resolving LC–LC system can be implemented by
employing columns that operate by using a different separation mechanism (hetero-
modal LC–LC). Several combinations of mechanisms with great dissimilarity are
conceivable. These include the following: size exclusion–ion exchange; size exclu-
sion–reversed phase; ion exchange–reversed phase; reversed phase (alkyl
ligand)–reversed phase (ion-pairing eluent); reversed phase (alkyl ligand)–reversed
phase (electron-pair acceptor or donator ligand); reversed phase–affinity (biospecific
interactions); normal phase (plain silica)–normal phase (electron-pair acceptor or
donator ligand (41). In addition, a significant number of applications describing the
coupling of immunoaffinity chromatography and reversed-phase HPLC have been
reported over the last ten years. A specific antibody is immobilized on a appropriate
sorbent to form a so-called immunosorbent (IS) for packing into a HPLC precolumn.
The antibodies are selected in order to involve antigen–antibody interactions, thus
providing selective extraction methods based on molecular recognition. Samples or
extracts from biological matrices are introduced on to this immunoaffinity system
with little or no sample pretreatment. The analytes are then eluted from the
immunoaffinity column and analysed directly by suitable on-line HPLC methods.
Immunoaffinity columns can be packed with chemically activated sepharose beads,
and antibodies will then covalently bind to these beads (42). However, Sepharose-
based immunosorbents are not pressure resistant and therefore direct connection of
the precolumn to the analytical column could not be achieved. When using these
immunosorbents, analytes are usually desorbed at low pressure in a second precol-
umn packed with C 18 , which subsequently can be coupled on-line to the LC system
(43–45). Antibodies have also been immobilized on silica-based sorbents. The par-
ticular advantage of silica is its pressure resistance, which means that it can be used
directly in on-line LC–LC systems (46–48). The on-line set-up using a silica-based
immunosorbent precolumn is very simple and does not differ to any great extent
from that which uses a single reversed-phase precolumn. Heteromodal LC–LC cou-
pling has also been widely employed as a chiral separation technique, which usually
involves sequential chromatography on a chiral and an achiral column. The consecu-
tive order in which the columns are combined can be varied (e.g. first a chiral col-
umn, then an achiral column or vice versa), depending on the problem to be solved
and the main restrictions involved. Such restrictions may be a low sample amount, a
low analyte concentration or a complex sample matrix, as well as a high degree of
optical purity to be monitored (49–53). Enantiomeric separations can also be easily
achieved by a two-dimensional HPLC system using achiral columns in both dimen-
sions (54). Separation of unmodified amino acids in complex mixtures was achieved
by employing two different separation methods. First, the amino acid separation was
carried out by means of a cation-exchange column by elution with a lithium chlo-
ride–lithium citrate buffer, and then each peak corresponding to an individual amino
acid was switched to an achiral reversed-phase column where the chiral discrimina-
tion was achieved by using a mobile phase containing a chiral copper (II) complex.
LC–LC coupling systems are also employed to perform separations requiring
very large plate numbers. However, it has been demonstrated (see equation (5.20)
that for coupled columns peak capacity increases linearly with the square root of n