Reversed Phase
Reversed phases are dispersive in character and the term, reversed phase has a curious history. The first chromatography columns (Tswett and later Martin) used calcium carbonate and silica as the stationary phase (which were polar) and a hydrocarbon or chlorinated hydrocarbon as the mobile phase (which were dispersive). Thus, the solutes were predominantly retained by polar interactions and predominantly eluted by dispersive interactions. Martin wanted to separate some dispersive substances (long chained fatty acid esters) and so wanted dispersive interactions to dominate in the stationary phase. He, thus, reversed the original system making the stationary phase a high molecular weight hydrocarbon (liquid paraffin) coated on a support and used water or water-alcohol mixtures as the mobile phase. As a result the dispersive hydrocarbon phase was called the reversed phase and, as a consequence, the concept persisted and all dispersive substances have been called reversed phases. Typical reversed phases (dispersive phases) are hydrocarbons, chlorinated hydrocarbons, naphthenes and C8, C16 and C24 bonded phases etc. The use of this term is becoming less common with time
Author: RPW Scott
Book:Liquid Chromatography
Section:HPLC Bonded-Phases Interactions
Between 'Brush' and 'Bulk' Reverse Phases and Aqueous Solvents
The
interactions between aqueous solvents and brush reverse phases
differ very significantly from those with a bulk reverse phase at very low concentrations
of solvent. This difference has been investigated by a number of workers
(25-27) and the basic difference between the two types of phase are shown in
the curves relating retention volume of methanol to the concentration of
methanol in the mobile phase in figure 35. The phases shown are the RP-18
brush, reverse phase manufactured by E. M. Laboratories, which had a C18 (dimethyloctadecyl) chain and ODS-3 a
bulk reverse phase which had a C18
(octadecyl) chain and was manufactured by Whatman Inc. The curves relating
retention volume with solvent composition for the two phases show very
different behavior patterns.
The ODS-3 bulk
reverse phase behaves in the expected manner, as the concentration of methanol
increases the retention volume of the ethanol
HPLC Bonded-Phases Interactions
Author: RPW Scott
Book:Liquid Chromatography
Section:HPLC Bonded-Phases Types Brush
two methyl
groups next to the silicon atom hinder the reaction of adjacent hydroxyl groups
with the reagent and thus there will be a considerable amount of unreacted
hydroxyl groups remaining even after capping. In the extreme, it has been
suggested that there is a hydroxyl group situated between each bonded chain.
There is certainly evidence of some polar interactions with reverse phases
which if completely covered with hydrocarbon chains should only exhibit
dispersive interactions. However, reverse phases are predominantly dispersive
in character and it would appear that if there are any hydroxyl groups still
present on the surface it is likely that they would be relatively few in number
compared with the bonded moieties
HPLC Bonded-Phases Types Brush
Author: RPW Scott
Book:Liquid Chromatography
Section:HPLC Bonded-Phases Retention-Properties
commercially
available reverse phases and determined the carbon content of each phase and
the retention volume of a series of solutes on columns packed with each
adsorbent. The retentive properties of the five reverse phase are shown in figure
37 where the corrected retention volume (V'r) of 2-ethyl
anthraquinone is plotted against carbon content of the reverse phase. It is
seen, somewhat surprisingly, that there is a linear relationship between
retention volume and carbon content of the brush phases (R2, R8, R18). This
relationship can only be expected to occur if all the stationary phase is
available to the solute and the packing procedure is very reproducible so that
each column contains the same amount of packing.
Figure 37. Graph of Retention Volume against Carbon
Content (%w/w)
It should
again be stressed that all three reverse phases were produced from base silicas
of very different surface areas and, despite this, the linear relationship
between carbon content and
HPLC Bonded-Phases Retention-Properties
Author: RPW Scott
Book:Liquid Chromatography
Section:HPLC Macroporous-Polymers
Short chain
reverse phases reduce the extent to which proteins are denatured in the
separation of substances of biological origin, it is seen by the chromatogram
from the C2 reverse phase, that a serious price must be paid in loss of
resolution if the nature of the
separation demands the use of such material. However, the development of the
polymer packings have, at last, partly solved this problem.
In general,
because the brush type phases can be synthesized in a more reproducible manner,
particularly if carried out in a fluidized bed, the brush phases are generally
recommended for the majority of applications. For high retentive capacity and
for systems that will be operated with aqueous solvent mixtures having a very
high water content, the bulk phases might be preferred. The partially reacted,
low carbon content bulk phase also have special areas of application
particularly in sample preparation.
Macroporous
HPLC Macroporous-Polymers
Author: RPW Scott
Book:Liquid Chromatography
Section:HPLC HPLC-Mobile-Phases Reversed-Phase-Surface
reverse phase than the
layer of solvent molecules then the solute will displace the solvent and
interact directly with the surface by displacement. In, general, those
solutes that elute early in the chromatogram will interact by sorption, those
that elute late in the chromatogram will interact by displacement and at some
intermediate point in the elution scale, solute stationary phase interactions
will probably involve both sorption and displacement. Bi-layer adsorption is
also possible with reverse phases but, at this time, experimental evidence of
this does not appear to be available in the literature
HPLC HPLC-Mobile-Phases Reversed-Phase-Surface
Author: RPW Scott
Book:Liquid Chromatography
Section:HPLC Chiral-Stationary-Phases
The spatial character of the solute
will determine the degree of entry and consequently the proximity of
interaction which, in turn, will determine the energy of interaction and the
magnitude of the retention. Finally, the fifth group contains the cyclodextrin based materials that control
retention in a similar manner to that previously described for GC. In LC, the
cyclodextrin stationary phases are bonded to a support such as silica and are
prepared using similar techniques to those for making reverse phases. The more
recent and most effective stationary phases are without doubt those based on
the macrocyclic glycopeptides and the cyclodextrins
HPLC Chiral-Stationary-Phases