HPLC
Uptisphere, the different C18 Chemistries
C18-ODB Uptisphere
C18-HDO Uptisphere
C18-NEC, C18-HSC, C18-TF Uptisphere
Selectivity Guide
Presentation of the C18 Column Test
Classification Graph for ODS stationnary phases
+ 120 commercial columns tested
Our laboratory staff are always concerned to meet the different
requirements of our customers. They are searching and
developing new technologies on a daily basis.
The Uptisphere ODB and HDO, our first C18 chemistries, were created by our chemists in the mid-nineties, are meeting an ever-growing
success.
A large number of analysis methods of pharmaceuticals compounds requiring the most sensitive techniques such as LC/MS, MS/MS...
have been validated with one of these
2 phases.
Monofunctional bonding processes, combined with our proprietary end-capping techniques, guarantee perfect reproducibility for these
C18 phases.
C18-ODB Uptisphere
Representing the C18 benchmark. Its characteristics meet the majority of analysts daily requirements. The excellent mechanical
resistance of our silica-Upti-prep base combined with the perfect ODB chemistry means our silica can be used under basic pH conditions
(-8), the
lifetime of the column being unchanged.
The kinetic dissolution of the silica is lowered when the traditional phosphate buffers are replaced by organic buffers (tris, 1-methyl
piridine, pyrrolidone..). This allows you to work under Ph 10-11 with a good lifetime (a silica-saturator upstream the injector, favours the
protection of the silica of the analytic column.
We particularly recommend our C18-ODB Uptisphere for the separation of moderately polar and non-polar compounds.
C18-HDO Uptisphere
The last recovering steps in its manufacturing process give it a very special selectivity. This is not a "polar linked alkyl phase " type
(PLAP) and it does not have any hydrophylic end-capping.
The propietary technology developed by our chemists leads to a unique bonded-silica structure. It enables you to perform perfect
separation of moderately polar and non-polar compounds, Uptisphere C18 HDO also allows you to use 100 % acqueous mobile phase.
C18-NEC, C18-HSC, C18-TF Uptisphere
Our intention was not to create new C18 but rather a kit of tools with complementary properties to meet the current requirements of
analysis methods. This is how and when our new C18 Uptisphere phases were created.
We have very close links with the" Laboratoires des Techniques et Instruments d’Analyses Moléculaires" (LETIAM), which is part of the
analytical chemistry group in Paris-Sud within the Orsay Technology and Academic Institute; this has fundamentally influenced our
thoughts about developing new C18 phases.
This laboratory has made an excellent reputation in the characterization of silica in HPLC chromatography for years.
A simple and quick test has been developed in subcritic chromatography from the analysis of carotenoid pigments.
It enables the comparison of the 3 major criteria of stationnary phases :
Hydrophobicity (related to the carbon content)
Shape recognition (depending on the bonding density, on the nature of the bonding and on the surface area of the silica)
Accessibility to the polar interaction sites (due to the silica base, end-capping technologies or the type of C18 bonded phase).
Our C18-NEC, C18-HSC and C18-TF uptisphere phases have been synthesized to match the different areas of interests shown on the
test chart.
C18-NEC Uptisphere : similar to the ODB but not end-capped, NEC keeps moderately polar and non-polar compounds away from the
solvent front.
C18-HSC Uptisphere : thanks to a strong end capping process, HSC has a very good selectivity for non-polar compounds
This phase is very stable up to pH : 10
Uptisphere C18-TF : polyfunctional end-capped. TF is particularly recommended for aromatics, polyphenols, PAHs...
Selectivity Guide
Bonding
Code USP
C18-NEC
C18-HDO
C18-ODB
C18-HSC
C18-TF
L1
L1
L1
L1
L1
120A
n.a.
n.a.
320 m2/g
n.a.
Pore size
Surface area
Bonding technology
End-capping
Monofunctional
n.a.
Trifunctional
no
n.a.
yes
yes
yes
16%
17%
18%
20%
14%
3µm
-
x
x
x
-
5µm
x
x
x
x
x
7µm
x
-
x
-
-
10µm
x
x
x
-
-
15µm
x
x
x
-
-
20µm
x
-
x
-
-
Carbon Content
-
Presentation of the C18 column test
This test has been carried out in the LETIAM (Orsay techn & acad.institute), in the laboratory of the analytic chemistry group, PARISSUD.EA3343.
It was performed in subcritical chromatography, a fast and performing technique, offering an undeniable potential for the elaboration of
this test. Great eluent strength of the modified subcritical fluid (mixture of carbon dioxide and methanol), low fluid viscosity leading both
to a reduced molecular kinetic dispersion (thin peaks) and to the use of high mobile phase flows (fast analysis).
These results have been correlated with the ones obtained with hydro organic mobile phases. They provide complementary information.
This test shows that we can get more than 10 classes of stationnary phases with inverted polarities phases from a carotenoid pigments
analysis; their properties are sometimes very different.
StudyS conditions
Injection of 15 microliters of a beta-carotene diluted mixture partially isomerated and containing all-trans forms, 9-cis, 13-cis (major
cis),15-cis and zeaxanthine (beta-carotene di-hydroxylated).
The mobile phase is a carbon dioxide/methanol mixture (85/15, v/v) temperature : 25 °C, back pressure : 15 Mpa, flow rate :
3ml/min, U.V. : 440nm.
Présentation of the results
(see classificiation graph) : 2 selectivities are calculated and shown on the classification graph :
One of them is shown on the X axis, between 13-cis isomer (bend shape) and the all-trans form (linear shape) of the beta-carotene.
The separation of cis/trans isomers of beta-carotene is based on form recognition, i.e. on the ability of the stationnary phase
to distinguish a linear molecule and a bend molecule in space; these will not interact in the same way with the stationnary
phase because of the tridimentional geometry.
Conclusions drawn from the comparison of the results with other tests (especially with NIST 869 test) with stationnary phases
of identified structures show that for a cis/trans selectivity between :
o
o
o
1 and 1.1: the phase is monomeric (mono layer bonded onto the base silica) with a low bounding density
1.1 and 1.2 : the phase is monomeric with a high bonding density or more rarely with partial polymeric character
(polymolecular layers bounded onto the base silica)
More than 1.2 : the phase is polymeric with large pores ; polymeric phases generally show a special ability to separate
polyaromatic hydrocarbons and rigid isomers from different stereochemistry.
Monomeric phases can be obtained by reaction of the base silica with mono silanes ; di or trifunctional in the absence of
water ; polymeric phases can be obtained by reaction with trifunctional silanes in the presence of water traces.
The other selectivity, on the Y-axis of the graph is calculated between the all-trans beta-carotene and the zeaxanthine ; the structure
of the zeaxanthine is that of the beta-carotene with a hydroxyl group on each final cycle.
Other polar interactions can therefore be observed : silanophilic interactions which are developed between the zeaxanthine hydroxyl
and
the residual silanols of the stationnary phase, or,
with other polar sites from the stationnary phase (i.e. siloxane for the polyfunctional phases, amide, urea, ether-oxide, quaternary
ammonium or carbamate for inserted polar grouped phases called "shielded phases").
The higher this selectivity value is, the lower the interactions are ; the stationnary phase,therefore, carries fewer polar groups capable
of interacting with the zeaxanthine, which has a low retention.
This selectivity varies from 0.3 for phases with an inserted polar group, to almost 20 for the most protected phases.
From 0.3 to 1 : the access to polar sites is high, from 1 to 5 it is average and from 5 to 10 it is low, above this it is very low.
VariSous end-capping treatments (uniques or combined) lead most of the time to an increased selectivity, therefore showing the
covering of residual silanols. Other non-end-capped phases with a high bonding density show a high selectivity it's means a reduced
access to the polar sites of the stationnary phase.
The hydrophobicity values cannot be shown on the
same graph, they are given separately in the form of
a histogramme.
The Y-axis shows the selectivity between the
13 cis isomer (bended shape) and the all-trans
form (linear shape) of the beta-carotene.
The X-axis shows the K log hydrophobicity, alltrans beta-carotene.
The combination of the 3 probes allows you to define a set of groups, columns belonging to the same group will exhibit similar
chromatographic results, even if the chemistry in support manufacture differs.
Classification Graph for ODS stationnary
phases
+ 120 commercial columns tested
Courtesy of E. Lesellier et A. Tchapla
Selectivity 13 cis/all-trans b-carotene
Courtesy of E. Lesellier et A. Tchapla
For more information on the results of this test, the name of the different columns tested and their positions on the graph, please contact
LETIAM : [email protected]
INTERCHIM: [email protected]
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