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NEWS FROM HPLC 2000
Britt Erickson and Veronika Meyer report from Seattle, WA.
High-temperature HPLC
makes a comeback
As new stationary phases designed for
use with high temperatures become
available, more and more researchers
are beginning to rethink of tempera-
ture as a variable in HPLC. Judging
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by the large number of talks on the
subject presented at HPLC 2000,
high-temperature HPLC seems to be
making a comeback.
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The use of temperature as a vari-
able in HPLC is not new. Several re-
ports appeared throughout the 1970s
that described the use of temperature
programming in HPLC; in the 1980s,
papers began to appear showing the
benefits of temperature as a variable
in micro-HPLC. But temperature pro-
gramming never really caught on in
LC as it did in GC. Perhaps the big-
gest reason was the lack of suitable
LC columns. Most of the early stud-
ies found that silica-based stationary
phases deteriorate at temperatures
above 70 °C in an aqueous environ-
ment. Today, however, as more robust
columns emerge, the benefits of tem-
perature programming in HPLC are
once again beginning to be realized.
Peter W. Carr of the University of
Minnesota, who runs a side business
selling zirconia-based LC columns, is
particularly interested in high-temper-
ature LC for ultrafast applications, as
zirconia is known for its ability to with-
stand high temperatures. Carr showed
that LC separation times can be signif-
icantly reduced by operating at higher
temperatures and flow rates compared
with conventional LC separations. For
the analysis of long-chain alkylphenones,
a 50-fold improvement in time was
achieved at 150 °C (15 mL/min flow
rate, 5 cm 3 4.6 mm i.d. zirconia-
based column) compared with 25 °C.
Perhaps one of the most exciting
aspects of high-temperature LC is the
ability to use pure water as the eluent.
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Carr demonstrated that the separation
of 4-cyanobenzoic acid, 4-nitrobenzoic
acid, benzoic acid, and an impurity
could be performed in 20 s at 210 °C
(flow rate of 16 mL/min) using super-
heated water as the eluent. It would
be impossible to separate that mixture
at 25 °C using only water as the elu-
ent, he said.
Developing an ultrafast, high-tem-
perature LC system, however, is not
just a simple matter of replacing your
Hunting healthy
molecules
Many plants could be considered
philanthropists because of their abili-
ty to synthesize molecules that keep
us healthy. These compounds, which
we cannot manufacture by ourselves,
include numerous amino acids, vita-
mins, lipids, and antioxidants. It is
not a surprise that many research
groups are working to identify and
quantify these food constituents in
all kinds of plants and plant prod-
ucts. Here is just a snapshot of some
of the healthy food research present-
ed at HPLC 2000.
Günther Stecher and co-workers
at Leopold-Franzens University
(Austria) and Bionorica GmbH
(Germany) have been seeking res-
veratrols, piceids, and
flavonoids in grapes
and red wine. So far,
the research is done
by capillary elec-
trochromatography
with UV detection.
An ammonium acetate
buffer turned out to
be superior to a phos-
phate buffer. Stecher
wants to develop his
method further to be
compatible with fluo-
silica-based column with one made of
a more robust material, such as zirco-
nia, and cranking up the temperature.
“You must carefully consider relevant
heat transfer, band broadening, and
pressure-drop issues,” warns Carr. For
example, if the eluent and column are
not at the same temperature, the re-
sulting thermal mismatch leads to
band broadening.
Other manufacturers have also
been developing LC columns that are
rescence and MS detection and final-
ly be performed on a chip. In con-
trast to Stecher, Raimund Grüner
of the University of the Saarland
(Germany) has been using HPLC
to solve the same analytical problem.
He is able to perform the separation
within 5 min and differentiate be-
tween wines of different origin.
If you do not like wine, evidence
suggests that freshly squeezed cran-
berry juice may also do the trick.
Hao Chen and Yuegang Zuo of the
University of Massachusetts found
phenolic antioxidants (e.g., catechin,
epicatechin, benzoic acid, and p-
anisic acid) in fresh cranberry juice,
but not always in canned cranberry
juice. These compounds were shown
to have anticarcinogenic properties
several years ago. Chen and Zuo
used a protocol
of filtration, solid-
phase extraction,
and reversed-phase
HPLC for quantita-
tive analysis.
Another class of
antioxidants, the
carotenoids, can be
found in orange
juice and have re-
cently been investi-
PHOTODISC
gated by Hyoung
Lee of the Universi-

capable of withstanding high tempera-
tures. As Brian Jones of Selerity Tech-
nologies showed, going to high tem-
peratures does not necessarily mean
giving up on silica phases. He de-
scribed the development of new phas-
es that are bonded to a silica base.
These ruggedized C8 and C18 phases
have similar lifetimes under high-tem-
perature conditions, as more tradi-
tional LC columns have at ambient
temperature.
ty of Florida. With a ternary gradient
on a C30 HPLC stationary phase, Lee
was able to separate more than 25
saponified carotenoids within 40 min.
With this method at hand, Lee could
track the appearance and disappear-
ance of the different compounds dur-
ing fruit maturation.
... and those that are not
so healthy
For many, summer just wouldn’t
be the same without the backyard
barbecue. What outdoor cooks don’t
realize is that too much charcoal-
grilled meat can be harmful. That was
the take-home message of more than
one presentation at HPLC 2000.
Mark Knize and co-workers at
Lawrence Livermore National Labo-
ratory have been investigating the
role of dietary constituents in the de-
velopment of human cancer. In par-
ticular, they’ve been investigating
whether there are differences in the
way individuals metabolize 2-amino-
1-methyl-6-phenylimidazo[4,5-b]
pyridine (PhIP), an aromatic amine
that forms in meat during cooking.
According to Knize, PhIP has been
shown to be mutagenic in short-term
tests and the cause of colon, breast,
and prostate tumors in rats.
Human urine samples were col-
lected from individuals before and
news
Not to be outdone, Roger Trones
and co-workers at G&T Septech, a
Norwegian manufacturer specializing
in micro-HPLC columns, along with
Thomas Andersen and colleagues at
the University of Oslo (Norway),
showed that silica-based columns can
be used with aqueous mobile phases
above 90 °C, if boiling is suppressed
through temperature programming
and the column is exposed to high
temperatures for only a short amount
after eating well-done chicken. Fol-
lowing consumption of the chicken,
samples were collected every 6 h for
a 24-h period. Each 5-mL sample
was spiked with a deuterium-labeled
metabolite, which served as the inter-
nal standard. Metabolites were re-
moved from the sample using solid-
phase extraction, passed through an
ion-exchange column, and concen-
trated on a C18 column.
Using LC/MS/MS, the re-
searchers detected four major PhIP
metabolites in the
urine samples.
However, they
were unable to de-
tect the metabo-
lites using only
LC/MS. The
amounts and
times of metabo-
lite excretion var-
ied significantly
among individu-
als, suggesting
that metabolism
may have an effect on whether an in-
dividual develops cancer or that other
dietary constituents, such as fruits
and vegetables, influence the absorp-
tion and types of metabolites pro-
duced from PhIP.
Reseachers in Austria have also
been investigating PhIP. Siegfried
S E P T E M B E R 1 , 2 0 0 0 / A N A LY T I C A L C H E M I S T R Y
of time. In nonaqueous mobile phas-
es, the packing material was stable up
to about 200 °C. The researchers at-
tribute the ability of the columns to
withstand high temperatures to very
dense packing, which is achieved by
using supercritical CO2 and sonication
during the packing process. “We sus-
pect that many liquid-packed columns
are too loosely packed to retain their
original structure when subjected to
temperature ramping,” said Trones.
Zöchling and co-workers at Graz
University of Technology studied
the formation of PhIP during the
cooking of meat and fish by varying
the temperatures, heating times, and
molar ratios of reactants. In addition,
they tested various antioxidants for
their ability to suppress the forma-
tion of PhIP. According to the re-
searchers, the clean-up step is critical
in the analysis of PhIP. They used
liquid–liquid extraction but occasion-
ally needed an additional blue cotton
COREL
adsorption technique for further pu-
rification. Samples were then analyzed
using HPLC with fluorescence detec-
tion. Results suggest that antioxidants,
such as ascorbic acid, do indeed de-
crease the concentration of PhIP,
whereas pro-oxidants, such as FeSO4,
increase the formation of PhIP.
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