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NEWS FROM HPLC 2000 Britt Erickson and Veronika Meyer report from Seattle, WA.
As new stationary phases designed for use with high temperatures become available, more and more researchers are beginning to rethink of temperature as a variable in HPLC. Judging by the large number of talks on the subject presented at HPLC 2000, high-temperature HPLC seems to be making a comeback. The use of temperature as a variable in HPLC is not new. Several reports 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 programming never really caught on in LC as it did in GC. Perhaps the biggest reason was the lack of suitable LC columns. Most of the early studies found that silica-based stationary phases deteriorate at temperatures above 70 °C in an aqueous environment. Today, however, as more robust columns emerge, the benefits of temperature 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-temperature LC for ultrafast applications, as zirconia is known for its ability to withstand high temperatures. Carr showed that LC separation times can be significantly 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. zirconiabased 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 superheated water as the eluent. It would be impossible to separate that mixture at 25 °C using only water as the eluent, he said. Developing an ultrafast, high-temperature LC system, however, is not just a simple matter of replacing your
Hunting healthy molecules Many plants could be considered philanthropists because of their ability to synthesize molecules that keep us healthy. These compounds, which we cannot manufacture by ourselves, include numerous amino acids, vitamins, 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 products. Here is just a snapshot of some of the healthy food research presented at HPLC 2000. Günther Stecher and co-workers at Leopold-Franzens University (Austria) and Bionorica GmbH (Germany) have been seeking resveratrols, piceids, and flavonoids in grapes and red wine. So far, the research is done by capillary electrochromatography with UV detection. An ammonium acetate buffer turned out to be superior to a phosphate buffer. Stecher wants to develop his method further to be compatible with fluo-
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silica-based column with one made of a more robust material, such as zirconia, 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 resulting thermal mismatch leads to band broadening. Other manufacturers have also been developing LC columns that are
rescence and MS detection and finally be performed on a chip. In contrast 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 between wines of different origin. If you do not like wine, evidence suggests that freshly squeezed cranberry 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 panisic 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, solidphase extraction, and reversed-phase HPLC for quantitative analysis. Another class of antioxidants, the carotenoids, can be found in orange juice and have recently been investigated by Hyoung Lee of the UniversiPHOTODISC
High-temperature HPLC makes a comeback
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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 disappearance of the different compounds during 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 charcoalgrilled 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 Laboratory have been investigating the role of dietary constituents in the development of human cancer. In particular, they’ve been investigating whether there are differences in the way individuals metabolize 2-amino1-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 collected from individuals before and
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. Following 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 internal standard. Metabolites were removed from the sample using solidphase extraction, passed through an ion-exchange column, and concentrated on a C18 column. Using LC/MS/MS, the researchers detected four major PhIP metabolites in the urine samples. However, they were unable to detect the metabolites using only LC/MS. The amounts and times of metabolite excretion varied significantly among individuals, suggesting that metabolism may have an effect on whether an individual develops cancer or that other dietary constituents, such as fruits and vegetables, influence the absorption and types of metabolites produced from PhIP. Reseachers in Austria have also been investigating PhIP. Siegfried
of time. In nonaqueous mobile phases, the packing material was stable up to about 200 °C. The researchers attribute 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 suspect 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 formation of PhIP. According to the researchers, the clean-up step is critical in the analysis of PhIP. They used liquid–liquid extraction but occasionally needed an additional blue cotton
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capable of withstanding high temperatures. As Brian Jones of Selerity Technologies showed, going to high temperatures does not necessarily mean giving up on silica phases. He described the development of new phases that are bonded to a silica base. These ruggedized C8 and C18 phases have similar lifetimes under high-temperature conditions, as more traditional LC columns have at ambient temperature.
adsorption technique for further purification. Samples were then analyzed using HPLC with fluorescence detection. Results suggest that antioxidants, such as ascorbic acid, do indeed decrease the concentration of PhIP, whereas pro-oxidants, such as FeSO4, increase the formation of PhIP.
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