Exploring the Full Range of Temperature When Faced With a Difficult Separation

Carotenoids such as beta-carotene, lutein, zeaxanthin, and lycopene are believed by many scientists to act as powerful antioxidants that protect against heart disease and cancer and boost the immune system. Antioxidants play an important role in health maintenance by helping to reduce the impact and minimize oxidative damage caused by surplus-free radicals. It is believed that the antioxidant capabilities of carotenoids may reduce the risk of developing cancer. The separation of phytochemicals in natural products—including carotenoids, powerful antioxidants—has seen renewed interest because of their potential therapeutic effects and use in nutraceuticals.

Until recently, analysis required complex mobile phases with up to six components. Traditional approaches have been compared with the use of a column temperature programmer to evaluate whether subambient and thermal gradient separations are more suitable than isothermal analysis. The following experiments illustrate HPLC separations of carotenoids, Vitamin A, and Vitamin E under ambient temperature conditions using a solvent gradient, and then retested using a temperature program. The separation of each of these nutrient groups into its components is important to determine their effect on the body’s cells and tissues more accurately. The aim of the experiments was to see whether separations could be performed more efficiently or peak shape made sharper by using a temperature program. In addition to more efficient peaks, the comparative tests described here evaluate whether temperature programming can also reduce analysis time.

Vitamin E was examined since it provides the greatest total antioxidant capability in the lipoproteins of the blood. Additionally, there is evidence that forms other than α-tocopherol may be more effective at some biochemical functions. While vitamins are important for maintaining health, excessive levels of certain vitamins can lead to vitamin poisoning (hypervitaminosis). The HPLC analysis of retinol and retinyl esters will assist in the detection of the major form of Vitamin A in blood plasma (retinol) and its long-chain fatty acid esters (predominantly retinyl palmitate and retinyl stearate) to detect whether patients have hypervitaminosis A.

This research was conducted by Craft Technologies (Wilson, NC) using a Polaratherm Series 9000 total temperature controller (Selerity Technologies, Salt Lake City, UT). The Polaratherm is an HPLC column compartment capable of controlling the temperature from subzero to 200 °C benefiting from two modes of operation: temperature programming and isothermal. It allows and regulates high, ambient, low, or dynamic temperatures for selectivity tuning and improved HPLC separations.

Fast, subambient separation of carotenoids using a temperature program

Typically, the separation of carotenoids is conducted under isothermal conditions. To examine whether temperature programming would have an improved effect on separations, a temperature program with a simple mobile phase was used. The HPLC system consisted of a solvent degasser, autosampler maintaining samples at 20 °C, gradient pump, programmable UV-VIS detector, and computer data system.

Figure 1 - Chromatogram showing the separation of carotenoid mixture isothermally at 25 °C. The analysis takes about 40 min and the lutein and zeaxanthin are not well resolved.

Figure 2 - Chromatogram showing the separation of carotenoid mixture using a temperature program. The analysis time was less than 30 min and lutein and zeaxanthin are nearly baseline resolved, while the lycopene and beta-carotene peaks are sharper at the elevated temperature.

The separation was conducted with a simple mobile phase of tetrahydrofuran (THF) and acetonitrile at 25 °C. The isothermal analysis took about 40 min (see Figure 1); under these conditions, the lutein and zeaxanthin are not well resolved. The same separation using a temperature program from 15 to 35 °C (Figure 2) has an analysis time of less than 30 min. The lutein and zeaxanthin are nearly baseline resolved, while the lycopene and beta-carotene peaks are sharper using the thermal gradient. Temperature programming conclusively simplified method development, improved resolution, and greatly reduced analysis time.

Separation of retinol and retinyl esters using a subambient temperature program

Retinyl acetate does not occur naturally in biological tissues, but it is often used as an internal standard in the HPLC analysis of Vitamin A because it has the same absorbance characteristics, is inexpensive, and is readily available. Retinyl acetate and retinyl palmitate are both used for food fortification and for Vitamin A supplementation. Although the compounds are lipophilic, the polarity range is large and typically requires a solvent gradient to resolve all forms of Vitamin A in a single HPLC analysis.

The major form of Vitamin A in blood plasma is retinol, and high concentrations of the esters in plasma may be diagnostic of hypervitaminosis A. Hypervitaminosis A can result from acute high doses of Vitamin A during a short period of time or chronic elevated levels of Vitamin A intake over an extended period of time. If left undetected, a patient suffering from hypervitaminosis A can suffer from bone remodeling, headaches, nausea, and diarrhea. It can also lead to skeletal deformities, skin disorders, and psychiatric side effects. High doses of Vitamin A are potentially teratogenic.

Figure 3 - Overlay of two isocratic separations of retinoids. The lower trace was run at ambient temperature and the upper trace employed a temperature program.

The first separation was done at ambient temperature and a second separation using a temperature program. The temperature program was started at 0 °C, holding for 1 min, ramping to 35 °C over 6 min, holding for 0.1 min, then ramping to 45 °C over 10 min, and holding at 45 °C until the end of the run. Using the temperature program, a 60% reduction in analysis time and much sharper peaks for all analytes were attained. Figure 3 illustrates an overlay of the two isocratic separations of retinoids. The upper trace used the temperature program, and the lower trace was run at ambient temperature. In the separation of retinol and retinyl esters, a subambient temperature program has been an effective measure to improve speed, resolution, and peak efficiency.

Optimization of the separation of tocopherols using subzero temperature

Vitamin E provides the greatest total antioxidant activity in the lipoproteins in blood; in addition, these compounds have been implicated in the reduced risk of many diseases including cancer, cardiovascular disease, Alzheimer’s disease, and cataracts. Compounds containing the six hydroxychroman ring and possessing the biological activity of α-tocopherol are described using the term “Vitamin E.” There are eight naturally occurring homologues comprised of four tocopherols (α-, β-, σ-, γ-) and four tocotrienols (α-, β-, σ-, γ-). While α-tocopherol has the greatest amount of Vitamin E activity, the other homologues may be more effective at specific activities. For example, γ-tocopherol is thought to be more effective at reducing the risk of prostate cancer. Again, two separations were run—one at ambient temperature and one performed at –20 °C.

Figure 4 - Separation of tocopherols at ambient temperature. Several of the isomers coelute. Column from Alltech Associates, Deerfield, IL.

Figure 5 - Separation of tocopherols at –20 °C. β-tocopherol and γ-tocopherol are now well-resolved; β-tocotrienol and γ-tocotrienol are partially resolved.

No separations of all eight Vitamin E homologues have been previously reported using isocratic reversed-phase HPLC conditions. In the ambient isocratic separation, all eight homologues were injected on a C18 column (see Figure 4). Note the coelution of the β- and γ-tocotrienol and β- and γ-tocopherol. In Figure 5, the separation is performed at –20 °C using the Polaratherm. The low temperature influences the column selectivity, resulting in partial separation of the two tocotrienols and near-baseline separation (α = 1.065) of the two tocopherols. The separation showed improved resolution of β- and γ-isomers when compared to the analysis performed at ambient temperature. An isocratic separation of tocopherols at –20 °C was achieved.

Conclusion

From these experiments it was concluded that temperature programming can simplify method develop development, greatly reduce analysis time, and improve peak resolution using subambient temperatures and temperature gradients. In each of these experiments, a temperature program using the Polaratherm has had a positive result in achieving more efficient separations and/or peak resolution.

Dr. Craft is President, Mr. Tucker is a Chemist, and Dr. Estes is Head of Research & Development, Craft Technologies, Inc., Wilson, NC, U.S.A. Dr. Marin is Marketing Manager, Selerity Technologies, 2484 W. Custer Rd., Salt Lake City, UT 84104, U.S.A.; tel.: 801-978-2295; fax: 801-978-2298; e-mail: [email protected].

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