Better HPLC Methods Using Temperature Programming

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1 Better HPLC Methods Using Temperature Programming Stephanie J. Marin Brian A. Jones, W. Dale Felix Selerity Technologies, Inc. Salt Lake City, UT 1

2 Abstract Temperature programmed liquid chromatography (TPLC) has been demonstrated to provide faster and more efficient separations. Method development time is reduced, and many complex methods can be replaced with an isocratic method and a simple temperature program. The dielectric constant of water changes with increasing temperature, and water becomes less polar and mimics the effect of adding an organic modifier. Because of this, temperature programming also opens the door to HPLC methods with the reduction or complete elimination of organic modifier. This paper will showcase several examples of temperature programmed HPLC, including water-only reversed phase separations, and separations using "green" solvents. 2

3 Introduction Temperature Programmed Liquid Chromatography (TPLC) offers several advantages to the separation scientist. Temperature programming can replace binary solvent gradients. Water exhibits non-polar character at elevated temperatures, reducing the amount of organic modifier needed. Increased diffusion rates and lower back pressure permit higher flow rates without sacrificing efficiency. Adequate preheating of the mobile phase is critical to avoid thermal mismatch band broadening. New commercially available equipment and columns stable at high temperatures have made temperature programmed HPLC possible in any laboratory. 3

4 Better Chromatography with Temperature Programming Less organic modifier is needed Water less polar at higher temperatures and behaves more like methanol so less organic modifier needed Change retention and selectivity with temperature programming Replace solvent gradients with temperature gradients 4

5 Solvent Polarity as a Function of Temperature % Methanol/Water or % Acetonitrile/Water at 25 C 0% 20% 40% 60% 80% 100% 0% 20% 40% 60% 80% 100% Dielectric Constant, ε Pure Water (at 50 bar) Methanol/Water Acetonitrile/Water C 75 C 125 C 175 C 225 C Pure Water Temperature, C Data from Y. Yang et al. J. Chromatogr. A 810 (1998)

6 Faster and More Efficient Separations Higher efficiency - better resolution Increased diffusion rates provide a reduction in plate height at higher temperature Lower viscosity and back pressure permits higher flow rates with smaller particle size packings Speed Flatter van Deemter curves allow operation at flow rates many times optimal velocity 6

7 Temperature Effects on Plate Height h (x 10-4 ) ºC 80 ºC 120 ºC 150 ºC u (cm/s) Effect of temperature on plate height. The Van Deemter curve flattens out as the temperature is increased. This means that operation at higher linear velocities at elevated temperature does not sacrifice efficiency as significantly as it does at ambient temperature. 7

8 Selerity Polaratherm Series 9000 Total Temperature Controller Forced air oven and chiller Isothermal and temperature programmed operation Sub-zero to 200 C Flow rates up to 10.0 ml/min Temperature programs up to 30 C/min Mobile phase preheating Peltier effluent temperature control Vapor sensor Compatible with any HPLC system 8

9 Aquachrom TM Series 8000 Applies gas chromatography theory to HPLC separations Superheated water is the mobile phase Temperature programmable HPLC oven FID for universal detection Compatibility with any LC system and detector Green Machine 9

10 Examples The following examples show several different samples separated using Temperature Programmed Liquid Chromatography (TPLC). The first example shows the separation of Retinol and Retinyl Esters using a sub-ambient temperature program to improve resolution and reduce analysis time. The next two sets of chromatograms show temperature induced selectivity changes of sulfonamides, and how the optimum separation was obtained using a temperature program. Acetonitrile was also replaced with ethanol to make the mobile phase more environmentally friendly or green. The last example is the separation of glycols using superheated water as the mobile phase and flame ionization detection. Water, the greenest of mobile phases, also allows the use of a universal detector for these analytes that lack a strong chromophore. 10

11 Separation of Retinol and Retinyl Esters using Thermal Gradient by Reversed-Phase HPLC Vitamin A in biological samples exists as the free alcohol (retinol) and its esters with long-chain fatty acids (predominantly retinyl palmitate and retinyl stearate). The major form of vitamin A in blood plasma is retinol; high concentrations of the esters in plasma are diagnostic of hypervitaminosis A. In most other biological tissues (for example, liver and kidney), the esters dominate. Retinyl acetate does not occur naturally in biological tissues, but it is often used as an internal standard in HPLC analysis of vitamin A because it has the same absorbance characteristics and it is inexpensive and readily available. Retinyl acetate and retinyl palmitate are both used for food fortification and for vitamin A supplementation. Although all of these compounds are lipophilic, the polarity range is rather large and typically requires a solvent gradient to resolve all of these forms of vitamin A in a single HPLC analysis. Here a subambient temperature program was used. 11

12 Separation of Retinol and Retinyl Esters Using a Temperature Program Chromatographic conditions: Column: Genesis C18 column, 4um, 150 x 4.6 mm (Jones Chromatography) Mobile phase: 97.5% acetonitrile:2.5% tetrahydrofuran Flow Rate: 1.0 ml/min Detection: Absorbance at 325 nm Temperature gradient: as listed on figure. Separation was performed by John Estes at Craft Technologies, Wilson, NC. 60% reduction is analysis time and improved peak shape and resolution! 12

13 Separation of Sulfonamides Using a Temperature Program and Green Mobile Phase Green chemistry is the use of chemistry for pollution prevention. Since the Pollution Prevention Act of 1990, many organizations such as the U.S. Environmental Protection Agency (EPA) and the International Union of Pure and Applied Chemistry (IUPAC) have instituted green chemistry programs. Because of copious amounts of hazardous chemicals generated when performing HPLC separations, the technique is a good candidate to investigate the use of green chemicals such as water and ethanol to replace some of the existing mobile phases. In this example temperature induced selectivity changes are observed in the separation of sulfonamides. The separation was optimized using a temperature program and a green mobile phase of ethanol and water replaced acetonitrile and water. 13

14 mau mau mau mau Changes in Selectivity - Separation of Sulfonamides at Different Temperatures 40 C, 0.6 ml/min C, 0.6 ml/min C, 0.6 ml/min C, 1.2 ml/min min min min min Chromatographic conditions: Column: Zorbax Stablebond C18, 150 x 3.0 m, 3.5um. Mobile phase: A: 0.1% acetic acid in water, B: 0.1% acetic acid in acetonitrile. Gradient 20 to 50% B in 2 min. Flow Rate: 0.60 or 1.2 ml/min Detection: UV at 270 nm Temperature: as listed on figure Sample: Sulfonamide test mix (Agilent) 1. Sulfamethazone 2. Sulfamethazine 3. Sulfachloropyridazine 4. Sulfametoxine Separation provided by Prof. Dr. Pat Sandra and his group at the Research Institute for Chromatography in Kortrijk, Belgium 14

15 Separation of Sulfonamides Using a Temperature Program and Green Mobile Phase mau mau mau mau C C C to 90 C (20 C/min) min min min min Chromatographic conditions: Column: Zorbax Stablebond C18, 150 x 3.0 m, 3.5um. Mobile phase: A: 0.1% acetic acid in water, B: 0.1% acetic acid in ethanol. Gradient 17 to 50% B in 3 min Flow Rate: 0.60 ml/min Detection: UV at 270 nm Temperature: as listed on figure Sample: Sulfonamide test mix (Agilent) 1. Sulfamethazone 2. Sulfamethazine 3. Sulfachloropyridazine 4. Sulfametoxine Separation provided by Prof. Dr. Pat Sandra and his group at the Research Institute for Chromatography in Kortrijk, Belgium 15

16 Separation of Glycols using Superheated Water and Flame Ionization Detection Traditionally, the analysis of glycols has been difficult to achieve because they are polar compounds. Furthermore, they have a very high boiling point and are only soluble in alcohol and water. They also lack a chromophore and cannot be detected by UV. When analyzed by GC or HPLC they must be derivatized. This makes them ideal candidates for superheated water analysis with flame ionization detection. Using the Aquachrom Green Machine, glycols were analyzed by using temperature programming with the FID and pure water. Water presents an attractive mobile phase for these types of separations because it shows no significant response in the FID and has the added benefit of being an environmentally-friendly solvent. 16

17 Separation of Glycols using Superheated Water and Flame Ionization Detection mv Chromatographic conditions: Column: Thermo Electron Hypercarb, 100 x 1.0 mm, 3 um. Mobile phase: Water Flow Rate: 75 ul/min Detection: FID at 400 C Temperature: 50 C ramp to 165 C at 25 /min, hold ten minutes Sample: Glycol mixture: Ethylene Glycol Diethylene Glycol Triethylene Glycol 2-Methoxy Ethyl Ether Tetra(ethylene glycol) Minutes 17

18 Conclusions Temperature programming can replace solvent gradients for analysis of many analytes Temperature programming at sub-ambient or elevated temperatures can improve resolution and reduce analysis time Temperature induced selectivity changes can offer a new way to attain optimal separations Temperature opens the door to separations using more environmentally friendly mobile phases Water-only separations are possible which allow for universal flame ionization detection (FID). 18

19 Acknowledgements The authors Dr. Neal Craft, Harold Furr and John Estes of Craft Technologies Inc., in Wilson, NC and Prof. Dr. Pat Sandra and his group at the Research Institute for Chromatography in Kortrijk, Belgium for applications used in this poster. Striking when hot! Visit us at booth

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