Analyte Focusing in Elevated Temperature HPLC

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1 Analyte Focusing in Elevated Temperature HPLC Jody Clark Brian Jones, Stephanie J. Marin, Dale Felix Selerity Technologies, Inc. Salt Lake City, UT

2 Introduction HPLC at extreme temperatures offers shorter run times and faster method development to the separation scientist. The real power of High Temperature Liquid Chromatography (HTLC) is the ability to do temperature programming. This allows the use of an isocratic mobile phase and a thermal gradient to alter retention and selectivity. One critical parameter in HTLC is proper preheating of the mobile phase to eliminate thermal mismatch band broadening. There is also some evidence that preheating the mobile phase independently of the oven temperature can lead to peak focusing (1). In this work, a new dynamic preheater was used to heat the mobile phase independently of the column oven program at three different flow rates to look for evidence of peak focusing.

3 Better Chromatography with Temperature Gradient Programming Change retention through temperature gradient programming Replace solvent gradients with temperature gradients Water less polar and behaves more like methanol so less organic modifier needed 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

4 Selerity Polaratherm Series 9000 Total Temperature Controller Forced air oven and chiller Isothermal and thermal gradients Sub-zero to 200 C Flow rates up to 10.0 ml/min Thermal gradient up to 30 C/min Mobile phase preheating and precooling Peltier effluent cooling/heating (outlet control) Vapor sensor Compatible with any HPLC system

5 Why is Mobile Phase Preheating so Important? No Preheating Parabolic flow caused by mobile phase heating up faster along column wall With Preheating Mobile phase at column temperature eliminates parabolic flow Flow Flow

6 Low Voltage Preheater Design Voltage from 60 mv to 22 V in 4000 increments under continuous microprocessor control Demand is only 6 V for 1 ml/min mobile phase flows heated to 150 C Very responsive and non-invasive Low-mass and low-volume: <2 grams mass (including the tubing),<1 µl totally swept volume 0.005, and ID available Can respond to fast temperature ramps used in thermal gradients

7 Mobile Phase Preheating Improves Chromatography Separation of Barbiturates 40 Barbital 30 Preheater Off - Thermal Mismatch mv Butabarbital Carbromal Secobarbital Preheater On Sharp Peaks Minutes Zirchrom PBD, 80 C

8 Axial Heat Profile for Selerity Preheater 1. Nichrome wire wrapped around stainless steel tube 2. Mobile phase flow through stainless steel tube 1 2 Heat Input Length in inches Heat transfer into the fluid path in a linear fashion over the length of the nichrome wire wrapped section (drawing not to scale).

9 Radial Heat Flow Into the Q 1. Nichrome wire 2. Outer tubing wall 3. Transition zone 4. Mobile phase Mobile Phase Heat flux (and temperature) transfer through the stainless steel tubing into the mobile phase. At the inner tubing wall a very thin transition zone is apparent with the bulk fluid reaching a constant uniform temperature a short distance later. The spirals represent turbulence within the mobile phase that assists in rapidly reaching a uniform state. The inner diameter of the tubing is only to (drawing not to scale).

10 Mobile Phase Temperature Offsetting Useful in programmed runs for peak focusing Preheater was set to match, lead, and lag the oven temperature at three different flow rates Peak width and asymmetry were measured to look for differences Independent preheater control allows mobile phase temperature to more closely match the internal column temperature

11 Analgesics Using a Hypercarb Column and a Thermal Gradient mv Minutes Column: Thermo Hypersil-Keystone Hypercarb, 7 µm, 100 x 4.6 mm Mobile Phase: 35:65 acetonitrile:water with 0.1% TFA Flow Rate: 4.0 ml/min Detection: UV 220 nm Temperature Program: thermal gradient from 125 to 200 C at 30 /min, hold five min. Elution Order: Caffeine Aspirin Salicylic Acid Ibuprofen Phenacetin Acetaminophen Naproxen

12 Analysis of Acetaminophen at Three Flow Rates with Different Preheater Settings Flow Rate (ml/min) Preheater Temperature Retention Time Peak Width Asymmetry C

13 Result The best peak width and asymmetry values were obtained with the preheater lagging the oven temperature by 10 C. Conclusions The Selerity low mass mobile phase preheater is effective in controlling power distribution without overheating damage to sensitive analytes It is independently controllable and can be used to focus solutes and improve peak shape It enables the extension of elevated temperature HPLC beyond the isothermal range, allowing temperature gradient separations in large bore (4.6 mm) columns

14 Future Work Explore the new capability of precision mobile phase temperature matching in sub-ambient HPLC Determine optimal preheater program profiles as a function of column dimensions and oven ramp rates Acknowledgements Thermo Hypersil-Keystone ZirChrom Separations Reference 1. M. Tebrake, R. Smith, S. Wren, I. Wilson, Improving Efficiency by Cooling the Eluent in Reversed Phase High Performance Chromatography, HPLC 2002 Symposium.

15 Turn up the Heat Bring on the Cold Selerity Technologies Inc W. Custer Rd. Salt Lake City, UT Patent applications have been filed relative to the new technologies presented in this work.