Replacing Solvent Gradient HPLC Methods with Temperature Programmed HTLC

Transcription

1 Replacing Solvent Gradient HPLC Methods with Temperature Programmed HTLC Stephanie J. Marin Brian Jones, Dale Felix, Jody Clark Selerity Technologies, Inc. Salt Lake City, UT

2 Introduction Introducing temperature as an active parameter in HPLC method development opens new possibilities for improving the speed and effectiveness of separations. The ability to utilize temperature programming provides the potential of replacing solvent gradients with temperature gradients. This opens the possibility of much faster method development. Temperature programming offers some of the same functions provided by solvent gradient programming. Temperature dependent changes in both the mobile phase and the stationary phase results in significant changes in the solute partition coefficients. Changes in the diffusion rates in both phases will also impact resolution.

3 The polarities of most organic solvents such as acetonitrile do not show significant temperature dependence. Water, on the other hand, shows a significant shift in polar to non-polar character as the temperature increases. Solvent gradients in reversed-phase systems are typically programmed to go from polar to less polar solvent conditions. Temperature programming of aqueous based reversed-phase solvents can therefore result in similar polarity changes. We examine this phenomenon in more detail and show the application of temperature programming in HPLC to replace solvent gradient programming. Adequate preheating of the mobile phase is critical to achieving quality separations during temperature programming. A highly responsive preheater capable of maintaining mobile phase temperature during fast temperature ramps is required for thermal gradient work.

4 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

5 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 gradients up to 30 C/min Mobile phase preheating and pre-cooling Peltier effluent temperature control Vapor sensor Compatible with any HPLC system

6 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

7 Mobile Phase Preheater 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

8 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

9 Examples The first example shows the separation of a group of aromatic hydrocarbons isothermally at three temperatures and then using a thermal gradient. The second example compares a group of aromatic hydrocarbons separated using a fast solvent gradient isothermally at three temperatures and then isocratically using a thermal gradient. In both examples, the thermal gradient provides the superior separation. The next example is the isocratic separation of proteins using a thermal gradient, and the last example shows a selection of over-the-counter analgesics using a thermal gradient instead of a binary solvent gradient.

10 mv Aromatic Hydrocarbons at Three Minutes Temperatures 50 o C Chrysene elutes at 22 min 100 o C Chrysene is better but resolution of early eluting peaks is lost. 150 o C Chrysene looks good but all resolution of other components is lost! 10 Column: ZirChrom PBD 100 x 4.6 mm Mobile Phase: 35:65 Acetonitrile:Water Flow Rate: 2.0 ml/min Detection: UV 254 nm Elution Order: Uracil Benzene Toluene Ethylbenzene Naphthalene Biphenyl Tetrahydronaphthalene Anthracene Phenanthrene Chrysene (+impurity)

11 mv 50 0 Aromatic Hydrocarbons on PBD Column Using a Temperature Gradient All ten components baseline resolved in six minutes! Baseline corrected Minutes Column: Zirchrom PBD 100 x 4.6 mm Mobile Phase: 35:65 Acetonitrile:Water Flow Rate: 2.0 ml/min Detection: UV 254 nm Temperature Program: 50 C to 150 C at 20 /min Elution Order: Uracil Benzene Toluene Ethylbenzene Naphthalene Biphenyl Tetrahydronaphthalene Anthracene Phenanthrene Chrysene (+impurity)

12 Aromatics on PRP-1 Isothermally Using a Fast Solvent Gradient 100 o C Column: PRP-1, 3µm, 100 x 2.1 mm Mobile Phase: 75:25 acetonitrile:water Flow Rate: 0.5 ml/min Detection: UV 254 nm Solvent Gradient: 75-90%ACN over five minutes, hold two minutes. mv o C 50 o C Minutes Elution Order: Uracil (off scale) Benzene Toluene Ethylbenzene Naphthalene Tetrahydronaphthalene Phenanthrene

13 mv 50 0 Aromatics on PRP-1 Using a Thermal Gradient at 20 C/min Column: PRP-1, 3µm, 100 x 2.1 mm Mobile Phase: 75:25 acetonitrile:water, isocratic Flow Rate: 0.5 ml/min Detection: UV 254 nm Temperature Program: 50 C to 150 C at 20 /min, hold two minutes Elution Order: Uracil (off scale) Benzene Toluene Ethylbenzene Naphthalene Tetrahydronaphthalene Phenanthrene Minutes

14 Separation of Proteins Using a Hamilton PRP-3 Column and a Thermal Gradient mv Isocratic! Column: Hamilton PRP-3, 3 µm, 100 x 2.1 mm Mobile Phase: 25:75 acetonitrile:water with 0.1% TFA Flow Rate: 1.0 ml/min Detection: UV 215 nm Temperature Program: 50 C to 150 C at 30 /min, hold five min. Elution Order: Ribonuclease A Cyrochrom C Lysozyme Myoglobin Bovine Serum Albumin Minutes

15 mv Separation of Analgesics on a Selerity Blaze C 8 Using a Thermal Gradient Minutes Column: Selerity Blaze C 8, 3 µm 100 x 4.6 mm Mobile Phase: 40:60 acetonitrile:water with 0.1%TFA Flow Rate: 1.5 ml/min Detection: UV 220 nm Temperature Program: hold at 50 C for one minute, ramp to 100 C at 30 C/min, hold six min. Elution Order: Acetaminophen Caffeine Salicylamide Aspirin Salicylic acid Ibuprofen Naproxen

16 Conclusion No more solvent gradients! Acknowledgements The authors thank Thermo Hypersil-Keystone and Hamilton Company for providing columns used in this work Turn up the Heat!