1 The world leader in serving science Dr. Frank Steiner Manager HPLC Solutions Marketing Co-Authors: Michael Heidorn, Melanie Neubauer, Dr. Markus M. Martin, Dr. Tony Edge, Dr. Luisa Pereira Facts and Fictions About Temperature Control in UHPLC – Adiabatic vs. Isothermal Operation or Trading Method Portability Against Ultimate Efficiency
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Facts and Fictions About Temperature Control in Ultra High Performance Liquid Chromatography – Adiabatic vs. Isothermal Operation or Trading Method Portability Against Ultimate Efficiency
Modern liquid chromatography hardware and software embrace larger parts of our laboratory workflows than ever before. From sample preparation to sample vial labeling, from setting-up Liquid Chromatography runs to instant result calculation – everywhere along the workflow software and hardware automate work steps which have required manual action before. Next to better productivity, the automation and improved technologies also result in enhanced quality and result consistency. The seminar reviews very practical examples which all users can relate too. It covers an attractive variety of application areas and analytical challenges.
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Transcript
1
The world leader in serving science
Dr. Frank Steiner
Manager HPLC Solutions Marketing
Co-Authors: Michael Heidorn, Melanie Neubauer,Dr. Markus M. Martin, Dr. Tony Edge, Dr. Luisa Pereira
Facts and Fictions About Temperature Control in UHPLC – Adiabatic vs. Isothermal Operation or Trading Method Portability Against Ultimate Efficiency
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Outline
• Introduction: Column thermostatting and thermal mismatch
• Van Deemter curves and thermostatting modes
• Van’t Hoff plots and thermostatting modes
• Thermostatting and method transfer
• Conclusions and recommendations
3
Radial Thermal Mismatch in Columns
60 °C COLUMN COMPARTMENT
60 °C COLUMN COMPARTMENT
60 °C 60 °CSAMPLE ATAMBIENT TEMPERATURE
ELUENTPRE-HEATER
SAMPLE AT AMBIENT TEMPERATURE
40 °C 60 °C
Mismatch:• Centre of column below oven temperature
• Higher viscosity, lower linear velocity in centre• Higher retention in centre
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Isothermal and Adiabatic Operation, Frictional Heating
AdiabaticIsothermal
70 °C 70 °C 70 °C 70 °C
70 °C
70 °C 70 °C 70 °C 70 °C
70 °C
Ideal HPLC case (≤ 400 bar) Ideal HPLC case (≤ 400 bar)
22 °C 45 °C 65 °C 70 °C 70 °C
70 °C
22 °C 22 °C
70 °C
Cold incoming solvent Cold incoming solvent cools column near to solvent temp. over time
70 °C 80 °C73 °C 76 °C
70 °C
70 °C 90°C
70 °C
Frictional heating (≥ 600 bar)
Radial temperature gradient
Frictional heating (≥ 600 bar)
Axial temperature gradient
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Instrumental Setup for Both Experimental Series
• Prototype UHPLC System with Diode Array Detector• Optimized for minimum extra column effects• System variance of σ² = 5.2 µL2 (by FIA experiments with acetone)
• Special prototype column thermostat• Small air volume around columns• Adjustable fan for controlled air circulation• Actively controlled pre-heater (independent temperature)• 2nd pre-heater can be used as temperature sensor behind column
Active pre-heater at column inlet
2nd pre-heater (off) as column temperature sensor
Thermostatted bent around column ground plate
Fan with adjustable speed
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Statements on Modern UHPLC – Facts or Fictions?
4 Statements:
• UHPLC instruments account for excellent column temperature control
• UHPLC thermostats account for best possible column efficiency
• Column thermostatting is of minor relevance for method transfer
• Retention factors in UHPLC are independent of column length and flow
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Outline
• Introduction: Column thermostatting and thermal mismatch