what efficient temperature control can teach us in … efficient temperature control can teach us in...

What Efficient Temperature Control Can Teach Us in Liquid Chromatography Frank Steiner,1 David Thomas,2 Evert-Jan Sneekes,1 and Michael Heidorn1 1Thermo Fisher Scientific, Germering, Germany; 2Thermo Fisher Scientific, Chelmsford, MA, USA

2 What Ef� cient Temperature Control Can Teach Us in Liquid Chromatography

Temperature Gradients by Frictional Heat

In Figure 6, the axial temperature increase within a typical UHPLC column is plotted against the flow rate. It is shown for different isocratic mobile phase compositions.

With a mobile phase containing 50% acetonitrile at a flow rate of 1.04 mL/min or linear velocity of 8 mm/s, e.g., an axial temperature gradient of 6 °C results with forced air mode and 11.5 °C with still air mode. Consequently, 5.5 °C of frictional heat are dissipated through the column wall under forced air condition, thereby forming a radial temperature gradient. As shown in Figure 7, the chromatographic results are enormously altered.

Frictional Heat Effects on Chromatography As depicted in Figure 6, the column thermostatting mode has a big influence on the axial temperature increase as soon as frictional heat is dissipated. Then, radial temperature gradients are formed within the column. Figures 5 a-c demonstrate theoretic temperature distributions inside an LC column under typical, different column thermostatting conditions. The effect on the separation efficiency (theoretical number of plates, N) as well as analyte retention is shown by authentic chromatograms.

What Efficient Temperature Control Can Teach Us in Liquid Chromatography

Frank Steiner1, David Thomas2, Evert-Jan Sneekes1, and Michael Heidorn1 1Thermo Fisher Scientific, Germering, Germany; 2Thermo Fisher Scientific, Chelmsford, MA, USA

Temperature Control in UHPLC With still air mode, frictional heat is not actively removed from the column. The separation temperature inside the column increases axially, depending on the flow rate. Thereby, a retention factor does not stay constant when using different flow rates (Figure 8). Retention factor changes can be critical for certain applications, especially for samples with very polar compounds. For such compound classes, retention factors often change irregular to each other which might result in a change of selectivity and resolution (Figure 1).

Hence, chromatographic results may change if the frictional heat contribution is not addressed adequately during a method transfer from HPLC to UHPLC, between two UHPLC systems with a different column oven, or when a method speed-up is applied especially in UHPLC. Retention factors, selectivity, and resolution do not change to that extent under forced air conditions, if the efficiency loss due to frictional heat does not compromise chromatographic resolution.

Temperature Settings in Method Transfer If a method is transferred from HPLC to UHPLC conditions, the flow rate is adjusted but the actual linear velocity is kept constant. Ideally, uopt is used in the HPLC method and maintained for the UHPLC method. In this case, the use of the forced air mode does not significantly compromise the separation efficiency (Figure 7). Retention factors and selectivity are retained while the analysis is generally speeded-up and the resolution is often improved. The still air mode can also be used but retention control might become an issue (Figure 8).

During a method speed-up, when the flow rate is accelerated beyond Fopt, the frictional heat contribution to the separation temperature increases. Retention factors cannot be kept constant anymore (Figure 8), even when using the forced air mode. Furthermore, a loss of separation efficiency would occur with the forced air mode. Here, the still air mode is the better choice as the separation efficiency is not affected. To maintain the retention factors and selectivity with the still air mode, frictional heating has to be taken into account. For this purpose, either the mobile phase or the column compartment temperature must be lowered down by half the nominal value of the axial temperature increase resulting from frictional heating (Figure 5a, see comment in the note).

Conclusion In each case, mobile phase pre-heating is essential for proper temperature

control in LC and to obtain best chromatographic results.

To maintain proper detector operation, a post-column mobile phase cooler needs to be used when elevated temperatures are applied.

At column backpressures above 400 bar, frictional heating effects on retention and column efficiency needs to be addressed.

Retention factors, selectivity, and resolution do not change if uopt and the forced air mode are applied for a method transfer from HPLC to UHPLC conditions.

For a method transfer from HPLC to UHPLC conditions where the linear velocity is above uopt or when the linear velocity is increased above uopt during a method speed-up, the still air mode should be used and either the mobile phase or the column compartment temperature must be lowered down by half the nominal value of the axial temperature increase resulting from frictional heating.

References 1. Heidorn, Michael. The Role of Temperature and Column Thermostatting in LC.

2015. Thermo Scientific White Paper 71499.

2. Heidorn, Michael. A More Flexible Column Thermostatting Technique in LC Method Transfer. 2015. Thermo Scientific White Paper 71500.

Overview Purpose: Influences of the mobile phase temperature or frictional heat in combination with the column thermostat type on the chromatographic result shall be evaluated.

Methods: Same method settings compared under different thermostatting conditions.

Results: Proper temperature control is more than setting a value for the column thermostat, especially for UHPLC methods or when using elevated temperatures.

Introduction Each good chromatographer wants to implement proper temperature control. For this purpose, column thermostats have evolved from simple units just providing a fixed temperature to high-tech devices. Elevated temperatures, well above the boiling point of the mobile phase, become more and more applied. Consequently, mobile phase pre-heating prior to entering the column as well as post-column mobile phase cooling prior to entering the detector becomes mandatory. In addition, viscous heating is a much discussed topic in Ultra High Performance Liquid Chromatography (UHPLC). When pressures increase to values far above 400 bar, friction between column and mobile phase generates high amounts of viscous heat and, thereby, an additional temperature increase inside the column.

In this work, various elements of the entire temperature control (e.g., mobile phase pre-heating or column thermostat type) are evaluated to determine effects of the different thermostatting conditions on chromatographic performance. Furthermore, the amount of frictional heat was measured for a typical UHPLC column (1.9 m, 2.1 x 100 mm). It is discussed how chromatographic results can be maintained or even improved during a method transfer or speed-up.

Importance of Temperature Control in LC

A rule of thumb for isocratic reversed-phase LC predicts 1—2% retention time decrease for each 1 °C column temperature increase. The percentage decrease in retention time is usually not the same for all compounds of a sample mixture and changes in peak spacing are common. In that case, retention factors of sample compounds change, sample compounds may co-elute, or their selectivities can be inverted by a small variation of the column temperature (Figure 1).

For most molecules and separation conditions, the retention factor k of an analyte changes proportional with temperature (c.f. Van´t Hoff equation). In Van´t Hoff plots, a ln(k) value is plotted against the reciprocal value of the corresponding separation temperature (Figure 2). An intersection point of two Van´t Hoff curves indicate a change in selectivity for the two corresponding analytes at the given temperature.

Mobile Phase Pre-Heating

With a still air thermostat, retention time shifts and selectivity changes might occur at elevated temperatures if the mobile phase is not pre-heated accurately (figure 3).

With a forced air thermostat, peak broadening or even peak distortion might occur at elevated temperatures if the mobile phase is not pre-heated accurately (Figure 4).

Post-Column Mobile Phase Cooling When running separations at elevated temperatures, boiling of organic mobile phases might appear or vapor bubbles might outgas in the detector. During separation, both is suppressed by a high system pressure. Frictional heating at ultra high pressures might lead to similar situations. Increased baseline noise, ghost peaks or even complete light absorption can appear in the detector flow cell. A post-column mobile phase cooler should be used. It also maintains sufficient backpressure for proper detector operation.

The Effect of Frictional Heat on Efficiency

An efficiency drop is indicated by the slope of a Van Deemter curve. Figure 7 shows Van Deemter curves recorded with increasing viscous heating effect by accelerating the linear velocity. The Vanquish UHPLC system provides a still air and a forced air mode. Except for those two complementary thermostatting modes, identical conditions were used to acquire each Van Deemter curve. At the linear velocity of 8 mm/s, the column efficiency with forced air mode was by 40% lower than with still air mode.

The Effect of Frictional Heat on Retention

In figure 8, retention factors were investigated with increasing viscous heating by accelerating the linear velocity. With still air mode, the retention factor decreased constantly. With forced air mode, the retention factor remained constant until a linear velocity of 3.5 mm/s or a flow rate of 0.5 mL/min was reached. Beyond this flow rate, the retention factor began to decrease. The same slope for the retention factor decrease indicates that an equal amount of frictional heat was produced per step of flow rate increase under both thermostatting modes.

Thermo Scientific™ Dionex™ UltiMate™ 3000 RS System Sample: 1. Uracil 2. Propranolol

3. Dipropylphthalate 4. Butylparaben 5. Naphthalene 6. Amitriptyline 7. Acenaphthene

Inj. Volume: 5 µL of model sample Eluent: CH3OH:20 mM Na2HPO4 buffer at pH 7 Isocratic: 65:35 (v/v), mixed by pump Flow Rate: 1.0 mL/min

FIGURE 1. Influence of the temperature on the chromatographic result.

Thermo Scientific™ Vanquish™ UHPLC System Sample: 1. Dimethylphthalate 2. Methylparabene 3. Propranolol 4. Naphthalene 5. Biphenyl Inj. Volume: 1 µL Eluent: CH3OH:20 mM Na2HPO4 buffer at pH 7 Isocratic: 65:35 (v/v), mixed by pump Flow Rate: 0.65 mL/min

FIGURE 2. Van´t Hoff plots for some neutral and polar analyte molecules.

Vanquish UHPLC System Sample: 1. Uracil 2. Dimethylphthalate 3. Propranolol 4. Naphthalene 5. Biphenyl Inj. Volume: 1 µL Eluent: CH3OH:20 mM Na2HPO4 buffer at pH 7 Isocratic: 65:35 (v/v), mixed by pump Flow Rate: 0.45 mL/min

FIGURE 3. Influence of mobile phase pre-heating with a still air thermostat.

Vanquish UHPLC System Sample: 1. Uracil 2. 4-Nitroaniline 3. Methyl Benzoate 4. Phenetole 5. o-Xylene Inj. Volume: 2.5 µL Eluent: CH3CN:H2O Isocratic: 40:60 (v/v), mixed by pump Flow Rate: 1.5 mL/min

FIGURE 4. Influence of mobile phase pre-heating with a forced air thermostat.

Vanquish UHPLC System Column: Hypersil GOLD, 1.9 m, 2.1 x 100 mm Sample: Hexanophenone Inj. Volume: 2.7 L Eluent: CH3CN:H2O Isocratic: 50:50 (v/v), mixed by pump Flow Rate: Up to 1.04 mL/min Temperature: 30 °C (column compartment and mobile phase pre-heater) Note: The same experimental setup was used for both thermostatting modes by applying the same Vanquish UHPLC system without changing fluidics or anything else.

FIGURE 8. Influence of frictional heat on retention factor.

Thermo Scientific Vanquish UHPLC System Column: Thermo Scientific Hypersil GOLD, 1.9 µm, 2.1 x 100 mm Sample: Hexanophenone Inj. Volume: 2.7 µL Eluent: CH3CN:H2O Isocratic: 50:50 (v/v), mixed by pump Flow Rate: Up to 1.04 mL/min Temperature: 30 °C (column compartment and mobile phase pre-heater) Note: The same experimental setup was used for both thermostatting modes by applying the same Vanquish UHPLC system without changing fluidics or anything else.

FIGURE 7. Influence of frictional heat on column efficiency. Vanquish UHPLC System Column: Thermo Scientific™ Hypersil GOLD™, 1.9 m, 2.1 x 100 mm Eluent: CH3CN:H2O Flow Rate: Up to 1.40 mL/min Temperature: 30 °C (column compartment and mobile phase pre-heater) Note: The Vanquish UHPLC system provides a still and forced air mode. Hence, the same experimental setup was used with both thermostatting modes without changing fluidics or anything else.

FIGURE 6. Axial temperature increase in correlation to the flow rate.

Vanquish UHPLC System Column: Thermo Scientific™ Accucore™ Vanquish™ C18, 1.5 m, 2.1x100 mm Sample: Uracil, acetanilide, and 8 phenones Inj. Volume: 1 L Eluent: 55:45 CH3CN:H2O (v/v) Flow Rate: 0.65 mL/min Pressure: 1450 bar Note: To mimic a “no frictional heat” situation, the mobile phase was actively pre-heated to an inlet temperature of 23 °C. Caused by frictional heat, the mobile phase had a temperature of 39 °C at the column outlet. Hence, an average separation temperature of approximately 30 °C can be assumed, theoretically leading to a similar result as with a thermal equilibration state at 30 °C.

FIGURE 5a. Temperature distribution inside an LC column without frictional heat and chromatogram resulting with thermal conditions at equilibrium.

Thermo Scientific Vanquish UHPLC System Column: Accucore Vanquish C18, 1.5 m, 2.1x100 mm Sample: Uracil, acetanilide, and 8 phenones Inj. Volume: 1 L Eluent: 55:45 CH3CN:H2O (v/v) Flow Rate: 0.65 mL/min Pressure: 1430 bar Note: For a quasi-isothermal situation, the forced air mode with the highest settable fan speed of the Vanquish UHPLC system was applied. The Vanquish UHPLC system allows to set seven different fan speeds for method transfer reasons.

FIGURE 5b. Influence of frictional heat under quasi-isothermal conditions (forced air mode) on temperature distribution and resulting chromatogram.

Vanquish UHPLC System Column: Accucore Vanquish C18, 1.5 m, 2.1x100 mm Sample: Uracil, acetanilide, and 8 phenones Inj. Volume: 1 L Eluent: 55:45 CH3CN:H2O (v/v) Flow Rate: 0.65 mL/min Pressure: 1385 bar Note: Quasi-adiabatic column thermostatting can be supposed when still air condition is used. The Vanquish UHPLC system provides a still air mode. For a fast column heat-up even with this still air mode, Thermo Scientific™ Dionex™ Chromeleon™ Chromatography Data System automatically uses forced air during the temperature equilibration phase, hence, until the set temperature is reached. Afterwards, the fan of the column thermostat is turned off for a certain period of time. The separation is performed under still air condition.

FIGURE 5 c. Influence of frictional heat under quasi-adiabatic conditions (still air mode) on temperature distribution and resulting chromatogram.

TABLE 1. Axial temperature increase for a 1.9 m, 2.1 x 100 mm column with a mobile phase containing 50% acetonitrile related to backpressure and flow rate.

50% Acetonitrile Still Air Mode Forced Air Mode

Flow Rate [mL/min] System

Backpressure [bar] Axial Temperature

Increase [°C] System


Increase [°C] 0.2 173 0.0 177 0.0 0.4 343 2.3 356 0.2 0.6 506 5.4 527 1.7 0.8 659 8.4 685 3.7 1.0 809 11.3 830 5.8 1.2 958 14.2 975 8.1 1.4 1079 17.0 1099 10.3

All trademarks are the property of Thermo Fisher Scientific Inc and its subsidiaries.

This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. PO71517-EN 03/15S

3Thermo Scienti� c Poster Note • PITTCON • PN71517-EN 0215S































































Increase [°C] 0.2 173 0.0 177 0.0 0.4 343 2.3 356 0.2 0.6 506 5.4 527 1.7 0.8 659 8.4 685 3.7 1.0 809 11.3 830 5.8 1.2 958 14.2 975 8.1 1.4 1079 17.0 1099 10.3



4 What Ef� cient Temperature Control Can Teach Us in Liquid Chromatography































































Increase [°C] 0.2 173 0.0 177 0.0 0.4 343 2.3 356 0.2 0.6 506 5.4 527 1.7 0.8 659 8.4 685 3.7 1.0 809 11.3 830 5.8 1.2 958 14.2 975 8.1 1.4 1079 17.0 1099 10.3



5Thermo Scienti� c Poster Note • PITTCON • PN71517-EN 0215S































































Increase [°C] 0.2 173 0.0 177 0.0 0.4 343 2.3 356 0.2 0.6 506 5.4 527 1.7 0.8 659 8.4 685 3.7 1.0 809 11.3 830 5.8 1.2 958 14.2 975 8.1 1.4 1079 17.0 1099 10.3




In Figure 6, the axial temperature increase within a typical UHPLC column is plottedagainst the flow rate. It is shown for different isocratic mobile phase compositions.

With a mobile phase containing 50% acetonitrile at a flow rate of 1.04 mL/min or linearvelocity of 8 mm/s, e.g., an axial temperature gradient of 6 °C results with forced airmode and 11.5 °C with still air mode. Consequently, 5.5 °C of frictional heat aredissipated through the column wall under forced air condition, thereby forming a radialtemperature gradient. As shown in Figure 7, the chromatographic results areenormously altered.

Frictional Heat Effects on ChromatographyAs depicted in Figure 6, the column thermostatting mode has a big influence on theaxial temperature increase as soon as frictional heat is dissipated. Then, radialtemperature gradients are formed within the column. Figures 5 a-c demonstratetheoretic temperature distributions inside an LC column under typical, different columnthermostatting conditions. The effect on the separation efficiency (theoretical numberof plates, N) as well as analyte retention is shown by authentic chromatograms.


Frank Steiner1, David Thomas2, Evert-Jan Sneekes1, and Michael Heidorn1

1Thermo Fisher Scientific, Germering, Germany; 2Thermo Fisher Scientific, Chelmsford, MA, USA







To maintain proper detector operation, a post-column mobile phase cooler needsto be used when elevated temperatures are applied.

At column backpressures above 400 bar, frictional heating effects on retentionand column efficiency needs to be addressed.

Retention factors, selectivity, and resolution do not change if uopt and the forcedair mode are applied for a method transfer from HPLC to UHPLC conditions.

For a method transfer from HPLC to UHPLC conditions where the linear velocityis above uopt or when the linear velocity is increased above uopt during a methodspeed-up, the still air mode should be used and either the mobile phase or thecolumn compartment temperature must be lowered down by half the nominalvalue of the axial temperature increase resulting from frictional heating.



2. Heidorn, Michael. A More Flexible Column Thermostatting Technique in LCMethod Transfer. 2015. Thermo Scientific White Paper 71500.

OverviewPurpose: Influences of the mobile phase temperature or frictional heat in combinationwith the column thermostat type on the chromatographic result shall be evaluated.


Results: Proper temperature control is more than setting a value for the columnthermostat, especially for UHPLC methods or when using elevated temperatures.

IntroductionEach good chromatographer wants to implement proper temperature control. For thispurpose, column thermostats have evolved from simple units just providing a fixedtemperature to high-tech devices. Elevated temperatures, well above the boiling pointof the mobile phase, become more and more applied. Consequently, mobile phase pre-heating prior to entering the column as well as post-column mobile phase cooling priorto entering the detector becomes mandatory. In addition, viscous heating is a muchdiscussed topic in Ultra High Performance Liquid Chromatography (UHPLC). Whenpressures increase to values far above 400 bar, friction between column and mobilephase generates high amounts of viscous heat and, thereby, an additional temperatureincrease inside the column.

In this work, various elements of the entire temperature control (e.g., mobile phase pre-heating or column thermostat type) are evaluated to determine effects of the differentthermostatting conditions on chromatographic performance. Furthermore, the amountof frictional heat was measured for a typical UHPLC column (1.9 m, 2.1 x 100 mm). Itis discussed how chromatographic results can be maintained or even improved duringa method transfer or speed-up.


A rule of thumb for isocratic reversed-phase LC predicts 1—2% retention timedecrease for each 1 °C column temperature increase. The percentage decrease inretention time is usually not the same for all compounds of a sample mixture andchanges in peak spacing are common. In that case, retention factors of samplecompounds change, sample compounds may co-elute, or their selectivities can beinverted by a small variation of the column temperature (Figure 1).

For most molecules and separation conditions, the retention factor k of an analytechanges proportional with temperature (c.f. Van´t Hoff equation). In Van´t Hoff plots, aln(k) value is plotted against the reciprocal value of the corresponding separationtemperature (Figure 2). An intersection point of two Van´t Hoff curves indicate a changein selectivity for the two corresponding analytes at the given temperature.


With a still air thermostat, retention time shifts and selectivity changes might occur atelevated temperatures if the mobile phase is not pre-heated accurately (figure 3).

With a forced air thermostat, peak broadening or even peak distortion might occur atelevated temperatures if the mobile phase is not pre-heated accurately (Figure 4).

Post-Column Mobile Phase CoolingWhen running separations at elevated temperatures, boiling of organic mobile phasesmight appear or vapor bubbles might outgas in the detector. During separation, both issuppressed by a high system pressure. Frictional heating at ultra high pressures mightlead to similar situations. Increased baseline noise, ghost peaks or even complete lightabsorption can appear in the detector flow cell. A post-column mobile phase coolershould be used. It also maintains sufficient backpressure for proper detector operation.


An efficiency drop is indicated by the slope of a Van Deemter curve. Figure 7 showsVan Deemter curves recorded with increasing viscous heating effect by acceleratingthe linear velocity. The Vanquish UHPLC system provides a still air and a forced airmode. Except for those two complementary thermostatting modes, identical conditionswere used to acquire each Van Deemter curve. At the linear velocity of 8 mm/s, thecolumn efficiency with forced air mode was by 40% lower than with still air mode.


In figure 8, retention factors were investigated with increasing viscous heating byaccelerating the linear velocity. With still air mode, the retention factor decreasedconstantly. With forced air mode, the retention factor remained constant until a linearvelocity of 3.5 mm/s or a flow rate of 0.5 mL/min was reached. Beyond this flow rate,the retention factor began to decrease. The same slope for the retention factordecrease indicates that an equal amount of frictional heat was produced per step offlow rate increase under both thermostatting modes.

Thermo Scientific™ Dionex™ UltiMate™ 3000 RS System

Sample: 1. Uracil2. Propranolol3. Dipropylphthalate4. Butylparaben 5. Naphthalene6. Amitriptyline7. Acenaphthene

Inj. Volume: 5 µL of model sampleEluent: CH3OH:20 mM Na2HPO4 buffer at pH 7Isocratic: 65:35 (v/v), mixed by pump Flow Rate: 1.0 mL/min


Thermo Scientific™ Vanquish™ UHPLC System

Sample: 1. Dimethylphthalate2. Methylparabene3. Propranolol4. Naphthalene5. Biphenyl

Inj. Volume: 1 µLEluent: CH3OH:20 mM Na2HPO4 buffer at pH 7Isocratic: 65:35 (v/v), mixed by pump Flow Rate: 0.65 mL/min


Vanquish UHPLC System

Sample: 1. Uracil2. Dimethylphthalate3. Propranolol4. Naphthalene5. Biphenyl

Inj. Volume: 1 µLEluent: CH3OH:20 mM Na2HPO4 buffer at pH 7Isocratic: 65:35 (v/v), mixed by pump Flow Rate: 0.45 mL/min



Sample: 1. Uracil2. 4-Nitroaniline3. Methyl Benzoate4. Phenetole5. o-Xylene

Inj. Volume: 2.5 µLEluent: CH3CN:H2OIsocratic: 40:60 (v/v), mixed by pump Flow Rate: 1.5 mL/min



Column: Hypersil GOLD, 1.9 m, 2.1 x 100 mm

Sample: HexanophenoneInj. Volume: 2.7 LEluent: CH3CN:H2OIsocratic: 50:50 (v/v), mixed by pump Flow Rate: Up to 1.04 mL/min Temperature: 30 °C (column compartment and mobile

phase pre-heater)

Note: The same experimental setup was used for both thermostatting modes by applying the same Vanquish UHPLC system without changing fluidics or anything else.


Thermo Scientific Vanquish UHPLC System

Column: Thermo Scientific Hypersil GOLD, 1.9 µm, 2.1 x 100 mm

Sample: HexanophenoneInj. Volume: 2.7 µLEluent: CH3CN:H2OIsocratic: 50:50 (v/v), mixed by pump Flow Rate: Up to 1.04 mL/min Temperature: 30 °C (column compartment and mobile

phase pre-heater)

Note: The same experimental setup was used for both thermostatting modes by applying the same Vanquish UHPLC system without changing fluidics or anything else.

FIGURE 7. Influence of frictional heat on column efficiency.Vanquish UHPLC System

Column: Thermo Scientific™ Hypersil GOLD™, 1.9 m, 2.1 x 100 mm

Eluent: CH3CN:H2OFlow Rate: Up to 1.40 mL/min Temperature: 30 °C (column compartment and mobile

phase pre-heater)

Note: The Vanquish UHPLC system provides a still and forced air mode. Hence, the same experimental setup was used with both thermostatting modes without changing fluidics or anything else.



Column: Thermo Scientific™ Accucore™ Vanquish™ C18, 1.5 m, 2.1x100 mmSample: Uracil, acetanilide, and 8 phenonesInj. Volume: 1 L Eluent: 55:45 CH3CN:H2O (v/v) Flow Rate: 0.65 mL/minPressure: 1450 bar

Note: To mimic a “no frictional heat” situation, the mobile phase was actively pre-heated to an inlet temperature of 23 °C. Caused by frictional heat, the mobile phase had a temperature of 39 °C at the column outlet. Hence, an averageseparation temperature of approximately 30 °C can be assumed, theoretically leading to a similar result as with a thermalequilibration state at 30 °C.


Thermo Scientific Vanquish UHPLC System

Column: Accucore Vanquish C18, 1.5 m, 2.1x100 mmSample: Uracil, acetanilide, and 8 phenonesInj. Volume: 1 L Eluent: 55:45 CH3CN:H2O (v/v) Flow Rate: 0.65 mL/minPressure: 1430 bar

Note: For a quasi-isothermal situation, the forced air mode with the highest settable fan speed of the Vanquish UHPLC system was applied. The Vanquish UHPLC system allows to set seven different fan speeds for method transfer reasons.



Column: Accucore Vanquish C18, 1.5 m, 2.1x100 mmSample: Uracil, acetanilide, and 8 phenonesInj. Volume: 1 L Eluent: 55:45 CH3CN:H2O (v/v) Flow Rate: 0.65 mL/minPressure: 1385 bar

Note: Quasi-adiabatic column thermostatting can be supposed when still air condition is used. The Vanquish UHPLC system provides a still air mode. For a fast column heat-up even with this still air mode, Thermo Scientific™ Dionex™

Chromeleon™ Chromatography Data System automatically uses forced air during the temperature equilibration phase, hence, until the set temperature is reached. Afterwards, the fan of the column thermostat is turned off for a certain period of time. The separation is performed under still air condition.




Flow Rate [mL/min]System

Backpressure [bar]Axial Temperature

Increase [°C]System

Backpressure [bar]Axial Temperature

Increase [°C]0.2 173 0.0 177 0.00.4 343 2.3 356 0.20.6 506 5.4 527 1.70.8 659 8.4 685 3.71.0 809 11.3 830 5.81.2 958 14.2 975 8.11.4 1079 17.0 1099 10.3


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