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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

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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

• Van Deemter curves and thermostatting modes

• Van’t Hoff plots and thermostatting modes

• Thermostatting and method transfer

• Conclusions and recommendations

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Experimental Plan of van Deemter Tests

• Method fundamentals:• Stationary phase: Thermo Scientific™

Hypersil™ GOLD™ 1.9 µm column

• Mobile phase: 50/50 v/v H2O/ACN isocratic

• Test sample: phenones + uracil

• Detection: UV @ 240 nm

• Aim was to study efficiency and retention behavior at• different column lengths and diameters (2.1 x 20 , 2.1 x 100, 3.0 x 100 mm)

• different linear velocities (0.3 -11.7 mm/s for 100 mm column, 2.5 - 14.5 mm/s for 20 mm col.)

• different temperatures (30 and 50 °C)

• different thermostatting modes (still air and forced air)

• All presented data are solely for Hexanophenone

0 3 6 min

Uracil

Acetanilide

Acetophenone

Propiophenone

Butyrophenone

Benzophenone

Valerophenone

Hexanophenone

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Van Deemter Curves on 20 mm Column Length

30 °C

forced air

still air

50 °C

forced air

still air

• Still air and forced air mode are close in their optimum efficiency• Slope of C-term 8—10% steeper in forced air mode (less different at higher T)

• At higher T the C-term bends down from 2 x uopt on (data not precise)

• Difference between both thermostatting mode is minor on short column

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Van Deemter Curves on 100 mm Column Length

30 °C

forced air

still air

• Still air mode is ~10% more efficient and 20% faster at curve minimum

• At u = 2 x uopt, still air mode is 40% better at 30 °C and 25% better at 50 °C

• Difference between thermostatting modes decreases with increasing T• Fast separations on long UHPLC columns require close to adiabatic

thermostatting in order to preserve efficiency

880 bar

860 bar

40 %

25 %

50 °C

forced air

still air

825 bar

810 bar

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Temperature Influence in Different Modes

Forced Air

30 °C

50 °C

30 °C

50 °C

Still Air

• Apparent effect of temperature decreases in still air mode• At ambient temperature “adiabatic” thermostatting is particular critical in

order to run long columns fast

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Influence of Column Diameter (Forced Air Mode)

30 °C

3.0 mm i.d.

2.1 mm i.d.

50 °C

3.0 mm i.d.

2.1 mm i.d.

• At both temperatures, 2.1 mm i.d. shows better efficiency (6-8%) and higher optimal linear velocity (10-15%) than 3 mm i.d.

• With increasing linear velocity the C-term of the 3 mm column slows down and efficiency comes close to that of the 2.1 mm column

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Retention vs. Linear Velocity on a 100 mm Column

30 °Cforced air

still air

uopt

2x uopt

uopt

2x uopt

50 °Cforced air

still air

• Forced air:• Retention constant in B-term (up to

H/u minimum, or beyond @ 30 °C)

• Retention drops by 2.5% from uopt to 2 x uopt (both temperatures)

• Still air:

• Retention drops by 2% up to uopt (both temperatures)

• Retention drops by 5% up to 2 x uopt (both temperatures)

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Column Diameter and Total Effects on Retention

30 °C3.0 mm i.d.

2.1 mm i.d.

• Even in forced air mode, the long 3.0 mm i.d. column showed almost no constant retention range

• At linear velocity beyond van Deemter minimum, retention drop is similar for both diameters

Forced air mode!

Mode100 mmk (uopt)

100 mmk (2.5uopt)

Δ%20 mm

k·εT (uopt)100 mmk·εT (uopt)

Δ %

Still air 7.7 7.5 - 2.6 4.6 4.8 + 4.3

Forced air 7.9 7.6 - 3.8 4.6 4.9 + 6.5

Behavior of 2.1 mm columns on retention of hexanophenone at 30 °C:

Temperature effect

Pressure effect

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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

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Experimental Plan of Van’t Hoff Plot Study

• Method fundamentals:• Stationary phase: Thermo Scientific™ Acclaim™ RSLC™

PA2 column, 2.2 µm

• Mobile phase: 20 mM phosphate buffer pH 7 / methanol 35/65 v/v (Neue Test eluent)

• Test sample: modified Neue Test (see below in corner) dissolved in mobile phase

• Detection: UV @ 210 nm

• Aim was to study efficiency and selectivity behavior at• different column lengths (30, 150 mm, 2.1 mm i.d.)

• different linear velocities (450 – 900 µL/min, flow or pressure constant series)

• different temperatures (10, 20, 30, 40, and 50 °C)

• different thermostatting modes (still air and forced air)

1. uracil2. dimethylphthalate3. methylparabene4. naphthalene5. propranolol6. biphenyl

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Van‘t Hoff Plots and Thermostatting Mode

• Van’t Hoff plots indicate elution inversion propranolol / naphthalene at 40 °C and methylparabene / dimethylphthalate at 60 °C

• With long column thermostatting mode significantly influences slope ofvan’t Hoff plot (example biphenyl)

~200 bar @ 30 °C ~650 bar @ 30 °C

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Thermostatting Mode and Selectivity at High Pressure

• Critical selectivity changes with switch in thermostatting mode

• Effect increases with pressure as expected

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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

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Axial Thermal Mismatch (Lack of Pre-Heating)

0.0 0.6 1.2

Minutes

2.1 x 30 mm50 °C still Air37 °C pre-heater45 °C post column220 bar

12 + 3

45

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2.1 x 30 mm50 °C still Air50 °C pre-heater50 °C post column200 bar

12 + 3

45

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0.0 0.6 1.2

Minutes

• Thermostatting compartment is on elevated temperature, but pre-heater switched off (only passive)

• Eluent hardly reaches compartment temperature at column outlet (short column!)

• Significant selectivity change observed

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Transferring Forced Air Selectivity to Still Air Mode

Forced air 30 °C, Pre-heater 30 °C 700 µL/min, 1200 bar

N = 4600

tR = 4.8 min

Still air 30 °C, Pre-heater 30 °C 700 µL/min, 1100 bar

N = 7700tR = 4.6 min

Still air 30 °C, Pre-heater 30 °C 760 µL/min, 1200 bar

N = 7300 tR = 4.5 min

Still air 30 °C, Pre-heater 27 °C (off) 700 µL/min, 1170 bar

N = 7600

tR = 4.9 min

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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

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What are Facts? What are Fictions?

• UHPLC instruments account for excellent column temperature control• Fiction: even forced air thermostats are not fully capable to dissipate viscous

heating, but most UHPLC thermostats (still air) fail completely with columns of 2.1 mm i.d. or wider (deviations in retention of up to 5%)

• UHPLC thermostats account for best possible column efficiency• Fact: (if still air type and as close as possible to adiabatic behavior)

• Column thermostatting is of minor relevance for method transfer• Fiction: Selectivities can change with thermostatting mode and peaks can

even merge (that were separated with different mode at nominally identical column temperature)

• Retention factors in UHPLC are independent of column length and flow• Fiction: retention changes with column temperature and pressure which

(under viscous heating) both change with flow and/or column length

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Conclusions and Recommendations

• Modern UHPLC instruments trade method portability and best possible column temperature control for best efficiency (but they have to)

• Effective column temperature at elevated pressures with still air thermostatting is unknown

• Could be a concern from a regulatory standpoint• No easy solution available, but forced air capability is helpful

• To mimic the selectivity as achieved at the absence of viscous heating, independent control of incoming eluent temperature is helpful

• If you can measure the column outlet temperature, set pre-heater atTcompartment minus 0.5 x ΔTcolumn outlet – compartment

• If you cannot measure the column outlet temperature, set pre-heater temperature at a T that generates the same pressure as forced air mode

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Thank You!