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.  

1. 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

2. 2
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. 3
Radial Thermal Mismatch in Columns
60 °C COLUMN COMPARTMENT
60 °C COLUMN COMPARTMENT
60 °C 60 °C
SAMPLE AT
AMBIENT
TEMPERATURE
ELUENT
PRE-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

4. 4
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

5. 5
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

6. 6
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

7. 7
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

8. 8
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

9. 9
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

10. 10
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

11. 11
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

12. 12
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

13. 13
Retention vs. Linear Velocity on a 100 mm Column
30 °Cforced air
still air
uopt
2x uopt
uopt
2x uopt
50 °C
forced 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)

14. 14
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!
Mode
100 mm
k (uopt)
100 mm
k (2.5uopt)
Δ%
20 mm
k·εT (uopt)
100 mm
k·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:

15. 15
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

16. 16
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. uracil
2. dimethylphthalate
3. methylparabene
4. naphthalene
5. propranolol
6. biphenyl

17. 17
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 of
van’t Hoff plot (example biphenyl)
~200 bar @ 30 °C ~650 bar @ 30 °C

18. 18
Thermostatting Mode and Selectivity at High Pressure
• Critical selectivity
changes with switch in
thermostatting mode
• Effect increases with
pressure as expected

19. 19
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

20. 20
Axial Thermal Mismatch (Lack of Pre-Heating)
0.0 0.6 1.2
Minutes
2.1 x 30 mm
50 °C still Air
37 °C pre-heater
45 °C post column
220 bar
1
2 + 3
4
5
6
2.1 x 30 mm
50 °C still Air
50 °C pre-heater
50 °C post column
200 bar
1
2 + 3
4
5
6
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

21. 21
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 = 7700
tR = 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

22. 22
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

23. 23
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

24. 24
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 at
Tcompartment 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

25. 25
Thank You!