The Effect of Temperature on Selectivity in HPLC€¦ · The Use of Temperature in HPLC Temperature...
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The Effect of Temperature on Selectivity in HPLC Brian Jones , Jody Clark, Dale Felix and Stephanie J. Marin Selerity Technologies, Inc. 2484 W. Custer Road Salt Lake City, UT 84104 www.selerity.com
Transcript of The Effect of Temperature on Selectivity in HPLC€¦ · The Use of Temperature in HPLC Temperature...
The Effect of Temperature on Selectivity in HPLC
Brian Jones, Jody Clark, Dale Felix and Stephanie J. Marin
Selerity Technologies, Inc.2484 W. Custer Road
Salt Lake City, UT 84104
www.selerity.com
The Use of Temperature in HPLC
Temperature is considered to be the overlooked or forgotten optimization parameter in HPLC by many of the experts.
“Although nearly all of the physical parameters that play a role in liquid chromatographic separation are a function of temperature, temperature has not yet been adequately explored as a parameter to tune separation and shorten analysis times in LC .” *
* Nebojsa M. Djordjevic, Patrick W.J. Fowler, Fabice Houdiere J. Microcolumn Separations 11(6) (1999) 403-413
Typical Retentive Behavior
• Retention uniformly decreases with temperature
• Linear van’t Hoff plots – log k vs. 1/T• A 1% methanol increase is equivalent to
~4°C temperature increase
Change in Retention with Temperature
k1’ / k2’ = exp(∆H(T2 – T1) / (RT1T2)
• Effect of temperature on retention factor depends on enthalpy of the solute. The larger the enthalpy, the greater the change in k’
F. FF. Houdiere, P.W.J. Fowler, N.M. Djordjevic, Anal. Chem., 69, 2589-2593 (1997)
Analyte Families Giving Reversed Retention with Increasing Temperature
• Polyethers• Reverse retention due to apparent decreasing
polarity due to hydrogen bond strength reduction
• Some dipeptides• Decreasing polarity with conformational change
Factors Affecting Retention That Are Influenced by Temperature
• Hydrogen bonding• Solvation sphere around analytes• Hydration extent of column surface• Functional group interaction• Ordering, shape and hydration transitions
– Conformation changes• Dielectric constant of mobile phase
Temperature Affects Hydrogen Bonding
• Increasing temperature-– Increases intermolecular distance– Weakens hydrogen bonds
O
Si
O
OO
Si
O
OHO
H
H
O H
H O
H
O
Si
O
OO
Si
O
OHO
H
OH
HH O
H
Column Surface Hydration
OSi
OSi
OSi
OSi
OSi
OSi
OSi
OSi
O OH O O O OH O O
SiOH
OHSiHO Si
HO
OH HO SiOH
HO
H
HO
HH
OH
H
OH
H
OH
H
OH
H
O H
HO
H
HO
H H
OH
H
OH
H
OH
Functional Group ConformationOctadecyl ordering at low temperatures
O
Si
O
Si
O
Si
O
Si
O
Si
O
Si
O
Si
O
Si
O
O
OH
O
O
O
OH
O
O
O
O
O
O
O
O
O
Si
Si
Si
Si
Si
Si
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Functional Group ConformationOctadecyl disorder at higher temperatures
O
Si
O
Si
O
Si
O
Si
O
Si
O
Si
O
Si
O
Si
O
O
OH
O
O
O
OH
O
O
O
O
O
O
O
O
O
Si
Si
Si
Si
Si
Si
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Functional Group Phase Transitions
• N-Isopropylacrylamide coated silica
OHN
O NHO NH O NH
OHN
Analyte Conformational Changes
• Heteroduplex and homoduplex oligonucleotides separable by HPLC with temperature gradients– Axial or spatial gradients– Temperature programming of the entire
column
From R.E. Gerber and R.G. Hatch in US Patent 6,486,309
Solvent Polarity as a Function of Temperature
Data from Y. Yang et al. J. Chromatogr. A 810 (1998) 149.
% Methanol or Acetonitrile in Water at 25°C
20
30
40
50
60
70
80
90
25 75 125 175 225
Acetonitrile/Water
Methanol/Water
Pure Water(at 50 bar)D
iele
ctric
Con
stan
t, ε
0 20 40 60 80 100
Pure Water Temperature, °C
High Temperature LiquidChromatography Advantages
•The advantages of using temperature to optimize separations in HPLC are well documented in the literature. •The major advantages are
– Increased speed – Higher efficiencies and resolution– Ability to tune selectivity with temperature– Decreased organic solvent usage
• Use less organic in solvent ratio• Perform isocratic separations and recycle solvent
Increased Diffusivity
• Increasing the temperature increases the enthalpy of solute transfer from mobile phase to stationary phase*
– Improves efficiency, particularly for large analytes
– Allows operation at higher flow rates without penalty
*F.D. Antia and Cs. Horvath, J. Chromatogr. 435 (1988) 1-15.
*B. Yan, J. Zhao, J.S. Brown, J. Blackwell, P. W. Carr, Anal. Chem. 72 (2000) 1253-1262
Decreased Viscosity
• As the temperature increases the viscosity of the eluent decreases thus lowering the system back pressure– Perform analysis at higher flow rates
without over-pressurizing the pump– Use smaller size packing materials in
columns increasing efficiency
Temperature and Elution Strength
• Increasing temperature 4 to 5 ºC is comparable to increasing the methanol or acetonitrile concentration by 1% in a reversed phase system
• Viscosity is reduced 1 to 2 % per ºC increase– Result – a significant reduction in back pressure
P = ηµL/dp2
T. Greibrokk, et al,, Chapter 8 in ACS Symp. Ser., 748 (ed. Parcher and Chester), 2000, page 121
Elution Strength Illustration: Separation of Steroids Using Water as the Mobile Phase
Flow Rate: 6.0 mL/minMobile Phase: WaterTemperature: 200°C
Flow Rate: 3.0 mL/minMobile Phase: 25:75 acetonitrile:waterTemperature: 50°C
Elution Order:UracilAndrostadienedioneAndrostenedioneEpitestosterone
Column: ZirChrom PBD, 3 µm 100 X 4.6 mmDetection: UV 254 nm
2.0Minutes
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.80
100
200
300
400
500
600
700
mV
Prototype C18 Silica Column Separation of Steroids
Seconds0 10 20 30 40 50 60 70 80 90 100 110 120
AU
0.00
0.05
0.10
0.15
0.20
0.25
0.30
Elution Order:UracilAndrostadienedioneAndrostenedioneEpitestosterone
Flow Rate: 4.0 mL/minMobile Phase: 5:95 acetonitrile:waterTemperature: 200°C
Column: 3 µm, 100A,50 X 2.1 mmDetection: UV 254 nm
Efficiency with High Speed• Temperature effect on plate height
12
14
16
18
20
22
24
26
0 0.5 1 1.5 2 2.5Linear Velocity (cm/s)
Hei
ght
(mic
ron
s)
25 ºC80 ºC120 ºC150 ºC
Historical Obstacles in the Pathway of Extended Range HPLC
•Most commonly listed reasons why temperature has not been utilized as an optimization tool in reversed phase
– Poor temperature stability of silica based columns – Lack of adequate column heating system– Mobile phase composition is easily adjusted and
provides the flexibility to handle a wide range of samples
Mobile Phase Pre-heating
Separation of Barbiturates
Minutes0 2 4 6 8 10 12 14
mV
10
20
30
40
Barbital
Butabarbital
SecobarbitalCarbromal
Preheater Off
Preheater On
Stable Column Phases
– Silica (Selerity Blaze C8)
– Zirconia* (ZirChrom PBD, DiamondBond, CARB)
– Carbon (Thermo Hypercarb)
– Polymeric (Jordi, Hamilton with SS fittings)
* Not recommended for temperature programmed conditions because of severe column bleed
Blank Runs with Thermal GradientsThermo Hypersil-Keystone Hypercarb 40 to 200°C at 15°/min hold 5 minSelerity Blaze C8 40 to 100°C at 15°/min hold 5 minHamilton PRP-1 40 to 150°C at 15°/min hold 5 minZirchrom DiamondBond 40 to 200°C at 15°/min hold 5 minZirchrom CARB 40 to 200°C at 15°/min hold 5 minZirchrom PBD 40 to 150°C at 15°/min hold 5 min
Minutes0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0
0
200
400
600
800
1000
1200
1400
50:50 Acetonitrile:Water at 254 nm
Abs
orba
nce
Polaratherm ™ DesignTemperatures from sub-zero to 200 °CForced air circulationIsothermal and thermal gradient operationIntegrated solvent preheatingEffluent temperature controlFlammable vapor sensorCompatible with any HPLC system
Diesel Range Aromatics -Columns
• Partisil Amino-Cyano (PAC) column– 5µm, 250 x 4.6 mm
• All stainless steel hardware• Hexane mobile phase
Retention Data for PAC Column Isothermal Conditions
Analyte0°C 30°C 60°C 100°C
toluene 4.56 4.23 3.89 3.54tetralin 4.59 4.29 4.00 3.69thiophene 5.02 4.46 4.08 3.69naphthalene 6.81 5.69 4.99 4.32acenaphthene 7.49 5.89 4.92 4.47benzothiophene 7.81 5.82 5.22 4.49dibenzothiophene 11.25 8.38 6.73 5.47anthracene 12.14 9.04 7.30 5.86
Retention Time
3
4
5
6
7
8
9
10
11
12
13
0 2 4 6 8 10
Compound #
Ret
entio
n (M
inut
es)
0°
30°
60°
100°
Retention Data for PAC Column Isothermal Conditions
β-Carotene and Isomers at 24 ºC
ACN/THF 90:10Isocratic1 mL/minColumn: Genesis C18
β-Carotene and Isomers at 10 ºC
β-Carotene and Isomers at 0 ºC
Separation of Carotenoid Mixture at 15°C
Column: Jones Chromatography Genesis C18, 150 x 4.6 mmMobile Phase:1.25% THF in acetonitrileFlow Rate: 1.0 mL/minDetection: UV 450 nmTemperature: 15°C, isothermal
70 minute run time0
minutes
AU
30 40 50 60 7010 20
0
2
4
6
8
10
12
Lycopene
β-carotene
Zeaxanthin
Lutein
Separation of Carotenoid Mixture at 25°C
Column: Jones Chromatography Genesis C18, 150 x 4.6 mmMobile Phase:1.25% THF in acetonitrileFlow Rate: 1.0 mL/minDetection: UV 450 nmTemperature: 25°C, isothermal
0minutes
15 20 25 30 455 10
AU
0
2
4
6
8
10
12
35 40
18
16
14
Lycopene
β-carotene
Zeaxanthin
Lutein
Separation of Carotenoid Mixture at 35°C
Column: Jones Chromatography Genesis C18, 150 x 4.6 mmMobile Phase: 1.25% THF in acetonitrileFlow Rate: 1.0 mL/minDetection: UV 450 nmTemperature: 35°C, isothermal
AU
0
5
10
15
20
25
0 5 10 15 20 25 30
Lycopene
β-carotene
Zeaxanthin
Lutein
minutes
0
2
4
6
8
10
12
0 5 10 15 20 25 30
mAU
minutes
Lycopene
β-carotene
Zeaxanthin
Lutein
Separation of Carotenoid Mixture Using a Temperature Program
Column: Jones Chromatography Genesis C18, 150 x 4.6 mm
Mobile Phase:1.25% THF in acetonitrile
Flow Rate: 1.0 mL/min
Detection: UV 450 nm
Temperature Program: hold at 15°C for five minutes, ramp to 35°C over two minutes, hold 15 minutes.
Separation of Analgesics on a Selerity BlazeTM C8 Using a Thermal Gradient
Column: Selerity Blaze C8, 3 µm 100 x 4.6 mmMobile Phase: 40:60 acetonitrile:water with 0.1%TFAFlow Rate: 1.5 mL/minDetection: UV 220 nmTemperature Program: hold at 50°C for one minute, ramp to 100°C at 30°C/min, hold six min.
Elution Order:AcetaminophenCaffeineSalicylamideAspirinSalicylic acidIbuprofenNaproxen
Minutes0 1 2 3 4 5 6 7 8 9
mV
0
100
200
300
400
500
Analgesics Using a Hypercarb Column and a Thermal Gradient
Column: Thermo Electron Hypercarb, 7 µm, 100 x 4.6 mmMobile Phase: 35:65 acetonitrile:water with 0.1% TFAFlow Rate: 4.0 mL/minDetection: UV 220 nmTemperature Program: thermal gradient from 125° to 200°C at 30°/min, hold five min.
Elution Order:CaffeineAspirinSalicylic AcidIbuprofenPhenacetinAcetaminophenNaproxen
Minutes0 1 2 3 4 5 6 7
mV
0
25
50
75
100
125
150
175
Conclusions
• Temperature is powerful but under-used tool for selectivity tuning.
• Increasing the temperature reduces the amount of organic modifier needed and generally speeds elution.
• Temperature adjustment can be a fast way to optimize a separation.
• Shorter analysis times with better efficiency usually result from higher temperatures.
The authors thank Thermo Electron and Hamilton Company for providing columns for this work.Appreciation is also given to Craft Technologies for the carotenoid data.
Acknowledgements
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