Applications of Ultra-Stable Phases for HPLC: High Temperature Ultra-Fast Liquid Chromatography and

Thermally Tuned Tandem Column (T3C) Liquid Chromatography

Yun Mao, Merck & Co.Jon Thompson, Peter W. Carr, University of Minnesota

Clayton V. McNeff, ZirChrom Separations, Inc.Richard Henry, Angelos Kyrlidis, Greg Gaudet, Cabot Corporation

HPLC Columns

TM

Outline

Development of Ultra-Stable Stationary Phases– Theoretical and Practical Benefits of High Temperature

HPLC – Stability of Zirconia-based HPLC Columns (Z-phases)– Examples of Ultra-Fast High Temperature Separations

Using Temperature to Control Selectivity– Importance of Selectivity in HPLC Optimization– Selectivity of Zirconia-based HPLC Columns (Z-phases)– Thermally Tuned Tandem Columns (T3C) Concept– Examples of T3C Applications

Conclusions

Retention and Selectivity Variables

HPLC Column (Stationary Phase)

Solvent Composition (Both Type and %)

Temperature (ambient-200 oC)

Additive (Both Type and Concentration)

pH

• Once an HPLC column is selected, there are four main variables to adjust.

• Temperature is the least often used, but may be the most convenient to try.

•Temperature affects both retention and selectivity.

Effect of Temperature on Retention Time*

50 150100 200

0.2

0.4

0.6

0.8

1.0

0.2

0.4

0.6

0.8

1.0

Temperature (oC)

t Ana

lysi

s,T /

t Ana

lysi

s, 25

oC 20-fold improvement!

*R. D. Antia and Cs. Horvath, J. Chromatogr., 435, 1-15 (1988).

Keeping Pressure and Plate Count Constant

Effect of Temperature on Column Efficiency

25 oC

75 oC

125 oC

175 oC

udp/Dm,25

0 10 20 30 40 50 60 70 80 90 100

H/d

p

0

2

4

6

8

10

25 oC

75 oC

125 oC

175 oC

Ways that temperature increases efficiency and speed:– Increased temperature

increases diffusivity, thus decreasing the reduced velocity

– Increased temperature accelerates sorption kinetics

– The result is speed without loss of resolution and sensitivity, even at high flow rates

van Deemter Plot h AB

C DDk d

m

d p= + + + +

νν ν ν2 3

2

38

/

R. D. Antia and Cs. Horvath, J. Chromatogr., 435, 1-15 (1988).

Estimated Effect of Temperature on Viscosity*

T ( oC)

0 20 40 60 80 100 120 140 160 180 200 220

η (cP)

0.0

0.2

0.4

0.6

0.8

1.0

ACN

Water50 % ACNMeOH

η∝Nt

* H. Chen and Cs. Horvath, "Rapid Separation of Proteins by RP-HPLC at Elevated Temperatures," Anal. Methods Instrum., 1, 213-222 (1993).

Ways that increased temperature increases efficiency and speed:1. Increased temperature

increases diffusivity, thus decreasing the reduced velocity

2. Increased temperature accelerates sorption kinetics

3. Increased temperature reduces k values

4. Increased temperature decreases mobile phase viscosity which enables higher flow rates

Why Stable Phases?

Advantages of ExtraordinaryChemical Stability

pH < 1

pH Stability

pH > 13

Clean withconc. Acids

Suppress Ionsfor Acids

Suppress Ionsfor Amines

SanitationDepyrogenation

LowerPressure Drop

Thermal Stability

Less OrganicSolvent

ThermallyOptimize

Selectivity

Less Wearand Tear

Higher FlowFast Analysis

More RobustAnalysis

Easier MethodDevelopment

Stable stationary phases have advantages in terms of analysis time, selectivity and column lifetime. If LC/MS is employed, stable phases exhibit low bleed background.

ZirChrom® Particle Properties

Characteristic PropertySurface Area (m2/g) 22Pore Volume (cc/g) 0.13Pore Diameter (Å) 250-300Porosity 0.45Density (g/cc) 5.8 (2.5x silica)Particle Diameters (µ) 3.0, 5.0, 10.0ZirChrom®-Carb and

DiamondBond particles are prepared by coating base particles with a thin layer of carbon using a chemical vapor deposition process

NH2 Y + 2 HA + NaNO2 = AN≡N Y + 2 H2O + NaA

Diazonium SaltCarbon Clad Zirconia Modified Carbon Clad Zirconia

+ N+

N Y

Bonding Reaction on Carbon Clad Zirconia

- C18

ZirChrom®-PBD and -PS particles are prepared by coating with a layer of highly crosslinked polymer

DiamondBond-C18 Stabilityk'

0

1

2

3

4

5

6

7

8

0 250 500 750 1000 1250 2000

Column Volumes

k' Butylbenzene (0.5M Nitric Acid)

k' Butylbenzene (1.0M NaOH)

k' Butylbenzene (200 oC)

LC Conditions: Base Stability—DiamondBondTM Phase A, 30 x 4.6 mm id; Mobile phase, 50/50 ACN/Water; Flow rate, 1.0 ml/min.; Temperature, 30 oC; Injection volume, 5ul; Detection at 254nm. Acid Stability—DiamondBondTM Phase A, 50 x 4.6 mm id; Mobile phase, 50/50 ACN/Water; Flow rate, 1.0 ml/min.; Temperature, 30 oC; Injection volume, 5ul; Detection at 254nm. Temperature Stability-- DiamondBondTM Phase B, 50 x 4.6 mm id; Mobile phase, 50/50 ACN/Water; Flow rate, 1.0 ml/min.; Temperature, 30 oC; Injection volume, 5ul; Detection at 254nm.

Stable phases allow retention and selectivity variables to be fully explored and exploited.

Increasing Separation Speed

Temperature

Ret

entio

n Ti

me

Cold HotColumn Length

Ret

entio

n Ti

me

Long Short

T increases 50 oC k’ decreases 3-fold

Increasing only flow rate to reduce retention time is the least desirable option because pressure increases and performance drops. Increasing temperature and decreasing column length are better ways to decrease retention time and speed up analysis; however, decreasing length reduces efficiency and resolution, while increasing temperature doesn’t.

Column Temperature vs. Column Length vs. Flow Rate

Antihistamines- Fast Isocratic

min0 2 4 6 8 10 12 14

mAU

0

10

20

30

40

50

60

VWD1 A, Wavelength=254 nm (P102501\TEST0146.D)

1

2

3

4

5min2.4 2.6 2.8 3 3.2

2

3

Temperature: 21 oCFlow rate: 1.35 ml/min.Pressure drop: 195 barResolution (2,3): 2.1Analysis time: 12.5 minutes

min0 2 4 6 8 10 12 14

mAU

0

20

40

60

80

VWD1 A, Wavelength=254 nm (P102501\TEST0151.D)

Temperature: 80 oCFlow rate: 3.00 ml/min.Pressure drop: 195 barResolution (2,3): 2.1Analysis time: 2.5 minutes

3

24

5

1

min0. 9 0. 95 1 1 . 05 1 . 1 1 . 1 5 1 . 2 1 . 25

3

2

LC Conditions: (A) Mobile Phase, 29/71 ACN/50mM Tetramethylammonium hydroxide, pH 12.2; Flow Rate, 1.35 mL/min.; Injectionl, 0.5 ul; 254 nm detection; Column Temperature, 21°C; Pressure drop = 195 bar; Solutes: 1=Doxylamine, 2=Methapyrilene, 3=Chlorpheniramine, 4=Triprolidine, 5=Meclizine4=Triprolidine, 5=Meclizine, 100 x 4.6 ZirChrom-PBD (B) same as A, except Mobile Phase, 26.5/73.5 ACN/50mM Tetramethylammonium hydroxide, pH 12.2; Flow Rate, 3.00 mL/min.; Column Temperature, 80°C; Pressure drop = 195 bar.

80 oC

21 oC

Beta-Blockers- Fast Isocratic

0.40 min0.6

0

200

400

600

800

1000

1200

mAU

CHCH2NHCH

H2NOC

HO

OHCH3

CH2CH2

OCH2CHCH2NHCH(CH3)2CH3OCH2CH2

OH

Labetalol

Metoprolol

AlprenololOCH2CHCH2NHCH(CH3)2

CH2CH CH2

OH

0

200

400

600

800

1000

1200

mAU

CHCH2NHCH

H2NOC

HO

OHCH3

CH2CH2

CHCH2NHCH

H2NOC

HO

OHCH3

CH2CH2

OCH2CHCH2NHCH(CH3)2CH3OCH2CH2

OH

OCH2CHCH2NHCH(CH3)2CH3OCH2CH2

OH

Labetalol

Metoprolol

AlprenololOCH2CHCH2NHCH(CH3)2

CH2CH CH2

OH

OCH2CHCH2NHCH(CH3)2

CH2CH CH2

OH

Separation in 0.4 minutes!

Column Temperature = 150 oC, pH = 11

LC Conditions: Column, 50 x 4.6 Diamondbond-C18, OD0121601A; Mobile phase, 45/55 ACN/20mM Ammonium Phosphate pH11.0; Flow rate, 3.0 ml/min; Temperature, 150 oC; Injection volume, 1.0 ul; Detection at 210 nm;

Nitrosamines- Fast Gradient

min0 0.5 1 1.5 2 2.5 3

mAU

0

50

100

150

200

250

Column: -C18, 100 × 4.6mm Mobile Phase: 2.5-90%B from 1-3 minutesA: 10mM Ammonium hydroxide, pH 9.5B: 100% Acetonitrile

Flow rate: 4.0 mL/min.Temperature: 75 ºCInjection volume: 1.0 µLDetection: 230 nmBack Pressure: 200 bar

pH 9.5, 75 ºC

Non-Steroidal Anti-inflammatories

min0 1 2 3 4 5 6 7 8

Norm.

0

250

500

750

1000

1250

1500

1750

2000 Column Temperature = 80 oC12

3

4

56

LC Conditions: Column, 50 x 4.6 DiamondBondTM-C18; Mobile phase, 50-80 A over 10 minutes, A=ACN, B=40mM ammonium phosphate, 5mM ammonium fluoride, pH 2.0; Flow rate, 1.0 ml/min.; Temperature, 80 oC; Injection volume, 5ul; Detection at 254nm; Solute concentration, 0.15 mg/ml.; Solutes, 1=Acetaminophen, 2=Ketoprofen, 3=Ibuprofen, 4=Naproxen, 5=Oxaprofen, 6=Indemethacin.

Non-Steroidal Anti-inflammatories (FAST)

1

Column Temperature = 150 oC

min0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8

Norm.

0

50

100

150

200

250

300

350

400

2

3 45

Separation in 1 minute!

LC Conditions: Column, 50 x 4.6 DiamondBondTM-C18; Mobile phase, 25/75 ACN/40mM phosphoric acid, pH 2.3; Flow rate, 5.5 ml/min.; Temperature, 150 oC; Injection volume, 1ul; Detection at 254nm; Solute concentration, 0.15 mg/ml.; Solutes, 1= Acetaminophen, 2=Ketoprofen, 3=Naproxen, 4=Ibuprofen, 5=Oxaprofen.

PTH-Amino Acids

LC Conditions: Column, 50 x 4.6 DiamondBondTM-C18; Mobile phase, 20/80 ACN/0.1% TFA, pH 2.1; Flow rate, 1.0 ml/min.; Temperature, 80 oC; Injection volume, 1ul; Detection at 254nm; Solute concentration, 0.15 mg/ml.; Solutes, 1=PTH-Arginine, 2=PTH-Serine, 3=PTH-Glycine, 4=PTH-Alanine, 5=PTH-Isoaminobutyric acid, 6=PTH-Aminobutyric acid, 7=PTH-Valine, 8=PTH-Norvaline.

Column Temperature = 80 oC

min0 1 2 3 4 5 6 7 8 9

mAU

0

20

40

60

80

100

1

2

3

4

5

6

7 8

The Impact of Selectivity on Resolution

N

Efficiency SelectivityRetention

R=k’

k’+1α-1α4

α =kj’ki’

Selectivity (α) has the greatest impact on improving resolution.

1.00 1.05 1.10 1.15 1.20 1.250.0

0.5

1.0

1.5

2.0

2.5

3.0

0 5000 10000 15000 20000 25000

0 5 10 15 20 25

N

k’

α

αNk’

Res

olut

ion

(R)

Comparing and Adjusting Selectivity in HPLC

Mobile Phase Composition (B%)**

Mobile Phase Type (ACN, MeOH, THF) Stationary Phase Type (C18-SiO2, C-ZrO2, PBD-ZrO2)Temperature

log k' (condition 1)0 1 2 3

log k’ (condition 2)

0

1

2

3

log k' (condition 1)0 1 2 3

0

1

2

3

α1=1.03

α2=1.03

α1=1.00

α2=2.00

κ−κ plots1

r2 = 1.00, s.d. = 0.00 r2 = 0.66, s.d. = 0.70

log k’ (condition 2)

Poor correlations in the κ−κ plot indicate changesin selectivity.

** Only works in mixed-mode

1. Horvath, Snyder and Carr

22 Non-Electrolyte Solutes

NonpolarBenzene Toluene Ethylbenzene p-xylene Propylbenzene Butylbenzene

PolarBr Cl

Cl

O O

O

CN NO2 NO2Cl

NO2

OBromobenzene p-Dichlorobenzene Anisole Methylbenzoate Napthalene Acetophenone

Benzonitrile Nitrobenzene p-Nitrotoluene p-Nitrobenzyl Chloride

O

Benzophenone

HB DonorOH OH NH H

OOH OH

Cl

Benzylalcohol 3-Phenyl Propanol N-Benzyl Formamide Phenol p-Chlorophenol

The selectivity of the stationary phase becomes paramount for non-ionic solutes. Temperature and mobile phase variables become less important.

Selectivity Comparison

-1.5

-1

-0.5

0

0.5

1

1.5

2

Diamondbond-C18

ODS-1

Unmodified Carbon coated zirconia

Mobile phase, 40/60 Acetonitrile/Water; Flow rate, 1.0 ml/min.; Temperature, 30 oC; Detection at 254nm; 5ml Injection volume.

Comparison of Variables Affecting Selectivity

logk' (50/50 ACN/H2O)

0.00 1.00

0.00

1.00

2.00

logk' (THF/H 2O)0.00 1.00

0.00

1.00

2.00

logk' (PBD-ZrO2)-2.00 -1.00 0.00 1.00

0.00

1.00

logk' (C18, 30 oC)0 1 2 3

0

1

2

logk' (PBD-ZrO2)-1.00 0.00

0.00

1.00

C18-SiO2 vs. PBD-ZrO2

Carbon-ZrO2 vs. PBD-ZrO2

30% ACN vs. 50% ACNMeOH vs. THF

R2=0.896SD=0.17R2=0.989

SD=0.05

R2=0.98SD=0.09

R2=0.385SD=0.42

80oC vs. 30oC

R2=0.995SD=0.03

Stationary Phase Type

Stationary phase type has a large effect on selectivity.

Carbon Surface Has Unique Selectivity

Mobile phase: 35/65 A/B

A: ACNB: Water

Flow rate: 1.0 mL/min.Temperature: 30 oCInjection volume: 5 µLDetection: 254 nm

LC Conditions

Column: ZirChrom®-PBD, 100 x 4.6 mm

Mobile phase: 37.5/5/57.5 A/B/C

A: ACNB: THFC: Water

Flow rate: 1.0 mL/min.Temperature: 60 oCInjection volume: 5 µLDetection: 254 nm

LC Conditions

Column: DiamondBond-C18, 100 x 4.6 mm

Mobile phase: 32.5/67.5 A/B

A: ACNB: Water

Flow rate: 1.0 mL/min.Temperature: 60 oCInjection volume: 5 µLDetection: 254 nm

LC Conditions

Column: ZirChrom®-CARB, 100 x 4.6 mm

1,2

min0 1 2 3 4 5 6 7 8 9

mAU

0

20

40

60

80

min0 1 2 3 4 5 6 7 8 9

mAU

0

20

40

60

80

min0 1 2 3 4 5 6 7 8 9

mAU

0

10

20

30

40

min0 1 2 3 4 5 6 7 8 9

mAU

0

10

20

30

40

12

min0 1 2 3 4 5 6 7 8 9

mAU

0

20

40

60

80

min0 1 2 3 4 5 6 7 8 9

mAU

0

20

40

60

80

12

Analytes1 - Ethylbenzene

2 - p-XylenePBD or ODSα=1.03

DB-C18α=1.22

CARBα=1.58

Bonded Carbon

Carbon

Selectivity Difference vs ODS (Orthogonality)

For non-ionizable solutes:– ZirChrom-Carb and Diamondbond-C18 columns have

very different selectivity from traditional C18-silica columns, exceeding that of embedded polar phases.

– ZirChrom-PBD has selectivity similar to C18-silica.For ionizable solutes:– The selectivity difference is even greater on zirconia

phases due to mixed mode possibilities.

Column R2 SelectivityDifference*

ZirChrom-PBD 0.985 12DiamondBond-C18 0.889 33ZirChrom-Carb 0.549 67

*S=100(1-R2)0.5, as described by U. Neue at FACSS 2002

Zirconia Has Unique Surface Chemistry

OZr

OZr

OZr

OZr

OZr

OZr

OZr

OZr

O

OOHO-

H2O OH OHH2O H2OO-P OHOO-

Hydrophobic Coating (PBD, Carbon)A+

A+

A+A+

Lewis acid

Bronsted baseand acid

Zirconia by itself has very rich surface chemistryCoated zirconia phases (Carbon and PBD) have mixed surface propertiesThe retention of various basic and acidic analytes can be fine tuned by changing pH, buffer and salt concentration, in addition to mobile phase modifier concentration and type

Zirconia Features Tunable Surface Properties

The choice of buffer and pH on zirconia columns affects the surface charge and the elution properties of ionizable analytes. Mixed-mode (RP plus CEX) selectivity can be created.

- - - - - -0 0 - - - - - - - - - - - - - - -0 0 0 0 0 0 0 0 0 0 - - - - - - -

3 90 14

0 0 0 0 - - - - - - - - - - - - -0 0 0 0 0 0 0 0 0 0 - - - - - - -

3 90 14

+ + - -+ + + + 0 0 0 0 0 0 0 0 0 - - - -+ + + + + + + 0 0 0 - - - - - - -

3 90 14

Silica Surface Charge State

Net Zirconia Surface Charge State withstrong Lewis Base Buffers

Net Zirconia Surface Charge State withweak Lewis Base Buffers

pH

pH

pH

Antihistamine Drug Selectivity Comparison

log k'(PBD-ZrO2 at 40oC)0.0 0.5 1.0 1.5

0.5

1.0

1.5

17

2 6

3

log

k' (O

DS

at 4

0o C)

54

8

9A

bsor

banc

e (m

AU

)

0 5 1 0 1 5 2 0 2 5

0

5

1 0

1 5

2 0

2 50 5 1 0

0

5

1 0

1 5

2 0

2 5

3 0 C18-SiO230 oC1

45

6

9 8

7

2+3

ZirChrom-PBD30 oC

1+7

623 2 4 8

9

Abs

orba

nce

(mA

U)

R2=0.147Mobile Phase: 40/60 Acetonitrile/25 mM Phosphate, pH=7

The selectivity of zirconia based columns towards ionizablecompounds becomes very different from that of traditional silica columns. Buffer type and pH have an effect on mixed-mode retention (RP and CEX) by Z-phases.

S=92

Thermally Tuned Tandem Columns

What if you could continuously adjust the selectivity of your HPLC column?Think of temperature as a replacement for solvent strength in adjusting retention time.

3 4Column 1

1 23,4

Column 2

1 2 34

OptimizedT3C

Thermally Tuned Tandem Columns (T3C)

T3C is a method to continuously adjust HPLC selectivity using tandem, orthogonal columns and dual, independenttemperature control.

DetectorColumn 1Temperature 1

Column 2Temperature 2

e.g. C18-SiO2 e.g. C-ZrO2

1,2

InjectorPump

Requirements for T3C

Two columns with different (ideally orthogonal) selectivityAt least one thermally stable column (e.g. Zirconia-based)Thermally stable compounds Temperature control for at least one column (both preferred)Easy method development

Theory and practice of T3C (references 1-3)Guidelines for method development

Effect of Temperature on T3C Selectivity

DetectorColumn 1Temperature 1

Column 2Temperature 2

InjectorPump

Low TLow T

netα 2α

High T

High T Low T1αnetαLow T

'2

'1

'1

1 kkkf+

=

=netα 11αf 22αf+'2

'1

'2

2 kkkf+

=

Temperature continuously changes the T3C selectivity between α1 and α2.

Method Development for T3C

DetectorColumn 1T1

Column 2T2

InjectorPump

log k1’ =A1+B1/T

tRnet (T1,T2) = tR1(T1) + tR2(T2)

)'1( 1011 kttR += )'1( 2022 kttR +=

2 trial runs 2 trial runs

log k2’ =A2+B2/T

Guidelines for Optimizing T3C

Choose Two Stationary Phases

Choose Mobile Phase (1<k’<20)

Different Selectivity?

Two More Runs atHigher Temperatures

Calculate T3C Retentionsln k’=A+B/T trnet=tr1+tr2

Window DiagramOptimization

No Yes

Separation of Ten Triazine Herbicides by T3C

0 5 10 15 20 25 30

0

10

20

Time (min)

0

10

20

30

0

5

10

15

20

25

24

3

5

76

8 9 10

12

3

5

4 67

8 9 10

2+7

354

6

8

19

10

C18-SiO230 oC

T3C

Abs

orba

nce

(mA

U)

C-ZrO260 oC

1

30 oC 125 oCC18-SiO2 C-ZrO2

N

NN

N

NCH3S, CH3O, Cl =R

H R1

H

R2

Solutes:1. Simazine2. Cyanazine3. Simetryn4. Atrazine5. Prometon

6. Ametryn7. Propazine8. Terbutylazine9. Prometryn10. Terbutryn

Other conditions:30/70 ACN/water1ml/min; 254 nm detection

T3C can improve separation without increasing analysis time. Extra length is offset by shorter k values.

0.30 0.35 0.400.0

0.4

0.8

1.2

0.4 0.5 0.6

Min

imum

Rs

0.0

0.2

0.4

0.20 0.25 0.300.0

0.2

0.4

0.6

0.8

2 4 6 8 10

0

20

40

0 2 4 6

0

20

40

60

0 2 4 6 8 10 12 14 16

0

10

20

30

ACN R<1.0

MeOH R<0.3

THF R<0.5

40%ACN

50%MeOH

30% THF12

53

4

67+8

9+10

12 3+4

6+7

58 9 10

1+2

3

45

67

8 9 10

Time (min) Percentage of organic modifier

Comparison of T3C with Solvent Optimization

T3C is more powerful than mobile phase optimization on ODS

More T3C Separations

Time (min)0 1 2 3 4 5

0

20

40

0 2 4 6 8 10 12

0

20

40

60

800 2 4 6 8 10 12 14 16 18

0

5

10

15

20

13

5

46 7

8 9 10

2

15

46 7

8 910

2+3

1 34

5+7

6 8 9 10

2

Abs

orba

nce

(mA

U)

0 4 8 12 16

0

4

8

0 2 4 6 8

0

10

20

T3C 39oC+89oC

0 1 2 3 4 5 6 7 80

4

8

123 4

5 67 8

9

10 1112*

123 46

7

8

5+9

10 11+12

*

123

54

768 9

10+11

12*

C18-SiO2 30oC

C-ZrO2 90oC

Abs

orba

nce

(mA

U)

Urea and Carbamate Pesticides Barbiturates

C18-SiO2 30oC

C-ZrO2 30oC

T3C 80oC+40oC

Time (min)

Basic Drugs: C18-Silica and PBD-Zirconia

Temperature 1 Temperature 2

C18-SiO2 Carbon-ZrO2

Partition Mechanism Adsorption Mechanism

Phosphate BufferpH=7

Temperature 1 Temperature 2

C18-SiO2

Reversed Phase Mode+

Cation-Exchange Mode

Cation-Exchange Mode+

Reversed Phase Mode

PBD-ZrO2

Even more orthogonal for ionics

orthogonal for nonionics

Separation of Antihistamine Drugs by T3C

log k'(PBD-ZrO2 at 40oC)0.0 0.5 1.0 1.5

0.5

1.0

1.5

17

2 6

3

log

k' (O

DS

at 4

0o C)

54

8

9

0 5 1 0 1 5 2 0 2 5 3 0

0

5

1 0

1 5

T3C40 oC +35 oC

Abs

orba

nce

(mA

U)

Time (min)

0 5 1 0 1 5 2 0 2 5

0

5

1 0

1 5

2 0

2 50 5 1 0

0

5

1 0

1 5

2 0

2 5

3 0 C18-SiO230 oC1

45

6

9 8

7

2+3

PBD-ZrO230 oC

1+7

623 2 4 8

9

1 2

3 45

6 78

9

Abs

orba

nce

(mA

U)

R2=0.147

Temperature on PBD-ZrO2 column304050607080

Tem

pear

ure

on O

DS

colu

mn

20

25

30

35

40

45

50

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2

(a)

Resolution

Mobile Phase: 40/60 Acetonitrile/25 mM Phosphate, pH=7

S=92!

Advantages and Disadvantages of T3C

Advantages:– Provides much better separation– Can improve selectivity without big change in analysis

time– Relatively easy method development– Good selection of orthogonal stationary phases available

Disadvantages:– Stationary phases and sample must be thermally stable– Potential for higher pressure drop and longer run time – Need to carefully select stationary and mobile phase– Need well-designed column oven; two ovens or dual-zone

oven provide more selectivity

Conclusions

Zirconia-based phases are ultra-stable to extreme conditions in both temperature and pHThe durability of Zirconia-based phases allows for ultra-fast separations at elevated temperaturesZirChrom® zirconia-based phases offer selectivity that can be very different from traditional silica-based stationary phasesThe combination of unique (orthogonal) selectivity and exceptional thermal stability allows the development of novel separations using the Thermally Tuned Tandem Column (T3C) approachFor a given analysis, T3C requires two stationary phases with orthogonal selectivity and different critical pairs.

References

1. Jun Mao and Peter W. Carr, Anal Chem. 2000, 72, 110-118.2. Jun Mao and Peter W. Carr, Anal. Chem. 2001, 73, 4478-

4485.3. Jonathan D. Thompson and Peter W. Carr, Anal. Chem.

2002, 74, 1017-1023.

Acknowledgements

the technical support group at ZirchromSeparations, Inc.the research group at Cabot Corporationthe research group under Peter Carr at University of Minnesota

Richard A. Henry

rhenry@psualum.com

The author wishes to thank the following: