Ultra-Fast High Temperature

Liquid Chromatography

EAS 2008

C.V. McNeff1*, B. Yan1, D. R. Stoll2, R.A. Henry3, Dan Nowlan1

1 ZirChrom Separations, Inc., 617 Pierce Street, Anoka, MN 553032 Department of Chemistry, University of Minnesota, 207 Pleasant St. Minneapolis,

MN 55455.3 Independent Consultant, 983 Greenbriar Drive, State College, PA 16801

2008 ZirChrom Separations, Inc.

Outline

• Advantages of High Temperature HPLCTheoretical Effects of High Temperature HPLCPractical Analytical Advantages of Using High Temperature HPLC

• Using Temperature to Control SelectivityImportance of Selectivity in HPLC Optimization

• Using Temperature in 2D SeparationsBasic Design Elements of 2D SeparationsExample of 2D-UFHTLC Capability

2

Theoretical Advantages to High Temperature LC

3/13/2max

3/2

)'1(TP

LkNt η

∆+∝

Practical Limit Temperature Dependence

Guiochon, Georges, Anal. Chem., 52, 2002-2008 (1980).

van Deemter Plot h AB

C DD

k dm

d p= + + + +

νν ν ν2 3

2

38

/

Three ways that temperature increases efficiency and speed•Increased temperature increases diffusivity, thus decreasing the reduced velocity•Increased temperature accelerates sorption kinetics•Increased temperature decreases mobile phase viscosity

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

3

Theoretical 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

F. D. Antia and Cs. Horvath, J. Chromatogr., 435, 1-15 (1988). 4

T ( oC)

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

η (c

P)

0.0

0.2

0.4

0.6

0.8

1.0

ACN

Water50 % ACNMeOH

Estimated Effect of Temperatureon Viscosity*

η∝Nt

*H. Chen and Cs. Horvath, "Rapid Separation of Proteins by RP-HPLC at Elevated Temperatures," Anal. Methods Instrum., 1, 213-222 (1993). 5

Effect of Temperature on Theoretical Analysis Time at Constant Pressure

and Plate Count*

50 150100 200

0.2

0.4

0.6

0.8

1.0

0.2

0.4

0.6

0.8

1.0

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

Temperature (oC)

t Ana

lysi

s,T /

t Ana

lysi

s, 25

oC 20-fold improvement!

6

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=0.973SD=0.09

R2=0.385SD=0.42

80oC vs. 30oC

R2=0.995SD=0.03

Stationary phase type has a very large effect on selectivity.

Stationary Phase Type

7

Fast Separations Non-Steroidal Anti-Inflammatories

1

Column Temperature = 150oC

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 DiamondBond®-C18; Mobile phase, 25/75 ACN/40mM phosphoric acid, pH 2.3; Flow rate, 5.5 mL/min.; Temperature, 150 oC; Injection volume, 1µL; Detection at 254nm; Solute concentration, 0.15 mg/mL.; Solutes, 1= Acetaminophen, 2=Ketoprofen, 3=Naproxen, 4=Ibuprofen, 5=Oxaprofen.

8

Fast β-Blockers Separation

0.4LC Conditions: Column, 50 x 4.6 DiamondBond®-C18; Mobile phase, 45/55 ACN/20mM Ammonium Phosphate pH11.0; Flow rate, 3.0 mL/min; Temperature, 150 oC; Injection volume, 1.0 µL; Detection at 210 nm; Solutes, 1=Labetalol, 2=Metoprolol, 3=Alprenolol

0 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 minute!

Column Temperature = 150oC, pH = 11

9

Fast Steroid Separation at 125oC Using a ZirChrom®-CARB Column

min0 0.5 1 1.5 2 2.5 3

0

min0 0.5 1 1.5 2 2.5 3

0

min0 0.5 1 1.5 2 2.5 3

0

0 0.5 1 1.5 2 2.5 3

mAU

0

100

200

300

400

500

600

700

LC Conditions: Column, 100 mm x 3.0 mm i.d. ZirChrom®-CARB, gradient elution 2-90% B from 0.3-3.9 minutes, A = 40/60 ACN/25 mM ammonium fluoride, pH 5.6, B = 40/60 ACN/THF, Flow rate: 2.5mL/min., Temperature, 125 °C (using Metalox 200C), Injection volume, 2 ml, UV Detection at 215 nm.

1

2

34

56

7 8 9

H3C O

HO

SO

OO

H3C OH

OSO OO

H3C O

OSO OO

H3C O

OSO OO

H3C O

OSO OO

H3C OH

OSO OO

H3C

O

OHCH3

H3C

H3C OH

HO

H3C O

HO

1, Estradiol sulfate 2, Dihydroequilin sulfate 3, Equlilin sulfate

4, Estrone sulfate 5, Equilenin sulfate 6, Dihydroequilenin sulfate

7, Methyltestosterone 8, Equilin 9, Estrone

10

Two Minute “Green” Separation of Chlorophenols at 200 oC

0 0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.25

mAU

min0 0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.25

mAU

50

40

30

20

10

0

Chromatographic conditions: Column, ZirChrom®-PBD, 150 x 4.6 mm i.d., Mobile Phase, 100% Water, Flow Rate, 3.0 mL/min., UV detection at 280 nm, Column Temperature, 200 °C using a Metalox® 200C column heater (ZirChrom Separations, Anoka, MN).

OH

OH

ClOH

ClOH

OHCl Cl

Cl

100% Water!

11

Thermally Tuned Tandem Columns (T3C)

DetectorColumn 1Temperature 1

Column 2Temperature 2

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

1,23 4

Column 1

1 23,4

Column 2

1 2 34

OptimizedT3C

A Mechanism to Continuously Adjust the Stationary Phase

InjectorPump

12

0 5 10 15 20 25 30

0

10

20

0

10

20

30

0

5

10

15

20

25

2

43

5

76

8 9 10

12

3

5

4 67

8 9 10

2+7

35

4

6

8

19

10

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

C18-SiO230 oC

T3C

Time (min)

Abs

orba

nce

(mA

U) C-ZrO2

60 oC

1

T3C can improve separation without increasing analysis time.

Separation of Ten Triazine Herbicides by T3C

13

Schematic of a Complete LC ×UFHTLC System

10-port, 2-position

valve

Binary Pump #1

Binary Pump #2

Heating Jacket1st Dimension

Column

UV Detector

Eluent Preheater

Eluent Preheater

Eluent Preheater

Heating Jacket2nd Dimension

Column

Counter-Current Heat Exchanger

S a m ple Lo o p # 1

S a m ple Lo o p # 2

T1

T7

T6 T5

T4

T3

T2Auto-

Injector

Temperature Controlled

Standard HP1090 Pump and Autosampler

2.1 mm column

High flow pump 14

2DLC Separation Of A 26 Component Indolic Metabolite Mixture

1st Dimension Conditions: Column, 50 mm x 2.1 mm i.d. Discovery® HS-F5; Mobile phase, A: 20 mM Na3PO4 20 mM NaClO4pH=5.7 B: ACN; Gradient: 95/5 A/B to 60/40 20 min, hold 2 min, 60/40 to 30/70 1 min, 30/70 to 95/5 0.01 min, hold 7 min. Flow rate, 0.10 mL/min.; Injection volume, 10 µL; Temperature, 40 oC; UV 220 nm

2nd Dimension Conditions: Column, 50 mm x 2.1 mm i.d. ZirChrom®-CARB; Mobile phase, A: 20 mM HClO4 B: ACN, Gradient: 0-70% B in 17.4 s ; Flow rate, 3.0 mL/min.; Injection volume, 13.4 µL; Temperature, 110 oC; UV 220 nm

Stoll, D. R.; Cohen, J. D.; Carr, P. W. Journal of Chromatography A, 2006, 1122, 123-137.15

Conclusions

(1) Zirconia Based Stationary Phases are ultra-durable and efficient , stable at the extremes of pH and at column temperatures as high as 200oC.

(2) High Temperature Liquid Chromatography (HTLC) is a powerful technique that can be used as a routine analytical tool in the development of separation methods.

(3) HTLC is a unique tool in altering chromatographic selectivity (T3C method), increasing analysis speed.

(4) HTLC capability will become an important part of HPLC system design in order to fully utilize the benefits of columns prepared withultra-small particles and utrafast analyses.

(5) Fast 2D-LC separations have been successfully achieved allowing for the rapid analysis of complex mixtures.

16