by

Jacek Nawrocki, Jon Thompson, Yun Mao,

Bingwen Yan, Dwight R. Stoll and Peter W. Carr

Analytical Potential of Stable Phases for Reversed-Phase Liquid

Chromatography

An Overview of the the Development of Stationary Phases for Reversed-Phase

Liquid Chromatography

1. A. Wehrli, J.C. Hildenbrand, H.P. Keller, R. Stampfli, R.W. Frei,"Influence of organic bases on the stability and separation properties of reversed-phase chemically bonded silica gels", J. Chromatogr. 149 (1978), 199-210.

2 . J.J. Kirkland, J.L. Glajch, R.D. Farlee, "Synthesis andcharacterization of highly stable bonded phases for high-performance liquid chromatography column packings", Anal. Chem. 61 (1989), 2-11.

Key Papers in History of Stable Reversed-Phases:

OutlinePart I. Overview of analytical potential of high phase

stability. • Chemical stability. • Thermal stability.

Part II. Using stability to achieve selectivity.• Thermally tuned tandem columns in HPLC.

Part III. Using stability to speed up HPLC.• High temperature ultrafast liquid chromatography.• High temperature fast two dimensional liquid

chromatography.

Advantages of Highly StableStationary Phases

Allows Cleaningwith Conc. Acid

Ion Suppressionof COOH

pH < 1

SanitizationDepyrogenation

Ion Suppressionof NH2

pH >13

pHStability

Less Wareand Tare

Faster analyses

HigherFlowRate

LowerPressureDrop

LessOrganicSolvent

MoreRobustAnalysis

EasierMethod

Development

ThermallyOptimizedSelectivity

ThermalStability

High ChemicalStability

Temperature The Third Dimension

in HPLC

Temperature

MobilePhase

StationaryPhase

Role of Temperature in LC

“High-Performance Liquid Chromatography at Elevated Temperatures: Examination of Condition for the Rapid Separation of Large Molecules”, R. D. Antia and Cs. Horvath,J. Chromatogr., 435, 1-15 (1988).

“Temperature as a Variable in Reversed –Phase High-Performance Liquid Chromatographic Separations of Peptide and Protein Samples”, W. S. Hancock, R. C. Chloupek, J. J. Kirkland and L. R. Snyder, J. Chromatogr. A, 686, 31-43 (1994)

“Superheated Water: A New Look at a Chromatographic Eluent for Reversed-Phase Liquid Chromatography”,R. M. Smith and R. J. Burgess, LC-GC, 17, 938-945 (1999)

Part II.

The Thermally Tuned Tandem Column (T3C) Concept

Importance of Selectivity in HPLC Optimization Thermally Tuned Tandem Column (T3C) Concept

An Example – Ten Triazine HerbicidesApplications

Conclusions

TheoryOptimization

Urea and Carbamate PesticidesBarbituratesAntihistamine Drugs

T3C WorksIt Can Save Time or Do Difficult SeparationsOnly Four or Five Initial Runs Are Needed

Outline

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

The Ultimate Goal of Separation: Resolution (R)

N

Efficiency Selectivity Retention

R= k’k’+1

α-1α4

N

k’

α

Selectivity (α) has the greatest impact on improving resolution

αNk’

Res

olut

ion

(R)

Comparison of Variables Affecting Selectivity

logk' (50/50 ACN/H2O)

0.00 1.00

0.00

1.00

2.00

30% ACN vs. 50% ACNMeOH vs. THF

2O)

0.00

1.00

2.00

logk' (THF/H0.00 1.00

R2=0.896SD=0.17R2=0.989

SD=0.05 Carbon-ZrO2 vs. PBD-ZrO2

logk' (PBD-ZrO2)-1.00 0.00

0.00

1.00

R2=0.385SD=0.42

80oC vs. 30oC

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

0

1

2 R2=0.995SD=0.03

C18-SiO2 vs. PBD-ZrO2

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

0.00

1.00

R2=0.973SD=0.09

Stationary phase type can have a very large effect on selectivity.

DetectorColumn 1Temperature 1

Column 2Temperature 2

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

1,2 34Column 1

1 2 3,4Column 2

1 234OptimizedT3C

A Mechanism to Continuously Adjust the Stationary Phase

Requirements for T3C:

Two columns with different(ideally orthogonal) selectivity

One very thermally stable columnMethod development must be easy

The Concept: Thermally Tuned Tandem Columns (T3C)

InjectorPump

0 5 10 15 20 25 30

0

10

20

0

10

20

30

0

5

10

15

20

25

Separation of Ten Triazine Herbicides by T3C

2

43

5

76

8 9 10

12

3

5

4 67

8 9 10

2+7

35

4

6

8

19

10

30oC 125oCC18-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-SiO230oC

T3C

Time (min)

Abs

orba

nce

(mA

U) C-ZrO260oC

1

T3C can improve separation without increasing analysis time

Guidelines for Optimizing T3C

Choose Two Stationary Phases

Choose Mobile Phase (1<k’<20)

Different Selectivity?

Two More Runs atHigh Temperatures

Calculating T3C Retentionsln k’=A+B/T

trnet=tr1+tr2

Window DiagramOptimization

No Yes

0.8 1.2 1.6

0.8

1.2

1.6

1

2 4

3

5

6

7

8

9

10

Log

k’ (C

-ZrO

2, 60

o C)

log k’ (ODS, 30oC)

R2=0.107, sd=0.342

0 1 2 3 4 5 6 7 8 9 1 0

0

1 0

2 0

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

30

40

50

60

70

80

6080

100120

140

Min

imum

Res

olut

ion

Tempe

rature

on O

DS Colu

mn

Temperature on C-Zr Column

1 23

5

4

67

8 9 10

T3C, 3ml/min

Time (min)

Steps in T3C Optimization of Triazine Herbicides

TODS=30oCTC-ZrO2=125oCRs=3.30

R=2.02

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

0 1 2 3 4 5 6 7 8

0

4

8

123 4

5 67 8

9

10

T3C 39oC+89oC

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)

Time (min)

Applications of T3C Method

Urea and Carbamate Pesticides BarbituratesC18-SiO2 30oC

C-ZrO2 30oC

T3C 80oC+40oC

Time (min)

0 5 10 15 20 25

0

5

10

15

20

250 5 10

0

5

10

15

20

25

30 C18-SiO240oC1

45

6

9 8

7

2+3

Separation of Anti-Histamines by T3C

PBD-ZrO230oC

1+7

623 2 4 8

9

0 5 10 15 20 25 30

0

5

10

15

T3C35oC +40oC

1 2

3 45

6 7

8

9

Abs

orba

nce

(mA

U)

T3C offers unique selectivity for the separation of complex mixtures.

T3C requires that on the two phases the critical pairs must be different.

Optimization needs only 4 or 5 trial runs.

In many cases, T3C:is superior to mobile phase optimization.provides better resolution than a single phase.improves analysis speed.

Conclusions

Carbon Phase+

Aliphatic Phase

Reversed Phase+

PBD-ZrO2 PhasePhosphate Buffer

Neutral Compounds

Basic Compounds

Part III. High Temperature Ultra-Fast Liquid Chromatography

Why Fast HPLC?• Monitor reaction rates with half-lives on order of

minutes not hours.• Monitor prep scale chromatography.• Increase sample through-put thus lower cost.• Increase screening rate in combinatorial chemistry

(speed up LC side of LC-MS).• Make 2D-HPLC practical and thus greatly

enhance peak capacity of HPLC.

HPLC is Slow Compared to Other Methods*

CE**Open Capillary GCPacked GC Current HPLCEarly HPLC Open Column LCTLC

Technique

100dc = 0.1-.05 mm100dc= 0.03 mm4010152-5220-500.021500.01150

Neff/t (plates/s)

dp (µm)

*L.R. Snyder; J.J. Kirkland, Introduction to Modern Liquid Chromatography; Wiley: New York, 1979.**R. Kennedy et al., Chem. Rev., 99, 3081-3140 (1999).

Fast HPLC at High TemperatureFast HPLC at High TemperatureAb

sorb

ance

(mAU

)

Time (min)0 3 6 9 12 15

0

100

200

300

400

500

A

5

4

3

21

0.0 0.2 0.4 0.6 0.8 1.0

0

100

200

300

400 B

5

43

2

1

30 oC1 ml/min

100 oC5 ml/min

Effect of Temperature on Analysis Timeat Constant N and P

“High-Performance Liquid Chromatography at Elevated Temperatures: Examination of Condition for the Rapid Separation of Large Molecules”, R. D. Antia and Cs. Horvath, J. Chromatogr., 435, 1-15 (1988).

50 150100 200

0.2

0.4

0.6

0.8

1.0

t R/t R

,25

TEMPERATURE ( O C)

0.2

0.4

0.6

0.8

1.0

Theoretical and Practical Limits of Speed in HPLC

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

2)'1(p

m

dhD

kNt

ν+

=

max

3

max PD

dk

m

po ∆=η

ν

3/2max

3/23/1

3/1

)'1(PL

DkA

Nt

m ∆+

≅ η

3/13/2max

3/2

)'1(TP

LAkNt η

∆+∝

Fixed Pressure*

Theoretical Limit*

Practical Limit

Reduced Velocity Limit

Practical Limit Temperature Dependence

2)'1(p

m

dD

kCNt +

≅∞→ν

Temperature (oC)

40 65 90 115 140 165 190

Visc

osity

(cp)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Water 50/50 ACN-waterMeOH ACN

Solvent Viscosity vs. Temperature

Data from Horvath and Chen.

“Influence of Thermal Conditions on the Efficiency of High-Performance Liquid Chromatography.”

H. Poppe and J. C. Kraak, J. Chromatogr., 282, 399-412 (1983).

Thermal Mismatch Broadening

*H. Poppe and J.C. Kraak

Peak Shapes Observed for Various Mobile-Phase Feed Temperatures*

LC conditions: Column water jacket, 30 oC; 6.2 mm IDx8cm; 3µ Zorbax ODS; at 5 mL/min; 50/50 (v/v) ACN,H2O; nitrobenzene

2222mismatchthermalcolumnnextracolumnobs −− ++= σσσσ

LC conditions: 2.1 x 5 cm, C-18 INERT, 55 % ACN, 5 cm preheater, 60 oC4.6 x 5 cm, C-18 INERT, 60% ACN, 5 cm preheater, 60 oC.

Peaks: 1. toluene, 2. ethylbenzene, 3. propylbenzene,4. butylbenzene

Comparison of the Effect of Incomplete Thermal Equilibration on Column Performance

Time (min)0.0 0.5 1.0

Abso

rban

ce (m

AU)

0

2

4

6

8

10

12

14

161

2

3

4

4.4.6 mm ID6 mm ID2.1 mm ID

Time (min)0.0 0.5 1.0

Abso

rban

ce (m

AU)

0

20

40

60

801 2

34

105 cm/min 105 cm/min

u ( cm/s )0.0 0.5 1.0 1.5 2.0 2.5

Plat

e He

ight

( x1

0-4

cm )

12

14

16

18

20

22

24

26

25 oC80 oC

120 oC

150 oC

Effect of Temperature on Column Efficiency in HTUFLC

Conclusion: Resistance to mass transfer is greatly reduced as the column temperature is increased . ∆ , 25 oC (decanophenone, k’=12.23), ∇, 80 oC (dodecanophenone, k’=7.39), , 120 oC (tetradecanophenone, k’=12.32).

Fast Separations NSAIDs at High Temperature

1

Column Temperature = 150 oCF = 5.5 ml/min.

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.

High Speed HPLC

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

5

LC Conditions: Mobile Phase, 29/71 ACN/50mM Tetramethylammonium hydroxide, pH 12.2; Flow Rate, 1.35 mL/min.; Injection volume, 0.5 ul; 254 nm detection; Column Temperature, 21°C; Pressure drop = 195 bar; Solutes: 1=Doxylamine, 2=Methapyrilene, 3=Chlorpheniramine, 4=Triprolidine, 5=Meclizine 100 x 4.6 ZirChrom-PBD

min2.4 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 min

LC Conditions: Mobile Phase, 20.5/79.5 ACN/50mM Tetramethylammonium hydroxide, pH 12.2; Flow Rate, 4.20 mL/min.; Injection volume, 0.5 ul; 254 nm detection; Column Temperature, 140°C; Pressure drop = 194 bar; Solutes: 1=Doxylamine, 2=Methapyrilene, 3=Chlorpheniramine, 4=Triprolidine, 5=Meclizine 100 x 4.6 ZirChrom-PBD

min0 2 4 6 8 10 12 14

mAU

-10

0

10

20

30

40

50

60

70

VWD1 A, Wavelength=254 nm (G:\HPCHEM\DATA\P102501\TEST0158.D)

min0 0.25 0.5 0.75 1 1.25 1.5 1.75

mAU

010203040506070

5

41

2

3 Temperature: 140 oCFlow rate: 4.20 ml/min.Pressure drop: 194 barResolution (2,3): 2.1Analysis time: .85 minutes

min0.48 0.5 0.52 0.54 0.56 0.58 0.6 0.62 0.64

Courtesy ZirChrom

( )1'ln4

1 ++= ns

c kRNn

One-dimensional HPLC has low peak capacity

1 2 3 4 5 6 7 8 9

10

5000

17000290000

10

20

30

40

50

60

Peak

Cap

city

(nc)

Retention Factor(N)

Comprehensive two-dimensional HPLC has high peak capacity

21 cccTotal nnn ×=

( ) ( )[ ]( )2

2max211max 1'1'υ

++=

kLNkt c

rtotal

A major limitation is low speed related to the second dimension linear

velocity, u2

Giddings, J. C. Multidimensional Chromatography: Techniques and Applications; Marcel Dekker: New York, 1990

Fast, Comprehensive Two-Dimensional HPLC

LC × UFHTLC Separation of Ten Triazine Herbicides

0

20

40

60

80

100

0 5 10 15 20T im e (m in .)

mA

U

1st Dimension Conditions: Column, 50 mm x 2.1 mm I.d. PBD-ZrO2; Flow rate, 0.08 ml/min.;Temperature, 40 oC

2nd Dimension Conditions: Column, 50 mm x 2.1 mm I.d. PBD-C-ZrO2; Flow rate, 7.0 ml/min.;Temperature, 150 oC; 1st dimension sampling frequency, 0.1 Hz

N N

NHN CH3

HNH3C

ClSimazine

Total LC × UFHTLC peak capacity = 185

A single column would be 2.5 meter and take 44 hours to generate same peak capacity

Fast Chromatography on the 2nd Column

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

0.18

0.2

0 2 4 6 8 102nd Dimension Time (seconds)

mV

210220230240250300310320330340350360370380390

Time (s)

Conclusions:(1)Heat transfer, pressure drop and extra-column

broadening considerations are key to design HTUFLC.

(2)Tubing pressure drop is important.

(3) HTUFLC can be as much as 50 times faster thanroom temperature HPLC.

(4) HTUFLC can be done with 100% water as the eluent.

(5) Fast (< 0.5 hr.) 2D-LC can be done.

National Institutes of Health