High Speed Two-Dimensional Liquid Chromatography Through the Use of Ultra-Fast

High Temperature Liquid Chromatography as the Second Separation Dimension

Dwight Stoll and Peter W. Carr

University of MinnesotaDepartment of Chemistry

AbstractA fundamental limitation of High Performance Liquid Chromatography (HPLC) is its relatively low peak capacity compared to other techniques. By far the most powerful approach to improving peak capacity is two-dimensional separations (2DLC), where the ideal peak capacity of the two-dimensional separation is the product of the individual peak capacities of the first and second dimension separations. While comprehensive 2DLC separations were reported almost a decade ago, the demands placed on the speed of the second dimension separation of these systems make the technique very slow, typically about one half-day per full 2D chromatogram. A solution to this extreme impediment to the widespread use of 2DLC is to apply Ultra-Fast High-Temperature Liquid Chromatography (UFHTLC) to the second dimension separation to allow fast and comprehensive sampling of the first dimension separation, thereby allowing full 2DLC analyses at ten-thirty minutes per analysis. The concomitant decrease in eluent viscosity and increase in analyte diffusivity under UFHTLC conditions allows very efficient separations to be performed at extremely high column linear velocities in the second dimension separation.

In this work we will show one-dimensional LC separations on the ten-second timescale; these are enabled by using UFHTLC conditions. We will then show how these ultra-fast separations can be used in the second dimension of a fast, comprehensive 2DLC system, which we call LC ×UFHTLC. This fast 2DLC system can be used to greatly improve the resolution of components in complex samples, which are difficult to resolve using conventional HPLC.

A Common Problem in HPLC

Sample composed of 20 components with

randomly distributed k’ values

150 mm x 4.6 mm i.d. column

0

0.1

0.2

0.3

0.4

0.5

0.6

0 5 10 15 20 25 30Time (min.)

AU

N = 10,000 plates/columnnc = 38, 12 unresolved

components

0

0.2

0.4

0.6

0.8

1

0 5 10 15 20 25 30Time (min.)

AU

N = 25,000 plates/columnnc = 60, 9 unresolved

components

Even with state-of-the-art HPLC, only

50% of the components in this

sample can be resolved !!!

Peak capacity (nc)

0 50 100 150 200

Sing

le c

ompo

nent

pea

ks (s

)

0

5

10

15

20

Peak Capacity Limitations of One-Dimensional HPLC

#1a - Low peak capacity (nc) #1b – The number of components observable as single

peaks is even lowercnmmes /2−=

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

Comprehensive two-dimensional HPLC is the most efficient way to greatly increase the peak capacity of HPLC

m (number of components)=

20

0

10

20

30

40

50

60

500010000

1500020000

250003000

2

46

8

Peak

Cap

acity

(nc)

Plate Number (N)

Retention Factor (k')

( )1'ln4

1 ++= ns

c kRNn

For Rs=2

Requirements and Advantages in Conventional Two-Dimensional HPLC

Two conditions must be met for the technique to be considered “two-dimensional”1. Orthogonality of separation mechanisms – This is a requirement imposed primarily on the

stationary phase chemistry

2. Separation gained in one dimension must not be diminished by separation in the other dimension

If these two conditions are satisfied, the maximum total

peak capacity of the two-dimensional system will be:

21 cccTotal nnn ×=

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

0

0.05

0.1

0.15

0.2

0.25

0 1 2 3 4 5 6Time (min.)

AU

( ) ( )[ ]( )2

2max211max 1'1'υ

++=

kLNkt c

rtotal

Murphy, R. E.; M. R. Schure; J. P. Foley Anal. Chem., 1998; Vol. 70, pp 1585-1594

Comparison of Peak Capacity Production

TechniquePeak Capacity

Limit (nc)Analysis Time

(hr)Peak Capacity

Production (nc/hr)

Capillary GC 103 100-101 102

GC x GC 104-105 101 103-104

HPLC 102-103 100-101 101-102

LC x LC 103-104 101-102 102

LC x UFHTLC 103-104 100-101 ?? 103 ??2D-Gel Electrophoresis 103-104 102 101-102

Ultra Fast High Temperature LC has the potential to significantly improve the rate of peak capacity production in

HPLC

Temperature (oC)

40 60 80 100 120 140 160 180 200 220

η/η

(25

o C)

0.0

0.2

0.4

0.6

0.8

1.0 Water20/80 ACN/WaterMethanolAcetonitrile

Improving the Speed of 2DLC Through the Use of UFHTLC – Effect of Temperature on Eluent Viscosity

Improving the Speed of 2DLC Through the Use of UFHTLC – Implications of Decreasing Viscosity

2pd

uLP ηϕ=∆

ϕη

LPd

u p2∆

=

As the viscosity decreases, the linear velocity through the column is allowed to

increase when the pressure is held constant

1.

As the viscosity decreases, the

diffusivity of the analyte in the eluent increases,

thereby relaxing resistance to mass

transfer

2.η1

, ∝mAD

Requirements for Fast 2DLC

Improving the speed of 2DLC is not possible without columns that have ALL of the following three characteristics for use in the second dimension separation:

1. The stationary phase used must be thermally and chemically stable under the conditions of UFHTLC

2. The columns (narrow-bore or smaller i.d.) packed with the stable stationary phase must be thermo-mechanically stable

3. The stationary phase must provide selectivities that are orthogonal to existing phases for the analytes of interest

Also, the column and eluent must be heated properly to avoid “thermal mismatch” band broadening

Effect of Temperature on the Shape of the van Deemter Curve

0.000

0.002

0.004

0.006

0.008

0.010

0.012

0.014

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0

Interstitial Linear Velocity (ue, cm/s)

Plat

e H

eigh

t (cm

)

UFHTLC velocity range

ee

CuuBAH ++=

C-ZrO2• 40 oC• 150 oC

For small molecules there is roughly a 4-fold lowering1 of the

van Deemter C-term as the temperature is raised from 40 to

150 oC

This allows practically useful column efficiencies to be obtained at extremely high column linear

velocities

(1) Yan, B.; Zhao, J.; Brown, J. S.; Blackwell, J.; Carr, P. W. Analytical Chemistry 2000, 72, 1253-1262.

Column Flow Rate (ml/min.)

ue

(cm/s)tm (sec.) k'max

N (Plates/column)

Peak Capacity (nc)

C-ZrO2 5.00 6.3 1.6 5.25 2000 11

Current Column Technology for Fast 2DLC

30

35

40

45

50

55

60

65

70

0 2 4 6 8 10

Time (sec.)

mA

U

Column: 50 mm x 2.1 mm i.d. C-ZrO2

Temperature: 150 oC

Flow rate: 5.0 ml/min.

Solutes: Alkylphenones

Mobile: 30/70 ACN/WaterPhase

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

An Example Application – Initial Work on a Screening Tool for Drugs of Abuse

Goal – To significantly improve the speed and comprehensiveness of a single HPLC method for screening for presence of opiates and amphetamines in biological samples

Background• Opiates and amphetamines are two major classes of abused drugs that are routinely assayed

for in public and private forensic labs.

• The International Olympic Committee has identified pseudoephedrine as a significantly abused stimulant1

• Immunoassay-based techniques are commonly employed as qualitative screening techniques because of relatively broad compound coverage, and the ability to significantly improve throughput. However, these techniques are not perfect and have problems such as cross-reactivity and the potential for false positives.

• GC/MS is commonly used for compound confirmation and quantitation.

• A main reason LC is not used is because it is not efficient. It takes multiple methods (runs) to attain broad compound coverage because of low peak capacity.

1) Gmeiner, G.; Geisendorfer, T.; Kainzbauer, J.; Nikolajevic, M.; Tausch, H. Journal of Chromatography, B: Analytical Technologies in the Biomedical and Life Sciences 2002, 768, 215-221

The Opiates

O

N

OH3C

HO

O

N

OH3C

O

O

N

HO

HO

O

N

O

HO

O

NCH3

OH

H3CO

O

O

NCH3

OH

HO

O

O

N

HO

O

O

Codeine (9)

Hydromorphone (3) Oxymorphone (2)

Oxycodone (12)Hydrocodone

6-acetylmorphine Morphine (1)

The Amphetamines

Amphetamine (10)

CH3

NH2

HN

CH3

OH

CH3

HN

CH3

CH3

NH2

H3C CH3

HN

CH3

OH

CH3

NH2

OH

CH3

NH

CH3

O

CH3

CH3

NH2O

CH3

CH3

NH2

O

O

CH3

HN

O

O CH3

NH2

O

CH3

HNO

CH3

CH3

CH3

HN

O

O CH2

CH3

CH2

HN

O

O CH3

CH3

MBDBMDEA

MDA (11)

PMMA

Phentermine Phenylpropanolamine (4)

PMA

Cathinone (5)

MDMA

Methcathinone (8)Methamphetamine

Ephedrine (6)

Pseudoephedrine (7)

An Initial Column Selectivity Evaluation –First Dimension Column

Condition 1: Column, ACE-C18; Mobile phase, 20/80 ACN/20mM ammonium acetate, pH 5.0; Temperature, 40 oC

0

5

10

15

20

25

0.0 2.0 4.0 6.0 8.0 10.0

Retention Factor (k')

Sol

ute

Inde

x

Ephedrine/Pseudoephedrine

MDA/Oxycodone

Minimum Resolution = 0

An Initial Column Selectivity Evaluation –Adding the Second Dimension Column

Condition 1: Column, ACE-C18; Mobile phase, 20/80 ACN/20mM ammonium acetate, pH 5.0; Temperature, 40 oC

Condition 2: Column, ZirChrom C-ZrO2; Mobile phase, 20/80 ACN/30mM formic acid, 15mM octylamine, 50µM EDTPA, pH 3.6; Temperature, 150 oC

0.0

1.0

2.0

3.0

4.0

5.0

0.0 2.0 4.0 6.0 8.0 10.0

ACE-C18 Retention Factor (k')

C-Z

rO2 R

eten

tion

Fact

or (k

')

MDA/Oxycodone

Ephedrine/Pseudoephedrine

Minimum Resolution = 0.52

Thermal Instability of Amphetamines at pH - 5

LC Conditions: Solute = Cathinone, 100 ug/ml; Flow rate varied from 1.0 to 5.0 ml/min (see figure).; Temperature, 150 oC; 10 µl injection; Detection at 254 nm; Mobile phase, 40/60 ACN/20mM ammonium acetate, pH 5.0; Column, 50 mm x 2.1 mm i.d. ZirChrom C-ZrO2

0

200

400

600

0 0.1 0.2 0.3 0.4 0.5 0.6

Time (min.)

mA

U

1.02.03.04.05.0

NH2

O

Cathinone

Degradation as a Function of Eluent pH

-10

40

90

140

190

0 0.1 0.2 0.3 0.4

Time (min.)

mA

U

Ammonium acetate pH 5.0

-10

40

90

140

190

0 0.1 0.2 0.3 0.4

Time (min.)

mA

U

Ammonium bicarbonate pH 7.0

0

50

100

150

200

0 0.1 0.2 0.3 0.4

Time (min.)

mA

U

TFA pH 2.1

0

50

100

150

200

250

300

0 0.1 0.2 0.3 0.4

Time (min.)

mA

U

Formic acid pH 3.6

Cathinone and Methcathinone show the same behavior, however, their reactivities are insignificant on the chromatographic timescale of UFHTLC in the pH range of 2.1-3.6.

Strategy for Optimizing the Two-Dimensional Separation

1. Experimentally determine the retention of all solutes on the ACE-C18 column at three different eluent strengths

2. Fit retention data to Pade approximation

2'log φφ CBAk ++=

3. Experimentally determine the retention of all solutes on the C-ZrO2 column at seven different eluent strengths at 150 oC

4. Fit retention data to a parabolic function of φ

5. Optimize the minimum two-dimensional resolution by varying the eluent strength in each separation dimension independently

22,

21,2, ssDs RRR +=

φφC

BAk+

+=1

'ln

Experimental vs. Predicted Retention

2'log φφ CBAk ++=

ZirChrom C-ZrO2Retention

0.00

0.20

0.40

0.60

0.80

1.00

0 0.1 0.2 0.3 0.4 0.5

φ

log

k'

MDBD FitMDBD ExptCathinone FitCathinone Expt

ACE-C18 Retention

φφC

BAk+

+=1

'ln

-1.20

-0.60

0.00

0.60

1.20

1.80

2.40

0.05 0.10 0.15 0.20 0.25

φ

ln k

'

MDBD FitMDBD ExptCathinone FitCathinone Expt

A Prediction of the First Half of the Two-Dimensional Separation

0.00

0.05

0.10

0.15

0.20

0.25

0 4 8 12 16 20 24

ACE-C18 Retention time (min.)

C-Z

rO2 R

eten

tion

time

(min

.)

1st Dimension ConditionsColumn – 150 mm x 2.1 mm I.d. ACE-C18, 5 micronFlow rate – 0.08 ml/min.Mobile phase – 10/90 ACN/20mM Ammonium

acetate, pH 5.0Temperature – 40 oCInjection volume – 5 µl of 100 µg/ml of each solute

2nd Dimension ConditionsColumn – 33 mm x 2.1 mm i.d. ZirChrom C-ZrO2, 3 micronFlow rate – 5.00 ml/min.Mobile phase –ACN/30mM Formic acid, 15mM

Octylamine, 50µM EDTPA, pH 3.6

Temperature – 150 oCInjection volume - 15 µlDetection at 215 nm

Time (min.) % ACN0 60

15.8 6519.0 50

1 Morphine 7 Pseudoephedrine2 Oxymorphone 8 Methcathinone3 Hydromorphone 9 Codeine4 Phenylpropanolamine 10 Amphetamine5 Cathinone 11 MDA6 Ephedrine 12 Oxycodone

• Opiates• Amphetamines

1 5

3

4

2

8

9 10

11

126

7

100 1200 1300 1400 1500960 980 1000 1020 1040 1060 10800

2

4

6

8

0

0 200 400 600 8000

2

4

6

8

10

5 10 15 20 25

ACE-C18 Retention Time (min.)

C-Z

rO2

Re t

e nt io

n T

i me

( se c

.)

-200

-195

-190

-185

-180

-175

-170

0 2 4 6 8 10

C-ZrO2 Ret. time (sec.)

1

1 – Methcathinone2 – Ephedrine3 - Pseudoephedrine

2

3

An Initial Attempt at the First Half of the Separation

1

3

2

6

7

8

12

10 119

5

4

Extreme Column Overloading of Amines Severely Degrades the Two-Dimensional

Resolution

0

5

10

15

0 0.5 1 1.5

Time (min.)

mAU

0

20

40

60

80

100

0 0.5 1 1.5

Time (min.)

mAU

0

100

200

300

400

500

0 0.5 1 1.5

Time (min.)

mAU

0

200

400

600

800

0 0.5 1 1.5

Time (min.)

mAU

Cathinone

2000 ng1000 ng

100 ng20 ngLC Conditions

Column – 50 mm x 4.6 mm i.d. ACE-C18, 5 micron

Flow rate – 2.00 ml/min.

Mobile phase – 10/90 ACN/20mM Ammonium acetate, pH 5.0

Temperature – 40 oC

Injection volume – 20 µl

Mass PlateInjected (ng) Symmetry Count (N)

20 0.83 2800100 0.80 29501000 0.59 24002000 0.47 1700

Conclusions and Future Work1. There is a great potential for significantly increasing the speed of

comprehensive two-dimensional liquid chromatography through the implementation of UFHTLC as the second separation dimension.

2. Ultra-fast one-dimensional separations on the ten-second timescale are feasible using narrow-bore columns at high temperatures. This allows extremely high column linear velocities to be reached using practical volumetric flow rates, without excessive diminishing of the optimum efficiency of the column.

3. There remains significant room for improvement of these ultra-fast separations. These gains will likely be brought about through the use of smaller particles, and columns packed better than those currently in use.

4. An initial attempt at the first half of a two-dimensional LC separation of 21 commonly abused opiates and amphetamines has been successful. The problem of severe overloading of the C18-silica column must first be solved before the full two-dimensional LC separation can be satisfactorily demonstrated.

Acknowledgements

ZirChrom Separations, Inc.

Mac-Mod Analytical

Systec, Inc.

Minnesota Bureau of Criminal Apprehension

Lakeview Associates