High Speed 2D-HPLC Through the Use of

Ultra-Fast High Temperature HPLC as the

Second Dimension

Minnesota Chromatography Forum

Spring Symposium

Dwight Stoll and Peter W. Carr

Department of Chemistry
University of Minnesota
 A Common Problem in HPLC

0.6 N = 10,000 plates/column

Sample composed of 20 0.5 nc = 38, 12 unresolved
components with 0.4 components
random k’ values

AU
0.3

0.2
150 mm x 4.6 mm i.d.
0.1
column
0
0 5 10 15 20 25 30
Time (min.)

1 N = 25,000 plates/column
Even with state-of- 0.8
nc = 60, 9 unresolved
the-art HPLC, only 0.6 components
half of the components
AU

0.4

in this sample can be 0.2

resolved !!! 0
0 5 10 15 20 25 30
Time (min.)
LC × UFHTLC Separation of Ten Triazine Herbicides
H H
H3C N N N CH3
100

80 N N
1st Dimension Conditions: Column, 50 mm x
60 Simazine 2.1 mm i.d. PBD-ZrO2; Mobile phase, 20/80
mAU

40
Cl
ACN/Water; Flow rate, 0.08 ml/min.;
20
Injection volume, 20 µl; Temperature, 40 oC
0
0 5 10 15 20
T im e (m in .)

2nd Dimension Conditions: Column, 50 mm x

2.1 mm i.d. PBD-C-ZrO2; Mobile phase, 20/80
ACN/Water; Flow rate, 7.0 ml/min.; Injection
volume, 15 µl; Temperature, 150 oC; 1st
dimension sampling frequency, 0.1 Hz

Total LC × UFHTLC peak

capacity = 185
Using a single column, it would
take a 2.5 meter long column and
44 hours to generate the same
peak capacity
 Outline
• Background
– Limitations of one-dimensional HPLC (1DLC)
– Requirements and advantages in two-dimensional HPLC (2DLC)
– Improving 2DLC by applying UFHTLC (Ultra Fast High Temperature Liquid
Chromatography) to the second dimension separation

• Results
– Assembly of a preliminary LC × UFHTLC instrument
– Fast 1DLC separations using narrow-bore columns
– A preliminary, fast LC × UFHTLC separation

• Conclusion – Implementation of UFHTLC in the second dimension

separation of 2DLC will increase both the practical limit and the
rate of peak capacity production in HPLC.
 Limitations of One-Dimensional HPLC
#1a - Low peak capacity #1b – The number of
components observable as single
N
ln (k 'n +1)
peaks is even lower
nc = 1 + −2 m / nc
4 Rs s = me
60 20
For Rs=2 For m=20
50

Single component peaks (s)

15
Peak Capacity (nc)

40

30
10

20

10 5

0
Re 8 3000
te nti
o 25000
nF 6 20000
0
ac 4
to r (k 15000 0 50 100 150 200
)
') 2 10000 e r (N
umb Peak capacity (nc)
5000 te N
Pla

Comprehensive two-dimensional HPLC is the most efficient way to

greatly increase the peak capacity of HPLC
Giddings, J. C. Multidimensional Chromatography: Techniques and Applications; Marcel Dekker: New York, 1990
 Requirements and Advantages in
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 on the stationary
phase chemistry
2. Separation gained in one dimension cannot be diminished by separation in the other dimension
0.25

0.2 If these two conditions are

0.15 satisfied, the maximum total
AU

0.1 peak capacity of the two-

0.05
dimensional system is:
0
0 1 2 3 4 5 6
Time (min.)

(k 'max1 +1)( N1 [Lc 2 (k 'max 2 +1)] ) ncTotal = nc1 × nc 2

t rtotal =
υ2
Murphy, R. E.; M. R. Schure; J. P. Foley Anal. Chem., 1998; Vol. 70, pp 1585-1594
Giddings, J. C. Multidimensional Chromatography: Techniques and Applications; Marcel Dekker: New York, 1990
 Comparison of Peak Capacity Production

Peak Capacity Analysis Time Peak Capacity

Technique
Limit (nc) (hr) 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

Goal: To increase the speed of peak capacity production in HPLC

such that 10-20-fold increases in peak capacity can be
achieved for separations under 60 minutes
 Potential Approaches to Improving the
Speed of HPLC

Approach Advantage Disadvantage

Works with most equipment,
Shorter Columns Low plate count and resolution
stationary phases

Narrow-bore columns are not

Monolithic Columns Low backpressure
available, high sovent useage

Ultra-High Pressure High plate counts with small Specialized equipment needed,
LC particles losses in N at high velocity

Low backpressure, high Requires adequate heating,

High Temperature LC
efficiency at high velocity stable phases, stable analytes.

High temperature LC is the only approach that allows a significant

fraction of the column plate count to be retained as the column linear
velocity is increased to values that allow significantly faster HPLC
 Improving Two-Dimensional HPLC by Applying
UFHTLC to the Second Dimension Separation

#1 – High column temperatures #2 – High column temperatures

dramatically lower eluent dramatically increase analyte
vicosities allowing higher diffusivity in the eluent producing
column linear velocities more efficient separations at high
linear velocities
1.0

0.8

(k 'max 1 + 1)( )
N 1 [Lc 2 (k ' max 2 + 1)]
tR/tR,25

0.6

t rtotal =
0.4
υ2
0.2

50 100 150 200

TEMPERATURE, [OC]

Antia, F. D.; C. Horvath In J. Chromatogr., 1988; Vol. 435, pp 1-15

 Outline
• Background
– Limitations of one-dimensional HPLC (1DLC)
– Requirements and advantages in two-dimensional HPLC (2DLC)
– Improving 2DLC by applying UFHTLC (Ultra Fast High Temperature Liquid
Chromatography) to the second dimension separation

• Results
– Assembly of a preliminary LC × UFHTLC instrument
– Fast 1DLC separations using narrow-bore columns
– A preliminary, fast LC × UFHTLC separation

• Conclusion – Implementation of UFHTLC in the second dimension

separation of 2DLC will increase both the practical limit and the
rate of peak capacity production in HPLC.
 Developing Instrumentation: Guidelines for
System Parameters in LC × UFHTLC
Target second dimension peak capacity = 20 (within 10 seconds)
Flow rate required (ml/min.)
Theoretical work by Thompson
Column Inner Diameter (mm)
suggests the 2.1 mm column ue (cm/s) 1.0 2.1 4.6
diameter is most suitable for 0.1 0.02 0.08 0.38
UFHTLC 1.0 0.18 0.79 3.8
10.0 1.8 7.9 38
Column Temperature (oC) 40a,b 150c,d
For a maximum of 10% Retention Factor 1 5 1 5
decrease in column Vinjection (µl) 3.8 11.4 5.1 15.4

efficiency due to extra- Vdetector (µl)

Detector response time (s)
3.8
0.3
11.4
1.0
5.1
0.02
15.4
0.05
column broadening: Lconnecting tubing (cm)e 17.2 155 1.3 11.3
a. hypothetical van Deemter coefficients are A=1.5, B=5, C=.03
b. assumes flow rate of 0.2 ml/min.
c. hypothetical van Deemter coefficients are A=1.5, B=5, C=.0075
d. assumes flow rate of 5.0 ml/min.
-5 2 o
e. hypothetical diffusion coefficient = 1.0 x 10 cm /s at 30 C

Thompson, J. D.; Carr, P. W. Analytical Chemistry 2002, 74, 4150-4159

Schematic of a Complete LC × UFHTLC System

Standard HP1090 Pump and

Autosampler Heating Jacket
1st Dimension T2
Auto- T1
Binary Eluent Column
Pump #1 Injector Preheater

Temperature
S a m p le
Lo o p # 1
Controlled
Heating Jacket
10-port,
T6 2nd Dimension T5 Eluent 2-position
Column Preheater valve
T4
S a m p le
Lo o p # 2

Counter-
T7 UV Detector T3
Current Heat
Exchanger Eluent Binary
Preheater Pump #2

All components are controlled using Labview

 Developing Instrumentation -
Sample Transfer in 2DLC
Position 1 Position 2
Column 1 Column 1

1 1
2 10 2 10
Column 2

Column 2
9 9
3 3
8 8
4 4
7 7
5 6 5 6

Waste Pump 2 Waste Pump 2

Detector Detector
 A Very Fast One-Dimensional Separation

3
4 ue = 6.0 cm/s
160
140
120
2
k’max = 4.6
100
m AU

80
5
6 N = 1400
60
40
1 nc = 8
20
0
0 5 Tim e (se c.) 10 15
∆P = 300 bar

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

Temperature: 150 oC
Flow rate: 4.75 ml/min.
Injection volume: 1 µl
Detection at 254 nm with 6 µl flow cell and 50 ms detector response time
Solutes: Acetone, propiophenone, butyrophenone, valerophenone, hexanophenone,
and heptanophenone
LC × UFHTLC Separation of Ten Triazine Herbicides
1st Dimension Conditions: Column, 50 mm x
2.1 mm i.d. PBD-ZrO2; Mobile phase, 20/80
ACN/Water; Flow rate, 0.08 ml/min.;
Injection volume, 20 µl; Temperature, 40 oC
2nd Dimension Conditions: Column, 50 mm x
2.1 mm i.d. PBD-C-ZrO2; Mobile phase, 20/80
ACN/Water; Flow rate, 7.0 ml/min.; Injection
volume, 15 µl; Temperature, 150 oC; 1st
dimension sampling frequency, 0.1 Hz

0.2
210
0.18
220
0.16 230
240
0.14
250
0.12 300
310
mV
0.1
320
0.08 330
340
0.06 350

0.04 360
370
0.02 380

0 390

0 2 4 6 8 10

2nd D imension Time (seconds)

 Conclusions
• Implementation of UFHTLC in the second dimension
separation of 2DLC will allow comprehensive 2DLC
separations on a practical timescale. This will…
– Extend the peak capacity limit of practical HPLC
– Increase the rate of peak capacity production in HPLC

• The dramatically increased peak capacity will allow faster

separations of complex pharmaceutical and biological
samples
Acknowledgements

Professor Peter W. Carr

ZirChrom Separations, Inc.
Systec Inc.