high speed two-dimensional liquid chromatography …giddings, j. c. multidimensional chromatography:...
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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