Developments in

Multidimensional- and
Comprehensive-
ChromatographyBeyond
Boxcars and Deans
Pittcon 2007
Ronald E.Majors
Agilent Technologies
Wilmington, DE
Early On-Line 2D GC Publication
Pages 32-35
Early Instrument Designed for
2D GC
Early 2D GC On-Line
Fractionation of Hydrocarbons
Outline of Presentation
Off-Line and On-Line MD
Chromatography: Definitions,
advantages and disadvantages
Multi-dimensional LC
Comprehensive LC (LCXLC)
Multi-dimensional GC
Comprehensive GC (GCXGC)
Conclusions
What is Multidimensional Chromatography?
The selective transfer of a fraction (or fractions) from one
chromatographic medium (usually a column) to a secondary (or
additional) chromatographic media for further separation
Technique used for:
Further resolution of complex mixture that cannot be separated on
a single medium (increased peak capacity)
Sample cleanup by removing matrix or interfering compounds
Increased sample throughput
Trace enrichment of minor compounds of interest
Various names have been used (with variations):
Column switching, multiphase chromatography, coupled column
chromatography, sequential analysis, boxcar chromatography and
others
Simplified Schematic of 2D MDC
LC Separation
1
0
Separation Mode: Size Exclusion Chromatography
2
0
Separation Mode: Affinity Chromatography
1D GC Information Capacity
Heartcut 2D GC Information
Capacity
Comprehensive GCxGC
Information Capacity
Sampling
Adahchour, M.; Beens, J.; Vreuls, R. J. J.; Brinkman, U. A. T. "Recent developments in comprehensive two-
dimensional gas chromatography (GC*GC). Introduction and instrumental set-up." TrAC, Trends in Analytical
Chemistry 2006, 25, 438-454.
Basic Concept of Comprehensive 2D Chromatography
(thanks to Pete Carr for loan of slide)
Peak Capacity
The maximum number of peaks that
can be resolved side-by-side into the
available separation space.
Assumes each dimension is totally orthogonal
Peptide Map of BSA on Three SB-C18
Columns with Different Particle Sizes
Gradient Time = 30 min
Temp. = 80C
238
Peak Capacity
391
540
Starting
Pressure
51
103
340
min 4 6 8 10 12 14 16 18 20
mAU
0
5
10
15
20
25
30
min* 4 6 8 10 12 14 16 18 20
mAU
0
10
20
30
40
50
min* 4 6 8 10 12 14 16 18 20
mAU
0
10
20
30
40
50
60
SB-C18, 2.1x150mm, 5m
SB-C18, 2.1x150mm, 3.5m
SB-C18, 2.1x150mm, 1.8m
[P
c
using eqn. of Neue, J.Chromatogr. A, 1079(1-2), 153-161(2005)]
Separation Space Utilization by Orthogonal
and Correlated Mechanisms
Separation Axis 2
Separation Axis 2
Separation Axis 2
S
e
p
a
r
a
t
i
o
n

A
x
i
s

1
Orthogonal Separations
Separations
with correlation
Separations
With total correlation
Two Dimensional Chromatographic Techniques

GC SFC

SFC SFC

LC SFC

LC or GC SPE, SPME or
SBSE

GC LC

LC LC

GC GC
X

GC or LC Preparative TLC

TLC (or HPTLC) TLC (or HPTLC)
On-Line Off-Line Secondary
Technique
Primary
Technique
Off-Line Multidimensional
Chromatography
On-Line Multidimensional
Chromatography
Coupled HPLC Modes
++ ++
Basic Setup for an On-Line MD LC-LC
Use of Coupled Column RAM-
RPC System for Analysis of Drugs in Plasma
Column A: RAM Column
Mobile phase: water, 0.5 mL/min
Column B: LiChrospher 60 RP Select B
Mobile phase: Trichloroacetic acid,
Acetonitrile, 0.1%
Triethanolamine,
pH2, 1.0 mL/min
B.
A.
100-uL plasma
BioTrap C8
LiChrospher RP-4 ADS
ISRP C8
SPS C8
Peaks:
1. Epirubicinol
2. Epirubicinal aglycone
3. Epirubicin
4. Epriubicin aglycone
5. 7-deoxyepirubicinal aglycone
(compounds in 5.6-8.2 ng/mL range)
A. Rudolphi and K.-S. Boos, LC/GC 15, 814-823 (1997).
Switching time
Detector: Fluorescence, 445 nm ex
560 nm em
(70/30 V/V)
Proteomics With 2D-LC/MS - Workflow
HPLC:
1st dimension
Ion-exchange
2nd dimension
Reversed phase
1. cell disruption
2. prefractionation
3. solubilization
4. sample clean up
cell
free proteins
(10
4
to 10
5
)
tryptic digest
Peptides
(10
5
to 10
6
)
Mass Spec
Data Analysis
Separation &
Isolation
identification
Theoretical Resolving Power of 2D-LC/MS
e.g. reversed phase
Peak capacity n
c
= L /(4 )
L total elution time
average standard deviation of peaks
15 fractions
e.g. cation exchange,
gel filtration
Gradient run time L = 90 min,
peak width 4 ~25s
n
c
= 216
Total Peak capacity : 1. Dim * 2. Dim 15 * 216 = 3240
3. Dimension Mass Spec
1
0
Dimension
2
0
Dimension
(15 x 2
0
Dimension)
7 * 3240 = 23,000 peptides
(Wolters Et Al.)
2-D HPLC: Cation Exchange and
Reversed Phase Chromatography
Waste
Mass Spec
Data Analysis
SCX)
Peptides
RP
Protein mixture
Digest pH < 3
MS/MS Data
1) Load peptides on SCX at 0% salt
2) Elute w/ increments of salt (0.1 M - 1 M)
3) a. Collect fractions and re-inject on RP
column (OFF-LINE approach)
or
b. Inject directly on RP column
(ON-LINE approach)
2D approach results in more resolved
peptides than either single dimension
ICAT
Eluted and separated peptides
are directly analyzed by MS/MS
HPLC-Chip Platform for Nanospray LC/MS
1200 NanoLC System
6000 Series Mass Spectrometer
(Ion trap, SQ, QQQ, TOF, Q-TOF)
HPLC-
Chip/MS
interface
Protein 2D
2D HPLC-Chip/MS: 1
st
D SCX; 2
nd
D RP C18
Sample
1
2
3
4
5
6
Through hole
Waste
Nano electrospray
1
2
3
4
5
6
Open to bottom
Open to top
1
st
D SCX column
2
nd
D RP column
Nanoflow LC Pump
Low to high organic/H20 gradient
Prototype
2D Separation of 16 Protein Tryptic Digest Mixture using
BioSCX II Column in-Line with Protein ID Chip
Peptides were eluted from
SCX column using series of
salt injections
Comprehensive 2D-LC Mode: NPLC-RPLC-UV/MS
1
st
Dimension NPLC Condition:
Column: Diol Phase, 250 1mm, 5
um; Mobile Phase: 85/15 hexane/1-
butanol + 0.2% ethanolamine,
isocratic. Flow rate: 0.03 ml/min, 30
o
C, Sample: pharmaceutical
compounds B-mix, 254 nm.
2
nd
Dimension RPLC Condition:
Column: E. Merck Chromolith RP
18e 100 4.6mm. Mobile Phase: A:
H
2
O, B:AcCN, Gradient: 25%B for 3
s, to 50%B in 3 s, to 100%B in 15 s,
100%B for 9 s, back to 25%B in 3 s.
Flow rate: 5 ml/min (170 Bar), 30
o
C,
Sample: pharmaceutical compounds
B-mix, response time 0.1 s.
Injection of immiscible phase onto
RPLC column is possible without loss
of efficiency! And reproducibility is
found good also
min 10 20 30 40 50
mAU
0
50
100
150
200
250
300
350
B3
B6*
B7*
B6
B2
B1
B4
B5
B7
B4*
min 10 20 30 40 50
mAU
0
50
100
150
200
250
300
350
min 10 20 30 40 50
mAU
0
50
100
150
200
250
300
350
B3
B6*
B7*
B6
B2
B1
B4
B5
B7
B4*
min
0.2 0.4 0.6 0.8
mAU
-40
-20
0
20
40
60
80
100
B4
B1+B5
B2
B6
B7
B3
B7*
min
0.2 0.4 0.6 0.8
mAU
-40
-20
0
20
40
60
80
100
min
0.2 0.4 0.6 0.8
mAU
-40
-20
0
20
40
60
80
100
B4
B1+B5
B2
B6
B7
B3
B7*
1
st
D: NPLC method:
50 min run
2
nd
D: RPLC method:
1 min run
(courtesy of Yining Zhou, Pfizer)
Comprehensive 2D-LC-UV
Chromatogram
Low correlation (orthogonal dimensions)
More info than in either 1D separation
(courtesy of Yining Zhou, Pfizer)
GC Analysis in Complex Matrices
In complex sample matrices, there are often too many
overlapping compounds to allow resolution of the
compound(s) of interest, even with the highest resolution
columns available.
Must use some approach that gives selectivity
Selective sample prep like SPE
Selective stationary phase like Carbowax
Selective element detector like FPD, AED, NPD etc.
Spectral detector like GC-MS or GC-IR
Multidimensional (2-D) GC
Multidimensional (2-D) GC
Very old (>25 yrs) but powerful separation technique
Based on cutting peak(s) from one GC column onto
another with stationary phase of different selectivity
Compounds that co-elute with analyte on first
column separate from analyte on second column
Example pairs of complimentary phases:
DB-1 (non-polar) with Innowax (polar)
TCEP (very polar) with DB-1
DB-5 (low polarity) with Cyclosil (chiral)
Early 2-D GC Had Some Challenges
Early systems were difficult to use. 2-D
often implied 2- difficult
Column connections: inertness, dead volume
Balancing gas flows: complex flow system, needle valves
Retention time drift: wide cut windows, lower resolution
Inertness problems: loss of polar analytes
High cost:
Multiple GC ovens
Cryogenic focusing devices
Heart-Cutting 2-Dimensional GC
Overview
Cut
Deans Switch
7683
Auto-
sampler
6890N
GC
FID1
FID2
Column 1 Column 2
Why 2-D GC? Whats Changed?
Modern 2-D GC systems (e.g. Agilent 7890A) are
much easier to use:
Column connections are easier, zero dead volume, inert,
and reliable
Balancing gas flows done with EPC and Flow Calculator
Retention time drift greatly reduced with modern oven
and EPC
Inertness problems with switch hardware eliminated with
surface coatings (Sulfinert)
Because RT control is so tight and the switch is so quick,
multiple ovens and cryo focusing devices can often be
avoided
Original Design of Deans
Hardware for Agilent 6890
New Deans Switch Design
Photolithography and chem-milling technologies used to
produce a Gas Phase Micro-Fluidic* Deans Switch
4x less thermal mass than traditional hardware
* Capillary flow technology
9.78 psi
11.14 psi
FID A
S/S Inlet
FID B
PCM
Restrictor
6.54 mL/min
4.54 mL/min
<< 1mL/min
2mL/min
purge
restrictor
8.54 mL/min
Off
6.54 mL/min
BP<Benzene
Benzene&
unresolved
hydrocarbons
Column 1: HP-1
Column 2: Innowax
Sample BP>Benzene
Heart Cutting 2-D GC How It Works
Valve off, no heart cutting inject sample, initial separation on column 1
FID A
S/S Inlet
FID B
PCM
On
BP<Benzene
6.54 mL/min
4.54 mL/min
6.54 mL/min
purge
restrictor
2mL/min
8.54 mL/min
<< 1mL/min
9.78 psi 11.14 psi
BP>Benzene
Benzene&
unresolved
hydrocarbons
Restrictor
Column 1: HP-1
Column 2: Innowax
Heart Cutting 2-D GC How It Works
Valve on start heart cut from column 1 to column 2
Benzene&
unresolved
hydrocarbons
FID A
S/S Inlet
FID B
PCM
6.54 mL/min
4.54 mL/min
<< 1mL/min
9.78 psi 11.14 psi
2mL/min
purge
restrictor
8.54 mL/min
Off
6.54 mL/min
BP<Benzene
BP>Benzene
BP>Benzene
Hydrocarbon, Benzene
Restrictor
Column 1: HP-1
Column 2: Innowax
Heart Cutting 2-D GC How It Works
Valve off end heart cut, perform 2
nd
separation on column 2
Connections for Capillary Flow
Technology Switch
Simple, easy to make connectors
A single, special design metal ferrule
More inert than graphite/vespel
Does not leak at high oven temperature (>400
o
C)
Primary
Column
UDFS
Restrictor
Plate
Metal
Ferrule
Nut
Secondary
Column
Channel
4,6-Dimethyldibenzothiophene (4,6-
DMDBT) at Low ppm in Diesel with FID
Most difficult sulfur compound to hydro-treat
Used to monitor overall trace sulfur in diesel
Does not require Sulfur Chemiluminescence
Detector or Atomic Emission Detector
Old Method for 4,6-DMDBT in Diesel Fuel
0 5 10 15 20
C 179
S 181
426 ppm wt/wt total sulfur, run on GC-AED
4,6-
Dimethyldibenzothiophene
(162 ng/uL)
Diesel Fuel Deans Setup
Used to heart cut 4,6-DMDBT from HP-5 to Innowax
column
FID1
S/S Inlet
FID2
PCM
solenoid valve
restrictor
HP-5
Innowax
30m x 0.25 mm x 0.25
um
15m x 0.25 mm x 0.25
um
0.77m x .1 mm UDFS
4,6-DMDBT in Diesel Fuel
0 2 4 6 8 10 12 14 16 18
Cut window 6.40-6.65 min
4,6-DMDBT
165 ng/uL
(162 on AED)
4,6-DMDBT is completely resolved using FIDs.
Method good to low ppm level and comparable to AED.
HP-5
Innowax
Method Developers Tools
Calculator to correctly set flows and restrictor size
GC x GC Background Information
Why is the Application Important ?
Real-world samples can be too complex for sufficient
separation on one chromatographic phase
Target analysis is very complex samples
Excellent visualization of the sample
Powerful technique for hydrocarbon class determination
Issues with Current Solutions
Non-integrated hardware
Lack of good data reduction software
Many based on thermal modulation requiring large
quantities of cryogenic media
Comprehensive 2-D GC (GCxGC) Basics
Consists of four parts:
1. A primary column (conventional separation)
2. A modulator
3. A second column (very fast separation)
4. Fast detector
The modulator does two jobs:
1. It collects effluent from the
primary column
2. It transfers the collected
effluent (in whole) to the
secondary column
This process is repeated
approximately every 1.5
seconds, synchronized with
the start of data acquisition
GC x GC Chromatogram
The peak capacity of the system is the
product of the peak capacities of the two
columns: result a lot of separation power
Basic System Layout
7683
Auto-sampler
7890A GC
FID
Column 1
Column 2
Flow modulator
s/s inlet
PCM
Switching valve
modulated
2
nd
column
1
st
column
7683
Auto-sampler
7890A GC
FID
Column 1
Column 2 Column 2
s/s inlet
PCM
Switching valve
modulated
2
nd
column
1
st
column
Agilents Flow Modulator : Differential Flow
Using Design by Prof. John V. Seeley,
Oakland University
Modulation
Valve
FID
Split/Splitless
Inlet
Column 1 (25 30 M)
Column 2 (5M)
Collection
channel
Flow Modulator
H2
Flush Flow
direction
Collect Flow
direction
Flow modulator eliminates the need for cryo. Sample compression controlled
by flow ratios occurs in the collection loop and is quickly injected into the second
column, resulting in very narrow and tall peaks.
Capillary Flow Technology- Design
Photolithographic chemical milling for low dead volume
Diffusion bond two halves to form a single flow plate
Small, thin profile provides fast thermal response
Projection welded connections for leak tight fittings
Deactivation of all internal surfaces for inertness
Flow Modulation Device
Load or Collect Step
Modulation
Valve
FID
Split/Splitless
Inlet
Column 1 (25 30 M)
Column 2 (5M)
Collection
channel
Flow Modulator
H2
Collect Flow
direction
1 ml/min
20 ml/min
Load time must not be
longer than time to fill
collection channel
Work in constant flow mode
Inject Step
Modulation
Valve
FID
Split/Splitless
Inlet
Column 1 (25 30 M)
Column 2 (5M)
Collection
channel
Flow Modulator
H2
Inject Flow
direction
1 ml/min
20 ml/min
Collection channel is
quickly injected into
second column in about
100 milliseconds
20 ml/min
200 Hz
Typical times: Load 1.4 sec; Inject 0.12 sec
N-Butylbenzene
11.8 11.85 11.9 11.95
11.75 11.8 11.85 11.9 11.95
unmodulated
modulated
Kerosene Raw Data
Alkane, mono-aromatic, and di-aromatic
separated in 1.5 seconds
Zoom from 15.8 to 16 min
Heavy Gasoline Raw 2D Data
Note hydrocarbons being separated
in each 1.5 second modulation
GC X GC Modulation Basics
1.Acquisition
2. Transformation
1D-GC chromatogram
(at the end of the 1
st
column in blue)
9 modulations shown
Coelution of 3 compounds!
Raw 2D-GC
chromatograms
(at the end of the 2
nd
column)
Take slices of the co-eluting
peaks and inject quickly to
another column of different
selectivity
red peak elutes last now and green peak elutes first and all 3 completely separated!
1
2
3
4
5
6
7
8
9
GC X GC Visualization
2
nd
Dimension
chromatograms stacked
Reconstitute the peaks by
combining each of them from
each chromatogram
2D Image
1
st
Dimension
2
n
d
D
i
m
e
n
s
i
o
n

(
f
a
s
t

G
C
)
Chromatograms produced
by 8 modulation cycles
System Performance Check
Mixture: 7890A
1
2
1
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
1
2
1
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
1. Octane
2. Fluorobenzene
3. Propylbenzene
4. Bromo-2-fluorobenzene
5. Indane
6. Butylbenzene
7. Tetralin
8. Dodecane
9. Naphthalene
10. Tridecane
11. Tetradecane
12. Fluorobiphenyl
13. 1,3,5-Tributylbenzene
14. Acenaphthalene
15. Fluorene
16. Terphenyl
17. 2-Methyl anthracene
18. Eicosan
Mixture of wide boiling point and polarity
Flow Modulation:
(GC x GC) of Diesel Fuel: 7890A
GC x GC Image:
Showing the normal B.P. distribution (1
st
dimension)
Also shows the hydrocarbon class clusters
Consistent RT for alkanes in 1
st
dimension showing precise modulation
Comparable peak in 2
nd
dimension band shows minimum peak broadening
with flow modulation
Naphthalene
Toluene
p-xylene
o-xylene
C9
C12 C16
Alkanes
mono-Aromatics
di-Aromatics
Methyl-naphthalenes
Conclusions
Both 2D and comprehensive 2D gas and liquid
chromatography can be useful for handling complex
mixtures that cannot be adequately separated by 1D
chromatography.
In HPLC, phases of higher peak capacity and fast LC
columns can be combined to provide LCXLC
separations
In GC, more advanced capillary flow technology
hardware, rapid electronics, Deans calculator and
transformation software can combine to provide
powerful separation capability.
Acknowledgements
GC Work-Jim McCurry, Roger Firor,
and Bruce Quimby in Wilmington, DE
LC-Chip Work-Georges Gauthier and
LC Chip Team in Waldbronn and Santa
Clara