Developments in Multidimensional- And Comprehensive- Chromatography—Beyond Boxcars and Deans

8/12/2019 Developments in Multidimensional- And Comprehensive- ChromatographyBeyond Boxcars and Deans

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Developments in

Multidimensional- andComprehensive-

ChromatographyBeyondBoxcars and Deans

Pittcon 2007

Ronald E.Majors

Agilent TechnologiesWilmington, DE


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Early On-Line 2D GC Publication

Pages 32-35


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Early Instrument Designed for

2D GC


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Early 2D GC On-Line

Fractionation of Hydrocarbons


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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


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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


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Simplified Schematic of 2D MDC

LC Separation

10 Separation Mode: Size Exclusion Chromatography

20 Separation Mode: Affinity Chromatography


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1D GC Information Capacity


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Heartcut 2D GC Information

Capacity


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Comprehensive GCxGC

Information Capacity


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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

Chemistry2006, 25, 438-454.

Basic Concept of Comprehensive 2D Chromatography

(thanks to Pete Carr for loan of slide)


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Peak Capacity

The maximum number of peaks thatcan be resolved side-by-side into the

available separation space.

Assumes each dimension is totally orthogonal


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Peptide Map of BSA on Three SB-C18

Columns with Different Particle SizesGradient Time = 30 min

Temp. = 80C

238

Peak Capacity

391

540

Starting

Pressure

51

103

340

min4 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

[Pc using eqn. of Neue, J.Chromatogr. A, 1079(1-2), 153-161(2005)]


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Two Dimensional Chromatographic Techniques

GCSFC

SFCSFC

LCSFC

LC or GCSPE, SPME or

SBSE

GCLC

LCLC

GCGC

XGC or LCPreparative TLC

TLC (or HPTLC)TLC (or HPTLC)

On-LineOff-LineSecondaryTechnique

PrimaryTechnique


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Off-Line Multidimensional

Chromatography

On-Line MultidimensionalChromatography


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Coupled HPLC Modes

++ ++


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Basic Setup for an On-Line MD LC-LC


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Use of Coupled Column RAM-

RPC System for Analysis of Drugs in PlasmaColumn 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 C8Peaks:

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)


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Proteomics With 2D-LC/MS - Workflow

HPLC:

1st dimensionIon-exchange

2nd dimension

Reversed phase

1. cell disruption

2. prefractionation

3. solubilization

4. sample clean up

cell free proteins

(104 to 105)

tryptic digest

Peptides

(105 to 106)

Mass Spec

Data Analysis

Separation &

Isolation

identification


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Theoretical Resolving Power of 2D-LC/MS

e.g. reversed phase

Peak capacity nc = 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

nc

= 216

Total Peak capacity : 1. Dim * 2. Dim 15 * 216 = 3240

3. Dimension Mass Spec

10 Dimension

20 Dimension

(15 x 20 Dimension)

7 * 3240 = 23,000 peptides

(Wolters Et Al.)


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2-D HPLC: Cation Exchange andReversed 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 RPcolumn (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 peptidesare directly analyzed by MS/MS


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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


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Protein 2D

2D HPLC-Chip/MS: 1st D SCX; 2nd D RP C18

Sample

1 2

3

45

6

Through holeWaste

Nano electrospray1 2

3

45

6

Open to bottom

Open to top1st D SCX column

2nd D RP column

Nanoflow LC Pump

Low to high organic/H20 gradient

Prototype


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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 ofsalt injections


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Comprehensive 2D-LC Mode: NPLC-RPLC-UV/MS

1st Dimension NPLC Condition:

Column: Diol Phase, 250 1mm, 5um; Mobile Phase: 85/15 hexane/1-

butanol + 0.2% ethanolamine,

isocratic. Flow rate: 0.03 ml/min, 30oC, Sample: pharmaceutical

compounds B-mix, 254 nm.

2nd Dimension RPLC Condition:

Column: E. Merck Chromolith RP

18e 100 4.6mm. Mobile Phase: A:

H2O, B:AcCN, Gradient: 25%B for 3s, 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 oC,

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

min10 20 30 40 50

mA U

0

50

100

150

200

250

300

350

B3

B6* B7*

B6

B2

B1

B4

B5

B7B4*

min10 20 30 40 50

mA U

0

50

100

150

200

250

300

350

min10 20 30 40 50

mA U

0

50

100

150

200

250

300

350

B3

B6* B7*

B6

B2

B1

B4

B5

B7B4*

min0.2 0.4 0.6 0.8

mAU

-40

-20

0

20

40

60

80

100

B4

B1+B5

B2

B6

B7

B3B7*

min0.2 0.4 0.6 0.8

mAU

-40

-20

0

20

40

60

80

100

min0.2 0.4 0.6 0.8

mAU

-40

-20

0

20

40

60

80

100

B4

B1+B5

B2

B6

B7

B3B7*

1st D: NPLC method:

50 min run

2nd D: RPLC method:

1 min run

(courtesy of Yining Zhou, Pfizer)


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Comprehensive 2D-LC-UV

Chromatogram

Low correlation (orthogonal dimensions)

More info than in either 1D separation

(courtesy of Yining Zhou, Pfizer)


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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 resolutioncolumns 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


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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)


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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

H t C tti 2 Di i l GC


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Heart-Cutting 2-Dimensional GC

Overview

Cut

Deans Switch

7683Auto-sampler

6890NGC

FID1 FID2

Column 1 Column 2


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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


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Original Design of Deans

Hardware for Agilent 6890


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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

C GC


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9.78 psi11.14 psi

FID A

S/S Inlet

FID B

PCM

Restrictor

6.54 mL/min

4.54 mL/min


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FID A

S/S Inlet

FID B

PCM

On

BP


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FID A

S/S Inlet

FID B

PCM

6.54 mL/min

4.54 mL/min

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


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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 oC)

Primary

Column

UDFS

Restrictor

Plate

Metal

Ferrule

Nut

Secondary

Column

Channel


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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


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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)


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Diesel Fuel Deans Setup

Used to heart cut 4,6-DMDBT from HP-5 to Innowaxcolumn

FID1

S/S Inlet

FID2

PCM

solenoid valverestrictor

HP-5

Innowax

30m x 0.25 mm x 0.25um

15m x 0.25 mm x 0.25

um

0.77m x .1 mm UDFS


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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


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Method Developers Tools

Calculator to correctly set flows and restrictor size


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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


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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 theprimary column

2. It transfers the collected

effluent (in whole) to thesecondary column

This process is repeatedapproximately every 1.5seconds, 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


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Basic System Layout

7683

Auto-sampler

7890A GC

FID

Column 1 Column 2

Flow modulator

s/s inlet

PCM

Switching valve

modulated

2nd column

1st column

7683

Auto-sampler

7890A GC

FID

Column 1 Column 2Column 2

s/s inlet

PCM

Switching valve

modulated

2nd column

1st column

Agilents Flow Modulator : Differential Flow


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Agilent s 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)

Collectionchannel

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


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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


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Flow Modulation Device

C S


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Load or Collect Step

ModulationValve

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

I j S


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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 about100 milliseconds

20 ml/min

200 Hz

Typical times: Load 1.4 sec; Inject 0.12 sec


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N-Butylbenzene

11.8 11.85 11.9 11.9511.75 11.8 11.85 11.9 11.95

unmodulated modulated

Kerosene Raw Data


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Kerosene Raw DataAlkane, mono-aromatic, and di-aromatic

separated in 1.5 seconds

Zoom from 15.8 to 16 min


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Heavy Gasoline Raw 2D Data

Note hydrocarbons being separated

in each 1.5 second modulation

GC X GC M d l ti B i


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GC X GC Modulation Basics1.Acquisition

2. Transformation

1D-GC chromatogram(at the end of the 1st column in blue)

9 modulations shown

Coelution of 3 compounds!

Raw 2D-GC

chromatograms

(at the end of the 2nd column)

Take slices of the co-eluting

peaks and inject quickly toanother 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


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GC X GC Visualization2nd Dimension

chromatograms stackedReconstitute the peaks by

combining each of them from

each chromatogram

2D Image

1st Dimension

2nd

Dime

nsion(fa

stGC)

Chromatograms produced

by 8 modulation cycles

System Performance Check


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Syste e o a ce C ec

Mixture: 7890A

1

21 3

45

6

7

8

9

10 11

12

13

14

15

16

17

181

21 3

45

6

7

8

9

10 11

12

13

14

15

16

17

18

1. Octane

2. Fluorobenzene3. Propylbenzene

4. Bromo-2-fluorobenzene

5. Indane

6. Butylbenzene

7. Tetralin

8. Dodecane9. Naphthalene

10. Tridecane

11. Tetradecane12. Fluorobiphenyl

13. 1,3,5-Tributylbenzene

14. Acenaphthalene

15. Fluorene

16. Terphenyl

17. 2-Methyl anthracene18. Eicosan

Mixture of wide boiling point and polarity

Flow Modulation:


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(GC x GC) of Diesel Fuel: 7890A

GC x GC Image:

Showing the normal B.P. distribution (1st dimension)

Also shows the hydrocarbon class clusters

Consistent RT for alkanes in 1st dimension showing precise modulation

Comparable peak in 2nd 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


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Conclusions

Both 2D and comprehensive 2D gas and liquidchromatography can be useful for handling complex

mixtures that cannot be adequately separated by 1Dchromatography.

In HPLC, phases of higher peak capacity and fast LCcolumns can be combined to provide LCXLCseparations

In GC, more advanced capillary flow technologyhardware, rapid electronics, Deans calculator andtransformation software can combine to provide

powerful separation capability.


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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

Developments in Multidimensional- And Comprehensive- Chromatography—Beyond Boxcars and Deans

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