Evolution

of Peak Capacity in

Fluid-based Separation

Techniques

T

SUPER-

CRITICAL

FLUID

P

SOLID LIQUID

GAS

31.1°C

73.8 bar

Fluid-based

T

P

SOLID

GAS

31.1°C

73.8 bar

Fluid-based + CE

FLUID

21st Century Triangle

Throughput 10.000 N in < 3 min

Productivity 25.000 N in < 10 min

Resolution > 100.000 N

Robust

21st Century Triangle

Throughput

Productivity Resolution

Applicable in a

GLP-GMP Environment

Fundamentals

- Resolution

- Plate Number

- Peak Capacity

- Giddings Statements

- Importance of Mass Spectrometry

- …

Resolution

N 0.5 a - 1 k2 Rs = 4 a 1 + k2

tR = L/u (k + 1) = Nd/u (k + 1) N = L/xdp

X = 1.8 – 2.2 porous particles

X = 1.4 – 1.6 core shell particles

Re-evaluating

Resolution:

“The” Master Equation of

Separation Science -

Kinetic Plots

Fundamentals Van Deemter Plots - DP equation- Kinetic Plots (30°C)

-4

-3.5

-3

-2.5

-2

-1.5

-1

-0.5

0

3.5 4 4.5 5 5.5 6

Log

(H/u

0)

Log (N)

5 µm, 400 bar

1.8 µm, 400 bar

1.8 µm, 1200 bar

Tim

e

Efficiency

691,500 273,000 90,900

10

2.7

1.6

2.5

10

1.8

3.6

10

10,000 25,000 100,000

Fundamentals Kinetic Plots (30°and 80°C) – Dm and h

DP 1200 bar – 1.8 µm

-4

-3.5

-3

-2.5

-2

-1.5

-1

-0.5

0

3.5 4 4.5 5 5.5 6

Log

(H/u

0)

Log (N)30°C

80°C

273,000

10

10 10

342,000

4.2

5.2 5

Peak Capacity

nc ~ fcon x N 0.5 fcon < 2

tfirst tlast

Peak Capacity

nc ~ fcon x N 0.5 fcon < 2

tfirst tlast

𝑛𝑐 = 1 +𝑡𝑔𝑤

𝑛𝑐 = 1 +𝑡𝑙 − 𝑡𝑓𝑤

𝑛𝑐 ≅ 1 + 𝑁4𝑅𝑠

𝑡𝑙𝑡𝑓− 1

High Resolution 1D-LC K. Sandra et al. Journal of Separation Science, 30 (2007) 241

Human serum tryptic digest

(depleted for the 6 most abundant proteins)

Peak capacity: 1230

Time ( min )

50 100 150 200 250 300 350 400 450 Time ( min )

50 100 150 200 250 300 350 400 450

Run 1 Run 3

Run 2

Reproducibility/Profiling

- 3 samples treated in parallel

- 3 m x 2.1 mm RPLC separation – 60°C

- Fraction collection every 15 s (3072 fractions)

- MALDI target spotting (3072 spots)

- MALDI-MS analysis

Int Std Int Std Int Std Int Std

RP

LC

Rete

nti

on

Tim

e (

min

)

RP

LC

Rete

nti

on

Tim

e (

min

) R

PL

C R

ete

nti

on

Tim

e (

min

)

m/z m/z

m/z

Reproducibility/Profiling

High Resolution: Peptide map

,

Reversed-phase HPLC: Peptide map

Giddings Statements

… using the statistical theory of peak overlap …

J.M. Davis, J.C. Giddings, Anal. Chem. 55 (1983) 418

… in order to resolve 98% of the components, the peak capacity must

exceed the number of components by a factor of 100.

J.C. Giddings, J. Chromatogr. A 703 (1995) 3

… 100 peptides … peak capacity should be 100 x 100 = 10.000 …

or N ca. 100.000.000 plates !!!

… yes but … nowadays overruled by the selectivity of mass spectrometers

Dynamic MRM of 300 Pesticides 2 Transitions Each

September, 2009

2.1 mm x 100 mm C18 Eclipse PLUS 1.8 µm

Insight into the skin ceramides Profiling and Characterizing Skin Ceramides Using RPLC-QTOF-MS

R. t’Kindt, L. Jorge, E. Dumont, P. Couturon, F. David, P. Sandra, K. Sandra

Anal. Chem. 84 (2012) 403

Tackling the lipidome by LC-MS

• Chromatographic conditions: - C18 (2.1 mm ID x 100 mm L x 1.7 µm POROUS)

- A: 20 mM NH4-formate pH 5 and B: MeOH

- Flow rate: 0.5 mL/min

- Column temperature: 80C - Polaratherm

• Mass spectrometric conditions: - Q-TOF

- Jetstream ESI source (pos/neg)

- Extended dynamic range mode (105)

- Resolution: 10,000 (m/z 1000)

- Mass accuracy: < 2ppm

Data handling Construction of a skin ceramide map (feature plots)

500

Ma

ss (

Da

)

700

900

1300

Retention time (min)

30 40 50 60

Ester-linked

103

105

107

1100

Non-ester linked

701172 features (100% frequency, n=3)

Insight into the skin ceramides

• Identified species

400

500

600

700

800

900

1000

1100

1200

1300

10 20 30 40 50 60 70

mas

s

retention time (min)

CER[NDS]CER[ADS]CER[EODS]CER[NS]CER[AS]CER[OS]CER[EOS]CER[NP]CER[AP]CER[OP]CER[EOP]CER[NH]CER[AH]CER[OH]CER[EOH]CER[NT]

• CER [N(29) P(16)]

• CER [N(28) P(17)]

• CER [N(27) P(18)]

• CER [N(26) P(19)]

• CER [N(25) P(20)]

• CER [N(24) P(21)]

• CER [N(23) P(22)]

• CER [N(22) P(23)]

264 unique lipid

spots (100% frequency, n=3)

…. 1680 identified

Skeletal

isomerism

PAPER AND TLC

R. Consden, A. H. Gordon, and A. J. P. Martin. Biochem. J. 38, 224-232 (1944) R. E. Liesegang. Naturwissenschaften 31, 348 (1943) M. S. Shraiber. J. Chromatogr. 73, 367-370 (1972)

0.0

0.2

0.4

0.6

0.8

1.0

R f

R1 R2

Multidimensional separations

Multidimensional Chromatography • combination of different chromatographic separation

mechanisms

• inject the effluent or a part of the effluent of one

column to a second column, ideally with high

orthogonality

• increase total separation power

• improve peak capacity of separation

Different modes:

Off-line or sequential

Heart-cutting 2D-LC (LC-LC)

Comprehensive 2D-LC (LCxLC)

Coupling RPLC-HILIC !

Diagram of experimental set-up

x mL/min RPLC – 2 mm

I.D.

≥10 x x mL/min acetonitrile Isocratic pump

HILIC - 4.6 mm I.D.

UV ELSD

Gradient pump

Serial coupling of reversed-phase

and hydrophilic interaction liquid

chromatography to broaden the

elution window for the analysis of

genotoxic impurities

J. Chromatogr. A 1208 (2008) 90

Two highly efficient columns are used • Fraction collection or multiple injections: time-consuming • Complete development in the second dimension

First Column

Seco

nd

Co

lum

n

Off-line and sequential techniques

Shotgun – Multidimensional approach

Scatter Plot

2...

3...

3...

3...

3...

3...

4...

4...

4...

4...

1424 1426 1428 1430 1432 1434 1436 1438 1440 1442

RPLC

RPLC Direct

spotting MALDI-MS

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115 119

-20

-10

0

10

20

30

40

50

60

70

80

90

100

110

120

130

140

150

160mAU

min

33

32

31

30

29

28

27

26

25

24

23

22

21

20

19

18

17

16

15

14

13

12

11

10

9

8

7

6

5

4

3

2

1

WVL:214 nm

RPLC (15 cm x 75 µm ID x 3 µm dp Pepmap C18)

Sample

preparation SCX

Fraction

collection

m/z

SCX

40 µg Top 6 depleted human serum tryptic digest

Peak capacity: 104

30

40

1430

1440

min

m/z #

Two highly efficient columns are used • A selected fraction is transferred to the second column • Enrichment of selected fraction can be done by multiple injections

First Column

Seco

nd

Co

lum

n

Heart-cutting LC (LC-LC)

A highly efficient and a less efficient fast column are combined • Complete on-line development in the second dimension • High peak capacity

First Column

Seco

nd

Co

lum

n

Comprehensive LC or LC × LC

2n

d D

imen

sio

n

1st Dimension

Peak capacity first column nC1

Peak c

ap

acity

seco

nd

co

lum

n n

C2

2n

d D

imen

sio

n

1st Dimension

Peak capacity first column nC1

Peak c

ap

acity

seco

nd

co

lum

n n

C2

- ?

On the use of ionic liquid capillary columns for analysis of aromatic hydrocarbons

in low-boiling petrochemical products by one-dimensional and comprehensive

two-dimensional gas chromatography

J. Chromatogr. A 1301 (2013) 225

Ján Krupčík, Roman Gorovenko, Ivan Špánik, Ingrid Bočková, Pat Sandra, Daniel W. Armstrong

• Most widely used interface • 1 minute fraction collection 1 minute for analysis and regeneration

LC × LC Loop Interface

LC × LC

HILIC x IP-RP

di- to deca- oligonucleotides

JCA 1255 (2012) 237

Loop Interface with Parallel Second Dimension Columns

For higher peak capacity in second dimension: Extension with extra tools: binary pump, second 2nd dimension column, detector, and 2-position/10-port switching valve

Loop Interface with Parallel Second Dimension Columns

NPLC RPLC

Column Thermo Betasil Diol 2 x Zorbax SB C18

Dimensions 250 x 1 mm; 5 µm 50 x 4.6 mm; 3.5 µm

Mobile phase (A) n-Hexane

(B) Ethylacetate

(A) Water

(B) Acetonitrile

Gradient 0-40 min: 10% B; 50 min: 12% B; 60 min: 14% B; 80 min: 50% B; 90 min: 66% B

0-0.05 min: 0% B; 0.25 min: 45% B; 1.1 min: 80% B; 1.3 min: 90% B; 1.35-1.5 min: 100% B; 1.51-1.9 min: 0% B

Flow rate 30 µL/min 4 mL/min

Loop volumes 30 µL

Detection 315 nm @ 80 Hz

Loop Interface with Parallel Second Dimension Columns

0.3 min 1.1min

np = 437 np = 1095

NP

18

17

16

15

14 10

8

9

12

13

11

2 4

5

6

1 3

0 20 40 60 80

0.6

0.8

1.0

1.2

1.4

1.6

50 30 10 70

7

RP

min

min

50 70 20 40 60 80

0.6

18

16

15 17

14

11

12

13

10

8

1

6

3

9

3

5

2

0

0.4

0.5

10 30

0.7

7

RP

NP min

min

NP

18

17

16

15

14 10

8

9

12

13

11

2 4

5

6

1 3

0 20 40 60 80

0.6

0.8

1.0

1.2

1.4

1.6

50 30 10 70

7

RP

min

min

50 70 20 40 60 80

0.6

18

16

15 17

14

11

12

13

10

8

1

6

4

9

3

5

2

0

0.4

0.5

10 30

0.7

7

RP

NP min

min

Lemon oil extract

Loop Interface Loop Interface with Parallel Second Dimension Columns

np = 437 np = 1095

Comparison interfaces

BSA Tryptic digest

Coupling of 4 Halo columns at 45°C at pH 1.8

0 20 40 60 80 100 120 140

AU

-0.2

0

0.2

0.4

0.6

0.8

1

0 100 200 300

AU

-0.2

0

0.2

0.4

0.6

0.8

1

Gradient @ 0.3% B/min

np = 420

Gradient @ 0.1% B/min

np = 725

High Efficiency 1st dimension

BSA Tryptic Digest – Contour plot 150 mm x

0 50

0.4

0.5

0.6

0.7

0.8

Dim

en

sio

n 2

100 150 200 250 300

Dimension 1

min

min

1st dimension: 4 x HALO C18, 150 x 2.1 mm , 2.7 µm at 45°C

mobile phase at pH 1.8 and 100 µL/min

2nd dimension 2 parallel Zorbax 300 Extend C18, 50 x 4.6 mm, 3.5 µm

mobile phase at pH 10 and 4 mL/min Loop volume: 50 µL

725 x 35 = 25.375 !

SFC × RPLC interface

Low viscosity of SFs High efficient first dimension by coupling columns in series

RP

LC

0 10 20 30 40 50 60

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

RPLC min

min

18:0

16:0

14:0

20:1

18:1

20:2

18:2

16:1

14:1

16:2

20:3

18:3

16:3

20:4

18:4

16:4

22:4

22:5

20:5

22:6

15:0

17:0

Silver ion SFC

Silver ion SFC

FAMEs

Fish Oil

New 2D-LC-valve Single valve with fully

symmetric flow-paths and symmetric fill/flush-out

behavior

Allows co- and countercurrent flush-out of loops

Determination of Taxanes in Taxus sp.

Profiling of Citrus Oils and Determination of

Furocoumarins in Citrus Oils

Analysis of mAb Digests with the Agilent 1290

Infinity 2D-LC Solution

Application Notes – Agilent Technologies

LC × LC Taxanes

• Taxol® (Paclitaxel): Excellent antitumor activity (breast and lung cancer)

• 1971: first isolated from Taxus brevifolia

– Content is low > synthesis? – Too difficult thus not commercially interesting

• Semi-synthesis from more abundant precursors – 10-Deacetylbaccatin-III – Baccatin-III – Cephalomannine (Taxol B)

• Complex matrix

1 4

6 7

8

9

10 11

12 13

14

5 3 2

Peak Name 1 10-Deacetylbaccatin-III 2 Taxol side chain methylester 3 Baccatin III 4 7-Xylosyl-10-deacetyltaxol B 5 Taxinine M 6 7-Xylosyl-10-deacetyltaxol 7 7-Xylosyl-10-deacetyltaxol C 8 10-Deacetyltaxol 9 7-Xylosyltaxol 10 Cephalomannine (Taxol B) 11 7-epi-10-Deacetyltaxol 12 Paclitaxel (Taxol) 13 Taxol C 14 7-Epitaxol

UV spectrum MS spectrum APCI neg

(formate adduct)

Spectra of spot 12 (Taxol)

Sample

11 10

8

4

5

1

6

12

11 10

8

4

5

1

6

12

10 1

12

DAD, 228 nm

MS, negative, EIC

MS, negative, TIC

5

2D T

ime (s

) 10

15

5 10 15 20 25 1D Time (min) 30 35

5

10

15

20

5

10

15

20

Determination of Taxanes in Taxus sp.

Profiling of Citrus Oils and Determination of

Furocoumarins in Citrus Oils

Analysis of mAb Digests with the Agilent 1290

Infinity 2D-LC Solution

Application Notes – Agilent Technologies

1D RPLC 40 min

2D

RP

LC 2

0 s

UV at 330 nm !

Determination of Taxanes in Taxus sp.

Profiling of Citrus Oils and Determination of

Furocoumarins in Citrus Oils

Analysis of mAb Digests with the Agilent 1290

Infinity 2D-LC Solution

Application Notes – Agilent Technologies

Tryptic peptides Herceptin

Lig

ht

Ch

ain

B(1

-21

4)

Heavy C

hain

A(1

-449

)

Hc

Lc

EVQLVESGGGLVQPGGSLRLSCAAS

GFNIKDTYIHWVRQAPGKGLEW--

--NYKTTPPVLDSDGSFFLYSKLTVDKSRW

QQGNVFSCSVMHEALHNHYTQKSLSLSPG

• 62 identity peptides • Modifications • Incomplete and aspecific cleavages • ... > 100 peptides

SCX x RPLC - Herceptin

1D, SCX, 40 min

2D

, RP

LC, 3

0 s

T11

T5

T10

T58 T32

T7

T62 T15 T33

T57

T1

T21

T14

T45

(glycosylated)

T46

T9-T10 T40

T34 T56

T43 T41

T29 T54

T50

T22 T25 T30 T42

T31 T18 T26

T13

T3 T23

T8-T9

T2 T8

T59

T27

T38

T16

T24 T55

T6

T47

T19

T20

HILIC x RPLC - Herceptin

T62 T26

T30

T11 T32

T57

T10

T33

T46 T58

T25

T15 T40

T35 T7

T2

T13

T59

T34 T56 T22 T23

T29

T21

T1

T31 T54

T50

T43

T14

T45

T18

T3 T42

1D, RPLC, 55 min (13-68 min)

2D

, RP

LC, 2

7 s

RPLC x RPLC - Herceptin

1D, RPLC, 40 min

2D

, RP

LC, 2

4 s

T38

T45 (glycosylated)

T16 T24

T20

T42

T31

T19

T27

T59 T8

T14

T51 T47

T49

T21

T54

T29 T18

T13 T2

T41

T62 T1

T50

T3

T22 T23

T30 T25 T34

T57

T40

T33

T26

T58

T10

T1 T43

T32

T46

T11

T7

T6

T15

RPLC x RPLC - Method RPLC x RPLC

1D Bonus-RP, 150x2.1 mm, 3.5 µm @ 25°C, 80 µL/min A=10 mM NH4-bicarbonate pH 8.2 B=MeOH/ACN 50/50

2D (RPLC) Eclipse Plus C18, 50x4.6 mm, 3.5 µm @ 55°C, 3.5 mL/min DAD: A=0.1% phosphoric acid in water, B=ACN, Shifted gradient mode MS: A=0.1% formic acid in water, B=ACN, Shifted gradient mode

Modulation 40 µL loops

Detection DAD 214 nm MS: ESI positive, 8 spectra/s MS/MS: ESI positive, Data Dependent MS/MS, 8 spectra/s

Modifications (pH Stress)

DAD originator, not stressed

DAD originator, pH stress

T3

T3pH

T3

MS/MS, Deamidation T3 3 x10

0

0.5

1

1.5

2

2.5

3

3.5 852.93705

y 15

2+

916.96355

y 16

2+

611.64093

y 16

3+ 745.88418

y 13

2+

951.46840 553.28777

y 9

2+

1034.53588

y 8

717.31412 1105.56375

3 x10

0

0.5

1

1.5

2

2.5

3

853.42804

y 15

2+

611.97078

y 16

3+ 917.45770

y 16

2+

746.38118

658.66287

1105.57436

y 9 961.47753

1034.53353

y 8

554.29787

Counts vs. Mass-to-Charge (m/z) 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150

m/z 664,3369

[M+3H]3+

m/z 664,6649

[M+3H]3+

Injection 1 Injection 2 Injection 3 Injection 4 Injection 5

T11

T46

T3

T50

T13

T26

T42

T57

Repeatability LC x LC peptide map

Peak Volume RSD (%) 2D RT RSD (%) Peptide LC x LC LC x LC T3 1.51 0.05 T11 3.44 0.07 T13 1.06 0.06 T26 1.20 0.20 T42 4.15 0.04 T46 4.38 0.04 T50 3.16 0.07 T57 4.01 0.03

LC-CE

J.S. Mellors, J.M. Ramsey et al. Anal. Chem. 85 (2013) 4100

I

Love

2D-LC

Thanks to:

Koen Sandra

Gerd Vanhoenacker

Frank David

Isabelle François

Agilent Technologies

....

You for your attention