Comprehensive multidimensional

chromatography in natural product

analysis

aDepartment of Chemistry and Polymer Science, Stellenbosch UniversitybDepartment of Microbiology and Biochemistry, Hochschule Geisenheim University, Germany

bDepartment of Chemistry and Biochemistry, University of Namibia, NamibiadCentral Analytical Facility, Stellenbosch University

eDepartment of Biochemistry, Stellenbosch University

André de Villiersa, Gaalebalwe E. Ntlhokwea, Andreas G.J. Tredouxa,

Jochen Vestnerb, Kathithileni M. Kalilic, Chandré M. Willemsea, Pieter

Ventera, Maria A. Standerd,e

2

Introduction

• GC and HPLC find extensive use in this field.

The complexity of these samples dictates the need for

improvement of chromatographic performance.

• Need for improved analytical methods for natural product

characterisation informed by recognition of the complexity of these

samples and their derived commodities

• Requirement for fast, detailed and accurate chemical composition

data in support of research, product development and quality

control

• Important developments in MS have had a significant impact on

analytical performance (selectivity of high resolution systems,

tandem MS sensitivity)

• Performance of MS relies partially on chromatographic separation

(isomers, matrix effects…)

The comprehensive 2D approach

Limitations of 1-dimensional chromatography

Peak capacity (nc) for capillary GC, gradient HPLC limited to a few

hundreds. tg

To resolve 98% of n randomly distributed components, nc should be n

100

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

A. Felinger, Data Analysis and Signal Processing in Chromatography, Elsevier, 1998

3

G(L)C×G(L)C: For comprehensive combination of orthogonal separations, nc,2D is

multiplicative

1st dimension1st dimension

2n

dd

ime

ns

ion

Karger, Snyder and Horvath, An Introduction

to Separation Science, 1973

The challenges: An example

Wulf et al, Am. J. Enol. Vitic. 29

1978 42

de Villiers et al, J. Chromatogr. A 1218 2011

4660

Willemse et al, Anal. Chem. 87 2015 12006

Time10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00

%

0

100

3

46 7

9-12

15

16

19

24+25273133

40

44

48

53

5564

65

76

84

87

88

104

108

102

110

114116

125

127

133

135 13780

HPLC: 45 min, 21

compounds, nc ~ 45UHPLC: 1.7 mm, 90 min, 101

compounds nc ~ 290

HILICxRP-LC: 450 min, 94

compounds, nc ~ 900

Analysis of red wine pigments by HPLC

Analysis of wine volatiles by GC

Marais et al., S. Afr. J. Enol. Vitic. 2

1981 19

Packed GC: 53 min, 44 comounds,

nc ~ 92Ortega et al., J. Chromatogr. A 923 2001

205

Capillary GC: 65 min, 37

compounds, nc ~ 300Weldegergis et al., Anal. Chim. Acta

701 2011 98

GC×GC: 84 min, 276

compounds, nc ~ 6500

5

Two-dimensional chromatography

Necessary criteria for a two-dimensional system to be called

comprehensive:

(1) every part of the sample is subjected to two different separations;

(2) equal percentages of all sample components pass through both

columns and eventually reach the detector;

(3) the separation obtained in the first dimension is essentially maintained.

P. Schoenmakers, P. Marriott, J. Beens, LC-GC Europe 6

2003 1

Can be performed in one of two

modes:

(1) Heart-cutting mode: more information

on selected parts of the sample

(2) Comprehensive: complete sample

subjected to separation in both

dimensions

Comprehensive 2D chromatography:

considerations

Sampling rate

Sufficiently short fraction collection times (sampling rates) are

essential to maintain the first dimension separation.

..each first dimension peak should be

sampled at least three times …..

Murphy, Schure and Foley. 1998.

Anal. Chem. 70 (1998) 1585

1D tR

Orthogonality

The degree of mutually exclusive information that may be obtained

from each separation dimension

In GC: different stationary

phases

In LC: different modes,

stationary mobile phases

Experimental: HS-SPME-GCxGC-TOF-MS

Part I:

GC×GC in natural

product analysis

8

GC×GC: Principles and instrumentation

1D column

2D columnmodulator

injector

oven

detector1D column: conventional2D column: short, high speed

Modulation: 2-10 seconds

Detection: > 100 Hz (FID, TOF-

MS)

Orthogonal separations obtained

using apolar and (mid)-polar

columns

sign

al

time

sign

al

Data representation

Animation courtesy of T. Gorecki

9

Modulation in GC×GC

Thermal modulation

• heater based

• cryogenic

Flow modulationValve modulation

U.J. Meinert et al., Ang. Chem. Int. Ed. 51 (2012)

10460

P.J. Marriott et al., TrAC 34 (2012) 1F.C. Wang, J. Chromatogr. A 1188 (2008) 274

Analysis of wine volatiles: the challenges

Analysis of wine volatiles essential, but complicated:

• More than 800 volatiles identified in wine

J.S. Camara et al., Anal. Chim. Acta 563 (2006) 188–197.

% - mg/L mg/L mg/L – ng/L

Isoamyl

alcohol

estersacids phenols terpenes pyrazines

ng/L mg/L g/L - mg/L

• Wide range of concentrations (<ng/L - % level)

• Diverse physicochemical properties (polarity, volatility, reactivity)

alcohols

Isobutyl

methoxypyrazine

b-ionone

m-cresol

Isoamyl

acetate

Hexanoic

acid

Wine aroma is primarily determined by volatile content

11

GC×GC-TOF-MS in wine analysis

48 compounds identified (standards); 155 compounds tentatively

identified (deconvoluted mass spectra, 1D retention index)

Sample prep:

Columns:

Modulation:

Detection:

HS-SPME (CAR/PDMS),

10 mL, 10min, s/less inj.1D: 30 m x 0.25 mm VF-12D 1.5 m x 0.25 mm Wax

Single stage cryogenic (N2)

4 sec

TOF-MS, 50 Hz

isopropyl

methoxypyrazine

sec-butyl

methoxypyrazineOverloading/wraparound of high-level polar compounds

J. Harynuk, T. Gorecki, J. Chromatrogr. A 1019 (2006) 53B.T. Weldegergis et al., Food Chem. 129 (2011) 188

12

Secondary fermentation with lactic acid bacteria (Oenococcus oeni)

Primary goal is deacidification:

MLF also results in:

• Improvement of “mouth feel”, better microbial stability and

changes in aroma

A detailed description of the effect of MLF on wine volatile

composition is still lacking, in part due to analytical

limitations.

+

L-Malic acid L-Lactic acid

Investigating the effect of malolactic

fermentation using GC×GC

13

Investigating the effect of malolactic

fermentation using GC×GC

linalool

2-nonanol

nonanal

Ethyl heptanoatePropyl

hexanoate

IBMP

Fenchone

HS-SPME (CAR/PDMS),

5 mL, 5/30 min, s/less inj.

1D: 30 m x 0.25 mm VF-12D 1.5 m x 0.25 mm Wax

Single stage cryogenic (N2)

4 sec

TOF-MS, 100 Hz

Sample prep:

Columns:

Modulation:

Detection:

J. Vestner et al., J. Agric Food Chem. 59 (2011) 12732

Statistical analysis

• Univariate analysis (ANOVA):

Levels of 43 compounds showed significant difference

• Multivariate analysis (principal component analysis, PCA)

Differentiation between all starter cultures and control

Differentiation of control wines from MLF wines

• hexanal (85)

• hexanol (61)

green, grassy,

herbaceous

aromas

Alcohols &

carbonyls

S. Malherbe et al., J. Ind. Microbiol. Biotechnol. 39 (2012) 477

Control

MLF Culture C

MLF Culture O

MLF Culture V

Decrease in these

properties in MLF

wines has been

reported

: PCA

Culture V lower

levels of:

• esters & alcohols

• diacetyl

(buttery aroma)

Statistical analysis: PCA

Culture C

Culture O

Culture V

Differentiation between MLF wines produced with different

starter cultures

In agreement

with sensory

data for these

wines.

S. Malherbe et al., J. Ind. Microbiol. Biotechnol. 39 (2012) 477

16

Focusing on minor wine volatiles

Sample prep:

Columns:

Modulation:

Detection:

SDVB SPE, rinse 50% MeOH

Elute DCM, s/less inj. 1D: 30 m x 0.25 mm Rxi2D 1.5 m x 0.25 mm Rtx

Dual stage cryogenic (N2)

4 sec

TOF-MS, 100 Hz

62 compounds

identified

(standards); 214

tentatively

identified (MS, 1D

RI)

B. T. Weldegergis et al., Anal. Chim. Acta 701 (2011)98

17

1 p-Cymen-8-ol2 a-Terpineol3 7-Methyl-3-methylene-6-octen-1-ol4 cis-Carveol5 b-Cyclocitral6 b-Citronellol7 Nerol8 b-Damascenone9 2-Propenal, 3-(2,6,6-trimethyl-1-

cyclohexen-1-yl)-10 trans-b-Ionone11 b-Farnesene12 (Z,E)-a-Farnesene13 (Z,E)-Nerolidol14 3-Hydroxy-b-damascone15 3-Oxo-a-ionol16 a-Bisabolol17 Blumenol C18 Nerolidyl acetate19 4-Oxo-7,8-dihydro-b-ionol20 (2E,6E)-Farnesol

16

1517

1820

1

2

3

4

67

9

11 1213

14

8

P2

2

36

7

9

13

14

15 17

204

19

811

P1

2

36

7

5 913

14

1517

1820

10

19

CS

CS

C

S

P1 & P2

P1 & P2

P2

P2

P2

Pinotage 1

Pinotage I1

Cabernet S.

Focusing on minor wine volatiles

18

GC×GC in indigenous tea analysis:

Honeybush tea

Honeybush is an indigenous South African

herbal tea, produced from Cyclopia species

(Fam. Fabaceae, tribe Podalrieae), endemic to

Western and Eastern Cape provinces.

Numerous Cyclopia species are used for the

production

+ Production: Blending depends on production yield and

availability of the species. Sensory profiles of individual

species not always taken into account.

E. Joubert et al, S.A. J. Bot. 77 2011 887

E. Joubert et al, J. Ethnopharmaco. 119 2008 376

Currently C. intermedia, C. subternata, and C.

genistoides are used for the production.

C. maculata and two others (C. sessiliflora, C.

longifolia) are to be included in production.

Single-stage thermal modulation

1D column

0.25 mm i.d.

Restrictor

0.05 mm i.d.

2D column

0.25/15 mm i.d.Coated trap

+/-

GCxGC trap

Restrictor

1-D (PDMS) column

2-D (WAX)

column

Passive

cooling fins

Ceramic

pads

19

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80

Tem

pe

ratu

re (

°C)

Time (min)

oven temp. (°C)Ceramic pads temp.(°C)

Carrier gas flow

Trap placed

between

ceramic pads

connected to

passive

cooling fins

20

GC×GC-FID analysis of honeybush tea

1D Retention time (min)

2D

Rete

nti

on

tim

e (

sec)

10 20 30 40 50 60

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

57

3

21

77

19

418

37

1

30

28

27

35

7

1670

69

9

49

68

73

13

45

7184

5811

32

262478

2325

33

47

18

75

7264

22.50 22.55 22.60 22.65 22.70 22.75 22.80 22.85 22.90

5.00 î 10 4

1.0 0î 10 5

1.5 0î 10 5

2.00 î 10 5

2.5 0î 10 5

3.00 î 10 5

3.5 0î 10 5

4.00 î 10 5

4.50î 10 5

5.00 î 10 5

U2

U1

U1+23U3

23

23

24

24

1D Retention time (min)

2D

Rete

ntion

tim

e (sec)

22.2 22.4 22.6 22.8 23 23.2 23.4 23.6 23.8

0.8

1

1.2

1.4

1.6

1.8

2

2.2

2.4

2.6

24

2523

unknown 3

unknown 1

unknown 2

1D column: HP-5MS, 0.25mm i.d., 30m L

2D column: StabilWax 0.15mm i.d., 0.6m L

Modulation: 5 seconds

Flow: 24 cm/s 1D Ramp: 3oC/min

GEN4

GEN6

GEN8

GENB5

GENC5

MAC2

MAC12MAC17

MACB5

MACC5

SUB2

SUB4

SUB8

SUBB5

SUBC5-3

-2

-1

0

1

2

3

4

5

6

-5 -4 -3 -2 -1 0 1 2 3 4 5 6

F2 (

29

.86

%)

F1 (34.23 %)

Observations (axes F1 and F2: 64.08 %)

Statistical analysis: PCA scoresComparison with sensory data

21

Se

ns

ory

da

ta

GEN4

GEN6GEN8

GENB5

GENC5

MAC2

MAC12MAC17

MACB5

MACC5

SUB2

SUB4

SUB8

SUBB5

SUBC5

A_FynbosFloral

A_RoseGeranium

A_RosePerfume

A_LemonLemongrassA_ApricotApricotJa

m

A_CookedApple

A_Woody

A_Pine

A_Fruitysweet

A_Caramel

A_Honey

A_FynbosSweet

A_CassiaCinnamon

A_Walnut

A_Coconut

A_Dusty

A_BurntCaramel

A_Rottingplantwater

A_HayDriedGrass

A_Greengrass

A_Cookedvegetable

-1.5

-1

-0.5

0

0.5

1

1.5

2

2.5

-2.5 -2 -1.5 -1 -0.5 0 0.5 1 1.5 2 2.5

F2 (2

9.86

%)

F1 (34.23 %)

Biplot (axes F1 and F2: 64.08 %)

22

GC×GC-TOF-MS for detailed qualitative

analysis of honeybush tea

3

4

5

9

10

12

14

18

20

23

64

35

41

28

29

30

31

33

36

38

47

48

48 69

35

56

57

58

61

62

71

63

73

76

78

92

101

146

150

132

156

165

/166

173

172

246

215

264

190

197

202

160

205

224

217

225

235

232

243

236

248

81

83

8782

91

88

90

108

105

24

111

114

119

125

127

127

135

140

136

162

171

176

181

188

208

209212

214216

219

237

244

247

250255

258

260

265

272

283

285301

308

309

284

Sample prep:

Columns:

Modulation:

Detection:

HS-SPME (PDMS/DVB),

10 mL, 30min, split inj.1D: 30 m x 0.25 mm Rxi2D 0.8 m x 0.25 mm Wax

dual stage cryogenic (N2)

5 sec

TOF-MS, 100 Hz

108 reference standards

Data obtained in Alvaro Viljoen’s lab (TUT)

23

Deconvolution

90Linalool

93

92

95

98

1835

3835

0840

2840

4840

1845

0

250000

500000

750000

1e+006

1.25e+006

1.5e+006

1.75e+006

2e+006

1st Time (s)

2nd Time (s) 71 109 119

90

92

93

90

40 60 80 100 120 140 160 180 200

500

1000 71

82

55

93 107

Peak True - sample "Mac-C5-R3 A2:1", peak 181, at 840 , 2.540 sec , sec

40 60 80 100 120 140 160 180 200

500

1000 71

82

43

55 91

Library Hit - similarity 931, "Hotrienol"

40 60 80 100 120 140 160 180 200

500

1000 109

81

53 124 65 91

Peak True - sample "Mac-C5-R3 A2:1", peak 180, at 840 , 2.420 sec , sec

40 60 80 100 120 140 160 180 200

500

1000 109

43 81

53 124 65 91

Library Hit - similarity 963, "6-Methyl-3,5-heptadiene-2-one"

GC×GC-TOF-MS: Identification

24

C.genistoides

C. subternata

C. maculata

GC×GC-TOF-MS: Comparison of

species

232 compounds

218 compounds

182 compounds

Total of 274 identified

161 new compounds

196

202

205

209

212

224

232

233

237

240

248 253

259

269 277

284

258

C. maculata

C. subternata

196

202

205

209

212

232

233

237

240

248 253

254

258

259

269277

284224

C. genistoides

25

196

202

205

209

212

224

232

233

237

240

248253

259

269277

284

258

200

cinnamaldehyde uniquely

detected in C. maculata -

cassia cinnamon aroma.

Eugenol associated with spicy

aroma was detected in all the 3

species but differ in

abundance.

O

OH

GC×GC-TOF-MS: Comparison of

species

K.A. Theron et al., Food Res. Int. 66 2014 12-22

C.M. Guedes et al. Eur. Food Res. Technol. 219 2004 460-464

Likely responsible

for the

characteristic

cassia/cinnamon

aroma of this

species

GEN4

GEN6GEN8

GENB5

GENC5

MAC2

MAC12MAC17

MACB5

MACC5

SUB2

SUB4

SUB8

SUBB5

SUBC5

A_FynbosFloral

A_RoseGeranium

A_RosePerfume

A_LemonLemongrassA_ApricotApricotJa

m

A_CookedApple

A_Woody

A_Pine

A_Fruitysweet

A_Caramel

A_Honey

A_FynbosSweet

A_CassiaCinnamon

A_Walnut

A_Coconut

A_Dusty

A_BurntCaramel

A_Rottingplantwater

A_HayDriedGrass

A_Greengrass

A_Cookedvegetable

-1.5

-1

-0.5

0

0.5

1

1.5

2

2.5

-2.5 -2 -1.5 -1 -0.5 0 0.5 1 1.5 2 2.5

F2 (2

9.86

%)

F1 (34.23 %)

Biplot (axes F1 and F2: 64.08 %)

26

GC×GC combined with high speed high

resolution TOF-MS: Rooibos tea

Sample prep:

Columns:

Modulation:

Detection:

HS-SPME (PDMS/DVB),

10 mL, 30min, split inj.1D: 30 m x 0.25 mm Rxi2D 2 m x 0.25 mm Wax

dual stage cryogenic (N2)

4 sec

TOF-MS, 120 Hz

acetophenone

1-octen-1-ol

1-octanol

C8H8O

Mass accuracy: -0.14ppm

Match factor: 94%

Experimental: HS-SPME-GCxGC-TOF-MS

Part II:

LC×LC in natural

product analysis

28

Considerations in LC×LC method development

For LC×LC, injection volume in the second dimension is

the product of 1D flow rate and sampling time

Injection of fractions in strong 2D eluents limited by injection band broadening

Compatibility determined by mobile phase characteristics

Separation modes dictated by sample properties

For polymers, SEC×RP-LC, SEC×LCCC etc

280 nmA

1a

1b

2a

2b

3a

3b

3c

4b

4a

3d

6a+b

4d

9

7a+b

4c

5b

5a 5c

6c

7c

8

For neutral organic compounds, especially HILIC×RP-LC has

proven effective

Ionisable compounds: IEX×RP-LC, RP-LC×RP-LC varying pH, etc.

S. Keuchkarian et al, J. Chromatogr. A 1119 2003 20

29

Practical aspects: Hyphenation mode

i. Off-line LC×LC

ii. On-line LC×LC

iii. Stop-flow LC×LC

Two dimensions operated

independently

Second dimension separation

completed during fraction

collection

First dimension flow stopped

during second dimension

separation

30

LC×LC considerations: Orthogonality

Orthogonality.

• HILIC and RP-LC characterised by high degree of orthogonality

• Different separation mechanisms required to exploit 2-dimensional

space.

*Lui, et al, Anal. Chem. 67 1995 3840 *Davis et al, Anal. Chem. 80 2008

8122

*Semard et al, J. Chromatogr. A 1217 2010

5449

0 0 0 0 1 0 0 0

0 0 0 0 0 0 0 0

1 0 2 0 1 1 0 0

0 0 2 0 3 1 0 0

1 0 2 0 2 4 4 0

0 0 1 0 3 1 4 0

0 0 0 0 1 2 5 3

0 0 1 0 1 1 5 70.2 0.4 0.6 0.8 1

0

0.2

0.4

0.6

0.8

1

“1. every part of the sample is subjected to two different separations”

M. Gilar et al, Anal. Chem. 77 2005 6426

surface coverage metrics preferred• Means to quantify orthogonality problematic;

31

First-dimension sampling time, 1ts

Determines 1st-dimension undersampling :

X. Li, D. Stoll, P.W. Carr, Anal. Chem. 81 2009 845

The volume of each fraction: Because HILIC eluents are strong solvents in

RP-LC, the maximum injection volume in the

second dimension (2Vinj) is limited

LC×LC considerations: Sampling time, 1ts

And the total analysis time (and second

dimension cycle time in on-line LC×LC)

“3. the separation (resolution) in the first dimension is essentially

maintained”

32 M. Schure, Anal. Chem. 71 1999 1645

LC×LC considerations: Dilution

“2. equal percentages (either 100% or lower) of all sample components

pass through both columns and eventually reach the detector”

2Dmodulation1D

is limited

Only a portion of each

fraction is injected:

if

Minimising dilution in HILIC×RP-LC

Off-line HILIC×RP-LC:• Minimise fraction volume: 1D i.d., flow rate

• Inject only portion of fraction (2Vinj < Vfrac; SR!)

• Evaporate and re-dissolve in weak solvent

• Dilute in weak solvent

Stop-flow and on-line HILIC×RP-LC:• Minimise fraction volume: 1D i.d., flow rate

• Inject only portion of fraction using a splitter (2Vinj < Vfrac; SR!)

• Use traps to trap analytes and remove mobile phase

• Dilute in weak solvent prior to 2D column

Loop 2

V

1

V

1

Waste

2nd D pump

2nd D column

Detector

Waste

1st D pump

1st D column

Loop 1

Trap 1

V

1

V

1

Waste

2nd D pump

Waste

Detector

Trap 2

1st D pump

1st D column

Make-up flow

2nd D column

Flow splitter

Weak diluent

34

Method development in LC×LC

• Complex relationship between several parameters complicates method development

• Method development procedures differ between separation modes; hyphenation mode

Some seminal papers on LC×LC method development

P. Schoenmakers et al, J. Chromatogr. A 1120 2006

282

Input: Minimum 1D i.d., DPmax, kinetic performance using

Poppe plots. Injection band broadening and dilution

minimised. SEC×RP-LC

F. Bedani et al, J. Chromatogr. A 1133 2006 126

Input: 2D separation, 1D conditions varied to

provide required nc,2D. Stop-flow SEC×RP-

LC

Vivo-Truyols et al, Anal. Chem. 82 2010 8525

Pareto optimisation of nc,2D, ttot and dilution.

Gradient and isocratic, variable column formats,

DPmax.

Horvath et al, J. Chromatogr. A 1216 2009

2511

Target required nc,2D. 1nc, 1ts,

2nc optimised.

SCX×RP-LC

35

HILIC×RP-LC method optimisation strategy

nc,1D of individual separations

Off-line and stop-flow:

• Fixed 1-D conditions (1tg=50min, 1F=50mL/min)

On-line:

• Fixed 1-D conditions (1tg=100min, 1F=25mL/min)

• Repeated for various 1ts, ttot calculated for each

• For fixed 1ts: 2tc varied, nc,2D calculated, corrected for undersampling &

orthogonality• Stop-flow: nc,1D corrected for 1st dimension broadening

• 2tc varied, nc,2D calculated, corrected for undersampling & orthogonality, ttot

calculated for each

K.M. Kalili, A. de Villiers J. Chromatogr. A 1289 2013

58; 69

0

100

200

300

0 20 40 60

(min)

C18, 50×4.6 mm, 1.8 mm dp, 1.5 mL/min, 50°C Diol, 150×1 mm, 5 mm dp, 25-50 mL/min

36

Optimisation results

ii. On-line LC×LC

Second dimension separation

completed during fraction

collection

SR=62:1

0

100

200

300

400

500

600

0 5 10 15 200

0.2

0.4

0.6

0.8

1

1.2

1/b

2tc (min)

1/b

25 mL/min

n’c,2D

Optimal 1ts = 3

min

n’c2,D = 570

SR=124:1

SR=37:1

SR=24:1

• Higher 2nd dimension

peak capacity at longer 2tc

• 1st dimension under-

sampling dominates for long 2tc

37

Wine tannins

37

Complexity increases with degree of polymerisation (DP)

"polymeric procyanidins.....cannot be separated through conventional high-

performance liquid chromatography”.

*Tourino, et al, Rapid Comm. Mass Spectrom. 22 2008 3489

Tannins: proanthocyanidins, oligomeric phenolics comprising several

constituent units and a large number of isomeric

structures

On-line HILIC×RP-LC-UV-FL-

Q-TOF-MS analysis of grape seed tannins

1D: 80 min gradient, 25 mL/min2D: 2 min cycle time, 1.5 mL/min

1ts: 2 min

11

HILIC pump(UPLC )System 1

HILIC column(Devlosil Diol column:

250 x 1 mm, i.d., 5 mm dp)

RP column(KinetexC18 column:

50 x 4.6 mm, i.d., 1.8

UPLC-PDA DetectorSystem 2

RP pump(UPLC)

System 2

Waste

Loop 1

Loop 2

Q-TOF-MS DetectorSystem 3

Flow

splitter

Flow splitter

UPLCFLD DetectorSystem 2

, mm dp)

fluorescenc

eBase peak

chromatogram

Retention Time - HILIC [min]

Rete

ntion T

ime -

RP

LC

[m

in]

20 40 60 80 100

0.5

1

1.5

1

2

3

4

8

6

7

9

1011

16

17

1819

2122

32 3334

3536

37

38

4647

49

50

61,62

63

64

65

66

48

20

Retention Time - HILIC [min]

Rete

ntion T

ime -

RP

LC

[m

in]

20 40 60 80 100

0.6

0.8

1

1.2

1.4

1.6

1

2

†

†

5

3

4

†

6

7

9

10

11

12

13

14

15

1617

18

1920

2122

23

24

25

27

28

29

30

3132

3334

45

3536

37

38

39

40

42

43

44

26

41

8

†

†

FL MS

HILIC×RP-LC-UV-FL-Q-TOF-MS

analysis of grape seed tannins

A B

DC

F=10 F=50 F=100 F=20

F=10 F=5 F=2

n = 0-3

Extracted ions: gallated procyanidins

41

Wine anthocyanins and their alteration

O+

OH

HO

OH

OCH3

O

OCH3

O

OHOHHO

OH

O+HO

OH

O O

OHOHHO

OH

OH

O

HO

OH

OH

HO

OH

OCH3

OCH3

R1R2

direct

condensationRemy et al, J. Sci. Food

Agric. 80 2000 745

Proanthocyanins

(tannins)

Acetadehyde-

mediated

condensationRivas-Gonzalo et al, J. Agric.

Food Chem. 431995 14444

Proanthocyanins

(tannins)

O+HO

OH

O O

OHOHHO

OH

OH

OCH3

OCH3O OH

HO

HOOH

HO

R1

R2

O+HO

OH

O O

OHOHHO

OH

O

OCH3

O

HO

OH

OH

HO

OH

OCH3

R1R2

O+HO

OH

O O

OHOHHO

OH

O

OCH3

O

HO

OH

OH

HO

OH

OCH3

R1R2

A+-F

configuratio

n

Vinylflavano

l adductsFrancia-Aricha et

al, J. Agric. Food

Chem. 45 1997

2262

vinylflavanols

(tannins)

Vitisin A

derivativesBakker et al, Phytochem.

44 1997 1375

Pyruvic acid,

cycloaddition

O+

O

HO

OH

O O

OHOHHO

OH

OCH3

OCH3

COOH

Vinylphenol

derivativesSchwarz et al, J. Agric. Food

Chem. 51 2003 3682

O+

O

HO

OH

O O

OHOHHO

OH

OH

R1

OCH3

OCH3

R2

Hydroxy-

cinnamic

acids

0

2

4

6

8

1 0

400

800

1200

1600

0

10

20

30

40

50

17:1 (223min)

24:1 (155min)

1 mL/min

2 mL/min

6 mL/min

10 mL/min

7:1 (225min)

n* c

,2D

Split ratio

2tc (m

in)

4:1 (416min)

Minimising dilution: experimental conditions

Effect of 1D flow rate, gradient time:

0 2 4 6 8 10

500

750

1000

1250

1500

1750

2000

2250

n* c

,2D

2tc(min)

1tg = 376 min

2mL/min

1tg = 217 min

1tg = 156 min

0 2 4 6 8 10

500

750

1000

1250

1500

1750

2000

n* c

,2D

2tc(min)

6mL/min

1tg = 217 min

1tg = 126 min

1tg = 72 min

Lower 1F and longer 1tg provide:

• Higher n'c,2D

• lower split ratios

Solvent implications: required split ratio’s:

2tc,opt

C.M. Willemse et al., Anal. Chem. 87 2015 12006

11

HILIC pump

(CapLC)

System 1

HILIC column

(BEH Amide column: 150 × 1 mm, i.d., 1.7 mm dp)

RP column(Kinetex C18 column:

50 × 2.1 mm, i.d., 1.3 mm dp)

UPLC-PDA Detector

System 2

RP pump

(UPLC)

System 2

Waste

Loop 1

Loop 2

Q-TOF-MS

DetectorSystem 3

Flow

splitter

BEH Amide, 150×1 mm, 1.7 mm dp, 1-10 mL/min

Kinetex C18, 50×4.6 mm, 1.3 mm dp, 0.86

mL/min, 60°C

On-line HILIC×RP-LC analysis of red wine pigments

1D: 315 min gradient, 1 mL/min2D: 2 min cycle time, 0.86 mL/min

1ts: 2 min

To avoid the need for flow splitting:1F = 1 mL/min, 315 min gradient

1ts = 2tc = 2 min

SR =1

Sacrifice 2D performance for maximum sensitivity

Cap-LC

PDAQ-TOF

On-line HILIC×RP-LC-UV-

Q-TOF-MS analysis of red wine pigments

On-line HILIC×RP-LC analysis if wine pigments

6-year old red wine

Red wine

HILIC (n1) 22

RP-LC (n2) 63

orthogonalit

y

0.68

β 1.02

n'c,2D 890

Comparison of pigment profiles

1-year old 6-year old

2013 and 2008 Pinotage wines produced using standard winemaking

procedures at the Welgevallen experimental cellar (Stellenbosch) using grapes

from the same vineyard. 94 compounds tentatively identified based on 1tR, 2tR,accurate

mass and fragmentation information

m/z200 300 400 500 600

%

0

100493.1350

479.0844

303.0509

494.1381

495.1422

MS

m/z200 250 300 350 400 450 500

%

0

100331.0815

315.0496

287.0562

332.0853

333.0889493.1278

MSE

On-line HILIC×RP-LC: 2008 Pinotage

Retention time - HILIC (min)

Re

ten

tio

n t

ime -

RP

-LC

(m

in)

100 150 200 250 300 350 400 4500.4

0.6

0.8

1.0

1.2

1.4

26

265

34

73 72

7748

39

10

20

58

7469

9

92

1314

91 90

88 84 82

85 64

68

6267

61

93

66

m/z 781

2008 Pinotage

266.0 267.0 268.0 269.0 270.0

%

100

26

2728

26

27

28

26

27

28

26

27

28

120 140 160 180 200 220 240 260 2800.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

Re

ten

tio

n t

ime

-R

P-L

C (

min

)

Retention time - HILIC (min)

(b)

120 140 160 180 200 220 240 260 2800.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

Re

ten

tio

n t

ime

-R

P-L

C (

min

)

Retention time - HILIC (min)

(a)

Comparison of pigment profiles

□ Anthocyanin-glucosides (15)

○ Anthocyanin-di-glucosides (5)

Δ Oligomeric Anthocyanins (5)

□ Anthocyanin-tannin

adducts (15)

○ Acetaldehyde-mediated

tannin adducts (16)

Δ Vinylflavanol

condensation products

(12)□ Oxovitisins (3)

○ Pyruvic acid derivatives (6)

Δ Acetaldehyde derivatives

(4)□ Anthocyanin-catechol

derivatives (5)

○ Anthocyanin-phenol derivatives

(3)

Δ Anthocyanin-guaicol derivatives

(4)

◊ Anthocyanin-syringol derivative

(1)

2013

2008

Retention Time - HILIC [min]

Rete

ntion T

ime -

RP

LC

[m

in]

20 40 60 80 100

0.6

0.8

1

1.2

1.4

1.6

1

2

†

†

5

3

4

†

6

7

9

10

11

12

13

14

15

1617

18

1920

2122

23

24

25

27

28

29

30

3132

3334

45

3536

37

38

39

40

42

43

44

26

41

8

†

†

48

The future: some perspective…

PC10G3

PC11G3

PC12G396.0236 96.0214

PC11G2

PC11G4

PC11G5

50.6716 50.6715

50.6704

50.6632

PC11G1

49

The future: some perspective…

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

0 2 4 6 8 10 12 14 16 18

Co

un

t p

er s

eco

nd

Retention time (min)

Chestnut - Reverse phase

RP-LC-MS analysis of tara hydrolysable tannins

1

2

x x

x x x

x x x x x x

3 5

4

11 14

6 8 10

12-2

7

9

13

9.1

16

17.1-2

17.2-2

19.3-2

17-2

20

18-2

18.1-2

19-2

19.1-2

21.1-2

21-2

22-2

23-2

24

19.2-2

26

272-

262-

282-

252-

22.1-2

292-

302-

30

22.2-2

312-

32

34

33

26.12-

35

27.1-2

28.12-

26.1

35.12-

36-2

37 37.1

34.1

6692-

x

39

909-2 m/z, RT 6.56

39

26.12-

34.2

38.32

x x

41 422-

42

34.3

x

44

43.1

34.4

34.5

34.6

45.10 37.2

46

47 82.4

48.2

47.22-

43.2

47.32-

51

522-

82.32-

53

48.

48.6

48.7

61

59.1

59.2

562-

552-

67

682-

83

65.3 65.2

69.2

75

79

Retention time (min)

Dri

ft t

ime

(m

se

c)

50

Conclusions

The complexity of natural products demands powerful analytical methods

for detailed chemical analysis.

• Chromatographic separation complementary to MS in this endeavor.

GC×GC with cryogenic modulations by now an established technique

offering higher resolving power, better sensitivity and improved

compound identification

• Developments in cheaper modulation strategies on-going

• Combination with HR-TOFMS extends the capability of the technique

• Current focus on improved data analysis techniques

LC×LC not established, but instrumental and theoretical developments

support fast-growing field

• Choice of orthogonal methods essential; implications for ease of

hyphenation

• Method development still complex

• Hyphenation to various detectors extends applicability

• HILIC×RP-LC in particular a powerful methods for flavonoid analysis

51

Acknowledgements

Post-graduate students: Elizabeth Ntlhokwe, Martha Kalili, Chandré Willemse, Jochen

Vestner, Pieter Venter, Magriet Muller

Collaborators: Andreas Tredoux, Marietjie Stander, Elizabeth Joubert, Dalene de Beer

(ARC), Nina Muller (Food Science), Wessel du Toit (DVO Stellenbosch), Harald Pash

(Stellenbosch), Frederic Lynen (Gent), Taduesz Gorecki (Waterloo)

Stellenbosch University, SASOL, Restek, Agilent Technologies, NRF, IFS, Restek, TWAS

for financial support

Dr. Peter Gorst-Allman (LecoAfrica), Prof. Alvaro Viljoen, Guy Kamatou (TUT)