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Journal of Chromatography A, 1340 (2014) 90–98

Contents lists available at ScienceDirect

Journal of Chromatography A
journal homepage: www.elsevier.com/locate/chroma

Development of on-line comprehensive two-dimensional liquid


chromatography method for the separation of biomass compounds
Agnès Le Masle a , David Angot b , Cécile Gouin a , Amélie D’Attoma b , Jérémie Ponthus a ,
Alain Quignard a , Sabine Heinisch b,∗
a
IFP Energies Nouvelles–Lyon, Direction Physique et Analyse, Rond-Point de l’échangeur de Solaize, BP3–69360 Solaize France
b
Institut des Sciences Analytiques, CNRS UMR 5280, Université de Lyon, 5 rue de la Doua, 69100 Villeurbanne France

a r t i c l e i n f o a b s t r a c t

Article history: Comprehensive on-line two-dimensional liquid chromatography (on-line LC × LC) was used for the char-
Received 1 November 2013 acterization of bio-oils obtained by fast pyrolysis of lignocellulosic biomass. The resulting bio-oil contains
Received in revised form 10 February 2014 a large number of oxygenated chemical families and must therefore be upgraded before being used
Accepted 6 March 2014
as drop-in transportation biofuels. The good knowledge of its complex composition is essential for
Available online 13 March 2014
optimizing the mandatory bio-oil upgrading process to biofuels, thereby requiring powerful separa-
tion techniques designed to be hyphenated to mass spectrometry detection (LC × LC–MS). In this study,
Keywords:
reversed phase conditions were optimized in both dimensions for the RPLC × RPLC separation of the aque-
Practical peak capacity
Two-dimensional liquid chromatography
ous fraction of bio-oils. The first step of method development consisted in searching for a suitable set of
Biomass RP-conditions via the screening of a large number of RP-systems (made up of different stationary phases
Bio-oil and/or mobile phases and/or temperature). The practical peak capacity and the degree of orthogonality
RPLC × RPLC were calculated for a sample of 38 representative compounds, both descriptors having been considered
as selection criterion. Two different couplings were chosen and evaluated for the RPLC × RPLC separation
of the 38 representative compounds. The best of both, in terms of real practical peak capacity, was further
successfully applied to the separation of the aqueous phase of a partially dehydroxygenated bio-oil.
© 2014 Elsevier B.V. All rights reserved.

1. Introduction compared to fuel oil [3]. It cannot be directly used as basis for
biofuel manufacturing, nor be directly co-refined with petroleum
Because of the rarefaction of fossil fuels and CO2 overall bal- products because of its immiscibility. For these reasons, the bio-
ance, the incorporation of upgraded fast pyrolysis bio-oils in fuels oil has to be upgraded to reduce the proportion of oxygenated
is nowadays considered as a promising route to produce second molecules in order to be stabilized and to be treated with con-
generation (2G) biofuels that could be refined in a petroleum refin- ventional type refining processes and with petroleum feedstocks.
ery [1]. Pyrolysis is one of the main thermochemical processes used Different ways are possible to realize this upgrading step (gasi-
within the 200–550 ◦ C range to convert ligno-cellulosic biomass [2] fication to CO + H2 , zeolite cracking or hydroconversion [2]). The
in absence of oxygen. At high thermal level (450–550 ◦ C) and with optimization of the process scheme and operating conditions is
a very short residence time (a second or a few seconds) of the pro- complicated without a strong knowledge of the bio-oil’s compo-
duced vapors in the reactor in order to maximize the liquid yield sition. For acquiring this knowledge, and taking into account the
after condensation of the vapors, the process is called fast pyrolysis complexity of this product, multidimensional techniques have to be
and about 65–75 weight percent (wt%) of the resulted product is considered. Two-dimensional liquid chromatography is expected
a liquid [2]. This liquid, also called pyrolysis oil or bio-oil, is very to be complementary to two-dimensional gas chromatography
rich in oxygen compounds, corrosive because of the presence of since compounds with high polarities, low volatilities and poor
acids, not miscible with hydrocarbons and not stable at tempera- thermal stabilities are present in bio-oils, such as cracked sugars or
tures above 100–150 ◦ C. Furthermore, it has a low heating value molecules issued from the polycondensation of some oxygenated
compounds. In addition, the presence of compounds with very high
molecular weight was also demonstrated, probably proceeding the
∗ Corresponding author. Tel.: +33 4 37 42 35 51; fax: +33 4 72449319. condensation of molecules issued from the thermal cracking of
E-mail address: sabine.heinisch@univ-lyon1.fr (S. Heinisch). natural polymers (hemicelluloses, cellulose and lignin).

http://dx.doi.org/10.1016/j.chroma.2014.03.020
0021-9673/© 2014 Elsevier B.V. All rights reserved.
A. Le Masle et al. / J. Chromatogr. A 1340 (2014) 90–98 91

Over the last ten years, the interest for on-line comprehensive retention data obtained from 28 different sets of RP-conditions
two-dimensional liquid chromatography (LC × LC) has been grow- were used to determine the degree of orthogonality and the prac-
ing. On-line LC × LC has the advantage of dramatically decreasing tical sample peak capacity of each possible combination, hence
the analysis time compared to off-line LC × LC while avoiding any allowing the comparison of the separation power between 378 pos-
risk of contamination [4]. Different on-line LC × LC systems have sible 2D systems. In light of the results, two RPLC × RPLC systems
been developed, taking advantage of the great variety of sep- were selected and tested for the RPLC × RPLC separation of our rep-
aration techniques that are available in liquid chromatography resentative sample. The optimized 2D-conditions were applied to
including Reversed Phase Liquid Chromatography (RPLC), Normal the analysis of the aqueous phase of a bio-oil which is probably the
Phase Liquid Chromatography (NPLC), Steric Exclusion Chromatog- first 2D-separation in this field.
raphy (SEC), Ion Exchange Chromatography (IEC) and Hydrophilic
Interaction Liquid Chromatography (HILIC) [5]. Bio-oils are mainly
2. Material and methods
composed of small neutral compounds which makes SEC and IEC
less attractive. A great variety of monophenols, furans, ketones,
2.1. Chemicals and samples preparation
aldehydes were identified in bio-oils thanks to GC/MS analyses
[6–10] which makes the combination of NPLC and RPLC potentially
Organic solvents (acetonitrile, methanol) were HPLC grade from
very attractive. It was shown that the orthogonality between NPLC
SDS (Peypin, France). Water was obtained from an Elga water purifi-
and RPLC was very good for the analysis of polymers or pharmaceu-
cation system (Veolia Waters STI, Le Plessis Robinson, France). 0.1%
tical samples for example but the combination of both systems in
of formic acid (Sigma–Aldrich, Steinheim, Germany) was added to
2DLC was not easy to set up due to solvent incompatibility between
water (solvent A) and to the organic solvent (solvent B) to maintain
NPLC and RPLC mobile phases [11–13]. The same issue was recently
acidic solutes in a neutral state.
highlighted for the separation of peptides in on-line RPLC × HILIC
The 38 compounds selected as representative molecules of a
[14]. In addition, separations in NPLC are usually much less effi-
bio-oil were obtained from Sigma–Aldrich (Steinheim, Germany).
cient than those in RPLC making RPLC a key technique for LC × LC
Their physico-chemical properties are listed in Table 1. For the pre-
separations. RPLC × RPLC is therefore often preferred despite the
liminary search of 2D-systems, each compound was diluted in a
lower degree of orthogonality which is usually observed between
mixture of water/acetonitrile (87.5/12.5 v/v) so that the final con-
RP-systems. Attractive results were obtained in RPLC × RPLC by just
centration was 50 ppm. All solutions were stored at 5 ◦ C before
changing, between the two dimensions, either the organic solvent
injection. For the 2D-separations, a mixture of the 38 compounds
for the analysis of a mixture of phenolic compounds [15] or the pH
was prepared in the same way.
for charged compounds [14,16]. As a result, RPLC × RPLC was suc-
The real sample used in this study was a bio-oil obtained by
cessfully used for the analysis of small oxygenated molecules, such
conversion of red oak, white oak, ash and maple that has then
as phenolic compounds [17–20].
been partially dehydroxygenated. It was converted and upgraded
The development of a RPLC × RPLC method must start with
by IFP Energies Nouvelles (Solaize, France). The upgrading process
the search for two different RP-systems (stationary phase/mobile
consisted in hydro-reforming followed by hydrotreatment of the
phase/temperature) capable of providing a good degree of orthog-
bio-oil. The resulting product at the end of the hydro-reforming
onality [21] and a sufficient practical sample peak capacity [22].
step was spontaneously separated into two distinct liquid phases,
This step requires the choice of a limited number of representative
organic and aqueous. The aqueous phase was collected, centrifuged
compounds and the further collection of their retention data from
and filtered before being analyzed by two-dimensional liquid chro-
one-dimensional separations. The practical sample peak capacity
matography without any further dilution.
was first introduced by Liu et al. [23] in order to obtain a more accu-
rate estimation of the number of resolvable compounds. Whereas
the theoretical sample peak capacity is the product of the calcu- 3. Instruments
lated sample peak capacities in each dimension (n2D = n1 × n2 ), the
practical sample peak capacity represents the actual sample peak Two different 2D instruments were used to perform 2D analyses.
capacity that can be achieved for a given sample in view of the The first two-dimensional instrument was used for the LC × LC
restricted available separation space. analysis of the 38 representative compounds. Both dimensions
Full orthogonality is an easily understood concept. It is achieved consisted in a Prominence HPLC system (Shimadzu, Kyoto, Japan).
when retention data are not at all correlated which often fails to The first dimension includes two LC-20AD pumps, a SIL-30A
occur in 2DLC. The degree of orthogonality aims at assessing to what autosampler with a 5 ␮L sample loop, a CTO-20A column oven
extent the selectivity between two considered systems is different. with a maximum temperature of 85 ◦ C and a SPD-20A UV detec-
Different methods have been proposed to evaluate the degree of tor equipped with a 210 nL flow-cell. The instrument control
orthogonality. Some of them are based on linear regression of reten- was performed by LC Solution software Version 1.23. The wave-
tion data [24,25] or more complex chemometric tools [26], while length was set at 254 nm with a sampling rate of 5 Hz. The
others make use of a geometrical approach [23,27,28]. Recently, extra-column volume was estimated at 12 ␮L and the measured
new descriptors have been proposed to evaluate both degree of dwell volume was approximately 100 ␮L. The second dimension
orthogonality and practical peak capacity between two RP-systems includes two LC-20ADXR pumps with a maximum back-pressure
differing in at least one parameter [29]. Both descriptors were of 660 bar, a CTO-30A column oven with a maximum temperature
proved to be quite complementary, the degree of orthogonality of 150 ◦ C and a SPD-M20A PDA detector with a 2.5 ␮L flow-cell.
being directly related to the concept of selectivity in gradient elu- The instrument control was performed by LCMS Solution soft-
tion and the practical sample peak capacity assessing the expected ware Version 3.60.361. The detection wavelengths were ranged
overall separation power of a given RPLC × RPLC system. This within 250–275 nm with a sampling rate of 40 Hz. The extra-
method was successfully applied to the separation of charged com- column volume of this second dimension was estimated at 25 ␮L
pounds [14]. In the present study, we applied the same method to and the measured dwell volume was 150 ␮L. The two dimensions
the selection of two one-dimensional systems capable of generat- were connected by a 10-ports high pressure two-positions switch-
ing a significant practical peak capacity for the 2D-separation of ing valve (IDEX Health & Science, Oak Harbor, United States). It
biomass compounds. 38 representative molecules were selected was equipped with two 20 ␮L sample loops. Flow splitting was
according to the current knowledge of bio-oil composition. Their realized between first and second dimensions by using a zero
92 A. Le Masle et al. / J. Chromatogr. A 1340 (2014) 90–98

Table 1
Physical properties of bio-oil representative compounds.

Compound Chemical family Molecular formula MW (g/mol) log P

1 acrylic acid acid C3 H4 O2 72.06 0.149


2 benzoic acid acid C7 H6 O2 122.12 1.559
3 trans-cinnamic acid acid C9 H8 O2 148.16 1.212
4 pyruvalehyde aldehyde C3 H4 O2 72.06 −0.414
5 2,5-hexanedione ketone C6 H10 O2 114.14 −0.091
6 ␣-angelica lactone lactone C5 H6 O2 98.10 0.236
7 2-phenylethanol alcohol C8 H10 O 122.16 1.504
8 1-indanol alcohol C9 H10 O 134.18 1.180
9 Furfural furan C5 H4 O2 96.08 0.712
10 2-(hydroxymethyl)furan furan C5 H6 O2 98.10 0.213
11 5-methylfurfural furan C6 H6 O2 110.11 0.670
12 Phenol phenol C6 H6 O 94.11 1.540
13 o-cresol phenol C7 H8 O 108.14 1.962
14 m-cresol phenol C7 H8 O 108.14 2.043
15 p-cresol phenol C7 H8 O 108.14 2.066
16 2,4-dimethylphenol phenol C8 H10 O 122.16 2.496
17 2,4,6-trimethylphenol phenol C9 H12 O 136.19 2.935
18 1,2,4-benzenetriol phenol C6 H6 O3 126.11 −0.163
19 Catechol phenol C6 H6 O2 110.11 0.844
20 4-ethylphenol phenol C8 H10 O 122.16 2.576
21 ␣-hydroxycumene phenol C9 H12 O 136.19 2.861
22 Eugenol guaiacol C10 H12 O2 164.20 2.403
23 Isoeugenol guaiacol C10 H12 O2 164.20 3.081
24 Guaiacol guaiacol C7 H8 O2 124.14 1.341
25 Apocynin guaiacol C9 H10 O3 166.17 1.511
26 ferulic acid guaiacol C10 H10 O4 194.18 0.963
27 Syringaldehyde syringol C9 H10 O4 182.17 1.304
28 Syringol syringol C8 H10 O3 154.16 1.218
29 1-indanone enone C9 H8 O 132.16 1.419
30 cyclopent-2-en-1-one enone C5 H6 O 82.10 −0.114
31 3,4-dimethylacetophenone enone C10 H12 O 148.20 2.029
32 ␣-methylstyrene hydrocarbon C9 H10 118.18 3.364
33 naphthalene hydrocarbon C10 H8 128.17 3.310
34 m-cymene hydrocarbon C10 H14 134.22 4.205
35 Anisol aromatic ether C7 H8 O 108.14 2.170
36 2,5-dimethoxytoluene aromatic ether C9 H12 O2 152.19 2.559
37 kaempferol aromatic ether C15 H10 O6 286.24 2.685
38 2-hydroxyquinoline nitrogen compound C9 H7 NO 145.16 1.174

dead volume T-connector. It was connected to the ten ports valve Houston, USA). It was equipped with two 20 ␮L sample loops. 2D-
with a polyether ether ketone tube (60 mm × 0.05 mm) achiev- data were processed using calculation tools developed under Excel
ing 1/5 as split ratio. 2D-data were processed using 2DChrom 2007 and Matlab V7.12.0635.
software (V2.2.9, Thermo Fisher Scientific, Waltham, United The preliminary screening of possible 2D-systems was per-
States). formed with the Acquity UPLC instrument (see above) with
The second two-dimensional instrument was used to analyze wavelengths ranging from 220 nm to 300 nm.
the bio-oil sample. The first dimension consisted in a LC Pack-
ings ultimate chromatograph (Dionex, Amsterdam, Netherlands). It
includes two micro-pumps with minimum and maximum delivery
flow-rates of 1 and 500 ␮L/min, respectively, an autosampler with
a 5 ␮L sample loop, a column oven with a maximum temperature 4. Columns and gradient conditions
of 70 ◦ C and a UV detector with a 160 nL flow-cell. The wave-length
was set at 254 nm with a sampling rate of 2 Hz. The instrument A large variety of reversed-phase columns was studied. Most
control was performed by Chromeleon software. The extra-column of them were silica-based, but stationary phases based on porous
volume was estimated at 10 ␮L and the measured dwell volume graphitic porous carbon (Hypercarb column), zirconia and organic
was 110 ␮L. The second dimension was an Acquity UPLC chromato- polymers were also considered. Conditions used for the prelimi-
graph (Waters, Milford, United States) consisted in a high-pressure nary screening of chromatographic systems are listed in Table 2.
binary solvent manager with a maximum delivery flow-rate of The 38 selected compounds were injected separately in each con-
2 mL/min, an autosampler with a 5 ␮L sample loop, a column oven sidered system using linear gradient conditions (from 1% to 99%
with a maximum temperature of 90 ◦ C and a diode array detector of organic solvent). Optimum flow-rates being dependent on both
with a 500 nL flow-cell. It was controlled by Empower software. The column dimensions and column temperature, they were chosen so
wavelength was set at 254 nm with a sampling rate of 40 Hz. The that linear velocities were close to Van Deemter optima. For each
maximum backpressure is 1000 bar for flow-rates up to 1 mL/min, compound, two gradient runs were performed with two different
800 bar for flow-rates up to 1.5 mL/min and 630 bar for flow-rates normalized gradient slopes (5% and 1.5%, the normalized gradi-
up to 2 mL/min. The mobile phase was pre-heated thanks to a ent slope being given by S = C × t0 /tG , where C is the gradient
coiled stainless steel tube (50 cm × 0.127 mm) located between the composition range, t0 , the column dead time and tG , the gradient
injection valve and the column inlet. The extra-column volume of time). The normalized gradient slope was kept constant by chang-
this UPLC system was 15 ␮L and the measured dwell volume was ing the gradient time when the column dead time varied so that
120 ␮L. The two dimensions were connected by a ten ports high the average retention factor at elution was the same for all studied
pressure two-positions switching valve (Vici Valco Instruments, RP-systems.
A. Le Masle et al. / J. Chromatogr. A 1340 (2014) 90–98 93

5. Calculations

Organic solvent

ACN, MeOH
ACN, MeOH

ACN, MeOH

ACN, MeOH

ACN, MeOH

ACN, MeOH
ACN, MeOH

ACN, MeOH
Two preliminary gradient runs allowed to determine the S value
for each compound in a given RP-system, S being the slope of the
ACN relationship between the logarithm of the retention factor and the
solvent composition [30]. The calculations were performed using
Osiris software (V4.2, Datalys, Grenoble, France) [31]. The composi-
tion at elution (Ce ) was calculated using the following relationship:
Temperature (◦ C)

 
Cf − Ci
30 ◦ C, 65 ◦ C

30 ◦ C
30 ◦ C, 65 ◦ C

30 ◦ C, 90 ◦ C

30 ◦ C, 90 ◦ C

90 ◦ C

90 ◦ C
30 ◦ C, 90 ◦ C

30 ◦ C, 90 ◦ C
Ce = Ci + × (tR − tD − t0 ) (1)
tG

where tR is the retention time, Ci and Cf , the initial and final gra-
dient compositions respectively and tD the instrument dwell time.
Experimental sample peak capacities were calculated according to
dP c (␮m)

tn − t1
n=
2.6

1.7

1.7

(2)
5
5

3
3

5
w
tn and t1 , being the retention times of the most and the least
LC b (cm)

retained compound, respectively, and w, the average 4 peak width


(13.4% of peak height). The sample peak capacity in the first dimen-
15

15
15

10

5
5

15

sion was normalized to both 5 cm column length and 5 ␮m particle


size for a more suitable comparison of the different stationary
dC a (mm)

phases. Similarly, the sample peak capacity in the second dimen-


sion was normalized to 5 cm column length. According to the
4.6

4.6
4.6

2.1

2.1

4.6
4.6

2.1

procedure previously developed [29], the degree of orthogonality


3

Polystyrene/divinylbenzene

and the practical sample peak capacity were calculated for each
combination of RP-systems using
Carbon-coated zirconia
C18-bonded silica with

Polybutadiene-coated
Cyanopropyl-bonded
(core-shell particles)

phenylhexyl groups
Pentafluorophenyl-

Od = S1 S2 Ce,1 Ce,2


Silica bonded with

(3)
C18-bonded silica
Stationary phase

Porous graphitic
embedded polar

n∗2D
bonded silica

= n1 (1 − ) × n1 n2 (4)
chemistry

zirconia
groups

carbon

where Ce is the difference in composition at elution between the


silica

most and the least retained compounds, subscripts “1” and “2” refer
to first and second dimension, respectively, and , is the correction
factor, corresponding to the ratio of the effective to the theoretical
Polymer Laboratories

retention area. Its calculation is detailed in [29].


According to the recommendations of Davis et al. [32], the
Thermo Fischer
Manufacturer

undersampling was taken into account for evaluating the effective


Phenomenex

sample peak capacity in the first dimension (n1,effective ) by means


Supelco
Supelco
Waters

Waters

Waters
Agilent

of
n1
n1,effective =   2
(5)
1 + 0.21 6⁄
Abbreviation

where  is the sampling rate of the 2D-separation (number of frac-


KinPFP

PhHex
AcqSh

ZCarb

tions sent to D2 per 6 peak width in D1). The sampling rate was
ZPBD

PLRP
Hyp
XBr
CN

determined by the average 6 peak width divided by the sampling


time, t2 (w6 /t2 ).
The practical sample peak capacity in the second dimension was
Acquity CSH Phenyl-Hexyl

Discovery Zr-Carbon C18


Acquity BEH Shield RP18
Zorbax StableBound-CN

evaluated according to

n∗2 = 1 +  × (n2 − 1)
Columns characteristics and operating conditions.

(6)
Discovery Zr-PBD
Column name

XBridge C18

The dilution factor, Df was calculated in each dimension with


Kinetex PFP

Hypercarb


PLRP-S

v 2
Df = (7)
Vi

 v being the peak standard deviation in volume units and Vi , the


Column internal diameter.

injected volume.
Silica-based columns

The conditions of 2D-separations (presented in Table 5 and in


Particles diameter.
Non silica-based

Table 6 for the bio-oil) were optimized using a home-made calcu-


Column length.

lation tool aiming at maximizing the effective peak capacity while


columns

decreasing both analysis time (first dimension analysis time) and


dilution factor. This calculation tool developed under Excel 2007
Table 2

(Microsoft, Redmont, USA) made use of the classical relationships


for LC and LC × LC. The column plate number was determined from
b
a

c
94 A. Le Masle et al. / J. Chromatogr. A 1340 (2014) 90–98

Table 3 especially in case of high molecular weight molecules present in


Average S values (with organic solvent content expressed as volumetric fraction) for
the water non-soluble fraction (pyrolytic lignin). Information on
the 30 studied RP-systems.
detailed molecular composition of the water soluble fraction (our
Organic solvent Acetonitrile Methanol study) is easier although limited to liquid extraction and GC/MS
Temperature 30 ◦ C 65 ◦ C 30 ◦ C 65 ◦ C analysis which can provide a rough idea of its composition. This
complex mixture consists in very different compounds, includ-
Xbridge C18 5.7 5.2 4.0 4.0
Kinetex PFP 3.9 4.1 ing alcohols, esters, carboxylic acids, aldehydes, ketones, furans,
ZorbaxSDB-CN 3.2 3.1 3.0 3.1 lignin monomers stemming from the conversion of lignin (phenols,
Acquity Shield-C18 4.0 4.2 3.3 3.2 methoxy or di-methoxy phenols, anisoles, guaiacols, syringols,
Acquity CSH-phenyl hexyl 4.9 4.6 3.4 4.1 catechols, . . .), molecules stemming from the conversion of holo-
cellulose (anhydrosugars, oligo/polysaccharides, aliphatic hydroxy
Organic solvent Acetonitrile Methanol
acids. . .) and unsaturated molecules such as olefins and nitrogen
Temperature 30 ◦ C 90 ◦ C 30 ◦ C 90 ◦ C compounds [33]. We chose 38 representative compounds (Table 1)
Non silica-based columns among the different expected chemical families with the aim of
Hypercab 3.8 3.3 comparing 28 different RP-systems. The 28 studied RP-systems
Discovery Zr(Carbon C18 4.2 4.1 led to 378 possible combinations for LC × LC. Only those using
Discovery Zr-PBD 3.4 3.1 1.7 2.6
lower temperature in the first dimension and higher temperature
PLRP-S 4.4 4.7 3.9 3.4
in the second dimension were evaluated since high temperature
in the second dimension offers numerous advantages including
Table 4
Comparison of the separation power of RPLC × RPLC systems for the 38 representa- a decrease in retention, a decrease in mobile phase viscosity and
tive compounds of biomass by-products. hence in analysis time and sometimes a significant improvement of
column efficiency [34,35]. The Hypercarb column was used at high
N◦ D1 D2 Od n*2D
temperature in both dimensions in order to decrease the retention
Non-silica-based × non-silica-based and to improve peak shapes, usually very poor on this column. As a
1 PLRP-ACN-30 Hyp-ACN-90 7.9 899
2 PLRP-ACN-30 ZCarb-Me-90 6.5 810
result, 14 1D-systems were considered for the first dimension and
3 PLRP-Me-30 Hyp-ACN-90 7.0 348 17 for the second dimension thereby leading to 237 possible com-
4 PLRP-Me-30 ZCarb-Me-90 6.2 327 binations, considering the same temperature in both dimension
5 Hyp-ACN-90 PLRP-Me-90 5.9 602 for the Hypercarb column. Two gradient runs with two different
6 Hyp-ACN-90 ZCarb-Me-90 5.6 509
gradient slopes made it possible to calculate the average S val-
7 Hyp-ACN-90 PLRP-ACN-90 5.5 696
8 Hyp-Me-90 ZCarb-Me-90 5.0 560 ues for each studied systems (S being the absolute slope value of
the relationship between the logarithm of the retention factor and
Non silica-based × silica-based
9 PLRP-ACN-30 AcqSh-Me-65 2.9 1707
the organic solvent composition). The obtained values are listed in
10 PLRP-ACN-30 PhHex-Me-80 2.9 1612 Table 3. It is interesting to notice that whereas the average S values
11 Xbr-ACN-30 ZCarb-ACN-90 5.0 1499 seem to be similar at low and high temperature, for a given RP-
12 ZPBD-ACN-30 AcqSh-Me-65 2.2 1428 system, they are significantly higher with acetonitrile (in the range
13 PLRP-ACN-30 AcqSh-ACN-65 2.6 1410
3.1–5.7) than with methanol (in the range 1.7–4.0). With silica-
14 ZPBD-ACN-30 PhHex-Me-80 2.1 1337
15 Xbr-Me-30 ZCarb-ACN-90 3.4 1317 based stationary phases, longer bonding chains (more retentive
16 Hyp-ACN-90 AcqSh-Me-65 3.6 1283 columns) lead to higher S values. Conversely, S values are signif-
17 PLRP-ACN-30 KinPFP-ACN-65 2.7 1274 icantly higher with the PLRP-S phase, a polymer-based material
18 ZPBD-ACN-30 AcqSh-ACN-65 2.4 1246 than with the Hypercarb phase, made of porous graphitic carbon,
19 PLRP-ACN-30 PhHex-ACN-80 3.1 1190
whereas both stationary phases are strongly retentive. Average S
20 Hyp-ACN-90 AcqSh-ACN-65 4.0 1188
21 Hyp-Me-90 AcqSh-Me-65 2.7 1188 values are of prime importance because they have a direct impact
22 Hyp-ACN-90 PhHex-Me-80 3.3 1173 on the degree of orthogonality [Eq. (3)] and of course on the prac-
23 ZPBD-ACN-30 KinPFP-ACN-65 2.0 1122 tical sample peak capacity [Eqs. (4) and (6)] which is dependent on
24 Hyp-Me-90 PhHex-Me-80 2.5 1117
the former one. Both criteria are expected to reflect the quality of a
25 AcqSh-Me-30 ZCarb-ACN-90 2.8 1102
26 Xbr-ACN-30 ZCarb-Me-90 6.6 1090 two-dimensional separation for a given sample. However, as high-
27 AcqSh-ACN-30 ZCarb-ACN-90 3.0 1077 lighted in a previous study [29], these criteria are complementary,
28 Hyp-ACN-90 PhHex-ACN-80 4.5 1038 the degree of orthogonality being directly related to the concept
29 ZPBD-ACN-30 PhHex-ACN-80 2.5 1033 of selectivity in gradient elution while the sample practical peak
30 PhHex-ACN-30 ZCarb-ACN-90 3.6 1032
capacity represents the overall separation power of the considered
31 Xbr-Me-30 Hyp-ACN-90 5.1 1030
32 Hyp-ACN-90 KinPFP-ACN-65 3.2 1015 2D-system for a given sample. The 237 possible 2D-systems were
33 AcqSh-ACN-30 Hyp-ACN-90 5.4 1004 therefore compared by means of these two descriptors. In order
34 Hyp-MeOH-90 PhHex-ACN-80 3.2 1002 to achieve an objective comparison, the 38 selected compounds
Conditions are expressed as “stationary-phase-organic modifier-temperature”. (see were injected separately in each system using linear gradient con-
Table 2 for stationary phase abbreviation). ditions. The normalized gradient slope was set at 5% in order to
yield similar values for the retention factor at elution (close to 2) in
reduced Van Deemter plots of ethylparaben obtained at 30 ◦ C. Dif- all studied conditions. Fig. 1 shows the obtained practical sample
fusion coefficients were calculated according to the Wilke-Chang peak capacities, n* 2D , as a function of the degree of orthogonal-
equation, considering a value of 0.8 m2 /s at 20 ◦ C in water. ity, Od , each spot corresponding to one possible two-dimensional
system. n* 2D varies from 0 to 1800 while Od varies from 0 to 8.
6. Results and discussion Whereas the correlation between the two descriptors seems to be
fairly good in the range of low values (i.e. n* 2D < 600 and Od < 2), it
6.1. Comparison of the possible 2D-systems is interesting to notice the poor dependence of the practical peak
capacity on the degree of orthogonality beyond that, with data scat-
Choosing compounds for suitable representation of a fast pyrol- tering in both directions. Similar observation was previously made
ysis bio-oil, or a partially upgraded bio-oil, is not an easy task, for charged compounds [29] suggesting that these two descriptors
A. Le Masle et al. / J. Chromatogr. A 1340 (2014) 90–98 95

Table 5
Experimental conditions and experimental results for the RPLC × RPLC separations of the 38 representative compounds using two sets of different conditions (2D-systems
#5 and #34 in Table 4.

2D-system #5 2D-system #34

D1 D2 D1 D2

Stationary phase PLRP-S Acquity Shield C18 Hypercarb Kinetex Phenyl Hexyl
Column geometry 50 mm × 2.1 mm; 3 ␮m 50 mm × 2.1 mm; 1.7 ␮m 50 mm × 2.1 mm; 3 ␮m 50 mm × 2.1 mm; 1.7 ␮m
Organic solvent Acetonitrile Acetonitrile Methanol Acetonitrile
Gradient conditions 1% to 75%B in 150 min 1% to 61%B in 45s 1% to 99%B in 150 min 1% to 81%B in 45s
Normalized gradient slope 1% 5.5% 1.1% 6.1%
Temperature 30 ◦ C 65 ◦ C 60 ◦ C 60 ◦ C
Flow-rate 50 ␮L/min (split ratio 1:5) 1500 ␮L/min 60 ␮L/min (split ratio 1:5) 1500 ␮L/min
Injected volume 5 ␮L 10 ␮L 5 ␮L 12 ␮L
Run number 1 151 1 151
Sampling rate (6␴) 2.2 3.6
Average peak width (4␴) 1.48 min 0.71s 2.4 min 0.8s
Dilution factora 9 1.1 18 1
Effective practical peak capacity 64b 11.5c 51b 17.5c
n*2D d 740 890

Solvent A: water + 0.1% formic acid; solvent B: organic solvent + 0.1% formic acid; 1 min as sampling time.
a
According to Eq. (7).
b
According to Eqs. (2) and (5).
c
According to Eqs. (2) and (6).
d
According to Eq. (4).

1800 Table 6
n*2D Experimental conditions and experimental results for the RPLC × RPLC separation
1600 of the aqueous phase of a partially dehydroxygenated bio-oil using the coupling of
Hypercab and Acquity CSH Phenyl-Hexyl columns.
1400
D1 D2
1200 Stationary phase Hypercarb Acquity CSH
Phenyl Hexyl
1000 Column geometry 100 mm × 1 mm; 50 mm × 2.1 mm;
5 ␮m 1.7 ␮m
800
Mobile phase A: H2O + 0.1% A: H2O + 0.1%
600 formic acid formic acid
B: ACN + 0.1% B: MeOH + 0.1%
400 formic acid formic acid
Gradient conditions 1%–99%B in 1%–81%B in 44s
200 285 min
Normalized gradient 1.6% 8.0%
0 slope
0 1 2 3 4 5 6 7 8 9 Temperature 60 C ◦
80 ◦ C
Od Flow-rate 10 ␮L/min 1400 ␮L/min
Injected volume 1 ␮L 11 ␮L
Fig. 1. Practical sample peak capacity versus degree of orthogonality for the 237 Run number 1 240
considered 2D-systems (for interpretation of the references to color in this figure Sampling rate (6) 3.6
legend, the reader is referred to the web version of the article). Average peak width 2.7 min 0.85s
(4)
Dilution factora 17 1.1
were complementary to assess the potential of a given 2D-system. Effective practical peak 81b 24c
Surprisingly, our present study showed that changing either the capacity
type of organic solvent (acetonitrile and methanol) or the temper- n*2D d 1940
ature while using similar silica-based stationary phase in the two 1.15 min as sampling time.
dimensions had no strong impact on the two descriptors. However, a
According to Eq. (7).
b
two groups of data, represented by dotted frames in Fig. 1 are note- According to Eqs. (2) and (5).
c
According to Eqs. (2) and (6).
worthy. The first one (dotted red frame) corresponds to high values d
According to Eq. (4).
for the degree of orthogonality (from 5 to 8) but to rather low val-
ues for the practical peak capacity (from 300 to 900). These results
mainly concern data obtained by using non silica-based stationary
phases in both dimensions. The corresponding conditions are listed for certain studied compounds, especially with the Hypercarb col-
in Table 4 (#1–#8). Thus, it seems that non-silica based stationary umn. Much longer columns should therefore be used with such
phases such as Discovery Zr-Carbon C18 (zirconia-based material), stationary phases, at least in the first dimension, to gain in efficiency
PLRP-S or Hypercarb are enough different from another to produce and hence in practical peak capacity. The second group (dotted
a significant change in selectivity from D1 to D2 for the studied green frame) includes 2D-systems (#9–#34 in Table 4) exhibiting
representative sample. Compounds are strongly retained on PLRP- high practical peak capacity (from 1000 to 1700) but lower degree
S, Hypercarb and ZrCarb which results in broadening the range of of orthogonality (from 2 to 5). They are characterized by the pres-
compositions at elution and hence the degree of orthogonality [Eq. ence of a non-silica-based phase, usually in the first dimension,
(3)]. Conversely the Discovery Zr-PBD phase being poorly retentive and an efficient silica-based column in the second dimension. The
due to its short chain of carbon is not included in this group. For all change in selectivity between the two dimensions is not as signifi-
these 2D-systems, the practical sample peak capacity does not seem cant as between two different non silica-based columns as shown
to be very attractive. This is likely to be related to poor peak shapes by the degree of orthogonality. Thus, a major advantage of these
96 A. Le Masle et al. / J. Chromatogr. A 1340 (2014) 90–98

combinations is that they can be designed with short columns in 29000


(a)
both dimensions while yielding attractive practical peak capacity.
19000
µAu
7. Application to 2D-separations 9000

- 1000
For preliminary 2D-separations, we used the commercially 2D- 0 20 40 60 80 100 120 140
instrument described in the experimental section which delivered min
660 bar as maximum pressure in both dimensions. It was there- 29000
fore better to use acetonitrile in the second dimension rather than (b)
methanol. Among the different possible conditions listed in Table 4, 19000
µAu
we favored silica-based columns in the second dimension which
9000
offer the possibility of using sub-2 ␮m particles and hence reduc-
ing the analysis time. For similar reasons we preferred columns - 1000
which can withstand high temperatures. As a result, two different 0 20 40 60 80 100 120 140
2D-conditions were selected in Table 4. However, it is important min
to remember that all 2D-systems listed in Table 4 are potential 40
candidates for the 2D-separation of biomass compounds. 30
(c)
According to our criteria, the best 2D-system (#13 in Table 4)
concerns the use of a PLRP-S column at 30 ◦ C in the first dimension, mAU 20
and an Acquity BEH Shield RP18 at 65 ◦ C in the second dimension, 10
using acetonitrile in both dimensions. The predicted peak capacity
was 1400 under the conditions described in the preceding sec- 0
tion. Such a large value is likely to be due to different retention 0 50 100 150 200 250
min
mechanisms between silica-based phases and PLRP-S, in particular
the specific charge transfer interaction of the co-polymer styrene Fig. 2. First dimensional chromatograms obtained with the 2D-separations of (a) the
divinyl benzene [36] as well as to relative good efficiencies of PLRP- 38 representative compounds using 2D-system #13 in Table 4 with experimental
conditions given in Table 5; (b) the 38 representative compounds using 2D-system
S in addition to very high efficiencies of Acquity BEH columns. We
#34 in Table 4 with experimental conditions given in Table 5 and (c) a bio-oil sam-
also selected 2D-system #34 with a view to changing stationary ple with experimental conditions given in Table 6. The corresponding RPLC × RPLC
phases in both dimensions. This 2D-system combined Hypercarb chromatograms are shown in Figs. 3, 4 and 5, respectively.
with methanol in D1 and Acquity CSH phenyl-hexyl with acetoni-
trile in D2. As displayed in Table 4, this second set of conditions
acceptable peak capacity. For neutral compounds, it was found that
exhibit a higher degree of orthogonality compared with the first
two column volumes were usually sufficient to afford good run-to-
one (3.2 vs 2.6) with a lower practical sample peak capacity (1000
run repeatability [37]. We therefore chose these latter conditions
vs 1400). This is clearly due to the fact that the difference in selectiv-
for column equilibration. Similarly, the ratio of the linear veloci-
ity between Hypercarb and silica-based columns is more attractive
ties (D2–D1) has to be as high as possible to maintain a suitable
than between PLRP-S and silica-based columns. However column
peak capacity in the second dimension [34]. As a result, high lin-
efficiency is poorer on Hypercarb than on PLRP-S which explains
ear velocities (much higher than the optimum value) were used
the decrease in practical peak capacity between 2D-systems #13
in the second dimension whereas low linear velocities (slightly
and #34. It is interesting to notice that various terms are involved
below the optimum value) were used in the first dimension. Simi-
in Eq. (3). Although the product S1xS2 is lower for 2D-system #34
larly, the selected normalized gradient slope was low in D1 (about
(Table 3), both  coefficient and composition range, C1 are higher
1%) while high in D2 (about 6%). For both 2D-systems, the column
thereby making the degree of orthogonality higher.
diameter in D1 was 2.1 mm. The flow-rate was therefore splitted
The temperature in the first dimension was limited by the max-
before entering the ten-port switching valve in order to reduce
imum temperature allowed by the oven, namely 60 ◦ C. For this
the injected volume in the second dimension (i.e. 10 ␮L and 12 ␮L,
second studied 2D-system, we used a 2.7 ␮m Kinetex phenyl-hexyl
respectively). Flow splitting is convenient since it allows the use
column in the second dimension instead of the previously studied
of columns of identical internal diameter in both dimensions [38].
1.7 ␮m Acquity CSH phenyl-hexyl, in order to decrease backpres-
However, it may generate significant additional band broadening
sure while keeping similar selectivity. It is important to note that
due to dispersion into the split [39].
the predicted practical sample peak capacities in Table 4 were cal-
The first dimensional chromatograms for the coupling of PLRP-
culated with a normalized gradient slope of 5% in both dimensions
S and Acquity Shield C18 and for the coupling of Hypercarb and
and with optimum flow-rate conditions. However, these conditions
Kinetex phenyl-hexyl are shown in Figs. 2a and 2b respectively. The
cannot be used in on line LC × LC. As a result, the resulting practi-
corresponding 2D-separations are shown in Figs. 3 and 4, respec-
cal sample peak capacity cannot be the same. It depends on the
tively. Conditions and experimental results are listed in Table 5.
used 2D-conditions that must be optimized. In on-line LC × LC, the
Large difference in selectivity between PLRP-S and Hypercarb for
sampling time corresponds to the total analysis time in the sec-
biomass compounds is highlighted in Fig. 2. Wider peaks on Hyper-
ond dimension, t2 . The second dimension has to be fast to permit
carb than on PLRP-S can also be observed in this figure.
enough sampling of the first dimension peaks. In this study, the
With the first 2D-system (Fig. 3), retention data are much dia-
sampling time was set at 1 min so that the predicted sampling rate
gonalized thereby leading to a poor surface coverage ( = 0.21).
was close to 3. The sampling time is given by
However, a fairly high effective peak capacity (n1 = 64) in D1 com-
t2 = tG,2 + t0,2 + tD,2 + tEq,2 (8) bined with very thin peak widths (about 0.7s) in D2 allowed to
reach an acceptable value for the practical sample peak capacity
tG,2 , t0,2 , tD,2 and tEq,2 being the gradient time, the column dead (n* 2D = 740). As expected from our preliminary study, the occu-
time, the instrument dwell time and the column equilibration pation of the retention space is much larger ( = 0.55) with the
time, respectively. The column equilibration time should be as second system (Fig. 4) but the effective practical peak capac-
short as possible to have a gradient time high enough to provide ity is only slightly higher (n* 2D = 890). Indeed, the effective peak
A. Le Masle et al. / J. Chromatogr. A 1340 (2014) 90–98 97

Fig. 3. RPLC × RPLC separation of the 38 representative compounds using the coupling of PLRPS and Acquity Shield C18 columns. Experimental conditions are given in Table 5.
(a) contour plot representation with dotted lines representing the retention surface coverage and (b) 3D-chromatogram.

Fig. 4. RPLC × RPLC separation of the 38 representative compounds using the coupling of Hypercarb and Kinetex phenyl-hexyl column. Experimental conditions are given
in Table 5. (a) Contour plot representation with dotted lines representing the retention surface coverage and (b) 3D-chromatogram.

capacity in D1 is lower (n1 = 51) due to the combined effect of lower pressure instrument (HT-UHPLC); (3) use of a 10 cm column length
column efficiency and lower S values in case of Hypercarb com- in D1 in order to increase peak capacity in D1; (4) use of 1 mm col-
pared to PLRP-S. Furthermore, peak distortion occurred for some umn internal diameter in D1 in order to inject small amounts of
compounds strongly retained in D1 while poorly retained in D2. sample and to avoid extra-column band broadening arising from
This is due to the fact that these compounds are eluted from D1 splitting. Experimental conditions and obtained results are listed
with a high content of methanol in the mobile phase (strong eluent in Table 6. It is interesting to observe that, similarly to the two pre-
strength), thus providing a strong injection solvent in D2 whereas ceding 2D-systems, the dilution factor in D2 was close to 1 which
they are eluted from D2 with a low content of acetonitrile (weak means that the sample was not subjected to additional dilution in
eluent strength). This situation usually gives rise to very bad peak the second dimension. The contour plots and the 3D-chromatogram
shapes in gradient elution [40], sometimes with two distinct peaks, are illustrated in Fig. 5. The contour plot (Fig. 5a) clearly shows a
particularly for large injection volumes as is the case here [41]. It large occupation of the separation space, delimited by dotted lines.
is important to note that additional peak band broadening both The similarity between the contour plots shown in Figs 4 and 5
in the first dimension due to undersampling and in the second is evident, thereby highlighting the satisfactory representativeness
dimension due to injection effects should ideally be considered of our test sample. The peaks, observed in the 3D-chromatogram
in the preliminary search for two suitable LC systems in a global (Fig. 5b), are symmetrical for almost all detected compounds. Yet, a
optimization of all LC × LC parameters. This task is very challeng- few compounds are still subjected to injection effects as discussed
ing. It should require a true knowledge of peak band broadening above. The related peaks are located close to the upper dotted line
depending on the different possible factors so that the prediction in Fig. 5a. The larger the retention area, the higher the risk it is to
of practical sample peak capacity should be correct. broaden the peaks. The injection volume in D2 was 11 ␮L, proba-
Despite this issue, the use of Hypercarb in the first dimension bly somewhat too high in these conditions. However, the injection
was selected for the separation of the aqueous phase of a partially volume should be easily tuned by splitting the flow prior to the ten-
dehydroxygenated bio-oil. In order to maximize the practical peak port switching valve, yet increasing dilution. Further optimization
capacity, some adjustments were made: (1) use of acetonitrile in is probably required to further gain in peak capacity. In the present
D1 and methanol in D2 (#28 in Table 4); (2) use of 1.7 ␮m parti- conditions, a large number of separated compounds (>120) can be
cle size in D2 at 80 ◦ C, possible with a high-temperature ultra high observed in Fig. 5a. The obtained results highlight the applicability
98 A. Le Masle et al. / J. Chromatogr. A 1340 (2014) 90–98

Fig. 5. RPLC × RPLC separation of the aqueous phase of a partially dehydroxygenated bio-oil using the coupling of Hypercarb and Acquity CSH phenyl-Hexyl columns.
Experimental conditions are given in Table 6. (a) Contour plot representation with dotted lines representing the retention surface coverage and (b) 3D-chromatogram.

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