Subscriber access provided by CMU Libraries - http://library.cmich.edu
Article
Reducing Dilution and Analysis Time in On-line Comprehensive Two-Dimensional Liquid Chromatography by Active Modulation Andrea F. G. Gargano, Mike Duffin, Pablo Navarro, and Peter J Schoenmakers Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b04051 • Publication Date (Web): 28 Dec 2015 Downloaded from http://pubs.acs.org on January 4, 2016
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 25
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Analytical Chemistry
1
Reducing Dilution and Analysis
2
Time in On-line Comprehensive
3
Two-Dimensional Liquid
4
Chromatography by Active
5
Modulation
6
Andrea F.G. Gargano1,2 Mike Duffin3, Pablo Navarro3, Peter J. Schoenmakers2
7 8 9 10
1
11 12
2
Van 't Hoff Institute for Molecular Sciences, Science Park 904 1098 XH Amsterdam, Netherlands
13
3
Syngenta, Jealott's Hill International Research Centre, Bracknell, Berkshire RG42 6EY, UK
TI-COAST, Van 't Hoff Institute for Molecular Sciences, Science Park 904, 1098 XH Amsterdam, Netherlands
14 15 16 17 18 19 20 21
*
[email protected] 22 23 24
Tel: +31-20-525 7040 Science Park 904 |1098 XH Amsterdam Building C2, room 248A
Dr. Andrea Gargano Analytical Chemistry Van 't Hoff Institute for Molecular Sciences Universiteit van Amsterdam
25 26
Keywords
27
On-line comprehensive two-dimensional liquid chromatography, LC×LC, Active Modulation,
28
UHPLC, Surfactant Analysis, Tristyrylphenol ethoxylate
29
1 ACS Paragon Plus Environment
Analytical Chemistry
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 2 of 25
30
Abstract
31
On-line comprehensive two-dimensional liquid chromatography (LC×LC) offers ways to
32
achieve high-performance separations in terms of peak capacity (exceeding 1000) and
33
additional selectivity to realize applications that cannot be addressed with one-dimensional
34
chromatography (1D-LC). However, the greater resolving power of LC×LC comes at the
35
price of higher dilutions (thus reduced sensitivity) and, often, long analysis times (>100 min).
36
The need to preserve the separation attained in the first dimension (1D) causes greater
37
dilution for LC×LC, in comparison with 1D-LC, and long analysis times to sample the 1D with
38
an adequate number of second dimension separations. A way to significantly reduce these
39
downsides is to introduce a concentration step between the two chromatographic
40
dimensions. In this work we present a possible active-modulation approach to concentrate
41
the fractions of 1D effluent. A typical LC×LC system is used with the addition of a dilution
42
flow to decrease the strength of the 1D effluent and a modulation unit that uses trap columns.
43
The potential of this approach is demonstrated for the separation of tristyrylphenol ethoxylate
44
phosphate surfactants, using a combination of hydrophilic-interaction and reversed-phase
45
liquid chromatography. The modified LC×LC system enabled us to halve the analysis time
46
necessary to obtain a similar degree of separation efficiency with respect to UHPLC based
47
LC×LC and of 5 times with respect to HPLC instrumentation (40 compared with 80 and 200
48
min, respectively), while at the same time reducing dilution (DF of 142, 299 and 1529,
49
respectively) and solvent consumption per analysis (78, 120 and 800 mL, respectively).
2 ACS Paragon Plus Environment
Page 3 of 25
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Analytical Chemistry
50
Introduction
51
Two dimensional liquid chromatography is a technique that allows the characterization of
52
samples that, due to their complexity, cannot be resolved using one-dimensional
53
chromatography. The higher resolving power is a direct consequence of the consecutive
54
separation of the sample components using two distinct chromatographic processes that
55
exploit different selectivity principles. In comprehensive two-dimensional LC (LC×LC) the
56
entire sample is subjected to the two different separations. In off-line LC×LC there are no
57
restrictions on the speed of analysis in the second dimension, making this the best way to
58
exploit the full resolving power of both the separation dimensions1,2. However, the resulting
59
long overall analysis times make this technique impractical for routine analysis of large
60
numbers of samples.
61
Online LC×LC has the advantage of significantly decreasing the analysis time necessary to
62
obtain a high peak capacity. However, the tight constraints imposed by the coupling of the
63
two chromatographic processes seriously limit the separation efficiency. The second
64
dimension separations (2D) must be fast to allow frequent sampling of the first separation
65
(1D), while providing sufficient resolving power. Studies have demonstrated that the optimal
66
number of fractions per 1D peak is somewhere between 2 and 43–6.Moreover, to reduce the
67
volumes collected for injection onto the 2D column, the 1D separation has to be run at low,
68
often suboptimal7, linear flow velocities and thus long run times are required to deliver
69
efficient gradients (spanning at least five column volumes). Other restrictions are related to
70
the compatibility of the mobile phases used in the two dimensions8 and to the volume of
71
sample that can be injected on the 2D column9,10.
72
If online LC×LC is to become more frequently adopted for routine analysis, robust high-
73
throughput analysis exploiting orthogonal selectivities will need to be developed. In this
74
context it is important to develop strategies that enable fast and efficient separations.
75
Significant shortening of the analysis time can be achieved by reducing the 2D cycle time
76
using ultra-high-pressure LC (UHPLC)11–13, increased temperatures14 or parallel 2D15,16
77
technologies that are now available in commercialized LC×LC systems. However, to reduce
78
the injection band broadening in the second dimension, these systems use combinations of
79
long and narrow 1D columns (e.g. 1 or 2.1 mm I.D.) and short and wide 2D columns (e.g. 4.6
80
mm I.D). Using such configurations it is possible to minimize the ratio between the volume of
81
the fractions collected from the first dimension and the 2D column volume5, typically injecting
82
less than 10% of the 2D column volume. As a consequence, high peak capacities are
83
obtained at the price of high dilutions and thus loss in sensitivity.
84
Alternative column couplings have been suggested to limit the injection band broadening
85
without increasing the dilution and allow combinations of chromatographic modes that make 3 ACS Paragon Plus Environment
Analytical Chemistry
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 4 of 25
86
use of incompatible solvents. Examples include interfaces with trap columns instead of
87
loops17–23, vacuum evaporation24 and thermally-assisted modulation21,25. These technologies
88
have proven, with different degrees of success, their effectiveness at reducing problems
89
connected with the injection of strong or incompatible solvents in the 2D system, but they
90
have not yet reached a level of maturity that would allow their use in routine LC×LC
91
applications.
92
In this work we present an active-modulation approach (LC/a×m/LC), in which the fractions
93
collected from the 1D are concentrated prior to injection in the 2D. This is achieved using a
94
HPLC×UHPLC system that was modified to include a dilution flow to decrease the mobile
95
phase strength of the 1D effluent and a modulation unit that uses trap columns. This
96
configuration allows, in comparison with the conventional loop interface (“passive-
97
modulation”), linear velocities closer to the optimum in the 1D without using flow splitters7,26.
98
The LC/a×m/LC system also reduces the effects of the 1D dead times (consequences of low
99
flow rates in the first dimension), dwell volumes and 2D injection band broadening (thanks to
100
the reduction of the volumes collected from the 1D). Together these result in a considerably
101
shorter time necessary to obtain a specified separation efficiency.
102
The feasibility of LC/a×m/LC is demonstrated for the separation of tristyrylphenol ethoxylate-
103
phosphate (TSP) surfactants (polymeric compounds), using a combination of hydrophylic
104
interaction LC and reversed-phase LC (HILIC×RPLC). Thanks to the independent
105
selectivities offered by this method the sample can be separated according to its ethoxyate
106
chain length (HILIC) and the degree of styrene and phosphate substitution (RP).
107 108 109
Experimental Section Chemicals
110
The samples used for this study are: tristyrylphenol ethoxylate (TSP; CAS 99734-09-05)
111
commercially available under the names Agent 3152-90 and Termul 3150; tristyrylphenol
112
ethoxylate phosphate (TSP phosphate CAS 90093-37-1) as Agnique PE TSP 16A, Agent
113
3152-92 and Soprophor 3D33. The chemical structure of the compounds is reported in
114
Figure 1. The solvents used were Milli-Q grade water (18.2 mΩ) and liquid-chromatography-
115
grade acetonitrile (ACN), methanol (MeOH), tetrahydrofuran (THF) and 1-butanol, obtained
116
from Avantor Performance Materials B.V. (Deventer, NL). Ammonium acetate (Bioxtra
117
grade, 99% purity) was purchased from Sigma-Aldrich (Zwijndrecht, NL). All materials were
118
used as received and the mobile phases were not filtered prior to use.
119
Samples were prepared by dissolving the surfactant in a mixture of 94% ACN 5% THF and
120
1% 12.5-mM ammonium acetate at the concentrations reported in Table 1 for the HILIC×RP 4 ACS Paragon Plus Environment
Page 5 of 25
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Analytical Chemistry
121
separations or at 1 mg/ mL for the HILIC study reported in Figure 2, 3. The performance of
122
the different RP materials (Figure 4) was studied with a 1 mg/mL solution of surfactants in
123
H20/ACN 1/1 v/v.
124 125
Instrumentation & Data Analysis
126
HPLC×HPLC Instrumentation and data analysis.
127
An LC×LC instrument composed of an Agilent (Waldbronn, Germany) 1100 quaternary (1D)
128
pump (Model G1311A) and a 1260 quaternary (2D) pump (Model G1311B), two degassers
129
(Model G1322), thermostats (Model G1316A), detectors (Model G4212B with 13 µL flow
130
cell), one autosampler (Model G1329B) and a 10-port two-position switching valve (Agilent
131
G4232A and G1170A support unit) with 60-µL loops was used for our experiments. The
132
valve was configured for back-flush injection27 and all Agilent modules were controlled using
133
Agilent Chemstation B.04.03 software on two different computers, the first was used to
134
control the autosampler, 1D pump and thermostat, and detector while the second controlled
135
the switching valve, 2D pump and detector. The data were exported as comma-separated
136
files and processed using a recently published Matlab-2014b (The Mathworks, Inc., Natick,
137
MA, USA) script28,29.
138
A Kromasil 60 Si-OH 1D column (250 × 3 mm i.d.; 5-µm particles; 60-Å pore size; Hichrom
139
Ltd, Reading, United Kingdom) was used, followed by a stainless-steel tee (Model U-428,
140
IDEX Corp; DaVinci, Rotterdam NL) to split the flow before the switching valve. Stainless-
141
steel tubing of the same I.D. and length (75 µm I.D. x 200 mm) were used on the two sides
142
of the tee, to approach a split ratio of 1:1. As 2D column we used a Phenomenex Kinetex
143
C18 (50 x 4.6 mm; 2.6-µm; 100-Å, Phenomenex, Macclesfield, United Kingdom). Operation
144
conditions and further details are reported in Table 1.
145
HPLC×UHPLC and HPLC/a×m/UHPLC
146
An Agilent 1200 degasser (G1379), a 1200 SL binary pump (Model G1312, with the original
147
mixer replaced by a 50-µL mixer from Waters) an autosampler (Model G4226A) and column
148
thermostat compartment (Model G1316C) were used for the 1D. The 2D was composed of
149
UHPLC binary pumps (Model G4220A configured with a JetWeaver V35 mixer) a
150
thermostatted column compartment (Model G1316C) and a diode-array UV detector (DAD,
151
Model G4212A) with a standard flow cell (13 µL volume). The valve schematically shown in
152
Figure 5 is a 4-port duo 2-position valve (Model 5067-4214) installed in the 2D thermostat.
153
Loops of 40 µL were used with valve configuration for back-flush injections.
5 ACS Paragon Plus Environment
Analytical Chemistry
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 6 of 25
154
We used an Acquity UPLC BEH HILIC (150 x 2.1 mm; 1.7-µm; 130-Å; Waters, Elstree, UK)
155
1
156
Additionally, we tested a Hypersil GOLD C-18 (50 x 1 mm; 1.9-µm; ThermoFisher Scientific,
157
Breda, NL) column. The comparison between the different columns is reported in Figure 4.
158
The HPLC/a×m/UHPLC set-up used the same hardware described above and, in addition, a
159
stainless-steel tee connection (Model U-428, IDEX Corp) to add a dilution flow after the first-
160
separation dimension using an isocratic pump (Model G1310B). The modulation interface
161
was configured for forward-flush operation and the loops where substituted by two C-18 pre-
162
column cartridges (2.1 x 2 mm with sub-2 µm particles, SecurityGuard Ultra AJ0-8782,
163
Phenomenex, Utrecht, NL) directly installed in the switching valve and connected to the port
164
receiving effluent from the 1D by a 50 mm x 50 µm I.D. connection. A schematic
165
representation of the setup is shown in Figure 5.
166
All Agilent modules of this configuration were controlled and programmed using Agilent
167
Chemstation C.01.04. Data analysis was performed using GC image R 2.5 software
168
(GCimage, Lincoln, USA). Operation conditions are reported in the Chromatographic
169
Conditions section and Table 1.
170
D column and an Acquity UPLC HSS C18 2D column (50 x 2.1 mm; 1.8-µm; 100-Å).
Chromatographic Conditions
171
The 1D separations were performed using a mobile phase A: 94% ACN, 5% THF, 1% 12.5
172
mM ammonium formate, to mobile phase B: 12.5 mM ammonium acetate. In the 2D the
173
mobile phase used was A: 99% 12.5 mM ammonium formate, 1% 1-butanol and B: 99%
174
MeOH, 1% 1-butanol30. Butanol was used to reduce the equilibration time. The separation
175
performed using the LC/a×m/LC set-up used a dilution flow composed of 12.5-mM
176
ammonium acetate.
177
The conditions used for the HPLC×HPLC (graphical abstract and Table 1) were: 1D flow rate
178
of 0.1 mL/min at T=10˚C and gradient elution from 0% B to 60% B in 140 min, 20 min at 60%
179
B. At 180 min the flow rate was set to 1 mL/min and run at 0% B till 200 min. For the 2D
180
separation we used a flow rate of 4 mL/min, T=40˚C using, gradient elution from 40% B to
181
80% in 0.01 min, from 80% to 100% at 0.76 min, kept at 100% B till 0.86 min and
182
equilibrated at 40% B for 0.14 min. Modulation volume= 50 µL. The HPLC×UHPLC
183
separation described in Table 1 was performed using a 1D flow rate of 0.04 mL/min, T= 5˚C,
184
gradient elution from 2% B to 20% B in 20 min, then to 50% B at 50 min, 55% B at 60 min,
185
2% B at 70 min. Flow rate increased to 0.2 mL/min at 78 min. The 2D was run at 50˚C at a
186
flow rate of 1.5 mL/min, gradient elution from 35% B to 80% in 0.01 min, 85% to 100% at
187
0.35 min, 0.03 min 100% B and at 0.4 min re-equilibration at 40% B. Modulation volume= 20
188
µL. The HPLC×UHPLC and HPLC/a×m/UHPLC reported in Figure 6, 7 runs were performed
6 ACS Paragon Plus Environment
Page 7 of 25
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Analytical Chemistry
189
using a 1D flow rate of 0.08 mL/min, T= 5˚C, gradient elution from 2% B to 20% B in 10 min,
190
then to 50% B at 25 min, 55% B at 30 min, 2% B at 39.99 min. Flow rate increased to 0.2
191
mL/min at 40 min. In the 2D T=50˚C, gradient elution from 35% B to 80% in 0.01 min, then to
192
100% at 0.29 min, 100% B at 0.30 min, followed by 0.06 min re-equilibration at 35% B (0.35
193
min modulation time). The 2D flow rate was of 1.5 mL/min in the HPLC×UHPLC setup and
194
1.35 mL/min in the HPLC/a×m/UHPLC. The dilution flow in used in the /a×m/ setup was 0.6
195
mL/min and this incremented the 1D back pressure off approximately 100 bar. All the LC×LC
196
separation where monitored at 220 nm using 80 Hz scan rate.
197
Calculations
198 199
The following paragraph describes the equations adopted to obtain the results reported in
200
Table 1.
Peak Capacity
201 202 203 204
The peak capacity (nc) was determined from the average width at half height ( ) applying Eq. (1).
=
+ ) . ∙(
1
(1)
205
For the calculation of the 1D peak capacity the average width was obtained from the
206
integration of five peaks at the beginning of the chromatogram (indicated in red in Figure 2b)
207
and five peaks in the area of elution of the phosphate species (green in Figure 2b). The 2D
208
peak capacity was obtained as average of all the (5 to 15, depending on tG) peaks present in
209
the chromatograms.
210
The predicted peak capacity for each LC×LC system was calculated as:
211
, = ∙
212
(2)
Using values obtained from one dimensional runs under the same conditions as the
214
LC×LC runs (see Table 1 for the respective parameters) and .calculated from the
215
Equation (2) is not taking into account 1D undersampling and band broadening associated
213
average of multiple 2D run (2D nc parameter of Table 1)
216
with the modulation process3,4,6.
217
Two different approaches were used to correct for these effects, using the equation
218
proposed by Davis-Li et al.6,31,32
219
,
!
=
"# ∙ "# $
=
"# ∙ "#
%&'.'(∙)
* + # # , *
(3)
7 ACS Paragon Plus Environment
Analytical Chemistry
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
220 221 222
223 224
Page 8 of 25
where - is the undersampling correction factor . is the modulation cycle and ./ the
dimension gradient time; and that proposed by Vivó et al. for loop-based LC×LC5 ,
!
=
123 45
0
5 *# ; 6789: ; &
