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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

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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.

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Analytical Chemistry

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Reducing Dilution and Analysis

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Time in On-line Comprehensive

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Two-Dimensional Liquid

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Chromatography by Active

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Modulation

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Andrea F.G. Gargano1,2 Mike Duffin3, Pablo Navarro3, Peter J. Schoenmakers2

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Van 't Hoff Institute for Molecular Sciences, Science Park 904 1098 XH Amsterdam, Netherlands

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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

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*[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

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Keywords

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On-line comprehensive two-dimensional liquid chromatography, LC×LC, Active Modulation,

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UHPLC, Surfactant Analysis, Tristyrylphenol ethoxylate

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 Abstract

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On-line comprehensive two-dimensional liquid chromatography (LC×LC) offers ways to

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achieve high-performance separations in terms of peak capacity (exceeding 1000) and

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additional selectivity to realize applications that cannot be addressed with one-dimensional

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chromatography (1D-LC). However, the greater resolving power of LC×LC comes at the

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price of higher dilutions (thus reduced sensitivity) and, often, long analysis times (>100 min).

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The need to preserve the separation attained in the first dimension (1D) causes greater

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dilution for LC×LC, in comparison with 1D-LC, and long analysis times to sample the 1D with

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an adequate number of second dimension separations. A way to significantly reduce these

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downsides is to introduce a concentration step between the two chromatographic

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dimensions. In this work we present a possible active-modulation approach to concentrate

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the fractions of 1D effluent. A typical LC×LC system is used with the addition of a dilution

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flow to decrease the strength of the 1D effluent and a modulation unit that uses trap columns.

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The potential of this approach is demonstrated for the separation of tristyrylphenol ethoxylate

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phosphate surfactants, using a combination of hydrophilic-interaction and reversed-phase

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liquid chromatography. The modified LC×LC system enabled us to halve the analysis time

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necessary to obtain a similar degree of separation efficiency with respect to UHPLC based

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LC×LC and of 5 times with respect to HPLC instrumentation (40 compared with 80 and 200

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min, respectively), while at the same time reducing dilution (DF of 142, 299 and 1529,

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respectively) and solvent consumption per analysis (78, 120 and 800 mL, respectively).

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Analytical Chemistry

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 Introduction

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Two dimensional liquid chromatography is a technique that allows the characterization of

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samples that, due to their complexity, cannot be resolved using one-dimensional

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chromatography. The higher resolving power is a direct consequence of the consecutive

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separation of the sample components using two distinct chromatographic processes that

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exploit different selectivity principles. In comprehensive two-dimensional LC (LC×LC) the

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entire sample is subjected to the two different separations. In off-line LC×LC there are no

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restrictions on the speed of analysis in the second dimension, making this the best way to

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exploit the full resolving power of both the separation dimensions1,2. However, the resulting

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long overall analysis times make this technique impractical for routine analysis of large

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numbers of samples.

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Online LC×LC has the advantage of significantly decreasing the analysis time necessary to

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obtain a high peak capacity. However, the tight constraints imposed by the coupling of the

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two chromatographic processes seriously limit the separation efficiency. The second

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dimension separations (2D) must be fast to allow frequent sampling of the first separation

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(1D), while providing sufficient resolving power. Studies have demonstrated that the optimal

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number of fractions per 1D peak is somewhere between 2 and 43–6.Moreover, to reduce the

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volumes collected for injection onto the 2D column, the 1D separation has to be run at low,

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often suboptimal7, linear flow velocities and thus long run times are required to deliver

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efficient gradients (spanning at least five column volumes). Other restrictions are related to

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the compatibility of the mobile phases used in the two dimensions8 and to the volume of

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sample that can be injected on the 2D column9,10.

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If online LC×LC is to become more frequently adopted for routine analysis, robust high-

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throughput analysis exploiting orthogonal selectivities will need to be developed. In this

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context it is important to develop strategies that enable fast and efficient separations.

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Significant shortening of the analysis time can be achieved by reducing the 2D cycle time

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using ultra-high-pressure LC (UHPLC)11–13, increased temperatures14 or parallel 2D15,16

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technologies that are now available in commercialized LC×LC systems. However, to reduce

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the injection band broadening in the second dimension, these systems use combinations of

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long and narrow 1D columns (e.g. 1 or 2.1 mm I.D.) and short and wide 2D columns (e.g. 4.6

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mm I.D). Using such configurations it is possible to minimize the ratio between the volume of

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the fractions collected from the first dimension and the 2D column volume5, typically injecting

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less than 10% of the 2D column volume. As a consequence, high peak capacities are

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obtained at the price of high dilutions and thus loss in sensitivity.

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Alternative column couplings have been suggested to limit the injection band broadening

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without increasing the dilution and allow combinations of chromatographic modes that make 3 ACS Paragon Plus Environment

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use of incompatible solvents. Examples include interfaces with trap columns instead of

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loops17–23, vacuum evaporation24 and thermally-assisted modulation21,25. These technologies

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have proven, with different degrees of success, their effectiveness at reducing problems

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connected with the injection of strong or incompatible solvents in the 2D system, but they

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have not yet reached a level of maturity that would allow their use in routine LC×LC

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applications.

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In this work we present an active-modulation approach (LC/a×m/LC), in which the fractions

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collected from the 1D are concentrated prior to injection in the 2D. This is achieved using a

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HPLC×UHPLC system that was modified to include a dilution flow to decrease the mobile

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phase strength of the 1D effluent and a modulation unit that uses trap columns. This

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configuration allows, in comparison with the conventional loop interface (“passive-

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modulation”), linear velocities closer to the optimum in the 1D without using flow splitters7,26.

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The LC/a×m/LC system also reduces the effects of the 1D dead times (consequences of low

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flow rates in the first dimension), dwell volumes and 2D injection band broadening (thanks to

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the reduction of the volumes collected from the 1D). Together these result in a considerably

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shorter time necessary to obtain a specified separation efficiency.

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The feasibility of LC/a×m/LC is demonstrated for the separation of tristyrylphenol ethoxylate-

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phosphate (TSP) surfactants (polymeric compounds), using a combination of hydrophylic

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interaction LC and reversed-phase LC (HILIC×RPLC). Thanks to the independent

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selectivities offered by this method the sample can be separated according to its ethoxyate

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chain length (HILIC) and the degree of styrene and phosphate substitution (RP).

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 Experimental Section Chemicals

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The samples used for this study are: tristyrylphenol ethoxylate (TSP; CAS 99734-09-05)

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commercially available under the names Agent 3152-90 and Termul 3150; tristyrylphenol

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ethoxylate phosphate (TSP phosphate CAS 90093-37-1) as Agnique PE TSP 16A, Agent

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3152-92 and Soprophor 3D33. The chemical structure of the compounds is reported in

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Figure 1. The solvents used were Milli-Q grade water (18.2 mΩ) and liquid-chromatography-

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grade acetonitrile (ACN), methanol (MeOH), tetrahydrofuran (THF) and 1-butanol, obtained

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from Avantor Performance Materials B.V. (Deventer, NL). Ammonium acetate (Bioxtra

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grade, 99% purity) was purchased from Sigma-Aldrich (Zwijndrecht, NL). All materials were

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used as received and the mobile phases were not filtered prior to use.

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Samples were prepared by dissolving the surfactant in a mixture of 94% ACN 5% THF and

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1% 12.5-mM ammonium acetate at the concentrations reported in Table 1 for the HILIC×RP 4 ACS Paragon Plus Environment

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Analytical Chemistry

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separations or at 1 mg/ mL for the HILIC study reported in Figure 2, 3. The performance of

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the different RP materials (Figure 4) was studied with a 1 mg/mL solution of surfactants in

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H20/ACN 1/1 v/v.

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Instrumentation & Data Analysis

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HPLC×HPLC Instrumentation and data analysis.

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An LC×LC instrument composed of an Agilent (Waldbronn, Germany) 1100 quaternary (1D)

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pump (Model G1311A) and a 1260 quaternary (2D) pump (Model G1311B), two degassers

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(Model G1322), thermostats (Model G1316A), detectors (Model G4212B with 13 µL flow

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cell), one autosampler (Model G1329B) and a 10-port two-position switching valve (Agilent

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G4232A and G1170A support unit) with 60-µL loops was used for our experiments. The

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valve was configured for back-flush injection27 and all Agilent modules were controlled using

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Agilent Chemstation B.04.03 software on two different computers, the first was used to

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control the autosampler, 1D pump and thermostat, and detector while the second controlled

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the switching valve, 2D pump and detector. The data were exported as comma-separated

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files and processed using a recently published Matlab-2014b (The Mathworks, Inc., Natick,

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MA, USA) script28,29.

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A Kromasil 60 Si-OH 1D column (250 × 3 mm i.d.; 5-µm particles; 60-Å pore size; Hichrom

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Ltd, Reading, United Kingdom) was used, followed by a stainless-steel tee (Model U-428,

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IDEX Corp; DaVinci, Rotterdam NL) to split the flow before the switching valve. Stainless-

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steel tubing of the same I.D. and length (75 µm I.D. x 200 mm) were used on the two sides

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of the tee, to approach a split ratio of 1:1. As 2D column we used a Phenomenex Kinetex

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C18 (50 x 4.6 mm; 2.6-µm; 100-Å, Phenomenex, Macclesfield, United Kingdom). Operation

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conditions and further details are reported in Table 1.

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HPLC×UHPLC and HPLC/a×m/UHPLC

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An Agilent 1200 degasser (G1379), a 1200 SL binary pump (Model G1312, with the original

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mixer replaced by a 50-µL mixer from Waters) an autosampler (Model G4226A) and column

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thermostat compartment (Model G1316C) were used for the 1D. The 2D was composed of

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UHPLC binary pumps (Model G4220A configured with a JetWeaver V35 mixer) a

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thermostatted column compartment (Model G1316C) and a diode-array UV detector (DAD,

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Model G4212A) with a standard flow cell (13 µL volume). The valve schematically shown in

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Figure 5 is a 4-port duo 2-position valve (Model 5067-4214) installed in the 2D thermostat.

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Loops of 40 µL were used with valve configuration for back-flush injections.

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We used an Acquity UPLC BEH HILIC (150 x 2.1 mm; 1.7-µm; 130-Å; Waters, Elstree, UK)

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1

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Additionally, we tested a Hypersil GOLD C-18 (50 x 1 mm; 1.9-µm; ThermoFisher Scientific,

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Breda, NL) column. The comparison between the different columns is reported in Figure 4.

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The HPLC/a×m/UHPLC set-up used the same hardware described above and, in addition, a

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stainless-steel tee connection (Model U-428, IDEX Corp) to add a dilution flow after the first-

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separation dimension using an isocratic pump (Model G1310B). The modulation interface

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was configured for forward-flush operation and the loops where substituted by two C-18 pre-

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column cartridges (2.1 x 2 mm with sub-2 µm particles, SecurityGuard Ultra AJ0-8782,

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Phenomenex, Utrecht, NL) directly installed in the switching valve and connected to the port

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receiving effluent from the 1D by a 50 mm x 50 µm I.D. connection. A schematic

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representation of the setup is shown in Figure 5.

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All Agilent modules of this configuration were controlled and programmed using Agilent

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Chemstation C.01.04. Data analysis was performed using GC image R 2.5 software

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(GCimage, Lincoln, USA). Operation conditions are reported in the Chromatographic

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Conditions section and Table 1.

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D column and an Acquity UPLC HSS C18 2D column (50 x 2.1 mm; 1.8-µm; 100-Å).

Chromatographic Conditions

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The 1D separations were performed using a mobile phase A: 94% ACN, 5% THF, 1% 12.5

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mM ammonium formate, to mobile phase B: 12.5 mM ammonium acetate. In the 2D the

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mobile phase used was A: 99% 12.5 mM ammonium formate, 1% 1-butanol and B: 99%

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MeOH, 1% 1-butanol30. Butanol was used to reduce the equilibration time. The separation

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performed using the LC/a×m/LC set-up used a dilution flow composed of 12.5-mM

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ammonium acetate.

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The conditions used for the HPLC×HPLC (graphical abstract and Table 1) were: 1D flow rate

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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%

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B. At 180 min the flow rate was set to 1 mL/min and run at 0% B till 200 min. For the 2D

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separation we used a flow rate of 4 mL/min, T=40˚C using, gradient elution from 40% B to

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80% in 0.01 min, from 80% to 100% at 0.76 min, kept at 100% B till 0.86 min and

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equilibrated at 40% B for 0.14 min. Modulation volume= 50 µL. The HPLC×UHPLC

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separation described in Table 1 was performed using a 1D flow rate of 0.04 mL/min, T= 5˚C,

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gradient elution from 2% B to 20% B in 20 min, then to 50% B at 50 min, 55% B at 60 min,

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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

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flow rate of 1.5 mL/min, gradient elution from 35% B to 80% in 0.01 min, 85% to 100% at

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0.35 min, 0.03 min 100% B and at 0.4 min re-equilibration at 40% B. Modulation volume= 20

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µL. The HPLC×UHPLC and HPLC/a×m/UHPLC reported in Figure 6, 7 runs were performed

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Analytical Chemistry

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using a 1D flow rate of 0.08 mL/min, T= 5˚C, gradient elution from 2% B to 20% B in 10 min,

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then to 50% B at 25 min, 55% B at 30 min, 2% B at 39.99 min. Flow rate increased to 0.2

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mL/min at 40 min. In the 2D T=50˚C, gradient elution from 35% B to 80% in 0.01 min, then to

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100% at 0.29 min, 100% B at 0.30 min, followed by 0.06 min re-equilibration at 35% B (0.35

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min modulation time). The 2D flow rate was of 1.5 mL/min in the HPLC×UHPLC setup and

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1.35 mL/min in the HPLC/a×m/UHPLC. The dilution flow in used in the /a×m/ setup was 0.6

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mL/min and this incremented the 1D back pressure off approximately 100 bar. All the LC×LC

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separation where monitored at 220 nm using 80 Hz scan rate.

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Calculations

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The following paragraph describes the equations adopted to obtain the results reported in

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Table 1.

Peak Capacity

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The peak capacity (nc) was determined from the average width at half height (    ) applying  Eq. (1).

 =

+    ) . ∙( 

1

(1)

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For the calculation of the 1D peak capacity the average width was obtained from the

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integration of five peaks at the beginning of the chromatogram (indicated in red in Figure 2b)

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and five peaks in the area of elution of the phosphate species (green in Figure 2b). The 2D

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peak capacity was obtained as average of all the (5 to 15, depending on tG) peaks present in

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the chromatograms.

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The predicted peak capacity for each LC×LC system was calculated as:

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, =  ∙ 

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(2)

Using  values obtained from one dimensional runs under the same conditions as the

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LC×LC runs (see Table 1 for the respective parameters) and  .calculated from the

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Equation (2) is not taking into account 1D undersampling and band broadening associated

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average  of multiple 2D run (2D nc parameter of Table 1)

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with the modulation process3,4,6.

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Two different approaches were used to correct for these effects, using the equation

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proposed by Davis-Li et al.6,31,32

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,

!

=



"# ∙ "# $

=



"# ∙ "#

%&'.'(∙)

* +  # # , *

(3)

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220 221 222

223 224

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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: ; &