Research Article

pubs.acs.org/journal/ascecg

Cocktail Chromatography: Enabling the Migration of HPLC to

Nonlaboratory Environments
Christopher J. Welch,* Timothy Nowak, Leo A. Joyce, and Erik L. Regalado*
Process & Analytical Chemistry, Merck Research Laboratories, Rahway, New Jersey 07065, United States

ABSTRACT: HPLC and HPLC-MS are powerful and wide

ranging analytical tools employed in every aspect of modern
chemical and biomedical research. The use of these tools is
currently restricted to formal laboratory environments, partly due
to cost and complexity but also owing to the special handling
requirements for the solvents consumed and waste generated by
these instruments. Ongoing innovations targeting the decrease in
cost, size, and complexity of HPLC and HPLC-MS instrumenta-
tion raise the intriguing possibility that such tools may soon
become both mobile and widespread in usage, breaking free of
traditional laboratory boundaries. However, the dependence of
these techniques on regulated organic solvents currently limits
such mobility. In this study, we investigate the use of distilled
alcohol spirits (cachaça, rum, vodka, aguardiente, etc.) as well as other household items typically available in a supermarket
(vinegar, ammonia) as mobile phases and additives for carrying out HPLC and HPLC-MS experiments, showing that in many
cases excellent analytical performance can be obtained using these low cost, universally available, green alternatives to the organic
solvents typically used in such studies.
KEYWORDS: Food chemistry, Green chromatography, Ethanol-based mobile phases, Microfluidic chromatography,
High performance liquid chromatography

■ INTRODUCTION
The technique of high performance liquid chromatography
(Figure 1a). Given the omnipresence of HPLC instrumentation
within research laboratories, the cumulative solvent use and
(HPLC) is a dominant method for analysis in modern chemical waste production of analytical HPLC has been identified as a
and biomedical research,1−6 especially when coupled with mass significant target for green chemistry intervention.29−37 Most
spectrometry detection (HPLC-MS).7−15 HPLC and HPLC- HPLC analysis is carried out in the so-called reversed phase
MS instruments line the laboratory benches wherever research mode, with hydrophobic stationary phases and an eluent mixture
into pharmaceuticals, biomedicine, agrochemistry, specialty of water and organic solvent, typically acetonitrile or methanol,
chemicals, and food science is carried out. Currently, the use containing small amounts of additives such as trifluoracetic acid
of these tools is restricted to formal laboratory environments, or ammonium formate.38,39 Consequently, the incoming stream
partly due to cost and complexity, but also owing to the special of solvents and additives for HPLC analysis must be purchased
handling requirements for the solvents consumed and waste from specialty chemical suppliers, shipped, stored, and
generated by these instruments (often mixtures of water with inventoried under strict handling requirements. Typical purchase
acetonitrile or methanol and small amounts of additives such costs for methanol and acetonitrile HPLC-grade solvents are
as triflouoroacetic acid, ammonium formate, etc.). Innovations about $50−130/L. In addition, the controlled on-site storage
targeting decreased cost, size, and complexity of HPLC and MS and delivery of regulated solvents, as well as the collection and
instrumentation16−28 raise the intriguing possibility that such disposal of hazardous waste solvents, adds significantly to the
tools may soon become both mobile and widespread in usage, overall cost of operating LC-MS instrumentation.
breaking free of traditional laboratory boundaries. However, the Replacement of acetonitrile or methanol with ethanol has
dependence of these techniques on regulated organic solvents been identified as a greener alternative for improving the
currently limits such mobility. solvent use profile of HPLC (Figure 1b). While chromato-
Figure 1 outlines the current state and several future scenarios graphic efficiency and overall performance typically suffers
for greener HPLC analysis. Current practice centers around a with the switch to ethanol, significant recent improvements in
standard developed nearly 50 years ago: an HPLC column with HPLC column and instrumentation technologies40−44 mean
an inner diameter of 4.6 mm and a length of 25 cm, operating at
a flow rate of about 1.5 mL/min, and leading to the consump- Received: February 20, 2015
tion of about a liter of solvent per instrument per day, with Revised: March 17, 2015
the concomitant production of an equivalent amount of waste Published: April 2, 2015

© 2015 American Chemical Society 1000 DOI: 10.1021/acssuschemeng.5b00133

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Figure 1. Developing new green alternatives to perform liquid chromatography experiments is not only a way to protect the environment but is also
a pathway to point of use applications beyond the traditional laboratory.

that there is often “performance to spare”, making the use of costs about $120/L, azeotropically derived 95% alcohol
ethanol-based mobile phases a viable option in some instances.33 (containing 5% water) is often quite suitable for chromatog-
In addition to the green chemistry advantages of moving from raphy and can cost as little as $8/L. Furthermore, distilled
a petrochemical-derived feedstock to a renewable resource, alcohol is available at a relatively inexpensive price from local
the price and availability of ethanol offers compelling incen- markets in virtually all regions of the world, offering the
tives for changeespecially given the volatile price fluctuations possibility of local, sustainable production of a green, nontoxic
for acetonitrile. While HPLC-grade absolute ethanol typically eluent for HPLC-MS studies.
1001 DOI: 10.1021/acssuschemeng.5b00133
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Figure 2. Ethanol composition, cost in $ (a) and UV spectra (b) of all mobile phases used in the study.*These spirits were purchased in a
United States liquor store; costs can drastically change from store to store or country to country.

Use of microscale columns allows significant reduction monitoring of therapeutic drug levels. Similarly, point of use
in overall solvent use, either when conventional solvents are applications in the agricultural experimental station or even
used or when alternative solvents such as ethanol are used individual farms could allow monitoring of crop maturation, or
(Figure 1c). The use of microscale columns has been shown to levels of key pest or disease biomarkers, pesticide levels, or
reduce solvent usage by several orders of magnitude45−50 to other factors relating to optimal timing of planting and harvest.
only a few milliliters per instrument per day. Further reductions Mobile use in law enforcement could allow for the possibility
can easily bring the level of solvent consumption and waste of immediate point of use forensics, allowing for applications
generation to well below 1 mL per instrument per day. In in narcotics investigations or counterterrorism activities. The
addition to the trend toward narrower HPLC columns, recent freedom from toxic and regulated chemicals means that the
improvements in stationary phase performance are enabling mobile HPLC-MS instrumentation could potentially be usable
a move to shorter columns, with many separations and in pristine remote environments, enabling the search for natural
high-throughput analyzes now being possible with very short products in remote jungle locations, islands, wetlands, etc.
columns in the 1−5 cm range.51−54 Together, these trends without fear of contamination. Similarly, environmental monitor-
make possible the scenario depicted in Figure 1c, where a ing of point sources of pollution (oil spills, waste discharge
column, solvent reservoir, and waste reservoir can all fit into a outlets, etc.) could take place in real time or on an ongoing basis
prefabricated unit the size of an ink jet printer cartridge. Current with the use of semipermanently deployed equipment. Military
technology allows for a fully functional LC-MS instrument the uses ranging from personal war gas detection units to deployed
size of a carry-on suitcase, but there is clear possibility that hand- battlefield sensors could also be imagined. Minigreen LC-MS
held instruments the size of a laptop computer or even a smart instrumentation would be well suited to classroom use, and
phone could one day be possible. While a contained solvent/ recent experience has shown that high school students readily
waste reservoir offers an attractive option for increasing the grasp the concepts of LC-MS operation and interpretation of
mobility of HPLC-MS, the reliance on conventional organic results.55,56 Such tools would increase the availability of this
solvents will limit the convenience and ease of use of dealing important equipment at the high school and possibly even the
with such cartridges. Alternatively, the use of ethanol-based grade school level, leading to use in classroom investigations,
systems could allow greater freedom of use in nontraditional science fair projects, etc. (clearly, safeguards surrounding the use
unregulated laboratory spaces, especially if all additives and of a potential intoxicant in the classroom would be required).
components used for the assay are generally regarded as safe, Given recent interest in “molecular gastronomy” and mixol-
perhaps even allowing for the spent cartridges to be disposed or ogy,57−61 the use of minigreen LC-MS in restaurants and cocktail
recycled as common unregulated waste. lounges can easily be imagined, and if the price point can be
The availability of a miniaturized hand-held HPLC-MS driven low enough, a big but not insurmountable challenge,
instrument operating with nontoxic solvents and reagents could the use of minigreen HPLC-MS in the home may eventually be
have a transformative effect on science, allowing application in possible.
environments far beyond the current range of use (Figure 1d). In this study, we investigate the use of distilled alcohol spirits
If cost and performance are sufficiently attractive, application in (cachaça, rum, vodka, aguardiente and grain alcohol) as well
physician and dentist offices can be possible, helping to allow as other household items typically available in a supermarket
point of use assays to aid in rapid diagnosis of disease and (vinegar, ammonia) as mobile phases and additives for carrying
1002 DOI: 10.1021/acssuschemeng.5b00133
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ACS Sustainable Chemistry & Engineering Research Article

Figure 3. HPLC-DAD analysis of test mixture 1 using different spirit alcohol-based mobile phases and column temperatures. Column: Eclipse XDB-
C8 (4.6 cm × 15 cm, 5 μm). A flow rate of 1.2 mL/min was used in experiments performed at 25 °C column temperature, while the flow rate was
increased to 1.5 mL/min when working at 60 °C. Mixture 1: five components of a wide polarity spectrum from uracil commonly used as void marker
in RP separations to antracene, an apolar compound strongly retained in RP mode.*All spirit alcohol-based mobile phase compositions were kept as
listed in Figure 2 (≈ 40%), except for aguardente (38.3%) HPLC-grade ethanol was diluted to 95%.

out HPLC and HPLC-MS experiments, showing that in many Alcohol (Luxco, Inc., St. Louis, MO, U.S.A.), Aguardente Bagaceira
cases excellent analytical performance can be obtained using (Caves do Solar de SãO Domingos, S.A., Anadia, Portugal), Cachaça
these low cost, universally available, green alternatives to the 51 (Compahia Müller de Bebidas, Pirassunuga, Brasil), Bacardi Ron
solvents and chemicals typically used in such studies. Carta Blanca (Bacardi Corporation, San Juan, Puerto Rico), and

■
Smirnoff Vodka No. 21 (Smirnoff Co., Norwalk, CT, U.S.A.), were all
purchased from a local liquor store (Rahway, NJ, U.S.A.) and directly
EXPERIMENTAL SECTION used for analysis after filtration. Components of mixtures 1-3,
Instrumentation. Reversed phase HPLC experiments were hydrobenzoin, 4-flavanone, and omeprazole were all obtained from
performed on two different instruments: ExpressLC-100 microfluidic Fisher Scientific (Fairlawn, NJ, U.S.A.) and Sigma-Aldrich, Inc. (St. Louis,
HPLC instrument (Eksigent Technologies, Dublin, CA, U.S.A.), with MO, U.S.A). Protocatechuic aldehyde, catechol, syringaldehyde, vanillin,
a number of modifications that are described in detail in previous guaiacol, propyl gallate, p-anisaldehyde, cinnamaldehyde, eugenol, and
publication,62 and an Agilent 1100 system (Agilent, Palo Alto, methyl salicylate were obtained from the Merck Building Block Collec-
CA, U.S.A.). The Agilent stack comprised a G1312A binary pump, tion (Newark, NJ, U.S.A.). Ultrapure water was obtained from a Milli-Q
G1367A WPALS autosampler, G1315B diode array detector, and a Gradient A10 from Millipore (Bedford, MA, U.S.A.). The 4.6 mm i.d. by
6120 quadrupole LC/MS detector with electrospray ionization in the 50 mm length, 2.5 μm XBridge phenyl column was purchased from
positive mode. The system was controlled by Chemstation software, Waters Corporation (Milford, MA, U.S.A.). The Eclipse XDB-C8 and
with the FIA mode enabled. UV spectra were measured on a Cary XDB-C8 (4.6 cm × 15 cm, 5 μm) were purchased from Agilent. The
300 Bio UV−visible spectrometer (Agilent, Palo Alto, CA, U.S.A.) by AD-3R (4.6 mm × 100 mm, 3 μm) column was obtained from Chiral
scanning 190 to 600 nm. Technologies (West Chester, PA, U.S.A.).
Beverage Mobile Phases, Chemicals, Reagents, and Stationary Determination of Ethanol, Acetic Acid, and Ammonium
Phases. Ethanol (HPLC grade) was purchased from Acros Organics Hydroxide. Determination of ethanol and acetic acid was performed
(Fair Lawn NJ, U.S.A.). Clear ammonia and distilled white vinegar on an Agilent 6890N GC-FID with an Agilent 7683B injector.
were distributed by Wakefern Food Co. (Elizabeth, NJ, U.S.A.), The system was controlled by Empower 2 Chromatographic System
orange juice, lemon, vitamins, pure vanilla extract, and whole and (Waters, Milford, MA, U.S.A.). Ethanol samples were separated
ground peppercorns were all purchased at a ShopRite store (Rahway, on an Agilent DB-624, 20 m × 0.180 mm, 1.0 μm film thickness
NJ, U.S.A.). Spirits used as mobile phase, including Everclear Grain column. Determination of acetic acid was performed on a DB-FFAP,

1003 DOI: 10.1021/acssuschemeng.5b00133

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Figure 4. Reversed phase HPLC-UV analysis of several pharmaceutical and food-relevant compound mixtures using conventional HPLC instrumentation
with different spirit alcohol-based mobile phases. (a) Column: Eclipse XDB-C8 (4.6 cm × 15 cm, 5 μm). Sample: equal volumes of 1 mg/mL component
solutions in EtOH/H2O; flow rate, 1.2 mL/min; detection, 210 nm; eluent A, H2O; eluent B, vodka; mobile phase, 40% B isocratic. (b) Chromatographic
conditions as described for mixture 1; detection, 220 nm; eluent A, H2O; eluent B, grain alcohol; mobile phase gradient, 45% B for 2 min, 100% B at
4 min, keep 100% B for 3 min. (c) Column: Eclipse XDB-C18 (4.6 cm × 15 cm, 5 μm). Sample: equal volumes of 2 mg/mL component solutions in
EtOH; flow rate, 1.2 mL/min; detection, 220 nm; eluent A, H2O; eluent B, grain alcohol; mobile phase gradient, from 10 to 100% B in 8 min. (d)
Enantioseparation of three different racemic mixtures. Column: AD-3R (4.6 mm × 100 mm, 3 μm); temperature, 40 °C; detection, 210 nm; eluent A,
H2O; eluent B, cachaça or grain alcohol; mobile phase, isocratic 100% B; flow rate, 1.2 mL/min or 1.8 mL/min, respectively.
30 m × 0.320 mm, 1.00 μm film column. Ammonia was determined chromatographic analysis. The orange juice sample was diluted two
using potentiometric titration measurements. The titrations were times before the analysis. Twenty-five μL pure vanilla extract was
performed using a Metrohm LL Unitrode electrode with a Metrohm716 extracted with 2.5 mL EtOH/H2O.
DMS Titrino automatic titrator with a 728 stirrer system (Herisau, SUI). HPLC-MS Conditions. HPLC separations were carried out on a
The titrations were performed using the dynamic equivalence point 4.6 mm i.d. by 50 mm length, 2.5 μm Waters XBridge phenyl column
titration calculated by the Tiamo Software. at a flow rate of 1 mL/min. The LC eluents were 5 mM ammonium
Preparation of Buffer Solutions and Samples. Five mM acetate in both aqueous and organic eluents (pH 4.7). The column
ammonium acetate in H2O (pH 4.7) and 5 mM ammonium acetate in and samples were maintained at a temperature of 30 °C. The
alcoholic spirit solutions (pH 4.7): 8.333 mL clear ammonia (source of misergrams were obtained from periodic sample injections (0.5 μL)
every 1 min. The positive or negative ion ESI parameters were
NH4OH, 2.1%) and 5.884 mL distilled white vinegar (source of acetic
skimmer 45 V, desolvation gas (N2), temperature 350 °C, and flow
acid, 5.1%) were dissolved in 1 L solvent. The pH was adjusted by
rate 12 L/min; the nebulizer was adjusted to 35 psig, fragmentor, and
adding vinegar. the capillary voltage to 150 and 3000 V, respectively.

■
A total of 100 mg black pepper was extracted with 4 mL EtOH;
2260 mg lemon slices, 5 mL orange juice, and 20 mg vitamin pill
samples were extracted with 10, 15, and 10 mL of EtOH:H2O (1:1),
RESULTS AND DISCUSSION
respectively. The suspensions were sonicated for 15 min in an Distilled ethanol spirits are available worldwide in local markets,
ultrasonic bath and then centrifuged at 12000g for 5 min at 20 °C. but the quality and purity can vary widely. In this study, vodka,
The supernatants were filtered and transferred to HPLC vials for caçacha, rum, aguardente, and grain alcohol were obtained from
1004 DOI: 10.1021/acssuschemeng.5b00133
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Figure 5. LC-MS analysis of several food-relevant mixtures using 5 mM ammonium acetate as mobile phase additive in a grain alcohol-based eluent
and rum. Column: XBridge Phenyl (4.6 cm × 50 mm, 2.5 μm); flow rate, 1 mL/min; samples in EtOH/H2O (see details on buffer and sample
preparation in the Experimental section). (a) Conventional gradient separation of a black pepper extract. Eluent A, 5 mM ammonium acetate in H2O
(pH 4.7); eluent B, 5 mM ammonium acetate in 82% EtOH (grain alcohol, pH 4.7); mobile phase gradient, from 30 to 100% B in 12 min; detection,
ESI (+), the indicated EIC at m/z values of each piperamides as illustrated in the same figure. (b−d) MISER LC-MS analysis allows simple
visualization of relative amounts of piperine in several black peppers, vitamin C in different sources, and caffeine in a variety of teas. Detection: SIM
ESI-MS(±) at respective m/z values; 0.5 μL injection every 1 min. Isocratic mobile phases (pH 4.7): (b) 5 mM ammonium acetate in 82% EtOH
(grain alcohol). (c and d) 5 mM ammonium acetate in 36.4% EtOH (rum).

a local liquor store and compared with HPLC-grade ethanol We next investigated the use of the various alcohol spirits as
obtained from a chemical supply house (Figure 2). The cost chromatographic eluents, focusing initially on HPLC analysis of
and ethanol content varies considerably among these products, a mixture of five standard compounds by gradient elution
with grain alcohol (95%) being the best value at ∼$22/liter of reversed phase chromatography with UV detection (Figure 3).
ethanol and rum (40%) being the worst value at ∼$49/liter of Interestingly, the use of grain alcohol as an eluent shows
ethanol (Figure 2a). Independent testing of alcohol content by performance that is very comparable to that achieved with
gas chromatography (GC) generally substantiated label claims. HPLC-grade ethanol, even with UV detection at 210 nm. On
the other hand, the relatively poor UV transparency of vodka,
Figure 2b illustrates the UV spectrum of the various distilled
cachaça, rum, and aguardiente at low wavelengths required
spirits compared with HPLC-grade ethanol. The large UV analysis at wavelengths ≥220 nm, while the relatively low
absorbance of aguardente in particular renders this spirit of alcohol percentage of these spirits (nominally 40−46%)
limited value for investigations involving detection in the low resulted in very strong retention for the least polar component
UV range. On the other hand, the UV absorbance of rum, (5, anthracene). Performing the chromatographic separation at
caçacha, and vodka are significantly better, and the UV elevated temperature (60 °C) does enable elution of anthracene,
absorbance of grain alcohol closely approaches that of HPLC- albeit at an unacceptably long retention time of 16−20 min.
grade ethanol. While this shortcoming could potentially be mitigated by the use
1005 DOI: 10.1021/acssuschemeng.5b00133
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Figure 6. Chromatographic profiles of vanilla extract by two different LC-UV systems. (a) Conventional HPLC-UV analysis using 0.1% acetic acid in
30% EtOH (vodka) as mobile phase. (b) Eksigent Microfluidic HPLC-UV analysis using 0.1% acetic acid in vodka (40% EtOH) as mobile phase.
UV Detection at 280 nm.

of a less hydrophobic stationary phase (e.g., C4), grain alcohol would be negated by the addition of regulated additives such as
would seem to be the most generally suitable replacement for trifluoracetic acid, we investigated the use of LC-MS ionization-
HPLC-grade ethanol, especially when the analysis of highly enhancing additives derived from easily obtainable vinegar
hydrophobic and strongly retained components is important. On (acetic acid) and ammonia cleaner (NH4OH) obtained from
the other hand, the use of vodka, cachaça, or rum may potentially a local supermarket. Exact measurement of concentration
be of service when investigating analytes that are not strongly (849.7 mM for acetic acid and 599.3 mM for NH4OH) by
retained in reversed phase mode. titration allowed mixing a cocktail containing 5 mM ammonium
A variety of examples of the use of distilled alcohol spirits acetate, which afforded excellent ionization in the LC-MS
as chromatographic eluents for HPLC studies are shown in studies highlighted in Figure 5. Examination of the ethanolic
Figure 4. Separation of the relatively nonpolar purine alkaloids extract of whole grain black peppercorns using a grain alcohol-
(caffeine, theophiline, and theobromine) is easily accomplished based eluent showed the presence of piperine as well as a
using an isocratic vodka-based mobile phase (Figure 4a) with number of congeners depicted in Figure 5a. MISER LC-MS is a
detection at 210 nm, while the separation of the flavonoids simplified form of LC-MS that is well suited to fast analysis
resveratrol, quercetin, and emodin requires gradient elution of the presence of a given component (in this case, piperine,
using the stronger grain alcohol eluent at 220 nm. Clearly, the m/z = 286 amu) within multiple samples. Repeated injections
need to perform analysis at slightly higher UV wavelength in of different samples with continuous data recording provides a
gradient separations may present a problem for trace detection “misergram”, a graphical depiction of piperine levels in the
of some analytes with relatively poor UV chromophores; various commercial pepper extracts, allowing straightforward
however, the potential strength of the approach is highlighted interpretation of results with an analysis time of about one
by the excellent separation of a mixture of 11 plant-derived sample per minute (Figure 5b).63 Similarly, high-throughput
simple natural products using a grain alcohol-based eluent, MISER LC-MS using a rum-based eluent (Bacardi) containing
even with detection at 210 nm. In addition, the fast enantio- 5 mM ammonium acetate derived from household vinegar
separation of natural products and pharmaceutically relevant and ammonia cleaning solution can be used for the analysis of
compounds by chiral chromatography using cachaça- or grain vitamin C in various sources (Figure 5c) or the simultaneous
alcohol-based mobile phases presented in Figure 4d clearly measurement of caffeine and theanine present in brewed teas
illustrates the potential for using spirit alcohol-based mobile (Figure 5d).
phases for separation modes beyond simple reversed phase While these results are impressive, requiring, for example,
chromatography. only 1 min per sample for the LC-MS analysis of piperine,
We next turned our attention to the use of HPLC-MS using caffeine, theanine, or vitamin C using inexpensive rum or
spirit alcohol-based eluents. Mobile phases regularly used in grain alcohol, each analysis still consumes about 1 mL of spirits
LC-MS analysis contain additives to improve the MS ionization, (cost ∼$0.02) and generates an equivalent amount of waste.
for example, triflouoroacetic acid or ammonium salts, allowing Microcolumn HPLC and HPLC analysis provides an attractive
detection in both negative and positive ion modes. Reasoning alternative to conventional approaches, with the typical column
that the use of a sustainable nontoxic chromatographic eluent diameter being reduced from 4.6 to 0.5 mm or even smaller,
1006 DOI: 10.1021/acssuschemeng.5b00133
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and typical flow rate being reduced from 1 to 1.5 mL/min to used in this study. We also thank Sharon M. O’Brien (Merck
4−20 μL/min. Figure 6b shows an example of the use of Creative Studios) for helping with graphic design.
microflow chromatography using spirit alcohol-derived mobile
phases. Comparable chromatographic performance is obtained
relative to the conventional-sized experiment (Figure 6a), with
■ DEDICATION
Dedicated to our friend, Prof. Koji Nakanishi, on the occasion
considerable savings in eluent consumption. As a case in point, of his 90th birthday.

■
the high-throughput analysis of vanillin in a vanilla extract
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(15) Regalado, E. L.; Tolle, S.; Pino, J. A.; Winterhalter, P.;
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1007 DOI: 10.1021/acssuschemeng.5b00133

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