Comparison of three methods for the methylation of aliphatic and aromatic compounds

Article abstract of DOI:10.1002/rcm.7947

Rationale: Methylation protocols commonly call for acidic, hot conditions that are known to promote organic ¹H/²H exchange in aromatic and aliphatic C–H bonds. Here we tested two such commonly used methods and compared a third that avoids these acidic conditions, to quantify isotope effects with each method and to directly determine acidic-exchange rates relevant to experimental conditions. Methods: We compared acidic and non-acidic methylation approaches catalyzed by hydrochloric acid, acetyl chloride and EDCI (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide)/DMAP (4-dimethylaminopyridine), respectively. These were applied to two analytes: phthalic acid (an aromatic) and octacosanoic acid (an aliphatic). We analyzed yield by gas chromatography/flame ionization (GC/FID) and hydrogen and carbon isotopic compositions by isotope ratio mass spectrometry (GC/IRMS). We quantified the ¹H/²H exchange rate on dimethyl phthalate under acidic conditions with proton nuclear magnetic resonance (¹H-NMR) measurements. Results: The δ²H and δ¹³C values and yield were equivalent among the three methods for methyl octacosanoate. The two acidic methods resulted in comparable yield and isotopic composition of dimethyl phthalate; however, the non-acidic method resulted in lower δ²H and δ¹³C values perhaps due to low yields. Concerns over acid-catalyzed ¹H/²H exchange are unwarranted as the effect was trivial over a 12-h reaction time. Conclusions: We find product isolation yield and evaporation to be the main concerns in the accurate determination of isotopic composition. ¹H/²H exchange reactions are too slow to cause measurable isotope fractionation over the typical duration and reaction conditions used in methylation. Thus, we are able to recommend continued use of acidic catalysts in such methylation reactions for both aliphatic and aromatic compounds.

Full text of DOI:10.1002/rcm.7947

Comparison of three methods for the methylation of aliphatic and aromatic compounds
Hyejung Lee1*, Sarah J. Feakins1, Zhiyao Lu2, Arndt Schimmelmann3, Alex L. Sessions4,
Jessica E. Tierney5, Travis J. Williams2
1Department of Earth Sciences, University of Southern California, Los Angeles, CA 90089, USA
2Department of Chemistry, University of Southern California, Los Angeles, CA 90089, USA
3Department of Earth and Atmospheric Sciences, Indiana University, Bloomington, IN 47405-1405, USA
4Division of Geological and Planetary Sciences, California Institute of Technology, Pasadena, CA 91125,
USA
5Department of Geosciences, University of Arizona, Tucson, AZ 85721, USA
*Corresponding author: hyejungl@usc.edu
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doi: 10.1002/rcm.7947
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Abstract
Rationale
Methylation protocols commonly call for acidic, hot conditions that are known to promote
organic 1H/2H exchange in aromatic and aliphatic C—H bonds. Here we tested two such
commonly-used methods and compared a third that avoids these acidic conditions, to
quantify isotope effects with each method and to directly determine acidic-exchange rates
relevant to experimental conditions.
Methods
We compared acidic and non-acidic methylation approaches catalyzed by hydrochloric acid,
acetyl chloride and EDCI (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) / DMAP (4-
dimethylaminopyridine) respectively. These were applied to two analytes: phthalic acid (an
aromatic) and octacosanoic acid (an aliphatic). We analyzed yield by gas chromatography
flame ionization (GC/FID) and hydrogen and carbon isotopic composition by isotope ratio
mass spectrometry (GC/IRMS). We quantified the 1H/2H exchange rate on dimethyl phthalate
under acidic conditions with proton nuclear magnetic resonance (1H-NMR) measurements.
Results
The δ2H and δ13C values and yield were equivalent among the three methods for methyl
octacosanoate. The two acidic methods resulted in comparable yield and isotopic composition
of dimethyl phthalate; however, the non-acidic method resulted in lower δ2H and δ13C values
perhaps due to low yields. Concerns over acid-catalyzed 1H/2H exchange are unwarranted as
the effect was trivial over a 12-hour reaction time.
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Conclusions
We find product isolation yield and evaporation to be the main concerns in the accurate
determination of isotopic composition. 1H/2H exchange reactions are too slow to cause
measurable isotope fractionation over the typical duration and reaction conditions used in
methylation. Thus, we are able to recommend continued use of acidic catalysts in such
methylation reactions for both aliphatic and aromatic compounds.
INTRODUCTION
Stable hydrogen isotopic compositions (δ2H values) of organic compounds are widely used in
organic geochemistry, biochemistry, ecology, climate, and forensics. Applications include the
study of the hydrologic cycle and climate,[1–3] ecology,[4–6] biosynthetic pathways of lipids,[7,8]
paleoelevation,[9,10] diagenesis of organic matter,[11] and food authenticity and
provenance.[12,13] In many compounds, hydrogen positions include those in C—H bonds that
are non-exchangeable after biosynthesis and preserved under low temperature sedimentary
conditions. They carry the original biosynthetically fixed H, although even these H may be
exchanged under elevated temperatures and over geologic time.[14,15] In contrast, O—H bonds,
as found in cellulose or fatty acids, contain H atoms that are readily exchangeable with
ambient water in the plant,[16] soil, or sediments after burial.[11] In order to recover signals
relating to the original plant biosynthesis, analyses typically target only the carbon-bound
hydrogens, by masking exchangeable O—H hydrogens. The preparative steps for cellulose
involve ‘nitration’ (i.e. formation of nitric acid esters[17]), whereas derivatization of aliphatic
and aromatic acids typically involves esterification with methanol. Here, we examine isotopic
exchange associated with reaction conditions used for esterification prior to isotopic analysis.
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Acid catalyzed esterification
Organic compounds with carboxylic acid groups are commonly esterified with methanol
prior to gas chromatography (GC) analysis[18,19] using acidic catalysts. Hydrochloric acid
(HCl) and acetyl chloride (CH3COCl), which forms anhydrous HCl in methanol, are among
the most widely used catalysts of aliphatic and aromatic acids. The carboxylic acid is initially
protonated and undergoes an exchange reaction with the alcohol to give the reaction
intermediate. The intermediate can then lose a proton to become an ester. The equilibrium
point of this reaction is displaced so that esterification proceeds virtually to completion in
excess alcohol.[20] However, Brønsted acid catalyst can also promote hydrogens bound to an
aromatic ring to undergo 1H/2H exchange via electrophilic aromatic substitution (Figure
1).[21–23] Thus, during esterification, hydroxyl hydrogens in methanol can exchange with
aromatic hydrogens in an analyte molecule and the δ2H values can equilibrate.
Understanding acid-catalyzed isotope exchange during esterification is relevant to the study
of aromatic compounds, but also indirectly for the study of aliphatic compounds such as the
alkanoic acids derived from plant leaf waxes and microbial lipids, because aromatic
structures are commonly used as part of the isotopic determination.[24] One method to
determine the δ2H values and the carbon isotopic composition (δ13C values) of methyl groups
added in alkanoic acid esterification steps involves the use of phthalic acid.[25] Phthalic acid
of known isotopic composition (measured independently as a sodium salt) can be methylated
and measured by gas chromatography isotope ratio mass spectrometry (GC/IRMS) to
calculate the isotopic composition of the methyl hydrogens and carbon via mass balance.[19]
Because phthalic acid has an aromatic ring, conventional acidic esterification conditions
should enable 1H/2H exchange on the aromatic hydrogens (Figure 1).[21–23] Such exchange
may result in an incorrect estimation of the overall dimethyl phthalate isotopic composition
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and mass balance estimation of the δ2H value of methyl hydrogens, needed when calculating
the isotopic composition of the original fatty acid.[26] However, the magnitude of such an
effect is currently unknown.
Non-acidic coupling reagents to promote esterification
Alternatively, non-acidic approaches to methylation exist that may obviate the above
concerns. In this study, we tested a non-acidic method using 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide (EDCI) as a coupling reagent with 4-
dimethylaminopyridine (DMAP) as the catalyst (Figure 2).[27] Carbodiimides, due to their
accessibility and versatility, are ranked as one of the most important classes of compounds in
organic chemistry.[28] The most widely used carbodiimide reagent, dicyclohexylcarbodiimide
(DCC), makes insoluble urea byproducts requiring additional cumbersome purifications.[29]
Hence we chose EDCI, a water soluble carbodiimide with water soluble urea byproduct,[30]
combined with DMAP which has been shown to increase the efficiency of esterification by
104 fold.[31] We compared this approach with conventional acidic methylation reactions.
EDCI and DMAP are not expected to lead to 1H/2H exchange because the reaction conditions
are buffered, and the merits of these reagents for organic geochemical applications are tested
here.
Method comparison
In this study, we tested two acidic and one non-acidic methods of esterification as applied to
phthalic acid and octacosanoic acid to evaluate any isotopic fractionations and practical
issues arising with each using GC/IRMS and GC/FID approaches. We further directly
measured the 1H/2H exchange rate in phthalic acid methylation with 5 vol% HCl using proton
nuclear magnetic resonance (1H-NMR). Based on the exchange rate, we estimate the
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proportion of aromatic H substituted over 12 hours of reaction time from the initial δ2H
values of the reactants. The goal is to quantify the δ2H and δ13C perturbation possible with
each esterification method and to make practical recommendations for esterification prior to
GC/IRMS analyses.
EXPERIMENTAL
Phthalic acid and methanol of known isotopic composition
The hydrogen isotopic composition of phthalate was measured in Na-phthalate to exclude
exchangeable, carboxylic acid hydrogens and the δ13C value was measured from the free
diacid via offline combustion at Indiana University (Bloomington, IN, USA). Offline
combustion consisted of phthalic acid combustion in quartz ampoules and cryogenic
purification of combustion gases in a vacuum line. The aromatic hydrogen isotopic
composition was −95.5 ± 2.2 ‰ (, n = ) and the carbon had a δ13C value of −27.21 ± 0.02
‰ (n = ). The uncertainties reported represent replicate precision. Unquantified uncertainty
in the absolute value may exist, and the largest source of concern would be water inclusion in
the Na-phthalate crystals. In order to mitigate against the influence of water inclusions, the
crystals are warmed before combustion.
Methanol (MeOH) was 99.8 % anhydrous and the δ2H value of the methyl group was −141 ±
3 ‰ (n = 3), calculated via mass balance between bulk methanol and hydroxyl hydrogen at
Indiana University. Excess sodium metal and a small amount of methanol were reacted in an
evacuated, sealed ampoule to liberate H2 gas from the hydroxyl hydrogen for isotopic
determination. The uncertainties reported represent replicate precision. Unquantified
uncertainty in the absolute value may exist, the largest source of such error would be
associated with residual methanol vapor, although this is mitigated against by allowing the
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reaction to proceed for several days. The hydroxyl hydrogen has a δ2H value of −27 ‰ and
thus can provide the relatively 2H-enriched hydrogen source for exchange into the phthalate
aromatic ring during acid-catalyzed equilibration. The other H+ in the 5 vol% catalyst (from
HCl and water) can exchange freely with methanol’s hydroxyl hydrogen and their isotopic
compositions are unknown. Nonetheless, the dominant source of exchangeable hydrogens is
from methanol, which is 95 % of the reaction solvent. The δ13C value of the methanol was
−46.77 ± 0.04 ‰, from bulk measurement. The isotopic compositions of phthalic acid and
methanol were used to calculate the predicted isotope values of dimethyl phthalate via Eq. 1
for hydrogen and Eq. 2 for carbon.
(1)
(2)
The acidic methods: HCl and CH3COCl
For the HCl catalyzed methylation, 3 mg of phthalic acid or 3 mg of C28 n-alkanoic acid were
methylated with 1 mL of a mixture of 5 vol% HCl and 95 % MeOH in a tightly sealed 15-mL
culture tube at 70 °C overnight. For the acetyl chloride catalyzed methylation, the same
procedure was followed using CH3COCl (instead of HCl) and reacting at 50 °C overnight.
Practical guidance on methylation protocols are provided in the supplementary material, and
are briefly summarized here.
Following overnight methylation reactions, the samples were removed from the heat, and 1
mL of Milli-Q water (Millipore Milli-Q Plus QPAK 2; Millipore, Billerica, MA, USA) was
then added to produce an aqueous phase. The analyte was extracted using liquid-liquid
extraction with 1 mL of dichloromethane (DCM) for dimethyl phthalate and hexane for n-
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alkanoic acid methyl esters. For each extraction (repeated 3 or more times), the mixture was
vigorously shaken, left to partition into two phases and the organic phase, containing
dimethyl phthalate or fatty acid methyl ester (FAME), was extracted by pipette and dried by
passage through a column of anhydrous Na2SO4. The organic fraction was further purified by
passing through a silica column (5 cm ⅹ 40 mm Pasteur pipette, 5 wt% water-deactivated
silica gel, 100-200 mesh) eluting with three column volumes of DCM for dimethyl phthalate
and for FAME.
The non-acidic method: EDCI and DMAP
A general procedure for synthesis of ester derivatives in Lehmann et al [27] was followed, with
adjustments for scale. 22.5 mg of EDCI (8 mol ratio to phthalic acid or octacosanoic acid)
and 1.1 mg of DMAP (0.5 mol ratio) and 3 mg of phthalic acid or octacosanoic acid (C28 n-
alkanoic acid) were added to 1 mL of isotopically known methanol and the solution was kept
at room temperature overnight. The solution was diluted with 1 mL of Milli-Q water. The
methylated product was extracted through the addition of 1 mL of ethyl acetate. The mixture
was shaken vigorously to allow liquid:liquid extraction to proceed, and then allowed to settle
into two phases, and this process was repeated at least three times. The organic fraction,
containing dimethyl phthalate or methyl octacosanoate, was dried by passage through a
column of anhydrous Na2SO4. It was further purified by eluting the ester fraction through a
silica column with three column volumes of ethyl acetate.
GC/MS/FID analysis
The methylation products generated in this study, including dimethyl phthalate and C28
FAME, were identified and quantified using gas chromatography (Agilent 6890 series)
coupled with a single quadrupole mass-selective detector (Agilent 5973), using electron
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ionization with an ionization energy of 69.9 eV) and flame ionization detection (GC/MS/FID
instrumentation supplied by Agilent, Santa Clara, CA, USA). The instrument was equipped
with a Rxi-5 ms column (30 m ⅹ 0.25 mm, film thickness 0.25 m supplied by Restek
Corporation, Bellefonte, PA, USA) with a 4 mL min−1 constant column flow rate, split
passively 4:1 (based on theoretical calculations following Poiseulle’s law) between the mass
spectrometer (via a 80 cm ⅹ 0.25 mm silica capillary) and the flame ionization detector (via
a 120 cm ⅹ0.1 mm silica capillary). Helium was used as the carrier gas. The initial
temperature of 50 °C was held for 3.5 min, followed by a temperature ramp of 20 °C min−1 to
300 °C and maintained for 10 min. 1 L of sample was injected in splitless mode. Absolute
abundance was calculated using a calibration curve of an in-house standard (mixture of n-
alkanes and n-alkanoic acids) and their peak area response on the flame ionization detector.
Yields were calculated based on the chemical equation using masses of reagents, quantified
product abundances and are reported as % of predicted product. All uncertainties are reported
as standard deviations () unless otherwise stated.
GC/IRMS analysis
Compound-specific hydrogen and carbon isotopic compositions were measured using
GC/IRMS. Samples were introduced via a programmable temperature vaporizing (PTV)
injector in splitless mode at 50 C to a Thermo Scientific® Trace gas chromatograph. The gas
chromatograph was equipped with a Rxi-5 ms column (30 m ⅹ 0.25 mm, film thickness 0.25
m) and connected via an Isolink with a pyrolysis furnace at 1400 °C for hydrogen isotopic
analysis, and via a combustion furnace at 1000 °C for carbon isotopic analysis, through a
Conflo IV interface to a DeltaVPlus isotope ratio mass spectrometer (all supplied by Thermo
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Scientific, Bremen, Germany). The gas chromatograph was operated with a He flow rate of 1
mL min−1 from an initial temperature of 50 °C, followed by a ramp of 20 °C min−1 to 300 °C,
and held for 5 min.
+
+
For hydrogen, the H3 factor was measured daily to correct for the formation of H3 in the ion
source[32], and to check for linearity, with a mean value over the course of measurements
reported here of 3.6 ppm mV−1  0.35 (n = 6) across a range of 1−8 V. For carbon, the
standard deviation of CO2 pulses spanning 1–8 V was determined daily to check for linearity
and the standard deviation across that range averaged 0.02 ‰. Gas peaks of H2 or CO2 as
appropriate were co-injected bracketing the analyte peaks during the GC/IRMS run. Two of
these peaks were used for standardization between sample and standard, while the rest were
treated as unknowns to assess precision. Data were then normalized to the Vienna Standard
Mean Ocean Water (VSMOW)/Standard Light Antarctic Precipitation (SLAP) hydrogen
isotopic scale via comparison with an external standard with 15 n-alkanes (C16-C30 n-alkane
mixture of type A, isotopic standard from Indiana University), with δ2H values ranging from
−46 to −227 ‰. For carbon, the isotopic results were normalized to Vienna PeeDee
Belemnite (VPDB) / lithium carbonate prepared by H. Svec (LSVEC) by comparing with A
mix standard δ13C values ranging from −28.6 to −33.3 ‰. The standard was run daily
throughout the sequence. Measured δ2H values of the samples are normalized using a
regression between the known and measured values of the standards. The root-mean-square
error of replicate analysis of the external standard (A mix) averaged 4.6 ‰ for hydrogen and
0.2 ‰ for carbon. The results are reported using conventional delta (δ) notation in permil (‰).
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1H/2H exchange rate using 1H-NMR
We conducted a proton nuclear magnetic resonance (1H-NMR) kinetic experiment to measure
the rate of 1H/2H exchange on dimethyl phthalate in 5 vol% HCl (12 M) using a Varian
VNMRS-600 (supplied by Varian, Inc. now Agilent Technologies, Palo Alto, CA, USA) at
the University of Southern California (Loa Angeles, CA, USA). We reacted 30 mg of
phthalic acid in a solution of 0.60 mL of methanol-d4 (C2H3O2H) and 5 vol% HCl in a
Wilmad J Young NMR tube (type of NMR tube with a Teflon cap that can be attached to a
vacuum line, supplied by Wilmad-labglass, Vineland, NJ, USA) sealed with plastic cap and
parafilm, in an oil bath at 70 C. We measured the phthalate H peak areas daily for ten days
after the overnight esterification reaction. The tube was placed back in the oil bath after
measurement to maintain the reaction conditions over the ten days. The peaks were analyzed
with a relaxation delay of 10 sec. A decrease in peak area detected by 1H-NMR indicated
exchange of 1H on the phthalate with 2H from the methanol-d4 solvent.
RESULTS AND DISCUSSION
Product yield
We found that product yields varied between methods and analytes. For dimethyl phthalate,
the acidic methods resulted in > 70 % product yield, whereas the carbodiimide method
resulted in yields < 30 % (Fig. 3, Supplementary Table 1, supplementary material). For
methyl octacosanoate, all methods yielded > 80 % product yield, but again yields were
highest for the acidic methods > 94 % (Supplementary Table 2, supplementary material).
Low product yield (observed here for the dimethyl phthalate using the non-acidic method) is
a concern given the potential for isotopic fractionation associated with incomplete reaction or
recovery. Low yields are also problematic for many applications because sample size
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requirements increase. We investigate here the potential reasons for low yields and discuss
our attempts to improve them.
Yield for acidic methods
High yields were obtained using acidic methods for methylation, with acetyl chloride (mean
yield 80 %, = 5, n = 10) or HCl (mean yield 73 %, = 5, n = 13) as the starting reagent,
consistent with theoretical product yield for Fischer esterification.[20] The higher yield for the
acetyl chloride method is due to the lower water content of the reagent, compared with HCl
(12 M solution). These high yields were achieved after experimentation to optimize handling
to minimize loss (Supplementary Table 3, supplementary material). For example, the boiling
point of dimethyl phthalate is 283 °C and we observed significant removal of the product by
GC/FID under normal solvent blow-down on a hotplate; thus, we avoided using the hotplate.
We also improved our yield by up to 10 % by switching from hexane (used for aliphatic
esters) to DCM during liquid-liquid extraction, given the greater polarity of dimethyl
phthalate. Still, we found dimethyl phthalate has a lower isolated yield than aliphatic esters
(Supplementary Table 1, supplementary material), suggesting that extraction and purification
of dimethyl phthalate could be further improved. Less than 100 % isolated yield could be due
to either incomplete liquid-liquid extraction or evaporative loss prior to measurement.
Yield for carbodiimide method
The isolated yield of dimethyl phthalate was low (mean 22 %) using the carbodiimide method
and this could be due to incomplete reaction or loss during purification. We monitored the
reaction with thin layer chromatography (TLC) at the beginning and end of the reaction (after
12 hrs) and we confirmed that the product had formed while phthalic acid had been
completely consumed. Additional evidence for reaction completion was obtained by testing
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for the absence of phthalic anhydride, a reaction intermediate for the carbodiimide method, at
the end of the experiment by GC/MS. Thus, we can rule out incomplete reaction.
Low product yield using carbodiimide reagents for esterification has been previously noted,
due to the carboxylate being consumed during the formation of an N-acylurea byproduct[33]
.
Addition of DMAP as a catalyst inhibits the byproduct formation and facilitates the reaction
to form the ester, which has been shown to increase yield.[31] Following this protocol, we
obtained < 30 % expected yield of dimethyl phthalate, while obtaining > 80 % yield of
methyl octacosanoate. The low yield of dimethyl phthalate may be explained by its greater
volatility than methyl octacosanoate.
We tested yield response to various reaction conditions such as reagent concentration,
temperature and duration of reaction for the carbodiimide approach, although tests of
intermediates showed that reactions went to completion. In case isolation and purification
steps were the issues affecting low isolation yields we tested different solvents for extraction,
as well as citric acid washing and brine washing in efforts to improve the isolated product
yield. Each of these efforts failed to generate yield > 30 % (Supplementary Table 4,
supplementary material). Having experimented considerably with this approach, we find it is
not currently a pragmatic protocol for low concentration (3 mg) dimethyl phthalate
derivatization applications. In contrast, the method does have promise as a viable method for
derivatization of aliphatic acids.
Nevertheless, we note additional practical challenges for the use of the carbodiimide method
prior to GC analyses. We found it necessary to clean the syringe, inlet and trim the column
after running the carbodiimide samples by GC/IRMS probably due to urea persisting in the
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mixture even after liquid-liquid extraction. If these compounds were injected in measurable
quantity, due to their high polarizability and protic nature, they could interact with the GC
column. We do not, however, observe any humps or fronting and tailing of the analyte peak
in the chromatogram; our analytical results for both quantification and isotopic composition
therefore appear to be unaffected by any such polar contaminants. Regardless of analyte, the
carbodiimide method presented challenges for subsequent GC analyses that should be
addressed if this approach is to be adopted for aliphatic GC applications.
Isotopic Analyses
The expected isotopic ratios of dimethyl phthalate calculated from mass balance (Eq. 1 and
Eq. 2) were δ2H values of –123 ‰ and δ13C values of –31.1 ‰. For carbon isotopes, acidic
methods successfully resulted in values that are close to those expected (Figure 3). Acetyl
chloride and HCl methods produced δ13C values of –31.5 ± 0.0 ‰ (n = 3) and –31.3 ± 0.1 ‰
(n = 3). The non-acidic method resulted in a more negative value of –32.7 ± 0.4 ‰ (n = 4).
The hydrogen isotopic composition of dimethyl phthalate differed between the acidic and
non-acidic esterification methods (Figure 3). Dimethyl phthalate produced with acetyl
chloride and HCl resulted in δ2H values of −105 ± 2 ‰ (n = 3) and −107 ± 1 ‰ (n = 3),
respectively, thus being equal within uncertainty although +16 to +18 ‰ more 2H-enriched
than expected. The carbodiimide method resulted in a more negative δ2H value of −137 ± 1
‰ (n = 4). We calculated the initial methyl hydrogen and carbon isotopic composition of
methanol[25] by mass balance and found that none of the methods accurately assessed the
(known) initial methyl δ2H and δ13C values. Acidic methods produced 25 – 29 ‰ more
positive estimates for methyl δ2H values whereas the carbodiimide method produced 25 ‰
more negative estimates than the known value of the methanol −141 ± 3 ‰, n = 3 (Table 1).
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This inaccuracy is greater than analytical errors, the largest of which is the calibration to the
international VSMOW-SLAP isotopic scale (see Methods). This suggests an isotopic
fractionation during the methylation reaction and/or purification and one that differs between
the acidic and non-acidic methods.
Is acid-catalyzed exchange a cause of 2H-enrichment?
We directly measured 1H/2H exchange rates for dimethyl phthalate by 1H-NMR under
conditions relevant to the acidic methylation method. We found the same exchange rates
(slopes) within uncertainties for the hydrogens on the alpha and beta carbon positions of
dimethyl phthalate (Figure 4). Using the mean slope as the exchange rate constant (4.94
10-4 hr-1), this corresponds to an exchange half-life (the time to exchange 50 % of aromatic
hydrogens,
) of 1403 hours (58.5 days) at 70 °C. For a 100 ‰ disequilibrium in δ2H
values between aromatic H and methanol hydroxyl H, this would induce a change in the δ2H
value of the aromatic H of just 0.035 ‰ over a 1-hour reaction, which is not detectable by
GC/IRMS. For the reagents described here, with 70 ‰ disequilibrium in the δ2H values of
dimethyl phthalate and methanol hydroxyl H, and a 12-hour reaction time (0.4 % exchange),
we would expect 0.3 ‰ 2H-enrichment. Even with a 12-hour reaction (0.4 % exchange) and a
1000 ‰ disequilibrium, such as might be encountered working on extraterrestrial samples, a
shift of only 4 ‰ is predicted, which is still smaller than the analytical uncertainties for
GC/IRMS. Although 1H/2H exchange is taking place as expected, we directly establish, for
the first time, that the rates are too slow to cause any significant changes in δ-values over the
reaction conditions used in the HCl method. Still, when using strong acids as catalysts in
methylation, it is good practice to not extend reaction times.
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Is evaporation a cause of 2H-fractionation?
Guarding against evaporation is always a concern for volatile analytes intended for isotopic
analyses, requiring careful laboratory handling. Vapor pressure isotope effects vary between
molecules, depending on intermolecular vibrations (bonding) and frequency shifts on
condensation, both of which vary with temperature.[34] We directly tested fractionation for
dimethyl phthalate under worst-case scenario laboratory handling with evaporative losses to
demonstrate the scale of possible errors. To mimic extreme sample preparation error, we
evaporatively removed all solvent and ~45 % of the dimethyl phthalate using N2 blowdown,
redissolved the compound in solvent and observed +14 ‰ 2H-enrichment upon reanalysis.
We also tested GC inlet conditions: we compared δ2H values of dimethyl phthalate injected
via a PTV inlet in splitless mode with those from split-mode at 150 °C. With the inlet-
evaporation test, the peak area decreased by half and we measured a 2H-enrichment of +8 ‰.
These tests indicate that it is possible to fractionate dimethyl phthalate during handling, such
as complete solvent blow-down, or during analysis, via the use of heated, split injection
modes. To avoid such effects, we dried the solvents under a gentle N2(g) stream without the
use of heat and ran samples in splitless injection mode. Yields were still below 100 % for
acidic methods, leaving open the possibility that some product evaporated.
2H- and 13C-depletion using the carbodiimide method
The carbodiimide method resulted in more negative δ2H and δ13C values than expected for
dimethyl phthalate compared with the known isotopic composition. We infer a kinetic isotope
effect, with the preferential reaction of the lighter isotope, by the carbodiimide method.[35]
Although we have not fully resolved the cause of the isotopic offset using this method
(Figures 3 and 4) we suggest that securing higher yields and determining the potential for
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kinetic isotope effects with the carbodiimide reaction may be relevant questions for future
research if this method is to be used as preparative method prior to GC/IRMS analyses.
Implications for aromatic and aliphatic acid esterification
For methyl octacosanoate, prepared by each of three methods, the δ2H and δ13C values were
within 3 ‰ and 0.5 ‰ uncertainty envelopes, respectively (Figure 5). Thus, all three methods
are suitable preparative methods for GC/FID and GC/IRMS applications for long chain fatty
acids, although the acidic methods remain the recommended ones.
For phthalic acid, we found that the two acidic methods are equivalent, and δ13C values were
as predicted (within 0.4 ‰, similar within uncertainties), although we found that the δ2H
values were elevated by 15 ‰ (Figure 3). Although acidic exchange can lead to 2H-
enrichment, the rates established here are far too slow to explain observations. Small 2H-
enrichment can be reproduced through evaporation; however, evaporation cannot fully
explain the observed enrichment given the high yields obtained, given that ~50% of the
analyte would have to be lost to explain such an enrichment. Thus, the remaining offset may
reflect a fractionation associated with the methylation reaction. Nevertheless, from a practical
standpoint, we emphasize that volatility and evaporation are greater concerns than acidic
exchange on aromatic compounds, when pursuing phthalic acid methylation or methylation
of reagents with similar or lower boiling points. We recommend that analytical conditions be
carefully designed and monitored to minimize evaporative losses and further isotopic
enrichment, by testing quantitative yields by GC/FID in parallel with isotopic determinations
by GC/IRMS.
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In contrast, the non-acidic carbodiimide method performed poorly in phthalic acid
methylation experiments in terms of product yield and depletion in both 2H (by up to 15 ‰)
and 13C (by up to 2 ‰; Figure 3). We hypothesize that the low isolated yield is related to the
isotopic depletion but the yield could not be improved in our attempts shown here and thus
the consequent isotope effects could not be directly tested. Since acidic-exchange is not
quantitatively important for these applications, we have no reason to extend testing of the
non-acidic method here.
Phthalic acid methylation is commonly used to determine the isotopic composition of methyl
groups in methanol used for fatty acid esterification.[19] While the different methods induce
isotopic offsets from predicted values in this study (Table 1), the error in the isotopic
composition of the methanol becomes trivial for long chain n-alkanoic acids due to the small
proportion of methyl hydrogens and carbon. For example, a 54 ‰ offset in the methyl δ2H
value becomes 2 ‰ for the δ2H value of C28 FAME and 5 ‰ for C16 FAME and a 7 ‰ offset
in the methyl δ13C value becomes 0.2 ‰ and 0.3 ‰ for the δ13C values of C28 and C16 FAME.
These accuracy and precision errors contribute to methyl group uncertainty, and to the
propagated uncertainties in the calculation of analyte δ2H values.[26] Overall, the magnitude
of methyl hydrogen uncertainty on derivatized compounds remains small relative to other
sources of uncertainties in the estimation of high molecular weight aliphatic acids e.g., C28 n-
alkanoic acid, a constituent of plant leaf waxes (Table 1). For short chain lipids such as the
C16 n-alkanoic acid (common in microbial, plant and animal cells) the mean isotope values
differ more between methods (up to 5 ‰) and uncertainties increase. For practical guidance,
we provide detailed protocol and error propagations in the supplementary material.
CONCLUSIONS
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Methyl esterification is a commonly used derivatization procedure for both aromatic and
aliphatic compounds with carboxylic acid groups prior to GC analysis. We have studied
hydrogen and carbon isotopic fractionations during methylation using two widely used acidic
catalysts and one non-acidic method using a carbodiimide. We found practical advantages to
the acidic methods over the carbodiimide method, notably higher isolated yields. We
determined that the rates of 1H/2H exchange (4.94 10-4 hr-1) are too slow to be of
quantitative importance and thus find that the acidic conditions do not present problems for
most applications, even with aromatic compounds. We thus recommend following one of the
acidic protocols.
A phthalic acid standard is commonly used to determine the isotopic composition of the
methanol used to derivatize both the standard and the aliphatic or aromatic acid analyte for
GC/IRMS applications. Using phthalic acid and methanol standards of known values here,
we establish that for both carbon and hydrogen isotopic analyses the carbodiimide method
produces systematically lower values than the acidic methods. As we have shown that acidic-
exchange is not quantitatively significant, the greatest issues appear to be low yield
(carbodiimide method) and evaporation (under poor laboratory handling scenarios). Although
we observe a small systematic 13C- and 2H-enrichment in dimethyl phthalate from known
reagents using the acidic methods, the propagated errors for long chain n-alkanoic acid
applications are smaller than the analytical uncertainty of δ2H values by GC/IRMS at least for
high molecular weight analytes.
ACKNOWLEDGEMENTS
This work was supported by funding from the US American Chemical Society Award #PRF-
53747-ND2 to SF, from the US National Science Foundation Award CHE-1566167 to TW,
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and from the USC Provost fellowship to HJL. Federal support of NMR spectrometers (NSF
DBI-0821671, CHE-0840366; NIH S10 RR25432) is gratefully acknowledged. We thank
Elias Karkabi for discussions. This manuscript was improved with the comments from three
reviewers.
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TABLE
Table 1. Hydrogen and carbon isotope ratios calculated for C28 and C16 FAMEs and
propagated uncertainties based on the precision and accuracy of dimethyl phthalate analyses.
δ2H values (‰)
δ13C values (‰)
HCl CH3COCl EDCI/DMAP
HCl CH3COCl EDCI/DMAP
Mean dimethyl phthalate -107
Uncertainties
-105
-137
-31.3
-31.5
-32.7
i. Precision†
1
2
1
0.1
0.2
0.0
0.4
0.4
1.6
ii. Accuracy°
16
18
14
Estimated methyl group -115
Assigned FA value
-112
-100
-165
-47.9
-48.9
-20
-54.5
C28 FAME calculated
Propagated 
i. Precision
-101
-101
-103
-21.0
-21.0
-21.2
6
6
6
6
6
6
0.2
0.2
0.2
0.2
0.2
0.3
ii. Accuracy
C16 FAME calculated
Propagated 
i. Precision
-101
-101
-106
-21.6
-21.7
-22.0
6
7
6
7
6
7
0.2
0.2
0.2
0.2
0.2
0.5
ii. Accuracy
†
Reproducibility of phthalic acid methylation and preparative chemistry.
° Difference between known and measured dimethyl phthalate value from Figure 3.
* Propagated standard deviation calculated following the approach of Polissar and D’Andrea[26]
.
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Figure 1. Schematic diagram showing the risk of acid-catalyzed 1H/2H exchange in dimethyl
phthalate.
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Figure 2. The structures of non-acidic reagents EDCI and DMAP.
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Figure 3. Dimethyl phthalate isolated yield and isotopic results. Carbon and hydrogen
isotopic measurements are compared with known dimethyl phthalate values (i.e., target
values) calculated by isotopic mass balance. Diamonds represent carbon isotope results
whereas circles represent hydrogen isotopes.
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Figure 4. Pseudo-first order rate (knmr) of HCl-mediated 1H/2H exchange rate in dimethyl
phthalate. Hydrogens attached to the beta carbon position (red symbols, red line) and to the
alpha carbon (blue triangles, blue line).
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Figure 5. Methyl octacosanoate isolated yield, carbon and hydrogen isotopic composition.
Error bars are replicate instrument precision, results are the same within analytical
uncertainty.
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