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

PURPOSE This is a modification of a standard esterification procedure [1] to minimize isotopic fractionation and optimize yield for phthalic acid methylation.. The isotopic composition o

Supplementary Information 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 Geological 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

Protocol for phthalic acid methylation

A PURPOSE

This is a modification of a standard esterification procedure [1] to minimize isotopic

fractionation and optimize yield for phthalic acid methylation Fatty acids are derivatized prior to gas chromatography typically by the addition of a methyl group The isotopic

composition of that added group must be known to calculate the isotopic composition of the fatty acid It has previously been suggested that phthalic acid presents a useful reagent for determination of the isotopic composition of the added methyl group as phthalic acid is stable for long term storage and shipment, and its isotopic composition can be independently

measured offline relative to the international reference standards [2] In this way, the isotopic composition of the methyl group in methanol can be readily determined in-house without need for shipments and storage of a volatile compound Here we report a recommended method for the preparation of dimethyl phthalate yields for both carbon and hydrogen

isotopic analysis by gas chromatography flame ionization detection/isotope ratio mass

spectrometry (GC/FID/IRMS) using hydrochloric acid Other common catalysts include acetyl chloride and boron trifluoride (see text)

B NECESSARY MATERIALS

1 Isotopically-determined phthalic acid

2 5 wt% H2O deactivated silica gel

3 Solvents: ultra-pure water (Milli-Q or similar); dichloromethane (DCM) and hexane both gas chromatography (GC) grade

4 Hydrochloric acid

5 Combusted borosilicate glass Pasteur pipettes and glass wool for plugging

6 Sodium sulfate (Na2SO4) anhydrous

7 Hot plate

8 15-mL borosilicate glass culture tube and Teflon lined cap

9 Teflon tape

10 40-mL borosilicate glass vial

11 2-mL vial and Teflon-silicone-Teflon septum cap

12 N2 gas stream for evaporation

C PROCEDURE

1 Weigh out 3 mg of phthalic acid and record the weight (for yield calculations)

2 Prepare 5 vol% HCl in methanol

3 Put phthalic acid (1) and 1 mL of the mixture (2) to into a 15-mL culture tube

4 Cap vial with Teflon lined cap and wrap Teflon tape around the cap to ensure a good seal

5 Place in a hot block at 70 °C overnight

6 At completion, check no loss of reagent (solvent level unchanged)

7 Turn off the hot plate and allow to cool (Dimethyl phthalate will undergo ~ 0.3 ‰ 2 H-enrichment for every 12 hours, so should be promptly removed from the heat and acidic solution, and processed after the overnight reaction.)

8 Perform liquid-liquid extraction and pass desired fraction through sodium sulfate column

a Add 1 mL of MilliQ water to dilute the reagent to enable separation

b Add 1 mL of DCM, and cap tightly

c Shake vigorously for 30 s

d Leave to settle, until two distinct density-separated layers form: the (upper) aqueous layer and the (lower) DCM layer (Note if using hexanes for fatty acid extractions (b) hexanes will be the upper layer)

e Extract the bottom layer (DCM) with a short pipette

f Load the extracted fraction onto a column containing Na2SO4 anhydrous Collect the eluent in a 40-mL vial

g Repeat steps b – f 3 to 5 times

9 Silica gel

a Prepare a silica gel column and flush with DCM

b Load the fraction collected in 8f onto the silica gel column and elute with DCM

10 Dry and transfer

a Evaporate the solvent from 9b under gentle N2 gas stream (without heating) Do not dry completely to avoid evaporating dimethyl phthalate

b Transfer to a 2-mL GC vial with DCM

c Repeat a

d Add known volume of hexanes and analyze the dimethyl phthalate yield by GC/FID and isotopic composition by GC/IRMS Recap between measurements and store Teflon taped in solvent

D ISOTOPE MASS BALANCE

1 Dimethyl phthalate ((C2H3O2)2C6H4)

a

2 n-Alkanoic acid (CnH2n-1O2)

a

b

E ERROR PROPAGATION

1 GC/IRMS analytical uncertainties on an ester

The combined uncertainty on GC/IRMS δ2H determinations of an analyte, derivatized

as a methyl ester, is often ~5 ‰ As described in Polissar and D’Andrea [3],

propagated uncertainties include:

a instrument precision for replicate measurements of the analyte,

b the reference gas precision during sample and standard runs,

c the uncertainty when comparing measured and known standard values, and

d The uncertainty on the methyl group added during methylation (2)

2 Phthalic acid methyl ester chemical preparative uncertainties

The uncertainty on the methyl group added during methylation is often determined by derivatizing a phthalic acid (known isotopic composition), a molecule presenting two locations for methyl group additions, with an unknown methanol

a The uncertainty reported on Na-phthalate δ2H is 2.2 ‰ (1, n = 3)

b The reproducibility (reaction chemistry uncertainty) when methylating phthalic acid under normal, careful laboratory procedures is ~1 ‰ (1, n = 32) We recommend that at least 3 reactions are performed to check preparative reproducibility

c When reacting isotopically-known methanol and phthalic acid, we found the calculated isotopic compositions of methyl hydrogen to be offset from that of the known methanol value by 16 ‰ (n = 10; Figure 3) This inaccuracy points to an isotope effect during the methylation reaction or an isotopic fractionation during preparation

i We find negligible hydrogen isotope exchange under reaction conditions

ii We find yield to be only a problem for the non-acidic method; both acidic methods have quantitative yields

iii Evaporation during the preparation or measurement may lead to 2 H-enrichment of ~10 ‰ in extreme user-error scenarios This is avoidable and can be monitored by quantification

3 This study shows that the uncertainty on the methyl group added in methylation reactions

is underestimated when based on GC/IRMS instrument precision (1), Na-phthalate (2a) and chemistry-reproducibility (2b) The largest source of uncertainty arises from

inaccuracy in determinations (2c) and we find this is not due to acidic exchange We find

that phthalic acid is a particularly susceptible analyte due to its volatility The key

implication is that evaporation should be minimized and analyte yields should be

monitored, especially for volatiles

4 For the unknown analyte, the uncertainty in δ2H determination, introduced by the methyl group uncertainty will vary by mass balance (D) For long-chain compounds, e.g C28 n-alkanoic acid (from plant leaf wax), the weight of methyl group is small such that the combined, propagated errors are within 1 ‰ even if these of the methyl group using dimethyl phthalate are as large as 18 ‰, as identified here For shorter-chain compounds such as C16 n-alkanoic acid (a lipid present in many organisms) propagated errors are commensurately larger by 1 ‰ (Table 1)

F REFERENCES

[1] S G Wakeham, T K Pease, Lipid Analysis in Marine Particle and Sediment Samples A

Laboratory handbook Unpublished manuscript 1992

[2] A L Sessions, L L Jahnke, A Schimmelmann, J Hayes Hydrogen isotope

fractionation in lipids of the methane-oxidizing bacterium Methylococcus capsulatus

Geochim Cosmochim Acta 2002, 66, 3955.

[3] P J Polissar, W J D’Andrea Uncertainty in paleohydrologic reconstructions from

molecular δ2H values Geochim Cosmochim Acta 2014, 129, 146.