J Am Oil Chem Soc (2019)
DOI 10.1002/aocs.12165
ORIGINAL ARTICLE
Estolide Molecular Weight Distribution via Gel Permeation
Chromatography
Grigor B. Bantchev1 · Steven C. Cermak1 · Amber L. Durham1
Received: 30 May 2018 / Revised: 28 September 2018 / Accepted: 1 October 2018
Published 2019. This article is a U.S. Government work and is in the public domain in the USA
Abstract Using the universal calibration and the Mark-
Houwink equation (MHE) (½ηj ¼ KMjα ), three batches of
oleic estolide acids and their corresponding 2-ethylhexyl
esters were characterized using gel permeation chromatog-
raphy (GPC). The MHE parameters in tetrahydrofuran
(THF) at 40  C were determined (for acids: α =
0.442  0.003 and log10K=2.505  0.007, for esters: α
=0.531  0.006 and log10K =2.794  0.018). The fits of
the GPC chromatograms yielded also the oligomeric com-
position of the estolides, which can be used to calculate the
estolide number (EN) of an estolide mixture, and other
molecular-weight distribution parameters, such as number-
average molecular weight (Mn), weight-average molecular
weight (Mw), and dispersity (Ð). Using the Deming line fit,
we concluded that the GPC should be expected to be
Supporting information Additional supporting information may be
found online in the Supporting Information section at the end of the
article.
* Grigor B. Bantchev
grigor.bantchev@usda.gov
1 degradability (Bredsguard et al., 2016).
Bio-oils Research Unit, National Center for Agricultural
Utilization Research, Agricultural Research Service, United
States Department of Agriculture, 1815 N. University Street,
Peoria, IL 61604, USA
2
Renewable Product Technology Research Unit, National Center
for Agricultural Utilization Research, Agricultural Research
Service, United States Department of Agriculture, 1815
N. University Street, Peoria, IL 61604, USA
Mention of trade names or commercial products in this publication is
solely for the purpose of providing specific information and does not
imply recommendation or endorsement by the U.S. Department of
Agriculture. USDA is an equal opportunity provider and employer.
J Am Oil Chem Soc (2019)
· Neil P. J. Price2
approximately three times more sensitive than the currently
used methods for determination of EN values.
Keywords Estolides  Molecular weight distribution  Gel
permeation chromatography  Intrinsic viscosity
J Am Oil Chem Soc (2019).
Introduction
Estolides are a class of bio-based oligomeric compounds,
which have shown excellent properties as lubricants
(Cermak et al., 2017). Recently, estolides have been used
as a base oil to formulate a series of bio-based engine oils
(5W-20 and 5W-30), which has been certified by the Amer-
ican Petroleum Institute (API). Biodegradability tests on
the formulated estolide motor oils have shown that the esto-
lide base oils maintain a high degree of biodegradability
even while formulated or blended with a host of different
additives. The estolide-based engine oils were tested in a
series of Nevada taxi cabs in an automotive engine for
thousands of miles and maintained their high level of bio-
Estolides are formed when the carboxylic acid function-
ality of one fatty acid (FA) reacts at the olefinic site of
another FA to form a secondary ester linkage. The number
of these ester linkages represents the estolide number (EN).
The EN is one of the best means to describe the degree of
oligomerization of an estolide. The EN can be determined
using gas chromatographic (transesterification-GC) analysis
of the fatty acid methyl ester (FAME) composition after
methanolysis of the estolide, or by nuclear magnetic reso-
nance (Cermak and Isbell, 2001). The EN is defined as the

Scheme 1 Synthesis of estolide acids and estolide esters. Estolide
number (EN) of a molecule is equal to j−1 where j is the number of
starting FA. The estolide position was distributed from positions 5–13
(0 ≤ n ≤ 8) with the original Δ9 and Δ10 positions (n = 4 and 5) hav-
ing the greatest abundances
average number of FA added to the base FA (Scheme 1,
EN = j−1, where j is the total number of FA moieties in
the molecule). For example, j = 2 and EN = 1 would repre-
sent a dimer estolide ester (Scheme 2) or two FA linked
together with one ester linkage. EN values of two or more
indicate estolides with multiple ester linkages and are hence
called trimeric-plus estolides. The secondary ester linkages
of the estolide, resulting in a unique estolide structure, are
reported to be more resistant to hydrolysis than those found
in triacylglycerols, which yield materials with far superior
physical properties (Cermak and Isbell, 2003).
During the synthesis of estolides, the EN can easily be
altered or manipulated by changing any of the following
synthetic parameters: temperature, time, catalyst type,
and/or catalyst amounts. The ability to synthesize estolides
with a wide variety of EN has allowed researchers (Cermak
and Isbell, 2004) to discover predictable relationships
between EN and certain estolide properties (pour and cloud
points, viscosity, density, and/or wear/lubrication).
Gel permeation chromatography (GPC), also known as
size-exclusion chromatography, is a well-known method
for studying polymers and oligomers. Using this technique,
Scheme 2 2EH ester of oleic dimer estolide (EN = 1,
j = 2). 0 ≤ n ≤ 8
J Am Oil Chem Soc
the molecules are fractionated by their viscometric hydro-
dynamic size. The smaller molecules enter the pores of the
column resin, and this way they are retained, which leads
to their elution after the bigger molecules that cannot enter
the pores (Grubisic et al., 1967).
In the current article, we report the preparation of several
samples of oleic estolide acids (estolide acids) and their
corresponding oleic estolide 2-ethylhexyl esters (estolide
esters). The estolides differ by the synthesis reaction time
and the extent of distillation/purification, i.e., fractionation
by molecular weight (MW). The samples were analyzed by
GPC to determine the MW distribution and the average
MW. The limiting viscosity numbers (also known as intrin-
sic viscosity) for the oligomers in tetrahydrofuran (THF)
were also calculated, which can be used in future GPC
evaluations of estolides. The EN was calculated from the
number-average MW (Mn) and compared to the EN, deter-
mined from the transesterification-GC method.
Experimental Procedures
Materials
Hexanes, sodium sulfate, sodium chloride, sodium phos-
phate monobasic monohydrate, THF, and toluene were
from Fisher Scientific (Pittsburgh, PA, USA). Butylated
hydroxytoluene (BHT), methyl palmitate (99%) (MePa),
oleic acid (90%), perchloric acid (70%), and tristearin
(99%) (TriSt) were from Sigma-Aldrich (St. Louis, MO,
USA). Methanol, ethyl acetate, and potassium hydroxide
were purchased from EMD Millipore Co. (Billerica, MA,
USA). Filter paper was obtained from Whatman (Clifton,
NJ, USA). 2-ethylhexyl alcohol (2-EH alcohol) was pur-
chased from Univar (Downers Grove, IL, USA). Sulfuric
acid was purchased from LabChem Inc. (Zelienople, PA,
USA). Ethanol was purchased from AAPER Alcohol and
Chemical Company (Shelbyville, KY, USA). The FAME
standard mixtures were obtained from Nu-Check Prep
(Elysian, MN, USA). Solvents for chromatography and
extraction were HPLC grade or an equivalent, and were
used without further purification. Universal calibration
curve was prepared with EasiVial PS-L low-MW poly-
styrene standards from Agilent Technologies (Santa
Clara, CA, USA). The standards contained polystyrene
samples with peak MW (Mp) from 162 to 45,120 Da and
the log10(Mp[η]) between 0.42 and 4.0, where [η] was the
limiting viscosity number in dL g−1 (See the SI). The [η]
values of the polystyrene standards were supplied by the
manufacturer, except for the lowest MW (162 Da), which
was extrapolated from the higher MW values, using
the Mark-Houwink equation (MHE).
J Am Oil Chem Soc (2019)
J Am Oil Chem Soc

Estolide Synthesis

Simplified Scheme of the Procedures and Nomenclature ni j

(a) 13-ni
Scheme 3 represents a simplified information flowsheet of
the synthesis and the distillation procedures used to obtain
the different estolide batches and fractions. It also provides
(b)
the nomenclature that will be used throughout the manu-
script. Full details of the synthesis and the distillation pro-
cedures are given in the next sections. During the course of
the estolide acid synthesis, it is possible that some cyclic (c)
estolides are formed (Scheme 4). While dimers and higher
cyclic estolides from this kind of synthesis have never been
reported, the cyclic monomers: delta and gamma lactones
(Scheme 4 b and c) were reported previously by Cermak (d)
and Isbell (2001). The lactones were detected in the GPC
analysis of samples: Eb, Ec, E1b, and E1c. Scheme 4 Cyclic estolides. (a) general formula. i = 1 to j, (b) gamma
lactone (j = 1, n1 = 1 in figure 4a), (c) delta lactone (j = 1, n1 = 2),
and (d) cyclic dimer estolide (j= 2, n1 = 5, n2 = 8)
Oleic Estolide Acids (Estolide Acids)

An acid-catalyzed condensation reaction was conducted (7.5–10.9 kPa), and stirred with a Teflon-coated stir bar.
without solvent in a 500 mL round-bottom flask that had Once the desired temperature of 60  0.1  C was reached,
been pretreated with acidic wash. Oleic acid (100.2 g, perchloric acid (17.74 mmol, 1.5 mL, 0.05 eq.) was added,
354.7 mmol) was heated to 60  C under house vacuum vacuum restored, and stirred. After the corresponding time

Oleic H+ catalyst, ∆ Estolide acids “Ax”

acid Quenching the catalyst “Ax”= “Aa” (24h), “Ab” (96h) or “Ac”(168h)

Estolide Removal of monomer Estolide

acids “Ax” acid by distillation acids “A –1 x”

Estolide Esterification with 2EH alcohol, Estolide esters

acids “Ax” Quenching the catalyst “Ex”

Removal of monomer
ester by distillation

Monomer Estolide esters

esters “E1x” “E–1x”

Removal of dimer
ester by distillation

Dimer esters Estolide esters

“E2x” “E-2x”

Scheme 3 Simplified synthetic path and nomenclature in the current manuscript. “x,” depending on the starting estolide acid mixture, stands for
“a,” “b,” or “c” (see top of the Scheme). Capital “A” stands for estolide acid, capital “E” stands for estolide ester. “1” stands for mostly the mono-
mer mixture, “−1” for mixture with the removed monomer, “2” for mostly the dimer mixture, “−2”—for trimeric-plus estolide esters. “Ax” sam-
ples were not tested by GPC

J Am Oil Chem Soc (2019)


of reaction, i.e., 24, 96, or 168 h, the solution was allowed
to cool to room temperature with stirring. The reaction was
then quenched by the addition of KOH (1.144 g,
20.40 mmol, 1.15 eq. based on HClO4) in 90% ethanol/
water (20 mL) solution and allowed to stir for at least
30 min. The mixture was allowed to settle and was filtered
through a Buchner funnel using Whatman #54 filter paper.
The pH of the solution was adjusted to 5–6 using a pH 5
buffer (1 M NaH2PO4). The organic layer was then washed
with saturated sodium chloride solution, dried over sodium
sulfate, and filtered with Whatman #54 filter paper. All
reaction products were Kugelrohr-distilled at 180–200  C
at 0.013–0.067 kPa to remove any unreacted FA and by-
products, such as lactones (Cermak and Isbell, 2001). The
resulting estolide acid products (named A−1a, A−1b, and
A−1c) were then filtered with Whatman #54 filter paper.
Oleic Estolide 2-Ethylhexyl Esters (Estolide Esters)
Reactions were conducted without solvent in a 500 mL
round-bottom flask that had been pretreated with acidic
wash. Oleic acid (200.1 g, 708.3 mmol) was heated to
60  C under house vacuum (7.5–10.9 kPa), and stirred
with a Teflon-coated stir bar. Once the desired tempera-
ture of 60  0.1  C was reached, perchloric acid
(35.41 mmol, 3.1 mL, 0.05 eq.) was added, vacuum
restored, and stirred. After the corresponding time of
reaction, i.e., 24, 96, or 168 h, 2-EH alcohol (110.7 g,
849.9 mmol, 133.0 mL) was added to the vessel, vacuum
was restored, and the mixture was stirred for an addi-
tional 6 h. The completed reactions were quenched by
the addition of KOH (2.285 g, 40.7 mmol, 1.15 eq.
based on HClO4) in 90% ethanol/water (20 mL) solution
and allowed to stir for at least 30 min. After settling, the
mixture was filtered through a Buchner funnel using
Whatman #54 filter paper. The pH of the solution was
adjusted to 5–6 using a pH 5 buffer (1 M NaH2PO4).
The organic layer was then washed with saturated
sodium chloride solution, dried over sodium sulfate, and
filtered with Whatman #54 filter paper. All reaction prod-
uct mixtures were Kugelrohr distilled at 90–110  C at
0.013–0.067 kPa to remove any excess 2-EH alcohol. At
this point, the estolide ester samples, Ea, Eb, and Ec,
were collected. The remaining products were further dis-
tilled by Kugelrohr distillation at 180–200  C at
0.013–0.067 kPa to remove any unreacted FA and by-
products, such as lactones (Cermak and Isbell, 2001) and
oleic 2-EH esters (Monomer Esters, EN = 0). The final
products were filtered using Whatman #54 filter paper.
This final distillation resulted in the collection of estolide
esters (E−1a, E−1b, and E−1c) in the residue and Mono-
mer Esters (E1a, E1b, and E1c) in the distillate.
J Am Oil Chem Soc
MW Fractionation—Molecular Distillations
Fractional distillations from the previous experiment were
performed on the resulting set of estolide esters (E−1a,
E−1b, and E−1c) using a Myers Lab 3 short-path molecular
distillation unit (Myers Vacuum, Kittanning, PA, USA).
This step of distillation was performed to remove the dimer
estolide 2-EH esters (Dimer Esters, EN = 1) from the esto-
lide ester mixture leaving behind the larger oligomers
known as trimeric-plus estolides. Cermak et al. (2012) pro-
vided in-depth information on the capabilities, distillation
diagram, and exact parameters for fractional distillation
using the Myers Lab 3 unit. For this distillation, the con-
denser temperature was set at 30  C and cold tap water was
used to cool the diffusion pump and rotor bearing. Vacuum
was maintained at 1.60–3.73 Pa at both the chamber and
foreline pressure sensors. Rotor temperature was main-
tained between 224 and 228  C and the feed was adjusted
such that the estolide materials entered the rotor chamber at
about 40 drops min−1. Feedstock was fed onto a heated
rotor spinning at 28.75 Hz under a high vacuum
(1.60–3.73 Pa).
As the distillation proceeded, the feedstock of estolide
esters (E−1a, E−1b, and E−1c) was fractionated to yield a
mostly dimeric portion of the estolide esters (EN = 1) in
the distillate (dimer esters: E2a, E2b, and E2c) and a
trimeric-plus oligomer portion in the residue (EN >1) (tri-
meric-plus estolide esters: E−2a, E−2b, and E−2c). The
residual trimeric-plus estolide portion was a mixture of tri-
meric and larger estolide esters, including a smaller portion
of undistilled dimeric estolides. The short residence time at
high temperature of the Myers distillation unit helps reduce
degradation and color bodies of the residue unlike other
distillations where the sample remains at the distillation
temperature for several hours. The short residence time and
the high vacuum also make it possible for higher distilla-
tion temperatures, which are necessary for removal of the
heavy dimeric estolides.
Transesterification-GC–MS Method for the
Determination of the EN
The EN, which identifies the extent of oligomerization of
the product, is defined as the average number of FA
added to the base FA. Upon methanolysis of an estolide
sample with an EN = 2 ( j = 3, Scheme 1), the product
will have components: two hydroxy FAME and one
nonhydroxy FAME.
The EN is calculated from the transesterification-GC
method where the ratio of the areas is measured, i.e., the
hydroxy FAME peaks to the nonhydroxy FAME peaks as
described previously (Isbell and Kleiman, 1994). In the
example above, the ratio will be 2:1.
J Am Oil Chem Soc (2019)
J Am Oil Chem Soc
GC–MS Characterization of Low Estolides
The estolide esters with low MW (monomers and dimers)
were run without pretreatment (transesterification) on a
SPB-1 column. The temperature profile was a starting tem-
perature of 150  C, a ramp of 4  C min−1 for 40 min
(to 310  C), followed by a hold for 40 min at 310  C. The
helium flow rate was 1 mL min−1, the injection volume
was 2 μL, and the split ratio was 10:1. The sample concen-
tration was ~10 mg mL−1.
Matrix-Assisted Laser Desorption/Ionization Time-of-
Flight MS (MALDI MS)
MALDI MS spectra were obtained on a Bruker-Daltonic
Microflex (Billerica, MA, USA) instrument operating in the
reflectron mode, with lens and reflector voltages of 9.20 and
20.00 kV, respectively. Laser excitation (337.1 nm) was
typically at 75% of 150 μJ maximum output, and 1200 shots
were acquired. The matrix use was 2, 5-dihydrobenzoic acid
(2, 5-DHB).
GPC Runs
The unfiltered samples were run on a GPC consisting of a
precolumn PLgel 3 μm Guard (50 × 7.5 mm, Part
1110–1320), two PLgel 3 μm mixed E columns
(300 × 7.5 mm) from Agilent Technologies (Santa Clara,
CA, USA) connected in a series, a Wyatt Optilab REX dif-
ferential refractive index (dRI) detector, a Wyatt Treos
miniDAWN light-scattering detector from Wyatt Technol-
ogy (Santa Barbara, CA, USA), a Waters 717 autosampler,
and a Waters 1515 isocratic pump from Waters Corporation
(Milford, MA, USA). Forty to fifty milligrams of a sample
were dissolved in 1 mL of THF with 0.1% BHT (stabilizer)
and 0.5% toluene (internal standard for the flow rate). Half
of the estolide samples were prepared in duplicate on a sep-
arate day and rerun to determine the reproducibility of the
data. The EasiVial standards were prepared by dissolving
the content of the vial in 2 mL THF with 0. 1% BHT and
0.5% toluene. Hundred microliters were injected and the
solvent (THF with 0.1% BHT) was run at 1 mL min−1.
The columns were kept at 40  C during the runs. In addi-
tion to the estolide and calibration samples, MePa and TriSt
samples were run as monodisperse lipid samples.
Theoretical
Oligomer Distribution
If we assume that the carboxylic head of a FA is separate
from the rest of the estolide molecule and hence not influ-
enced by it, then the probability, p, of the carboxylic
J Am Oil Chem Soc (2019)
group being a part of the estolide chain, as opposed to an
end group, is the same for all of them. This assumption
leads us to the conclusion that during the synthesis of
estolide acids there is a geometric distribution of the olig-
omeric species:


Mj + 1

 ¼p ð1Þ
Mj
for every j ≥ 1, where 0 ≤ p < 1. j is the number of
monomeric units in the oligomer, the square brackets in this
case refer to the molar concentration of the species. This is
known as a Flory MW distribution (Kissin, 1995). The
Flory’s distribution is expected to be preserved during the
esterification of the estolide acids to estolide esters, but it
will be distorted during the distillations due to the higher
volatility of the monomers and lower oligomers. As we did
not detect (by GPC) any higher than tetramer oligomers to
distill, we assumed that the studied estolides obey the
Flory’s distribution for pentamers and higher oligomers.
The concentrations of the monomers to tetramers were
fitted individually, even in the cases (Ea, Eb, and Ec) where
all the oligomer concentrations were expected to obey the
Flory’s distribution (Eq. 1).
Jacobson and Stockmayer (1950) published a theory
(later refined by Flory and Semlyen, 1966) for the concen-
tration of cyclic (ring) polyesters. The Flory distribution for
linear polyesters (Eq. 1) can be written as:


Mj ¼ Bp j ð2Þ
where B is a constant. The Jacobson-Stockmayer distri-
bution can be written as:

 CVp j .
Rj ¼ ð3Þ
2:5 j
where Rj is a cyclic estolide with j FA, C is a constant,
and V is the volume of the system. From these distribu-
tions, it can be derived that the ratio of cyclic estolides with
j FA to the linear estolides with the same number of FA is:
. .
½Rj  ¼ CV=B 2:5 ð4Þ
½M j  j
and

 


 Mj + 1 j 2:5
Rj + 1 ¼ Rj
 ð5Þ
Mj j+1
The Jacobson and Stockmayer theory does not take
into account the ring strain, and work on cyclic oligo-
mers has shown that the cyclic oligomers with rings of
8 to 12 atoms to be strained (Illuminati and Mandolini,
1981). Correspondingly, the Eqs 3–5 are valid only for
j ≥ 2 and cyclic monomers (lactones, j = 1) do not con-
form them. There is an example in the literature that

rings with 9 to 13 atoms form in a lesser amount than
the corresponding dimer cycles (Ruddick et al., 1999),
due to the straining.
Universal Calibration
The principle of the universal calibration states that all
polymers with the same value of the product M[η] have the
same elution volumes (Grubisic et al., 1967):
log10 ðM ½ηÞanalyte ¼ log10 ðM ½ηÞcalibration standard
when
Vanalyte ¼ Vcalibration standard
In the above statement M is the MW, [η] is the limiting
viscosity number (also known as the intrinsic viscosity),
and V is the peak volume at which the material elutes from
the GPC column. Using the universal calibration is possible
if [η] of the molecules is known. Sometimes when [η] data
are not available, a simplification is made:
log10 ðM Þanalyte ¼ log10 ðM Þcalibration standard
when
Vanalyte ¼ Vcalibration standard
This approach, called “GPC calibration,” works when
the oligomers used for the calibration are similar to the ana-
lyzed ones. It is shown that using polystyrene standards for
the simpler calibration leads to wildly higher than expected
MW of lipid samples (Bantchev et al., 2016; Noor et al.,
2016). This is due to the large dissimilarities between the
two kinds of molecules. Universal calibration, which
requires knowledge of [η], is needed.
To determine the [η] values for a series of homologous
oligomers, the Mark-Houwink Equation (MHE) is used:
½ηj ¼ KMjα
where j is the number of monomer units in the oligomer,
K and α are constants, specific for each oligomeric series,
and Mj is the MW of the oligomer with j units.
Using well-characterized polystyrene standards, a uni-
versal calibration curve can be made:
z  log10 ðM ½ηÞ ¼ f ðVelutionÞ
It is quite typical for z to be a linear function of V. In our
case, we observed a slight nonlinear trend at higher MW,
so a cubic polynomial was used instead. zpj (the peak elu-
tion volumes for estolide oligomers with j units) were
determined from GPC chromatograms (see the “curve-
fitting of whole chromatograms” section).
Using the MHE and the universal calibration equation,
the following formula can be derived:
J Am Oil Chem Soc
   
zpj ¼ log10 Mj ½ηj ¼ log10 KMjα + 1

¼ log10 ðK Þ + ðα + 1Þlog10 Mj ð12Þ
This formula connects peak elution volumes with the
oligomer molar mass and two parameters, specific for the
oligomeric series and the experimental conditions (solvent
and temperature). It should not depend on the GPC column,
as long as the oligomers are within its range.
ð6Þ Computational
Preparation of Chromatograms
ð7Þ
The instrument was equipped with a light-scattering detec-
tor, but due to the relatively low MW of most of the esto-
lides, it did not produce usable signals. The dRI was
monitored as a function of the elution volume. All the com-
pounds at these instrument settings eluted after 12 mL, so
the signal between 1 and 10 mL was used to calculate a
baseline as an average value of the dRI signal. The baseline
ð8Þ was subtracted, and the chromatogram was smoothed with
a Savitzky–Golay filter of second order (Savitzky and
Golay, 1964). The optimal window size for the smoothing
(in most cases 11 datapoints, spanning ~0.096 mL, in one
ð9Þ
case 7 datapoints) was selected using the method described
by Vivó–Truyols and Schoenmakers (2006).
The calibration curve was determined to be
z  log10 ðM ½ηÞ ¼ −0:0039093328V 3 + 0:18410884V 2
−3:296576V + 23:009613
ð13Þ
where V is the elution volume in mL.
The chromatogram was recalculated to equally space
z ≡ log10(M[η]) from 3.3 to 0.74 axis with a step of 0.002
(11.8 to 18.2 mL, step 0.0034 to 0.005 mL). The dRI was
ð10Þ dz
recalculated inversely proportionally to the dv derivative
dz
− ¼ 3 × 0:0039093328v2 − 2 × 0:18410884v + 3:296576
dv
ð14Þ
The curve fitting was performed in recalculated dRI ver-
sus z coordinates.
ð11Þ
Curve Fitting of Individual Peaks
In a preliminary fitting, four fitting curves were tested:
Gaussian (three parameters per peak), log-normal (three
parameters), Poisson (four parameters), and generalized
exponentially modified Gaussian (EMG) (four parameters
per peak) (Di Marco and Bombi, 2001). The Gaussian and
the EMG curves were better fits. The Gaussian had the
J Am Oil Chem Soc (2019)
J Am Oil Chem Soc
advantage of being simpler and allowing easier calculations
of the curve characteristics (position of the peak maximum
and area under the curve) from the fitting parameters, while
EMG gave a better fit due to the present asymmetry of the
peaks. Finally, the EMG was selected as the best fit for the
data presented here.
The equation for the generalized EMG is (Di Marco and
Bombi, 2001):
Neglecting the higher oligomers (j > 20) was estimated to
lead to an error of less than 0.02% of the total area.
The three samples (A−1a, A−1b, and A−1c) of estolide
acids were run in duplicate. These runs yielded six chro-
matographic curves that were fitted simultaneously with
4 + 5*6 = 34 fitting parameters. The estolide esters ana-
lyzed were the undistilled esters (Ea, Eb, and Ec)
(in duplicate), the esters after the removal of the monomers
rffiffiffi  2    
s σ z −μ
hσ π σ z −μ
y¼ exp − erfc pffiffiffi − ð15Þ
jτj 2 2τ2 τ 2 τ σ There were a total of 15 chromatograms fitted simulta-
where μ is the position of the unmodified Gaussian peak,
σ is the Gaussian width of the peak, τ is the relaxation time,
related to the tailing of the peak, h is the Gaussian height of
the peak, π is the constant 3.14…, erfc() is the complemen-
tary error function, z ≡ log10(M[η]) is the abscissa value,
and s is =1 if τ >0 and = − 1 if τ <0.
The generalized EMG differs from EMG in that negative
values for τ are acceptable, which allows the generalized
EMG to represent fronted peaks. All the peaks were tailed
in the representation of the signal as a function of the eluted
volume, which corresponded to fronted peaks when the sig-
nal was presented as a function of the z (≡log10(M[η]) coor-
dinate, due to the negative slope between the two.
A computational complication arose from the fact that
the calibration was made based on the position of the maxi-
mums of the peaks, zp, while the fitting procedure did not
yield them directly. The formula to calculate them is not
explicit (see Appendix A).
Curve Fitting of Whole Chromatograms
Chromatograms were fitted using the EMG curves for each
individual peak. The positions of the estolide peaks were
assumed to obey the universal calibration and the MHE
(Eq. 12). log10(K) and (α + 1) were fitting parameters.
Assuming that most of the width of the peaks was due to
band broadening, we used the same values for σ and τ for
all the peaks in the fit. There are studies that have shown
weak or no dependence of these parameters on the elution
position of the peak (Busnel et al., 2001) when the MW is
not close to the cutoff limits of the column.
Several chromatographic curves were fitted simulta-
neously, using the same log10(K), (α + 1), σ, and τ as fitting
parameters. Five more fitting parameters were used per
chromatogram, and they were related to the area under each
peak (which is proportional to the concentrations of the
oligomer). As mentioned in the “oligomeric distribution”
section, the areas, Aj, of the first four peaks (1 ≤ j ≤ 4) were
fitted as independent parameters, while for the rest
(5 ≤ j ≤ 20) a fitting parameter p and the equation Aj = pAj − 1
were used according to the Flory’s distribution (Eq. 1).
J Am Oil Chem Soc (2019)
(E−1a, E−1b, and E−1c), and the esters after the partial
removal of dimers (E−2a, E−2b, and E−2c) (in duplicate).
neously with 4 + 5*15 = 79 fitting parameters.
The jackknife method (Caceci, 1989) was used to deter-
mine the average and the standard deviation of the common
fitting parameters: log10(K), (α + 1), σ and τ. log10(K) and
α were used with the MHE to calculate [η]j of the estolide
oligomers. Using the jackknife method, the averages and
the standard deviations are determined by fitting data sub-
sets with an omitted datapoint, and averaging the resulting
fitting parameters. In the current implementation of the
jackknife method, a GPC chromatogram was treated as a
datapoint.
The averages and the standard deviations of the oligo-
meric composition and related parameters were calculated
from the values for the duplicate runs.
Calculation of the MW Averages
The following formulas were used to calculate the MW
averages:
P 
w =M − 1
Mn ¼ Pi i ð16Þ
wi
P
wi Mi
Mw ¼ P ð17Þ
wi
where Mn and Mw are the number-average and weight-
average MW. The Eqs 16 and 17 can be used in two ways.
In the first, Mi refers to the MW of the ith estolide oligo-
mer, and wi is the mass amount of this oligomer (propor-
tional to the area of the peak). It can be used when the
composition is known. In the current manuscript, the com-
position is determined from fitting of the chromatograms.
The results will be denoted with a subscript “GPC1” if
needed.
In the second application, the chromatographic data are
used. Then, wi is the dRI signal at slice i, and Mi is deter-
mined from the universal calibration and the MHE:
pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
Mi ¼ α + 1 ð10zi =K Þ ð18Þ
In the above equation, zi is calculated from the calibra-
tion curve (Eq. 13). In this approach, if log10(K) and
 J Am Oil Chem Soc

(α + 1) are known, the MW averages can be calculated z=log(M[η])

3.0 2.5 2.0 1.5 1.0 0.5
without curve fitting the chromatogram. The summations
were carried out for z values of 0.9 to 3.3 for the estolide 4
10
acids and 0.956 to 3.3 for the estolide esters. The signal for

peak MW, Da
dRI, (a.u.)
z > 3.3 was essentially equivalent to the baseline. The sig-
3
nal for the low z values contained, in some cases, a lactone A-1b 10
Eb
peak that we wanted to treat separately (see results and dis- E-2b

cussion). In the current manuscript, the results obtained 2

using this approach will be indicated with a subscript
“GPC2,” if needed.
Other related parameters are dispersity (Ð) and EN:
Mw
Đ¼ ð19Þ
Mn
Ð is = 1 for monodisperse samples and >1 for polydis-
perse samples.
Mn −M0
EN ¼ −1 ð20Þ
ΔM
The EN is a measure of the average number of oligo-
meric units. M0 is the MW of the end groups (= 0 for esto-
lide acids, = 112 for estolide esters). ΔM is the MW of a
monomeric unit (=282 for estolides based on oleic acid).
Software
The calculations were performed with Microsoft Excel
2013. Some of the calculations used Visual Basic for
Applications. Curve fitting utilized the Solver Add-In to
find the minimum of the sum of squared differences
between the chromatograms and the fitting curves.
Results and Discussion
Representative Results
Fig. 1 shows three GPC chromatograms plotted as a func-
tion of the elution volume, V. We can see that the peaks of
10
12 13 14 15 16 17 18
Retention volume, (ml)
Fig. 1 Example of GPC chromatograms of estolides: differential
refractive index as a function of elution volume. Solid line: Estolide
acids formed after 96 h of synthesis (A−1b). Dotted line: the undis-
tilled estolide esters (Eb). Dashed line: the estolide esters, after
removal of monomers and partial removal of dimers by distillation
(E−2b). The markers show the Mp of the standards used to create the
universal calibration curve (right axis) vs. the peak retention volume
(bottom axis) or z (top axis)
Estolide Acids
The chromatograms from the GPC analysis are presented in
Fig. 2. It shows relatively small differences between the
samples. Nevertheless, there was a reproducible result that
the longer reaction times led to a decrease in the relative
proportion of the lower oligomers and increased the propor-
tions of the higher oligomers ( j > 5).
The chromatograms were well fitted (see Fig. 3 for an
example of the fit of one chromatogram and Table 1 for the
common fitting parameters and the R2 value).
The composition of the estolide acids, obtained from the
fitting of the GPC data, is given in Table 2. We can see that
lower oligomers ( j < 4) decrease with the length of the
reaction, while the higher oligomers ( j ≥ 5) increase.
Table 2 also includes the MW averages and derived
parameters.
a
b
c
Recalculated dRI, a.u.

4–5 lower oligomers can be easily identified; the higher

a
oligomer peaks overlap. While the peak positions of the b
estolide esters from the Eb sample are close to the corre- c

sponding ones from the other sample (E−2b), the ones from
c
the estolide acids (A−1b) are at different elution volumes. b
a
This is because the same oligomers have the same elution
volume, irrespectively of the run, or the exact composition.
It can be seen that for Eb, undistilled estolide esters, the 1.0 1.5 2.0 2.5 3.0
log(M[η])
area of the monomer peak (highest elution volume) is
higher, and it decreases with the increase of the oligomer
number. Fig. 2 GPC chromatograms of estolide acids (A−1a, A−1b, and A−1c).
Two separate chromatograms for each sample are run and shown.
On the top of the graph, the z-axis is plotted. It can be
Solid line: Estolide acids synthesized for 1 day (A−1a). Dotted line—
seen that the distortions from the linearity are very minor— estolide acids synthesized for 4 days (A−1b). Dashed line—estolide
it is hard to notice the unequal spacing of the z tick marks. acids synthesized for 7 days (A−1c)

J Am Oil Chem Soc (2019)

J Am Oil Chem Soc

E–1b (Monomer removed)

Data (A–1b) Eb (un-distilled)
Overall fit

Recalculated dRI, a.u.

Recalculated dRI, a.u.

Fit, individual peaks

E–2b (Dimer reduced)

j=7 E–1b (Monomer removed)
j=1 j=2 j=3 j=4 j=5 j=9 j=11 Eb (Undistilled)
L j=1 j=2 j=3 j=4 j=5

1.0 1.5 2.0 2.5 3.0 1.0 1.5 2.0 2.5 3.0
log(M[η]) log(M[η])

Fig. 3 Example of GPC chromatogram of estolide acids formed after Fig. 4 GPC chromatograms of the estolide esters: Eb, E−1b, and E−2b
4 days of synthesis (A−1b) (solid line), fit (dashed line), and the individ- (solid lines), and their fits (dashed lines). “L” denotes the lactone
ual fitting curves for the first six odd-numbered oligomers (dotted lines) peak. Its range is excluded from the fit

Estolide Esters
initial estolide esters were fractionated by distillation to
Evaluation of the Polydisperse Samples separate them into monomer-rich fractions (E1a, E1b, and
E1c) and monomer-free estolide esters (E−1a, E−1b and
Estolide esters (Ea, Eb, and Ec) were synthesized from E−1c). The monomer-free estolide esters were distilled fur-
estolide acids, by esterification with 2-EH alcohol. The ther into dimer-rich fractions (E2a, E2b, and E2c) and
Table 1 Fitting parameters for the chromatogram

Estolide acids Estolide esters MePa TriSt

n 6 15 4 3
(α + 1) 1.442  0.003 1.531  0.006 n.a. n.a.
log10K 2.505  0.007 2.794  0.018 n.a. n.a.
σ 0.0404  0.0005 0.0412  0.0001 0.0416  0.0013 0.0401  0.0001
−τ 0.0476  0.0006 0.0440  0.0003 0.0359  0.0044 0.0317  0.0010
R2 0.9962 0.9925 0.9987 0.9996

The parameters are determined with an abscissa z = log10(M[η]), where M is in Daltons and [η] in dL g−1 (units of the abscissa log10( mole−1 )).
n.a.; not applicable to compounds that are not a part of an oligomeric series. R2 is the squared coefficient of determination of the fits

Table 2 Composition of estolide acids and some descriptors, determined from fits of the GPC chromatograms

A−1a A−1b A−1c

1 day 4 days 7 days

% monomers 1.04  0.02 0.67  0.05 0.40  0.01

% dimers 29.4  0.1 28.0  0.2 27.3  0.1
% trimers 22.8  0.1 22.2  0.2 21.8  0.1
% tetramers 15.36  0.08 15.38  0.08 15.28  0.06
% pentamers 10.33  0.02 10.57  0.02 10.67  0.01
% higher oligomers 21.1  0.3 23.1  0.5 24.5  0.3
p 0.672  0.003 0.687  0.004 0.698  0.003
Mn (GPC1) 880  2 907  5 925  3
Mw (GPC1) 1157  5 1202  11 1232  7
Ð: (GPC1) 1.315  0.003 1.325  0.004 1.333  0.003
EN (GPC1) 2.12  0.01 2.22  0.02 2.28  0.01
Mn (GPC2) 851  1 876  6 894  1
Mw (GPC2) 1161  7 1172  51 1231  6
Ð: (GPC2) 1.364  0.006 1.34  0.05 1.377  0.005
EN (GPC2) 2.017  0.004 2.107  0.022 2.169  0.004

J Am Oil Chem Soc (2019)


trimeric-plus estolide esters with a reduced amount of
dimers (E−2a, E−2b, and E−2c). GPC chromatograms of the
estolide esters, obtained from 4-day-synthesized estolide
acids (b-series), are shown in Fig. 4. The chromatograms of
the undistilled estolide esters (Ea, Eb, and Ec), formed from
different estolide acids, are shown in Fig. 5.
The fitted compositions of the estolide esters are pre-
sented in Table 3. It can be seen that there is no statistically
significant difference between “b” esters and the corre-
sponding “c” esters (the lactone was excluded from the
calculations).
The log10 K, (α + 1), σ, and τ values, determined from
the fit, are included in Table 1. The table also includes the
σ and τ values for the two monodisperse lipids—MePa and
TriSt. The similarities between σ and τ values for estolide
acids, estolide esters, MePa, and TriSt suggest that the peak
widths are due to a large extent to the band broadening
from the column, and to a lesser extent to the dispersity
within the oligomers (from oligomers with the same num-
ber of units j, but different values of n in Scheme 1, for
example).
The values of (α + 1) for estolide acid and esters are low
compared to the slope of poly(hydroformylated methyl oleate)
(PHFMO), determined in THF at 30  C (Milic et al., 2012).
PHFMO has a structure that is close to (but different from)
the structure of the estolides. Similar to estolides, PHFMO
has a polyester backbone and a dangling hydrocarbon
chain. The dangling hydrocarbon chains are n-octyl or
n-nonyl for PHFMO, while in the estolides they can have
different lengths (the average side-chain lengths for the
estolides and PHFMO are expected to be close). PHFMO
also has an extra CH2 group in the backbone repeat
unit, and the end groups are different (methyl vs. 2-EH).
The slope for PHFMO in the Milic et al.’s study was
close to α=0.7 for the lower MW polymers, and decreased
at high MW. The authors explained the decrease of the
slope with the THF being a poor quality solvent for the
Ea (1 day)
Eb (4 days)
Recalculated dRI, a.u.
Ec
L
Ec
Eb
Ea j=1
j=2 j=3 j=4 j=5
1.0 1.5 2.0
log(M[η])
Fig. 5 GPC chromatograms of estolide esters Ea, Eb, and Ec (solid
lines), and the fit to Eb (dashed line). “L” denotes the lactone peak. Its
range is excluded from the fit
J Am Oil Chem Soc
dangling chains, which leads to their contraction. If this is
the case, THF should be even poorer quality solvent for the
estolides, because the low slope (α value) is observed at
low MW.
Presence of Cyclic Estolides
A potential complication is the possibility that cyclic esto-
lides can form during the estolide acid synthesis
(Scheme 4). The formation of cyclic oligomers is usually
favored when the starting concentration of the monomer
(oleic acid) is low. Lactones were reported earlier, and we
have detected them in Eb and Ec samples by GPC. To
determine the possible presence of cyclic dimers, some
samples were evaluated with MALDI and GC–MS (without
transesterification).
The 2-EH esters could be identified in the GC–MS sam-
ples by the presence of a prominent fragment with m/z
112 (2-ethyl 1-hexene ion) or 113. There was a group of
weak peaks that lacked these fragments. The presence of
fragment ions >400 Da suggested that they were dimers.
They were observed in E2b and E2c samples at slightly
above 4 area %.
The ions with m/z 587.85–587.88 are the expected dimer
estolide acids or dimer cyclic estolide (C36H68O4Na+
= 587.50) and are observed in the MALDI-MS data, where
they were a minor peak. Unfortunately, MALDI-MS
methods cannot definitely distinguish between linear esto-
lide acids and cyclic estolides, because they are isomers
and have identical molecular mass.
Taking Eq. 5 and the result from the GC–MS that the
ratio ½R2  ½M2  is approximately 4 area %, we can estimate
the concentration of all the cyclic estolides in the mix. Con-
centrations of estolide esters Eb and Ec were calculated to
be 1.6% and 1.9%, respectively. This evaluation was based
on the assumption that the peaks in the mixtures with ~4
area % were cyclic dimer estolide, and not something else,
like dimer estolide acids that remained during the esterifica-
tion with 2-EH alcohol.
Considering that, we should expect that the cyclic esto-
lides were present in a low amount and did not influence
significantly the α values of the mixtures.
Ec (7 days) Evaluation of Monomers and Dimers
Eb (4 days)
Ea (1 day)
When we fitted the GPC chromatograms of the monomer
2.5 3.0
esters (E1a, E1b, and E1c) and dimer esters (E2a, E2b, and
E2c) distilled away from the estolide mixture, we observed
that they contained more than 88% of the main component
(oleate 2-EH ester (EN = 0) or dimer estolide 2-EH ester
(EN = 1)). This led to overloading the column, and the
main peak was shifted relative to the corresponding one in
J Am Oil Chem Soc (2019)
 J Am Oil Chem Soc

J Am Oil Chem Soc (2019)

Table 3 Composition and related parameters of estolide esters, determined from fits of the GPC chromatograms

Ea Eba Ecb E−1a E−1b E−1c E−2a E−2b E−2c

1 day 4 days 7 days 1 day 4 days 7 days 1 day 4 days 7 days

% monomers 33.0  0.0 25.1  0.0 25.2  0.0 0.6 0.7 0.5 0.4  0.0 0.4  0.0 0.3  0.0
% dimers 26.2  0.1 23.4  0.1 23.5  0.1 39.2 31.4 31.6 24.1  0.1 21.4  0.8 21.0  0.0
% trimers 16.8  0.1 17.2  0.1 17.1  0.1 25.1 23.1 23.1 30.2  0.0 26.1  0.3 26.2  0.1
% tetramers 9.87  0.02 11.65  0.01 11.60  0.03 14.5 15.3 15.3 18.27  0.02 17.91  0.34 18.06  0.02
% pentamers 5.82  0.04 7.69  0.03 7.67  0.04 8.5 10.1 10.1 10.90  0.02 11.75  0.17 11.86  0.00
% higher oligomers 8.4  0.1 14.9  0.2 14.9  0.2 12.1 19.3 19.4 16.1  0.1 22.4  0.1 22.6  0.1
P 0.589  0.002 0.660  0.002 0.661  0.001 0.588 0.658 0.659 0.597  0.002 0.656  0.003 0.656  0.001
Mn (GPC1) 642  1 727  1 726  1 920 998 1002 997  1 1056  5 1065  0
Mw (GPC1) 863  3 1034  4 1032  3 1084 1232 1235 1201  5 1253  2 1314  3
Ð: (GPC1) 1.344  0.003 1.421  0.003 1.422  0.003 1.178 1.235 1.233 1.205  0.004 1.187  0.004 1.234  0.003
EN (GPC1) 0.880  0.002 1.182  0.004 1.177  0.003 1.865 2.141 2.155 2.235  0.005 2.443  0.022 2.471  0.003
Mn (GPC2) 626  2 712  2 710  2 891 971 976 999  1 1058  5 1066  0
Mw (GPC2) 854  2 977  4 1016  2 1070 1168 1222 1202  5 1254  2 1315  3
Ð: (GPC2) 1.364  0.000 1.372  0.003 1.432  0.002 1.20 1.20 1.25 1.203  0.004 1.185  0.004 1.233  0.003
EN (GPC2) 0.824  0.006 1.129  0.006 1.119  0.008 1.761 2.046 2.065 2.145  0.004 2.353  0.018 2.384  0.001
a
There was a lactone peak with area of 1.6% of the oligomer content.
b
There was a lactone peak with area of 3.0% of the oligomer content (the lactone amounts are not included in the total).
 J Am Oil Chem Soc

the other estolide mixtures toward lower z values (higher
elution volumes). This shift led to a poor fit, when a joint
fit was attempted (all the chromatograms with same param-
eters for (α + 1), log10K, σ, and τ).
To account for the effect of overloading the column,
chromatograms of the monomer esters and dimer esters
were fitted with the parameters (α + 1), log10K, σ, and τ,
obtained for the estolide esters (Table 2), but using an addi-
tional fitting parameter per chromatogram—the shifting
factor for the major peak. The results obtained from these
fits are listed in Table 4. While the use of a shifting factor
can be implemented in GPC1 calculations, it cannot be
implemented in the GPC2 calculations unless there is prior
research correlating with the loading of the column with
the shift factor. Because we applied the GPC2 calculation
without correction for overloading, the GPC2 results for
the MW and EN are shifted toward lower values.
Calculated Limiting Viscosity Values
The limiting viscosity numbers, [η], for the estolide oligo-
mers can be calculated from the parameters in Table 1 and
the MHE (Eq. 10). As the errors in the determination of the
constants (α + 1) and log10K are probably correlated,
Table 5 lists the values and the associated standard devia-
tion, derived using the jackknife method.
In this study, we also used MePa and TriSt. We calcu-
lated their [η] to be (26.4  0.1) × 0.001 and
(61.0  0.2) × 0.001 dL g−1, respectively. The lactone,
encountered in some of the estolide samples, had [η] =
(26.1  0.6) × 0.001 dL g−1.
We found some GPC data about oleic acid and triolein,
(Abidi and Warner, 2001) from which we were able to
Table 5 Limiting viscosity numbers, [η], in THF at 40  C for some
estolide oligomers. Values  standard deviation
j [η]j, 1000× dL g−1
Acids 2-EH esters
1 40.7  0.10 41.2  0.17
2 55.3  0.22 54.9  0.17
3 66.2  0.32 66.0  0.29
5 83.0  0.50 84.4  0.58
7 96.3  0.66 99.8  0.87
10 112.7  0.87 120  1.3
15 135  1.2 147  2.0
20 153  1.5 171  2.6
determine their [η] as 0.035 and 0.031 dL g−1, respectively.
The estolide acid monomer consists of oleic acids and their
isomers (the acid catalyst changes the configuration and the
position of the double bond). Its [η] of 0.0407 dL g−1 is
close to the value for the oleic acid (0.035 dL g−1). The
discrepancy between the values for TriSt (0.061 dL g−1)
and triolein (0.031 dL g−1) is much bigger, which indicates
a very strong effect of the double bonds on the limiting vis-
cosity number. The discrepancy between the present and
the previous research (Abidi and Warner, 2001) could be
due to the fact that the temperature of the GPC experiment
in the referenced study is not specified.
It should be noted that there are studies that cast in doubt
the principle of universal calibration for low MW polymers.
They have shown the universal calibration curves (M[η]
vs. elution volume) for three kinds of molecules (polysty-
rene, polyisobutene, and n-alkanes) deviate in the low
(<1000 Da) MW range (Chance et al., 1995). Attempts to
use for calibration a different than M[η] descriptor like the

Table 4 Composition and average values of monomer esters and dimer esters; no pentamers and higher mers were present
E1a E1ba E1cb E2a E2b E2c
1 day 4 days 7 days 1 day 4 days 7 days

% monomers 99.4 98.9 98.1 1.8 2.9 1.7

% dimers 0.5 1.0 1.4 91.1 88.0 89.3
% trimers 0.1 0.1 0.3 6.8 8.7 8.6
% tetramers 0.0 0.0 0.2 0.4 0.3 0.4
Mn (GPC1) 395 396 398 682 680 686
Mw (GPC1) 396 398 401 692 694 698
Ð (GPC1) 1.003 1.004 1.009 1.015 1.020 1.017
EN (GPC1) 0.004 0.007 0.013 1.022 1.014 1.036
Mn (GPC2) 374 380 385 648 649 655
Mw (GPC2) 379 382 409 663 663 673
Ð (GPC2) 1.013 1.005 1.062 1.022 1.022 1.028
EN (GPC2) −0.070 −0.050 −0.032 0.902 0.903 0.926
a
There was a lactone peak with area of 5.1% of the oligomer content.
b
There was a lactone peak with area of 9.5% of the oligomer content (the lactone amounts are not included in the total).

J Am Oil Chem Soc (2019)

J Am Oil Chem Soc

radius of gyration (Chance et al., 1995; Sun et al., 2004) or and

retention radius (Boyd et al., 1996) were only partially suc-
cessful. In light of these findings, the determined limiting RSGPC2=GC ¼ 3:0 ð24Þ
viscosity number values in this manuscript should be
regarded as apparent values, and a more direct method for The presence of a lactone peak (a low MW peak) leads
measuring (extrapolation from viscosity of solutions in to lowering of Mn, calculated by GPC, which leads to a
THF) should be used to confirm them. decrease in ENGPC. The deviations from one of the propor-
tionality constants in Eqs 21 and 23 could be due to the
presence of cyclic estolides. If higher cyclic estolides are
Comparison of the Methods for Determination of present, they will lead to an increase of ENGC and most
the EN probably, similar to lactones, will decrease the ENGPC
values. Calculations were made to determine the effect of
Fig. 6 compares the EN values, obtained by the the cyclic estolides on ENGC, assuming that the unidenti-
transesterification-GC and GPC1. It can be seen that there fied peaks in Eb and Ec are cyclic dimer estolides and
is a good correlation between the values. Because both Eq. 5 is valid for j ≥ 2. The result was that the cyclic esto-
methods for determining EN yield some errors in the lides will increase the ENGC values by 3.7–4.3%, which are
values, the method of Deming was used to calculate the close to the deviations of the proportionality constant from
regression coefficients (Billo, 2001). The result was: 1 (5.6% and 6.7%).
ENGPC1 ¼ 0:944 × ENGC + 0:0188 ð21Þ Therefore, the presence of cyclic estolides and lactones,
if not accounted for, will lead to opposing errors in determi-
nation of the EN.
From the slope of the regression line and standard devia- The lactones can be identified in the transesterification-
tions of the EN, determined by the two methods (σ GC and GC analysis, because they result in FAME with hydroxyl
σ GPC1), the relative sensitivity (RS) of the two methods was groups at the 4- and 5-positions, but we do not expect the
calculated according to Skiera et al. (2012): higher cyclic estolides to yield unique hydroxyl FAME
σ GC upon transesterification. In GPC2, the summations used in
RSGPC1=GC ¼ 0:944 ¼ 3:5 ð22Þ
σ GPC1 the calculation can exclude the low z interval, where the
lactone peak is observed. While the minimum between the
Because the RS > 1, it can be concluded that the GPC1
lactone peak and the monomer ester was at z ~0.954, for
method is more sensitive than the transesterification-GC for
the monomer acid the peak was ~1.0, so z = 0.900 looked
the determination of the EN.
like a better cutoff. In the current study, lactone was dis-
Comparing the GPC2 results with the transesterification-
tilled off and the monomer acid was in a low amount in the
GC (data not shown) resulted in:
investigated estolide acids, so the value of the low-cutoff
ENGPC2 ¼ 0:933 × ENGC −0:0443 ð23Þ limit had a negligible impact. In the future, the best cutoff
value for z may need to be addressed if the undistilled esto-
lide acids are analyzed.
Acids, monomer removed (A-1 a,b,c)
2.5 Un-distilled Esters (E a,b,c) In transesterification-GC, the hydroxy FAME with
Esters, monomer removed (E-1 a,b,c)
Esters, dimer reduced (E-2 a,b,c) hydroxy groups at the 4- or 5-position elute separately
2.0 Dimers(E2 a,b,c); Monomers(E1 a,b,c) and can be excluded from the calculations, too. Exclud-
EN from GPC1

1.5 ing all of the 4- and 5-hydroxy FAME in calculations

ignores estolides with these FA incorporated into the
1.0
Deming fit: estolides. A more elaborate study and reasoning needs to
of GPC1;
0.5 of GPC2. be applied to the analysis of the transesterification-GC
data in the cases where both lactones are present and
0.0
there are 4- and 5-hydroxy FA incorporated in the esto-
0.0 0.5 1.0 1.5 2.0 2.5
EN from GC lide oligomers. We are not aware of studies on such
cases.
In the case of overloading the column, the GPC1 method
Fig. 6 Comparison of the estolide numbers (EN), determined from
GC (x-axis), and the ones determined from GPC1 (y-axis). The error needs an additional adjustment parameter to account for the
bars show the standard deviations. We see that the error bars from the shift of the overloaded peak. In GPC2, there is no simple
GC are bigger than from GPC1. The symbols from the same sets from way to adjust for the overloaded peaks so the calculated
left to right correspond to a, b, and c samples. The best fit curve for
average MW and the EN are underestimates of the actual
the EN data for GPC2 is also shown (to avoid crowding the figure,
the individual datapoints from GPC2 are omitted) values.

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A source of error in the GPC2 stems from the band

broadening of the GPC peaks. The band broadening leads
to a decrease of the apparent Mn of the studied oligomers
(Busnel et al., 2001). A proportional fit between MnGPC2
and MnGPC1 (of all the datapoints, excluding the monomer
and dimer) showed that GPC2 underestimates Mn by 2.6%
compared to GPC1. The goodness of fit was R2 = 0.9996.
Mw and Dispersity
In addition to Mn (and the related EN), the GPC gives
information about the width of the distribution. The most
common method to express the distribution width is
through the dispersity Ð (Eq. 19). Fig. 7 displays the calcu-
lated Ð values as a function of EN. It can be seen that the
Dimer Esters, and the undistilled estolide esters, have close
EN values and it is hard to distinguish them by the EN
alone, but the Ð clearly differentiates the two samples.
Comparing the EN and Ð results from the same type of
mixture, but at different reaction times, shows that the EN
and Ð grow with the synthesis time. The largest changes
are seen during the first 24 h, while further reaction time
leads to less changes overall. In the case of the estolide
esters, the differences in EN and Ð between 96 and 168 h
were undetectable. A detectable difference was seen
between Eb and Ec in the amount of lactone formed, but it
is not included in these calculations.
Flory’s distribution (Eq. 1) and Eqs (16, 17, and 20) can
be used to derive the following formulas for the aver-
age MW:
ΔM
Mn ¼ + M0
1 −p
" #
 
1+p pð1 −pÞ
Mw ¼ ΔM + M0 1 −
1 −p M0
1 + ΔM ð1 −pÞ
Eb&Ec
1.4
Ea
a b
1.3
Ð from GPC1
p
EN ¼ ð27Þ
1 −p
Ð can be calculated from Eq. 19. The theoretical curve
from these formulas for the case of estolide esters is
plotted in Fig. 7. It can be seen that the experimental Ðs
for the undistilled estolide esters are higher than the the-
oretical ones. This cannot be easily explained, because
an inadvertent distillation of some of the monomers dur-
ing handling and esterification would lead to lower Ð
and increased EN values for the remaining estolide
esters. It can be seen that this was the case for the dis-
tilled esters.
Conclusions
In the current study, we demonstrated the feasibility of
using GPC to characterize estolide acids and estolide esters.
An important assumption for the GPC analysis was that the
MHE is valid for both the estolide acids and estolide esters.
The good fits support the validity of this assumption. In the
current study, we determined their MHE parameters in
THF at 40  C.
GPC yields EN for the samples that correlated well
with the EN, determined by the traditional method of
methanolysis and GC (transesterification-GC). Based on
the repeatability of the values, Deming analysis picked
the GPC as a more sensitive method than the traditional
one used for EN determination. Both methods do not
account for the possible presence of cyclic estolides. The
ð25Þ presence of cyclic estolides will influence the calculated
EN in the opposite direction, so the closeness of the
results from transesterification-GC and GPC indicates
ð26Þ that the amount of cyclic estolides was low in this study.
In addition to the EN, GPC analysis yields information
about the distribution of the oligomers and can identify
c the presence of a side product—lactone. Based on this,
we recommend using the GPC in cases when more in-

b&c
b&c depth information about the estolides is needed, and
a
1.2 a using both GPC and transesterification-GC when the
Acids, monomer removed (A-1 a,b,c) presence of cyclic estolides is suspected. Care must be
1.1 Un-distilled Esters (E a,b,c)
Esters, monomer removed (E-1 a,b,c) taken not to overload the GPC columns during the
Esters, dimer reduced (E-2 a,b,c)
1.0 Dimers(E2 a,b,c); Monomers(E1 a,b,c)
analysis.

0.0 0.5 1.0 1.5 2.0 2.5

EN from GPC1
Fig. 7 The dispersity (Ð) values as a function of EN. Both are deter-
mined using the GPC1 method. The standard deviations of the Ð
values are smaller than the marker size. The undistilled estolide esters
(Ea, Eb, and Ec) should lie on the solid line, if they follow the Flory’s
distribution
Acknowledgments We thank Daniel Knetzer for help with the GPC
instrument and Linda Manthey for the help with the runs. This work
is a part of the in-house research of the Agricultural Research Service
of the United States Department of Agriculture.
Conflict of interest The authors declare that they have no conflicts
of interest.

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Appendix
Generalized Exponentially Modified Gaussian (EMG)
Equation (11) was adequate to describe the data used in the
current manuscript
 J Am Oil Chem Soc

rffiffiffi  2    
hσ π σ z −μ s σ z −μ overflow/underflow. One possibility is to use the series
yðzÞ ¼ exp − erfc pffiffiffi − from Kalambet et al. (2011), and the other is to use the con-
jτ j 2 2τ2 τ 2 τ σ
tinued fraction presentation (Anon, 2018).
ð11Þ
Another question is to calculate the position of the peak
For the meaning of the symbols, see the explanation to maximum, zp. Using that at zp, the first derivative of Eq. 11
Eq. 11 in the main text. However, as it is pointed out is zero, it can be derived that:
(Di Marco and Bombi, 2001; Kalambet et al., 2011), no rffiffiffi
jτ j 2
single formula for EMG can be evaluated for all the combi- erfcxðuÞ ¼ when z ¼ zp ðA4Þ
nations of (z − μ), σ, and τ due to numerical overflow or σ π
underflow of the functions. Kalambet et al. (2011) pre- or:
sented an algorithm to evaluate the EMG for any combina-   qffiffiffiffiffiffiffi
σ pffiffiffi jτ j 2
tion of (z − μ), σ, and τ. Here, we expand their approach to zp ¼ μ + σ −s 2erfcxinv =π ðA5Þ
the generalized EMG. First, a variable u is calculated: τ σ
s σ z −μ where erfcxinv() is the inverse function of erfcx(). For
u ¼ pffiffiffi − ðA1Þ
2 τ σ |τ|/σ > 0.001, erfcxinv() can be evaluated numerically using
an iterative procedure adapted from Acklam (2018) to
If τ is equal to zero, then just a Gaussian is calculated Visual Basic for Excel. Since Kalambet et al. (2011)
from z − μ to σ. warned that this iterative procedure cannot converge for
For u < 0, the Eq. 11 is used. The erfc() function is avail- very small arguments, a crude approximation was made to
able in the Excel and other software programs (Appendix ensure good starting values for the iterations. The Eq. A5 is
S1, Supporting information). For u > 0, the Eq. (11) can be that it is not numerically stable for very small values of
rearranged into: |τ|/σ. This can be easily overcome by using the approximate
rffiffiffi  z −μ2  
hσ π Eq. A6 (Kalambet et al., 2011):
yðzÞ ¼ exp −0:5 exp u2 erfcðuÞ ðA2Þ
jτ j 2 σ zp ¼ μ + τ for jτj=σ < 0:001 ðA6Þ
The scaled complementary error function: The relative error of using A6 for |τ|/σ < 0.001 is less
 than 0.0001 %.
erfcxðuÞ  exp u2 erfcðuÞ ðA3Þ
An Excel file with the function needed for the calcula-
can be evaluated directly for values of u < 26.5. For tions of the generalized EMG is available upon request.
u > 26.5, a different approach is needed, due to numerical

J Am Oil Chem Soc (2019)