Analysis of Ethoxylated Fatty Amines.
Comparison of Methods
for the Determination of Molecular Weight
Russell F. Lang*, Dennisse Parra-Diaz, and Dana Jacobs
Beckman-Coulter Inc., Miami, Florida 33196-2500
ABSTRACT: Specific lengths of the fatty and polyoxyethylene
chains of ethoxylated fatty amines are critical to their perfor-
mance in specific applications, and thus the ability to charac-
terize these surfactants accurately is crucial. Normal-phase
high-performance liquid chromatography (HPLC) and matrix-
assisted laser desorption ionization-time of flight (MALDI-TOF)
mass spectrometry methods were developed to determine with
accuracy the molecular weight and degree of ethoxylation of
ethoxylated fatty amines. Ethoxylated fatty amines were ana-
lyzed using these methods, and comparison was made to mo-
lecular weight determinations using proton nuclear magnetic
resonance (NMR), neutralization equivalent weight, and hy-
droxyl value methods. Molecular weight results from normal-
phase HPLC analyses were in very good agreement with
MALDI-TOF results, typically varying less than one ethylene
oxide unit. A reversed-phase HPLC method was developed to
determine concentrations of polyethylene glycols (PEG) and
fatty homologs. PEG interfered with molecular weight determi-
nations by NMR, neutralization equivalent weight, and hy-
droxyl value methods. PEG caused no interference with molec-
ular weight determinations by normal-phase HPLC and MALDI-
TOF methods.
Paper no. S1134 in JSD 2, 503–513 (October 1999).
KEY WORDS: Degree of ethoxylation, ethoxylated fatty
amines, HPLC, hydroxyl value, MALDI-TOF, molecular weight,
neutralization equivalent weight, 1H NMR.
Ethoxylated fatty amines are used in different industrial
applications such as defoamers, textile-finishing agents,
corrosion inhibitors, emulsifiers (1), and dye promoters
(2,3). They enhance herbicidal activity of many pesticides
(1) and have potential uses in laundry products (4).
Ethoxylated fatty amines are produced by the reaction of a
fatty amine with ethylene oxide (EO) (1,5). The two-step
ethoxylation of a primary amine is shown in Equations 1
and 2, where R typically is C12–C18 fatty groups.
∆
RNH 2 + 2 ( C 2 H 4 O ) → RN ( CH 2 CH 2 OH ) 2
*To whom correspondence should be addressed at Beckman-Coulter
Inc., Mail Stop 11-A02, 11800 SW 147 Ave., Miami, FL 33196-2500.
E-mail: russell.lang@coulter.com
Copyright © 1999 by AOCS Press Journal of Surfactants and Detergents, Vol. 2, No. 4 (October 1999)
base, ∆
RN(CH2CH2OH)2 + (x + y)(C2H4O)
[2]
(CH2CH2O)x+1H
RN
(CH2CH2O)y+1H
The product is a polydisperse mixture of oligomers ap-
proaching a Poisson distribution (5). Polyethylene glycols
(PEG) are side products, formed from the reaction of EO
with residual water. Ethoxylated fatty amines are commer-
cially produced from coco, lauryl, tallow, oleyl, and stearyl
amines and typically contain from 2 to 50 moles of EO per
mole of amine hydrophobe. Specific lengths of the fatty
and polyoxyethylene chains are critical to performance in
a specific application, thus the ability to accurately charac-
terize these surfactants is crucial.
Analysis of ethoxylated fatty amines for estimating the
degree of ethoxylation (DOE) and average molecular
weight has historically been performed using the neutral-
ization equivalent weight (NEW) (6) and hydroxyl value
methods (6,7). These methods are still used almost exclu-
sively by the surfactant industry for determination of the
molecular weight of ethoxylated fatty amines. NEW is
often used during manufacturing as an in-process test to
determine the extent of ethoxylation. NEW is estimated by
titrimetric neutralization of the amine group with stan-
dardized acid. The hydroxyl value method involves de-
rivatization of terminal hydroxyl groups using either acetic
or phthalic anhydride followed by quantitative determina-
tion of excess anhydride.
Reversed- and normal-phase high-performance liquid
chromatography (HPLC) methods have been developed
for analysis of ethoxylated nonionic surfactants, most com-
monly for ethoxylated fatty alcohols, acids, sulfonates, and
alkylphenols (8–11). The one reported method applicable
to ethoxylated fatty amines was limited to a mean EO con-
[1] tent of only 15 moles owing to the ion-pair/fluorescence
detection system used (12). Use of an evaporative light-
scattering detector for HPLC applications allows for detec-
tion of polyalkoxylated compounds with no molecular
weight limitations (13). Matrix-assisted laser desorption
ionization-time of flight (MALDI-TOF) mass spectrometry
503
504 R.F. LANG ET AL.
has recently been used for the analysis of some synthetic
surfactants (14–18); however, ethoxylated fatty amines
have not been evaluated using this technique. Nuclear
magnetic resonance (NMR) spectroscopy has been an in-
valuable tool for the molecular structural analysis of or-
ganic compounds. This technique has been used to deter-
mine the degree of ethoxylation and characterize physical
properties of nonionic surfactants (19).
In our attempts to characterize ethoxylated fatty amines
and to estimate their degrees of ethoxylation and average
molecular weights accurately, new HPLC and MALDI-TOF
mass spectrometry methods were developed. These re-
versed-phase and normal-phase HPLC methods incorpo-
rated the use of an evaporative mass detector. The evapora-
tive mass detector is ideal for analytes lacking suitable chro-
mophores, and minimal baseline drift is observed when
used with gradient elution chromatography. The reversed-
phase HPLC method was developed to separate and quan-
tify PEG and fatty homologs of ethoxylated fatty amines.
The normal-phase method was developed to separate
oligomers of ethoxylated fatty amines and to determine
their average molecular weights. MALDI-TOF mass spec-
trometry was used to characterize ethoxylated fatty amines
and to assign mass values to oligomers of normal-phase
HPLC analyses. Ethoxylated fatty amines were also charac-
terized using 1H NMR, NEW, and hydroxyl value methods.
The results of these molecular weight determinations, using
these five different methods, showed that for many of the
ethoxylated fatty amines samples analyzed different meth-
ods yielded significantly different molecular weight esti-
mates. This paper describes the molecular weight charac-
terization of different ethoxylated fatty amines using these
five methods. A comparison of the results is reported.
EXPERIMENTAL PROCEDURES
Ethoxylated fatty amines were obtained from commercial
sources (Akzo Nobel Chemicals Inc., Chicago, IL; Ethox
Chemicals, Inc., Greenville, SC; Heterene Inc., Paterson, NJ)
and were used, as received, without further purification.
HPLC. The HPLC chromatographic system was a Wa-
ters 2690 (Milford, MA) with a Polymer Labs EMD 960
evaporative mass detector (Amherst, MA). The EMD 960
detector used industrial grade nitrogen (Air Products, Al-
lentown, PA) at a flow rate of 5 L/min. The detector was
operated at 65°C for normal-phase separations and 75°C
for reversed-phase separations. Sample solutions were pre-
pared by dissolving 100 mg of ethoxylated fatty amine into
10 mL of 2-propanol. Sample solutions were filtered
through 0.45 µm GH Polypro membrane filters (Gelman
Sciences, Ann Arbor, MI). A sample volume of 10 µL was
injected into the HPLC system for analysis. Mobile phases
were filtered through 0.45-µm GH Polypro membrane fil-
ters prior to use. For all HPLC analyses, the HPLC columns
were maintained at 40°C, and a mobile phase flow rate of
1 mL/min was used.
Journal of Surfactants and Detergents, Vol. 2, No. 4 (October 1999)
All reversed-phase HPLC analyses including PEG analy-
ses were performed using a Waters Nova-Pak 60Å C18, 4
µm, 150 × 3.9 mm column (Milford, MA). The isocratic mo-
bile phase was MeOH/H2O (85:15) containing 25 mM tri-
ethylamine and 50 mM glacial acetic acid. Normal-phase
HPLC separations were performed on a LiChrospher 100Å
Diol, 5 µm, 150 × 4.6 mm column (Alltech Associates, Deer-
field, IL). The mobile phase gradient program used for the
majority of ethoxylated fatty amines was a linear gradient
of hexane/2-propanol (both solvents contain 25 mM
triethylamine) from 95:5 to 70:30 over 140 min. This pro-
gram allowed for analysis of ethoxylated fatty amines over
a wide range of ethoxylate chain lengths, typically 5 to 60
EO units for an ethoxylated stearyl amine. As discussed
below, this mobile phase program can be modified to opti-
mize the analysis of a specific ethoxylated fatty amine.
MALDI-TOF mass spectrometry. MALDI mass spectra
were acquired on a PE-PerSeptive Biosystems (Framing-
ham, MA) Voyager-DE STR delayed extraction reflectron
time-of-flight mass spectrometer equipped with a Laser
Science nitrogen laser (337 nm, 3 ns pulse). Positive ion
spectra were acquired in the linear mode using an ac-
celeration voltage of 20 kV. The matrix used was a satu-
rated solution of α-cyano-4-hydroxycinnamic acid in 1:1
MeCN/H2O containing 0.1% trifluoroacetic acid (TFA).
Samples for MALDI-TOF analysis were prepared by dis-
solving 1 mg of sample in 1 mL MeCN/H2O (1:1) and fur-
ther diluted 1:20 with H2O containing 0.1% TFA. A 1-µL
aliquot of the sample solution was thoroughly mixed with
an equal volume of the α-cyano-4-hydroxycinnamic acid
matrix solution and analyzed.
1
H NMR. 1H NMR samples were analyzed at room tem-
perature on an NT-360 (360 MHz) spectrometer (Nicolet In-
struments Corporation, Madison, WI) with a wide-bore (89
mm) magnet or a 400 MHz spectrometer (Varian Analytical
Instruments, Valencia, CA). Samples were dissolved in
deuterated acetone or chloroform resulting in concentra-
tions of approximately 20 mM. NMR data were analyzed
using NUTS data analysis software (ACORN NMR Inc., Fre-
mont, CA). Proton NMR chemical shift assignments were
based on characteristic proton NMR shift tables. In addition,
NMR spectra of stearyl amine, PEG-900, and PEG-1500 were
used as guides to verify chemical shift values for the fatty
and polyoxyethylene moieties. Proton NMR molecular
weight calculations were based on the ratio of integrated
peak areas between the terminal methyl group of the alkyl
chain (δ = 0.872–0.888 ppm) to (i) the methylene protons of
the ethoxylate chains (δ = 3.580–3.595 ppm) excluding those
adjacent to the amine nitrogen, (ii) the hydroxyl terminal
protons (δ = 2.843–2.854 ppm), and (iii) the alkyl protons (δ
= 1.284–1.302 ppm) excluding the methyl protons, protons
adjacent to the methyl group, and protons adjacent to the
amine nitrogen. These ratios were verified by comparing ra-
tios of the terminal methyl protons to (i) its adjacent methyl-
ene protons (δ = 1.4343–1.581 ppm) and (ii) methylene pro-
tons adjacent to the nitrogen (δ = 2.512–2.522 ppm).
 ANALYSIS OF ETHOXYLATED FATTY AMINES 505

NEW. NEW was determined potentiometrically using
aqueous and nonaqueous titrations. Aqueous titrations
were performed to determine the concentration of base cat-
alyst remaining in the amine sample. Samples (≈2 g) were
dissolved in water/2-propanol (1:1) and titrated against
0.1 N HCl using a Mettler-Toledo DL-50 titrator (Hights-
town, NJ) equipped with a 20.0 mL buret and a DG111 elec-
trode. Nonaqueous titrations were performed by dissolv-
ing 2-g samples in 50 mL glacial acetic acid and titrating
against 0.1 N perchloric acid using a DG113 electrode.
Hydroxyl value. The American Oil Chemists’ Society hy-
droxyl value method (20) was followed to obtain the hy-
droxyl value of ethoxylated amines with the following
modifications. Samples for acetylation and two blanks
were refluxed for 1 h under constant stirring in a mineral
oil bath. The oil bath temperature was maintained within a
range of 95.0–110.0°C. The reaction was then quenched by
adding 15.0 mL of deionized water to the mixture followed
by a 20-min incubation in the oil bath. The mixtures were
allowed to cool to room temperature, then titrated with
ethanolic potassium hydroxide. Duplicate analyses were
performed for each sample, and the mean value was re-
ported. The method resulted in a coefficient of variation
(CV) of 7.0% for 14 determinations performed over a pe-
riod of several months.
RESULTS AND DISCUSSION
MALDI-TOF mass spectrometry. Ethoxylated fatty amines
with DOE values ranging from 10 to 50 were evaluated by
MALDI-TOF mass spectrometry. The α-cyano-4-hydroxy-
cinnamic acid/TFA matrix yielded reproducible mass
spectra with high abundances for all ethoxylated fatty
amines. A CV of 1.4% was obtained for Mn (number aver-
age molecular weight) values from three analyses of a 28-
mole EO stearyl amine in which the samples were pre-
pared and analyzed over a period of 4 mon. For these
ethoxylated fatty amines, the MALDI-TOF mass spectra
showed no fragmentation. The major mass peaks appear
as [M + H]+ ions with no multiply-charged ions observed.
The formation of sodium or potassium adducts was negli-
gible. In contrast, ethoxylated fatty alcohols analyzed
under identical conditions produced spectra in which the
predominant peaks appeared as sodium and potassium
adducts (Lang, R.F., and D. Parra-Diaz, unpublished re-
sults). The MALDI-TOF mass spectrum of a 25-mole EO
tallow amine is shown in Figure 1. The oligomer distribu-
tion is symmetrical with the highest abundant mass peak
(Mp) at 1122.9 Da (mono-isotopic) which represents the
monoprotonated oligomer, [C16N[EO]20H]+. Mn was calcu-
lated to be 1159 Da, where Mn = ∑(MiNi)/∑Ni, and Ni and
Mi are the abundance and mass of the ith oligomer, respec-
tively. The weight average molecular weight (Mw), defined
as ∑(Mi2Ni)/∑MiNi, was 1208 Da. The polydispersity index
(D), defined as Mw/Mn, was calculated to be 1.042, indicat-
ing a narrowly dispersed polymer. Figure 2 is an expanded
mass spectrum of the 25-mole EO tallow amine and shows
the saturated homologs, octadecyl ([C18N[EO]20H]+, m/z =
1150.9), hexadecyl ([C16N[EO]20H]+, m/z = 1122.9), and
tetradecyl ([C14N[EO]21H]+, m/z = 1138.9), and the mono-

FIG. 1. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrum of a 25-mole ethylene
oxide (EO) tallow amine. Mn, number average molecular weight; Mw , weight average molecular weight; D, poly-
dispersity index.

Journal of Surfactants and Detergents, Vol. 2, No. 4 (October 1999)

506 R.F. LANG ET AL.

FIG. 2. Expanded MALDI-TOF mass spectrum of 25-mole EO tallow amine. For abbreviations see Figure 1.

unsaturated octadecyl homolog ([C18eneN[EO]20H]+, m/z Mp and Mn values was within one mole of EO (44 Da), and
= 1148.8). D values were all very low, indicating that these ethoxy-
Table 1 is a summary of MALDI-TOF molecular weight lated amine polymers were all narrowly dispersed. For the
results for ethoxylated fatty amines over a DOE range of 28-mole EO stearyl amine Sample #2, the mass spectrum
10 to 50. For the samples listed, except the 28-mole EO showed that the oligomer distribution was slightly skewed
stearyl amine Sample #2, the difference between the toward higher masses. This resulted in a mass difference
of 81 Da between the Mp and Mn values and a slightly
higher D value of 1.07. The COA MW (certificates of analy-
TABLE 1
Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass
sis molecular weights) are the molecular weight stated on
Spectrometry (MALDI-TOF) Results for Fatty Amine Ethoxylates the products’ COA and were provided by the manufactur-
Ethoxylated fatty amine COA MWa Mpb Mnc Mwd De ers. The COA MW values listed in Table 1 show varying
degrees of agreement with the molecular weight values de-
10-mole EO stearyl amine,
termined by MALDI-TOF mass spectrometry. These dis-
nominal MW = 709 697 666 686 730 1.06
25-mole EO tallow amine, crepancies in molecular weight between the COA and the
nominal MW = 1369 1406 1122 1159 1208 1.04 MALDI-TOF values are discussed in detail below in con-
27-mole EO stearyl amine #1, junction with results of the PEG analyses.
nominal MW = 1457 1465 1458 1502 1564 1.04 Normal-phase HPLC. Both cyano and diol columns were
27-mole EO stearyl amine #2,
evaluated with various mobile phases to determine the op-
nominal MW = 1457 1450 1370 1391 1456 1.05
27-mole EO stearyl amine #3, timal analytical conditions for separation of individual
nominal MW = 1457 1450 1370 1372 1439 1.05 oligomers. The diol column consistently gave superior res-
28-mole EO stearyl amine #1, olution vs. the cyano column; thus all analyses were per-
nominal MW = 1501 1497 1546 1539 1616 1.05 formed using the diol column. The basic nature of the
28-mole EO stearyl amine #2,
amine group required addition of a modifier to the mobile
nominal MW = 1501 1605f 1282 1363 1452 1.07
50-mole EO stearyl amine, phase to eliminate peak tailing caused by silanol effects
nominal MW = 2469 2429 2647 2616 2666 1.02 (21). By using the diol column with a hexane/2-propanol
a
Molecular weight (MW) from manufacturer’s certificate of analysis (COA). mobile phase and no mobile phase modifier, ethoxylated
Determined by neutralization equivalent weight (NEW) analysis. fatty amines eluted as a broad tailing peak with no oligo-
b
Most probable molecular weight (unprotonated mono-isotopic mass). mer separation. Since the evaporative mass detector re-
c
Number average molecular weight (Mn = ∑(MiNi)/∑Ni).
d quires that only volatile buffers and modifiers be used in
Weight average molecular weight (Mw = ∑(Mi2Ni)/∑MiNi).
e
Polydispersity index (Mw/Mn). the mobile phase, triethylamine was initially evaluated.
f
MW from manufacturer’s COA. Determined by hydroxyl value analysis. Optimization experiments showed that good separation of

Journal of Surfactants and Detergents, Vol. 2, No. 4 (October 1999)

 ANALYSIS OF ETHOXYLATED FATTY AMINES 507

oligomers with minimal peak tailing was achieved with
the diol column using a hexane/2-propanol mobile phase
gradient containing 25 mM triethylamine. The ethoxylated
fatty amine oligomers elute in order of increasing EO units.
Figure 3 is a chromatogram of a sample containing three
components: a “15-mole EO” tallow amine, a “27-mole
EO” stearyl amine, and a “50-mole EO” stearyl amine.
These are nominal values of DOE, used for product identi-
fication, and are not the actual DOE values. The mobile
phase program was a linear gradient of hexane/2-
propanol (both solvents contain 25 mM triethylamine)
from 95:5 to 70:30 over 140 min. The chromatogram shows
the method is applicable up to approximately 60 moles EO
for stearyl amine. Good separation of oligomers is ob-
served as evidenced by almost complete baseline separa-
tion throughout the chromatogram. Some selectivity in the
separation of fatty groups of the 15-mole EO tallow amine
is observed as evidenced by split peaks due to the differ-
ent tallow amine fatty groups. Use of the evaporative mass
detector consistently resulted in negligible baseline drift
for the mobile phase gradients. This mobile phase gradient
is amenable to modification to meet the requirements for a
specific analysis. For example, as shown in Figure 4A, the
analysis time for a routine determination of a 27-mole EO
stearyl amine was reduced to less than 60 min using an ini-
tial mobile phase containing a higher concentration of 2-
propanol together with a steeper gradient. This mobile
phase program was hexane/2-propanol (both containing
25 mM triethylamine) from 90:10 to 60:40 over 80 min.
Chromatographic peak retention times were very repro-
ducible with this HPLC system, resulting in a CV = 0.40%
for analysis of five replicates of the 27-mole EO stearyl
amine.
Mass assignment of normal-phase HPLC oligomers. Mass
values were assigned to oligomer peaks of a normal-phase
HPLC analysis of a 27-mole EO stearyl amine. The high-
est-intensity HPLC peak (38.48 min) of a 27-mole EO
stearyl amine, shown Figure 4A, was isolated by repetitive
collection from nine analyses. These nine fractions were
pooled, the solvent was evaporated under a stream of N2,
and the residue was analyzed by MALDI-TOF mass spec-
trometry. As shown in Figure 4B, the m/z of the 38.48 min
peak was found to be 1503 Da (monoprotonated, mono-
isotopic mass), resulting in a DOE value of 28. Lower-
abundant peaks at 1459 and 1547 Da, which represent
oligomers with DOE values of 27 and 29, respectively, were
also observed in the fraction-collected sample. Presence of
these two other oligomers was due to the manual fraction
collection procedure, which required that the detector inlet
fitting be uncoupled to collect column effluent. To allow
for the time required for this procedure, the fraction collec-
tion process was intentionally started earlier and termi-
nated later relative to the peak valleys to ensure complete
collection of the 38.48 min peak. Mn value of the 27-mole
EO stearyl amine sample as determined by MALDI-TOF
(Fig. 4C) was calculated as 1502 Da. Thus, the m/z value de-

FIG. 3. Normal-phase high-performance liquid chromatography (HPLC) chromatogram of a three-component sam-

ple containing a 15-mole EO tallow amine, 27- and 50-mole EO stearyl amines. Stationary phase: LiChrospher
100Å Diol, 5 µm (150 × 4.6 mm column; Alltech Associates, Deerfield, IL). Mobile phase gradient elution:
hexane/2-propanol (both solvents containing 25 mM triethylamine) from 95:5 to 70:30 over 140 min. Flow rate: 1
mL/min. Column temperature: 40°C. Detector: evaporative mass detector. For abbreviation see Figure 1.

Journal of Surfactants and Detergents, Vol. 2, No. 4 (October 1999)

508 R.F. LANG ET AL.

m/z
FIG. 4. Assignment of mass values to oligomers of a normal-phase HPLC analysis. (A) Normal-phase HPLC chromatogram of a 27-mole EO stearyl amine.
The highest-intensity HPLC peak (38.48 min) was isolated by repetitive collection from nine analyses. Stationary phase: LiChrospher 100Å Diol, 5 µm (150 ×
4.6 mm column). Mobile phase gradient elution: hexane/2-propanol (both solvents containing 25 mM triethylamine) from 90:10 to 60:40 over 80 min. Flow
rate: 1 mL/min. Column temperature: 40°C. Detector: evaporative mass detector. (B) MALDI-TOF mass spectrum of the isolated 38.48 min HPLC peak. (C)
Complete MALDI-TOF mass spectrum of the 27-mole EO stearyl amine. For abbreviations and manufacturer see Figures 1 and 3, respectively.

Journal of Surfactants and Detergents, Vol. 2, No. 4 (October 1999)

 ANALYSIS OF ETHOXYLATED FATTY AMINES 509

termined for the highest-intensity HPLC peak, 1503 Da,

was in excellent agreement the MALDI-TOF Mn value of
1502 Da for the 27-mole EO stearyl amine. Once calibrated
using MALDI-TOF, normal-phase HPLC was used for de-
termination of the molecular weight of a variety of ethoxy-
lated fatty amines using retention times and mass assign-
ments of the 27-mole EO stearyl amine as reference values.
Reversed-phase HPLC. A C18 reversed-phase HPLC
method was developed for determination of PEG, and
alkyl homologs of ethoxylated fatty amines. Aqueous sol-
vent systems using MeOH, MeCN and tetrahydrofuran
(THF) were evaluated using acetic acid and triethylamine
modifiers. In the absence of any modifiers, a 28-mole EO
stearyl amine bound so strongly to the silica surface of the
C18 packing that even after 48 min of using 100% methanol
at a flow rate of 1 mL/min, the amine did not elute. Opti-
mization experiments showed that a mobile phase consist-
ing of MeOH/H2O (85:15) containing 25 mM triethylamine
and 50 mM glacial acetic acid gave a rapid analysis with
good separation of alkyl homologs with minimal peak tail-
ing of ethoxylated fatty amines. Figure 5 shows the chro-
matogram of a 27-mole EO stearyl amine. Complete sepa-

FIG. 6. Reversed-phase HPLC chromatogram of 15-mole EO coco

amine. HPLC conditions are the same as described in Figure 5. For ab-
breviations see Figures 1 and 3.

ration of C16 and C18 homologs was achieved. Hydrophilic

FIG. 5. Reversed-phase HPLC chromatogram of 27-mole EO stearyl
amine. Stationary phase: Waters Nova-Pak 60Å C18, 4 µm (150 × 3.9
mm column). Mobile phase isocratic elution: MeOH/H2O (85:15) con-
taining 25 mM triethylamine and 50 mM glacial acetic acid. Flow rate:
1 mL/min. Column temperature: 40°C. Detector: evaporative mass de-
tector. For abbreviations see Figures 1 and 3.
PEG are weakly retained by the C18 column and elute first,
as a single peak. Alkyl homologs elute in the order of in-
creasing alkyl chain length. Figure 6 shows the chromato-
gram of a 15-mole EO coco amine and the separation
achieved for C10, C12, C14, and C16 homologs. Analyses of
ethoxylated fatty amines with the same alkyl group con-
taining differing ethoxylate chain lengths showed that the
longer-chain ethoxylates eluted earlier than the shorter-
chain species. This observation is presumably due to an in-
crease in polarity as the ethoxylate chain length increases.
Under reversed-phase HPLC conditions, the analytes ex-
hibiting greater polarity elute first.
PEG quantitation. Ethoxylated fatty amines were ana-
lyzed for PEG content to determine how PEG influenced
molecular weight results for different methods. PEG con-
centrations in ethoxylated fatty amines were calculated
from a calibration curve prepared from analyses of stan-
dard solutions of PEG 1000, which in turn, were prepared
in acetonitrile and analyzed in triplicate. The calibration
plot is shown in Figure 7. Response from the evaporative
mass detector is linear when plotted logarithmically (22).
The coefficient of multiple determinations (R2) for the cali-
bration plot was 0.9987 over a concentration range of two
orders of magnitude. Sensitivity of the evaporative mass
detector allowed for detection of 100 ng of PEG 1000 with
a signal/noise ratio >5.

Journal of Surfactants and Detergents, Vol. 2, No. 4 (October 1999)

510 R.F. LANG ET AL.

FIG. 7. Calibration plot for polyethylene glycol (PEG) 1000. HPLC conditions are the same as
described in Figure 5. For abbreviation see Figure 3.

Molecular weight determinations—comparison of methods.
Ethoxylated fatty amines from multiple vendors were ana-
lyzed using MALDI-TOF mass spectrometry, 1H NMR, nor-
mal-phase HPLC, NEW, and hydroxyl value to determine
molecular weights. The ethoxylated fatty amines included
10-, 27-, 28-, and 50-mole EO stearyl amines and a 25-mole
EO tallow amine. Three different 27-mole EO stearyl amine
samples and two different 28-mole EO stearyl amine sam-
ples from two different manufacturers were analyzed.
Table 2 shows molecular weight and DOE values for the
ethoxylated amine samples. The COA MW for all samples
except the 28-mole EO stearyl amine Sample #2 were de-
rived from the NEW determination, and good agreement
between the COA MW and NEW values was found. The
COA MW for the 28-mole EO stearyl amine Sample #2 was
obtained from hydroxyl value determination.
MALDI-TOF Mn values were in good agreement with
molecular weight results from normal-phase HPLC mea-

TABLE 2
Comparison of Molecular Weight Results from Different Methods
Ethoxylated fatty amine COA Hydroxyl
MWa Mnb HPLCc NEWd 1
H NMR value MW
(DOE)e (DOE)e (DOE)e (DOE)e (DOE)e (DOE)e
f
10-mole EO stearyl amine 697 686 665 713 730
(nominal NW = 709) (9.7) (9.5) (9.0) (10.1) (10.5)
f
25-mole EO tallow amine 1406 1159 1184 1426 1526
(nominal MW = 1369) (25.8) (20.2) (21.0) (26.3) (28.6)
27-mole EO stearyl amine #1 1465 1502 1501 1496 1693 1300
(nominal MW = 1457) (27.2) (28.0) (28.0) (27.9) (32.4) (23.4)
27-mole EO stearyl amine #2 1450 1391 1369 1475 1558 1301
(nominal MW = 1457) (26.8) (25.5) (25.0) (27.4) (29.3) (23.5)
27-mole EO stearyl amine #3 1450 1372 1369 1465 1575 1190
(nominal MW = 1457) (26.8) (25.1) (25.0) (27.2) (29.7) (20.9)
28-mole EO stearyl amine #1 1497 1539 1589 1563 1791 1340
(nominal MW = 1501) (27.9) (28.9) (30.0) (29.4) (34.6) (24.3)
28-mole EO stearyl amine #2 1605g 1363 1325 1767 2003 1410
(nominal MW = 1501) (30.4) (24.9) (24.0) (34.0) (39.4) (25.9)
f
50-mole EO stearyl amine 2429 2616 2601 2462 2862
(nominal MW = 2469) (49.1) (53.3) (53.0) (49.8) (58.9)
a
MW from manufacturer’s COA. Determined by NEW analysis.
b
Mn (MALDI-TOF number average molecular weight) = ∑(MiNi)/∑Ni.
c
MW for stearyl amines calculated as 100% stearyl homolog. MW for 25-mole EO tallow amine based on homolog com-
position of 30% palmitic, 25% stearyl, and 45% oleic acid.
d
Neutralization equivalent weight (nonaqueous titration method).
e
Degree of ethoxylation.
f
Not performed.
g
MW from manufacturer’s COA. Determined by hydroxyl value analysis. HPLC, high-performance liquid chromatography;
NMR, nuclear magnetic resonance.

Journal of Surfactants and Detergents, Vol. 2, No. 4 (October 1999)

 ANALYSIS OF ETHOXYLATED FATTY AMINES 511

surements throughout the molecular weight range. Molec-
ular weight values from normal-phase HPLC analyses for
all ethoxylated stearyl amines were calculated as 100%
stearyl amine since the fatty homolog composition was
typically >95% stearyl. For the 25-mole EO tallow amine,
molecular weight was calculated based on a fatty homolog
composition of 30% palmitic, 25% stearic, and 45% oleic
acid. Molecular weights derived from normal-phase HPLC
analyses for all samples were within ±50 Da of the MALDI-
TOF molecular weight values. These results are consistent
with reports that the Mn and Mw values determined by
MALDI-TOF are in agreement with molecular weights
measured by chromatographic methods for polymers with
narrow molecular weight distributions (D ≤ 1.2) (16,18). In
addition, this agreement between normal-phase HPLC and
MALDI-TOF methods throughout the mass range indi-
cates that mass discrimination in the MALDI-TOF deter-
mination at the higher end of the mass range is not occur-
ring as was observed for some polydisperse polymers (18).
This presumably is due to the relatively low molecular
weights and narrow molecular weight distribution of the
ethoxylated fatty amine samples.
For the majority of the samples, the NEW and 1H NMR
determinations overestimated the molecular weight values
when compared to MALDI-TOF Mn results. The hydroxyl
value method generally underestimated the molecular
weight of ethoxylated fatty amines samples except for the
28-mole EO stearyl amine Sample #2. Side products pres-
ent in the ethoxylated fatty amine samples, which contain
terminal hydroxyl groups such as PEG, result in a lower
molecular weight value being obtained from the hydroxyl
value method. The determination of molecular weight by
1
H NMR uses the ratios of the fatty moiety to the poly-
oxyethylene and hydroxyl groups. Thus, the presence of
PEG results in an overestimation of molecular weight
when determined by 1H NMR. This was clearly evident in
the 28-mole EO stearyl amine Sample #2 where the signals
due to hydroxyl and ethoxylate protons were excessively
higher than theoretical values, and more than one signal
attributed to hydroxyl protons was observed.
NEW values are calculated from the quotient of sample
weight and moles of acid titrated. Any neutral compounds
present, such as PEG result in an overestimated NEW
value. The NEW values are also affected by residual base
catalyst present in the ethoxylated fatty amine samples.
The presence of base catalyst results in an underestimation
of NEW owing to the additional volume of acid titrated to
neutralize the base catalyst. This was observed in the NEW
determination of the 50-mole EO stearyl amine where
NEW was lower than the MALDI-TOF molecular weight.
Although this sample contained a low concentration of
PEG, the presence of 0.30% of base catalyst (calculated as
KOH) caused an underestimation of NEW. These trends
are illustrated in Table 3, which lists the concentration of
PEG, the differences between the MALDI-TOF Mn values
and the molecular weight estimates from normal-phase
HPLC, NEW, NMR and hydroxyl value determinations.
The percentage of PEG for ethoxylated fatty amines ranged
from 2.7 to 17.7% (w/w). In general, as the percentage of
PEG increased, the difference in molecular weight between
the MALDI-TOF Mn value and both NEW and 1H NMR
molecular weight values increased. The trend was not
clearly observed with the hydroxyl value results, presum-
ably owing to varying amounts of water present in the
samples (6) and greater method variability.
PEG containing a similar number of EO units as an
ethoxylated fatty amine sample did not significantly in-
terefere with normal-phase HPLC molecular weight deter-
mination. For PEG 400 and PEG 1000 it was observed that
the PEG eluted later than ethoxylated fatty amines contain-

TABLE 3
Concentrations of PEG and Differences in Molecular Weight (δ)
from MALDI-TOF Mna Values
δ
Percent PEG δ δ δ Hydroxyl
Ehtoxylated fatty amine (w/w) HPLC NEWb 1
H NMR value
c
10-mole EO stearyl amine 3.8 21 −27 −44
c
25-mole EO tallow amine 15.3 −25 −267 −367
27-mole EO stearyl amine,
sample #1 6.1 1 4 −191 202
27-mole EO stearyl amine,
sample #2 7.8 22 −84 −167 90
27-mole EO stearyl amine,
sample #3 9.8 3 −93 −203 182
28-mole EO stearyl amine,
sample #1 4.1 −50 −24 −252 199
28-mole EO stearyl amine,
sample #2 17.7 38 −404 −640 −47
c
50-mole EO stearyl amine 2.7 15 154 −246
a
Mn = ∑(MiNi)/∑Ni
b
From nonaqueous titration.
c
Not performed. PEG, polyethylene glycol; for other abbreviations see Tables 1 and 2.

Journal of Surfactants and Detergents, Vol. 2, No. 4 (October 1999)

512 R.F. LANG ET AL.
ing a similar number of EO units and thus resulted in no
significant interference in the oligomer distribution from
the normal-phase HPLC determination. PEG at low con-
centrations do not significantly interfere with the MALDI-
TOF analysis. MALDI-TOF spectra of samples containing
concentrations of PEG as high as 17.7% showed no mass
peaks attributed to PEG. This was confirmed by spiking
ethoxylated fatty amines with PEG-400, PEG-600, and
PEG-900 to give final PEG concentrations of 13.0%. For rea-
sons not presently understood, the combined sample
preparation method of using aqueous TFA together with
the α-cyano-4-hydroxycinnamic acid matrix resulted in
higher desorption/ionization yields for ethoxylated fatty
amines relative to PEG.
Both MALDI-TOF mass spectrometry and normal-
phase HPLC give accurate and reproducible molecular
weight results that correlate well with each other. A com-
bination of reversed-phase and normal-phase HPLC
methodologies offers a more comprehensive analysis since
PEG, fatty homologs, and molecular weight can be deter-
mined. In addition, HPLC instrumentation costs are signif-
icantly lower than those for MALDI-TOF. Once calibrated,
molecular weight determination by normal-phase HPLC
can be optimized for a specific amine polymer of interest
to yield short analysis times that are applicable for routine
in-process testing during manufacture.
ACKNOWLEDGMENTS
We wish to thank Dr. Yi Li for the numerous helpful discussions
and to Dr. Richard Milberg at the School of Chemical Sciences,
University of Illinois at Urbana-Champaign for collecting the
MALDI-TOF data.
REFERENCES
1. Reck, R., Cationic Surfactants Derived from Nitriles, in
Cationic Surfactants, edited by J. Richmond, Surfactant Science
Series, Marcel Dekker, Inc., New York, 1990, Vol. 34, p. 163.
2. Cegarra, J., J. Valldeperas, J. Navarro, and A. Navarro, Influ-
ence of Oxyethylenated Alkylamines in the Dyeing of Wool,
J. Soc. Dyers Colour 99:291 (1983).
3. Tsatsaroni, E., I. Eleftheriadis, and A. Kehayoglou, The Role
of Polyoxyethylenated Stearylamines in the Dyeing of Cotton
with Direct Dyes, Ibid. 106:245 (1990).
4. Arif, S., Fatty Amine Ethoxylates, HAPPI, 67 (1996).
5. Cross, J., Introduction to Nonionic Surfactants, in Nonionic
Surfactants, edited by J. Cross, Surfactant Science Series, Mar-
cel Dekker, Inc., New York, 1987, Vol. 19, p. 3.
6. Miwidsky, B.M., and D.M. Gabriel, Detergent Analysis, 1982,
John Wiley & Sons, New York, pp. 207, 208.
7. Cross, J., Aspects of Quality and Process Control, in Nonionic
Surfactants, edited by J. Cross, Surfactant Science Series, Mar-
cel Dekker, Inc., New York, 1987, Vol. 19, p. 371.
8. Marquez, N., R. Anton, A. Usubillaga, and J.L. Salager, Opti-
mization of HPLC Conditions to Analyze Widely Distributed
Ethoxylated Alkylphenol Surfactants, J. Liquid Chromatogr.
17:1147 (1994).
9. Miszkiewicz, W., and L. Szymanowski, Analysis of Nonionic
Surfactants with Polyoxyethylene Chains by High-Perfor-
Journal of Surfactants and Detergents, Vol. 2, No. 4 (October 1999)
mance Liquid Chromatography, Crit. Rev. Anal. Chem. 25:203
(1996).
10. Ban, T., E. Papp, and J. Inczedy, Reversed-Phase High-Perfor-
mance Liquid Chromatography of Anionic and Ethoxylated
Non-Ionic Surfactants and Pesticides in Liquid Pesticide For-
mulations, J. Chromatogr. 593:227 (1992).
11. Zeman, I., J. Silha, and M. Bares, Separation of Ethoxylates by
HPLC, Tenside Deterg. 23:181 (1986).
12. Schreuder, R., A. Martin, H. Poppe, and J.C. Kraak, Determi-
nation of the Composition of Ethoxylated Alkylamines in Pes-
ticide Formulations by High-Performance Liquid Chromatog-
raphy Using Ion-Pair Extraction Detection, J. Chromatogr.
368:339 (1986).
13. Martin, N., Analysis of Non-Ionic Surfactants by HPLC Using
Evaporative Light-Scattering Detector, J. Liquid Chromatogr.
18:1173 (1995).
14. Bahr, U., A. Deppe, M. Karas, F. Hillenkamp, and U. Geiss-
mann, Mass Spectrometry of Synthetic Polymers by UV-Ma-
trix-Assisted Laser Desorption/Ionization, Anal. Chem.
64:2866 (1992).
15. Thomson, B., Z. Wang, A. Paine, A. Rudin, and G. Lajoie, Sur-
factant Analysis by Matrix-Assisted Laser Desorption Time-
of-Flight Mass Spectrometry, J. Am. Oil Chem. Soc. 72:11
(1995).
16. Montaudo, G., M. Montaudo, C. Puglisi, and F. Samperi,
Characterization of Polymers by Matrix Assisted Laser Des-
orption/Ionization Time-of-Flight Mass Spectrometry: Mo-
lecular Weight Estimates in Samples of Varying Polydisper-
sity, Rapid Commun. Mass Spectrom. 9:453 (1995).
17. Bartsch, H., M. Strabner, and U. Hintze, Characterization and
Identification of Ethoxylated Surfactants by Matrix-Assisted
Laser Desoption/Ionization Mass Spectrometry, Tenside Surf.
Det. 35:94 (1998).
18. Wu, K., and R. Odom, Characterizing Synthetic Polymers by
MALDI MS, Anal. Chem. 70:456A (1998).
19. Montana, A., Nuclear Magnetic Resonance Spectrometry of
Nonionic Surfactants, in Nonionic Surfactants, edited by J.
Cross, Surfactant Science Series Vol. 19, Marcel Dekker, Inc.,
New York, 1987, p. 295.
20. AOCS Hydroxyl Value Determination, Official and Recom-
mended Practices of the American Oil Chemists’ Society, AOCS
Press, Champaign, 1993, Method Cd 13-60.
21. Snyder, L., J. Glajch, and J. Kirkland, Practical HPLC Method
Development, John Wiley & Sons, New York, 1988, pp. 60, 61.
22. Dreux, M., M. Lafosse, and L. Morin-Allory, The Evaporative
Light Scattering Detector-A Universal Instrument for Non-
Volatile Solutes in LC and SFC, LCGC International 14:148
(1996).
[Received February 26, 1999; accepted July 14, 1999]
Dr. Russell F. Lang is a Senior Scientist in the Reagents and Ap-
plications Development Group, in the Cellular Analysis Divi-
sion of Beckman-Coulter, Inc. His current research includes the
use of chromatographic and mass spectrometric techniques for
the characterization of surfactants, and the effect of surfactants
on cellular components. He received his B.S. in chemistry from
Florida International University and his Ph.D. in inorganic
chemistry from the University of Miami. Other areas of exper-
tise include marine, atmospheric, and organometallic chemistry.
Dr. Dennisse Parra-Diaz received her B.S. degree in chem-
istry from the University of Puerto Rico (1982) and her Ph.D.
degree in physical chemistry from the University of Miami
(1990). After completing postdoctoral training in biophysical
 ANALYSIS OF ETHOXYLATED FATTY AMINES 513

chemistry at Temple University (1991), she held a Research As-
sociate position at the United States Department of Agriculture
Eastern Regional Research Center. She began working for Beck-
man-Coulter, Inc. in 1996 and currently holds a Scientist posi-
tion in the Reagents and Application Development Group. Her
research interests include structural elucidation of peptides and
organic-alkali metal complexes using nuclear magnetic reso-
nance and molecular mechanics as well as the development of
hematology and immunology reagents.
Dr. Dana Jacobs is currently the Manager of the Controls and
Calibrators Group in the Cellular Analysis Division of Beckman-
Coulter, Inc. As an undergraduate, he studied chemistry, mathe-
matics, and zoology and received his B.A. from the University of
Vermont (1969). After serving in the military, he studied im-
munochemistry, lectin, and lymphokine biochemistry in the lab-
oratory of Dr. Ronald D. Poretz at Rutgers University and re-
ceived his Ph.D. in 1980.

Journal of Surfactants and Detergents, Vol. 2, No. 4 (October 1999)