analysis of ethoxylated fatty amines. comparison of methods for the determination of molecular...

ABSTRACT: Specific lengths of the fatty and polyoxyethylenechains 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-phasehigh-performance liquid chromatography (HPLC) and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF)mass spectrometry methods were developed to determine withaccuracy the molecular weight and degree of ethoxylation ofethoxylated fatty amines. Ethoxylated fatty amines were ana-lyzed using these methods, and comparison was made to mo-lecular weight determinations using proton nuclear magneticresonance (NMR), neutralization equivalent weight, and hy-droxyl value methods. Molecular weight results from normal-phase HPLC analyses were in very good agreement withMALDI-TOF results, typically varying less than one ethyleneoxide unit. A reversed-phase HPLC method was developed todetermine concentrations of polyethylene glycols (PEG) andfatty 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 fattyamines, HPLC, hydroxyl value, MALDI-TOF, molecular weight,neutralization equivalent weight, 1H NMR.

Ethoxylated fatty amines are used in different industrialapplications 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 afatty amine with ethylene oxide (EO) (1,5). The two-stepethoxylation of a primary amine is shown in Equations 1and 2, where R typically is C12–C18 fatty groups.

[1]

[2]

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 EOwith residual water. Ethoxylated fatty amines are commer-cially produced from coco, lauryl, tallow, oleyl, and stearylamines and typically contain from 2 to 50 moles of EO permole of amine hydrophobe. Specific lengths of the fattyand polyoxyethylene chains are critical to performance ina specific application, thus the ability to accurately charac-terize these surfactants is crucial.

Analysis of ethoxylated fatty amines for estimating thedegree of ethoxylation (DOE) and average molecularweight has historically been performed using the neutral-ization equivalent weight (NEW) (6) and hydroxyl valuemethods (6,7). These methods are still used almost exclu-sively by the surfactant industry for determination of themolecular weight of ethoxylated fatty amines. NEW isoften used during manufacturing as an in-process test todetermine the extent of ethoxylation. NEW is estimated bytitrimetric neutralization of the amine group with stan-dardized acid. The hydroxyl value method involves de-rivatization of terminal hydroxyl groups using either aceticor phthalic anhydride followed by quantitative determina-tion of excess anhydride.

Reversed- and normal-phase high-performance liquidchromatography (HPLC) methods have been developedfor analysis of ethoxylated nonionic surfactants, most com-monly for ethoxylated fatty alcohols, acids, sulfonates, andalkylphenols (8–11). The one reported method applicableto ethoxylated fatty amines was limited to a mean EO con-tent of only 15 moles owing to the ion-pair/fluorescencedetection system used (12). Use of an evaporative light-scattering detector for HPLC applications allows for detec-tion of polyalkoxylated compounds with no molecularweight limitations (13). Matrix-assisted laser desorptionionization-time of flight (MALDI-TOF) mass spectrometry

RN(CH2CH2OH)2 + (x + y)(C2H4O)base, ∆

RN(CH2CH2O)x+1H

(CH2CH2O)y+1H

RNH2 + 2 C2H4O( ) ∆ → RN CH2CH2OH( )2

Copyright © 1999 by AOCS Press Journal of Surfactants and Detergents, Vol. 2, No. 4 (October 1999) 503

*To whom correspondence should be addressed at Beckman-CoulterInc., Mail Stop 11-A02, 11800 SW 147 Ave., Miami, FL 33196-2500.E-mail: [email protected]

Analysis of Ethoxylated Fatty Amines. Comparison of Methods for the Determination of Molecular Weight

Russell F. Lang*, Dennisse Parra-Diaz, and Dana JacobsBeckman-Coulter Inc., Miami, Florida 33196-2500

has recently been used for the analysis of some syntheticsurfactants (14–18); however, ethoxylated fatty amineshave not been evaluated using this technique. Nuclearmagnetic 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 physicalproperties of nonionic surfactants (19).

In our attempts to characterize ethoxylated fatty aminesand to estimate their degrees of ethoxylation and averagemolecular weights accurately, new HPLC and MALDI-TOFmass 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 whenused 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 separateoligomers of ethoxylated fatty amines and to determinetheir average molecular weights. MALDI-TOF mass spec-trometry was used to characterize ethoxylated fatty aminesand to assign mass values to oligomers of normal-phaseHPLC analyses. Ethoxylated fatty amines were also charac-terized using 1H NMR, NEW, and hydroxyl value methods.The results of these molecular weight determinations, usingthese five different methods, showed that for many of theethoxylated 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 thesefive methods. A comparison of the results is reported.

EXPERIMENTAL PROCEDURES

Ethoxylated fatty amines were obtained from commercialsources (Akzo Nobel Chemicals Inc., Chicago, IL; EthoxChemicals, 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 960evaporative mass detector (Amherst, MA). The EMD 960detector used industrial grade nitrogen (Air Products, Al-lentown, PA) at a flow rate of 5 L/min. The detector wasoperated at 65°C for normal-phase separations and 75°Cfor reversed-phase separations. Sample solutions were pre-pared by dissolving 100 mg of ethoxylated fatty amine into10 mL of 2-propanol. Sample solutions were filteredthrough 0.45 µm GH Polypro membrane filters (GelmanSciences, Ann Arbor, MI). A sample volume of 10 µL wasinjected into the HPLC system for analysis. Mobile phaseswere filtered through 0.45-µm GH Polypro membrane fil-ters prior to use. For all HPLC analyses, the HPLC columnswere maintained at 40°C, and a mobile phase flow rate of1 mL/min was used.

All reversed-phase HPLC analyses including PEG analy-ses were performed using a Waters Nova-Pak 60Å C18, 4µm, 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-phaseHPLC separations were performed on a LiChrospher 100ÅDiol, 5 µm, 150 × 4.6 mm column (Alltech Associates, Deer-field, IL). The mobile phase gradient program used for themajority of ethoxylated fatty amines was a linear gradientof hexane/2-propanol (both solvents contain 25 mMtriethylamine) from 95:5 to 70:30 over 140 min. This pro-gram allowed for analysis of ethoxylated fatty amines overa wide range of ethoxylate chain lengths, typically 5 to 60EO units for an ethoxylated stearyl amine. As discussedbelow, this mobile phase program can be modified to opti-mize the analysis of a specific ethoxylated fatty amine.

MALDI-TOF mass spectrometry. MALDI mass spectrawere acquired on a PE-PerSeptive Biosystems (Framing-ham, MA) Voyager-DE STR delayed extraction reflectrontime-of-flight mass spectrometer equipped with a LaserScience nitrogen laser (337 nm, 3 ns pulse). Positive ionspectra 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 1:1MeCN/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-µLaliquot of the sample solution was thoroughly mixed withan equal volume of the α-cyano-4-hydroxycinnamic acidmatrix solution and analyzed.

1H 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 (89mm) magnet or a 400 MHz spectrometer (Varian AnalyticalInstruments, Valencia, CA). Samples were dissolved indeuterated acetone or chloroform resulting in concentra-tions of approximately 20 mM. NMR data were analyzedusing NUTS data analysis software (ACORN NMR Inc., Fre-mont, CA). Proton NMR chemical shift assignments werebased on characteristic proton NMR shift tables. In addition,NMR spectra of stearyl amine, PEG-900, and PEG-1500 wereused as guides to verify chemical shift values for the fattyand polyoxyethylene moieties. Proton NMR molecularweight calculations were based on the ratio of integratedpeak areas between the terminal methyl group of the alkylchain (δ = 0.872–0.888 ppm) to (i) the methylene protons ofthe ethoxylate chains (δ = 3.580–3.595 ppm) excluding thoseadjacent to the amine nitrogen, (ii) the hydroxyl terminalprotons (δ = 2.843–2.854 ppm), and (iii) the alkyl protons (δ= 1.284–1.302 ppm) excluding the methyl protons, protonsadjacent to the methyl group, and protons adjacent to theamine 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).

504 R.F. LANG ET AL.

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

NEW. NEW was determined potentiometrically usingaqueous and nonaqueous titrations. Aqueous titrationswere performed to determine the concentration of base cat-alyst remaining in the amine sample. Samples (≈2 g) weredissolved in water/2-propanol (1:1) and titrated against0.1 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 titratingagainst 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 followingmodifications. Samples for acetylation and two blankswere refluxed for 1 h under constant stirring in a mineraloil bath. The oil bath temperature was maintained within arange of 95.0–110.0°C. The reaction was then quenched byadding 15.0 mL of deionized water to the mixture followedby a 20-min incubation in the oil bath. The mixtures wereallowed to cool to room temperature, then titrated withethanolic potassium hydroxide. Duplicate analyses wereperformed 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 amineswith 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 massspectra with high abundances for all ethoxylated fattyamines. 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 theseethoxylated fatty amines, the MALDI-TOF mass spectrashowed no fragmentation. The major mass peaks appearas [M + H]+ ions with no multiply-charged ions observed.The formation of sodium or potassium adducts was negli-gible. In contrast, ethoxylated fatty alcohols analyzedunder identical conditions produced spectra in which thepredominant peaks appeared as sodium and potassiumadducts (Lang, R.F., and D. Parra-Diaz, unpublished re-sults). The MALDI-TOF mass spectrum of a 25-mole EOtallow 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 themonoprotonated oligomer, [C16N[EO]20H]+. Mn was calcu-lated to be 1159 Da, where Mn = ∑(MiNi)/∑Ni, and Ni andMi are the abundance and mass of the ith oligomer, respec-tively. The weight average molecular weight (Mw), definedas ∑(Mi

2Ni)/∑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 expandedmass spectrum of the 25-mole EO tallow amine and showsthe saturated homologs, octadecyl ([C18N[EO]20H]+, m/z =1150.9), hexadecyl ([C16N[EO]20H]+, m/z = 1122.9), andtetradecyl ([C14N[EO]21H]+, m/z = 1138.9), and the mono-

ANALYSIS OF ETHOXYLATED FATTY AMINES 505


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

unsaturated octadecyl homolog ([C18eneN[EO]20H]+, m/z= 1148.8).

Table 1 is a summary of MALDI-TOF molecular weightresults for ethoxylated fatty amines over a DOE range of10 to 50. For the samples listed, except the 28-mole EOstearyl amine Sample #2, the difference between the

Mp and Mn values was within one mole of EO (44 Da), andD values were all very low, indicating that these ethoxy-lated amine polymers were all narrowly dispersed. For the28-mole EO stearyl amine Sample #2, the mass spectrumshowed that the oligomer distribution was slightly skewedtoward higher masses. This resulted in a mass differenceof 81 Da between the Mp and Mn values and a slightlyhigher D value of 1.07. The COA MW (certificates of analy-sis molecular weights) are the molecular weight stated onthe products’ COA and were provided by the manufactur-ers. The COA MW values listed in Table 1 show varyingdegrees of agreement with the molecular weight values de-termined by MALDI-TOF mass spectrometry. These dis-crepancies in molecular weight between the COA and theMALDI-TOF values are discussed in detail below in con-junction with results of the PEG analyses.

Normal-phase HPLC. Both cyano and diol columns wereevaluated with various mobile phases to determine the op-timal analytical conditions for separation of individualoligomers. The diol column consistently gave superior res-olution vs. the cyano column; thus all analyses were per-formed using the diol column. The basic nature of theamine group required addition of a modifier to the mobilephase to eliminate peak tailing caused by silanol effects(21). By using the diol column with a hexane/2-propanolmobile phase and no mobile phase modifier, ethoxylatedfatty amines eluted as a broad tailing peak with no oligo-mer separation. Since the evaporative mass detector re-quires that only volatile buffers and modifiers be used inthe mobile phase, triethylamine was initially evaluated.Optimization experiments showed that good separation of



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

TABLE 1Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF) Results for Fatty Amine Ethoxylates

Ethoxylated fatty amine COA MWa Mpb Mn

c Mwd De

10-mole EO stearyl amine,nominal MW = 709 697 666 686 730 1.06

25-mole EO tallow amine,nominal MW = 1369 1406 1122 1159 1208 1.04

27-mole EO stearyl amine #1,nominal MW = 1457 1465 1458 1502 1564 1.04




28-mole EO stearyl amine #2,nominal MW = 1501 1605f 1282 1363 1452 1.07

50-mole EO stearyl amine,nominal MW = 2469 2429 2647 2616 2666 1.02

aMolecular weight (MW) from manufacturer’s certificate of analysis (COA).Determined by neutralization equivalent weight (NEW) analysis.bMost probable molecular weight (unprotonated mono-isotopic mass).cNumber average molecular weight (Mn = ∑(MiNi)/∑Ni).dWeight average molecular weight (Mw = ∑(Mi

2Ni)/∑MiNi).ePolydispersity index (Mw/Mn).fMW from manufacturer’s COA. Determined by hydroxyl value analysis.

oligomers with minimal peak tailing was achieved withthe diol column using a hexane/2-propanol mobile phasegradient containing 25 mM triethylamine. The ethoxylatedfatty amine oligomers elute in order of increasing EO units.Figure 3 is a chromatogram of a sample containing threecomponents: a “15-mole EO” tallow amine, a “27-moleEO” 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 mobilephase 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 showsthe method is applicable up to approximately 60 moles EOfor stearyl amine. Good separation of oligomers is ob-served as evidenced by almost complete baseline separa-tion throughout the chromatogram. Some selectivity in theseparation of fatty groups of the 15-mole EO tallow amineis observed as evidenced by split peaks due to the differ-ent tallow amine fatty groups. Use of the evaporative massdetector consistently resulted in negligible baseline driftfor the mobile phase gradients. This mobile phase gradientis amenable to modification to meet the requirements for aspecific analysis. For example, as shown in Figure 4A, theanalysis time for a routine determination of a 27-mole EOstearyl 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 mobilephase 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 stearylamine.

Mass assignment of normal-phase HPLC oligomers. Massvalues were assigned to oligomer peaks of a normal-phaseHPLC analysis of a 27-mole EO stearyl amine. The high-est-intensity HPLC peak (38.48 min) of a 27-mole EOstearyl amine, shown Figure 4A, was isolated by repetitivecollection from nine analyses. These nine fractions werepooled, 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 minpeak 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 representoligomers with DOE values of 27 and 29, respectively, werealso observed in the fraction-collected sample. Presence ofthese two other oligomers was due to the manual fractioncollection procedure, which required that the detector inletfitting be uncoupled to collect column effluent. To allowfor 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 completecollection of the 38.48 min peak. Mn value of the 27-moleEO 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: LiChrospher100Å 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: 1mL/min. Column temperature: 40°C. Detector: evaporative mass detector. For abbreviation see Figure 1.



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. Flowrate: 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.

m/z

termined for the highest-intensity HPLC peak, 1503 Da,was in excellent agreement the MALDI-TOF Mn value of1502 Da for the 27-mole EO stearyl amine. Once calibratedusing 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 HPLCmethod was developed for determination of PEG, andalkyl homologs of ethoxylated fatty amines. Aqueous sol-vent systems using MeOH, MeCN and tetrahydrofuran(THF) were evaluated using acetic acid and triethylaminemodifiers. In the absence of any modifiers, a 28-mole EOstearyl amine bound so strongly to the silica surface of theC18 packing that even after 48 min of using 100% methanolat 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 triethylamineand 50 mM glacial acetic acid gave a rapid analysis withgood 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-

ration of C16 and C18 homologs was achieved. HydrophilicPEG 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 separationachieved for C10, C12, C14, and C16 homologs. Analyses ofethoxylated fatty amines with the same alkyl group con-taining differing ethoxylate chain lengths showed that thelonger-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 influencedmolecular weight results for different methods. PEG con-centrations in ethoxylated fatty amines were calculatedfrom a calibration curve prepared from analyses of stan-dard solutions of PEG 1000, which in turn, were preparedin acetonitrile and analyzed in triplicate. The calibrationplot is shown in Figure 7. Response from the evaporativemass 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 twoorders of magnitude. Sensitivity of the evaporative massdetector allowed for detection of 100 ng of PEG 1000 witha signal/noise ratio >5.



FIG. 5. Reversed-phase HPLC chromatogram of 27-mole EO stearylamine. Stationary phase: Waters Nova-Pak 60Å C18, 4 µm (150 × 3.9mm 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.

FIG. 6. Reversed-phase HPLC chromatogram of 15-mole EO cocoamine. HPLC conditions are the same as described in Figure 5. For ab-breviations see Figures 1 and 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 determinemolecular weights. The ethoxylated fatty amines included10-, 27-, 28-, and 50-mole EO stearyl amines and a 25-moleEO tallow amine. Three different 27-mole EO stearyl aminesamples 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 theethoxylated amine samples. The COA MW for all samplesexcept the 28-mole EO stearyl amine Sample #2 were de-rived from the NEW determination, and good agreementbetween the COA MW and NEW values was found. TheCOA MW for the 28-mole EO stearyl amine Sample #2 wasobtained from hydroxyl value determination.

MALDI-TOF Mn values were in good agreement withmolecular weight results from normal-phase HPLC mea-



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

TABLE 2Comparison of Molecular Weight Results from Different Methods

Ethoxylated fatty amine COA HydroxylMWa Mn

b HPLCc NEWd 1H NMR value MW(DOE)e (DOE)e (DOE)e (DOE)e (DOE)e (DOE)e

10-mole EO stearyl amine 697 686 665 713 730 f

(nominal NW = 709) (9.7) (9.5) (9.0) (10.1) (10.5)25-mole EO tallow amine 1406 1159 1184 1426 1526 f

(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 = 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)50-mole EO stearyl amine 2429 2616 2601 2462 2862 f

(nominal MW = 2469) (49.1) (53.3) (53.0) (49.8) (58.9)aMW from manufacturer’s COA. Determined by NEW analysis.bMn (MALDI-TOF number average molecular weight) = ∑(MiNi)/∑Ni.cMW 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.dNeutralization equivalent weight (nonaqueous titration method).eDegree of ethoxylation.fNot performed.gMW from manufacturer’s COA. Determined by hydroxyl value analysis. HPLC, high-performance liquid chromatography;NMR, nuclear magnetic resonance.

surements throughout the molecular weight range. Molec-ular weight values from normal-phase HPLC analyses forall ethoxylated stearyl amines were calculated as 100%stearyl amine since the fatty homolog composition wastypically >95% stearyl. For the 25-mole EO tallow amine,molecular weight was calculated based on a fatty homologcomposition of 30% palmitic, 25% stearic, and 45% oleicacid. Molecular weights derived from normal-phase HPLCanalyses for all samples were within ±50 Da of the MALDI-TOF molecular weight values. These results are consistentwith reports that the Mn and Mw values determined byMALDI-TOF are in agreement with molecular weightsmeasured by chromatographic methods for polymers withnarrow molecular weight distributions (D ≤ 1.2) (16,18). Inaddition, this agreement between normal-phase HPLC andMALDI-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 molecularweights and narrow molecular weight distribution of theethoxylated fatty amine samples.

For the majority of the samples, the NEW and 1H NMRdeterminations overestimated the molecular weight valueswhen compared to MALDI-TOF Mn results. The hydroxylvalue method generally underestimated the molecularweight of ethoxylated fatty amines samples except for the28-mole EO stearyl amine Sample #2. Side products pres-ent in the ethoxylated fatty amine samples, which containterminal hydroxyl groups such as PEG, result in a lowermolecular weight value being obtained from the hydroxylvalue method. The determination of molecular weight by1H NMR uses the ratios of the fatty moiety to the poly-oxyethylene and hydroxyl groups. Thus, the presence ofPEG results in an overestimation of molecular weight

when determined by 1H NMR. This was clearly evident inthe 28-mole EO stearyl amine Sample #2 where the signalsdue to hydroxyl and ethoxylate protons were excessivelyhigher than theoretical values, and more than one signalattributed to hydroxyl protons was observed.

NEW values are calculated from the quotient of sampleweight and moles of acid titrated. Any neutral compoundspresent, such as PEG result in an overestimated NEWvalue. The NEW values are also affected by residual basecatalyst present in the ethoxylated fatty amine samples.The presence of base catalyst results in an underestimationof NEW owing to the additional volume of acid titrated toneutralize the base catalyst. This was observed in the NEWdetermination of the 50-mole EO stearyl amine whereNEW was lower than the MALDI-TOF molecular weight.Although this sample contained a low concentration ofPEG, the presence of 0.30% of base catalyst (calculated asKOH) caused an underestimation of NEW. These trendsare illustrated in Table 3, which lists the concentration ofPEG, the differences between the MALDI-TOF Mn valuesand the molecular weight estimates from normal-phaseHPLC, NEW, NMR and hydroxyl value determinations.The percentage of PEG for ethoxylated fatty amines rangedfrom 2.7 to 17.7% (w/w). In general, as the percentage ofPEG increased, the difference in molecular weight betweenthe MALDI-TOF Mn value and both NEW and 1H NMRmolecular weight values increased. The trend was notclearly observed with the hydroxyl value results, presum-ably owing to varying amounts of water present in thesamples (6) and greater method variability.

PEG containing a similar number of EO units as anethoxylated 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 thatthe PEG eluted later than ethoxylated fatty amines contain-



TABLE 3Concentrations of PEG and Differences in Molecular Weight (δ) from MALDI-TOF Mn

a Values

δPercent PEG δ δ δ Hydroxyl

Ehtoxylated fatty amine (w/w) HPLC NEWb 1H NMR value

10-mole EO stearyl amine 3.8 21 −27 −44 c

25-mole EO tallow amine 15.3 −25 −267 −367 c

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

50-mole EO stearyl amine 2.7 15 154 −246 c

aMn = ∑(MiNi)/∑NibFrom nonaqueous titration.cNot performed. PEG, polyethylene glycol; for other abbreviations see Tables 1 and 2.

ing a similar number of EO units and thus resulted in nosignificant interference in the oligomer distribution fromthe normal-phase HPLC determination. PEG at low con-centrations do not significantly interfere with the MALDI-TOF analysis. MALDI-TOF spectra of samples containingconcentrations of PEG as high as 17.7% showed no masspeaks attributed to PEG. This was confirmed by spikingethoxylated fatty amines with PEG-400, PEG-600, andPEG-900 to give final PEG concentrations of 13.0%. For rea-sons not presently understood, the combined samplepreparation method of using aqueous TFA together withthe α-cyano-4-hydroxycinnamic acid matrix resulted inhigher desorption/ionization yields for ethoxylated fattyamines relative to PEG.

Both MALDI-TOF mass spectrometry and normal-phase HPLC give accurate and reproducible molecularweight results that correlate well with each other. A com-bination of reversed-phase and normal-phase HPLCmethodologies offers a more comprehensive analysis sincePEG, 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 HPLCcan be optimized for a specific amine polymer of interestto yield short analysis times that are applicable for routinein-process testing during manufacture.

ACKNOWLEDGMENTS

We wish to thank Dr. Yi Li for the numerous helpful discussionsand to Dr. Richard Milberg at the School of Chemical Sciences,University of Illinois at Urbana-Champaign for collecting theMALDI-TOF data.

REFERENCES

1. Reck, R., Cationic Surfactants Derived from Nitriles, inCationic Surfactants, edited by J. Richmond, Surfactant ScienceSeries, 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 Roleof Polyoxyethylenated Stearylamines in the Dyeing of Cottonwith 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 NonionicSurfactants, 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 DistributedEthoxylated Alkylphenol Surfactants, J. Liquid Chromatogr.17:1147 (1994).

9. Miszkiewicz, W., and L. Szymanowski, Analysis of NonionicSurfactants with Polyoxyethylene Chains by High-Perfor-

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 EthoxylatedNon-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 byHPLC, 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 UsingEvaporative 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 andIdentification of Ethoxylated Surfactants by Matrix-AssistedLaser Desoption/Ionization Mass Spectrometry, Tenside Surf.Det. 35:94 (1998).

18. Wu, K., and R. Odom, Characterizing Synthetic Polymers byMALDI MS, Anal. Chem. 70:456A (1998).

19. Montana, A., Nuclear Magnetic Resonance Spectrometry ofNonionic 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, AOCSPress, Champaign, 1993, Method Cd 13-60.

21. Snyder, L., J. Glajch, and J. Kirkland, Practical HPLC MethodDevelopment, John Wiley & Sons, New York, 1988, pp. 60, 61.

22. Dreux, M., M. Lafosse, and L. Morin-Allory, The EvaporativeLight 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 theuse of chromatographic and mass spectrometric techniques forthe characterization of surfactants, and the effect of surfactantson cellular components. He received his B.S. in chemistry fromFlorida International University and his Ph.D. in inorganicchemistry 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



chemistry at Temple University (1991), she held a Research As-sociate position at the United States Department of AgricultureEastern 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. Herresearch interests include structural elucidation of peptides andorganic-alkali metal complexes using nuclear magnetic reso-nance and molecular mechanics as well as the development ofhematology and immunology reagents.

Dr. Dana Jacobs is currently the Manager of the Controls andCalibrators 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 ofVermont (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.



analysis of ethoxylated fatty amines. comparison of methods for the determination of molecular...

Documents