ABSTRACT: Speciﬁc lengths of the fatty and polyoxyethyleneSpeciﬁc lengths of the fatty and polyoxyethylene
chains of ethoxylated fatty amines are critical to
chains of ethoxylated fatty amines are critical to their perfor-their perfor-mance in specific applications, and thus the ability to mance in specific applications, and thus the ability to charac-terize these surfactants accurately is crucial. Normal-phase terize these surfactants accurately is crucial. Normal-phase high-performance liquid chromatography (HPLC) and high-performance liquid chromatography (HPLC) and matrix-assisted laser desorption ionization-time of ﬂight (MALDI-TOF) assisted laser desorption ionization-time of ﬂight (MALDI-TOF) mass spectrometry methods were developed to determine with mass spectrometry methods were developed to determine with accuracy the molecular weight and degree of ethoxylation of accuracy the molecular weight and degree of ethoxylation of ethoxylated fatty amines. Ethoxylated fatty amines were ethoxylated fatty amines. Ethoxylated fatty amines were ana-lyzed using these methods, and comparison was made to lyzed using these methods, and comparison was made to mo-lecular weight determinations using proton nuclear magnetic lecular weight determinations using proton nuclear magnetic resonance (NMR), neutralization equivalent weight, and resonance (NMR), neutralization equivalent weight, and hy-droxyl value methods. Molecular weight results from droxyl value methods. Molecular weight results from normal-phase HPLC analyses were in very good agreement with phase HPLC analyses were in very good agreement with MALDI-TOF results, typically varying less than
MALDI-TOF results, typically varying less than one ethyleneone ethylene oxide unit. A reversed-phase HPLC method was developed to oxide unit. A reversed-phase HPLC method was developed to determine concentration
determine concentrations of s of polyethylene glycols (PEG) andpolyethylene glycols (PEG) and fatty homologs. PEG interfered with molecular weight fatty homologs. PEG interfered with molecular weight determi-nations by NMR, neutralization equivalent weight, and nations by NMR, neutralization equivalent weight, and hy-droxyl value methods. PEG caused no interference with droxyl value methods. PEG caused no interference with molec-ular weight determinations by normal-phase HPLC and ular weight determinations by normal-phase HPLC and MALDI-TOF methods.
Paper no. S1134 in
Paper no. S1134 in JSD 2, JSD 2,503–513 (October 1999).503–513 (October 1999). KEY WORDS:
KEY WORDS: Degree of ethoxylation, ethoxylated fattyDegree of ethoxylation, ethoxylated fatty
amines, HPLC, hydroxyl value, MALDI-TOF, molecular weight, amines, HPLC, hydroxyl value, MALDI-TOF, molecular weight, neutralization equivalent weight,
neutralization equivalent weight,11H NMR.H NMR.
Ethoxylated fatty amines are used in different industrial Ethoxylated fatty amines are used in different industrial applications such as defoamers, textile-finishing agents, applications such as defoamers, textile-finishing agents, corrosion inhibitors, emulsifiers (1), and dye promoters corrosion inhibitors, emulsifiers (1), and dye promoters (2,3). They enhance herbicidal activity of
(2,3). They enhance herbicidal activity of many pesticidesmany pesticides (1) and have potential uses in laundry products (4). (1) and have potential uses in laundry products (4). Ethoxylated fatty amines are produced by the
Ethoxylated fatty amines are produced by the reaction of areaction of a fatty amine with ethylene oxide (EO) (1,5). The two-step fatty amine with ethylene oxide (EO) (1,5). The two-step ethoxylation of a primary amine is shown in Equations 1 ethoxylation of a primary amine is shown in Equations 1 and 2, where R typically is C
and 2, where R typically is C1212–C–C1818fatty groups.fatty groups.
The product is a polydisperse mixture of oligomers The product is a polydisperse mixture of oligomers ap-proaching a Poisson distribution (5). Polyethylene glycols proaching a Poisson distribution (5). Polyethylene glycols (PEG) are side products, formed from the reaction of EO (PEG) are side products, formed from the reaction of EO with residual water. Ethoxylated fatty amines are with residual water. Ethoxylated fatty amines are commer-cially produced from coco,
cially produced from coco, lauryl, tallow, oleyl, and stearyllauryl, tallow, oleyl, and stearyl amines and typically contain from 2 to 50 moles of EO per amines and typically contain from 2 to 50 moles of EO per mole of amine hydrophobe. Specific lengths of the fatty mole of amine hydrophobe. Specific lengths of the fatty and polyoxyethylene chains are critical to performance in and polyoxyethylene chains are critical to performance in a speciﬁc application, thus the ability to accurately a speciﬁc application, thus the ability to accurately charac- charac-terize these surfactants is crucial.
terize these surfactants is crucial.
Analysis of ethoxylated fatty amines for estimating the Analysis of ethoxylated fatty amines for estimating the degree of ethoxylation (DOE) and average molecular degree of ethoxylation (DOE) and average molecular weight has historically been performed using the weight has historically been performed using the neutral-ization equivalent weight (NEW) (6) and hydroxyl value ization equivalent weight (NEW) (6) and hydroxyl value methods (6,7). These methods are still used
methods (6,7). These methods are still used almost exclu-almost exclu-sively by the surfactant industry for determination of the sively by the surfactant industry for determination of the molecular weight of ethoxylated fatty amines. NEW is molecular weight of ethoxylated fatty amines. NEW is often used during manufacturing as an in-process test to often used during manufacturing as an in-process test to determine the extent of ethoxylation. NEW i
determine the extent of ethoxylation. NEW is estimated bys estimated by titrimetric neutralization of the amine group with titrimetric neutralization of the amine group with stan-dardized acid. The hydroxyl value method involves dardized acid. The hydroxyl value method involves de-rivatization of terminal hydroxyl groups using either acetic rivatization of terminal hydroxyl groups using either acetic or phthalic anhydride followed by quantitative or phthalic anhydride followed by quantitative determina-tion of excess anhydride.
tion of excess anhydride.
Reversed- and normal-phase high-performance liquid Reversed- and normal-phase high-performance liquid chromatography (HPLC) methods have been developed chromatography (HPLC) methods have been developed for analysis of ethoxylated nonionic surfactants, most co for analysis of ethoxylated nonionic surfactants, most com- m-monly for ethoxylated fatty alcohols, acids, sulfonates, and monly for ethoxylated fatty alcohols, acids, sulfonates, and alkylphenols (8–11). The one reported method applicable alkylphenols (8–11). The one reported method applicable to ethoxylated fatty amines was limited to a mean EO to ethoxylated fatty amines was limited to a mean EO con-tent of only 15 moles owing to the ion-pair/fluorescence tent of only 15 moles owing to the ion-pair/fluorescence detection system used (12). Use of an evaporative detection system used (12). Use of an evaporative light-scattering detector for HPLC applications allows for scattering detector for HPLC applications allows for detec- detec-tion of polyalkoxylated compounds with no molecular tion of polyalkoxylated compounds with no molecular weight limitations (13). Matrix-assisted laser desorption weight limitations (13). Matrix-assisted laser desorption ionization-time of ﬂight (MALDI-TOF) mass spectrometry ionization-time of ﬂight (MALDI-TOF) mass spectrometry
RN(CH22CHCH22OH)OH)22+ (+ (xx++ y y)(C)(C22HH44O)O) base, base,
∆ ∆ RN RN (CH(CH22CHCH22O)O)xx+1+1HH (CH (CH22CHCH22O)O) y y+1+1HH RNH RNH22++2 2 C( ( C22HH44OO)) → ∆∆→RN CHRN CH( ( 22CHCH22OHOH)) 2 2
*To whom correspondence should be addressed at Beckman-Coulter *To whom correspondence should be addressed at Beckman-Coulter Inc., Mail Stop 11-A02, 11800 SW 147 Ave., Miami, FL
Inc., Mail Stop 11-A02, 11800 SW 147 Ave., Miami, FL 33196-2500.33196-2500. E-mail: email@example.com
for the Determination of Molecular Weight
for the Determination of Molecular Weight
Russell F. Lang*, Dennisse Parra-Diaz, and Dana Jacobs Russell F. Lang*, Dennisse Parra-Diaz, and Dana Jacobs
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 using1H 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.
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.
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 ofα-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 theα-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
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 titratdissolv-ing against 0.1 N perchloric acid using a DG113 electrode.
Hydroxyl value. The American Oil Chemists’ Society 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 ( M p) at 1122.9 Da (mono-isotopic) which represents the monoprotonated oligomer, [C16N[EO]20H]+. Mnwas calcu-lated to be 1159 Da, where Mn=∑( M
iN i)/∑N i, andN iand
Miare the abundance and mass of the ith oligomer, respec-tively. The weight average molecular weight (Mw), deﬁned as∑( Mi2N i)/∑ MiN i, 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.
unsaturated octadecyl homolog ([C18eneN[EO]20H]+,m/z = 1148.8).
Table 1 is a summary of MALDI-TOF molecular weight results for ethoxylated fatty amines over a DOE range of 10 to 50. For the samples listed, except the 28-mole EO stearyl amine Sample #2, the difference between the
M pand Mnvalues was within one mole of EO (44 Da), and Dvalues were all very low, indicating that these ethoxy-lated amine polymers were all narrowly dispersed. For the 28-mole EO stearyl amine Sample #2, the mass spectrum showed that the oligomer distribution was slightly skewed toward higher masses. This resulted in a mass difference of 81 Da between the M p and Mn values and a slightly higherDvalue of 1.07. The COA MW (certiﬁcates of analy-sis molecular weights) are the molecular weight stated on the products’ COA and were provided by the manufactur-ers. The COA MW values listed in Table 1 show varying degrees of agreement with the molecular weight values de-termined by MALDI-TOF mass spectrometry. These dis-crepancies in molecular weight between the COA and the MALDI-TOF values are discussed in detail below in con- junction with results of the PEG analyses.
Normal-phase HPLC. Both cyano and diol columns were evaluated with various mobile phases to determine the op-timal analytical conditions for separation of individual oligomers. 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 the amine group required addition of a modiﬁer to the mobile phase to eliminate peak tailing caused by silanol effects (21). By using the diol column with a hexane/2-propanol mobile phase and no mobile phase modifier, ethoxylated fatty 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 in the 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.
Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF) Results for Fatty Amine Ethoxylates
Ethoxylated fatty amine COA MWa M
p b Mnc Mw d D e
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 27-mole EO stearyl amine #2,
nominal MW = 1457 1450 1370 1391 1456 1.05 27-mole EO stearyl amine #3,
nominal MW = 1457 1450 1370 1372 1439 1.05 28-mole EO stearyl amine #1,
nominal MW = 1501 1497 1546 1539 1616 1.05 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.
b Most probable molecular weight (unprotonated mono-isotopic mass). c Number average molecular weight (M
n=∑(Mi N i )/ ∑N i ). d Weight average molecular weight (M
w =∑(Mi 2N i )/ ∑Mi N i ). e Polydispersity index (M
w / Mn).
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/zof 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 intencollec-tionally started earlier and termi-nated later relative to the peak valleys to ensure complete collection of the 38.48 min peak. Mnvalue of the 27-mole EO stearyl amine sample as determined by MALDI-TOF (Fig. 4C) was calculated as 1502 Da. Thus, them/zvalue
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.
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.
termined for the highest-intensity HPLC peak, 1503 Da, was in excellent agreement the MALDI-TOF Mnvalue 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 C18packing 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-ration of C16and C18homologs was achieved. Hydrophilic PEG are weakly retained by the C18column 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 C16homologs. 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 inin-creases. 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.
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.
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.
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 Mnvalues were in good agreement with molecular weight results from normal-phase HPLC
mea-FIG. 7.Calibration plot for polyethylene glycol (PEG) 1000. HPLC conditions are the same as
described in Figure 5. For abbreviation see Figure 3.
Comparison of Molecular Weight Results from Different Methods
Ethoxylated fatty amine COA Hydroxyl
nb 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 = 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)
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. b M
n(MALDI-TOF number average molecular weight) = ∑(Mi N i )/ ∑N i .
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;
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 Mnresults. 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
1H 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 Mnvalues 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 Mnvalue 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
Concentrations of PEG and Differences in Molecular Weight (δ)
Percent 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
n=∑(Mi N i )/ ∑N i
b From nonaqueous titration.
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-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.
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.
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[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 a reas 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 (198 2) 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 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 Applicaposi-tion 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.