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

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 Beckman-Coulter Inc., Miami, Florida 33196-2500

Specific lengths of the fatty and polyoxyethylene chains of ethoxylated fatty amines are critical to their performance in specific applications, and thus the ability to characterize these surfactants accurately is crucial. Normal-phase high-performance liquid chromatography (HPLC) and matrixassisted 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 analyzed using these methods, and comparison was made to molecular weight determinations using proton nuclear magnetic resonance (NMR), neutralization equivalent weight, and hydroxyl value methods. Molecular weight results from normalphase 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 concentrations of polyethylene glycols (PEG) and fatty homologs. PEG interfered with molecular weight determinations by NMR, neutralization equivalent weight, and hydroxyl value methods. PEG caused no interference with molecular weight determinations by normal-phase HPLC and MALDITOF methods. Paper no. S1134 in JSD 2, 503–513 (October 1999). ABSTRACT:

RN(CH2CH2OH)2 + (x + y)(C2H4O)

 base, ∆ [2]

RN

(CH2CH2O)x+1H (CH2CH2O) y+1H

The product is a polydisperse mixture of oligomers approaching a Poisson distribution (5). Polyethylene glycols (PEG) are side products, formed from the reaction of EO with residual water. Ethoxylated fatty amines are commercially 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 characterize 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 neutralization equivalent weight (NEW) (6) and hydroxyl value methods (6,7). These methods are still used almost excluKEY WORDS: Degree of ethoxylation, ethoxylated fatty sively by the surfactant industry for determination of the amines, HPLC, hydroxyl value, MALDI-TOF, molecular weight, molecular weight of ethoxylated fatty amines. NEW is neutralization equivalent weight, 1H NMR. often used during manufacturing as an in-process test to determine the extent of ethoxylation. NEW is i s estimated by titrimetric neutralization of the amine group with stanEthoxylated fatty amines are used in different industrial dardized acid. The hydroxyl value method involves deapplications such as defoamers, textile-finishing agents, rivatization of terminal hydroxyl groups using either acetic corrosion inhibitors, emulsifiers (1), and dye promoters or phthalic anhydride followed by quantitative determina(2,3). They enhance herbicidal activity of many pesticides tion of excess anhydride. Reversed- and normal-phase high-performance liquid (1) and have potential uses in laundry products (4). Ethoxylated fatty amines are produced by the reaction of a chromatography (HPLC) methods have been developed co mfatty amine with ethylene oxide (EO) (1,5). The two-step for analysis of ethoxylated nonionic surfactants, most comethoxylation of a primary amine is shown in Equations 1 monly for ethoxylated fatty alcohols, acids, sulfonates, and alkylphenols (8–11). The one reported method applicable and 2, where R typically is C 12–C18 fatty groups. to ethoxylated fatty amines was limited to a mean EO con∆ RNH 2 + 2 ( C 2 H 4 O )    → RN(CH 2 CH 2 OH ) [1] tent of only 15 moles owing to the ion-pair/fluorescence 2 detection system used (12). Use of an evaporative lightscattering detector for HPLC applications allows for detection of polyalkoxylated compounds with no molecular *To whom correspondence should be addressed at Beckman-Coulter weight limitations (13). Matrix-assisted laser desorption Inc., Mail Stop 11-A02, 11800 SW 147 Ave., Miami, FL 33196-2500. ionization-time of flight (MALDI-TOF) mass spectrometry E-mail: [email protected] Copyright © 1999 by AOCS Press

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has recently been used for the analysis of some synthetic All reversed-phase HPLC analyses including PEG analysurfactants (14–18); however, ethoxylated fatty amines ses were performed using a Waters Nova-Pak 60Å C 18, 4 have not been evaluated using this technique. Nuclear µm, 150 × 3.9 mm column (Milford, MA). The isocratic momagnetic resonance (NMR) spectroscopy has been an in-  bile phase was MeOH/H 2O (85:15) containing 25 mM trivaluable tool for the molecular structural analysis of or- ethylamine and 50 mM glacial acetic acid. Normal-phase ganic compounds. This technique has been used to deter- HPLC separations were performed on a LiChrospher 100Å mine the degree of ethoxylation and characterize physical Diol, 5 µm, 150 × 4.6 mm column (Alltech Associates, Deerproperties of nonionic surfactants (19). field, IL). The mobile phase gradient program used for the In our attempts to characterize ethoxylated fatty amines majority of ethoxylated fatty amines was a linear gradient and to estimate their degrees of ethoxylation and average of hexane/2-propanol (both solvents contain 25 mM molecular weights accurately, new HPLC and MALDI-TOF triethylamine) from 95:5 to 70:30 over 140 min. This promass spectrometry methods were developed. These re- gram allowed for analysis of ethoxylated fatty amines over versed-phase and normal-phase HPLC methods incorpo- a wide range of ethoxylate chain lengths, typically 5 to 60 rated the use of an evaporative mass detector. The evapora- EO units for an ethoxylated stearyl amine. As discussed tive mass detector is ideal for analytes lacking suitable chro-  below, this mobile phase program can be modified to optimophores, and minimal baseline drift is observed when mize the analysis of a specific ethoxylated fatty amine. used with gradient elution chromatography. The reversed  MALDI-TOF mass spectrometry. MALDI mass spectra phase HPLC method was developed to separate and quan- were acquired on a PE-PerSeptive Biosystems (Framingtify PEG and fatty homologs of ethoxylated fatty amines. ham, MA) Voyager-DE STR delayed extraction reflectron The normal-phase method was developed to separate time-of-flight mass spectrometer equipped with a Laser oligomers of ethoxylated fatty amines and to determine Science nitrogen laser (337 nm, 3 ns pulse). Positive ion their average molecular weights. MALDI-TOF mass spec- spectra were acquired in the linear mode using an actrometry was used to characterize ethoxylated fatty amines celeration voltage of 20 kV. The matrix used was a satuand to assign mass values to oligomers of normal-phase rated solution of α-cyano-4-hydroxycinnamic acid in 1:1 HPLC analyses. Ethoxylated fatty amines were also charac- MeCN/H2O containing 0.1% trifluoroacetic acid (TFA). terized using 1H NMR, NEW, and hydroxyl value methods. Samples for MALDI-TOF analysis were prepared by disThe results of these molecular weight determinations, using solving 1 mg of sample in 1 mL MeCN/H 2O (1:1) and furthese five different methods, showed that for many of the ther diluted 1:20 with H 2O containing 0.1% TFA. A 1-µL ethoxylated fatty amines samples analyzed different meth- aliquot of the sample solution was thoroughly mixed with ods yielded significantly different molecular weight esti- an equal volume of the α-cyano-4-hydroxycinnamic acid mates. This paper describes the molecular weight charac- matrix solution and analyzed. 1 terization of different ethoxylated fatty amines using these  H NMR. 1H NMR samples were analyzed at room temfive methods. A comparison of the results is reported. perature on an NT-360 (360 MHz) spectrometer (Nicolet Instruments Corporation, Madison, WI) with a wide-bore (89 mm) magnet or a 400 MHz spectrometer (Varian Analytical EXPERIMENTAL PROCEDURES Instruments, Valencia, CA). Samples were dissolved in Ethoxylated fatty amines were obtained from commercial deuterated acetone or chloroform resulting in concentrasources (Akzo Nobel Chemicals Inc., Chicago, IL; Ethox tions of approximately 20 mM. NMR data were analyzed Chemicals, Inc., Greenville, SC; Heterene Inc., Paterson, NJ) using NUTS data analysis software (ACORN NMR Inc., Freand were used, as received, without further purification. mont, CA). Proton NMR chemical shift assignments were  HPLC. The HPLC chromatographic system was a Wa-  based on characteristic proton NMR shift tables. In addition, ters 2690 (Milford, MA) with a Polymer Labs EMD 960 NMR spectra of stearyl amine, PEG-900, and PEG-1500 were evaporative mass detector (Amherst, MA). The EMD 960 used as guides to verify chemical shift values for the fatty detector used industrial grade nitrogen (Air Products, Al- and polyoxyethylene moieties. Proton NMR molecular lentown, PA) at a flow rate of 5 L/min. The detector was weight calculations were based on the ratio of integrated operated at 65°C for normal-phase separations and 75°C peak areas between the terminal methyl group of the alkyl for reversed-phase separations. Sample solutions were pre- chain (δ = 0.872–0.888 ppm) to (i) the methylene protons of pared by dissolving 100 mg of ethoxylated fatty amine into the ethoxylate chains ( δ = 3.580–3.595 ppm) excluding those 10 mL of 2-propanol. Sample solutions were filtered adjacent to the amine nitrogen, (ii) the hydroxyl terminal through 0.45 µm GH Polypro membrane filters (Gelman protons (δ = 2.843–2.854 ppm), and (iii) the alkyl protons ( δ Sciences, Ann Arbor, MI). A sample volume of 10 µL was = 1.284–1.302 ppm) excluding the methyl protons, protons injected into the HPLC system for analysis. Mobile phases adjacent to the methyl group, and protons adjacent to the were filtered through 0.45-µm GH Polypro membrane fil- amine nitrogen. These ratios were verified by comparing raters prior to use. For all HPLC analyses, the HPLC columns tios of the terminal methyl protons to (i) its adjacent methylwere maintained at 40°C, and a mobile phase flow rate of ene protons (δ = 1.4343–1.581 ppm) and (ii) methylene pro1 mL/min was used. tons adjacent to the nitrogen ( δ = 2.512–2.522 ppm).  Journal of Surfactants and Detergents, Vol. 2, No. 4 (October 1999)

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NEW. NEW was determined potentiometrically using MALDI-TOF mass spectrometry. The α-cyano-4-hydroxyaqueous and nonaqueous titrations. Aqueous titrations cinnamic acid/TFA matrix yielded reproducible mass were performed to determine the concentration of base cat- spectra with high abundances for all ethoxylated fatty alyst remaining in the amine sample. Samples ( ≈2 g) were amines. A CV of 1.4% was obtained for  Mn (number averdissolved in water/2-propanol (1:1) and titrated against age molecular weight) values from three analyses of a 280.1 N HCl using a Mettler-Toledo DL-50 titrator (Hights- mole EO stearyl amine in which the samples were pretown, NJ) equipped with a 20.0 mL buret and a DG111 elec- pared and analyzed over a period of 4 mon. For these trode. Nonaqueous titrations were performed by dissolv- ethoxylated fatty amines, the MALDI-TOF mass spectra ing 2-g samples in 50 mL glacial acetic acid and titrating showed no fragmentation. The major mass peaks appear against 0.1 N perchloric acid using a DG113 electrode. as [M + H]+ ions with no multiply-charged ions observed.  Hydroxyl value . The American Oil Chemists’ Society hy- The formation of sodium or potassium adducts was neglidroxyl value method (20) was followed to obtain the hy- gible. In contrast, ethoxylated fatty alcohols analyzed droxyl value of ethoxylated amines with the following under identical conditions produced spectra in which the modifications. Samples for acetylation and two blanks predominant peaks appeared as sodium and potassium were refluxed for 1 h under constant stirring in a mineral adducts (Lang, R.F., and D. Parra-Diaz, unpublished reoil bath. The oil bath temperature was maintained within a sults). The MALDI-TOF mass spectrum of a 25-mole EO range of 95.0–110.0°C. The reaction was then quenched by tallow amine is shown in Figure 1. The oligomer distribuadding 15.0 mL of deionized water to the mixture followed tion is symmetrical with the highest abundant mass peak  by a 20-min incubation in the oil bath. The mixtures were ( M p) at 1122.9 Da (mono-isotopic) which represents the allowed to cool to room temperature, then titrated with monoprotonated oligomer, [C 16N[EO]20H]+. Mn was calcuethanolic potassium hydroxide. Duplicate analyses were lated to be 1159 Da, where  Mn = ∑( MiN i)/∑N i, and N i and performed for each sample, and the mean value was re-  Mi are the abundance and mass of the ith oligomer, respecported. The method resulted in a coefficient of variation tively. The weight average molecular weight (M w), defined (CV) of 7.0% for 14 determinations performed over a pe- as ∑( Mi2N i)/∑ MiN i, was 1208 Da. The polydispersity index riod of several months. (D), defined as  Mw/ Mn, was calculated to be 1.042, indicating a narrowly dispersed polymer. Figure 2 is an expanded mass spectrum of the 25-mole EO tallow amine and shows RESULTS AND DISCUSSION the saturated homologs, octadecyl ([C 18N[EO]20H]+, m/z =  MALDI-TOF mass spectrometry. Ethoxylated fatty amines 1150.9), hexadecyl ([C 16 N[EO]20 H] +, m/z = 1122.9), and with DOE values ranging from 10 to 50 were evaluated by 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.

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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  M p and Mn values was within one mole of EO (44 Da), and D values were all very low, indicating that these ethoxy= 1148.8). 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  M p and  Mn values and a slightly higher D value of 1.07. The COA MW (certificates of analyTABLE 1 sis molecular weights) are the molecular weight stated on Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass the products’ COA and were provided by the manufacturSpectrometry (MALDI-TOF) Results for Fatty Amine Ethoxylates ers. The COA MW values listed in Table 1 show varying Ethoxylated fatty amine COA MW a Mp b  Mnc  Mw d  D e  degrees of agreement with the molecular weight values de10-mole EO stearyl amine, termined by MALDI-TOF mass spectrometry. These disnominal MW = 709 697 666 686 730 1.06 crepancies in molecular weight between the COA and the 25-mole EO tallow amine, MALDI-TOF values are discussed in detail below in connominal MW = 1369 1406 1122 1159 1208 1.04 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 opnominal MW = 1457 1450 1370 1391 1456 1.05 timal analytical conditions for separation of individual 27-mole EO stearyl amine #3, oligomers. The diol column consistently gave superior resnominal MW = 1457 1450 1370 1372 1439 1.05 28-mole EO stearyl amine #1, olution vs. the cyano column; thus all analyses were pernominal 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 phase to eliminate peak tailing caused by silanol effects 50-mole EO stearyl amine, (21). By using the diol column with a hexane/2-propanol nominal MW = 2469 2429 2647 2616 2666 1.02 mobile phase and no mobile phase modifier, ethoxylated aMolecular weight (MW) from manufacturer’s certificate of analysis (COA). fatty amines eluted as a broad tailing peak with no oligoDetermined by neutralization equivalent weight (NEW) analysis. b  Most probable molecular weight (unprotonated mono-isotopic mass). mer separation. Since the evaporative mass detector rec  Number average molecular weight ( Mn = ∑(Mi N i )/ ∑N i ). quires that only volatile buffers and modifiers be used in d Weight average molecular weight ( M = ∑(M 2N )/ ∑M N ). w  i  i  i  i  e Polydispersity index ( M  / M ). the mobile phase, triethylamine was initially evaluated. w  n 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)

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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 identification, and are not the actual DOE values. The mobile phase program was a linear gradient of hexane/2propanol (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 observed as evidenced by almost complete baseline separation 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 different 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 initial mobile phase containing a higher concentration of 2propanol together with a steeper gradient. This mobile phase program was hexane/2-propanol (both containing

507

25 mM triethylamine) from 90:10 to 60:40 over 80 min. Chromatographic peak retention times were very reproducible 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 highest-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 N 2, and the residue was analyzed by MALDI-TOF mass spectrometry. As shown in Figure 4B, the m/z of the 38.48 min peak was found to be 1503 Da (monoprotonated, monoisotopic mass), resulting in a DOE value of 28. Lowerabundant 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 collection process was intentionally started earlier and terminated 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)

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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.

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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 determination of the molecular weight of a variety of ethoxylated fatty amines using retention times and mass assignments of the 27-mole EO stearyl amine as reference values. Reversed-phase HPLC. A C 18 reversed-phase HPLC method was developed for determination of PEG, and alkyl homologs of ethoxylated fatty amines. Aqueous solvent 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. Optimization experiments showed that a mobile phase consisting 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 tailing of ethoxylated fatty amines. Figure 5 shows the chromatogram 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 abbreviations see Figures 1 and 3.

FIG. 5. Reversed-phase HPLC chromatogram of 27-mole EO stearyl

amine. Stationary phase: Waters Nova-Pak 60Å C 18, 4 µm (150 × 3.9 mm column). Mobile phase isocratic elution: MeOH/H 2O (85:15) containing 25 mM triethylamine and 50 mM glacial acetic acid. Flow rate: 1 mL/min. Column temperature: 40°C. Detector: evaporative mass detector. For abbreviations see Figures 1 and 3.

ration of C16 and C18 homologs was achieved. Hydrophilic PEG are weakly retained by the C 18 column and elute first, as a single peak. Alkyl homologs elute in the order of increasing alkyl chain length. Figure 6 shows the chromatogram of a 15-mole EO coco amine and the separation achieved for C 10, C12, C14, and C 16 homologs. Analyses of ethoxylated fatty amines with the same alkyl group containing differing ethoxylate chain lengths showed that the longer-chain ethoxylates eluted earlier than the shorterchain species. This observation is presumably due to an increase in polarity as the ethoxylate chain length increases. Under reversed-phase HPLC conditions, the analytes exhibiting greater polarity elute first. PEG quantitation . Ethoxylated fatty amines were analyzed for PEG content to determine how PEG influenced molecular weight results for different methods. PEG concentrations in ethoxylated fatty amines were calculated from a calibration curve prepared from analyses of standard 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.

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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 analyzed using MALDI-TOF mass spectrometry, 1H NMR, normal-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 samples 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 derived 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

10-mole EO stearyl amine (nominal NW = 709) 25-mole EO tallow amine (nominal MW = 1369) 27-mole EO stearyl amine #1 (nominal MW = 1457) 27-mole EO stearyl amine #2 (nominal MW = 1457) 27-mole EO stearyl amine #3 (nominal MW = 1457) 28-mole EO stearyl amine #1 (nominal MW = 1501) 28-mole EO stearyl amine #2 (nominal MW = 1501) 50-mole EO stearyl amine (nominal MW = 2469) aMW b 

COA MWa (DOE)e 

Mnb 

(DOE)e 

HPLCc  (DOE)e 

NEWd  (DOE)e 

1H NMR

697 (9.7) 1406 (25.8) 1465 (27.2) 1450 (26.8) 1450 (26.8) 1497 (27.9) 1605 g  (30.4) 2429 (49.1)

686 (9.5) 1159 (20.2) 1502 (28.0) 1391 (25.5) 1372 (25.1) 1539 (28.9) 1363 (24.9) 2616 (53.3)

665 (9.0) 1184 (21.0) 1501 (28.0) 1369 (25.0) 1369 (25.0) 1589 (30.0) 1325 (24.0) 2601 (53.0)

713 (10.1) 1426 (26.3) 1496 (27.9) 1475 (27.4) 1465 (27.2) 1563 (29.4) 1767 (34.0) 2462 (49.8)

730 (10.5) 1526 (28.6) 1693 (32.4) 1558 (29.3) 1575 (29.7) 1791 (34.6) 2003 (39.4) 2862 (58.9)

(DOE)e 

Hydroxyl value MW (DOE)e  f  f 

1300 (23.4) 1301 (23.5) 1190 (20.9) 1340 (24.3) 1410 (25.9) f 

from manufacturer’s COA. Determined by NEW analysis.

Mn (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 composition 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.

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surements throughout the molecular weight range. Molecular 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 MALDITOF 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 indicates that mass discrimination in the MALDI-TOF determination at the higher end of the mass range is not occurring 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 present 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 polyoxyethylene and hydroxyl groups. Thus, the presence of PEG results in an overestimation of molecular weight

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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, presumably 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 interefere with normal-phase HPLC molecular weight determination. 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 Mn a Values δ

Ehtoxylated fatty amine 10-mole EO stearyl amine 25-mole EO tallow amine 27-mole EO stearyl amine, sample #1 27-mole EO stearyl amine, sample #2 27-mole EO stearyl amine, sample #3 28-mole EO stearyl amine, sample #1 28-mole EO stearyl amine, sample #2 50-mole EO stearyl amine a

Percent PEG (w/w)

δ

δ

δ

HPLC

NEW b 

1H NMR

3.8 15.3

21 −25

−27

−44

−267

−367

c 

6.1

1

4

−191

202

7.8

22

−84

−167

90

9.8

3

−93

−203

182

4.1

−50

−24

−252

199

17.7 2.7

38 15

−404

−640 −246

−47

154

Mn = ∑(Mi N i )/ ∑N i

b 

From nonaqueous titration. Not performed. PEG, polyethylene glycol; for other abbreviations see Tables 1 and 2.

c 

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Hydroxyl value c 

c 

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 concentrations do not significantly interfere with the MALDITOF 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 reasons 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 normalphase 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 determined. In addition, HPLC instrumentation costs are significantly 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.

mance Liquid Chromatography, Crit. Rev. Anal. Chem. 25 :203 (1996). 10. Ban, T., E. Papp, and J. Inczedy, Reversed-Phase High-Performance Liquid Chromatography of Anionic and Ethoxylated Non-Ionic Surfactants and Pesticides in Liquid Pesticide Formulations, 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, Determination of the Composition of Ethoxylated Alkylamines in Pesticide Formulations by High-Performance Liquid Chromatography 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. Geissmann, Mass Spectrometry of Synthetic Polymers by UV-Matrix-Assisted Laser Desorption/Ionization, Anal. Chem. 64:2866 (1992). 15. Thomson, B., Z. Wang, A. Paine, A. Rudin, and G. Lajoie, Surfactant Analysis by Matrix-Assisted Laser Desorption Timeof-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 Desorption/Ionization Time-of-Flight Mass Spectrometry: Molecular Weight Estimates in Samples of Varying Polydispersity, 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 Recommended 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 NonVolatile Solutes in LC and SFC , LCGC International 14:148 (1996).

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, Influence of Oxyethylenated Alkylamines in the Dyeing of Wool,  J. Soc. Dyers Colour 99:291 (1983). [Received February 26, 1999; accepted July 14, 1999] 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). Dr. Russell F. Lang is a Senior Scientist in the Reagents and Ap4. Arif, S., Fatty Amine Ethoxylates, HAPPI , 67 (1996).  plications Development Group, in the Cellular Analysis Divi5. Cross, J., Introduction to Nonionic Surfactants, in Nonionic Surfactants, edited by J. Cross, Surfactant Science Series, Marsion of Beckman-Coulter, Inc. His current research includes the cel Dekker, Inc., New York, 1987, Vol. 19, p. 3. use of chromatographic and mass spectrometric techniques for 6. Miwidsky, B.M., and D.M. Gabriel, Detergent Analysis , 1982, the characterization of surfactants, and the effect of surfactants  John Wiley & Sons, New York, pp. 207, 208. 7. Cross, J., Aspects of Quality and Process Control, in Nonionic on cellular components. He received his B.S. in chemistry from Florida International University and his Ph.D. in inorganic Surfactants, edited by J. Cross, Surfactant Science Series, Marcel Dekker, Inc., New York, 1987, Vol. 19, p. 371. chemistry from the University of Miami. Other a reas of exper8. Marquez, N., R. Anton, A. Usubillaga, and J.L. Salager, Opti- tise include marine, atmospheric, and organometallic chemistry. mization of HPLC Conditions to Analyze Widely Distributed Dr. Dennisse Parra-Diaz received her B.S. degree in chemEthoxylated Alkylphenol Surfactants , J. Liquid Chromatogr . istry from the University of Puerto Rico (198 2) and her Ph.D. 17:1147 (1994). 9. Miszkiewicz, W., and L. Szymanowski, Analysis of Nonionic degree in physical chemistry from the University of Miami Surfactants with Polyoxyethylene Chains by High-Perfor- (1990). After completing postdoctoral training in biophysical  Journal of Surfactants and Detergents, Vol. 2, No. 4 (October 1999)

ANALYSIS OF ETHOXYLATED FATTY AMINES chemistry at Temple University (1991), she held a Research Associate position at the United States Department of Agriculture Eastern Regional Research Center. She began working for Beckman-Coulter, Inc. in 1996 and currently holds a Scientist position in the Reagents and Application Development Group. Her research interests include structural elucidation of peptides and organic-alkali metal complexes using nuclear magnetic resonance and molecular mechanics as well as the development of  hematology and immunology reagents.

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Dr. Dana Jacobs is currently the Manager of the Controls and Calibrators Group in the Cellular Analysis Division of BeckmanCoulter, Inc. As an undergraduate, he studied chemistry, mathematics, and zoology and received his B.A. from the University of  Vermont (1969). After serving in the military, he studied immunochemistry, lectin, and lymphokine biochemistry in the laboratory of Dr. Ronald D. Poretz at Rutgers University and received his Ph.D. in 1980.

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