Preparation of benzalkonium salts differing in the length of a side alkyl Chain

Article abstract of DOI:10.3390/12102341

Benzalkonium salts are widely used as disinfectants, biocides and detergents, among a variety of other applications. The cationic surface-activity of these salts determines their potential to act as a biocide on both target and non-target organisms. In this study, a quick synthesis of a complete set of the benzalkonium salts differing in the length of an alkylating chain (from C₂ to C₂₀) is described. Moreover, their ¹H-NMR, HPLC-MS, TLC and HPLC analysis were recorded.

Full text of DOI:10.3390/12102341

Molecules 2007, 12, 2341-2347
molecules
ISSN 1420-3049
© 2007 by MDPI
www.mdpi.org/molecules
Communication
Preparation of Benzalkonium Salts Differing in the Length
of a Side Alkyl Chain
Kamil Kuca 1,2,3, Jan Marek 4,5, Petr Stodulka 1, Kamil Musilek 2,5,*, Petra Hanusova 4,
Martina Hrabinova 1 and Daniel Jun 1,2
1
Center of Advanced Studies, Faculty of Military Health Sciences, Hradec Kralove, Czech Republic
2
Department of Toxicology, Faculty of Military Health Sciences, Hradec Kralove, Czech Republic
3
Department of Chemistry, Faculty of Sciences, J.E. Purkinje University, Usti nad Labem, Czech
Republic
4
Vakos XT, Prague, Czech Republic
5
Faculty of Pharmacy in Hradec Kralove, Charles University in Prague, Hradec Kralove, Czech
Republic
* Author to whom correspondence should be addressed; E-mail: musilek@pmfhk.cz
Received: 3 September 2007; in revised form 3 October 2007 / Accepted: 4 October 2007 /
Published: 24 October 2007
Abstract: Benzalkonium salts are widely used as disinfectants, biocides and detergents,
among a variety of other applications. The cationic surface-activity of these salts
determines their potential to act as a biocide on both target and non-target organisms. In
this study, a quick synthesis of a complete set of the benzalkonium salts differing in the
1
length of an alkylating chain (from C2 to C20) is described. Moreover, their H-NMR,
HPLC-MS, TLC and HPLC analysis were recorded.
Keywords: Benzalkonium salt, detergent, surfactant, biocide, HPLC, TLC.
Introduction
Quaternary compounds, including the benzalkonium salts, constitute an economically important
class of industrial chemicals that are widely distributed among a diverse array of products and users

Molecules 2007, 12
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from an industrial to the household sector. Because of their strong cationic surface activity, quaternary
compounds are used primarily as disinfectants, biocides, and detergents, but also as anti-electrostatics,
and as phase transfer catalysts [1].
Very important features of benzalkonium salts are their bactericidal and antimicrobial properties.
The antimicrobial activity depends on a changing length of the side n-alkyl chain. It is well known that
the C12-homolog is most effective against yeast and fungi, the C14-homologue against gram-positive
bacteria and C16-homolog against gram-negative bacteria [2]. For these reasons they are widely used as
preservatives for ophthalmic, nasal and parenteral products. They are also used as topical antiseptics
and disinfectants for medical equipment [3-6].
These compounds are not generally used as single components, but rather as mixtures composed of
two or three benzalkonium members differing only in the length of the alkyl chains [7-8]. Such
mixtures are produced on a large-scale in industry. However, a targeted synthesis of pure individuals
could be of interest because of the above-mentioned specificity of each salt against different
pathogens.
Preparation of these compounds has been described previously [9-12], but a general synthesis
applicable to the whole series of benzalkonium salts is lacking. The synthesis of long chain derivatives
(C8-C20) was described earlier by our group [13]. To provide a synthetic route to such compounds, the
preparation of the whole set of these compounds (C2-C20) is now shown. A new set of the short chain
benzalkonium salts was developed and the preparation of the long chain benzalkonium salts was
improved.
Results and Discussion
The N-benzyl-N,N-dimethylalkyl bromides 3-12 were prepared by the general method shown in
Scheme 1.
Scheme 1. Preparation of benzalkonium salts.
EtOH
N
N
R-Br
2
R
Br
reflux
1
3 - 11
Yields of all reactions, together with the melting points of the prepared salts, are shown in Table 1.
Thin-Layer Chromatography (TLC) retention factors and High Pressure Liquid Chromatography
(HPLC) retention times for of all the salts are also listed in this table. The yields achieved in this study
were superior to those obtained earlier for C8-C20 derivatives [13]. Moreover, the C2-C6 derivatives
were added to complete the whole C2-C20 set. Yields achieved for those compounds were also
adequate. We have to point out that use of our method (mixture of the pure compounds) might
conceivably replace the synthetic “all in one” mixtures now used in industry. For example, Ajatine® (a
C12 analogue used as disinfectant) is generally a mixture of the parent substances (N-benzyl-N,N-

Molecules 2007, 12
2343
dimethylamine, C12 alkylating chain and solvent) and a reaction product (the appropriate
monoquaternary salt). Our pure products (without parent substances) could be used in more
sophisticated applications, where potential adverse effects of the parent substances would no longer be
a factor.
There are several articles which deal with HPLC analysis of benzalkonium derivatives [14-16].
None of these, however, describe HPLC analysis of the whole benzalkonium set. In this study, our
previously developed HPLC method was modified for a determination of each benzalkonium salt with
an aim of distinguishing them [17]. Such a method could be applicable not only for the benzalkonium
salts but also for other long chain quaternary compounds. For quick laboratory analysis of prepared
benzalkonium salts, a TLC method was also developed to distinguish each benzalkonium salt. Such a
method could be of interest in the laboratories not equipped by special expensive equipment (HPLC).
Table 1. Yields, melting points, retention factors and retention times of prepared bezalkonium salts.
Compound
Yield (%)
m.p. (°C)
TLC Rf
HPLC Rt
3
4
94
44
54
61
61
80
71
58
71
64
120.5-121.5
150.0-152.0
122.0-123.5
53.0-56.0
34.0-37.5
37.0-40.0
43.5-47.5
50.0-53.0
81.0-83.0
85.0-88.0
0.083
0.260
0.420
0.512
0.558
0.592
0.612
0.634
0.651
0.664
4.76
5.84
5
7.13
6
8.47
7
9.92
8
11.55
13.43
15.60
18.15
21.25
9
10
11
12
Conclusions
A quick and easy method for the preparation of pure benzalkonium salts in adequate yields was
described. Their purities were analyzed by 1H-NMR, HPLC-MS, TLC and HPLC techniques.
Experimental
General
All chemicals used in this study were obtained from Sigma-Aldrich (Czech Republic). Purity of all
products was tested by determination of their melting points (Boetius block) and were uncorrected,
TLC (Kieselgel Merck; mobile phase n-BuOH/CH3COOH/H2O (5:1:2); Detection: UV254,
Dragendorff´s reagent); HPLC-MS (HP1100 HPLC system - Agilent Technologies (Waldbronn,

Molecules 2007, 12
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Germany); quadrupole mass spectrometer MSD1456 VL; data were collected in positive ion mode
with an ESI probe voltage of 4000 V) and NMR analysis (Varian Gemini 300, 300 MHz, DMSO-d6).
Synthesis
Briefly N,N-dimethylbenzylamine (1; 7.4 mmol) in dry ethanol (25 ml) was mixed with an
equimolar amount of the appropriate 1-bromo-alkane (2; 10.4 mmol) and the mixture was refluxed for
28 hours. Solvent was evaporated and the crude N-benzyl-N,N-dimethylalkyl-1-ammonium bromides
(3-12) were recrystalized from acetone, washed with ether and allowed to dry at r.t. The corresponding
yields and melting points are summarized in Table 1. Spectral data (1H-NMR and MS spectra) are
listed below:
1
N-benzyl-N,N-dimethylethyl-1-ammonium bromide (3). H-NMR: δ 1.35 (t, 3H, CH3CH2N+); 2.93 (s,
6H, (CH3)2N+); 3.35 (t, J = 6.33 Hz, 2H, CH2N+); 4.52 (s, 2H, PhCH2N+); 7.53 (bs, 5H, Ph); ESI-MS:
m/z 164.2 [M+] (calc. for [C11H18N]+ 164.27).
1
N-benzyl-N,N-dimethylbutyl-1-ammonium bromide (4). H-NMR: δ 0.90 (t, J = 6.47 Hz, 3H, CH3);
1.28 (m, 2H, (CH2)3); 1.78 (m, 2H, CH2CH2N+); 2.96 (s, 6H, (CH3)2N+); 3.24 (t, J = 6.33 Hz, 2H,
CH2N+); 4.59 (s, 2H, PhCH2N+); 7.56 (bs, 5H, Ph); ESI-MS: m/z 192.2 [M+] (calc. for [C13H22N]+
192.32).
1
N-benzyl-N,N-dimethylhexyl-1-ammonium bromide (5). H-NMR: δ 0.88 (t, J = 6.47 Hz, 3H, CH3);
1.26 (bs, 6H, (CH2)3); 1.79 (m, 2H, CH2CH2N+); 2.95 (s, 6H, (CH3)2N+); 3.22 (t, J=6.33Hz, 2H,
CH2N+); 4.58 (s, 2H, PhCH2N+); 7.55 (bs, 5H, Ph); ESI-MS: m/z 220.2 [M+] (calc. for [C15H26N]+
220.38).
1
N-benzyl-N,N-dimethyloctyl-1-ammonium bromide (6). H-NMR: δ 0.86 (t, J = 6.47 Hz, 3H, CH3);
1.29 (bs, 10H, (CH2)5); 1.78 (m, 2H, CH2CH2N+); 2.95 (s, 6H, (CH3)2N+); 3.24 (t, J=6.33Hz, 2H,
CH2N+); 4.56 (s, 2H, PhCH2N+); 7.56 (bs, 5H, Ph); ESI-MS: m/z 248.2 [M+] (calc. for [C17H30N]+
248.44).
1
N-benzyl-N,N-dimethyldecyl-1-ammonium bromide (7). H-NMR: δ 0.83 (t, J = 6.60 Hz, 3H, CH3);
1.23 (bs, 14H, (CH2)7); 1.77 (m, 2H, CH2CH2N+); 2.92 (s, 6H, (CH3)2N+); 3.24 (t, J=7.15Hz, 2H,
CH2N+); 4.54 (s, 2H, PhCH2N+); 7.53 (bs, 5H, Ph); ESI-MS: m/z 276.3 [M+] (calc. for [C19H34N]+
276.50).
1
N-benzyl-N,N-dimethyldodecyl-1-ammonium bromide (8). H-NMR: δ 0.83 (t, J= 6.60 Hz, 3H, CH3);
1.22 (bs, 18H, (CH2)9); 1.77 (m, 2H, CH2CH2N+); 2.94 (s, 6H, (CH3)2N+); 3.24 (t, J=6.32Hz, 2H,
CH2N+); 4.55 (s, 2H, PhCH2N+); 7.52 (m, 5H, Ph); ESI-MS: m/z 304.3 [M+] (calc. for [C21H38N]+
304.55).

Molecules 2007, 12
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1
N-benzyl-N,N-dimethyltetradecyl-1-ammonium bromide (9). H-NMR: δ 0.83 (t, J = 6.60 Hz, 3H,
CH3); 1.22 (bs, 22H, (CH2)11); 1.78 (m, 2H, CH2CH2N+); 2.95 (s, 6H, (CH3)2N+); 3.23 (t, J=7.71Hz,
2H, CH2N+); 4.54 (s, 2H, PhCH2N+); 7.52 (m, 5H, Ph); ESI-MS: m/z 332.3 [M+] (calc. for [C23H42N]+
332.61).
N-benzyl-N,N-dimethylhexadecyl-1-ammonium bromide (10) 1H-NMR: δ 0.84 (t, J = 6.60 Hz, 3H,
CH3); 1.22 (bs, 26H, (CH2)13); 1.78 (m, 2H, CH2CH2N+); 2.94 (s, 6H, (CH3)2N+); 3.22 (t, J=8.39 Hz,
2H, CH2N+); 4.52 (s, 2H, PhCH2N+); 7.52 (bs, 5H, Ph); ESI-MS: m/z 360.4 [M+] (calc. for [C25H46N]+
360.64).
1
N-benzyl-N,N-dimethyloctadecyl-1-ammonium bromide (11). H-NMR: δ 0.84 (t, J = 6.47 Hz, 3H,
CH3); 1.24 (bs, 30H, (CH2)15); 1.75 (m, 2H, CH2CH2N+); 2.93 (s, 6H, (CH3)2N+); 3.25 (t, J=8.25Hz,
2H,CH2N+); 4.52 (s, 2H, PhCH2N+); 7.53 (bs, 5H, Ph); ESI-MS: m/z 388.4 [M+] (calc. for [C27H50N]+
388.71).
N-benzyl-N,N-dimethyleicosyl-1-ammonium bromide (12). 1H-NMR: δ 0.83 (t, J = 6.47 Hz, 3H, CH3);
1.24 (bs, 34H, (CH2)17); 1.74 (m, 2H, CH2CH2N+); 2.94 (s, 6H, (CH3)2N+); 3.24 (t, J = 8.26 Hz, 2H,
CH2N+); 4.54 (s, 2H, PhCH2N+); 7.52 (m, 5H, Ph); ESI-MS: m/z 416.4 [M+] (calc. for [C27H50N]+
416.78).
Subsequently, TLC and HPLC separations of all prepared compounds were developed (Table 1).
The HPLC system consisted of a P200 gradient pump (Spectra-Physics Analytical, Fremont, USA),
a 7125 injection valve – 10 μΛ loop (Rheodyne, Cotati, USA), a UV1000 detector (Spectra-Physics
Analytical, Fremont, USA) and a CSW Chromatography Station 1.5 software (Data Apex, Prague,
Czech Republic). For analyses a 250 × 4.6 mm I.D. Waters Spherisorb Cyano (5 μm) column was used
(Supelco Inc.). The mobile phase contained 45% acetonitrile and 55 % water. This mixture was
prepared as 0.1 M sodium acetate solution. Finally the pH was adjusted with acetic acid to 5.0. It was
delivered isocratically at a flow-rate of 1 mL/min. The absorbance was measured at 263 nm [18]. TLC
was performed on 140 mm × 140 mm plates coated with a 0.2 mm layer of silica gel 60 F254 (Merck,
Darmstadt, Germany). Plates was streaked with a CAMAG Linomat IV automatic applicator (Camag,
Berlin, Germany) and developed with a methanol/chloroform/acetic acid (25:5:0.5 v/v)) mobile phase
in a twin trough chamber with a stainless steel lid (Camag, Berlin, Germany). Compounds were
dissolved in acetonitrile/water (45:55 v/v) as 0.1 M acetate buffer, pH 5.0. Samples (5 μL from 2.5
mM solutions) were applied 1 cm from the bottom edge of the plate. Development was stopped when
the mobile phase front reached 1 cm from the top of the plate.
Acknowledgements
Support of the Grant FVZ0000501 (Ministry of Defence, Czech Republic) is gratefully
acknowledged.

Molecules 2007, 12
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Sample Availability: Samples of all prepared compounds are available from the authors.
© 2007 by MDPI (http://www.mdpi.org). Reproduction is permitted for noncommercial purposes.