Two new azamacrocycle-based stationary phases for high-performance liquid chromatography: Preparation and comparative evaluation

Journal of Chromatography A, 1270 (2012) 186–193

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Two new azamacrocycle-based stationary phases for high-performance liquid chromatography: Preparation and comparative evaluation Lijun He ∗ , Mingliang Zhang, Longhui Liu, Xiuming Jiang, Pu Mao, Lingbo Qu School of Chemistry and Chemical Engineering, Henan University of Technology, Zhengzhou 450001, China

a r t i c l e

i n f o

Article history: Received 15 August 2012 Received in revised form 31 October 2012 Accepted 2 November 2012 Available online 9 November 2012 Keywords: Azamacrocycle Stationary phase HPLC Multiple interactions Shape and planarity recognition

a b s t r a c t In this paper, two new azamacrocycle-bonded stationary phases for high-performance liquid chromatography are described. The new phases were prepared by respectively coupling a 14-membered Curtis macrocycle and a 30-membered hexaazaannulene to ␥-chloropropyltrimethoxylsilane-modified silica and characterized by elemental analysis and infrared spectroscopy. To understand the effects of the structures of the azamacrocyles and their functional groups upon the retention and separation, the chromatographic behaviors of the two stationary phases were compared by eluting alkylbenzenes, polycyclic aromatic hydrocarbons (PAHs) and aromatic amines. The two new phases demonstrated unique selectivity and fine chromatographic performance, which were guaranteed by multiple interactions, including hydrophobic, hydrogen-bonding, ␲–␲ and dipole-induced dipole interactions between stationary phase and solutes. On the other hand, the difference between the structures of embedded azamacrocyles did lead to remarkable dissimilarity in the shape and planarity recognition of isomers, which was highlighted by the 30-membered hexaazaannulene-bonded phases’ superiority over 14-membered tetraazamacrocyclebonded phases’ in discriminating specific PAHs. With a wide range of probes, the linear solvation energy relationship model was also applied to evaluate the chromatographic properties of the two stationary phases, further verifying the chromatographic results. © 2012 Elsevier B.V. All rights reserved.

1. Introduction During recent years, there has been a substantial growth in the development of specifically designed stationary phases (SPs) for high-performance liquid chromatography (HPLC), enabling the separation of a diversity of solutes. These SPs have chiefly incorporated those ligands with multifunctional groups such as cyclodextrin [1], polysaccharide [2], calixarene [3], crown ether [4] and ionic liquid [5]. With these new types of SPs, separation of desired solutes from complex matrix can be achieved due to the involvement of multiple interactions. It is noteworthy that toward certain kinds of analytes unsatisfactorily separated on conventional ODS phase, these new materials exhibit quite spectacular chromatographic retentivity and selectivity, thanks to the diversified moieties of the ligands, such as aromatic ring, amino group, hydroxyl group and halogen ions, entailing conventional multiple interactions covering ␲–␲, hydrogen-bonding, dipole-induced dipole interactions and acid–base equilibrium, and synchronously some special mechanisms involving the structure and rigidity of the ligand.

∗ Corresponding author. Tel.: +86 013526601910; fax: +86 371 67756718. E-mail addresses: [email protected], [email protected] (L. He). 0021-9673/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.chroma.2012.11.007

As a new generation of supramolecule, the azamacrocycles, which structurally resemble crown ether, have been widely applied in many fields, such as complex chemistry, biochemistry and pharmaceutical chemistry [6–9], where they serve the developments of new catalysts, new medicines and new techniques for targeting and labeling biomolecules. However, very scarce efforts have been made to exploit their potential for separation science, especially for HPLC [10]. In view of their distinctive structural characteristics, depending on the multiple interactions offered by alkyl, aromatic and amino groups, a combination of hydrophobic, ␲–␲, hydrogen bonding and dipole-induced dipole interactions could be utilized for purpose of diversifying the retention mechanisms and thus efficiently facilitating the separation of different kinds of solutes. As previously reported in our works [11,12], both a 26-membered and a 14-membered azamacrocycle-based HPLC SPs exhibited satisfactory chromatographic performances in separation of aromatic compounds, pesticides and phenols. Additionally, due to the tunability of the structure of the azamacrocycle, cavity size and rigidity vary with different azamacrocycle, it will be interesting to investigate this characteristic in separation of specific solutes concerning certain special mechanisms, such as shape and planarity selectivity, which are widely used to describe chromatographic behavior of SP can enhance separation of isomers having similar chemical properties [13].

L. He et al. / J. Chromatogr. A 1270 (2012) 186–193

In this paper, we reported the preparation of another two azamacrocyle-bonded SPs modified by two different kinds of azamacrocycles, namely 5,12-dimethyl-7,14-diphenyl-1, 4,8,11-tetraazacyclotetradecane (Me2 Ph2 [14]aneN4 ) and 1,4,11, 14,21,24-hexaaza-(2,3:12,13:22,23)-tributano-(6,9:16,19:26,29) -trietheno-(1H,2H,3H,4H,5H,10H,11H,12H,13H,14H,15H,20H, 21H,22H,23H,24H,25H,30H)-octadecahydro-(30)-annulene (cycHe3 Ph3 [30]aneN6 ), as well as the chromatographic evaluation by separating alkylbenzenes, PAHs and aromatic amines in an aim of further understanding the influences of incorporated multiple functional groups on the elution process and exploring the shape and planarity recognition capability of the two azamacrocycles-bonded SPs. Finally the chromatographic characteristics of both SPs were expressed and verified in a quantitative manner by the linear solvation energy relationship (LSER) model. 2. Experimental 2.1. General Doubly-distilled water, HPLC grade methanol and acetonitrile were used. Spherical silica (particle diameter: 5 ␮m, pore ˚ surface area: 440 or 220 m2 g−1 ) was purchased from size: 90 A, Lanzhou Institute of Chemical Physics, Chinese Academy of Science (Lanzhou, China). ␥-chloropropyltrimethoxysilane (CPTMS) was purchased from Sinopharm Group Chemical Reagent Co., Ltd. (Shanghai, China). Trans-1,2-cyclohexanediamine and terephthalaldehyde were purchased from Sigma–Aldrich (Steinheim, Germany). Benzylideneacetone of analytical grade were purchased from Aladdin Reagent Co., Ltd. (Shanghai, China). PAHs (naphthalene, acenaphthene, fluorine, biphenyl, phenanthrene, anthracene, pyrene, o-terphenyl, triphenylene, chrysene, perylene), aromatic amines (aniline, 2-methylaniline, 4-nitroaniline, N-methylaniline, 3,3 -dimethylbenzidine, 1-naphthylamine, 2,4,6-trimethylaniline, N,N-dimethylaniline, diphenylamine, 2,6-diisopropylaniline, Nphenyl-1-naphthylamine) and other analytes of analytical grade or better were obtained from different origins. All analytes were prepared in pure methanol. All the solvents for synthesis were of analytical grade, and dried by usual means prior to use. The 1 H NMR and 13 C NMR spectra were recorded using a Bruker DPX-400 (Bruker, Ettlingen, Germany). The Infrared spectra were recorded on a Prestige-21 spectrometer (Shimadzu, Kyoto, Japan) at 4000–400 cm−1 . Elemental analyses were performed on a Flash EA 1112 elemental analyzer (Thermo, Waltham, USA). ESI-MS of analyte was determined using 6310 ion trap LC/MS system (Agilent, SantaClara, USA). The prepared SP materials were in turn dispersed in tetrachloromethane and packed into stainless steel tube column (250 mm × 4.6 mm I.D.) using methanol as propulsive solvent by slurry packing technique. HPLC tests were carried out on a Shimadzu system (Shimadzu, Kyoto, Japan) equipped with a LC-10AT vp plus pump, a SPD-10A vp plus UV–vis detector and a CBM10A vp plus chromatographic station at room temperature. The flow rate was 1.0 mL min−1 and UV detection wavelengths was set at 254 nm. A Shimadzu VP-ODS column (150 mm × 4.6 mm, 5 ␮m, end-capped) was used. Mobile phases (MPs) were degassed ultrasonically prior to use. The column was stabilized for 30 min at every change of MP content. Dead time was marked by the signal of methanol. 2.2. Synthesis 2.2.1. Synthesis of Me2 Ph2 [14]aneN4 Synthesis of Me2 Ph2 [14]aneN4 was carried out according to the reported procedure [14]: equimolar benzylideneacetone (14.6 g, 0.1 mol) and ethylenediamine (6.0 g, 0.1 mol) were

187

heated in refluxed ether-cyclohexane (150 mL, v/v = 1/2) in presence of anhydrous potassium carbonate (13 g) for 3 h, and then the solution was filtered and the filtrate was evaporated under vacuum. The obtained yellow residue was allowed to stay overnight to solidify, and then suspended in ether; the obtained white precipitate by filtration was washed with ether (25 mL 3×). Recrystallization from petroleum ether afforded pure 5,12-dimethyl-7,14-diphenyl-1,4,8,11-tetraazacyclotetradeca4,11-diene (Me2 Ph2 [14]eneN4 ) (12 g, 65%). IR max. 3300 and 1656 cm−1 (KBr disc); 1 H NMR (400 MHz, CDCl3 ) ıH 7.23–7.44 (10H, m, CH aromatic), 4.07–4.10 (2H, m, CH), 4.10–4.20 (2H, broad, NH), 3.20–3.40 (4H, m, CH2 ), 2.46–2.76 (8H, m, CH2 ), 1.79 (6H, s, CH3 ). Me2 Ph2 [14]eneN4 (3.7 g) was dissolved in methanol (100 mL), to which potassium borohydride (4 g) was added in small portions over 1 h. The solution was stirred continuously for another 4 h at room temperature, then water (30 mL) was added and methanol was distilled under vacuum. The residue was extracted by chloroform (100 mL); the extract was dried by magnesium sulfate and evaporated under vacuum. The residual white solid was collected and recrystallized from cyclohexane, pure Me2 Ph2 [14]aneN4 (3 g, 80%) was obtained. IR max. 3270 cm−1 ; 1 H NMR (400 MHz, CDCl3 ) ıH 7.21–7.34 (10H, m, CH aromatic), 3.70–3.73 (2H, m, CH), 2.88–2.99 (8H, m, CH2 ), 2.49–2.53 (2H, m, CH), 2.31–2.53 (4H, broad, NH), 1.64–1.79 (4H, m, CH2 ), 1.02 (6H, d, CH3 ); 13 C NMR (400 MHz, CDCl ) ı 145.4, 128.6, 126.7, 126.4, 77.4, 3 C 77.1, 76.7, 66.1, 56.1, 48.4, 47.8, 46.8, 21.5 (see Supplementary Figs. 1 and 2). Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.chroma.2012.11.007.

2.2.2. Synthesis of cycHe3 Ph3 [30]aneN6 Synthesis of cycHe3 Ph3 [30]aneN6 was carried out according the reported procedure [15]: a solution of terephthalaldehyde (1.072 g, 8 mmol) in acetonitrile (150 mL) was added dropwise to a solution of trans-1,2-cyclohexanediamine (0.912 g, 8 mmol) in acetonitrile (250 mL) over 2 h. After stirring overnight at room temperature, a yellowish precipitate of 1,4,11,14,21,24hexaaza-(2,3:12,13:22,23)-tributano-(6,9:16,19:26,29)trietheno-(2H,3H,12H,13H,22H,23H)-hexahydro-(30)-annulene (cycHe3 Ph3 [30]eneN6 ) (1.62 g, 95%) was isolated and washed by acetonitrile and ether. IR max. 1642 cm−1 (KBr disc); 1 H NMR (400 MHz, CDCl3 ) ıH 8.10–8.17 (6H, m, CH N), 7.45–7.52 (12H, q, CH aromatic), 3.28–3.48 (6H, m, CH N), 1.47–1.87 (24H, overlapping m, CH2 ); 13 C NMR (400 MHz, CDCl3 ) ıC 160.3, 137.7, 127.9, 74.4, 32.7, 24.4; MS: 637.6 ([M+H]+ ). cycHe3 Ph3 [30]eneN6 (1.5 g) was dissolved in tetrahydrofuranmethanol (150 mL, v/v = 1/1), then sodium borohydride (2 g) was added in small portions over 1 h. The reaction continued for another 3 h at room temperature. The solution was evaporated under vacuum to dryness; the residue was extracted by dichloromethane (150 mL). The organic layer was separated after washing by water (20 mL 3×) and dried by anhydrous magnesium sulfate and evaporated under vacuum. The obtained green oil was dissolved in minimal ethanol, to which concentrated hydrobromic acid (wt.% = 47%) was added, a white precipitate was filtered and washed by methanol. This salt was dissolved in an aquatic solution of KOH, repetition of the above extraction and desiccation procedure afforded pure cycHe3 Ph3 [30]aneN6 (1.0 g, 67%) as an oil. IR ␯max. 3200 cm−1 (KBr disc); 1 H NMR (400 MHz, D2 O) ıH 7.38–7.40 (12H, q, CH aromatic), 4.03–4.46 (12H, m, CH2 Ar), 3.49 (6H, m, CH NH), 2.35(6 H, overlapping m, CH2 ), 1.33–1.77 (18 H, overlapping m, CH2 ) (see Supplementary Fig. 3). Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.chroma.2012.11.007.

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2.2.3. Preparation of Me2 Ph2 [14]aneN4 -bonded silica and cycHe3 Ph3 [30]aneN6 -bonded silica Silylation of silica was performed by adding CPTMS (5 mL) in toluene (20 mL) dropwise in 1 h to a suspension of activated silica (5 g) in toluene (30 mL) with stirring under reflux in nitrogen atmosphere. The mixture was stirred for 48 h, then filtered, washed by toluene, methanol, dichloromethane and ether (50 mL for each) and dried, CPTMS-modified silica (CPS) was obtained. CPS (4.0 g) was dispersed in dimethylformamide (100 mL), followed by addition of Me2 Ph2 [14]aneN4 (2 g) and a few drops of triethylamine. The mixture was heated at 90 ◦ C with magnetic stirring for 24 h in nitrogen atmosphere. The product was filtered off and rinsed successively by ethanol, chloroform, tetrahydrofuran and ether (50 mL for each) twice. The Me2 Ph2 [14]aneN4 -bonded silica (Me2 Ph2 [14]aneN4 CPS) was dried at 90 ◦ C under vacuum overnight. Preparation of cycHe3 Ph3 [30]aneN6 -bonded silica (cycHe3 Ph3 [30]aneN6 CPS) was conducted likewise, but replacing dimethylformamide with refluxing toluene. Illustration of the entire synthesis processes was given in Fig. 1.

NH2 2

+ 2

Reflux, K2 CO3 Cyclohexane-ether

NH

3. Results and discussion 3.1. Characterization of Me2 Ph2 [14]aneN4 CPS and cycHe3 Ph3 [30]aneN6 CPS Results of IR analysis revealed a small difference at 1450–1650 cm−1 (stretching frequency of benzene), CH2 ) in rela2850–2925 cm−1 (stretching frequency of tion to numbers and intensities of the bands for CPS and Me2 Ph2 [14]aneN4 CPS, despite the similarity of bands associated with the inorganic backbone. IR spectrum for cycHe3 Ph3 [30]aneN6 CPS showed much similarity with that for Me2 Ph2 [14]aneN4 CPS, except 804 cm−1 (p-disubstituted benzene) (see Supplementary Fig. 4). Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.chroma.2012.11.007. The elemental analysis of Me2 Ph2 [14]aneN4 CPS and cycHe3 Ph3 [30]aneN6 CPS gave quantitative information on the amounts of bonded azamacrocycles. The carbon contents of CPS, Me2 Ph2 [14]aneN4 CPS and cycHe3 Ph3 [30]aneN6 CPS were 3.941%, 6.594% and 9.233%, respectively. The bonding amount was ca.

N

N

NH HN

KBH4

NH HN

HN

H2N

O

(Me2 Ph2[14]eneN4)

N

(Me2Ph2[14]aneN4)

N

N H

CHO NH2 3

NaBH4

Acetonitrile

+ 3

NH2

THF-MeOH N

CHO

N H

N N

NH

HN H N

N

(cycHe3Ph3 [30]eneN6)

H N (cycHe3Ph3[30]aneN6 ) N H

OH SiO2

OH OH

CPTMS NEt3, Toluene, 110 ºC

SiO2

O O O

N6 ne 0]a ux [3 efl h3 ,r P e e3 en lu cH cy , to t 3 NE

Si (CPS)

Cl

SiO2

O O O

N H

Si N

HN H N

H N

(cycHe3Ph3[30]aneN6 CPS)

M

e NE 2 Ph [ t3 , 2 14] DM en F , eN 90 4 ºC

SiO2

O O O

NH HN Si

N

HN

(Me2 Ph2[14]aneN4CPS) Fig. 1. Illustration for preparation of azamacrocycles and corresponding stationary phases.

L. He et al. / J. Chromatogr. A 1270 (2012) 186–193 1.00

a

3

toluene ethylbenzene n-propylbenzene n-butylbenzene

50

Response(mv)

0.50

logk

a

75

0.75

0.25

0.00

189

6

1 4(5) 2 25

-0.25

-0.50

0 50

60

70

80

90

7.0

acetonitrile content (%) 1.00

10.5

b

17.5

b

toluene ethylbenzene n-propylbenzene n-butylbenzene

0.75

45

1

Response (mv)

0.50

logk

14.0

Time(min)

0.25

0.00

3

30

4

2

5

15

6

-0.25

-0.50

*

0 50

60

70

80

90

acetonitrile content (%) Fig. 2. Effect of different acetonitrile contents on log k of n-alkylbenzenes on Me2 Ph2 [14]aneN4 CPS (a) and cycHe3 Ph3 [30]aneN6 CPS (b).

0.092 mmol g−1 and 0.105 mmol g−1 for Me2 Ph2 [14]aneN4 CPS and cycHe3 Ph3 [30]aneN6 CPS, respectively, according to carbon content. 3.2. Chromatographic evaluation of Me2 Ph2 [14]aneN4 CPS and cycHe3 Ph3 [30]aneN6 CPS 3.2.1. Separation of alkylbenzenes It is universally acknowledged that chromatographic selectivity of a given SP toward solutes is important. Herein, to investigate the similitude and dissimilitude between Me2 Ph2 [14]aneN4 CPS and cycHe3 Ph3 [30]aneN6 CPS in hydrophobic selectivity, 4 alkylbenzenes were separated on two azamacrocycle-based SPs with varying mobile phases. As can be seen in Fig. 2, Me2 Ph2 [14]aneN4 CPS and cycHe3 Ph3 [30]aneN6 CPS both exhibited obvious hydrophobic characteristics and similar trend was observed in the change of log k values with the change of mobile composition, i.e. for a given mobile composition, log k values increase with the increase of length of solute’s aliphatic chain; for all the mobile compositions, log k values increase with the decrease of acetonitrile content. Based on the calculated selectivity factor (˛), cycHe3 Ph3 [30]aneN6 CPS was more potent in

7.0

10.5

14.0

17.5

time (min) Fig. 3. Separation of benzene (1), toluene (2), ethylbenzene (3), iso-propylbenzene (4), n-propylbenzene (5) and n-butylbenzene (6) on Me2 Ph2 [14]aneN4 CPS (a) and cycHe3 Ph3 [30]aneN6 CPS (b). Mobile phase: acetonitrile–water (50/50, v/v). *: impurity.

distinguishing methylene group than Me2 Ph2 [14]aneN4 CPS, except in 90% acetonitrile MP. For example, with 50% acetonitrile employed, greater ˛EB/T (ethylbenzene to toluene), ˛PB/EB (n-propylbenzene to ethylbenzene) and ˛BB/PB (n-butylbenzene to n-propylbenzene) were obtained, which were 1.319, 1.345 and 1.337 for cycHe3 Ph3 [30]aneN6 CPS respectively, while 1.257, 1.213 and 1.188 for Me2 Ph2 [14]aneN4 CPS respectively. Furthermore, iso-propylbenzene was added to assess the azamacrocycle-bonded phases’ capabilities for recognizing isomers. As displayed in Fig. 3, n- and iso-propylbenzene could be hardly discerned on Me2 Ph2 [14]aneN4 CPS, whereas on cycHe3 Ph3 [30]aneN6 CPS, this isomer pair could be differentiated and partially separated. In sum, two azamacrocycle-bonded phases showed evident hydrophobic selectivity toward alkylbenzenes, meanwhile cycHe3 Ph3 [30]aneN6 CPS could provide superior separation selectivity toward alkylbenzenes, especially toward positional isomer, over Me2 Ph2 [14]aneN4 CPS, presumably due to the more hydrophobic functional groups, larger cavity and greater structural rigidity of cycHe3 Ph3 [30]aneN6 .

190

L. He et al. / J. Chromatogr. A 1270 (2012) 186–193

Shape recognition

a

60

5

Response (mv)

48

Anthracene

Phenanthrene

2(3)

36

1

24

6

4

7 9(10)

12

Triphenylene

8

Chrysene

11

0

Planarity recognition

0

8

16

24

32

40

time (min)

b

50

5

Fluorene

Biphenyl

40

Triphenylene

Naphthalene

Acenaphthene

Response (mv)

o-terphenyl

30

1

2 4

20

3

6 7 9

10

8

10

11

0 0

Pyrene

Perylene

Fig. 4. Structure of PAHs used in this study.

3.2.2. Separation of PAHs According to the chemical structures of Me2 Ph2 [14]aneN4 and cycHe3 Ph3 [30]aneN6 , it could be easily noticed that the former possesses a flexible 14-membered aliphatic ring having phenyl substituents, the latter a rigid 30-membered aromatic-aliphatic-mixed ring. It was anticipated that the cavity size and rigidity of attached ligand would exert impact upon its shape and planarity recognition capability. In order to verify this special mechanism for specific analytes, a set of 11 PAHs (Fig. 4) was used to preliminarily evaluate the shape and planarity selectivity of both SPs. It is well recognized that examining the chromatographic selectivity toward phenanthrene and anthracene, as well as toward triphenylene and chrysene is a reliable route to measure SP’s shape selectivity capability, meanwhile planarity selectivity capability is determined by comparing the retentions of o-terphenyl with that of triphenylene [16,17]. Separation of these designated PAHs was specified as follows. From the chromatograms (Fig. 5) for the separation of all PAHs using both SPs, it could be evidently observed that shape recognition capability of cycHe3 Ph3 [30]aneN6 CPS was greater than that of Me2 Ph2 [14]aneN4 CPS by demonstrating a greater ˛ for phenanthrene and anthracene, as well as for triphenylene and chrysene (co-eluted on Me2 Ph2 [14]aneN4 CPS). The above two pairs of coplanar solutes possess same lipophilicity and numbers of ␲ electrons, still they could be discriminated, though not completely, the reason resided in the difference between their

15

30

45

60

75

time (min) Fig. 5. Separation of PAHs mixture composed of naphthalene (1), biphenyl (2), fluorene (3), acenaphthene (4), phenanthrene (5), anthracene (6), oterphenyl (7), pyrene (8), triphenylene (9), chrysene (10) and perylene (11) on Me2 Ph2 [14]aneN4 CPS (a) and cycHe3 Ph3 [30]aneN6 CPS (b). Mobile phase: acetonitrile–water (50/50, v/v).

molecular shape parameter, i.e. length/breadth ratio (L/B ratio), which actively propelled the separation rather than hydrophobic and ␲–␲ interaction at this point, v.z. the more “rod-like” the solute is, the longer its retention time is. For instance, anthracene was more strongly retained than phenanthrene due to the former’s larger L/B ratio (1.57) than latter’s (1.46), so did chrysene’s (L/B = 1.72) than triphenylene’s (L/B = 1.11); furthermore, inversion of the elution order of pyrene (L/B = 1.27, log k(o/w) = 4.93) and o-terphenyl (L/B = 1.11, log k(o/w) = 5.52) occurred on new phases, which could be ascribed to the dominant role played by L/B ratio on cycHe3 Ph3 [30]aneN6 CPS, whereas the decisive factor is hydrophobicity on Me2 Ph2 [14]aneN4 CPS. Likewise, a greater planarity recognition capability of cycHe3 Ph3 [30]aneN6 CPS could be confirmed by much higher ˛ (1.536) for planar triphenylene and non-planar o-terphenyl with similar molecular mass and same lipophilicity than that on Me2 Ph2 [14]aneN4 CPS (1.075). It was believed that planar triphenylene was able to penetrate easier into the SP than the non-planar o-terphenyl, therefore interacted more energetically with the SP [13]. On the separation of above-mentioned PAHs, besides the shape and planarity influences, effects of hydrophobic and ␲–␲ interaction could never be neglected. Here, the term “product selectivity factor (˛p )” [18] was used to measure the overall selectivity of two

L. He et al. / J. Chromatogr. A 1270 (2012) 186–193 Table 1 Selectivity (˛) and product selectivity (˛p ) factors for PAHs calculated for each SP. cycHe3 Ph3 [30]aneN6 CPS

1.37 1.23 1.10 1.39 1.18 3.03

1.69 1.34 1.43 1.48 1.55 7.47

4

azamacrocycle-based SPs in a quantitative manner. The term was defined as below:

50

9

8

11

5(6)

1

75

NAP: naphthalene, ACE: acenaphthene, ANT: anthracene, PYR: pyrene, CHR: chrysene, PER: perylene.

2(3) 7

10

25

0

(1)

3.2.3. Separation of aromatic amines It was well-known that separation of polar analytes, e.g. amines, on conventional ODS was less pleasant than that of non-polar ones, due to the hydrogen-bonding interaction between residual silanols on the silica surface with the polar amino groups. In consideration of the amino group-rich structure of azamacrocycles, they may have the potential for inhibiting the hydrogen-bonding interaction by competing for silanols with polar solutes. A set of 11 aromatic amines was used to examine whether and to what extent Me2 Ph2 [14]aneN4 CPS and cycHe3 Ph3 [30]aneN6 CPS were capable of improving the separation of amines by alleviating negative hydrogen-bonding interactions and offering multiple interactions. Fig. 6a–c were typical chromatograms for separations of these amines on Me2 Ph2 [14]aneN4 CPS, cycHe3 Ph3 [30]aneN6 CPS and end-capped ODS columns, respectively. As shown in Fig. 6 there was a clear difference between the chromatographic behaviors of azamacrocycle-based SPs and ODS, and several solutes, which can be separated on cycHe3 Ph3 [30]aneN6 CPS, but was co-eluted on Me2 Ph2 [14]aneN4 CPS. In general, the performance of cycHe3 Ph3 [30]aneN6 CPS was superior to either Me2 Ph2 [14]aneN4 CPS or ODS. The reason for the difference between the chromatographic behaviors could be found in the diverse structures of the aza-ligands. For 2methylaniline and 4-nitroaniline, retention of 4-nitroaniline on cycHe3 Ph3 [30]aneN6 CPS was enhanced by stronger

0

6

12

18

24

time (min)

b

100

Response (mv)

where ˛1 was the selectivity factor for the first peak pair, ˛2 for the second peak pair, so on, a larger ˛p value denoted a higher selectivity. Table 1 reported the individual ˛ and ˛p obtained on both SPs for separable PAHs. It could be evidently observed that cycHe3 Ph3 [30]aneN6 CPS demonstrated higher individual ˛ value and also a much higher ˛p value than Me2 Ph2 [14]aneN4 CPS. Hereby cycHe3 Ph3 [30]aneN6 CPS was more excellent than Me2 Ph2 [14]aneN4 CPS in separation of PAHs. In addition, it should be noted that two azamacrocycle-bonded phases possessed high column efficiency and good stability under reversephase condition. Using naphthalene, acenaphthene and anthracene as representative probes, the plate number is above 34,000 per meter and above 25,000 per meter for cycHe3 Ph3 [30]aneN6 CPS and Me2 Ph2 [14]aneN4 CPS, respectively, and the plate number decreased about 6.6% after elution for 1800 h. Actually, both of two azamacrocycle-bonded phases had potential for separation of PAHs. In conclusion, apart from hydrophobicity, the rigidity and cavity size of the aza-ligands were undeniably in connection with the selectivity toward PAHs. The L/B ratio and/or planarity of PAHs molecule exerted a tremendous influence upon the separation of solutes possessing the same hydrophobicity. CycHe3 Ph3 [30]aneN6 CPS expressed superiority in recognizing the selected PAHs over Me2 Ph2 [14]aneN4 CPS.

4

8

1

75

2 3

50

11 57 6

9 10

25

0 0

7

14

21

28

time (min)

c

100

4 1 8

2

75

Response (mv)

˛p = ˛1 ˛2 ˛3 × · · · × ˛n

a

100

Response (mv)

˛ACE/NAP ˛ANT/ACE ˛PYR/ANT ˛CHR/PYR ˛PER/CHR ˛p

Me2 Ph2 [14]aneN4 CPS

191

36

7

50

11 5

9

25

10

0 0

10

20

30

40

time (min) Fig. 6. Separation of aromatic amines composed of aniline (1), 2-methylaniline (2), 4-nitroaniline (3), N-methylaniline (4), 2,4,6-trimethylaniline (5), 3,3 dimethylbenzidine (6), 1-naphthylamine (7), N,N-dimethylaniline (8), 2,6diisopropylaniline (9), diphenylamine (10) and N-phenyl-1-naphthylamine (11) on Me2 Ph2 [14]aneN4 CPS (a), cycHe3 Ph3 [30]aneN6 CPS (b) and ODS (c). Mobile phase: acetonitrile–water (50/50, v/v).

L. He et al. / J. Chromatogr. A 1270 (2012) 186–193 1.0

0.6

0.0 0.0

˛H 2 +ˇ



0.2

0.4

0.6

0.8

1.0

logk(exp) 1.0

b

0.8

0.6

0.4

0.2

0.0

-0.2 0.0

0.2

0.4

0.6

0.8

1.0

logk(exp)

To gain a deeper insight into chemical origins of the (dis)similarity between the chromatographic behaviors of two azamacrocycle-based SPs, we had resorted to the LSER model, which is regarded as an effective method to characterize reversephase HPLC SPs [19–23] and expressed as below:



0.4

0.2

3.3. LSER analysis of Me2 Ph2 [14]aneN4 CPS and cycHe3 Ph3 [30]aneN6 CPS

log k = c + rR2 + s2H + ˛

a

0.8

logk(calc)

hydrogen-bonding and dipole-induced dipole interactions between nitro group and hexaaza-ligand than that between methyl group and aza-ligand; co-elution of the two on Me2 Ph2 [14]aneN4 CPS revealed weaker hydrogen-bonding and dipole-induced dipole interactions between nitro group and tetraaza-ligand, therefore the retention of 4-nitroaniline was albeit stronger but not enough to surpass that of 2-methylaniline. Toward 2-methylaniline and 3,3 -dimethylbenzidine, ODS exhibited the lowest ˛ and cycHe3 Ph3 [30]aneN6 CPS had the highest ˛, as a result of stronger ␲–␲ interaction between biphenyl moiety of 3,3 -dimethylbenzidine and aza-ligand. Remarkably, for 3,3 -dimethylbenzidine and 1-naphthylamine containing more ␲ electrons, their retention on cycHe3 Ph3 [30]aneN6 CPS was enhanced so greatly that they were eluted after more hydrophobic N-methylaniline and 2,4,6-trimethylaniline. The observed change in elution order on cycHe3 Ph3 [30]aneN6 CPS could be attributed to the impacts caused not only by hydrophobicity, but also significantly by ␲–␲ and dipole-induced dipole interactions. Toward 2,6-diisopropylaniline and diphenylamine, cycHe3 Ph3 [30]aneN6 CPS demonstrated a greater ˛ (1.307) than Me2 Ph2 [14]aneN4 CPS (1.106). Based on Fig. 6 and above chromatographic results, conclusions could be drawn that (1) though not end-capped like the ODS, the azamacrocycle-based SPs, especially cycHe3 Ph3 [30]aneN6 CPS, were meritorious in separation of basic analytes, as supported by the chromatographic results: good selectivity, high symmetry of peaks, no occurrence of peak distortion; (2) cycHe3 Ph3 [30]aneN6 CPS outperformed Me2 Ph2 [14]aneN4 CPS in aspect of selectivity, even exceeded the ODS from the viewpoint of overall performance; (3) selectivity toward polar solutes was influenced by more than hydrophobic interaction, other interactions such as hydrogen-bonding, dipole-induced dipole and ␲–␲ interactions could synergically contribute to the improvement of selectivity.

logk(calc)

192

ˇ2H + vVx

(2)

Meanings of each term were described elsewhere [24]. In total 30 probe solutes (see Supplementary Table 1) were used in the LSER study. Evaluation of the LSER model was performed by comparing the experimentally-determined log k values (log k(exp) ) with the calculated values (log k(calc) ). As displayed in Fig. 7, using the results in 50% acetonitrile as representative example, there was

Fig. 7. Plot depicting log k(calc) versus log k(exp) in 50% Me2 Ph2 [14]aneN4 CPS (a) and cycHe3 Ph3 [30]aneN6 CPS (b).

acetonitrile

for

good consistence between log k(exp) and log k(calc) on each column (correlation coefficient > 0.97), none of the solutes appeared to be significant outliers, confirming the competence of selected probe solutes in specifying the interactions between solutes and the SPs under reverse-phase condition. Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.chroma.2012.11.007. The coefficients of LSER equation derived from the descriptors of these solutes and corresponding retention data were listed in Table 2, high correlation coefficient (R > 0.96) was obtained for each

Table 2 System coefficients obtained on three stationary phases in varying mobile phase compostion. Stationary phase

Acetonitrile/water

c

r

s

a

b

v

R

F

SE

n

Me2 Ph2 [14]aneN4 CPS

70:30 60:40 50:50

−0.203 −0.157 −0.078

0.125 0.163 0.252

−0.093 −0.105 −0.143

−0.270 −0.272 −0.319

−0.385 −0.427 −0.759

0.365 0.594 0.721

96.36% 97.28% 97.34%

126 172 175

0.026 0.025 0.038

32 32 32

CycHe3 Ph3 [30]aneN6 CPS

70:30 60:40 50:50

−0.510 −0.435 −0.316

0.158 0.165 0.169

−0.115 −0.129 −0.162

−0.092 −0.115 −0.107

−0.690 −0.814 −1.130

0.547 0.699 1.002

97.44% 97.81% 97.96%

182 214 303

0.026 0.030 0.034

32 32 32

Shimadzu VP-ODS

70:30 60:40 50:50

−0.389 −0.291 −0.226

0.057 0.079 0.021

−0.276 −0.298 −0.265

−0.409 −0.432 −0.445

−1.211 −1.404 −1.818

1.145 1.342 1.615

97.97% 97.96% 99.22%

231 230 613

0.047 0.053 0.038

32 32 32

R: overall correlation coefficient, F: statistic, SE: standard error in the estimate, n: number of solutes.

L. He et al. / J. Chromatogr. A 1270 (2012) 186–193

SP. It was noticeable that the magnitude of coefficients r, s, a, b and v varied greatly with the type of the SPs. Generally, the positive r and v for Me2 Ph2 [14]aneN4 CPS and cycHe3 Ph3 [30]aneN6 CPS exhibited their reverse-phase characteristics, showing more favorable ␲–␲ and hydrophobic interactions between SP and solutes than that between MP and solutes. The r coefficient reflected the capability of the chromatographic system to interact with the solute through ␲- and ␴-electron pairs. A slightly larger r value was observed for cycHe3 Ph3 [30]aneN6 CPS in 70% and 60% acetonitrile. It was surprising to observe that cycHe3 Ph3 [30]aneN6 CPS possessed a smaller r value than Me2 Ph2 [14]aneN4 CPS in 50% acetonitrile, since cycHe3 Ph3 [30]aneN6 has more aromatic rings and amino groups than Me2 Ph2 [14]aneN4 . This unusual case could be ascribed to the fact that certain contributions of some special mechanisms such as shape recognition were not covered in LSER model, accordingly system error would inevitably occur [24], for example: n- and iso-propylbenzene have nearly identical solute descriptors, their log k(calc) (0.673 and 0.663) were almost the same, but difference between their corresponding experimental log k(exp) (0.679 and 0.647) was significant. Both the azamacrocycle-based SPs owned a larger r value than ODS, which could be explained by the electronegativity of the amino groups and aromatic ring’s ␲-electrons of Me2 Ph2 [14]aneN4 CPS and cycHe3 Ph3 [30]aneN6 CPS. As the concentration of acetonitrile dropped, the magnitude of r increased, since acetonitrile was more capable of suppressing ␲–␲ interaction between solute and SP than water [25]. The coefficient v was in close relation to the “hydrophobic selectivity” of SP, and originated in a combination of cavity effects and dispersive interactions. The positive v coefficients for two azamacrocyclebased SPs highlighting their hydrophobic properties, were smaller than that for ODS. The larger v values for cycHe3 Ph3 [30]aneN6 CPS than that for Me2 Ph2 [14]aneN4 CPS indicated the former’s higher hydrophobicity, which was likely to confirm the observation that cycHe3 Ph3 [30]aneN6 CPS possessed greater hydrophobic selectivity capability than Me2 Ph2 [14]aneN4 CPS in separation of alkylbenzenes and PAHs. The s, a and b coefficients maintained negative at all acetonitrile concentrations, demonstrating the higher affinity of MP for solutes in association with the dipolarity/polarizability and hydrogen-bonding interactions. For both azamacrocycle-based SPs, the little differences between the s values could be resulted from their similar chemical structure. The smaller s values for Me2 Ph2 [14]aneN4 CPS and cycHe3 Ph3 [30]aneN6 CPS than that for ODS suggested that their dipolarity/polarizability must be higher than ODS’s. The smaller a and b coefficients for azamacrocyclebased SPs than that for ODS were due to the fact the polar amino group was much more inclined to not only accept, but also donate protons than alkyl chain. Due to the electronegativity of amino groups, the hydrogen-bonding basicity of azamacrocycle-based SPs was stronger than corresponding acidity, as reflected by smaller a values and larger b values. A smaller negative a value obtained on cycHe3 Ph3 [30]aneN6 CPS indicated its greater capability for accepting hydrogen-bonding than Me2 Ph2 [14]aneN4 CPS. As noticed in the separation of aromatic amines, cycHe3 Ph3 [30]aneN6 CPS exhibited the extraordinary selectivity toward this type of polar analytes, which could be in relationship with the strong hydrogen-bonding interaction. It was obviously demonstrated by the interpretations of the LSER parameters that hydrophobic interaction on cycHe3 Ph3 [30]aneN6 CPS and Me2 Ph2 [14]aneN4 CPS phases greatly impacted the chromatographic retention of solutes, involvements of ␲–␲ and hydrogen bonding interactions simultaneously

193

played a significant role in the retention process. Furthermore, cycHe3 Ph3 [30]aneN6 CPS exceeded Me2 Ph2 [14]aneN4 CPS with regard to hydrophobic, hydrogen-bonding and ␲–␲ interactions. 4. Conclusions new HPLC SPs, Me2 Ph2 [14]aneN4 CPS and Two cycHe3 Ph3 [30]aneN6 CPS were prepared via covalently attaching azamacrocycles to silica and characterized by IR spectroscopy and elemental analysis. The chromatographic performance of two new phases was evaluated and compared by separating alkylbenzenes, PAHs and aromatic amines, both Me2 Ph2 [14]aneN4 CPS and cycHe3 Ph3 [30]aneN6 CPS showed multiple-interaction mechanism, including hydrophobic, ␲–␲, hydrogen-bonding and dipole-induced dipole interactions, all of which were testified by conducting LSER analysis. The rigidity and cavity size of the ring structure of azamacrocycle were in close connection with the chromatographic behaviors of the new phases. It was noteworthy that cycHe3 Ph3 [30]aneN6 CPS outperformed Me2 Ph2 [14]aneN4 CPS by demonstrating superior hydrophobic selectivity and relatively higher shape and planarity selectivity toward PAHs, and even exceeded conventional ODS phase in separation of aromatic amines through multiple interactions. Further research into the shape and planarity selectivity of this type of azamacrocycle-based SP is currently underway. Acknowledgements The authors gratefully acknowledge financial supports from the National Natural Science Foundation of China (No. 20905020) and Plan for Scientific Innovation Talent of Henan Province. References [1] Z.M. Zhou, X. Li, X.P. Chen, M. Fang, X. Dong, Talanta 82 (2010) 775. [2] H. Nelander, S. Andersson, K. Öhlén, J. Chromatogr. A 1218 (2011) 9397. [3] M. Sliwka-Kaszynska, G. Gorczyca, M. Slebioda, J. Chromatogr. A 1217 (2010) 329. [4] I. Ilisz, Z. Pataj, R. Berkecz, A. Misicka, D. Tymecka, F. Fülöp, H.J. Choi, M.H. Hyun, A. Péter, J. Chromatogr. A 1217 (2010) 1075. [5] H.D. Qiu, K.M. Abul, M. Takafuji, X. Liu, S.X. Jiang, H. Ihara, J. Chromatogr. A 1232 (2012) 116. [6] S. Lacerda, M.P. Campello, F. Marques, L. Gano, V. Kubíˇcek, P. Fousková, E. Tóth, I. Santos, J. Chem. Soc. Dalton Trans. (2009) 4509. [7] J.H. Wen, C.Y. Li, Z.R. Geng, X.Y. Ma, Z.L. Wang, Chem. Commun. 47 (2011) 11330. [8] V.K. Bansal, P.P. Thankachan, R. Prasad, Appl. Catal. A 381 (2010) 8. [9] C. Bianchini, G. Giambastiani, F. Laschi, P. Mariani, A. Vacca, F. Vizza, P. Zanello, Org. Biomol. Chem. 1 (2003) 879. [10] T. Shinbo, Y. Shimabukuro, T. Kanamori, T. Iwatsubo, Y. Nagawa, K. Hiratani, J. Chromatogr. A 877 (2000) 61. [11] L.J. He, J. Zhang, Y.J. Sun, J. Liu, X.M. Jiang, L.B. Qu, J. Chromatogr. A 1217 (2010) 5971. [12] L.J. He, M.L. Zhang, W.J. Zhao, J. Liu, X.M. Jiang, S.S. Zhang, L.B. Qu, Talanta 89 (2012) 433. [13] L.C. Sander, M. Pursch, S.A. Wise, Anal. Chem. 71 (1999) 4821. [14] K. Hideg, D. Lloyd, J. Chem. Soc. (1971) 3441. ´ J. Hodaˇcová, J. Závada, P.C. Junk, Tetrahedron: Asym[15] M. Chadim, M. Bud˘esˇ ínsky, metry 12 (2001) 127. [16] C. Perruchot, M.M. Chehimi, M. Delamar, F. Dardoize, J. Chromatogr. A 969 (2002) 167. [17] E. Lesellier, J. Chromatogr. A 1218 (2011) 251. [18] S. Kayillo, G.R. Dennis, R.A. Shalliker, J. Chromatogr. A 1126 (2006) 283. [19] P.B. Ogden, J.W. Coym, J. Chromatogr. A 1218 (2011) 2936. [20] C. West, E. Lesellier, J. Chromatogr. A 1115 (2006) 233. [21] T. Liu, I.A. Nicholls, T. Öberg, Anal. Chim. Acta 702 (2011) 37. [22] Y. Sun, B. Cabovska, C.E. Evans, T.H. Ridgway, A.M. Stalcup, Anal. Bioanal. Chem. 382 (2005) 728. [23] P.R. Fields, Y. Sun, A.M. Stalcup, J. Chromatogr. A 1218 (2011) 467. [24] M. Vitha, P.W. Carr, J. Chromatogr. A 1126 (2006) 143. [25] M.G. Kiseleva, P.N. Nesterenko, J. Chromtogr. A 898 (2000) 23.