preparation, characterization and application of a new...

Research Article

Preparation, characterization andapplication of a new 25,27-bis-[2-(5-methylthiadiazole)thioethoxyl]-26,28-dihydroxy-para-tert-butyl calix[4]arenestationary phase for HPLC

A new para-tert-butylcalix[4]arene column containing thiadiazole functional groups was

prepared and used for the separation of polycyclic aromatic hydrocarbons, phenolic

compounds, aromatic amines, benzoic acid and its derivatives by high-performance liquid

chromatography (HPLC). The effect of organic modifier content in the mobile phase on

retention and selectivity of these compounds were investigated. The results indicate that the

stationary phase behaves like reversed-phase packing. However, hydrogen bonding, p–p and

inclusion interactions seem to be involved in the separation process. The column has been

successfully employed for the analysis of clenbuterol in pork and pig casing; the limit of

detection and the limit of quantitation for this method by HPLC-UV detection was 0.03 and

0.097 mg/mL, respectively; the method is demonstrated to be suitable and a competitive

alternative analytical method for the determination of clenbuterol.

Keywords: Aromatic amines / Clenbuterol / para-tert-Butylcalix[4]arene /Stationary phase / ThiadiazoleDOI 10.1002/jssc.201100733

1 Introduction

Calixarenes, following cyclodextrines and crown ethers, are

considered to be a typical representative of the third

generation of host supramolecules. They consist of phenol

units linked via methylene bridges and can also form

inclusion complexes like the other host supramolecules.

The peculiar configurations lead to the formation of typical

host–guest interaction between calixarenes and numerous

compounds, and result in widely varied applications in ion-

selective membranes and electrodes [1–5], electrophoresis

[6–9] and chromatography [10–17] and other fields [18–20].

In the field of chromatography, calixarene-bonded

stationary phases are preferable to the use as mobile-phase

additives, because the UV detection of analytes is prevented

by strong absorbance of calixarenes. Additionally, poor

solubility of most calixarenes precludes their applications as

additives in aqueous eluents. With various methods for

functionalizing calixarenes have been developed, more and

more applications of different calixarene-bonded stationary

phases have been reported. The resulting interactions of

different calixarenes stationary phase influence the reten-

tion factors and improve the selectivity of the solutes. The

modification of the calixarenes, for instance, by varying

the ring size, substitutents and conformations, enables a

more enhanced interaction spectrum and can improve the

specificity for guest molecules. For example, Sliwka-

Kaszynska’s group synthesized twelve 1,3-alternate

calyx[4]arene–silica-bonded stationary phases and char-

acterized them in terms of their surface coverage, hydro-

phobic selectivity, aromatic selectivity, shape selectivity,

hydrogen bonding capacity and ion-exchange capacity [21].

Serkan and Mustafa prepared a new 1,3-alternate-calix[4]-

arene-bonded HPLC stationary phase and investigated the

chromatographic performance by using phenols, aromatic

amines and drugs [22]. In 2007, our group prepared six

calixarene-bonded silica gel stationary phases, including

calixarene of different cavity sizes (calix[4, 6 or 8]arene)

and with or without para-tert-butyl groups, and investigated

their chromatographic performance [23]. Very recently, we

have reported the preparation of a new para-tert-butylca-

lix[4]arene-1,2-crown-4-bonded silica stationary phase

and the evaluation of its chromatographic performance [24].

These previous reports have shown that calixarene-bonded

Kai Hu1

Junwei Liu1

Chao Tang1

Caijuan Wang1

Ajuan Yu2�

Fuyong Wen1

Wenjie Zhao1,3

Baoxian Ye1

Yangjie Wu2

Shusheng Zhang1

1Chemistry Department,Zhengzhou University,Zhengzhou, P. R. China

2Key Laboratory of ChemicalBiology and Organic Chemistryof Henan, Zhengzhou,P. R. China

3School of Chemistry andChemical Engineering, HenanUniversity of Technology,Zhengzhou, P. R. China

Received August 17, 2011Revised October 19, 2011Accepted October 20, 2011

Abbreviations: GBS, c-glycidoxypropyl-bonded silica gel;MeOH, methanol; PAH, polycyclic aromatic hydrocarbon

�Additional correspondence: Dr. Ajuan Yu

E-mail: [email protected]

Correspondence: Dr. Shusheng Zhang, Chemistry Department,Zhengzhou University, Daxue Road 75, Zhengzhou 450052,P. R. ChinaE-mail: [email protected]: 186-371-67766667

& 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com

J. Sep. Sci. 2012, 35, 239–247 239

stationary phases featuring inclusion capability exhibit a

promising prospect in HPLC.

In the present work, we describe the synthesis of a

para-tert-butylcalix[4]arene derivative containing thiadiazole

group and the preparation of its bonded silica

stationary phase (TBS4). The newly developed stationary

phase has been characterized by using elemental analysis,

FT-IR and thermal gravimetric analysis. The

chromatographic performance was investigated by using

polycyclic aromatic hydrocarbons (PAHs), phenols,

aromatic amines, benzoic acid and its derivatives as

probes in comparison with ODS, and the influence of

methanol (MeOH) content in mobile phase on the chro-

matographic behavior of the solutes was also investigated. A

method for the determination of clenbuterol in pork and pig

casing samples was set up by using our newly prepared

column.

2 Materials and methods

2.1 Apparatus and materials

Chromatographic analyses were carried out by using an

Agilent 1200 series system equipped with a 1200 model

quaternary pump, a G1314A model multiple wavelength

UV–Vis detector, a G1316A model thermostated column

compartment, a 1322A model vacuum degasser and an

Agilent Chemstation B.03.02 Patch data processor. The

home-made calixarene column was filled using a packing

machine (Kerui Tech., Dalian, China) under the pressure

of 50 MPa. An Eclipse XDB-C18 column (Agilent,

150 mm� 4.6 mm id, 5 mm) was used as a comparison with

the home-made calixarene column. Elemental analysis

was performed with a Flash EA 1112 elemental analyzer.1H-NMR spectrum was recorded with a Bruker 400 MHz

spectrometer in CDCl3. IR spectra were recorded with a

Bruker Vector 22 instrument. Thermal gravimetric analysis

(TGA) was carried out with a Shimadzu DT-40 thermal

analyzer; the analysis was performed from 40 to 6501C at a

heating rate of 101C/min in argon atmosphere with a gas

flow rate of 20 mL/min.

All analytes and solvents used were of analytical grade

and obtained from Beijing Chemical Plant (Beijing,

China) unless specially mentioned. Silica gel (with the

particle size of 5 mm, pore size of 100 A and specific

surface area of 300 m2/g) was provided by Lanzhou Institute

of Chemical and Physics of Chinese Academy of

Sciences (Lanzhou, China). g-Glycidoxypropyltrimethoxy-

silane (KH-560) was purchased from Wuhan University

Chemical Plant (Wuhan, China). A phosphate buffer

(0.5%, w/w, pH 4.5) was prepared by mixing KH2PO4

with ultra-high-quality pure water and filtered through a

0.45-mm filter before use. HPLC-grade methanol was

purchased from the Luzhong Reagent Plant of Shanghai

(Shanghai, China). Water was purified by using Milli-Q

purification equipment.

2.2 Synthesis of 25,27-bis-[2-(5-methylthiadiazole)-

thioethoxyl]-26,28-dihydroxy-para-tert-butyl

calix[4]arene

Scheme 1 shows the synthesis scheme of 25,27-bis-[2-(5-

methylthiadiazole)thioethoxyl]-26,28-dihydroxy-para-tert-butyl calix[4]arene. para-tert-Butylcalix[4]arene was prepared

in good yield according to the previous literature [25], then

para-tert-butylcalix[4]arene was alkylated with 1,2-dibro-

moethane to give calix[4]arene derivatives (compound 2) in

the presence of potassium carbonate [26], and the final

product (compound 3) with appending thiadiazole groups at

the lower rim was synthesized in good yield by the reaction

of compound 2 with 5-methyl-2-mercapto-1,3,4-thiadiazole

[27]; the specific procedures are as follows.

To give 25,27-dihydroxy-26,28-bis-(2-bromo-ethoxy)-

para-tert-butylcalix[4]arene (compound 2), the mixture of

para-tert-butylcalix[4]arene (1.62 g, 2.5 mmol), anhydrous

K2CO3 (0.345 g, 2.5 mmol) and 1,2-dibromoethane (2.5 mL,

29 mmol) were added to freshly distilled acetonitrile

(50 mL). After stirring under reflux for 24 h, the solvent was

removed under reduced pressure; the residue was then

purified by column chromatography over silica gel (CHCl3/

hexane, 1:2) to afford products 1.07 g (50%, yield).

25,27-bis-[2-(5-Methylthiadiazole)thioethoxyl]-26,28-

dihydroxy-para-tert-butyl calix[4]arene was obtained by

reacting compound 2 with 5-methyl-2-mercapto-1,3,4-thia-

diazole [23]. NaOH (0.16 g, 4 mmol) and 5-methyl-2-

mercapto-1,3,4-thiadiazole (0.53 g, 4 mmol) were added to

anhydrous THF (120 mL); the mixture was stirred at reflux

for 0.5 h under the protection of argon, and then compound 2(0.86 g, 1 mmol) was added. The reaction mixture was

stirred under reflux for an additional 48 h. The resulting

solution was evaporated to dryness and the residue was

taken up with CH2Cl2 (10%, 100 mL), washed with aqueous

hydrochloric acid (5%, 100 mL), brine and water. The

organic layer was dried by anhydrous Na2SO4 and evapo-

rated to give the raw product. The product was purified by

column chromatography with EtOAc–petroleum ether (2:1,

v/v) as the eluent to give 0.63 g (65%, yield) white power.

MS/ESI m/z: 987.5 [M1Na1]. 1H-NMR (CDCl3, 400 MHz).

d: 7.14 (s, 2 H, OH), 7.05 (s, 4 H, ArH), 6.79 (s, 4 H, ArH),

4.33 (t, 4 H, OCH2CH2), 4.32 (d, 4 H, ArCH2Ar), 3.92–3.95

(t, 4 H, OCH2CH2), 3.30–3.33 (d, 4 H, ArCH2Ar), 2.71 (s,

6 H, CH3), 1.28 [s, 18 H, C(CH3)3], 0.96 [s, 18 H, C(CH3)3].

2.3 Preparation of stationary phase (TBS4)

Scheme 1 shows the synthesis process of a new calix[4]ar-

ene-bonded silica gel stationary phase. Details of the

bonding procedure are as follows. Active silica gel (5.0 g)

was suspended in 50 mL dry toluene (freshly distilled) and

then 6.0 mL of KH-560 and 1.0 mL of triethylamine (used as

a catalyst) was added to this suspension. The mixture was

stirred and heated to 801C under the protection of nitrogen

atmosphere for 8 h. After the reaction finished, the solid was

J. Sep. Sci. 2012, 35, 239–247240 K. Hu et al.


filtered by 1.5 mm filter and washed in sequence with

toluene and acetone, and then dried at 801C under vacuum

for 8 h. Finally, g-glycidoxypropyl-bonded silica gel (GBS,

Scheme 1) was obtained and used as a precursor in the

following reaction.

25,27-bis-[2-(5-Methylthiadiazole)thioethoxyl]-26,28-dihy-

droxy-para-tert-butyl calix[4]arene (3.0 g, 3.1 mmol), NaH

(0.6 g) and toluene (60 mL, freshly distilled) were stirred at

801C for 30 min, the supernatant liquid was transferred to a

100-mL three-neck flask, 3.0 g GBS was added and the

mixture was refluxed with the catalyst for 48 h. The whole

process was carried out under nitrogen atmosphere. After

the reaction finished, the product was filtered and washed in

sequence with toluene, acetone, MeOH and distilled water.

Subsequently, TBS4 was obtained and dried at 1001C under

vacuum for 8 h, and then cooled to room temperature in a

desiccator.

2.4 Characterization of TBS4

As can be seen from Scheme 1, the new calixarene-bonded

stationary phase (TBS4) was prepared by the reaction of 25,27-

bis-[2-(5-methylthiadiazole)thioethoxyl]-26,28-dihydroxy-para-tert-butyl calix[4]arene (compound 3) and GBS in the presence of

NaH, and with toluene as the solvent. The characterization of

the developed stationary phase was carried out by elemental

analysis, IR and thermal gravimetric analysis.

Table 1 shows the elemental analysis results of GBS and

TBS4. The result indicates that TBS4 owns higher content

of carbon, nitrogen and sulfur than that of GBS, which

confirmed that the calixarene was successfully immobilized

onto the silica gel. The bonded amount of compound 3 onto

the silica gel was calculated by subtracting that of GBS.

According to the carbon content of the bonded silica gel

stationary phases, the resulting stationary phase contains

10.51% carbon corresponding to 0.052 mmol calixarene per

gram silica gel.

Also, the bonded stationary phase was characterized by

infrared spectroscopy. From IR spectra, the characteristic

absorption band of the benzene ring appears at 1627, 1550

and 1485 cm�1; the peaks at 2959 and 2867 cm�1 are

assigned to C–H stretching frequency. The peak at

1710 cm�1 is assigned to the absorption frequency of the

Table 1. Elemental analysis results of the bonded phase

Bonded phase C% N% S% H% Bonded amount (mmol/g)

GBS 7.13 0 0 1.21 0.743

TBS4 10.51 0.75 0.66 1.67 0.052

iv

HO

OSi

O

O

OSilica gel

+ OO Si O

O

Silica gelO

Silica gelO

OSi

O

O

Oiii

GBS

OHOH HOOHOO HOOH

i

1 2

+

Br Br

OO HOOH

S S

S N

N

S N

N

OO HOO

S S

S N

N

S N

N

TBS4

3

ii

Scheme 1. Preparation of compound3 and TBS4 stationary phase. (i)K2CO3, dibromoethane, MeCN,refluxed; (ii) 5-methyl-2-mercapto-1,3,4-thiadiazole, NaOH, THF,refluxed; (iii) toluene, catalyst, N2,801C stirred for 24 h; (iv) toluene,catalyst, compound 3, N2, refluxedfor 48 h.

J. Sep. Sci. 2012, 35, 239–247 Liquid Chromatography 241


–C==N– group. The peak at 1106 cm�1 corresponds to the

groups of Si–O–Si and C–O–C. All IR spectra indicate that

the organic ligands were bonded onto silica gel.

The thermal stability of GBS and TBS4 has been

investigated by TG analysis. The results show that both the

temperatures of weight loss for GBS and TBS4 are more

than 3001C. It indicates that the new TBS4-bonded phase

possesses high thermal and chemical stability. Moreover,

the weight losses in 25–6501C are 8.75 and 14.22% for GBS

and TBS4, respectively, which was in line with the results of

elemental analysis.

The stability of the column was evaluated over 3 months

of being used under different chromatographic conditions.

The relative standard deviations (RSDs) of retention time of

biphenyl were o2.0% (n 5 10) during that time. The

prepared column showed high chemical stability when

water (phosphate buffer, pH from 3.5 to 7.5) and MeOH

mixtures were used as mobile phases.

2.5 Chromatographic procedures

The column (150 mm� 4.6 mm i.d.) was packed with

modified calix[4]arene-silica gels according to a slurry

packing procedure by using MeOH as the displacing

agent (50 MPa, 2 h). The mobile phases used were

MeOH/water and MeOH/phosphate buffers (0.5%, w/w,

pH 4.5).

Analytes were dissolved in the mobile phase at the

concentration in the range of 5–100 mg/mL, and 20 mL of the

solution was injected into the chromatographic column. The

void time (t0) for the calculation of the retention factor was

determined by injecting 0.05 M sodium nitrate (NaNO3) at

UV detection 210 nm, with MeOH–H2O (70:30, v/v) as the

mobile phase. All measurements were carried out at 301C

and repeated three times.

3 Results and discussion

Solutes with non-polar and polar characteristics and

possessing basic, acidic and neutral properties were selected

to evaluate the chromatographic characteristics of TBS4

stationary phase. The selected analytes include six PAHs,

phenols, aromatic amines, benzoic acid and its derivatives.

Their retention factors (k0) and the separation factors (a1,2)

were calculated and listed in Table 2. The influence of

MeOH content in mobile phase on retention and selectivity

of same analytes was investigated. And, moreover, the

chromatographic behaviors of the analytes on TBS4 were

compared with these on ODS.

3.1 The separation of PAHs on TBS4

In this section, the separations for six PAHs on TBS4 and

ODS columns were carried out. The chromatogram (Fig. 1)

was obtained under the same chromatographic conditions

on TBS4 and ODS, respectively. As can be seen from Fig. 1,

the elution order of PAHs on TBS4 is the same as

that on ODS, indicating that TBS4 and ODS have

similar hydrophobic interactions with PAHs. However,

the retention time of aromatic hydrocarbons on TBS4 is

less than that on ODS. This implies that hydrophobic

interaction plays an important role in the separation, and

the hydrophobic property of TBS4 is weaker than that of

ODS.

3.2 The separation of phenols on TBS4

In this section, three phenolic compounds (phenol, 1,3-

benzenediol and 1,3,5-trihydroxybenzene) were used as

probes for the investigation of the chromatographic

characteristics of the new calixarene stationary phase. From

Table 2, it can also be observed that both TBS4 and ODS

exhibited high selectivity for phenolic compounds. With

Table 2. The retention factors (k0) and the separation factors (a)

of phenols on TBS4 and ODS

Column 1,3,5-Trihydroxybenzene 1,3-Benzenediol Phenol

TBS4 k0 3.12 4.34 6.50

a 1.39 1.50

C18 k0 1.31 2.39 6.95

a 1.82 2.91

Chromatographic conditions: Mobile phase, MeOH/water (35:65,

v/v); flow rate, 0.5 mL/min; detection wavelength, 254 nm;

column temperature, 301C.

Figure 1. The chromatograms of six aromatic hydrocarbons onTBS4 and ODS. Chromatographic conditions: Mobile phase,MeOH/H2O (70:30, v/v); flow rate, 0.8 mL/min; detection wave-length, 260 nm; column temperature, 301C; injection volume,10 mL. Peaks: 1, benzene; 2, biphenyl; 3, acenaphthene; 4,anthracene; 5, pyrene; 6, chrysene.

J. Sep. Sci. 2012, 35, 239–247242 K. Hu et al.


more hydroxy groups attached to benzene ring, the analyte

became a stronger polarity and was first eluted out on both

the TBS4 and ODS. The elution order of the

three phenolic compounds on TBS4 is the same as that

on ODS, indicating that TBS4 and ODS have similar

hydrophobic interactions with the analytes. From the

retention time of the analytes, we can see that the

analytes are more strongly retained on TBS4, specifically

for 1,3,5-trihydroxybenzene and 1,3-benzenediol, which

indicates that hydrophobic interaction is not the only

factor in the separations. The thiadiazole groups and the

cavity of the calixarene play the additional roles on the

separation mechanism for the phenols. These additional

interactions are possibly p–p and hydrogen bonding

interactions.

3.3 The separation of aromatic amines on TBS4

Reversed-phase liquid chromatography is widely used for

separation and quantitative analysis of aromatic amines. To

exploit the chromatographic potential of the new calixarene

stationary phase for basic solutes, three groups of aromatic

amines were separated on both TBS4 and ODS. For each

group, the separation was optimized and obtained better

separation; the results were compared with their separation

on ODS column. The typical chromatograms and the

retention factors of aromatic amines on TBS4 and ODS

are shown in Fig 2–5.

3.3.1 The separation of aniline, N-methyl aniline and

N,N-dimethyl aniline

Many experimental results showed that the substituted

anilines and their quaternary ammoniums were main

guests of the calixarenes in solutions [28]. Therefore, to

understand the effect of –NH2 group in the process of

separation on TBS4, we selected three aniline derivatives

which own –NH2, –NH(CH3), –N(CH3)2 as probes. As can

be seen in Fig. 2, both the stationary phases exhibited good

separation abilities for the above solute probes, and the

probes were eluted in the same order on the two columns.

This case shows that the hydrophobic interaction is mainly

responsible for the retention behavior of the aniline

derivatives, and TBS4 exhibits good hydrophobic interaction

property. At the same time, from the chromatogram we can

find another interesting phenomenon, the retention of

aniline is stronger on TBS4 column than that on ODS, but

for N-methyl aniline and N,N-dimethyl aniline, the reten-

tions are weaker on TBS4 column. The reason can be

concluded as that aniline can easily form hydrogen bond

with the thiadiazole group on TBS4; moreover, the electron-

rich cavity of calixarene can attract the –NH2 groups on the

aniline, while N-methyl aniline and N,N-dimethyl aniline do

not own a polarity group of –NH2, so the retentions of

N-methyl aniline and N,N-dimethyl aniline are weaker on

TBS4 column.

3.3.2 The separation of five aniline derivatives

In this section, five aniline derivatives were separated on

both the TBS4 and ODS column; the typical chromatograms

are shown in Fig. 3. From the chromatograms, we can see

that baseline separation of the five aniline derivatives were

obtained on both the columns, and the elution order for the

solutes was identical on both phases, which indicates that

the two columns own the similar chromatographic selectiv-

ities for the aniline derivatives.

However, it can also be noticed in the chromatograms

that the retention of m-phenylenediamine, p-phenylenedi-

amine and aniline on TBS4 was a little stronger than those

on ODS; this behavior indicates that the hydrophobic

interaction is effective in the separation on TBS4. The

explanation should be that the additional hydrogen-donor

(NH2) of the solutes and the heteroatom atoms (N, S) of the

thiadiazole groups on TBS4 existed. In addition to this, 2,5-

dimethylaniline and 4,40-diaminodiphenyl were retained

stronger. That is, because the electron-donor substituents

(the methyl) on the 2,5-dimethylaniline increase the elec-

tron density of NH2, which can have the role of inclusion

interaction with the cavity of calixarene. As for 4,40-diami-

nodiphenyl, the existence of large delocalized p-bond leads

to stronger interaction. In conclusion, the synergistic effects

of calixarene increase the retention of the analytes.

Figure 4 illustrates the plot of logarithmic retention

factor of phenols against the volume percentage of MeOH

in mobile phase. As the MeOH content of the mobile phase

increases, the retention factors’ k0 values of the solutes are

decreasing. This result indicates that the new stationary

phase can behave as an excellent reversed-phase perfor-

mance, and the hydrophobic interaction is one of the factors

playing a role in the separation of aniline and its derivatives.

However, it is obvious that the relationship between lgk0 and

the content of MeOH is not linear. Moreover, by increasing

Figure 2. The chromatogram of aniline, N-methyl aniline andN,N-dimethyl aniline on TBS4 and ODS. Mobile phases: MeOH/water (45:55, v/v); flow rate, 0.8 mL/min; detection wavelength,254 nm; temperature, 301C; injection volume, 10 mL. Peaks: 1,aniline; 2, N-methyl aniline; 3, N,N-dimethyl aniline.



the MeOH contents to 45%, the relationship line of lgk0 and

MeOH content tends to flatten, which means that there exist

other interactions in the separation process when the

MeOH content was in the low levels. Hence, it indicates that

hydrophobic was not the only factor in the separation of the

solutes, and the thiadiazole functional groups may also be

responsible for the retention behavior.

3.3.3 The separation of nitroanilines

According to the chromatogram of the nitroanilines

shown in Fig. 5, hydrogen bond interaction between the

nitroanilines and TBS4 existed besides hydrophobic inter-

action; obviously, the elution order on TBS4 is mopoo; in

contrast to this, the order is pomoo on ODS. This is

because the separation of nitroanilines on ODS is based on

only hydrophobic interaction. However, other interaction

can also contribute to the above chromatographic process on

TBS4, and the results mainly ascribe to the hydrogen bond

interaction between nitroanilines and the calixarene.

With the pKa values of protonated nitroaniline lessening

from 2.45, 1.11 to �0.28 for m-, p-, o-nitroaniline,

respectively, the proton-donor capability increased, the

hydrogen bonding interaction between protonated nitroani-

lines and the thiadiazole groups on calixarene was

enhanced, which leads to the retention time increasing

from m- to p- and o-nitroaniline. On the other hand, the

space steric hindrance of nitroaniline solutes can also

influence the retention, instead of a linear structure

(p-nitroaniline); m-nitroaniline was more difficult of being

attracted by the cavity of calixarene and the former to be

eluted than p-nitroaniline.

Figure 6 illustrates the plots of logarithmic retention

factor of nitroanilines against the volume percentage

of MeOH in mobile phase. It is obvious from the

plots that the increase in organic modifier content in mobile

phase led to the decrease in the retention of the analytes.

This is another evidence that TBS4 behaves as a reversed-

phase, and hydrophobic interaction is one of the most

important factors playing roles in the retention of the

analytes.

From the above discussion, it is noteworthy that the

separations of aromatic amines were synergistic effects

including hydrophobic interaction, hydrogen bonding

interaction and inclusion interaction. With all these effects

and the separation results, it implies that the separation

mechanism was different from ODS.

Figure 3. The chromatogram of aniline and its derivatives onTBS4 and ODS. Chromatographic conditions: Mobile phase,MeOH/0.1% triethylamine (30:70, v/v); flow rate, 0.8 mL/min;detection wavelength, 254 nm; column temperature, 301C;injection volume, 10 mL. Peaks: 1, m-phenylenediamine; 2, o-phenylenediamine; 3, aniline; 4, 2,5-dimethylaniline; 5, 4,40-diaminodiphenyl.

Figure 4. Effect of the methanol content of mobile phases on thelogarithmic retention factor of aniline and its derivatives onTBS4.

Figure 5. The chromatogram of nitroanilines on TBS4 and ODSchromatographic conditions: Mobile phase, MeOH/water (30:70,v/v); flow rate, 0.8 mL/min; detection wavelength, 254 nm;column temperature, 301C; injection volume, 10 mL. Peaks: 1,m-nitroaniline; 2, p-nitroaniline; 3, o-nitroaniline.

J. Sep. Sci. 2012, 35, 239–247244 K. Hu et al.


3.4 The separation of benzoic acid and its

derivatives on TBS4

To further study the retention mechanism, the separation of

solutes representing acidic compound was carried out using

a buffered mobile phase at pH 4.5, at which all of the

analytes exist in non-ionized forms. As can be seen in Fig. 7,

under the given condition, the baseline separation of benzoic

acid and its four derivatives was achieved on TBS4, instead of

ODS. Therefore, it is obvious that TBS4 exhibits a better

selectivity for these compounds, which may be ascribed to

other interactions besides hydrophobic interaction, and these

effects affected the results on the stationary phase.

At the same time, we can see that benzoic acid and its

derivatives gave comparatively stronger retention on calixar-

ene column than those on ODS; for example, the retention

times of p-nitrobenzoic acid and benzoic acid were about 3.2

and 4.5 min on ODS, however, which changed to 8 and

14 min on TBS4. Moreover, the value of 3,5-dinitrobenzoic

acid changed from 4.5 min on ODS to 26 min on TBS4 which

further confirmed that there were the additional interactions

with the exception of the hydrophobic interaction between

calixarene and analytes. This is because that, on one hand,

the carboxyl group in the benzoic acid and its derivatives can

easily form hydrogen bond with the thiadiazole groups on the

TBS4, which may importantly influence the retention time of

the analytes. On the other hand, the group of –NO2 can affect

with the electron-rich cavity of the calixarene in the form of

dipole–dipole interaction; at the same time, p–p interaction

was existing between the analytes and the benzene skeleton

of calixarene.

3.5 The determination of clenbuterol in pork and pig

casing on TBS4 column

Clenbuterol is a synthetic b2-agonist, which is used for the

treatment of asthma both in humans and animals. In high

doses, clenbuterol exhibits a metabolic effect, which results

in an increase in muscle mass and a decrease in adipose

tissue. Therefore, the compound is also misused as nutrient

repartitioning agent in livestock by diverting nutrients from

fat deposition in animals to the production of muscle tissues

[29]. This misuse had caused some severe accidental

poisonings in humans [30, 31]. Therefore, all b2-agonists

are banned for growth promotion in animal production in

China [32]. To protect consumers, specific and sensitive

methods for the identification and quantification of

clenbuterol in meat and other food are required.

The stock solution of clenbuterol (250 mg/mL)

was prepared by dissolving the reference substance in

acetonitrile and stored in the refrigerator. The standard

working solutions were prepared by diluting aliquots

of the stock solution with 0.2% formic acid solution/acet-

onitrile (90:10, v/v) to obtain concentrations ranging

from 0.1 to 2.0 mg/mL. The calibration graph was

constructed by plotting the peak areas obtained at

wavelength 243 nm versus the corresponding injected

concentrations.

The samples were treated according to the following

methods: 2.000 g tissue samples (accurately weighed to

0.001 g) was transferred to a 50-mL polypropylene centrifuge

tube with a stopper, 10 mL ammonium acetate (20 mmol/L)

buffer solution was added to the tube, then 50 mL b-glucur-

onidase-aryl sulfatase (containing 134 600 U/mL b-glucur-

onidase and 5200 U/mL aryl sulfatase) was added, after that,

allowed to oscillate on the vortex oscillator for 2 min and

sonicated for 20 min by using a KQ-3300 Kunshan ultrasonic

(Kunshan, Jiangsu Province, China) processor at 50 W model,

and then kept in a incubator for 16 h at 371C. After the tube

was centrifuged at 4000 rpm/min for 10 min, the supernatant

Figure 6. Effect of the methanol content of mobile phases on thelogarithmic retention factor of nitroanilines on TBS4.

Figure 7. The chromatogram of benzoic acid and its derivativeson TBS4 and on ODS. Chromatographic conditions: Mobilephase, MeOH/0.5% KH2PO4 (40:60, v/v), flow rate, 0.8 mL/min;detection wavelength, 254 nm; column temperature, 301C;injection volume, 10 mL. Peaks: 1, benzoic acid; 2, m-methoxy-benzoic acid; 3, p-nitrobenzoic acid; 4, p-chlorobenzoic acid; 5,3,5-dinitrobenzoic acid.



was transferred into another tube, and then 5.0 mL chloroform

were added, oscillated and sonicated again.

The supernatant was applied to Waters Oasis MCX

cartridge (6 mL, 100 mg), which was activated with 5 mL of

MeOH, 5 mL of water, followed by 5 mL hydrochloric acid

(0.01 mol/L) solution. The cartridge was washed with 6 mL

ethyl acetate/ammonium hydroxide (97:3, v/v), the eluent

was collected into a glass tube and evaporated until dryness

under a stream of nitrogen at 401C water bath, and the

residue was reconstituted in 1.0 mL of 0.2% formic acid in

water/acetonitrile (90:10, v/v). The resulting solution was

filtered through a 0.22-mm filter and 10 mL of the filtrate

were ready for the analysis.

The graph of the peak area (y) against concentration

(x, mg/mL) proved linear in the range from 0.1 to 20.0 mg/mL,

and the linearity equation was: y 5 101.1x�12.02 and

regression coefficient R2 5 0.9994. The limit of detection

(LOD) defined as the injected quantity giving S/N of 3 (in

terms of peak area) was found to be 0.03 mg/mL, for

which the corresponding LOD of the sample is 0.015 mg/g.

The limit of quantitation (LOQ) defined as the injected

quantity giving S/N of 10 (in terms of peak area) was found to

be 0.097 mg/mL, for which the corresponding LOQ of the

sample is 0.049 mg/g. Inter-day precision was assessed by

injecting the standard solution of different concentrations

(0.2, 2.0 and 20 mg/mL) and on each day for 5 days. The

results show that there were high inter-day precisions, the

RSD% of retention times was within 0.046 and the RSD% of

peak areas was within 2.64. Intra-day precisions were asses-

sed by injecting the standard solution at three concentrations

five times during a day, and the intra-day RSD% of

retention time was within 0.040 and the RSD% of peak area

was within 2.04.

The accuracy of the method was determined by recovery

experiments. The analysis of clenbuterol in pork and pig

casing showed high accuracy when the spiked concentra-

tions were 0.1 and 0.2 mg/mL, and the recovery is

93.5–108.5%. The chromatogram of clenbuterol in pork and

pig casing is shown in Fig. 8, from which we can see that

clenbuterol obtained better separation from the matrix in

the pork and pig casing. The residues of clenbuterol in pork

and pig casing are 102 and 94 ng/g, respectively. These

clenbuterol residual values exceed the tolerable limits of

China National Standard (clenbuterol should not be detec-

ted) [33]. It clearly demonstrated that the clenbuterol resi-

dual levels in the meat samples would be expected to pose

health risks to the consumers. Thus, these food-stuffs

containing clenbuterol should be destroyed.

4 Concluding remarks

A new para-tert-butylcalix[4]arene column containing thia-

diazole functional groups was prepared and characterized by

elemental analysis, thermal analysis and FT-IR. The

chromatographic behaviors of this new developed stationary

phase were investigated by using PAHs, phenols, aromatic

amines, benzoic acid and its derivatives. The retention

behaviors were compared with those on ODS. It can be

concluded that the column behaves as a reversed-phase

material with weaker hydrophobicity as compared with

ODS; additionally, various chromatographic retention

mechanisms occur in the separation of the above analytes,

such as p–p interaction, hydrogen bonding interaction

and inclusion complexation. The TBS4 column was

successfully used for the analysis of clenbuterol in pork

and pig casing; the LOD and LOQ for this method were 0.03

and 0.097 mg/mL, and the recovery is between 93.5 and

108.5%. This method using a new TBS4 stationary phase

has been proven suitable for routine determination of

clenbuterol in meat.

The authors acknowledge the support of NSF of China(20875083, 20775073 and 21077095), State Key Laboratory ofEnvironmental Chemistry & Ecotoxicology (KF2008-22) andthe Innovation Scientists & Technicians Troop ConstructionProjects of Zhengzhou City (10LJRC192).

The authors have declared no conflict of interest.

Figure 8. Chromatogram of clenbuterol in pork and pig casingon TBS4 column. Chromatographic conditions: Mobile phase,0.2% formic acid solution/acetonitrile (90:10, v/v); pH, 5.0; flowrate, 0.6 mL/min; detection wavelength, 243 nm; columntemperature, 301C; Peak: 1, clenbuterol.

J. Sep. Sci. 2012, 35, 239–247246 K. Hu et al.


5 References

[1] Cadogan, F., Kane, P., McKervey, M.-A., Diamond, D.,Anal. Chem. 1999, 71, 5544–5550.

[2] Demirel, A., Dogan, A., Akkus, G., Yilmaz, M., Kilic, E.,Electroanalysis 2006, 18, 1019–1027.

[3] Kivlehan, F., Mace, W.-J., Moynihan, H.-A., Arrigan,D.-W. M., Anal. Chim. Acta 2007, 585, 154–160.

[4] Sustrova, B., Stulik, K., Marecek, V., Janda, P., Electro-analysis 2010, 22, 2051–2057.

[5] Wang, F., Wei, X.-H., Wang, C.-B., Zhang, S.-S., Ye,B.-X., Talanta 2010, 80, 1198–1204.

[6] Shohat, D., Grahka, E., Anal. Chem. 1994, 66, 747–750.

[7] Xu, H., Yu, X.-D., Chen, H.-Y., Electrophoresis 2003, 24,4254–4263.

[8] Wu, X., Liu, H.-X., Liu, H., Zhang, S.-S., Haddad, P.-R.,Anal. Chim. Acta 2003, 478, 191–197.

[9] Mori, M., Hirayama, A., Tsue, H., Tanaka, S., ActaChromatogr. 2007, 19, 73–80.

[10] Lim, H.-J., Lee, H.-S., Kim, L.-W., Chromatographia1998, 48, 422–426.

[11] Schneider, C., Jira, T., J. Sep. Sci. 2010, 33, 2943–2955.

[12] Huai, Q.-Y., Zuo, Y.-M., Liq, J., Chromatogr. Relat.Technol. 2006, 29, 801–814.

[13] Li, L.-S., Da, S.-L., Feng, Y.-Q., Liu, M., J. Liq. Chroma-togr. Relat. Technol. 2004, 27, 2167–2188.

[14] Sliwka-Kaszynska, M., Jaszcolt, K., Anusiewicz, I., J.Sep. Sci. 2009, 32, 3107–3115.

[15] Sliwka-Kaszynska, M., Karaszewski, S., J. Sep. Sci. 2008,31, 926–934.

[16] Schneider, C., Meyer, R., Jira, T., Anal. Sci. 2008, 24,1157–1164.

[17] Mokhtari, B., Pourabdollah, K., Dalali, N., Chromato-graphia 2011, 73, 829–847.

[18] Mokhtari, B., Pourabdollah, K., Dalali, N., J. Radioanal.Nucl. Chem. 2011, 287, 921–934.

[19] Mokhtari, B., Pourabdollah, K., Dalali, N., J. InclusionPhenom. Macrocyclic Chem. 2011, 69, 1–55.

[20] Mokhtari, B., Pourabdollah, K., Dalali, N., J. Coord.Chem. 2011, 64, 743–794.

[21] Sliwka-Kaszynska, M., Gorczyca, G., Slebioda, M., J.Chromatogr. A 2010, 1217, 329–336.

[22] Serkan, E., Mustafa, Y., Talanta 2010, 82, 1240–1246.

[23] Ding, C.-H., Qu, K., Li, Y.-B., Hu, K., Liu, H.-X., Ye, B.-X.,Wu, Y.-J., Zhang, S.-S., J. Chromatogr. A 2007, 1170, 73.

[24] Hu, K., Zhao, W.-J., Wen, F.-Y., Liu, J.-W., Zhao, X.-L.,Xu, Z.-H., Niu, B.-L., Ye, B.-X., Wu, Y.-J., Zhang, S.-S.,Talanta 2011, 85, 317–324.

[25] Gutsche, C.-D., Iqbal, M., Steward, D., J. Org. Chem.1986, 51, 742–745.

[26] Chen, C.-F., Zheng, Q.-Y., Zheng, Y.-S., Huang, Z.-T.,Synth. Commun. 2001, 31, 2829–2836.

[27] Zhao, B.-T., Wang, L., Ye, B.-X., Youji Huaxue 2006, 26,1562–1565.

[28] Shinkai, S., Araki, K., Matsuda, T., Nishiyama, N., Ikeda,H., Takasu, L., Iwamoto, M., J. Am. Chem. Soc. 1990,112, 9053–9058.

[29] Shishani, E., Chai, S.-C., Jamokha, S., Aznar, G., Hoff-man, M.-K., Anal. Chim. Acta 2003, 483, 137–145.

[30] Martinez-Navarro, J.-F., Lancet 1990, 336, 1311.

[31] Maistro, S., Chiesa, E., Angeletti, R., Brambilla, G.,Lancet 1995, 346, 180.

[32] Ministry of Agriculture of the People’s Republic ofChina, No. 235 Bulletin of the Ministry of Agriculture ofthe People’s Republic of China, Beijing 2003.

[33] General Administration of Quality Supervision, Inspec-tion and Quarantine of the People’s Republic of China(AQSIQ), GB 18406.3-2001, Beijing 2001.



preparation, characterization and application of a new...

Documents