Hybrid polyacrylamide chiral stationary phases for HPLC prepared by surface-initiated photopolymerization

Research Article

Hybrid polyacrylamide chiral stationaryphases for HPLC prepared by surface-initiated photopolymerization

Two hybrid polyacrylamide chiral stationary phases (CSPs) for HPLC have been synthe-

sized by a new surface-initiated photo-induced radical polymerization approach of enan-

tiopure N,N0-diacryloyl derivatives of (1R,2R)-diaminocyclohexane (CSP1) and (1R,2R)-

diphenylethylenediamine (CSP2). This system is based on the activation of mesoporous

silica microparticles by chemically bonded trichloroacetyl groups and dimanganese

decacarbonyl as catalyst. UV irradiation was performed using a lab-made quartz photo-

chemical reactor, ad hoc designed for the photo-induced polymerization process on the

surface of microparticles. The two phases were evaluated and compared as chromato-

graphic supports for the enantioselective HPLC of model chiral compounds. Their

physico-chemical properties and chromatographic performances were also evaluated in

comparison with those exhibited by the homologue CSPs obtained by the grafting-fromthermal-induced process (CSP3 and CSP4). The new photopolymerization approach

yielded higher grafting density than the thermal-induced one, especially in the case of the

less reactive monomer (the diacryloyl derivative of (1R,2R)-diphenylethylenediamine),

good chromatographic efficiency and a broad application field under normal phase and

polar organic mode conditions.

Keywords: Chiral polymers / Chiral stationary phases / HPLC / Photopoly-merizationDOI 10.1002/jssc.201000355

1 Introduction

Investigation on chiral stationary phases (CSPs) based on

totally synthetic, optically active polymers linked to a

chromatographic matrix is continuously evolving. Since

their introduction in 1980, polyacrylamide and polymeth-

acrylamide CSPs were used for the separation of a wide

range of chiral compounds, including benzodiazepines,

barbiturates, and hydantoins [1, 2]. Diverse strategies are

indeed available for the grafting of polymers onto the

surface of ultra-fine particles for non-chromatographic

applications [3], based on surface-initiated polymerization

(also called grafting-from or g-from approach) from initiators

bound to surfaces. Uniform and well permeable polymeric

layers are available only if the degree of polymerization and

the grafting density are carefully controlled. Such polymeric

materials are therefore highly desirable for the preparation

of improved polymeric CSPs. We recently reported [4–6] a

new hybrid organic/inorganic CSP for HPLC synthesized by

the g-from radical polymerization of an enantiopure

diacryloyl derivative of trans-1,2-diaminocyclohexane in the

presence of mesoporous, azo-activated silica micro-particles.

This was the first example of application of the g-fromapproach to the synthesis of a CSP for HPLC applications.

However, the thermal grafting process could not be surface-

confined only, since concomitant solution polymerization

may lead to ungrafted polymer chains [3]. Thus, we moved

to study another way to induce polymerization of the above

monomer, and focused our attention on photo-induced

polymerization, which has been obtaining much attention

in recent years because of its numerous industrial applica-

tions [7–9]. The concept of photopolymerization is that the

initiators generate free radicals upon light irradiation, and

the resulting radical starts the polymerization process.

Recently, several photoinitiators in free radical promoted

cationic polymerization have successfully been used [10]. In

particular, several visible light-absorbing systems able to

generate oxidable radicals were reported. For instance,

radicals formed by the irradiation of systems containing a

xanthene dye and an aromatic amine, were oxidized by a

Alessia Ciogli1

Ilaria D’Acquarica1

Francesco Gasparrini1

Carmela Molinaro1

Romina Rompietti1

Patrizia Simone1

Claudio Villani1

Giovanni Zappia2

1Dipartimento di Chimica eTecnologie del Farmaco,Sapienza Universita di Roma,Roma, Italy

2Istituto di Chimica Farmaceutica,Universita di Urbino ‘‘Carlo Bo’’,Urbino, Italy

Received May 18, 2010Revised July 22, 2010Accepted July 22, 2010

Abbreviations: 3-APSG, 3-aminopropyl silica gel; CSP, chiralstationary phase; DACH, (1R,2R)-diaminocyclohexane;

DPEDA, (1R,2R)-diphenylethylenediamine; DRIFT, diffusereflectance infrared Fourier transform; NP, normal phase;

POM, polar organic mode

Correspondence: Professor Francesco Gasparrini, Dipartimentodi Chimica e Tecnologie del Farmaco, Sapienza Universita diRoma, P.le A. Moro 5, 00185 Roma, ItalyE-mail: [email protected]: 1390649912780

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com

J. Sep. Sci. 2010, 33, 3022–30323022

diphenyliodonium salt [11]. Similarly, the dimanganese

decacarbonyl Mn2(CO)10-organic halides combination is an

efficient co-initiator for visible light cationic polymerization,

when used in conjunction with onium salts [12, 13].

Polymerization of vinyl monomers, such as methyl meth-

acrylate, styrene, and glycidyl methacrylate, was successfully

initiated by the system consisting of molibdenum hexa-

carbonyl Mo(CO)6 and surface-linked trichloroacetyl groups

as well [13, 14]. A possible mechanism of photoinitiation

involves a preliminary interaction between the metal

carbonyl and the trichloroacetyl functionalities, yielding

initiating alkyl radicals, that later react with the monomer

[15, 16].

In this work we describe for the first time the pre-

paration of two hybrid polyacrylamide CSPs by the

surface-initiated photo-induced radical polymerization of

enantiopure N,N0-diacryloyl derivatives of (1R,2R)-diamino-

cyclohexane (DACH) and (1R,2R)-diphenylethylene-

diamine (DPEDA), initiated by the system consisting

of trichloroacetyl groups on mesoporous silica particles

and dimanganese decacarbonyl Mn2(CO)10 under UV

irradiation.

2 Materials and methods

2.1 Chemicals and reagents

Spherical Daisogel SP-300-5P (5 mm particle size, 115 m2/g

specific surface area) silica gel was purchased from Daiso

(Osaka, Japan). Diisopropylethylamine, DACH, acryloyl

chloride, (3-aminopropyl)triethoxysilane, phosphorous

pentachloride, ammonium acetate, and dry toluene were

purchased from Fluka (Sigma-Aldrich, Buchs, Switzerland);

dimanganese decacarbonyl (Mn2(CO)10), dry tetrahydro-

furan, 2,2,2-trichloroacetyl isocyanate, DPEDA, and 4,40-

azobis-4-cyanopentanoic acid were purchased from Aldrich

(Sigma-Aldrich).

1-Methoxy-2-methyl-1-(trimethylsilyloxy)-1-propene was

purchased from Lancaster (Clariant group, UK). HPLC-

grade solvents were purchased from Merck (Darmstadt,

Germany). Chloroform (analytical grade) was dried by

filtration through an open glass column filled with neutral

alumina under inert atmosphere and then degassed with

helium. Chiral solutes (compounds 1�12) were available

from previous studies.

2.2 Apparatus

Diffuse reflectance infrared Fourier transform (DRIFT) and

transmission IR (potassium bromide pellets or liquid

paraffin dispersion) spectra were recorded on a Jasco 430

Fourier transform (FT) IR spectrometer (Jasco Europe,

Cremella, Italy) at a resolution of 4 cm�1. NMR spectra were

recorded on a Bruker Avance 400 spectrometer. Melting

points were determined on a Buchi B-545 instrument

(Flawil, Switzerland). Optical rotation values were obtained

on a Jasco P1030 polarimeter.

A lab-made quartz photochemical reactor, equipped

with a refrigerating chamber, an inert gas inlet, a mechanic

stirring and a high-pressure mercury (Hg) vapor lamp,

125 W (Helios Italquartz srl, Milan, Italy) was used for the

synthesis of CSP1 and CSP2.

Analytical liquid chromatography was performed on a

Waters chromatograph equipped with a Rheodyne model

7725i 20 mL loop injector, a 1525 binary HPLC pump and a

2487 dual wavelength absorbance detector (Waters, Milford,

MA, USA). Chromatographic data were collected and

processed using Empower software.

2.3 Preparation of N-(2-acryloylamino-(1R,2R)-cyclo-

hexyl)-acrylamide (R,R-DACH-ACR)

N-(2-Acryloylamino-(1R,2R)-cyclohexyl)-acrylamide or (R,R)-

DACH-ACR was prepared starting from DACH and acryloyl

chloride as previously described [5]. Elemental analysis: found

% C, 64.68; % H, 8.21; % N, 12.46; calculated for C12H18N2O2

% C, 64.84; % H, 8.16; % N, 12.60. Mp: 2331C. ½a�20D 5 185.4

(c 5 1.0; DMSO). 1H-NMR (d6-DMSO) d (ppm): 1.20–1.30

(m, 4H), 1.60–1.70 (m, 2H), 1.85–1.95 (m, 2H), 3.60–3.70

(m, 2H), 5.52 (dd, J 5 9.90 Hz, 2.44 Hz, 2H), 6.02

(dd, J 5 17.09 Hz, 2.44 Hz, 2H), 6.15 (dd, J 5 17.09 Hz,

9.90 Hz, 2H), 7.85 (d, 2H). 13C-NMR (d6-DMSO) d (ppm):

23.93, 31.30, 52.24, 124.74, 130.28, 166.14. FT-IR (KBr): 3284,

3075, 3033, 1656, 1625, 1550, 1410 cm�1.

2.4 Preparation of N-[2-acryloylamino-(1R,2R)-di-

phenylethyl]-acrylamide (R,R-DPEDA-ACR)

To a solution of DPEDA (1.0 g, 4.7 mmol) in 17.5 mL of dry

toluene was added diisopropylethylamine (1.6 mL,

9.4 mmol). To the cooled (01C, ice bath) solution was added

dropwise over a period of 1 h, with magnetic stirring under

an argon atmosphere, a solution of acryloyl chloride

(0.8 mL; 9.9 mmol) in 25 mL of dry toluene. The reaction

mixture was kept at 01C for 1 h, with magnetic stirring

under an argon atmosphere. The whitish precipitate was

collected by filtration, washed with toluene and hexane and

dried at reduced pressure (0.1 mbar, 251C) to yield 1.1 g of a

crude solid (73% yield), which was dissolved in 2-propanol

(20 mL) and precipitated by hexane (100 mL). The white

solid was filtered, washed with hexane and dried at reduced

pressure (0.1 mbar, 401C) to give 1.0 g of title compound

(66% overall yield). TLC: Merck plates Si-60-F254 eluent

CH2Cl2/MeOH 97:3 v/v, Rf 5 0.46. Elemental analysis:

found % C, 75.02; % H, 6.32; % N, 7.69; calculated for

C20H20N2O2 % C, 74.98; % H, 6.29; % N, 8.74. Mp: 2501C.

½a�25D 5 117.7 (c 5 1.0; DMSO). 1H-NMR (d6-DMSO) d

(ppm): 5.26 (dd, 2H), 5.54 (dd, J 5 9.95 Hz, 2.25 Hz, 2H),

6.08 (dd, J 5 17.05 Hz, 2.29 Hz, 2H), 6.31 (dd, J 5 17.05 Hz,

9.95 Hz, 2H), 7.20 (m, 10H), 8.70 (d, 2H). FT-IR (KBr):

J. Sep. Sci. 2010, 33, 3022–3032 Liquid Chromatography 3023


3305, 3063, 3033, 1655, 1628, 1604, 1537, 1496, 1407 cm�1.13C-NMR (d6-DMSO) d (ppm): 57.60, 126.00, 127.35, 127.87,

128.35, 132.10, 140.96, 164.62. FT-IR (KBr): 3305, 3063,

3033, 1655, 1628, 1604, 1537, 1496, 1407 cm�1.

2.5 Preparation of 3-aminopropyl silica gel (3-APSG)

Spherical Daisogel SP-300-5P silica (10.0 g) was dried at

reduced pressure (0.1 mbar) at 1501C for 1 h. Dried silica

was placed in a 500 mL three-necked round bottom flask

equipped with a Dean Stark trap, reflux condenser and inert

gas inlet, and 240 mL of toluene were added. The slurry was

heated to reflux temperature with mechanical stirring under

an argon atmosphere, and 25 mL of distillate were collected

over a 1 h period.

After cooling to room temperature, 5.0 mL of

(3-aminopropyl)triethoxysilane (21.5 mmol) was added at

once and the slurry was heated to reflux temperature for 4 h,

with mechanical stirring under an argon atmosphere. After

cooling to room temperature, modified silica gel (3-APSG)

was collected by filtration, washed with 200 mL portions of

toluene, methanol, dichloromethane, and dried at reduced

pressure (0.1 mbar, T 5 601C) up to a constant weight

(weight increment: 4.0%). Elemental analysis: % C, 1.47; %

H, 0.46; % N, 0.44, corresponding to 325 mmol/g of starting

silica (2.82 mmol/m2) or 314 mmol/g of final matrix (based

on nitrogen). FT-IR (DRIFT): 3644, 2978, 2940, 1874, 1614,

1099, 948, 809 cm�1.

2.6 Activation of 3-aminopropyl silica gel with 2,2,2-

trichloroacetyl isocyanate (3-APSG-COCCl3)

3-Aminopropyl silica gel (3-APSG) obtained as described in

Section 2.5 (3.0 g) was dispersed in 10 mL of tetrahydrofur-

an, with mechanical stirring and under an argon atmo-

sphere. To the slurry was added at once 2,2,2-trichloroacetyl

isocyanate (1.0 g; 5.3 mmol), the stirring was continued at

room temperature for 1 h, and then the slurry was heated to

701C for 2 h. Modified silica gel (3-APSG-COCCl3) was then

isolated by filtration, washed with 50 mL portions of

tetrahydrofuran, methanol, acetone, dichloromethane, and

dried at reduced pressure (0.1 mbar, T 5 251C) up to a

constant weight (weight increment: 3.9%). Elemental

analysis: % C, 2.60; % H, 0.46; % N, 0.83, corresponding

to 324 mmol g�1 of starting silica (2.82 mmol m�2) or

296 mmol g�1 of final matrix (based on nitrogen). FT-IR

(DRIFT): 2947, 2885, 1732, 1707, 1548, 1479 cm�1.

2.7 General preparation of photo-induced polyacryl-

amide stationary phases (CSP1 and CSP2)

Into a lab-made quartz photochemical reactor methanol

(220 mL) was first placed and degassed by helium, and then

heated to 401C, under mechanical stirring (Fig. 1). The

diacryloyl monomer (1R,2R)-DACH-ACR (0.87 g; 3.9 mmol)

or (1R,2R)-DPEDA-ACR (0.87 g; 2.7 mmol) was charged into

the reactor under continuous stirring until complete

dissolution, followed by dimanganese decacarbonyl (0.1 g).

3-APSG-COCCl3 (2.9 g) was then added, under mechanical

stirring, and the slurry was degassed by helium. The reactor

was purged with argon for 5 min and then irradiated with a

125 W high-pressure Hg lamp with continuous stirring at

401C for 3 h. Modified silica gels (CSP1 and CSP2) were

isolated by filtration, washed with 100 mL portions of

methanol, tetrahydrofuran, dichloromethane, and dried at

reduced pressure (0.1 mbar, T 5 601C).

CSP1 (CSP-hn-poly-(R,R)-DACH-ACR): weight incre-

ment 22%. Elemental analysis: % C, 15.12; % H, 2.19; % N,

2.95, corresponding to 1082 mmol/g of starting silica

(9.41 mmol/m2) or 810 mmol/g of final matrix (based on

carbon). FT-IR (DRIFT): 2941, 2863, 1713, 1653, 1543,

1453 cm�1.

CSP2 (CSP-hn-poly-(R,R)-DPEDA-ACR): weight incre-

ment 20%. Elemental analysis: % C 17.25; % H, 1.99; % N,



carbon). FT-IR (DRIFT): 3072, 3032, 2941, 1725, 1660, 1538,

1457 cm�1.

2.8 Activation of 3-APSG with 4,40-azobis-4-cyano-

pentanoic acid dichloride (3-APSG-AZO)

3-APSG was activated with 4,40-azobis-4-cyanopentanoic

acid dichloride as previously described [5] to yield 3-APSG-

AZO silica gel.

2.9 General preparation of thermal-induced poly-

acrylamide stationary phases (CSP3 and CSP4)

To a heated (601C) solution of the diacryloyl monomer

(1R,2R)-DPEDA-ACR in anhydrous, degassed chloroform

(0.45 g; 1.4 mmol in 40 mL) was added 3-APSG-AZO (3.0 g)

with mechanical stirring and under an argon atmosphere.

The slurry was heated and kept at reflux temperature for 6 h,

with continuous stirring. After cooling to room tempera-

ture, modified silica (CSP4) was isolated by filtration,

washed with 100 mL portions of methanol, acetone,

dichloromethane and dried at reduced pressure (0.1 mbar,

T 5 601C).

CSP3 (CSP-D-poly-(R,R)-DACH-ACR) was prepared as

previously described [5]. Elemental analysis: % C, 10.90; %

H, 1.76; % N, 2.30, corresponding to 590 mmol/g of starting

silica (5.14 mmol/m2) or 488 mmol/g of final matrix (based

on carbon). FT-IR (DRIFT): 3078, 2941, 2863, 2237, 1713,

1653, 1543, 1453 cm�1.

CSP4 (CSP-D-poly-(R,R)-DPEDA-ACR): weight incre-

ment: 5.6%. Elemental analysis: % C, 7.99; % H, 1.13; % N,



J. Sep. Sci. 2010, 33, 3022–30323024 A. Ciogli et al.


carbon). FT-IR (DRIFT): 3065, 3034, 2941, 2244, 1654, 1532,

1455 cm�1.

2.10 Columns packing and geometry

Stainless steel columns (250 mm� 4.6 mm id for CSP3;

250 mm� 4.0 mm id for CSP1, CSP2, and CSP4) supplied

by Alltech (IL, USA) were packed with CSP1�CSP4 at

9000 psi by using a previously described slurry packing

procedure [17]. The column dead times (t0) were determined

from the elution times of an unretained marker (1,3,5-tri-

tert-butylbenzene), using as eluent a mixture made up of

97% dichloromethane (amylene stabilized) and 3% metha-

nol, at T 5 251C, a flow-rate of 1.0 mL/min, and UV

detection at 254 nm. To maintain the same linear velocity,

flow-rate for CSP3 was adjusted to 1.3 mL/min.

2.11 Chromatographic conditions

HPLC separations were carried out under normal phase

(NP) and polar organic mode (POM) conditions on

CSP1�CSP4. In the first case, the mobile phase was made

up of 97% dichloromethane stabilized with ethanol

(�0.25%) and 3% methanol (v/v) for compounds 1�5. For

compound 6, a mobile phase consisting of 10% ethanol in

hexane was used. In the second case (POM mode), a

mixture of acetonitrile–methanol 70:30 v/v plus 20 mM

ammonium acetate was used as mobile phase for

compounds 7�9. For compounds 10�12, a mobile phase

made up of acetonitrile–methanol 85:15 v/v plus 20 mM

ammonium acetate was instead used. The flow-rate was set

to 1.0 mL/min for CSP1, CSP2, and CSP4, and adjusted to

1.3 mL/min for CSP3, to maintain the same linear velocity.

All the columns were thermostated at 251C. Samples were

dissolved in mobile phase and aliquots of 10–20 mL were

injected. Chromatograms were recorded by monitoring the

UV trace at 254 nm.

3 Results and discussion

3.1 Choice of a new synthetic strategy to hybrid

polyacrylamide CSPs

We recently reported a new hybrid organic/inorganic CSP

for HPLC synthesized by the g-from radical polymerization

of an enantiopure diacryloyl derivative of trans-1,2-diamino-

cyclohexane in the presence of mesoporous, azo-activated

silica micro-particles [4–6]. Hybrid CSPs based on synthetic

polymers as chiral selectors linked to inorganic supports

have gained attention and are proposed as synthetic

counterparts of the CSPs based on derivatized polysaccha-

rides. The expected advantages are increased mechanical,

thermal, and chemical stability accompanied by large

chromatographic efficiencies [18–22].

With this experience in hand, we moved to study

another way to induced polymerization of the above

monomer, and focused our attention on photo-induced

polymerization, which has been obtaining much attention

in the recent years because of its numerous industrial

Figure 1. Pictures of empty photo-chemical reactor (left) and filled withthe slurry of silica gel microparticlesbefore the photo-induced polymeri-zation (right).



applications [7–9]. We thus prepared two hybrid poly-

acrylamide stationary phases, CSP1 and CSP2, by a new

photo-induced polymerization approach based on the acti-

vation of mesoporous silica microparticles by chemically

bonded trichloroacetyl groups and using dimanganese

decacarbonyl Mn2(CO)10 as catalyst. The chiral polymeriz-

able monomers were enantiopure N,N0-diacryloyl derivatives

of trans-DACH (DACH-ACR) and DPEDA (DPEDA-ACR),

for CSP1 and CSP2, respectively.

3.2 Photochemical lab-made reactor

The surface-initiated photochemical grafting process was

carried out in a lab-made reactor tailored to address some

particular requirements of an heterogeneous reaction. A

photoreactor system was in fact sought in which all the

silica particles could be homogeneously exposed to the

irradiating region of the UV lamp and where mechanical

damage and gravitational segregation of the silica particles

could be avoided. On the basis of such design requirements

we developed the photoreactor shown in Fig. 1. We used a

quartz jacketed photoreactor system equipped with a high-

pressure mercury (Hg) vapor lamp and a propeller blade

stirrer for efficient, uniform motion of the reaction mixture.

The reactor is also equipped with gas inlet and sampling

ports, to provide easy degassing and O2 depletion of the

reaction medium by He sparging, to keep an inert atmo-

sphere (argon) during UV irradiation, and to allow for

sample withdrawing for reaction progress monitoring.

3.3 Outcome of the polymerization and character-

ization of the new polyacrylamide CSPs

The whole synthetic process (Fig. 2) starts with the

derivatization of 3-APSG with trichloroacetyl isocyanate to

generate the activated silica with surface-linked R-NH-CO-

NH-COCCl3 moieties (R 5 propyl, 3-APSG-COCCl3) which,

under irradiation with a mercury (Hg) lamp in the presence

of Mn2(CO)10, initiates the co-polymerization of the chiral

diacrylamide [(R,R)-DACH-ACR or (R,R)-DPDEA-ACR]

directly from the silica surface, yielding the final CSP1

and CSP2, respectively, containing the covalently grafted

chiral polymers [5]. A plausible mechanism of photoinitia-

tion involves a preliminary decomposition of Mn2(CO)10

into Mn(CO)5 species that react with trichloroacetyl groups,

yielding carbon-centered, surface-confined radicals [12, 13].

The latter, in the presence of chiral vinyl monomers, initiate

the polymerization yielding the final surface-grafted chiral

polymeric stationary phase [15, 16]. Grafting reactions were

monitored by DRIFT spectroscopy (Fig. 3), and the final

CSPs were fully characterized by elemental analysis (see

Table 1) and DRIFT. Activation of 3-APSG by trichloroacetyl

isocyanate is accompanied by some changes in the DRIFT

spectra (Fig. 3, top), which clearly indicate the presence of

additional absorption bands, diagnostic of the presence of

two different carbonyl groups on the silica surface (1732,

1707, 1548, and 1479 cm�1), in agreement with the

proposed structure containing the 1-trichloroacetyl-3-propyl-

urea fragment.

In the DRIFT spectrum of CSP1 (Fig. 3, middle),

intense signals arising from the chiral polymer of DACH-

ACR are clearly visible in the amide stretching region (1713,

1653, 1543 cm�1) and in the aliphatic stretching and bend-

ing regions (2941, 2863, 1453 cm�1). Similar stretching and

bending vibration bands are evident in the DRIFT spectrum

of CSP2 (Fig. 3, bottom), as they are diagnostic of the

presence of the chiral polymer of DPEDA-ACR (2941, 1725,

1660, 1538, 1457 cm�1); additional bands above 3000 cm�1

are symptomatic of phenyl groups (3072 and 3032 cm�1,

aromatic C–H stretching).

For comparative purposes, we prepared two homologue

CSPs (CSP3 and CSP4) by the thermal-induced approach

described in [5] from DACH-ACR and DPEDA-ACR,

respectively. Surface coverage characterization of the poly-

acrylamide CSP1–CSP4 and of aminopropyl silica gel

precusors are collected in Table 1. Carbon content data on

CSP1 show an average surface density of monomer units of

NH2NH2 NH2

NH

NH

CCl3

CHCl2 CHCl2 CHCl2

silica

B

NH

NH

O

O

NH

NH

O

O

or

O

O

NH

NH

CCl3

O

O

NH

NH

CCl3O

ONH

NH

O

O

NH

NH

O

O

NH

NH

O

O

hν, cat.

A

Figure 2. Synthetic pathway to polyacrylamide CSPs by surface-initiated photopolymerization. (A) 2,2,2-trichloroacetyl isocya-nate, tetrahydrofuran, r.t., 1 h-701C, 2 h; (B) Mn2(CO)10, metha-nol, irradiation with a 125 W high-pressure Hg lamp, 401C, 3 h.



9.41 mmol/m2, corresponding to 810 mmol of monomer

units per gram of matrix. These values are larger than those

obtained on the thermal triggered CSP3, prepared from the

same chiral monomer and azo-activated silica (5.14 mmol/

m2, corresponding to 488 mmol/g of matrix).

The surface coverage of CSP2 based on carbon

content is 6.95 mmol/m2, corresponding to 591 mmol/g of

matrix. Again, these values are higher when compared to

those found on the homologue thermal triggered CSP4

(1.99 mmol/m2, corresponding to 196 mmol/g of matrix).

Further inspection of elemental analysis data yields the

average number of chiral monomer units of the new CSPs.

Thus, considering the number of mol of monomer units per

square meter of matrix (last column in Table 1), it is clearly

seen that all the aminogroups are converted to trichlor-

oacetyl derivatives, and that the averaged number of

monomer units in CSP1 and CSP2 is about 3.3 and 2.5,

respectively.

These findings clearly indicate that the photo-induced

grafting reaction yields higher polymer loading and grafting

efficiency in a shorter time, compared to the thermal

process, and no radical fragments escape from the silica

surface. Moreover, activation of the aminopropyl silica is

much easier in this case, as no by-products are generated.

Notably, the photo-induced process turned out to be the

method of choice for the grafting polymerization of DPEDA-

ACR, which showed low reactivity under thermal conditions

(6.95 versus 1.99 mmol/m2). Moreover, the solvent change

from chlorofom (the solvent used in the thermal process)

to methanol (the low wavelength UV cut-off solvent

used in the photo-triggered process) has a negligible effect

on the outcome of the DPEDA-ACR thermal grafting

polymerization.

With regard to the temperature of photochemical reac-

tion, we chose 401C to be sure that both chiral monomers

would be soluble and yield a suitable final concentration in

the reaction solvent.

3.4 Enantioresolution capabilities of the new poly-

acrylamide CSPs

In an effort to better understand the relationship between

structural features of the analytes and their retention and

enantioselectivity, a preliminary investigation was carried

out by screening a set of chiral compounds on the analytical

columns packed with the new CSPs. For comparison

purposes, retention and enantioselectivities were also

checked, under the same experimental conditions, on the

CSPs prepared by the thermal process.

The structures of the investigated analytes are shown in

Fig. 4. They can be divided into two sets, according to their

relative polarity and the elution mode chosen to analyze

them. Compounds 1�6 have no easily ionizable fragments

and can be eluted under NP conditions, using a single polar

mobile phase for 1�5 (dichloromethane/methanol 97:3 v/v)

and a less polar eluent for 6 (hexane/ethanol 90:10 v/v).

Compounds 7�12 are N-protected amino acids with differ-

ent fragments on the amino group and structural variations

on the side chains, and all of them have a free carboxyl

group that required polar organic solvents containing

20 mM ammonium acetate for their HPLC elution.

Inspection of CSPs and analytes structures suggests

that CSP-solute H-bonding is the dominant interaction

governing retention and, to some extent, enantioselectivity

4000 20000

20

40

60

80

100

120

% T

20

40

60

80

100

120

% T

0

20

40

60

80

100

% T

wavenumber / cm-1

3600 3200 2800 2400 1600 1200 800 400

4000 20003600 3200 2800 2400 1600 1200 800 400

4000 20003600 3200 2800 2400 1600 1200 800 400

Figure 3. FT-IR (DRIFT) spectra of 3-APSG-COCCl3 silica gel,CSP1 (CSP-poly-(R,R)-DACH-ACR), and CSP2 (CSP-poly-(R,R)-DPEDA-ACR) (from top to bottom).



in the examined systems. Amide C=O groups of our CSPs

may accept H-bonds from alcoholic, phenolic, amidic, or

carboxylic groups of the analytes, whereas amide NH groups

on the CSPs can act as H-bond donors towards H-bond

acceptor sites of the analytes. Dipole–dipole interactions

and, for CSP2 and CSP4, p–p interactions with aromatic

substrates are also expected to modulate both retention and

enantioselectivity.

Chromatographic data collected on the four CSPs 1�4

using NP elution mode for compounds 1�6 are listed in

Table 2. Inspection of retention (k1 and k2) and enantio-

selectivity (a) data disclosed marked differences between the

chromatographic behavior of photo- and thermal-induced

poly-DACH-ACR CSPs. Retention of both the enantiomers

is always larger on CSP1 (with the exception of the second

eluted enantiomer of compound 3), as a result of the higher

polymer loading obtained in the photo-initiated process (see

Table 1). An additional aspecific contibution to retention on

CSP1 can in principle arise from the polar acylurea frag-

ments present on the surface of the photo-initiated CSP.

Enantioselectivity is in favor of CSP3 for compounds 2�4and 6: for instance, compound 2 is resolved with a5 4.36 on

CSP3 and a5 2.61 on CSP1, and the difference in enan-

tioselectivity in favor of CSP3 is due to the decreased

Table 1. Surface coverage characterization of the polyacrylamide CSP1�CSP4 and of aminopropyl silica gel precusors

Support Chiral selector C (%) H (%) N (%) mmol/gc) mmol/m2

3-APSGa) � 1.47 0.46 0.44 325 (314) 2.82

3-APSG-COCCl3a) � 2.60 0.46 0.83 324 (296) 2.82

CSP1 (hn-induced)b) poly-(R,R)-DACH-ACR 15.12 2.19 2.95 1082 (810) 9.41

CSP3 (D-induced)b) poly-(R,R)-DACH-ACR 10.90 1.76 2.30 590 (488) 5.14

CSP2 (hn-induced)b) poly-(R,R)-DPEDA-ACR 17.25 1.99 2.33 799 (591) 6.95

CSP4 (D-induced)b) poly-(R,R)-DPEDA-ACR 7.99 1.13 1.39 229 (196) 1.99

a) Calculation was based on nitrogen content.

b) Calculation was based on carbon content.

c) mmol of monomer units per gram of starting silica (in parentheses are reported the same data relative to final matrix).

Figure 4. Chemical structures of chiral compounds.



retention of both the enantiomers, compared to CSP1.

Compound 5, on the other hand, is better resolved on the

photo-induced CSP1 (a5 3.19) than on the thermal-induced

CSP3 (a5 2.07). A similar picture is observed for retention

data collected on the photo- and thermal-induced poly-

DPEDA-ACR CSPs, with the analytes showing larger affi-

nities for the photo-induced CSP2 compared to the corre-

sponding thermal-induced CSP4. Enantioselectivity in these

cases is in favor of CSP4 for compounds 1�3, whereas

compounds 5 and 6 are better resolved on CSP2, and

compound 4 shows no enantioseparation at all on the two

CSPs (a5 1.00). Figures 5 and 6 show the chromatographic

resolutions of compounds 6 and 4, respectively, on the four

different CSPs, under the same experimental conditions. In

most cases, narrow and symmetric peaks are observed for

the resolved enantiomeric analytes, indicating a fast and

efficient solute mass transfer between mobile phase and

polymeric stationary phases.

Taken together, these data clearly indicate that the two

CSPs prepared by photo-initiated polymerization have

increased retention under NP elution for compounds 1�6,

compared to the CSPs prepared by thermal-initiated poly-

merization. The higher polymer loading and the polar

acylurea moiety from which the polymer grows in CSP1 and

CSP3 may account for the observed differences in retention.

Enantioselectivity has no clear cut dependence on the

analyte structure and on the polymerization conditions:

here, the interplay of several factors including polymer

loading, polymer structure, conformation, and mobility,

aspecific solute–CSP interactions established which of the

Table 2. Chromatographic data obtained for the enantioresolution of compounds 1–6 (Fig. 4) by polyacrylamide CSPs under NP

conditions

Normal phase (NP) poly-DACH-ACR-CSPs poly-DPEDA-ACR-CSPs

hn-induced CSP1 D-induced CSP3 hn-induced CSP2 D-induced CSP4

Compound Eluent k1 k2 a2,1 k1 k2 a2,1 k1 k2 a2,1 k1 k2 a2,1

1 CH2Cl2/MeOH 97:3 0.10 0.19 1.90 0.09 0.16 1.78 0.19 0.48 2.53 0.04 0.14 3.50

2 CH2Cl2/MeOH 97:3 3.41 8.89 2.61 1.25 5.45 4.36 1.98 2.72 1.37 0.61 0.95 1.56

3 CH2Cl2/MeOH 97:3 7.99 9.54 1.19 6.03 9.81 1.63 8.27 15.02 1.82 3.77 7.36 1.95

4 CH2Cl2/MeOH 97:3 3.55 4.88 1.37 2.00 3.49 1.75 0.35 0.35 1.00 0.15 0.15 1.00

5 CH2Cl2/MeOH 97:3 0.21 0.67 3.19 0.15 0.31 2.07 0.33 0.59 1.79 0.28 0.33 1.18

6 Hexane/EtOH 90:10 11.59 11.59 1.00 4.85 4.98 1.03 6.35 12.14 1.91 3.80 4.56 1.20

0

Minutes

0

Minutes

0

Minutes

0

Minutes

Δ Δ

5 10 15 20 25 10 20 30 40 50

5 10 15 20 255 10 15 20 25

Figure 5. NP HPLC of compound 6 onhn-induced – (top right) and D-induced – (bottom right) CSPs-poly-(R,R)-DACH-ACR, together with hn-induced – (top left) and D-induced –(bottom left) CSPs-poly-(R,R)-DPEDA-ACR. Eluent: n-hexane/etha-nol, 90:10 v/v; flow-rate: 1.0 mL forCSP1, CSP2, CSP4 and 1.3 mL/min forCSP3; T 5 251C; detection: UV at254 nm.



two phases performs better in terms of enantiomeric

recognition capability.

Chromatographic data collected on the four CSPs 1�4

using POM for compounds 7�12 are listed in Table 3.

Compounds 7�12 all have a free carboxyl group whose

presence required the addition of ammonium acetate

in the mobile phase to have short analysis times and

acceptable peak shapes (see Fig. 7 for compound 7).

Retention of the analytes under these conditions is

mainly controlled by the ionizable carboxyl fragment, and

retention differences between photo- and thermal activated

CSPs are less pronounced compared to NP conditions. In

most cases, retention is higher on the CSPs prepared by

thermal-initiated polymerization. The differences in enan-

tiorecognition abilities between photo- and thermal-induced

poly-DACH CSPs are smaller compared to the NP elution

mode, and here are always in favor of CSP3, except for

compound 6. Comparison of enantioselectivity data of

photo- and thermal-induced poly-DPEDA CSPs shows, on

the other hand, that the photo-induced CSP2 has larger

values and that CSP4 fails to resolve five out of six

compounds.

Examination of the enantioselective properties of the two

CSPs prepared by photo-initiated polymerization suggests

that their selectivities are influenced by a combination of

different factors, thus offering rather complementary appli-

0

Minutes

0

Minutes

0

Minutes

Minutes

Δ Δ

5 10 15 20 251 2 3 4 5

0 1 2 3 4 5 5 10 15 20 25

Figure 6. NP HPLC of compound 4 onhn-induced – (top right) and D-induced – (bottom right) CSPs-poly-(R,R)-DACH-ACR, together with hn-induced – (top left) and D-induced –(bottom left) CSPs-poly-(R,R)-DPEDA-ACR. Eluent: dichloro-methane/methanol, 97:3 (v/v); flow-rate: 1.0 mL for CSP1, CSP2, CSP4and 1.3 mL/min for CSP3; T 5 251C;detection: UV at 254 nm.

Table 3. Chromatographic data obtained for the enantioresolution of compounds 7–12 (Fig. 4) by polyacrylamide CSPs under POM

conditions.

Polar organic mode (POM) poly-DACH-ACR-CSPs poly-DPEDA-ACR-CSPs

hn-induced CSP1 D-induced CSP3 hn-induced CSP2 D-induced CSP4

Compound Eluent k1 k2 a2,1 k1 k2 a2,1 k1 k2 a2,1 k1 k2 a2,1

7 ACN/MeOH 70:30 1 20 mM AcONH4 0.67 0.82 1.22 0.73 0.97 1.33 0.53 0.68 1.28 0.73 0.73 1.00

8 ACN/MeOH 70:30 1 20 mM AcONH4 0.63 0.78 1.24 0.72 0.90 1.25 0.63 0.63 1.00 0.87 0.87 1.00

9 ACN/MeOH 70:30 1 20 mM AcONH4 0.51 0.71 1.40 0.56 0.90 1.61 0.46 0.46 1.00 0.51 0.51 1.00

10 ACN/MeOH 85:15 1 20 mM AcONH4 1.90 2.17 1.14 1.26 1.53 1.22 1.08 1.20 1.11 1.30 1.30 1.00

11 ACN/MeOH 85:15 1 20 mM AcONH4 1.89 2.47 1.31 1.25 1.66 1.33 1.20 1.33 1.11 1.28 1.28 1.00

12 ACN/MeOH 85:15 1 20 mM AcONH4 2.43 2.74 1.13 1.71 2.10 1.23 1.50 1.98 1.32 1.53 1.64 1.07



cation fields. This is highligthed by the comparative plots of

Figs. 5 and 6, where enantioselectivity towards compound 6is observed only on poly-(R,R)-DPEDA-ACR CSP (see Fig. 5),

whereas compound 4 was resolved only by poly-(R,R)-DACH-

ACR CSP (see Fig. 6). Additionally, the two polymeric

selectors may have different enantioselectivities under the

same experimental condition, as shown in Fig. 7: a non-

racemic mixture ad hoc prepared by mixing the two enan-

tiopure enantiomers of Fmoc-Phe (compound 7) was resolved

on both CSP1 and CSP2, but an elution order inversion took

place by changing the selector moiety.

4 Concluding remarks

The surface-initiated photopolymerization of chiral, enan-

tiopure diacryloyl derivatives of 1,2-diamines on mesopor-

ous silica yielded hybrid polyacrylamide CSPs that have

been characterized by elemental analysis and DRIFT

spectroscopy. The new photopolymerization process is

highly efficient in terms of polymer loading and reaction

time, and leads to very stable hybrid polymeric organic–

inorganic CSPs. The photopolymerization process is

succesfull also for those monomers (DPEDA-ACR) that

are less reactive in the thermal polymerization. HPLC

columns packed with the above CSPs show good enantios-

electivities towards a broad range of compounds under NP

and POM elution conditions. Enantioselectivity is accom-

panied by high chromatographic efficiency and permeabil-

ity, as a result of a polymerization process that generates a

uniform polymer layer on the silica surface.

We thank Sapienza Universita di Roma, Italy (Funds forselected research topics 2008�2010) and Istituto Pasteur-Fondazione Cenci Bolognetti, Roma, Italy, for financial support.

The authors have declared no conflict of interest.

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0

Minutes

0

Minutes

Δ

Minutes

Minutes

Δ

2 4 6 8 10 0 2 4 6 8 10

0 2 4 6 8 102 4 6 8 10

Figure 7. POM HPLC of compound 7

(non-racemic mixture ad hocprepared by mixing the two enantio-pure enantiomers of Fmoc-Phe) onhn-induced – (top right) and D-induced – (bottom right) CSPs-poly-(R,R)-DACH-ACR, together with hn-induced – (top left) and D-induced –(bottom left) CSPs-poly-(R,R)-DPEDA-ACR. Eluent: acetonitrile/methanol, 70:30 v/v 1 20 mM ammo-nium acetate; flow-rate: 1.0 mL forCSP1, CSP2, CSP4 and 1.3 mL/min forCSP3; T 5 251C; detection: UV at254 nm.



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Hybrid polyacrylamide chiral stationary phases for HPLC prepared by surface-initiated photopolymerization