Adsorption of cytochrome c on new mesoporous carbon molecular sieves

Article abstract of DOI:10.1021/jp035246f

Carbon mesoporous molecular sieves have been prepared from SBA-15 materials synthesized at different temperatures. This allows the synthesis of hexagonally arranged mesoporous carbons with pore diameters between 3.0 nm (CMK-3-100) and 6.5 nm (CMK-3-150). The novel materials have been studied in the adsorption of horse heart cytochrome c from solutions with different pH. A maximal adsorption capacity of 18.5 μmol/g was found for CMK-3-130 (d_p = 4.5 nm) at pH 9.6, which is near the isoelectric point of cytochrome c (pI = 9.8).

Full text of DOI:10.1021/jp035246f

J. Phys. Chem. B 2003, 107, 8297-8299
8297
Adsorption of Cytochrome C on New Mesoporous Carbon Molecular Sieves
A. Vinu,†,‡ Carsten Streb, V. Murugesan, and Martin Hartmann*
‡
†
,‡
Kaiserslautern UniVersity of Technology, Department of Chemistry, Chemical Technology, P.O. Box 3049,
D-67653, Kaiserslautern, Germany, and Anna UniVersity, Chennai 600 025, India
ReceiVed: May 7, 2003; In Final Form: June 16, 2003
Carbon mesoporous molecular sieves have been prepared from SBA-15 materials synthesized at different
temperatures. This allows the synthesis of hexagonally arranged mesoporous carbons with pore diameters
between 3.0 nm (CMK-3-100) and 6.5 nm (CMK-3-150). The novel materials have been studied in the
adsorption of horse heart cytochrome c from solutions with different pH. A maximal adsorption capacity of
1
8.5 µmol/g was found for CMK-3-130 (d
p
) 4.5 nm) at pH 9.6, which is near the isoelectric point of
cytochrome c (pI ) 9.8).
In recent years, porous carbon materials have been used for
many applications such as catalysts, battery electrodes, capaci-
tors, gas storage, biomedical engineering, and adsorbents. For
and sucrose as the carbon source. The obtained materials were
designated CMK-3-100, CMK-3-130, and CMK-3-150,
where the number indicates the synthesis temperature of the
2
1
such applications, the carbon materials should possess a high
mesoporosity allowing the adsorption of molecules and ions that
are too large to enter micropores. Ordered mesoporous carbons
prepared from mesoporous silica templates possess large specific
surface areas, large specific pore volumes, and electrical
conductivity and are of general interest owing to their potential
applications, especially as adsorbents for relative bulky
SBA-15 template. In a typical synthesis of mesoporous carbon,
1
1
g of template was added to a solution obtained by dissolving
.25 g of sucrose and 0.14 g of H2SO4 in 5 g of water, and
keeping the mixture in an oven for 6 h at 373 K. Subsequently,
the oven temperature was raised to 433 K for another 6 h. To
obtain fully polymerized and carbonized sucrose inside the pores
of the silica template, 0.8 g of sucrose, 0.09 g of H2SO4, and 5
g of water were again added to the pretreated sample and the
mixture was subjected to the thermal treatment described above.
The template-polymer composites were then pyrolyzed in a
2
-5
molecules.
Synthesis of large pore mesoporous carbons is
extremely important for the envisaged applications in the
separation and adsorption of bulky molecules such as proteins
and enzymes.
-
1
nitrogen flow at a heating rate of 5 K min up to 1150 K and
kept under these conditions for 6 h to carbonize the polymer.
The mesoporous carbon was recovered by filtration after
dissolution of the silica framework in 5 wt % hydrofluoric acid,
washed several times with ethanol and dried at 393 K.
Adsorption of cytochrome c (cyt c) over porous glass and
sol-gels has been extensively studied due to potential applica-
6
,7
tions in the fields of bio-sensing and enzymatic catalysis.
However, porous glass materials are expensive and sol-gel
materials have a broad pore size distribution (PSD), and, hence,
cannot be used to selectively adsorb proteins with specific
molecular size. Some studies are dealing with the adsorption
For cyt c adsorption, a series of standard cyt c solutions with
concentrations ranging from 0.25 g/L to 4.0 g/L were prepared
by dissolving cyt c in different 25 mM buffer solutions (pH )
6.5 potassium phosphate buffer and pH ) 9.6 and 10.5 sodium
bicarbonate buffer). Horse heart cyt c (90% pure) was obtained
from Acros and used without further purification. In each
adsorption experiment, 20 mg of the mesoporous adsorbent were
suspended in 4 mL of cyt c solution. The resulting mixture was
continuously shaken at 293 K for 96 h to reach the adsorption
equilibrium. The amount of cyt c adsorbed was determined by
UV absorption at 409 nm.
of cyt c over mesoporous silica molecular sieves such as MCM-
-10
4
1, MCM-48, and SBA-15.8
However, for these materials
strong electrostatic interactions are observed between the surface
silanol groups and the surface charge on the amino acid residues
8
on the surface of cyt c, which affect the structure and orientation
of the active (heme) site of cyt c. Recently, we have reported
the synthesis of SBA-15 materials with different pore diameters
11
prepared by changing the synthesis temperature. We used these
materials as templates for the preparation of mesoporous carbons
with large pore diameter. Here we report the adsorption of cyt
c on (hydrophobic) mesoporous carbon molecular sieves.
Three different SBA-15 materials with pore diameters in the
range of 9 to 12 nm were prepared according to the method
reported by Hartmann and Vinu,11 and designated as SBA-15-
X, where X indicates the synthesis temperature (X ) 100, 130,
and 150 °C). Mesoporous carbons with different pore diameters
were prepared by using the SBA-15-X materials as templates
Figure 1 shows the powder X-ray diffraction patterns (XRD)
of CMK-3-100, CMK-3-130, and CMK-3-150. These ma-
terials are hexagonally ordered mesostructures, as is evident
from the presence of at least three XRD lines that can be indexed
as (100), (110), and (200) reflections. Consequently, the obtained
materials are a replica of the parent material SBA-15.2 The
low intensity of the higher order reflections in the case of CMK-
,6
3
-150 indicates that some structural changes occurred during
*
Author to whom correspondence should be addressed. Phone:
49-631-205-3559. Fax: +49-631-205-4193. E-mail: hartmann@
chemie.uni-kl.de.
silica template removal. This reduced stability is probably a
consequence of the changes in the number and size of the carbon
rods (formed from the micropores of SBA-15) that connect the
carbon rods formed from the main pores of SBA-15. The
+
†
Anna University.
Kaiserslautern University of Technology.
‡
1
0.1021/jp035246f CCC: $25.00 © 2003 American Chemical Society
Published on Web 07/23/2003

8298 J. Phys. Chem. B, Vol. 107, No. 33, 2003
Letters
TABLE 1: Textural Properties of the Mesoporous Carbon
Supports and the SBA-15 Parent Materials
pore
pore
unit cell specific specific
diameter diameter
a
b
c
size a0 surface area pore volume
dp,BJH
(nm)
dp,NLDFT
(nm)
2
3
sample
(nm)
(m /g)
(cm /g)
SBA-15-100 10.22
SBA-15-130 10.54
SBA-15-150 10.83
CMK-3-100 10.07
CMK-3-130 10.45
CMK-3-150 10.75
1100
546
393
1260
1250
1350
1.60
1.19
1.10
1.1 (0.41)
1.3 (0.46)
1.6 (0.49)
9.0
8.7
11.2
3.0
4.3
6.5
3.3
4.3
5.4
a
Total pore volume calculated at p/p
< 1.4 nm). b Calculated from the adsorption
branch of the isotherm. c Pore sizes obtained from the N
isotherm
following NLDFT (nonlocal density functional theory) analysis.
0
) 0.88. Values in parentheses
are micropore volumes (d
p
2
1
3
Figure 1. XRD powder patterns of the mesoporous carbon materials
CMK-3-150, CMK-3-130, and CMK-3-100.
Figure 3. Adsorption of cyt c on CMK-3-130 at various solution
pH. The solid lines are fits using the Langmuir isotherm.
shows a sharp initial rise (type L isotherm), suggesting a high
affinity between cyt c and the adsorbent surface. Finally, the
saturation level is reached at concentrations above 2 g/L. The
adsorption of cyt c is typically determined by electrostatic and
hydrophobic interactions (a kind of van der Waals attraction)
and the cohesive attraction and repulsion of the amino acids
present in the cyt c molecule. The adsorption maximum is also
influenced by the expansion and contraction of the molecular
diameter of the cyt c molecule at different pH. It is evident
from Figure 3 that the amount of adsorption is significantly
affected by the pH of the cyt c solution and increases from pH
6.5 to 9.6 and decreases at higher pH (10.5). The amount of
cyt c adsorbed is maximal (18.5 µmol/g; determined using the
monolayer capacity of the Langmuir fit) at pH 9.6, which is
near the isoelectric point (pI) of cyt c (pI ) 9.8). Near the
isoelectric point, the net charge of cyt c is around zero. Thus
the hydrophobic interactions between adsorbate and adsorbent
Figure 2. Nitrogen adsorption isotherms at 77 K (a) and BJH pore
size distributions (b) of mesoporous carbon materials (closed sym-
bols: adsorption; open symbols: desorption). The isotherms are shifted
3
by 300 cm /g for clarity.
number of micropores is drastically reduced in the case of SBA-
1
1,12
1
5-150, which was used as a template for CMK-3-150.
Figure 2a shows the nitrogen adsorption isotherms at 77 K
of CMK-3-130 and CMK-3-150 along with the BJH-PSD
Figure 2b) in comparison to CMK-3-100. All isotherms are
14
(carbon rods are hydrophobic ) are maximal. These hydropho-
bic interactions may either originate from attraction of the
nonpolar side chains of the amino acids residues on the surface
of the cyt c molecule by carbon species (chains, aromatic rings,
etc.) at the adsorbent surface or from cyt c-cyt c interaction
between the hydrophobic side chains of neighboring cyt c
molecules adsorbed on the surface of the mesoporous carbon.
As there is no repulsion between the amino acid residues on
the surface of adsorbed cyt c molecules near the pI, the cyt c
molecules are more densely packed on the adsorbent surface.
As a result, the amount of adsorption increases near the pI of
cyt c. The adsorption of cyt c is reduced below and above the
isoelectric point. This behavior is ascribed to the expansion of
the effective molecular diameter of cyt c since strong repulsive
forces between the amino acid residues of the molecule are
(
of type IV, according to the IUPAC classification, and exhibit
a H1 hysteresis loop. The materials obtained from SBA-15
prepared at 130 and 150 °C possess pores with diameters of
about 4.3 and 6.5 nm, high BET surface areas of 1250 and 1350
2
3
m /g, and large pore volumes of 1.3 and 1.6 cm /g, respectively
Table 1). Pore diameter and specific pore volume are signifi-
cantly larger than those of CMK-3-100 (dp ) 3.0 nm, ABET )
(
2
3
1
260 m /g, and Vp ) 1.1 cm /g) and facilitate the adsorption of
cyt c (size: 2.6 × 3.2 × 3.3 nm).
Adsorption isotherms of cyt c adsorbed onto CMK-3-130
with different solution pH ranging from 6.5 to 10.5 are shown
in Figure 3. As cyt c is denatured at pH above 11, we precluded
the adsorption study outside the pH range 3-11. Each isotherm

Letters
J. Phys. Chem. B, Vol. 107, No. 33, 2003 8299
present below and above the pI and more space is needed for
the expanded cyt c molecule.
Among all the mesoporous carbon materials studied, CMK-
Fe(III) is responsible for the observed increased peroxidative
activity of cyt c. Studies on the peroxidative activity of cyt c
adsorbed on the different CMK-3 materials are currently
underway.
3-130 shows maximum adsorption of cyt c (18.5 µmol/g)
whereas CMK-3-100 and CMK-3-150 exhibit adsorption
capacities of 10.9 and 14.5 µmol/g, respectively. The low cyt c
adsorption observed for CMK-3-100 is probably due to its
smaller pore diameter and lower pore volume. CMK-3-100
possesses a broad pore size distribution (Figure 2b) centered at
In summary, mesoporous carbons with tunable pore diameters
from 3 to 6.5 nm can be prepared by using SBA-15 silica with
various pore diameters as template. These materials allow the
adsorption and separation of bulky molecules such as proteins
and vitamins. A high adsorption capacity of 18.5 µmol/g has
been observed for CMK-3-130 which is significantly higher
than that reported for mesoporous silicas such as MCM-41 and
SBA-15.
3
3
nm (BJH) or 3.3 nm (NLDFT) (Table 1), showing that CMK-
-100 has pores which are a few angstroms smaller or bigger
than the size of the cyt c molecule (ca. 2.6 × 3.2 × 3.3 nm).
Pores which are a few angstroms smaller than the cyt c molecule
might not be accessible (molecular sieving). Thus, CMK-3-
Acknowledgment. Financial support of this work by Fonds
der Chemischen Industrie is gratefully acknowledged.
1
3
00 shows a lower adsorption capacity as compared to CMK-
-130 and CMK-3-150. Moreover, the adsorption capacity
References and Notes
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(
(
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(
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4
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4
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1
9
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(