Porous Carbon Nanostructured Sorbents for Biomedical Application

Almagul R. Kerimkulova,a,b Seitkhan Azat,a,b and Zulkhair A. Mansurov3,1*

aDepartment of Chemical Physics and Material Science,

Al-Farabi Kazakh National University,

  • 71 Al-Farabi Ave., Almaty 050040, Kazakhstan bInstitute of Combustion Problems,
  • 172 Bogenbay Batyr Str., Almaty 050012, Kazakhstan

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This work is devoted to the study of physicochemical characteristics of carbonized apricot kernels in the processes of sorption and desorption of biomolecules. The molecular sieve characteristics of protein-lipid complexes, adsorption chromatography of the biostimulator, sorption and desorption of such toxins as pesticides and cytokine on a nanostructured porous sorbent are investigated.

Carbon Nanomaterials in Biomedicine and the Environment

Edited by Zulkhair A. Mansurov

Copyright © 2020 Jenny Stanford Publishing Pte. Ltd.

ISBN 978-981-4800-27-3 (Hardcover), 978-0-429-42864-7 (eBook) www.jennystanford.com


Physical and Chemical Basis of Absorption, Hydrophobic and Gel Chromatography

Chromatography (from Greek chromos (color) andgrafos (to write)), the method for separation and analysis of mixtures is based on different distribution of their components between two phases: fixed (sorbent) and mobile (eluent). Such unusual name of this method is beholden to its discoverer, the Russian botanist Mikhail Semenovich Tsvet. In 1900, he conducted experiments with ethanolic extracts from leaf. He dropped these extracts on the pieces of chalk and over the time on the surface of chalk pieces there appeared colorful colors of bright green, yellow-green and orange colors. Which were conditioned by the separation of leaf pigments: chlorophylls and xanthophyll [1]. The author, after seeing this picture, called this method chromatography (color) and as a consequence, he skillfully entered his name to the method—Color (Tsvet). During the years 1900-1906, he improved this method and invented column chromatography for the separation of leaf pigments [2]. Currently, chromatography leads in various industries and formations.

Chromatography is able to bind with the sorbent. Various binding forms with the sorbent are used. It can be London dispersion forces (adsorption chromatography), electrostatic forces (ion-exchange chromatography), or molecular size differences (molecular-sieve chromatography). Depending on aggregative state of the eluent, there are distinguished gas and liquid chromatography. Chromatographic separation is carried out in tubes that filled with a sorbent (column chromatography); in capillaries with a length of several tens of meters, which walls are covered with sorbent (capillary chromatography); on plates that covered with adsorption layer (thin-layer chromatography); on paper (paper chromatography) [3-4]. Chromatography is widely used in laboratories and industry in order to control the production and isolation of individual substances.

Let's consider the main intermolecular forces that used in chromatographic separation. Adsorption chromatography is based on London dispersive forces [5]. For the first time the London forces were discovered half century ago by the Dutch physicist van der Waals. He noted that long and careful grinding of two bronze plates leads to the following effect. The combination of these two plates leads to their strong adhesion and these plates are difficult to separate. Because the bronze does not have magnetic properties, he introduced a concept of van der Waals radius, which is equal to 2-5 angstroms. Van der Waals suggested that between bodies that come close to such distance, attracting force occurs [6].

The scientific basis of this phenomenon was revealed by the English physicist London. In accordance with London technique, the convergence of atoms of two different molecules to van der Waals radius leads to synchronization effect of electrons motion, this is due to the fact that electrons of different molecules cannot be close to each other and therefore they are forced to rotate synchronously being apart to each other. If one atomic electron A is situated at a distance of the van der Waals radius from atomic nucleus В in another molecule, so an instantaneous attractive force arises between them. After a billionth of second, the atomic electron В will be at a distance of van der Waals radius from atomic nucleus A, and also there occurs an instantaneous attracting force. Alternately arising instantaneous forces were called dispersion forces by London [7]. Hydrophobic effect is the most common example of London dispersion forces. When London has created a theory of dispersion forces, he did not say what reason causes the molecules to contingence with van der Waals radius. In case of hydrophobic interaction, we can observe the contingence mechanism of molecules to van der Waals radius. This mechanism is explained by the antagonism of polar water molecules with uncharged hydrophobic molecules. As a result of Brownian motion, the water molecules cause the movement of hydrophobic molecules. Moreover, the water molecules situated between hydrophobic molecules due to antagonism penetrate easily to the aqueous medium. As a result, the hydrophobic molecules approach the van der Waals radius and as a consequence the London dispersion forces arising between them. Thus, the hydrophobic interaction is directly proportional to temperature, but this dependence can preserve to the temperature below 70°C. The more atoms will be approach the van der Waals radius, so the hydrophobic interaction will be stronger [8]. On the principles of hydrophobic interaction, there is formed a separate perspective type of chromatography: hydrophobic chromatography. Swedish firm "Pharmacia” has created hydrophobic sorbents (octyl-sepharose 4B and phenyl-sepharose 4B) [9]. These sorbents represent agarose gel balls to which the hydrophobic octyl or phenyl radicals are grafted. For good sorption of hydrophobic substances, the sorption is carried out at a temperature of 30-400°C. Desorption of the substance is carried out by washing the chromatographic column with ethanol solution of various concentrations or detergent solutions. Adsorption chromatography is a sort of chromatography is based on the ability of solid substance (stationary phase) to sorb the substances located in mobile phase. At the same time, the separation efficiency of substance is proportional to their adsorption amount under the experimental conditions. The interacting process can be accompanied by the chemical interaction of substances with stationary phase, it means chemisorption [10].

The separation of mixture components (sample) is based on their different sorbability using solid adsorbent. Activated charcoal, calcium phosphate gel as well as aluminum and silicon oxides are used as adsorbents. Adsorbent in the form of suspension with solvent (most often with buffer solution) is introduced into glass vertical tube (column), which is filled uniformly [11]. The sample with small amount of solvent is applied to the column as shown in Fig. 6.1.

Absorption chromatography

Figure 6.1 Absorption chromatography: (1) Application of sample to the column; (2) middle of the experiment; (3) experiment completion [12].

Separation of two different substances (A and B) moving across the column with different speeds happens. Components of separated mixture are adsorbed on adsorbent. Fractional collection is carried out using automatic fraction collector. Molecular-sieve chromatography (MSC) appeared in 1959 and was introduced into modern practice by Swedish scientists (J. Porath, P. Flodin) [13]. Until the present there is no common generally accepted term for this type of chromatography. It is known as "gel filtration”, "gel chromatography", and "permeation chromatography". Although this method is relatively young, it has received the widespread distribution and recognition among the technologists. Today, many studies are not possible without this method. For molecular-sieve chromatography, there are used two classes of carriers such as xerogels and aerogels. Xerogels are organic polymers swelling in solution and forming gel particles from three-dimensional polymer chains. Thus, the gel consists of three-dimensional grid of polymeric chains and contains solvent. In principle, aerogels are not gels, because do not contain a liquid phase [14]. An example are porous glasses and coal sorbents.

Structural differences in classes of gels are manifested primarily in their relation to the solvent. Xerogels give strongly swollen solvated gels that are not resistant to compression, and therefore do not withstand a large hydrostatic pressure, having also a low flow velocity. Aerogels do not have the above properties, and possessing large pores and biggest non-specific adsorption. These differences in sorbent types defined their application for fractionation of various substances with molecular weight [15]. Before proceeding to this, it is necessary to present a general mechanism for separation of substances on the columns with these sorbents. So, all sorbents types having porous structure, when molecules with different sizes passing through the sorbent, the molecules with smaller size (than pore size) can diffuse into sorbent particles, but largest one will move only in liquid volume is surrounded by sorbent particles. As a result, the smaller molecules will pass much larger way in liquid volume, and therefore they will stay longer on the column than larger molecules [16].

Distribution coefficient is an important characteristic for molecular-sieve chromatography that shows how the divided molecules distributed between free liquid volume, located between the sorbent particles and entire liquid volume of the column. Molecules that having large dimensions and escaping in free volume, which is equal to liquid volume is located between sorbent particles will have a coefficient is equal to V0, whereas the small molecules penetrating into all pores of sorbent will have a coefficient equal to Vt. Such numerical values of coefficients are observed during separation of substances that are very different in molecular weights, for example, at demineralization of large protein molecules [17]. In this case, the elution volumes for protein will be different very much from those of salts, this fact allows to apply large amounts of protein on the column—up to 15-20% of the total volume of sorbent. When fractionating the molecules that do not differ so much in molecular weight, their distribution coefficients will be closer to each other. This has made it possible to use effectively the molecular- sieve chromatography for determination of molecular mass of substances. At the same time, close values of distribution coefficient of molecular masses impose the restrictions on the volume of applied sample: it should be not more than 3% from column volume. Table 6.1 shows the xerogels and aerogels used in cleaning. Currently, the most widely used gel sorbents "Sephadex” produced by Pharmacia firm in Sweden. They derive their name from the initial syllables of English words Separation Pharmacia dextran, which means "separating dextran of Pharmacy” [18]. Dextran is starch oligomers. Sephadexes are extracted from natural dextran, cross-linked by epichlorohydrin in non-aqueous medium up to required porosity. Highly cross-linked gels like G-25 are differ by rigidity and can withstand high compression, whereas the gels like G-150 and G-200 have a friable structure and they are unstable under the influence of hydrostatic pressure. Negative side of weakly cross-linked gels: during their preparation they swell for a long time—up to three days. In order to accelerate the swelling, after suspending in water, it is incubated in water bath for 1-3 h, so the hydrogel is degassed in a vacuum. If the column does not fill completely by swollen gel, it may swell in the column and as a consequence the gel particles deforming greatly, and separation quality will be worse.

Table 6.1 Characteristics of sorbents for MSC

Name of sorbent and its type

Firm and country

Molecular weight separation range


Sephadex G-10



Sephadex G-15



Sephadex G-25


Sephadex G-50


Sephadex G-75


Sephadex G-100


Sephadex G-150


Sephadex G-200


Sephadex LH-20




Biogel P-10

United State



Biogel P-4


in non-aqueous

Biogel P-6



Biogel P-10


Biogel P-30


Biogel P-60


Biogel P-100


Biogel P-200


Biogel P-300


Ultragel AsA 22



Ultragel AsA 34



Ultragel AsA 44



Ultragel AsA 5



Sefacryl C-200,






Sefacryl C-300,







Sepharose 2B



marks like Cl are

Sepharose 4B



also produced

Sepharose 6B

The choice of column for molecular-sieve chromatography depends on the tasks facing the researcher. For fractionation, the sufficient ratio of the column diameter to the height of the gel is 1:10, but for determination of molecular masses of proteins, a necessary ration should be equal to 1:20 or even 1:30, at the same time, in narrow columns the wall effects are strong and impairing the separation quality. It would be better to take ready-to-use columns with cooling-water jackets. In the absence of alike you can use homemade items, but it is necessary to make it with free spaces. This applies especially for the lower part of the column, because the presence of the volume at least 2 ml can cause an eluent mixing and distorts the separation pattern, so the manufacturing of columns from glass burettes that filled by glass wool at the bottom end is not recommended [19].

Among the detectors that used in high-performance liquid chromatography (HPLC), the differential refractometers are most universal (allowing to measure the difference of refractory indexes of eluent and eluate) as well as streaming current detectors, commonly used in gas chromatography. The molecules of analytical substances contain chromophoric groups, which absorption region does not overlap with absorption region of eluent [20].

UV-detectors are most widely used, and with their help it is possible to detect the substances that absorbing the light in the region from 190 to 340 nm (the detection limit is several nanograms], it possess by high resistance to variations in flow rate of eluate and temperature. A wide range of measurements (about 105] give an opportunity to determine both large and trace amounts of substances in one experiment. UV detectors allow to make measurements either at the maximum of absorption band, or at the wavelength providing the maximum selectivity [21].

The Bouguer-Lambert-Beer law is a physical law is determining the weakening of parallel monochromatic light beam when it propagates in absorbing medium [22]. The law is expressed by the following formula:

where I0 is the intensity of input beam, / is the thickness of substance layer through which the light is passing, к/, is an absorption index (very often is incorrectly called absorption coefficient).

The absorption index is characterizing the substance properties and depends on wavelength A of absorbed light. This dependence is called absorbance spectrum of the substance.

The Bouguer-Lambert-Beer law was discovered experimentally by the French scholar Pierre Bouguer in 1729, and studied in details by the German scholar I. G. Lambert in 1760, as well as it was tested in practice on C concentration by German scholar A. Ber in 1852.

For solutions that absorbing substances in non-absorbent solvents, the absorption index can be written as:

where %X is a coefficient that characterizing an interaction of the absorbing substance molecule with light of wavelength Я, and C is the concentration of the soluble substance.

The assertion that does not depend on C is called Beer's law. Its meaning lies in the fact that the ability of a molecule to absorb the light does not depend on the state of other surrounding molecules. However, there are numerous deviations from this law, especially in the case of high concentrations of C [23].

In this work we used a flowing UV detector of "LKB firm” which measures the light adsorption at the wavelengths of 260 and 280 nm simultaneously.

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