Stationary phases based on the nanoparticles for pharmaceutical and biomolecule separations

Document Type : Review Paper


1 1 Central Research Laboratory, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran 2 Chemistry Department, Yasouj University, Yasouj, Iran

2 Department of Inorganic Chemistry, Chemistry and Chemical Engineering Research Center of Iran, Iran.


Because of impossibility to achieve a general column for separation purposes, columns must be amended in order to acquire specific chemical characteristics such as hydrophobic, hydrophilic or ionic interactions. In this regard, particular properties of nanoparticles such as large specific surface, high pore volume, narrow particle size- and pore size distribution, and excellent stability introduce them as perfect supports for electrochromatography and chromatographic applications, which enhance the retention and separation efficiency. On the other hand, because of disadvantages of silica stationary phases, it is noteworthy to design new stationary phases for the separation process. Apart from that, the applications of nanoparticles in separation process give rise to reach a suitable mass transfer, which is significant in chromatographic science. Hence, the present report studies various stationary phases based on the nanoparticles for the analysis of biomolecules and chiral drugs using capillary electrochromatography and liquid chromatography techniques, which provide rapid and efficient separations, short run time, high enantioseparation, and less consumption of expensive stationary phases.



Nanoparticles (NPs) with the particle size of 1 up to 100 nm have attracted a great attraction in sciences of chemistry, physics, materials, and medicine because of their special properties. Large surface area of NPs results in an appropriate mass transfer and increasing separation efficiency [1, 2] which are important factors in binding several functional groups onto the surface [3, 4]. Despite the benefits of NPs, their utilization as a stationary phase (SP) in electrophoretic and chromatographic separations is still a challange. Different NPs such as metal oxide NPs, magnetic nanoparticles (MNPs), gold nanoparticles (GNPs), and carbon nanotubes (CNTs) have been suggested for SPs. NPs are also used as SP for enantioseparation. However, proteins [5, 6], polysaccharides [7, 8], cyclodextrins and their derivatives [9, 10], macrocyclic antibiotic [11], chiral crown ethers [12], neutral donor-acceptor selectors [13], chiral ion-exchangers [14], and ligand-exchange selectors [15] have been developed for the enantioresolution of capillary electrochromatography (CEC) and high performance liquid chromatography (HPLC). Among them, with high ratio of surface-to-volume, high chemical stability, special physical and chemical properties, NPs provide improving separation performance. Therefore, they have been generally used in chemistry, physics, materials, medicine, and optics.

Pseudostationary phases (PSPs), monolithic columns, and silica columns have been modified using various NPs. PSPs are considered as interaction phases, which are moving with or contrary the mobile phase. In PSP, no column filling or frits are used, the PSP is continuously changed, and a new column is used for each new determination. The mentioned properties give rise to achieve a rapid equilibration and no carry-over effects [16]. Ionic micelles [17], microemulsions [18], cyclodextrins [19], dendrimers [20] and NPs [21] have been recommended as the packing of PSPs.

Monolithic capillary columns have shown a great attraction in enantioseparation process owing to thier high permeability, quick mass transfer, and easy preparation [22-26]. Copolymerization of functional monomers, the chemical amendments of monolithic surface, the incorporation of NPs into the polymeric scaffold, and grafting polymerization have been applied to modify surface chemistries of monolithic columns [1, 27-31]. Modification of monolithic columns with NPs provides a large surface-to-volume ratio leading to a specific chemistry. Some researchers have modified the monolithic columns with NPs because of unique porous structure, excellent hydrodynamic characteristics, and the ease of employing the high flow rates [32-36]. NPs can be loaded into the polymer monolith supports via polymerization of their dispersion in monomers and porogens [37, 38] or are joined to the pore surface of desired monoliths [39, 40].

To prepare a monolithic column with a desired permeability and a favorable surface area, a porogen is used to form preferred morphologies [41]. Cyclohexanol with 1-dodecanol [42] and a mixture of dioxane and water were applied as the porogens [43]. Monolithic columns including rigid organic polymer appeared two decades ago [44, 45] and have been recognized as a suitable SP for rapid liquid chromatography (LC) [46, 47]. They have a little resistance to flow which allows fast separation of proteins and other large molecules [1, 44, 47-50]. Aydoğan and Rassi suggested two chief benefits for monolithic columns: (i) easy preparation using in situ polymerization process in columns and (ii) providing excellent flow [35]. In the current review, monolithic columns were modified using mesoporous silica NP [32], methacryloyl fumed silica NP (MFSNP) [35], GNP [51, 52], hydroxyapatite NP [53], fumed silica nanoparticles (FSNPs) (36), poly(divinylbenzene) (polyDVB) [34, 43], poly(ethylene glycol dimethacrylate) (polyEDMA) [34, 43], and Ag-NPs [42].

Immobilization of NPs within the capillary columns leads to a stable SP [32]. Regarding characteristics of NPs, it is noteworthy to describe some applied NPs as SPs in separation techniques. Among different kinds of NPs, MNPs are a good subject owing to their superior properties such as good dispersion, outstanding biocompatibility, high field irreversibility, easy preparation, large surface area, and physicochemical stability [54, 55]. MNPs were extensively applied in the field of biotechnology such as protein selective separation [55-59], nucleic acid extraction [60], bacteria trapping [59, 61-63], biomedicine [54] and biosensing [63]. However, as indicated by the literature, a few MNPs have been utilized as the SP of CEC [64]. MNPs can be modified using biological molecules to definitely react with the biological compounds of interest [65]. Additionally, an external magnetic field can change their magnetic effects [65, 66]. MNPs are generally based on magnetic elements; for example, iron, nickel, cobalt and their oxides such as magnetite (Fe3O4), maghemite (γ-Fe2O3), cobalt ferrite (Fe2CoO4), and chromium dioxide (CrO2). In the current review, Wang et al. [64], Liang et al. [67], Yang et al. [68], Sun et al. [69], and Liu et al. [70] used MNPs for the separation of biomolecules and drugs.

Gold nanoparticles (GNPs) as other types of NPs will be discussed because of their compatibility with biomolecules. There is a high affinity between gold and thiol groups. Therefore, the surface of GNPs can be changed. Simple and cheap preparation, easy formation of active materials with biological compounds, tunable particle size with distribution of narrow size make GNPs very attractive in nanoscience [71]. Usually, applied GNPs in separation fields are synthesized in liquid phase using chemical reduction of gold(III) existing in the form of HAuCl4 or AuCl4 [72]. Chemical concentration, surfactants, pH, and temperature can affect the size and shape of gold particles [72]. In the present review, the attachment of GNPs to the surface of capillary columns of CEC, HPLC columns and TLC plates have been reported [73-75]. To the best of our knowledge, 47% of NP-based SPs contain GNPs, which is higher than the contribution of other NPs.

Since the discovery of carbon nanotubes (CNTs) in 1991, they have been generally known as ideal nanomaterials in nanoelectronic devices, catalyst supports, biosensors and hydrogen storage with high strength, unique topological and electronic properties [37, 76-80]. CNTs exist as single-walled carbon nanotubes (SWCNTs) or multi-walled carbon nanotubes (MWCNTs), which are formed by van der Waals interactions to create a dense, robust and high hydrophobic structure. SWCNT includes a sheet of graphene (G) turned around a cylinder with a typical diameter of 1 nm, but MWCNT as a concentric cylinder has shown an interlayer spacing of 3.4 Å and a diameter usually in the range of 10-20 nm [37]. Dispersion of CNTs in aqueous media and in some organic solvents is challenging [81], because dispersion of CNTs in aqueous solutions causes re-agglomeration, which can produce an unstable baseline, irreproducible retention times and column obstruction [82]. To overcome these limitations, mechanical or chemical methods are employed. In mechanical procedures, sonication of CNTs suspension is done to acquire a temporary dispersion. Physical preparation (non-covalent) can be performed via addition of a surfactant after sonication. However, chemical treatment (covalent) can be accomplished by surface modification of CNTs using acids, sonication and high temperature [83]. Since the application of CNT in chromatography is limited, due to insolubility in the recognized solvents [37], among the studied reports, SWCNTs and MWCNTs have been used as a SP of CEC [81, 84].

The packed atoms of carbon in a hexagonal crystalline array and as a sheet of two dimensional arrangement generate G material [85], which is considered the fundamental form of all graphitic structures [86, 87]. The applications of G for the extraction of benzenoid compounds have attracted considerable attention due to the high delocalized π-electron system of G, enabling the π-stacking interaction with the benzene molecules [88-90]. Recently, G, known as a mono-layer of graphite, has become the subject of intense research because of its high thermal and electronic conductivity, great mechanical characteristic, and large surface area [67]. Hence, many investigations have reported numerous applications of G in several fields such as nanocomposites [91], nanoelectronics [92], supercapacitors [93], ultrasensitive sensors and batteries [94]. The lamellar form of G and the absence of functional groups in its structure lead to its unsuccessful utilization as SP in LC technique [95]. Accordingly, in the current review, modification of G was carried out using MNPs for separation of several amino acids, alkylbenzenes, polycyclic aromatic hydrocarbons, amines, and phenolic compounds [67]. The chemically amended sheet of G gives rise to the formation of graphene oxide (GO) [96]. Unlike hydrofobic and nonpolar structure of G, GO structure contains a lot of oxygen functional groups such as epoxide, hydroxy, and carboxylic acid [97, 98]. This property makes GO a promosing support for its interaction with amino groups of biomolecules and amino acids. Consequently, some investigations used GO NPs to separate several proteins, amino acids, alkylbenzenes, polycyclic aromatic hydrocarbons, amines, and phenolic compounds [67, 95, 96].

Silica nanoparticles have received a great deal of attention because they show poor cytotoxicity, superior chemical stability, simple chemical modification, and mass transfer facilitation owing to their large surface-area. These properties give a platform for the attachment of numerous compounds for separation [16, 99, 100]. Therefore, the large number of investigations has been developed to modify columns using silica NPs. Mesoporous SiO2 NPs (MSNs) with 2D network of honeycomb-like porous structure include hundreds of vacant channels [101]. Large surface area, pore volume (>1 cm3g−1), stable mesopore structure, controllable pore diameter, two functional surfaces, and adjustable morphology have been described as the structural properties of MSNs [102].

Considering the significant applications of NPs and nanostructured materials, the current review describes various NPs as SPs of CEC, HPLC, and thin layer chromatography (TLC) techniques by focusing on separation of biomolecules and chiral drugs.


As a separation technique, CE is used to separate ionic components and is based on the difference in electrophoretic mobility of charged analytes under applying an electrical field [103]. Electrophoretic procedures are known as the effective methods for the analysis of peptides and proteins because applying a potential difference affects the separation process based on the ratio of charge-to-mass [104]. However, there are some drawbacks with CE; for instance, the incapability to separate nonionic compounds and analytes with the same ratio of size-to-charge. To resolve these limitations, CEC, which combines the benefits of high selectivity of HPLC and high efficiency of CE, is suggested [105-107]. Several types of columns comprising packed, monolithic, and open tubular (OT) columns are generally used in CEC technique. Among them, OT columns are of interest for the detection of complex compounds due to lack of bubble formation, easy preparation, and simple instrumental management [64]. In order to overcome the defects of open-tubular capillary electrochromatography (OT-CEC) such as inadequate amount of SP coating, low phase ratio and poor sample capacity of OT columns, new methods including coatings using polymer, layers of porous silica, etching, and sol-gel techniques have been developed, so that the surface area and the interaction between the compounds and the coated SP are improved [108, 109]. A brief description of the mentioned methods will be described here. In etching method, a novel chemical material, which influences both the electroosmotic flow (EOF) and the adsorption properties of the capillary, is formed at the inner wall of the capillary column. This process gives rise to achieve a large surface for the inner wall of the capillary column [110, 111].

In 1987, sol-gel technology, which is used as a separation column in LC, was applied to fill monolithic ceramic support of capillary with small diameter, [112]. The sol-gel method provides some advantages such as: (i) favorable homogeneity and high purity from raw materials; (ii) possibility to control the size, shape and properties of particles; (iii) possibility to create the material structure with new property via appropriate selection of sol-gel components; and (iv) high stability of SP and performance in chromatographic analyses [113]. To prepare SPs based on the sol-gel process, the chemical reagents are required: (1) one or more precursors, which is commonly a metal alkoxide [M(OR)x] [114], (2) a solvent for the dispersion of the precursor(s), (3) an acidic, basic or a fluoride catalyst [115-118], and (4) water [41].

Separation of chiral amino acids and drugs are continuously growing because L-enantiomers of amino acids show appropriate activity, whereas D-enantiomers are inactive molecules with different properties and toxic effects [9, 10]. On the other hand, the pharmacodynamic and pharmacokinetic of particular enantiomer influence the metabolism of chiral drugs, which can affect the progress of new drugs [13]. In separation of enantiomer, CEC compounds is a suitable technique. To do this, the internal wall of capillary column is coated with the chiral selector which resolves the problems of packed columns. Due to the specific properties of NPs in separation process, they are good for supports to load chiral selectors in OT-CEC [119]. In the current report, enantioseparation of chlorpheniramine, zopiclone, tropicamide [52] and enantioseparation of amino acids [75, 120] were performed using CEC.

So far, according to Qu et al., two chief methods have been applied to modify the capillary column using NPs [121]: (i) coating the pre-derivatized fused silica capillary with NPs via chemical bonding [122-124] and (ii) immobilization of one NP layer onto the capillary wall by a chemical reaction at high temperatures [125-127]. In the following sections we report the use of NPs as SP of CE technique for separation of biomolecules and drugs.


To further improve the separation process, functionalization and augmentation of the surface’s charge density of modified CNTs is necessary. Thus, loading SWNT into an organic polymer monolith containing vinylbenzyl chloride (VBC) and ethylene dimethacrylate (EDMA) provided a monolithic SP for micro-HPLC and CEC, which resulted in improving the peak efficiency and increasing the chromatographic retention of small neutral molecules in RP-HPLC due to the hydrophobic interaction. Separation of a peptide mixture comprising Gly-Tyr, Val-Tyr-Val, methionine enkephalin, leucine enkephalin, and angiotensin II was carried out by Li et al. using VBC-EDMA-SWNT SP [37]. MWCNTs provide a larger surface area than that of SWCNTs; therefore, the functionalization of the large surface area of MWCNTs can enhance the surface charge density because of high coverage of functional groups on the multiple layers of G [84]. Hence, modification of MWCNTs using carboxylated (COOH), hydroxylated (OH), and sulfonated (SO3H) groups coated with sodium dodecylsulfate (SDS) as PSPs in electrokinetic capillary chromatography (EKC) technique was performed by Alharthi and Rassi to study the retention of some molecules [81]. For the first time, MWCNTs-OH and MWCNTs-SO3H as PSPs were introduced in this study. Comparison of functionalized MWCNTs with unmodified type showed better results for electrophoretic force. In addition, it was discovered that MWCNT-COOH is a good choice for the separation of nucleic acid bases and nucleosides, owing to interactions of hydrophobic, hydrogen bond and π-π stacking between the analytes and the MWCNT-COOH [82, 128]. However, neutral solutes such as alkylbenzenes (ABs), phenyl alkyl alcohols (PAAs) and alanine derivatives in neutral types were not separated by MWCNT-COOH and MWCNTs-SO3H due to the insufficient surface charge density of the modified MWCNTs for AB and PAA compounds. Therefore, the SDS coated unmodified and functionalized MWCNTs to make better separation of neutral components. Also, based on the Cao et al.’s studies, application of SDS-coated SWCNTs gives a more efficient resolution than unmodified carboxylated SWCNTs [129]. Moreover, in another investigation, Alharthi and Rassi studied the retention behaviors of small and large solutes in nanoparticle capillary electrokinetic chromatography (NPEKC) [84]. MWCNTs-COOH, MWCNTs-SO3H, and MWCNTs-OH were coated using SDS as a good PSP for the separation of herbicides, barbiturates, dansyl-DL-amino acids, dipeptides and proteins by NPEKC. In addition, myoglobin, cytochrome C, and lysozyme were investigated, and high resolution, selectivity and efficiency were obtained for all of compounds.

Modified GNPs with poly(ethylene oxide) (PEO) were used as a filling of CE column by Huangʼs team to resolve the problems of long-stranded DNA separation using CE, which resulted in a quick and highly efficient separation [130]. The molecules of PEO bond to the surfaces of GNPs using hydrophobic groups, improving the accessibility of the hydrophilic groups of PEO to interact with the polar compounds [131, 132]. Similarly, in another study performed by Tseng et al., GNPs were used for the separation of long DNA molecules by CEC under the application of hydrodynamic and electrokinetic forces [133]. Compared to Huang et al.’s results [130], the proposed method provided a quick separation, high resolution, great reproducibility, simple preparation, and poor unfavorable peaks.

Analysis of acidic and basic proteins using the modified didodecyldimethylammonium bromide (DDAB) with GNPs as PSP resulted in a superior separation efficiency, excellent reproducibility, and high reversed EOF [134]. In this study, to acquire better interactions with proteins, the GNPs were modified by PEO through noncovalent bonding interactions to produce composites of GNPs/polymer (GNPPs). It was found an excellent peak efficiency for basic proteins. Therefore, it is possible to use the established method for the analysis of highly basic, macrophage proteins in mammalian cells [135], and myelin basic protein in cerebral spinal fluid [136].

Modification of GNPs by thiolated β-cyclodextrin (β-CD) as a PSP in CEC was performed by Yang and coworkers to analyze four couples of dinitrophenyl-labeled amino acid enantiomers namely, DL-Val, Leu, Glu, and Asp; and three couples of drug enantiomers including RS-chlorpheniramine, zopiclone and carvedilol [71]. A good theoretical plate number (up to
2.4 × 105 per meter) and separation resolution (up to 4.7) were achieved by GNPs as a chiral selector. Solubility of thiolated β-CD-GNP and its stability in both acidic and basic conditions give rise to an appropriate PSP for enantioseparation. To modify the fused silica capillaries of CEC, Qu et al. coated n-octadecanethiol onto the surface of GNPs via layer-by-layer (LBL) method for the separation of polycyclic aromatic hydrocarbons (PAHs), basic and acidic proteins [121]. To do so, positively charged GNPs were fabricated and immobilized onto the capillary wall modified via two layers of polyelectrolyte. The new modification not only was robust with an application of more than 810 analyses, but also demonstrated high chemical stability versus NaOH (0.01 M), HCI (0.01 M), and concentrations of electrolyte up to 70 mM. LBL method is a good subject to avoid the absorption of basic molecules onto the capillary column and has drawn considerable attention in OT-CEC [137-139]. In this method, a polyelectrolyte was used to modify the inner surface of fused silica capillary [140, 141].

Conjugation of bovine serum albumin (BSA)-GNPs as a chiral stationary phase (CSP) of CEC was performed for enantioseparation of amino acid derivatives with phenylisothiocyanate (PITC), which resulted in an analysis time of 18 min [75]. Enantiomers of tryptophan, tyrosine, leucine, phenylalanine, serine, β-phenylalanine, aspartic acid, alanine, threonine, and arginine were evaluated by CEC. The sol-gel method was applied to prepare the bare monolithic silica column and has been modified chemically with 3-mercaptopropyltrimethoxysilane to provide thiol groups, immobilization of GNPs through the fabrication of an Au-S bond, and modification with BSA as the chiral selector.

For excellent separations of synthetic peptides mixture, a modified fused silica capillary with GNPs was applied by OT-CEC technique [104]. It was proved that the obtained capillary can be reused almost 900 times and separation of peptides depends on the interaction between peptides and GNPs which expressively change the EOF.

OT-CEC using GNPs SP was used to analyze glycated proteins, BSA, and human transferrin [72]. The inner wall of the fused silica capillary was coated by bare GNPs. GNPs were prepared according to the reduction of Au(III) via trisodium citrate dehydrate. Sodium phosphate buffer 100 mmol/L at pH 2.5, separation voltage 10 kV per 47-cm long, inside diameter capillary of 50 µm, and temperature of 25 °C were described as the best separation of peptides.

Krenkova et al. used commercial hydroxyapatite NPs for the protein separation (ovalbumin, myoglobin, lysozyme, and cytochrome C) and selective enrichment of phosphopeptides via the monolithic capillary columns of CEC [53]. Hydroxyapatite as a SP of chromatography is a crystalline form of calcium phosphate with the structural formula of Ca10(PO4)6(OH)that was established by Tiselius [142]. Hydroxyapatite NPs as the commercial form with dimensions of 50 nm are very small to be applied directly as a SP. However, immobilization of hydroxyapatite NPs onto an ideal porous was suggested by Krenkova et al. to introduce SPs of capillary columns based on the hydroxyapatite. The mass transfer achieved was fast, which allows hydroxyapatite NPs to be used in in-line control of biotechnology procedures wherever throughput is serious.

For the enantiomeric separation of derivatives of amino acids with 9-fluorenylmethoxycarbonylchloride, Domínguez-Vega et al. synthesized cellulose tris(3-chloro-4-methylphenylcarbamate), recognized as Sepapak-2 or Lux Cellulose-2, as a polysaccharide-based CSP to study amino acids of Ser, Thr, Asn, Gln, Cys, Pro, Ala, Lle, Leu, Allo, Met, Phe, Trap, Val, Asp, Glu, Pipe, Pyro, Lys, Cit, Orn, Hys, and Arg [120]. The separation of 19 out of 23 enantiomeric amino acids by nano-LC represented high chiral detection for this novel CSP. A comparison of nano-LC and CEC under the similar conditions gives superior peak efficiencies and resolution by using CEC experiments, which made possible the chiral discrimination of 20 out of 23 amino acids tested. Compatibility with the organic and aqueous solvents, enantioselectivity properties towards various molecules, the easy accessibility of natural sources, and the possibility of high incorporation of a chiral selector onto an inert carrier were mentioned as CSPs properties of polysaccharide-type [143]. Short run times of analysis for derivatives of essentially uncharged amino acid at low pHs were performed by using aminopropylized silica as the support of CSP [120]. Usually, modification of the silica surface with coating or covalent-bonding of polysaccharide phenyl esters or phenylcarbamates leads to obtain polysaccharide-type CSPs [144].

In another investigation, Qu et al. evaluated nanosheets of GO and G for OT-CEC to assay ovalbumin, ovotransferrin, ovomucoid, ovoflavoprotein, lysozyme, and avidin in chicken egg white [96]. 3-aminopropyldiethoxymethyl silane as a coupling agent was used to immobilize nanosheets of GO onto capillary column. Coating of G onto the column was done via hydrazine reduction of GO. Investigations of Qu and coworkers showed a pH-dependent EOF from anode to cathode in the pH of 3-9 for G-column and a constant EOF for GO-column. Finally, GO-column showed a good separation for the neutral analytes, however the poor separation was achieved for G-column.

To improve the chromatographic separation of myoglobin, ribonuclease A, lysozyme and α-chymotrypsinogen A, Arrua et al. prepared cryopolymers using embedded polyDVB and polyEDMA by mini-emulsion polymerization [43]. The obtained cryopolymers with an open porous structure and considerable specific surface area are interesting compounds for the separation of biomolecules by hydrophobic interaction chromatography (HIC). The polymerization mixture was comprised of poly(ethyleneglycol) diacrylate (PEGDA) as the single monomer, a combination of dioxane and water as the porogen, N,N,N’,N’-tetramethylethylenediamine (TEMED) and ammonium persulfate as the initiator system. Although the peak capacity and resolution factor were lower than those described for conventional columns of methacrylate monolithic, the use of this polymerization method gives rise to a polymeric arrangement with a more open porous structure and higher permeability than conventional polymer monoliths. In another investigation made by Arrua et al., the incorporation of charged NPs such as NR4+ and SO3 onto the polymer surface was carried out using direct addition of their suspensions for the polymerisation mixture to analyze myoglobin, ribonuclease A, lysozyme and α-chymotrypsinogen A [34]. Compared with the previous study performed by Arrua et al. [43], this ionic modification allows the application of columns in ion-exchange chromatography. The results indicated that the modification of monolithic columns using the direct addition of NPs is a suitable choice to functionalize monolithic polymers without changing the polymeric scaffold. Arrua et al., in two different studies [34, 43], showed that although addition of neutral NPs enhanced the chromatographic separation of biomolecules, a lot of NPs were buried within the polymeric scaffold and, hence, the potential advantages of this methodology could not be exploited completely. In emulsion polymerization, dispersion of the monomer is performed in an aqueous solution of surfactant with the critical micelle concentration (CMC). In the following, an initiator agent, commonly a water-soluble compound, is used to begin the polymerization process [145]. Entry of particles into the micelle, growing oligomers precipitation in the aqueous phase, and entry of particles into droplets of monomer caused to form the polymer particles [145]. The expression of mini-emulsion was used to explain the submicron droplets of oil in water dispersions that are stable for a long time from several hours to months [146].

Immobilization of BSA onto GO-magnetic nanocomposites (GO-Fe3O4-BSA) as SP of OT-CEC was reported for the effective enantioseparation of tryptophan, threonine, and propranolol in less than 80 s with resolution factors of 1.22, 1.9, and 2.1, respectively [67]. The new NPs of GO-Fe3O4-BSA has magnetism properties of Fe3O4 NPs, larger surface, and good biocompatibility of G, leading to immobilize further biomolecules and well sustain their biological activity. GO-Fe3O4-BSA conjugation was packed into microchannels of poly(dimethylsiloxane) using the magnets, which facilitates the immobilization of protein and gives a pattern for high efficiency screening of enantiomer compounds.

Modification of MNPs with carboxyl group was applied for separation of amino acids, dipeptides and proteins using OT-CEC [64]. MNPs of Fe3O4-COOH, made through solvothermal reduction process, were loaded on the surface of positively charged poly(diallydimethylammonium chloride) (PDDA) via electrostatic self-assembly to evaluate Trp, Tyr, and Phe as amino acid, Gly-Trp, Gly-Tyr, and Gly-Phe as dipeptide, ConA, α-Lac, β-Lg, and BSA as protein. Separation of egg white as a real sample via OT column coated with PDDA@Fe3O4-COOH MNPs revealed a superior resolution for proteins compared with a bare fused-silica capillary. PDDA as a water-soluble cationic polyelectrolyte has been commonly used to make semipermanent coating in OT-CEC through electrostatic interaction [147-150]. PDDA with quaternary ammonium groups and positive charges make an innovative SP via electrostatic self-assembly with different NPs [64]. Furthermore, Yang et al. modified MNPs using β-CD (MNP-β-CD) and ionic liquid of mono-6-deoxy-6-(1-methylimidazolium)-β-cyclodextrin tosylate (MNP-β-CD-IL) for enantioseparation of dansylated forms of alanine, leucine, lsoleucine, valine, methionine, and glutamic acid using CEC technique [68]. In comparison with uncoated capillary, suitable reproducibility, stability, and high enantioseparation resolution were observed. The morphology of MNPs was studied by tunneling electron microscopy technique, through which the average diameter of 8-12 nm was obtained. Also, based on the scanning electron microscopy analysis, the coating thicknesses of MNP-β-CD and MNP-β-CD-IL were nearly 5-15 μm and 5-20 μm, respectively.

Another modification of MNPs was performed via GNP for separation of neutral compounds, isomers, ovalbumin, ovotransferrin, ovomucoid, lysozyme, and avidin [70]. The bifunctional dumbbell-like Janus GNP-MNP was fabricated using hydrothermal synthesis method. To do this, GNP with particle size of 5 nm was conjugated to MNPs with particle size of 11 nm. Also, it was indicated that modified capillary with MNPs and GNP-MNPs showed lower EOF than that of the uncoated silica column, and that modified capillary via MNPs had no enough resolution for thiourea, naphthalene, and biphenyl.

In another study, modification of silica NPs was done using 3-aminopropyltrimethoxysilane and N1-(3-trimethoxysilylpropyl)diethylenetriamine based on the hydrophilic interaction, resulted in monoamine- and triamine-bonded silica NPs with a particle size of 20 nm as PSP of CEC to separate oligosaccharide derivatives. These amendments produced quick EOF of 2.59 × 10-4 cm2v-1s-1 in the direction of the anode in an electrical field, which allows a rapid analysis [21]. Also, isomers of benzenediol, catechol, resorcinol, hydroquinone, uracil, adenine, cytosine, and guanine were separated via monoamine-bonded silica NPs. Separation of oligosaccharide derivatives is based on the polymerization degree (DP). Monoamine-bonded silica facilitates the separation of the saccharides with a DP of 1 up to 17, whereas the larger oligosaccharides co-migrated. Hence, to resolve the oligosaccharides separation, the triamine-bonded silica is a suitable selection.

For the first time, lysozyme, cytochrome C, and α-chymotrypsinogen A as the protein and PAH were separated using NPs of fibrous mesoporous silica (fSiO2) grafted with poly(2-(dimethylamino) ethyl methacrylate (PDMAEMA) as a SP for OT-CEC [99]. PDMAEMA is a polymer with positive charge, which its charges change against to pH of solution [151] and can be easily adsorbed to a silica capillary inner wall via strong interaction of electrostatic [99]. Moreover, functionalization of SP based on PDMAEMA polymer by phenylalanine [152] or polyethyleneimine (PEI) [153, 154] have been wildly explored for OT-CEC to increase phase ratio. Separation of egg white proteins such as ovalbumin, ovotransferrin, ovomucoid, lysozyme, and other proteins were investigated, which could not be easily separated on the bare capillary owing to interaction of the analytes with the wall of column. The relative standard deviations of run-to-run and day-to-day for EOF and retention time of analytes showed a reproducible method for the preparation of P-SiO2@C18 column.


Owing to stereoselectivity of interaction between drug and target, only one of the two enantiomers can provide the desired pharmacological effects, while the other one may exhibit less activity, minor effectiveness, poisonousness, and unwanted side effects. Hence, chirality is a crucial subject in many fields such as environmental, food, pharmaceutical, and especially the science of human health [155]. In this regard, among the proposed processes for analysis of enantiomers, proteins have been of particular interest because of various binding strengths with enantiomers. So recently, some investigations have been done to improve chiral separations using proteins. In this regard, discrimination of ephedrine and norephedrine isomers was investigated by BSA-GNP as a chiral SP in chip-based enantioselective OT-CEC [156], providing high speed, integration of sample preparation and introduction as on-line, controllable sample injection, high efficiency and low amount of sample [157-159]. A microchip is considered as an analytical device which all the steps of a measurement including preparation, separation, and detection of analytes are done on a small footprint [160-162].

Modification of GNPs with thiolated β-CD as a SP of OT-CEC was applied for enantioseparation of zopiclone, tropicamide and chlorpheniramine [119]. PDDA followed by self-adsorption of negatively charged CD-GNPs was used to fabricate enantioselective OT capillary column. In this report, Li’s team demonstrated that the column shows good repeatability for enantioseparation, and enantioselectivity of the column can preserve for more than 1 month provided that the column was kept in solution of CD-GNPs at 4 °C. Limit of detection (LOD) was reported as 8.8 for zopiclone, 6.3 for tropicamide and 5.4 µg/mL for chlorpheniramine. In another research, Li et al. amended GNPs with β-CD (β-CD-GNP) through covalent interaction as the SP of CEC for enantioseparation of chlorpheniramine, zopiclone, and tropicamide [52]. The new column with theoretical plate numbers of  displayed stable EOF over pH ranges from 4.6-9.7. Similar analysis using β-CD-GNP was done for enantioseparation of chlorphenamine, brompheniramine, pheniramine, and zopiclone [163]. The sol-gel strategy was used to prepare the chiral OT-CEC column via β-CD. It was indicated that the GNPs as the SP improved the phase ratio of column, which resulted in improving the selectivity of chiral column. Another investigation reported carboxymethyl-β-CD conjugated GNPs as SP of two pairs of α-tetralones derivatives enantiomers and enantiomers of tramadol hydrochloride and zopiclone [164]. LBL self-assembly method was applied to fabricate the OT-CEC column. Compared to the bare capillary, the suitable stability for EOF was achieved under the working pH range.

With regards to favorable properties of GNPs, the racemic β-blockers of propranolol, esmolol, bisoprolol and sotalol were analyzed using GNPs coated capillary [165]. L-cysteine and carboxymethyl-β-CD as chiral reagents were used to modify GNPs. Moreover, tetramethylammonium lactobionate was selected as a chiral marker. In this condition, based on the scanning electron microscopy analysis, L-cysteine provided insignificant change to the column, while, modification using carboxymethyl-β-CD created a bit bigger surface.

Enantioseparation of nefopam, chlortrimeton, azelastine, clenbuterol, ritodrine, esmolol, amlodopine, citalopram, propranolol, metoprolol, bisoprolol, salmeterol, atenolol, labetalol, and sotalol in less than ten minutes on pepsin-based poly(GMA-EDMA-NH2-MSN) monolithic column of CEC was done by Xu et al. [32]. Pepsin, as a type of aspartic protease, is usually utilized as a bioreactor and less attention has been paid to its enantioresolution characteristics [32]. NH2-MSN gets the required stability for enantioseparation purposes and functional groups to immobilize pepsin, which can augment the chiral resolution capacity.

Different literatures presented the benefits of porous polymeric materials as an innovative SP for selectivity improvement and chromatographic performances [166-168]. Grzywiński et al. changed the monolithic column in fused-silica capillaries with cystamine dihydrochloride, subsequently Ag-NPs immobilization, which was functionalized via cholesterol cysteamine to separate ribonuclease A, cytochrome C, and myoglobin using the RP-chromatographic performance [42]. The desired SP did not demonstrate selectivity for low molecular weight compounds owing to the modification in their porosity.

To separate enantiomers of propranolol, ofloxacin, amlodipine, chlortrimeton, tropicamide, and atenolol, Sun et al. modified magnetic microparticles (MMPs) using carboxymethyl-β-CD as SP of OT-CEC column [69]. Tunneling electron microscopy analysis demonstrated the mean diameter of 500 nm and spherical shape for bare MMPs and CD-MMPs. Moreover, compared to the bare capillary column, EOF variation was insignificant, leading to a high reproducibility for migration time and resolution.

The essential information for the mentioned methods is described in Table 1.


To achieve a high-resolution and quantitative separation, chromatographic analyses have been extensively used in separation, materials, environmental, and life sciences. After the introduction of NPs, new achievements have been obtained in chromatographic techniques using NPs as SP [169]. In the following sections we present the application of NPs as SP of HPLC technique for separation of biomolecules and drugs.


HPLC analysis equipped with GNPs postcolumn was developed by Lu and coworkers for cysteine, cysteinylglycine, homocysteine, glutamylcysteine, and glutathione with the LODs of 2.0 µM, 40, 40, 60 and 80 pmol, respectively in biological fluids. GNPs with the particle size of 12 nm were attached to Triton, Tween, and Brij, as commercially nonionic surfactants, and their interactions with biothiols and biomolecules were studied. Their results indicated a high selectivity for small biothiols and quick mass kinetics for Brij 35. Easy preparation, high stability in aqueous media over an extensive pH range at room temperature, and high selectivity against small biothiols were mentioned as the benefits of GNP colloids [170]. A porous polymer monolithic capillary column of micro-HPLC equipped with mass detector was modified using GNPs, which allowed the selective trapping of peptides including cysteine molecules [51]. The best conditions were found with HAuCl4 of 50 mmol/L and trisodium citrate of 200 mmol/L. A mixture of six peptides, three of them containing a cysteine residue, was tested using the commercial capillary column of C18 modified with GNPs, which resulted in retaining of peptides containing cysteine, whereas the other peptides passed [51].

CNPs, with size of 6-18 nm, obtaining from corn stalk soot via refluxing method in nitric acid has been applied as a SP of HPLC to separate five nucleosides, four sulfa compounds and safflower via both of hydrophilic interaction liquid chromatography (HILIC) and per aqueous liquid chromatography (PALC) conditions. The produced CNPs were soluble in water owing to the presence of OH and COOH groups and also they showed fluorescence property. Different nucleosides such as thymine, inosine, adenosine, cytosine, and adenine were studied and revealed stronger retention on the CNP column than on amino column, whereas the selected nucleosides were hard to retain on column of C18 in HILIC situation. The lowest and the highest retention factors were obtained for thymine and adenine, respectively in both of HILIC and PALC modes [171].

SP of GO@silica modified by octadecylsilane was used to separate alkylbenzenes, polycyclic aromatic hydrocarbons, amines, and phenolic compounds using HPLC [95]. Both of π-π- and hydrophobic interactions between analyte and new SP gave a better resolution in comparison with C18 commercial column. In the following, Liang et al. in another investigation applied GNPs onto the surface of GO@silica as a new SP of HPLC [74]. Compared with the GO@silica phase, GNPs@silica phase displayed good resolutions for alkylbenzenes, isomerides, amino acids (DL-leucine, L-tyrosine, proline, glycine, 3-nitro-L-tyrosine, and L-glutamic acid), nucleosides (thymidine, uridine, cytosine, inosine, and xanthine), 6-chlorouracil, and nucleobases due to the combination of hydrophilicity, large π-electron structures, hydrophobicity, and coordination groups, which resulted in a unique separation.

Incorporation of FSNPs into a monolithic column of polymethacrylate including glyceryl monomethacrylate (GMM) and EDMA was done to prepare a novel monolithic column for nucleosides of adenosine, uridine, cytidine, inosine and guanosine, hydroxybenzoic acids and nucleotides using HILIC [36]. Decreasing the pH of aqueous mobile phase was resulted in reducing the electrostatic repulsion and enhancing the retention time of acidic components. The best separation of nucleotides in less than 10 min was achieved via gradient elution with minimization of pH and ACN volume of mobile phase. Flame hydrolysis of silicon tetrachloride in a gas flame of oxyhydrogen at 1000 °C led to fabricate fumed silica with amorphous, extremely dispersed, nonporous, and hydrophilic properties [172, 173]. In the following, Aydoğan and Rassi in alternative investigation modified FSNPs with 3-(trimethoxysilyl)propylmethacrylate (TMSPM) to make the hybrid MFSNP monomer. The obtained MFSNP was combined with GMM and EDMA in a solvent comprising of cyclohexanol and dodecanol to initiate copolymerization of MFSNP, GMM and EDMA in a stainless steel column. Octadecyl ligand was used to incorporate the silanol groups of the hybrid monolith, which finally obtained the hybrid poly(GMA-EDMA-MFSNP)-octadecyl with hydrophobic interactions. Alkylbenzenes, phenolic compounds, aniline, 4-methylaniline, N-methylaniline, N-ethylaniline, 3,4-dichloroaniline, lysozyme, ribonuclease A, carbonic anhydrase isozyme II, cytochrome C, α-chymotrypsinogen A, and myoglobin were chromatographed on the monolithic column.


To analyze the chiral compounds, the application of analytical columns may be inappropriate because of high costs of chiral SP, great consumption of solvents, and a destructive environmental effect. Thus, various miniaturized methods such as capillary liquid chromatography (CLC) and nano-LC have been designed for the separation of chiral compounds [144, 174-176]. Recently, chiral selectors with different separation mechanisms have been demonstrated. For example, glycopeptide antibiotics as a chiral marker for amino acids, peptides, herbicides, and drugs displayed very high enantio resolution [177-179].

The LbL self-assembly procedure was applied to make NP of ZrO2-SiO2 as SP of HPLC [180]. Despite the silica support, zirconia is very stable over the pH of 1-14, whereas the availability and improvement of specific surface area and pore volume of zirconia is much lower than silica. Therefore, to enhance the surface area of zirconia-based support, Dun et al. used LbL self-assembly technique. Both of ZrO2-SiO2 support and SP of C18- ZrO2-SiO2 revealed outstanding chemical stability and good permeability. Moreover, cellulose tris(3,5-dimethylphenylcarbamate) polymer (CDMPC) was coated on ZrO2-SiO2 support for discrimination of chiral compounds.

In 2020, Li and coworkers prepared GNPs with particle size of 10-15 nm as a polysaccharide-based CSP for separation of ibuprofen using normal phase liquid chromatography. Also, six chiral pesticides, flavanone, and 6-hydroxyflavanone were investigated. It can be concluded that the -OH groups of cellulose provided a strong electrostatic attraction and hydrogen bond interaction, which resulted in long retention time of ibuprofen [181].

The important information of described analyses is presented in Table 1.


In addition to the mentioned methods, application of TLC has increased newly, because of low-cost, rapid, efficient resolution, and low consumption of solvents. Therefore, due to the advantages of NPs and TLC technique, some amendments for SPs have been applied to resolve peak tailing of chromatograms and give a better resolution. To do this, surface modification of a commercial TLC plate for direct detection of propranolol enantiomers was performed with GNPs as the first- and L-cysteine (L-cys) as the second layer, based on the ligand-exchange mechanism [73]. Ligand-exchange chromatography as one of the direct chromatographic techniques is used for a superior chromatographic enantioseparation. High attraction of thiol group of L-cys toward GNPs resulted in an excellent adsorption on the surface of plate.

Another method has been developed to modify silica gel with GNP grafted 3-triethoxysilyl propylamine as a novel SP for separation of progesterone and testosterone [182]. Modification of silica gel SP using GNPs provides less baseline interferences, causing a suitable interaction between SP and steroid hormones, and gives a good sensitivity. LOD values of 0.16 and 0.13 ng/spot and LOQ values of 0.51 and 0.40 ng/spot for progesterone and testosterone were achieved, respectively [182]. The main conditions are accessible in Table 1.


In the present literature survey, several NPs as SPs of electrophoretic and HPLC techniques were reviewed such as GNPs, CNTs, MNPs, GO, and G for separation of biomolecules and chiral drugs. Also, owing to importance of the analysis of biomolecules and chiral drugs, some studies have been allocated to modify columns using chiral selectors. Moreover, some separations were performed using miniaturized techniques because of high-throughput screening, inexpensive, great separation efficiency, and satisfied resolutions. It was revealed that high ratio of analyses has been performed using GNPs and electrophoretic procedures. GNPs in analysis of biomolecules have attracted a considerable attention, due to the formation of strong covalent bonds between GNPs and thiol groups in structure of biomolecules. Finally, it is concluded that NP-based SPs will play a significant role in separation studies as well as the bioanalytical identifications.


The authors are grateful to the support of Central Research Laboratory, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran, and Mr. M. Baghaie for editorial assistance.


The authors announce that there are no conflicts of interest.


The current study received no external funding.



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