Scholarly article on topic 'Evaluation of glycidyl methacrylate-based monolith functionalized with weak anion exchange moiety inside 0.5 mm i.d. column for liquid chromatographic separation of DNA'

Evaluation of glycidyl methacrylate-based monolith functionalized with weak anion exchange moiety inside 0.5 mm i.d. column for liquid chromatographic separation of DNA Academic research paper on "Chemical sciences"

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Abstract of research paper on Chemical sciences, author of scientific article — Aprilia Nur Tasfiyati, Elvina Dhiaul Iftitah, Setyawan Purnomo Sakti, Akhmad Sabarudin

Abstract In this study, the organic polymer monolith was developed as a weak anion exchanger column in high performance liquid chromatography for DNA separation. Methacrylate-based monolithic column was prepared in microbore silicosteel column (100 × 0.5 mm i.d.) by in-situ polymerization reaction using glycidyl methacrylate as monomer; ethylene dimethacrylate as crosslinker; 1-propanol, 1,4-butanediol, and water as porogenic solvents, with the presence of initiator α,α′-azobisisobutyronitrile (AIBN). The monolith matrix was modified with diethylamine to create weak anion exchanger via ring opening reaction of epoxy groups. The morphology of the monolithic column was studied by SEM. The properties of the monolithic column, such as permeability, mechanical stability, binding capacity and pore size distribution, were characterized in detail. From the results of the characterization, monoliths poly-(GMA-co-EDMA) with total monomer percentage (%T) 40 and crosslinker percentage (%C) 25 was found to be the ideal composition of monomer and crosslinker. It has good mechanical stability and high permeability, adequate molecular recognition sites (represented with binding capacity value of 36 mg ml−1), and has relatively equal proportion of flow-through pore and mesopores (37.2% and 41.1% respectively). Poly-(GMA-co-EDMA) with %T 40 and %C 25 can successfully separate oligo(dT)12–18 and 50 bp DNA ladder with good resolution.

Academic research paper on topic "Evaluation of glycidyl methacrylate-based monolith functionalized with weak anion exchange moiety inside 0.5 mm i.d. column for liquid chromatographic separation of DNA"

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Analytical Chemistry Research

journal homepage: www.elsevier.com/locate/ancr

Evaluation of glycidyl methacrylate-based monolith functionalized with weak anion exchange moiety inside 0.5 mm i.d. column for liquid chromatographic separation of DNA

Aprilia Nur Tasfiyati a, Elvina Dhiaul Iftitah a'c, Setyawan Purnomo Sakti b'c, Akhmad Sabarudin a'c' *

a Department of Chemistry, Faculty of Science, Brawijaya University, Jl Veteran Malang, 65145, Indonesia

b Department of Physics, Faculty of Science, Brawijaya University, Jl Veteran Malang, 65145, Indonesia

c Research Center for Advanced System and Material Technology, Brawijaya University, Jl Veteran Malang, 65145, Indonesia

ARTICLE INFO ABSTRACT

In this study, the organic polymer monolith was developed as a weak anion exchanger column in high performance liquid chromatography for DNA separation. Methacrylate-based monolithic column was prepared in microbore silicosteel column (100 x 0.5 mm i.d.) by in-situ polymerization reaction using glycidyl methacrylate as monomer; ethylene dimethacrylate as crosslinker; 1-propanol, 1,4-butanediol, and water as porogenic solvents, with the presence of initiator a,a'-azobisisobutyronitrile (AIBN). The monolith matrix was modified with diethylamine to create weak anion exchanger via ring opening reaction of epoxy groups. The morphology of the monolithic column was studied by SEM. The properties of the monolithic column, such as permeability, mechanical stability, binding capacity and pore size distribution, were characterized in detail. From the results of the characterization, monoliths poly-(GMA-co-EDMA) with total monomer percentage (%T) 40 and crosslinker percentage (%C) 25 was found to be the ideal composition of monomer and crosslinker. It has good mechanical stability and high permeability, adequate molecular recognition sites (represented with binding capacity value of 36 mg ml-1), and has relatively equal proportion of flow-through pore and mesopores (37.2% and 41.1% respectively). Poly-(GMA-co-EDMA) with %T 40 and %C 25 can successfully separate oligo(dT)12—18 and 50 bp DNA ladder with good resolution.

© 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND

license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Article history:

Received 19 September 2015 Received in revised form 23 November 2015 Accepted 25 November 2015 Available online 30 November 2015

Keywords: Ion exchange HPLC

Methacrylate Monolithic column Post-modification DNA

1. Introduction

Recently, DNA analysis has been widely applied to the diagnosis of various diseases. DNA analysis also provides information for effectively maintaining human health and gene therapy. Therefore, many efforts have been made to meet the needs of rapid, simple and efficient separation for DNA analysis. The primary method used to separate DNA fragments is gel electrophoresis. However, this method is complicated and requires great technical skill. On the other hand, high-performance liquid chromatography (HPLC) is shown to be attractive techniques for the separation of DNA fragments and oligonucleotides due to their high efficiencies, high

* Corresponding author. Department of Chemistry, Faculty of Science, Brawijaya University, Jl Veteran Malang, 65145, Indonesia.

E-mail addresses: sabarjpn@gmail.com, sabarjpn@ub.ac.id (A. Sabarudin).

throughput, and ease of automation [1—5]. Among the various chromatographic techniques used for DNA separation, anion-exchange method is most widely applied due to the fast binding between negatively charged phosphate groups in DNA backbone and positively charged groups of anion exchangers [1,2,6—8].

Since their discovery about two decades ago, monolithic columns have attracted much attention as the separation media in chromatography. Monolithic stationary phase is a continuous single piece porous structure prepared by in situ polymerization of monomers (organic/inorganic) inside the column tubing [9,10]. Uniformity of bed with no end frits, higher permeability and the ability to design to desired length are the main advantages of monolithic stationary phase [11]. The main characteristic of these materials is the presence of large through-pores which permits the use of high flow rates at low back pressure [12].

According to the IUPAC classification, micropores are pores with size smaller than 2 nm, mesopores have size between 2 and 50 nm,

http://dx.doi.org/10.1016/j.ancr.2015.11.001

2214-1812/© 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativec0mm0ns.0rg/licenses/by-nc-nd/4.0/).

and flowthrough pores are larger than 50 nm [13]. Micropores and mesopores at the surface of the skeletal structure provide sufficient surface area for efficient separation. On the other hand, macropores generate low-pressure drop and also enhance mass transfer kinetics, allow the use of higher flow rates for rapid separations [14]. Depending on their porosity and pore size distribution, monoliths have proven to be excellent chromatographic supports for the separation of a large variety of analytes, including low molecular weight molecules [15-17], and macromolecules such as peptides and proteins [18-20], DNA fragments [21], plasmid DNA [20,22,23], oligonucleotide [2,24], and oligodeoxythymidylic acids [14].

Organic polymer-based monolithic columns are produced by in situ polymerization. The polymerization reaction mixtures consist of a combination of an initiator, monomers, crosslinkers and porogens. The composition of the monomer, crosslinker, porogenic solvent, polymerization reaction time, polymerization temperature, and modification conditions would have great effect on the monolithic structure [11,25-27]. For anion exchange application, postpolymerization modification is the most widely used approach to afford anion exchange functionalities on the surface of the monolithic matrix. These two-step operations allow independent optimization of surface chemistry and porous properties of the monolith. Hence, leads to reproducible preparation of optimum monoliths with fully controlled pore sizes and exchange capacity

Among various types of monomers used in organic polymer monolith, methacrylate-based polymers have some advantages related with their use as stationary phase in monolithic columns, such as simple preparation, easy functionalization, various selectivity, and high stability under wide range pH conditions (pH 2-12) [28,29]. According to Vidic et al. [30], both mechanical and chemical stability of methacrylate monolith is high enough to withstand harsh conditions required during their implementation. Therefore, methacrylate-based monolith can be considered as the supports of choice for efficient purification of macromolecules under highly controlled conditions. Glycidyl methacrylate (GMA) was the most commonly used as monomer in organic polymer monolith fabrication, since it has highly reactive epoxy groups which could be easily converted into anion-exchange groups via ring opening reaction with various type of functional groups, such as diethylamine [2,14], triethylamine [31,32], trimethylamine [33], and sulfonate

In the present study, an anion exchange monolithic methacrylate-based column was prepared by in situ copolymeri-zation of glycidyl methacrylate (GMA) as monomer with ethylene dimethacrylate (EDMA) as crosslinker and ternary porogenic mixture of 1-propanol, 1,4-butanediol, and water, in the presence of radical initiator AIBN. The polymerization takes place inside a microbore silicosteel column (0.5 mm i.d. x 100 mm in length). The monolithic matrix was subsequently modified with diethylamine to obtain weak anion exchanger functionality. The morphology of the monolithic column was studied by SEM. The properties of the monolithic column, such as permeability, mechanical stability, binding capacity and pore size distribution, were characterized. The anion-exchange capacity was estimated from the dynamic binding capacity for BSA at 10% breakthrough. The pore size distribution of the monolithic column was determined by inverse-size exclusion chromatography (ISEC). Monolithic column with the best characteristic was then applied for separation of oligo(dT)12-18 and 50 bp DNA ladder to evaluate its chromatographic performance.

Previous work by Shu et al. described that the difference of the type housing column lead to different character of resulting monolith [34,35]. Therefore, we expect that the difference in column diameter also greatly affects the physical properties of the resulting monolith, such as porosity, permeability, and

homogeneity. Monolith polymerization in this work was performed in-situ in 0.5 mm i.d. microbore silicosteel column, which is smaller than those used by Sabarudin, 1.02 mm i.d [14]. Small diameter monolith column was expected to possess several advantages over large one, not only in better homogeneity, but also result in better separation efficiency especially for biological samples in small quantities. Moreover, low sample and low reagent consumption make it more efficient and also environmentally friendly. While large diameter monolith columns are less homogeneous, not only because of the unequal heating across the tube diameter but also because of the growing gravitational settling effect during the exothermic polymerization process [34-36].

Monolithic column fabricated in this work was successfully separate oligo(dT) fragments and 50 bp DNA ladder. All oligo(dT12-18) fragments are baseline resolved, showing better resolution than previous works reported so far [2,14]. This optimized monolith also offers the capability to perform separation of DNA fragments using a simple linear gradient elution. A linear gradient elution method is easy as DNA fragments are eluted during a linear increase of salt concentration. This method does not require a complicated setup, as often happened in step gradient elution mode. Moreover, the dynamic binding capacity (DBC) value of the optimized monoliths in this work were much higher than that of commercially available anion exchange monoliths, and the DNA fragments were baseline resolved revealing excellent chromato-graphic separation.

2. Material and methods

2.1. Instrumentation

All LC experiments were performed using HPLC unit prominence 20 from Shimadzu (Japan) equipped with Shimadzu's workstation software (LabSolutions) for system control and data acquisition. The system was composed of a communication bus module (CBM-20A), an HPLC pump (LC-20AD, a column oven (CTO-20AC), a UV/Vis detector (SPD-20A), and a Rheodyne 8125 injector with a home-made 2 mL PEEK sample loop. Scanning electron micrographic images for morphology observation of the monolithic column were obtained using SEM TM-3000 (Hitachi, Japan).

2.2. Materials

All chemicals used were of analytical grade and were used as received without further purification. Bovine serum albumin (BSA), glycidyl methacrylate (GMA), ethylene dimethacrylate (EDMA), 3-methacryloxypropyl-trimethoxysilane (MAPS), diethylamine, 1-propanol, 1,4-butanediol, tetrahydrofuran (THF), tris(hydrox-ymethyl)aminomethane (Tris), and polystyrene standard set (Mw 500-2,000,000) were purchased from Sigma-Aldrich Co. (USA). Oligo(dT)12-18 Primer and 50 bp DNA ladder were obtained from Invitrogen (Carlsbad, USA). NaCl, NaOH, and toluene were form Merck KGaA (Darmstadt, Germany). Silicosteel column (0.5 mm i.d. 1/16 inch o.d) was from Supelco (Bellefonte, Pennsylvania, USA) and a,a'-azobisisobutyronitrile (AIBN) was from Himedia (Mumbai, India). Methanol was purchased from Fulltime (Anhui, China), while ethanol, HCl, and acetone were from Smart Lab Indonesia.

2.3. Preparation of methacrylate-based anion-exchange monolithic column

Prior to the polymerization, the silicosteel column was pre-treated using MAPS in order to anchor the polymer monolith on the column wall, in a similar manner to that described by Shu et al. [34] with minor modification. The silicosteel column was pretreated by

successively filling the column with 0.2 M NaOH for 30 min twice, washing with water, filling with 0.2 M HCl for 30 min twice, and finally rinsing thoroughly with water and acetone. MAPS solution (MAPS: acetone: pyridine = 30: 65: 5) was used to fill the activated column. Thereafter, the silicosteel column was placed at room temperature for 12 h twice, with both ends sealed. Finally, the column was rinsed thoroughly with acetone to flush out the residual reagents. Thus Si—O—Si—C bonds were formed between the column wall and the reactive methacryloyl groups, which are available for subsequent attachment of monolith to the wall during the polymerization reaction. The pretreated column was then cut into the desired length (10 cm in length in this experiment).

The monolithic column was prepared by in situ polymerization, with procedure as described by Sabarudin et al. [14]. A polymerization mixture containing GMA, EDMA, 1-propanol, 1,4-butanediol, water, and AIBN (1% (w/v) of the total monomer amount). Six different batches of columns have prepared to examine the effect of total monomer percentage (%T) and crosslinker percentage (%C). Each batch contained two identical columns from which one column was chosen. Table 1 lists the different polymerization mixtures. This mixture solution was homogenized for 5 min using vortex and injected into the pretreated column. After the column was completely filled with the mixture, the column was sealed at both ends and placed in the oven to proceeds the polymerization at 60 °C for 24 h. The resulting monolith column was washed with ethanol and water for about 2 h respectively, to remove the unreacted monomers and remaining porogenic solvent present in the column.

Subsequently, epoxy groups in the monolith were reacted with diethylamine to obtain weak anion exchanger functional groups via ring opening reaction of epoxy groups, as the following procedure described by Sabarudin et al. [14]. Diethylamine solution (1 M in methanol) was passed through the monolithic column placed in oven with temperature maintained at 75 °C, at flow rate of 0.05 ml min-1 for 2 h. The anion exchange monolithic column produced was extensively washed with ethanol and water to ensure removal of unreacted reagent.

2.4. Pressure drop measurements

To investigate the permeability and mechanical stability of the monolithic columns, pressure drop measurements were made at room temperature using ethanol as permeating fluid at flow rates ranging from 0.01 to 0.1 ml min-1 for mechanical stability investigation, and kept constant at 0.05 ml min-1 (linear velocity of 4.24 mm s-1) for permeability measurements. Ethanol was pumped through the column and the back pressure was recorded when the pressure stabilized. The measurements were performed using the same LC system. Permeability (K, m2) was calculated according to Darcy's Law by using the following equation:

•L • u

•L • Fm

Dp •p,r2

where u (m s-1) is the linear velocity of mobile phase, h is the viscosity of mobile phase (1.095 x 10-3 Pa s at 20 °C with using ethanol in this experiment), L is the length of the monolithic column (m) and DP is the pressure drop across the monolithic column (Pa).

2.5. Swelling or shrinking

The experiment to observe the shrinking or swelling behavior was carried out as follows. A monolith, which was slipped out of the column tubing, was dried and divided into 3 parts. The two parts were immersed in THF and 0.02 M Tris—HCl pH 7.4 respectively. Then, the diameter of each monolith was measured using precision scale ruler with computer assistance.

2.6. Dynamic binding capacity (DBC)

DBC was determined by frontal elution with bovine serum albumin (BSA). Monolith columns were saturated with 0.02 M Tris—HCl pH 7.4 at first. Then a solution of BSA 2 mg ml-1 in 0.02 M Tris—HCl pH 7.4 was pumped through the column at constant flow rate of 0.05 ml min-1, and UV detection was carried out at 260 nm. DBC (mg ml-1) was calculated at 10% of the final absorbance value of the breakthrough curve using the following equation:

Vio% - Vo Vc

where V10% (mL) is the 10% breakthrough volume, V0 (mL) is the extracolumn volume of the HPLC system (was estimated to be 0.548 mL in this experiment), C0 is the concentration of BSA (mg ml-1), and Vc (mL) is total volume of column.

2.7. Inverse size exclusion chromatography (ISEC)

The porosity and pore size distribution of the produced monoliths were investigated by inverse size-exclusion described by Al-Bokari et al. [37]. ISEC utilizes a set of molecular probes with widely varying, but well-defined sizes to determine pore dimensions, that is toluene and polystyrene standard set (Mw 500—2,000,000) in this experiment. This examination is analogous to molecular mass calibration in SEC. All ISEC experiments were performed under isocratic elution conditions for each polymer standard sample using THF as mobile phase at constant flow rate of 0.05 ml min-1. The injection volume was 2 ml and UV detection was carried out at 254 nm.

Table 1

Polymerization mixtures used for fabrication of the monoliths.

No. %Ta (v/v) %Cb (v/v) GMA (mL) EDMA (mL) Porogenic solvent 1-propanol (mL) 1.4-butanediol (mL) Water (mL) AIBN 1% (w/v)

1 30 25 0.45 0.15 0.817 0.467 0.117 6 mg

2 30 35 0.39 0.21 0.817 0.467 0.117 6 mg

3 35 25 0.525 0.175 0.758 0.433 0.108 7 mg

4 35 35 0.455 0.245 0.758 0.433 0.108 7 mg

5 40 25 0.6 0.2 0.7 0.4 0.1 8 mg

6 40 35 0.52 0.28 0.7 0.4 0.1 8 mg

Total volume of polymer solution is 2 mL. a Total monomer proportion = ((vol. GMA + vol. EDMA)/total volume of polymer) x 100. b The proportion of crosslinker = (vol. EDMA/(vol. GMA + vol. EDMA)) x 100.

3. Results and discussion

3.1. Preparation of methacrylate-based anion-exchange monolithic column

Poly-(GMA-co-EDMA) anion exchange monolithic column was produced by a two-step procedure. First, the synthesis of a polymer matrix by using GMA as monomer, EDMA as crosslinker, and ternary porogen consists of 1-propanol, 1,4-butanediol, and water, with the presence of radical initiator AIBN. Then, it was followed by the introduction of diethylamine as weak anion exchange functional group via ring-opening reaction of the epoxy group. The morphology of the monolithic column was examined by SEM. As shown in Fig. 1, the obtained monolith displayed porous network with globular structure. The continuous porous channels in the

Table 2

Characterization data of poly-(GMA-co-EDMA) monolithic columns.

Monolith %T %C Pressure drop (MPa) Permeability (m2) DBC (mgml-1)

I 30 25 0.6 7.74 x 10~13 17.968

II 30 35 0.7 6.64 x 10~13 8.465

III 35 25 0.7 6.64 x 10~13 19.198

IV 35 35 0.8 5.81 x 10~13 33.877

V 40 25 2.5 1.86 x 10~13 36.804

VI 40 35 4.8 9.68 x 10~14 48.259

monolith bed which were formed by flow through pores can also be seen.

The permeability of monolithic column was examined by measure the back pressure at constant flow rate of 0.05 ml min-1 using ethanol as mobile phase. As shown in Table 2, column permeability

10 fim 10 Jim

Fig. 1. Scanning electron microphotographs of monoliths slipped out of the column tubing with 5000 x magnification. (a) monolith I, (b) monolith II, (c) monolith III, (d) monolith IV, (e) monolith V, (f) monolith VI.

is strongly affected by the amount of porogenic solvent. The larger the amount of porogenic solvent (indicated by the lower value of % T) would result in higher permeability, and the other way around. Permeability values of these 0.5 mm i.d. monolithic column are certainly lower than those of 1 mm i.d. used by Sabarudin et al. [14]. However, in comparison with particle-packed conventional column (Shimadzu Shim-pack VP-ODS 150 x 4.6 mm i.d. with permeability value of 6.17 x 10-14 m2 at linear velocity 4.24 mm s-1), the permeability of all six monolithic columns fabricated in this study was quite high with relatively low back pressure, which demonstrated that all of them have good permeability.

The low flow-resistance property of the poly-(GMA-co-EDMA) monolith makes it possible to apply high flow rate, allowing high speed separation. Thus, mechanical strength for high-pressure need to be examined. The mechanical stability of the monolithic column was evaluated by performed the pressure drop vs flow rate test. Good linear responses between back pressure and flow rate were observed. The back pressures dependence on flow rate was a straight line with a correlation coefficient (R2) above 0.979 (Fig. S1 in Supplementary information), which clearly indicated the good mechanical stability of the prepared monolithic columns, and capability to withstand pressures up to 12 MPa without any compression. In this experiment, we also examined a column-to-column reproducibility prepared from the same batch (n = 5), showing fairly good result since the relative standard deviation was within 7%.

Every stationary phase shrinks and swells to some extent when changes of the mobile phase composition occur. The swelling and shrinking behavior of monolithic bed influences the column permeability and can lead to problems such as poor column stability, which leads to reduced chromatographic efficiency and loss of resolution. Low swelling and shrinking tendency of a monolithic polymer material is a basic requirement for its HPLC applicability [30,38]. Therefore, this phenomenon should be extensively investigated. The experimental result for monolith V (%T 40 and %C 25), as indicated in Fig. S2 in Supplementary information, shows the shrinkage of 1.2% in Tris—HCl and 4.4% in THF. This indicates that some shrinking of the monolith occurs also with Tris—HCl, yet to a much lesser degree than with THF. Fortunately, THF is hardly ever used as mobile phase, and Tris—HCl, which is common solvent for DNA separation using HPLC, do not cause any considerable swelling of the monolithic bed. In this experiment, THF only used as mobile phase in the pore size distribution measurement using ISEC.

3.2. Dynamic binding capacity (DBC) and inverse size exclusion chromatography (ISEC)

Anion exchange functionality was obtained from postmodification of monolithic surface with diethylamine via ring opening reaction of epoxy groups in GMA. The dynamic binding capacity of anion-exchange monolithic column was estimated from the 10% breakthrough curves of bovine serum albumin (BSA) using frontal analysis. DBC measurement at all monolith columns produces very sharp breakthrough curve (Fig. S3 in Supplementary information). The sharpness of breakthrough profiles indicated the efficient mass transfer within the pores of the monolith. This result demonstrated highly efficient binding of proteins that could be expected to provide efficient peaks and high resolution.

From the DBCs data shown in Table 2, it was found that monolith V and VI had the highest DBCs, that is 36.804 and 48.259 mg ml-1 respectively at flow rate 0.05 ml min-1. The DBCs for BSA of monolith in this work was higher than those reported by Sabarudin and co-workers [14], that was found to be 21.4 and 20.6 mg ml-1 of column volume at flow rates of 0.1 ml min-1 and 0.5 ml min-1, respectively. Furthermore, the DBCs of our optimized monoliths are

also much higher than that of commercially available monoliths; ProSwift WAX-1S (http://www.dionex.com) has DBC of 18 mg ml-1, while CIM® DEAE-1 tube monolithic column (http://www. biaseparations.com) provides DBC of 18 mg ml-1. The good DBCs of these monoliths are attributed not only to a high incorporation of functional monomer, but also to their high-through pore structures that afford good pore accessibility by proteins.

Two monolith columns with the highest DBC value (monolith V and VI) was then assessed its pore size distribution using ISEC. Monolithic column in this study was used in liquid chromatography application, so determination of pore size distribution in the wet state should be more important than if it is measured in the dry state. ISEC provides an appropriate way to perform such measurements as it works at least under conditions similar to those used in actual HPLC separations [38,39]. As shown in Fig. 2a, total porosity (et) of monolith V derived from the retention volume of the tracer (toluene, Vt) was 0.57. This value is comparable to its porogen fraction (0.60). The interstitial/external (ee) porosity derived of the retention volume of the exclude molecular mass (Ve) and internal (ej) porosity obtained by subtraction of et with ee of this monolith were 0.20 and 0.37, respectively. The monolith VI, as shown in Fig 2b, provided the total porosity of 0.61, showing the commensurate value to the fraction of its porogen content (0.60), whereas ee and ei porosities of this monolith were found to be 0.23 and 0.38, respectively. Both monoliths possess larger internal porosities than their external porosities, implying predominant mesopore characters in these stationary phases.

From the ISEC plot shown in Fig. 3a, the volume fractions for macropores or flow-through pores (>50 nm), mesopores (2—50 nm), and micropores (<2 nm) were estimated to be 34.2%, 62.3%, and 3.5%, respectively for monolith V. And for monolith VI, as shown in Fig. 3b, the volume fractions were 37.2%, 60.3%, and 2.5%, respectively. Although the pore distribution of both monoliths is quite similar, but monolith V prevails mesopores size in the range of 2—10 (42.1%), while the monolith VI is dominated by mesopores size of 7—45 nm (49.6%). The difference in mesopores characters, may affect to the application of both monoliths.

A considerable surface area for chromatographic interactions, provided by mesopores, is necessary to obtain adequate functional groups for post-polymerization modification of the monolith, and to yield an acceptable binding capacity. Therefore, monolith must have an appropriate proportion between flow-through pore for efficient convective transport, and mesopores for effective surface area, to obtain a good binding capacity. Monolith V apparently is more suitable for separation of single strand DNA whereas monolith VI is probably well-suited for application to separation of double strand DNA sample. In this work, monolith V and VI was then applied to separation of oligo(dT) and 50 bp DNA ladder, respectively.

3.3. Separation of DNA samples

Oligo(dT)12—18 and 50 bp DNA ladder which consist of 7 and 16 fragments respectively, was used to evaluate the performances of the monolithic column. Oligo(dT) or oligodeoxythymine or oligo-deoxythymidylic acids is a series of short thymine nucleotide, usually consists of 12—20 nucleotides for each fragment. Separation of oligo(dT) and DNA ladder using anion exchanger was based on the difference in the amount of negatively charged phosphate groups in each fragment. The longer the fragment, the more phosphate groups exist, so the amount of negative charge becomes larger. Therefore, the fragment will attached stronger to the positively charged diethylamine groups on the stationary phase and retained longer in the column.

Fig. 4a and Fig. 4b demonstrate the separation of oligo(dT)12—18

0 0.005 0.01 0.015 0 0.005 0.01 0.015

Retention Volume / mL Retention Volume / mL

Fig. 2. Plot of the logarithm of molecular masses (MW) of polystyrene standards versus their elution volume for monolith V (a) and monolith VI (b).

Fig. 3.

.5 1.5 2.5 3.5

Logarithm of pore diameters / nm

Plot of the pore size distribution of poly-(GMA-co-EDMA) anion exchange monolith. Monolith V (a) and monolith VI (b).

Fig. 4. Separation of oligo(dT)12-18 with monolith poly-(GMA-co-EDMA) using monolith V (a) and 50 bp DNA ladder with monolith poly-(GMA-co-EDMA) using monolith VI (b).

Mobile phase (A): 0.02 M Tris-HCl pH 8, mobile phase (B): 1 M NaCl in (A), gradient elution: 0-100% B in 30 min, flow rate: 0.05 ml min" perature, detection UV at 260 nm.

injection volume: 2 mL, room tem-

and 50 bp DNA ladder, respectively. The separation for both samples was performed using a simple linear gradient elution isocratic mode, with NaCl 1 M in 0.02 M Tris-HCl buffer pH 8. As shown in Fig. 4a, seven fragments of oligo(dT) were successfully separated in short time within 6 min. It can be seen that all fragments are baseline resolved, with resolution ranging from 1.76 to 5 (Table 3). This resolution was much better than that in monolithic anion-exchange reported by previous works [2,14] where some fragments could be separated completely (Rs < 1.5). Meanwhile, in 50 bp DNA ladder separation shown in Fig. 4b, some of the peak are

quite close to each other (600 bp to 800 bp), but still can be identified as individual peaks. The fragments of 50-200 bp and 30-550 bp were baseline resolved with high resolution ranging from 1.58 to 3.87 (Table 3). While fairly good resolution ranging from 1.07 to 1.39 were obtained for the peaks of 250 bp and 600-800 bp. Interestingly, our optimized monoliths are capable of separate DNA fragments using a simple linear gradient elution, avoiding a complicated setup during experiments. This simple elution step is not found in the previous reports [2,14] in which step or shallow gradient elution mode was used.

Table 3

Separation efficiency for oligo(dT)12-1s and 50 bp DNA ladder fragments presented as retention times (tR) and resolution (Rs).

Oligo(dT) fragments tR (min) Rs DNA ladder fragments tR (min) Rs

dTi2 3.982 2.31 50 bp 24.274 2.00

dTi3 4.203 2.40 100 bp 24.946 2.84

dTi4 4.407 5.00 150 bp 26.362 2.69

dTi5 4.940 3.20 200 bp 27.s51 2.72

dTie 5.339 2.16 250 bp 28.978 1.39

dTiy 5.560 1.76 300 bp 29.364 2.46

dTis 5.697 - 350 bp 30.064 1.78

400 bp 30.608 1.58

450 bp 30.926 3.87

500 bp 31.618 2.84

550 bp 32.129 2.80

600 bp 32.575 1.18

650 bp 32.789 1.36

700 bp 33.022 1.17

750 bp 33.223 1.07

s00 bp 33.429 -

4. Conclusions

A poly-(GMA-co-EDMA) anion exchange monolithic column was successfully produced by a two-step procedure, involving the in situ polymerization and subsequent on-column chemical modification with diethylamine via ring opening reaction of epoxy groups. Morphology of the monolithic column was studied by SEM. From the results of the characterization of all six column variations, monolith poly-(GMA-co-EDMA) with %T 40, %C 25 and %T 40, %C 35 were found to be the ideal composition of monomer and cross-linker. Mechanical stability and permeability of the columns were both good. Adequate molecular recognition sites (represented with binding capacity), and appropriate composition amounts of flow-through pore and mesopores may contribute to successful separation of oligo(dT)i2_i8 and 50 bp DNA ladder with good resolution by employing simple gradient elution.

Acknowledgements

This work was supported by Directorate General of Higher Education, Ministry of Education and Culture, Indonesia through Penelitian Unggulan Perguruan Tinggi (PUPT) No. 023.04.2.414989/ 2013-2014. There is no any conflict of interest for all authors and also the funding institution.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ancr.2015.11.001.

References

[1] C.R. Smith, R.B. DePrince, J. Dackor, D. Weigl, J. Griffith, M. Persmark, Separation of topological forms of plasmid DNA by anion-exchange HPLC: shifts in elution order of linear DNA, J. Chromatogr. B 854 (2007) 121-127.

[2] D. Sykora, F. Svec, J.M.J. Frechet, Separation of oligonucleotides on novel monolithic columns with ion-exchange functional surfaces, J. Chromatogr. A 852 (1999) 297-304.

[3] M.C. Kelly, B. White, M.R. Smyth, Separation of oxidatively damaged DNA nucleobases and nucleosides on packed and monolith C18 columns by HPLC-UV-EC, J. Chromatogr. B 863 (2008) 181-186.

[4] T. Watanabe, K. Makitsuru, H. Nakazawa, S. Hara, T. Suehiro, A. Yamamoto, T. Hiraide, T. Ogawa, Separation of double-strand DNA fragments by highperformance liquid chromatography using a ceramic hydroxyapatite column, Anal. Chim. Acta 386 (1999) 69-75.

[5] W. Rozhon, T. Baubec, J. Mayerhofer, O.M. Scheid, C. Jonak, Rapid quantification of global DNA methylation by isocratic cation exchange highperformance liquid chromatography, Anal. Biochem. 375 (2008) 354-360.

[6] S. Yamamoto, M. Nakamura, C. Tarmann, A. Jungbauer, Retention studies of

DNA on anion-exchange monolith chromatography binding site and elution behavior, J. Chromatogr. A 1144 (2007) 155-160.

S. Yamamoto, N. Yoshimoto, C. Tarmann, A. Jungbauer, Binding site and elution behavior of DNA and other large biomolecules in monolithic anion-exchange chromatography, J. Chromatogr. A 1216 (2009) 2616-2620. J.C. Murphy, G.E. Fox, R.C. Willson, Enhancement of anion-exchange chro-matography of DNA usingcompaction agents, J. Chromatogr. A 984 (2003) 215-221.

I. Gusev, X. Huang, C. Horvath, Capillary columns with in situ formed porous monolithic packing for micro high-performance liquid chromatography and capillary electrochromatography, J. Chromatogr. A 855 (1999) 273-290. H. Zou, X. Huang, M. Ye, Q. Luo, Monolithic stationary phases for liquid chromatography and capillary electrochromatography, J. Chromatogr. A 954 (2002) 5-32.

T. Nema, E.C.Y. Chan, P.C. Ho, Applications of monolithic materials for sample preparation, J. Pharm. Biomed. 87 (2014) 130-141.

R.D. Arrua, M. Talebi, T.J. Causon, E.F. Hilder, Review of recent advances in the preparation of organic polymer monoliths for liquid chromatography of large molecules, Anal. Chim. Acta 738 (2012) 1-12.

J. Rouquerol, D. Avnir, C.W. Fairbridge, D.H. Everett, J.M. Haynes, N. Pernicone, J.D.F. Ramsay, K.S.W. Sing, K.K. Unger, Recommendations for the characterization of porous solids, Pure Appl. Chem. 66 (1994) 1739-1758. A. Sabarudin, J. Huang, S. Shu, S. Sakagawa, T. Umemura, Preparation of methacrylate-based anion-exchange monolithic microbore column for chro-matographic separation of DNA fragments and oligonucleotides, Anal. Chim. Acta 736 (2012) 108-114.

Y. Ueki, T. Umemura, J. Li, T. Odake, K.I. Tsunoda, Preparation and application of methacrylate-based cation-exchange monolithic columns for capillary ion chromatography, Anal. Chem. 76 (2004) 7007-7012.

J. Urban, F. Svec, J.M.J. Freechet, Efficient separation of small molecules using a large surface area hypercrosslinked monolithic polymer capillary column, Anal. Chem. 82 (2010) 1621-1623.

R. Koeck, M. Fischnaller, R. Bakry, R. Tessadri, G.K. Bonn, Preparation and evaluation of monolithic poly(N-vinylcarbazole-co-1,4-divinylbenzene) capillary columns for the separation of small molecules, Anal. Bioanal. Chem. 406 (2014) 5897-5907.

P.A. Levkin, S. Eeltink, T.R. Stratton, R. Brennen, K. Robotti, H. Yin, K. Killeen, F. Svec, J.M.J. Freechet, Monolithic porous polymer stationary phases in poly-imide chips for the fast high-performance liquid chromatography separation of proteins and peptides, J. Chromatogr. A 1200 (2008) 55-61. J. Krenkova, A. Gargano, N.A. Lacher, J.M. Schneiderheinze, F. Svec, High binding capacity surface grafted monolithic columns for cation exchange chromatography of proteins and peptides, J. Chromatogr. A 1216 (2009) 6824-6830.

V. Frankovic, A. Podgornik, N.L. Krajnc, F. Smrekar, P. Krajnc, A. Strancar, Characterisation of grafted weak anion-exchange methacrylate monoliths, J. Chromatogr. A 1207 (2008) 84-93.

S.H. Lubbad, M.R. Buchmeiser, Ring-opening metathesis polymerization-derived monolithic anion exchangers for the fast separation of double-stranded DNA fragments, J. Chromatogr. A 1218 (2011) 2362-2367. F. Smrekar, A. Podgornik, M. Ciringer, S. Kontrec, P. Raspor, A. SStrancar, M. Peterka, Preparation of pharmaceutical-grade plasmid DNA using meth-acrylate monolithic columns, Vaccine 28 (2010) 2039-2045. N.L. Krajnc, F. Smrekar, J. CSerne, P. Raspor, M. Modic, D. KrgovicS, A. SStrancar, A. Podgornik, Purification of large plasmids with methacrylate monolithic columns, J. Sep. Sci. 32 (2009) 2682-2690.

R. Koeck, R. Bakry, R. Tessadri, G.K. Bonn, Monolithic poly(N-vinylcarbazole-co-1,4-divinylbenzene) capillary columns for the separation of biomolecules, Analyst 138 (2013) 5089-5098.

R.D. Arrua, M.C. Strumia, C.I.A. Igarzabal, Macroporous monolithic polymers:

preparation and applications, Materials 2 (2009) 2429-2466.

E.C. Peters, M. Petro, F. Svec, J.M.J. Freechet, Molded rigid polymer monoliths as

separation media for capillary electrochromatography, Anal. Chem. 69 (1997)

3646-3649.

E.C. Peters, M. Petro, F. Svec, J.M.J. Freechet, Molded rigid polymer monoliths as separation media for capillary electrochromatography. 1. Fine control of porous properties and surface chemistry, Anal. Chem. 70 (1998) 2288-2295. D. Moravcova, P. Jandera, J. Urban, J. Planeta, Characterization of polymer monolithic stationary phases for capillary HPLC, J. Sep. Sci. 26 (2003) 1005-1016.

X. Shu, L. Chen, B. Yang, Y. Guan, Preparation and characterization of long methacrylate monolithic column for capillary liquid chromatography, J. Chromatogr. A 1052 (2004) 205-209.

J. VidicS, A. Podgornik, J. JanScar, V. FrankovicS, B. KoSsir, N. Lendero, K. CSucSek, M. Krajnc, A. Strancar, Chemical and chromatographic stability of methacrylate-based monolithic columns, J. Chromatogr. A 1144 (2007) 63-71.

A. Bruchet, V. Dugas, I. Laszak, C. Mariet, F. Goutelard, J. Randon, Synthesis and characterization of ammonium functionalized porous poly(glycidyl methacrylate-co-ethylene dimethacrylate) monoliths for microscale analysis and its application to DNA purification, J. Biomed. Nanotechnol. 7 (2011) 1-11.

C.M. Ongkudon, M.K. Danquah, Anion exchange chromatography of 4.2 kbp plasmid based vaccine (pcDNA3F) from alkaline lysed E. coli lysate using amino functionalised polymethacrylate conical monolith, Sep. Purif. Technol.

78 (2011) 303-310.

[33] N. Wang, S. He, W. Yan, Y. Zhu, Incorporation of multiwalled carbon nanotube into a polymethacrylate-based monolith for ion chromatography, J. Appl. Polym. Sci. 128 (2013) 741-749.

[34] S. Shu, H. Kobayashi, N. Kojima, A. Sabarudin, T. Umemura, Preparation and characterization of lauryl methacrylate-based monolithic microbore column for reversed-phase liquid chromatography, J. Chromatogr. A 1218 (2011) 5228-5234.

[35] S. Shu, H. Kobayashi, M. Okubo, A. Sabarudin, M. Butsugan, T. Umemura, Chemical anchoring of lauryl methacrylate-based reversed phase monolith to 1/16" o.d. polyetheretherketone tubing, J. Chromatogr. A 1242 (2012) 59-66.

[36] T. Umemura, Y. Ueki, K. Tsunoda, A. Katakai, M. Tamada, H. Haraguchi,

Preparation and characterization of methacrylate-based semi-micro monoliths for high-throughput bioanalysis, Anal. Bioanal. Chem. 386 (2006) 566—571.

[37] M. Al-Bokari, D. Cherrak, G. Guiochon, Determination of the porosities of monolithic columns by inverse size-exclusion chromatography, J. Chromatogr. A 975 (2002) 275—284.

[38] Y. Li, M.L. Lee, J. Jin, J. Chen, Preparation and characterization of neutral poly(ethylene glycol) methacrylate-based monolith for normal phase liquid chromatography, Talanta 99 (2012) 91—98.

[39] H. Oberacher, A. Premstaller, C.G. Huber, Characterization of some physical and chromatographic properties of monolithic poly(styrene—co-divinylben-zene) columns, J. Chromatogr. A 1030 (2004) 201—208.