Excess isotherms as a new way for characterization of the columns for reversed-phase liquid chromatography

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Journal of Chromatography A, 1191 (2008) 72–77

Excess isotherms as a new way for characterization of the columns for reversed-phase liquid chromatography B. Buszewski a,∗ , Sz. Bocian a , A. Felinger b a

Department of Environmental Chemistry & Bioanalytics, Faculty of Chemistry, Nicolaus Copernicus University, Gagarin Street 7, 87-100 Toru´n, Poland b Department of Analytical and Environmental Chemistry, Faculty of Science, University of P´ ecs, Ifj´us´ag u´ tja 6, H-7624 P´ecs, Hungary Available online 20 February 2008

Abstract In this work, we present the excess isotherm of acetonitrile for stationary phases with different coverage density. Data obtained with the minor disturbance method were compared with 29 Si cross-polarization/magic-angle spinning NMR spectra to find dependence between acetonitrile adsorption on C18 chemically bonded stationary phases and coverage density of stationary phase. The preferential adsorption of acetonitrile on the bonded phase and the adsorption of water on the silica surface can be well correlated with the coverage density. © 2007 Elsevier B.V. All rights reserved. Keywords: HPLC; NMR; Adsorption; Stationary phase; Coverage density

1. Introduction The knowledge of the structure of the stationary phase is very important for understanding the chromatographic separation. Stationary phases used in reversed-phase high-performance liquid chromatography (RP-HPLC) have different chemical properties. In the structure of bonded phase, two types of adsorption centers are included: hydrophobic organic bonded ligands and residual silanols. In this case, the type of bonded ligands, coverage density and surface homogeneity with the presence of accessible silanols are the most important parameters which determine the chromatographic properties of the packing [1–3]. Retention in RP-HPLC is governed by the hydrophobic effect, i.e. the interaction between organic bonded ligands and the solute [4,5]. Chemical modification of the silica gel surface allows preparing well-defined stationary phases. The chemical modification process can be controlled by changing the composition of the reaction components [6,7]. Despite the derivatization process of the silica gel, in the structure of the stationary phase, the residual silanols are always present. The presence of residual silanols can have a negative influence on the separation of polar analytes, especially basic compounds and biopolymers [8,9]. From the amount of residual silanols – which are not bonded with silanes – only about 5% exist as a polar strong adsorption center

∗

Corresponding author. Tel.: +48 56 6114308; fax: +48 56 6114837. E-mail address: [email protected] (B. Buszewski).

0021-9673/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2007.11.074

[10]. This effect is caused by shield properties of the methyl group connected to the silicon atom in the silanes [1,10,11]. In this case the bad homogeneity of the bonded ligands can uncover the surface of the silica gel and expose more accessible silanols which worsens chromatographic separation [12]. The commonly used mobile phases in RP-HPLC are binary mixtures of water and an organic modifier. Those components are selectively distributed between the mobile and the stationary phases so the stationary phase consists of adsorbed solvent molecules [4]. The adsorption behaviors of those components (water and organic modifiers) are totally different due to the polar and non-polar properties of the molecules. Differences of the adsorption of solvents, measured as excess isotherms, can indicate the packing properties as packing hydrophobic properties (coverage density, percent of surface coverage), polar properties (amount of accessible silanols) and homogeneity of bonded ligands [13]. Different instrumental techniques have been used to describe the surface of material packing. Elementary analysis (CHN) gives the possibility to determine the total percent value of carbon deposition and the value of individual elements which form the structure of the bonded ligands. Knowing the bare silica support surface area and percentage of carbon deposition it is possible to calculate the coverage density (αRP ). Spectroscopic analysis, mainly solid-state NMR and FT-IR, gives the information about types and character of bonded groups and also their distributions. Similarly, thermal analysis [thermogravimetric analysis (TGA) and differential scanning

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calorimetry (DSC)] and/or microscopy techniques, particularly atomic force microscopy (AFM), gives very interesting information about the subsurface film and the structure of the stationary phase [1,3,6,14]. Interesting information about surface character of RP-HPLC packings can be achieved based on the chromatographic study. Numbers of contributions related to this matter have been published. Authors also focused their interest on changes in stationary phase ligand conformation by determination of interaction and description of retention mechanism [4,15–18]. All those minds did not give univocal answer: how homogenous is the surface of the stationary phase? From that reason the subject of our research is focused on the physicochemical correlation (CHN analysis or solid-state NMR) that correlates the surface coverage of a stationary phase with the amount of adsorbed solvents. We found a relation between the preferential adsorption of water and acetonitrile molecules on C18 chemically bonded stationary phases and residual silanols and to the coverage density of stationary phase. For verification of our model two commercially available C18 columns were applied. 2. Theory In reversed-phase liquid chromatography, the surface of the stationary phase is in contact with individual components of a binary mobile phase. The composition of the liquid phase changes in an unknown manner from the adsorbent surface to the bulk liquid [19]. The organic modifier is preferentially adsorbed on the surface of hydrophobic stationary phase, thus it forms a layer in the immediate vicinity of the surface [20]. In isocratic chromatography, the stationary phase is in equilibrium with the mobile phase. A small volume of the mixture of the eluent components with different concentration injected onto the column introduces some perturbation and so-called minor disturbance peaks are observed. This perturbation moves through the column and elutes at a given retention volume. The retention volume of disturbance peak can be defined by Eq. (1) [20,21]. VR (C) = VM + S

dΓ (C) dC

(1)

where VR is the retention volume of the perturbation [ml], VM is the thermodynamic dead volume of the column [ml], S is the total surface area of the adsorbent in the column [m2 ] and Γ is the excess adsorption isotherm of the analyte [mol/m2 ] at concentration C [mol/l]. The integration of the Eq. (1) allows the calculation of excess adsorption values from the dependence of disturbance peak retention on the concentration [21]:  1 C Γ (C) = (VR (C) − VM )dC (2) S 0 The thermodynamic dead volume (VM ) of the column (total volume of the liquid phase in the column) is obtained by integrating the plot of the retention volumes of perturbations peaks obtained over whole concentration of mobile phase (from 0 to

100%) [21]: VM =

1 Cmax



C=Cmax

C=0

73

VR (C)dC

(3)

The maximum concentration of the preferentially adsorbed component (Cads ) can be found by extrapolating the slope of the excess isotherm in the linear region to the intercept y-axis [21–23]. The excess isotherm can be employed to represent the variation of an excessively adsorbed amount of organic solvent with the variation of the equilibrium concentration of this solvent in the mobile phase. In each point of the isotherm, the difference between the amount of organic solvent adsorbed on the stationary phase and the amount of solvent calculated from the volume of the adsorbed phase is shown [20]. The adsorbed layer has a finite volume or finite thickness. When the concentration of the organic solvent in the binary mobile phase increases, the excess amount of this solvent decreases in linear manner. It is caused by the effect of the adsorbed phase filling [20]. However, the stationary phase in RP-HPLC is heterogeneous. The presence of residual silanols causes the adsorption of water. When the binary hydro-organic mobile phase is used, the molecular exchange between the adsorbed and bulk mobile phase can appear [19]: [organic]adsorbed + [H2 O]mobile ⇔ [organic]mobile + [H2 O]adsorbed

(4)

Because of the finite volume of the adsorbed layer, some part of the organic solvent molecules can be displaced from the stationary phase and the water molecules can adsorb. It is shown as a negative part of the excess isotherm. 3. Experimental 3.1. Instruments A HP Model 1050 liquid chromatograph (Hewlett-Packard, Waldbronn, Germany) was used, equipped with four channels gradient pump, a manual injector with a 20-␮l loop, UV–vis detector Agilent 1100. Also a Shimadzu 10 (Kyoto, Japan liquid chromatograph) equipped with a multisolvent delivery system (LC-10AD) was used, with a manual injector with a 20-␮l loop, a diode-array detector (SPD-M10A), fluorescence detector (RF-10A) and computer data acquisition station. The porosity parameters characterizing the starting material were determined by low-temperature nitrogen adsorption–desorption method using a Model ASAR 2010 (version 2.0) sorptomatic apparatus (Micrometrics, Norcross, GA, USA). The degree of coverage of the surface by alkylsilyl ligands (αRP ) was calculated on the basis of the carbon percentage [15,24] determined with a Model 240 CHN analyzer (PerkinElmer, Norwalk, CT, USA).

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Table 1 Geometric parameters of bare porous silica Parameter

Abbreviation

Unit

Value

Mean particle size Particle shape Specific surface area Mean pore diameter Pore volume Concentration of OH groups

dp – SBET D Vp αSiOH

␮m – m2 /g ˚ A cm3 /g ␮mol/m2

5 Spherical 295 113 0.92 7.1

Solid-state NMR measurements were performed on a Bruker MSL 300 spectrometer (Bruker AG, Karlsruhe, Germany) with samples of 200–300 mg in double bearing rotors of ZrO2 . 29 Si cross-polarization/magic-angle spinning (CP/MAS) NMR were recorded with a pulse length of 5 ␮s together with a contact time of 5 ms and a pulse repetition time of 2 s. All spectra were externally referenced with liquid tetramethylsilane (TMS) and the chemical shifts (δ) were given in parts per million (ppm). 3.2. Materials The solid support of laboratory-prepared phases was Kromasil 100 AT 0191 (Akzo Nobel, Bohus, Sweden). Parameters of the silica are described in Table 1. The following reagents were used for chemical modification of the silica support material: octadecyldimethylchlorosilane (Johnson Matthey ALFA Products, Karlsruhe, Germany), trimethylchlorosilane (Sigma–Aldrich Chemie, Steinheim, Germany), morpholine (Reachim, Moscow, Russia). Organic solvents (methanol and acetonitrile) were high-purity “for HPLC” isocratic grade. Water was purified using Milli-Q system (Millipore, El Passo, TX, USA). All eluents were degassed in ultrasonic bath under vacuum. The silica was chemically modified with octadecyldimethylchlorosilane (Wacker, Munich, Germany). For residual silanols deactivation (end-capping) trimethylchlorosilane (Petrarch System, Levittown, PA, USA) was used. The bare silica surface chemical modification and end-capping conditions are described in detail in Ref. [12]. Parameters of the prepared stationary phases are described in Table 2.

Adsorbents were packed into 100 mm × 4.6 mm I.D. stainless steel columns using the slurry laboratory-made apparatus equipped with Haskel packing pomp (Burbank, CA, USA) under a constant pressure of 50 MPa. Two commercial C18 columns were studied. The physicochemical parameters of those columns are listed in Table 3. 3.3. Measurements Each column was equilibrated with mobile phase of decreasing concentration of organic solvent in water (100, 98, 94, 90, 80, 70, 60, 50, 40, 30, 20, 10, 8, 4, 0) by pumping at least 40 ml of solvent mixture. Perturbation of base line was done by 5 ␮l injection of the mixture with higher concentration of organic solvent than the plateau concentration. The signal was detected with a UV detector at 195 nm. For asymmetric peak observed in extreme mobile phase composition the retention volume was estimated from the extrapolation of the peak tail. Because the specific surface area of the volume of the stationary phase after derivatization is not known the excess amount of organic solvent was measured per product of volume of stationary phase (Vs ) and specific surface area of bare silica gel (SBET ). The volume of the stationary phase was calculated as a difference between the total volume (Vt ) and the dead volume (VM ) of the column calculated from Eq. (3). The parameters of tested columns are listed in Table 4. 4. Result and discussion Elementary analysis is the basic method used to describe the stationary phase. The amount of carbon and other elements can be measured by sample combustion. The knowledge of percent of the carbon enables to calculate the coverage density of the stationary phase. In this case the Berendsen equation is used [15]: αIRP =

1 106 PC × [␮mol/m2 ] 1200nC − PC (M1 − nX ) SBET

(5)

where αIRP is the coverage density [␮mol/m2 ], PC the percent of carbon [%], nC the number of carbon atoms in the ligand, M1

Table 2 Geometric parameters of chemically bonded phases Type of stationary phase C18L

M M C18L + EC M C18H M C18H + EC

Carbon load (%)

Coverage density (␮mol/m2 )

Surface coverage (%)

End-capping

10.66 12.68 17.10 17.45

1.75 2.13 3.10 3.17

23.3 28.5 41.5 42.4

No Yes No Yes

where EC, end-capping; L, low coverage density; H, high coverage density; M, monomer phases. Table 3 Properties of the columns tested Support

Bonded ligands

Producent

˚ Pore diameter (A)

SBET (m2 )

Carbon load (%)

End-capping

Symmetry Synergi Hydro

C 18 C18

Waters Phenomenex

100 80

344 475

19 19

Yes Yes (polar)

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Table 4 Characterization of used C18 packed columns Name of the column

Producer

Column length (mm)

Column diameter (mm)

Particle size (␮m)

Dead volume of the column (ml)

Symmetry Synergi Hydro

Waters Phenomenex

150 250

4.6 4.6

5 5

1.437 2.573

M C18L M C18L + EC M C18H M C18H + EC

Chair of Environmental Chemistry & Bioanalytics

100 100 100 100

4.6 4.6 4.6 4.6

5 5 5 5

1.099 1.048 0.949 0.934

the molar mass of the ligand, nx the number of functional group in reactive group of the silane and SBET the specific surface area [m2 /g]. For the stationary phase with end-capping the coverage density can be calculated from the equation modified by Buszewski et al. [24]: αIRP =

ing and it can adsorb on the residual silanols. Excess amount of the organic solvent adsorbed on the bonded phase can give some useful information about the structure and composition of the stationary phase [4]. However, our experiments demonstrate that more characteristic data was obtained for acetonitrile, as an organic modifier in mobile phase. The excess isotherms of ace-

PC(2) − PC(2) αIRP (M1 − 1) − 1200αIRP nC(1) 1200nC(2) − PC(2) (M2 − 1) ×

1 [␮mol/m2 ] SBET

(6)

2 where αII RP is the coverage density [␮mol/m ], PC(2) the percent of carbon after the second step of modification [%], nC(1) the number of carbon atoms in the ligand in the first step of modification, nC(2) the number of carbon atoms in the ligand in the second step of modification and M2 the molar mass of the ligand in the second step of modification. CP/MAS-NMR spectroscopy for 29 Si gives quantitative information about the density of silica gel coverage with ligands [7]. Spectra for bare silica contain three characteristic signals, which correspond to geminal (Q2 ) δ = −91 ppm and free/bonded (Q3 ) δ = −100 ppm) silanol groups and oxosilanes (Q4 ) δ = −108 ppm [1,3,6,14,25,26]. As seen in Fig. 1, the intensity of signals (Q2 ) and (Q3 ) decrease after derivatization and (Q4 ) increase. A signal M (δ = +2.5 ppm) corresponds with the monomeric structure of chemically bonded ligands. For the stationary phase with low coverage density of chemically bonded ligands, a signal of geminal silanols (Q2 ) is observed, even for an end-capped stationary phase. A part of geminal silanols is still unblocked and it can have a negative influence on the chromatographic separation. A signal of geminal silanols (Q2 ) is not observed for densely covered silica gel [7]. In this work, the retention of minor disturbance peaks of the eluent component and their excess isotherms were compared. Our investigation concerns methanol–water and acetonitrile–water mobile phases. From two tested solvents, acetonitrile exhibits much better adsorption properties. It is known [23,27,28] that the adsorption of methanol on alkyl chemically bonded phase is a monolayer and the adsorption of acetonitrile is a multilayer. Acetonitrile cannot create hydrogen bonding with silanol groups, so the adsorption of the acetonitrile on the residual silanols is very weak [10,11,28]. In acetonitrile–water conditions, water is preferentially adsorbed on residual silanols. Methanol is a protic solvent, which can create hydrogen bond-

Fig. 1. 29 Si CP/MAS-NMR spectra for the bare silica and four different bonded C18 stationary phases: (a) low coverage density, (b) high coverage density.

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Fig. 2. Excess isotherm of acetonitrile from water solution onto four different stationary phases vs. the concentration of acetonitrile in bulk mobile phase.

tonitrile on the stationary phases with different coverage density are shown in Fig. 2. The shape of the excess isotherms changes with coverage density of the chemically bonded phase. The adsorption of organic solvent depends also on the coverage density, not only on the length of organic chains. The shape of the excess isotherm and the parameters of the isotherm can give useful information about the coverage density of chemically bonded phase, homogeneity of the bonded ligands and about the amount of unblocked residual silanols. For the packing with low coverage without end-capping, the excess amount of acetonitrile is very small. It is connected with hydrophobic effect. In this stationary phase, 23% of the silica surface is covered, so there is a significant amount of accessible silanols, which can interact with water molecules, present in the binary mobile phase. When the density of the chemically bonded phase is small, the adsorption of the organic modifier is also limited. When the coverage density increases, the amount of hydrophobic adsorption centers increases and the number of residual silanols decreases, i.e. the adsorption of acetonitrile increases and that of water decreases. For the adsorption effect, two values were compared: the maximum concentration of the organic modifier on the stationary phase (Cads ) and the maximum of the solvent excess on the stationary phase, described as maximum of excess isotherm (Fig. 3). As was mentioned by Gritti et al. [28] the amount of the adsorbed acetonitrile on the stationary phase increases proportionally to the coverage density of the chemically bonded phase, the percent of surface coverage and to the carbon load (for the same ligand length) which were calculated from elementary analysis (CHN). From the two parameters, the maximum of the excess isotherm and the maximum concentration of the organic modifier in the stationary phase, the second one shows better correlation, and this parameter should be used in the comparison of solvent adsorption. Adsorption of water on residual silanols is visible in the negative part of the excess isotherm. In an acetonitrile–water mobile phase, the magnitude of the negative part of the excess isotherm can give information about the amount and concentration of

Fig. 3. Dependence between maximum of the isotherm and maximum concentration of acetonitrile on stationary phase vs. coverage density, percent of surface covered and carbon load of the stationary phase.

accessible residual silanols. Acetonitrile strongly interacts with the apolar organic ligands and its interaction with residual silanols is negligible. When the concentration of acetonitrile (or other organic solvent) increases, water interacts stronger with residual silanols. In this condition, water adsorption can be multilayer and it can create an excess of adsorbed water in comparison with the water concentration in the eluent (Fig. 4). Differences between low coverage density packings with and without end-capping are much higher than those for high coverage density. It is connected with steric effects. Low coverage density stationary phases have about 25% blocked silanols so the end-capping may be carried out with better efficiency. High-coverage-density stationary phases have about 44% of

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correlated with amount of adsorbed organic solvent. The maximum adsorbed amount of acetonitrile increases proportionally with the coverage density of chemically bonded phase. Excess isotherms can give some useful information about the presence of residual accessible silanols and coverage homogeneity of the silica gel by organic ligands. The amount of excess adsorbed acetonitrile shows the hydrophobic properties of the stationary phase. The negative part of the excess isotherm indicates the amount of polar adsorption centers–residual silanols. Acknowledgements Fig. 4. Negative part of the excess isotherm vs. coverage density of bonded phase.

This work was sponsored by Ceepus II scholarship CII-PL0004-01-0506-M-4479 and CII-PL-0004-02-0607-M-12188. This manuscript was prepared to the Special Issue J. Chromatogr. A as contribution presented at 13th ISSS conference in High Tatras. Financial support from the Hungarian National Science Foundation (Grant No. OTKA T048887) is gratefully acknowledged. References

Fig. 5. Acetonitrile excess isotherm from water solution onto the in-house prepared and commercial C18 stationary phases.

blocked silanols so the end-capping process is much more difficult (Fig. 4). Excess isotherms of in-house prepared stationary phases were compared with commercial C18 phases. The results are shown in Fig. 5. For a better comparison of the different stationary phases, the adsorbed amount was divided by the specific surface area of the bare silica. Adsorption on the commercial stationary phases exhibit a shape rather similar to the isotherm obtained on the in-house prepared stationary phases. The negative part of those two isotherms is negligible. The reason for that is that those packings have high coverage density and small amount of residual accessible silanols. It is known, that the coverage density of Waters C18 is αRP = 3.2 ␮mol/m2 , so it is very similar to the laboratory-made column M C18H + EC. 5. Conclusions In this work, the application of acetonitrile adsorption for the characterization of chemically bonded reversed phases is described. Data obtained from NMR spectroscopy and elementary analysis which characterized the stationary phases are

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