HPLC for Pharmaceutical Scientists 2007 (Part 3)

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The column is the only device in the high-performance liquid chromatography (HPLC) system which actually separates an injected mixture. Column packing materials are the “media” producing the separation, and properties of this media are of primary importance for successful separations. Several thousands of different columns are commercially available, and when selecting a column for a particular separation the chromatographer should be able to decide whether a packed, capillary, or monolithic column is needed and what the desired characteristics of the base material, bonded phase, and bonding density of selected column is needed. Commercial columns of the same general...

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Nội dung Text: HPLC for Pharmaceutical Scientists 2007 (Part 3)

3 STATIONARY PHASES Yuri Kazakevich and Rosario Lobrutto 3.1 INTRODUCTION The column is the only device in the high-performance liquid chromatogra- phy (HPLC) system which actually separates an injected mixture. Column packing materials are the “media” producing the separation, and properties of this media are of primary importance for successful separations. Several thousands of different columns are commercially available, and when selecting a column for a particular separation the chromatographer should be able to decide whether a packed, capillary, or monolithic column is needed and what the desired characteristics of the base material, bonded phase, and bonding density of selected column is needed. Commercial columns of the same general type (e.g., C18) could differ widely in their separation power among different suppliers. Basic information regarding the speciﬁc column provided by the manufacturer, such as surface area, % carbon, and type of bonded phase, usually does not allow prediction of the separation or for the proper selection of columns with similar separation patterns. Great varieties of different columns are currently available on the market. Four distinct characteristics could be used for column classiﬁcation: 1. Type (monolithic; porous; nonporous) 2. Geometry (surface area; pore volume; pore diameter; particle size and shape; etc.) HPLC for Pharmaceutical Scientists, Edited by Yuri Kazakevich and Rosario LoBrutto Copyright © 2007 by John Wiley & Sons, Inc. 75
76 STATIONARY PHASES 3. Surface chemistry (type of bonded ligands; bonding density; etc.) 4. Type of base material (silica; polymeric; zirconia; etc) All these characteristics are interrelated. Variations of porosity which include pore diameter can affect both the adsorbent surface area and the bonding density. The type of base material affects adsorbent surface chemistry. There- fore, in our discussion we combine these characteristics in two major classes: geometry and surface chemistry. Most geometry-related properties of packing materials are related to the column efﬁciency and ﬂow resistance: particle size, particle shape, particle size distribution, packing density, and packing uniformity. Surface-chemistry- related properties are mainly responsible for the analyte retention and sepa- ration selectivity. Adsorbent surface area, pore volume, and pore diameter are the properties of signiﬁcant importance. HPLC retention is generally proportional to the surface area accessible for a given analyte (Chapter 2). Surface area accessi- bility is dependent on the analyte molecular size, adsorbent pore diameter, and pore size distribution. The chemical nature of the ligands bonded on the surface of support mate- rial deﬁnes the main type of chemical interactions of the surface with eluent and analyte molecules. In essence, all C18-type columns should be similar with regard to their main interaction type, namely, hydrophobic interactions: Meth- ylene selectivity of all C18-type columns are virtually identical [1]. Bonded phases of the same type differ in their ability to suppress (or shield) other types of interactions (ionic; dipole) exerted by the base material (e.g., silica). Energy of these unwanted interactions is about 10 times greater than the energy of dispersive interactions [2]. Due to the exponential nature of the rela- tionship between retention and interaction energy even the presence of 1% or less of these active centers in the packing material surface can signiﬁcantly affect the analyte retention. Bonding density is the primary parameter in evaluation of the quality of the bonded material. Usually the higher the bonding density, the better the shielding effect, although care should be taken in cross-evaluation of similar columns on the basis of their bonding density. Surface geometry can also sig- niﬁcantly affect bonding density. Base material with smaller pores has higher surface area; however, bonding density is usually lower due to the smaller pores. All parameters of the packing material are interrelated in their inﬂuence on the chromatographic performance of the column. The quality of an HPLC column is a subjective factor, which is dependent on the types of analytes and even on the chromatographic conditions used for the evaluation of the overall quality. Long-term column stability (pH and temperature) and batch-to-batch reproducibility are probably the most important quality characteristics to be considered in column selection in the pharmaceutical industry. Nevertheless,
BASE MATERIAL (SILICA, ZIRCONIA, ALUMINA, POLYMERS) 77 these criteria should be evaluated with caution when selecting the column evaluation parameters. Long-term stability of retention and efﬁciency charac- teristics are usually different, depending on the testing conditions (mobile phase, temperature, and analyte probes). Efﬁciency is usually fairly stable at low mobile-phase pH, while retention of the probe analytes may show a drift in retention. However, the retention is generally stable at high pH while efﬁ- ciency could be deteriorated. 3.2 TYPE OF PACKING MATERIAL (POROUS, NONPOROUS, MONOLITHIC) Majority of packing materials used in HPLC are porous particles with average diameters between 3 and 10 µm. For most pharmaceutical applications, 3-µm particle sizes are recommended. Porosity provides the surface area necessary for the analyte retention (usually between 100 and 400 m2/g). Interparticle space is large enough to allow up to 1–3 mL/min ﬂow within acceptable pres- sure range (however, the pressure drop across the column depends on the particle size, length of column, temperature of separation, and type of mobile- phase composition). Introduction of small nonporous spherical particles in the mid-1990s [3, 4] was an attempt to increase efﬁciency by eliminating dual column porosity. In the column packed with porous particles, interparticle space is about 100-fold larger than pores inside the particles, and liquid ﬂow around the particles is also faster; this leads to the signiﬁcant band broadening. Unfortunately, elimi- nation of particle porosity dramatically decreases adsorbent surface area, thereby decreasing the column loading capacity. Columns packed with small (1.5 µm) nonporous particles also require ultra-microinjection volumes and a corresponding increase of detector sensitivity. The introduction of monolithic columns in the 1990s was another and more successful attempt to increase column permeability while decreasing the gap in column dual porosity. Macropores in the monolith are between 4000 and 6000 Å in diameter, and they occupy almost 80% of the column volume. Com- pared to the conventional packed column with 5- or even 3-µm particles, the silica skeleton in monolith is only approximately 1 µm thick, which facilitates accessibility of the adsorbent surface inside the mesopores of the skeleton (pores between 20 and 500 Å in diameter are usually called mesopores). Com- parison of the spherical packing material and monolithic silica is shown in Figure 3-1. 3.3 BASE MATERIAL (SILICA, ZIRCONIA, ALUMINA, POLYMERS) In modern liquid chromatography, almost all reversed-phase separations are performed on chemically modiﬁed adsorbents. Analyte interactions with the
78 STATIONARY PHASES Figure 3-1. SEM pictures of HPLC silica particles (5 µm) and silica monolith. (Reprinted with permission from reference 5.) stationary phase surface are the primary factor for successful separations. Most commercial adsorbents reﬂect their surface chemistry in their names (e.g., C18, C8, Phenyl, etc.) while the base material used usually is not speciﬁed, although its properties are very important. Speciﬁc parameters of the base of packing material are: • Surface area • Pore size • Pore volume • Pore size distribution • Particle shape • Particle size • Particle size distribution • Structural rigidity • Chemical stability • Surface reactivity • Density and distribution of the surface reactive centers Surface area is directly related to the analyte retention [Equation (2-47) in Chapter 2]. Generally, the higher the surface area, the greater the retention. Pore size is a critical parameter for the surface accessibility. Molecules of different size could have different accessible surface area due to the steric hin- drance effect (bigger molecules might not be able to penetrate into all pores). Pore size is also related to the surface area. Assuming that all pores of the base material are cylindrical and neglecting the networked porous structure (assuming straight and not interconnected pores), it is possible to write the following expressions for the surface area and pore volume [6]: S = 2pRL, V = pR 2 L (3-1) where S is a surface area of one gram of porous adsorbent; R is the average pore radius; V is a pore volume of one gram of adsorbent; and L is a total length of all pores in the same one gram of the adsorbent.
BASE MATERIAL (SILICA, ZIRCONIA, ALUMINA, POLYMERS) 79 It may be interesting for the reader to estimate an approximate length of all pores in 1 g of average adsorbent. Surface area of average HPLC adsorbent is on the level of 300 m2/g and average pore diameter is 100 Å. One gram of silica is an approximate amount that is usually packed into the standard 15-cm-long HPLC column (4.6-mm I.D.). If you calculate the length of all pores in this column [using equation (3-1) and express it in meters or kilometers], you will get a feeling of what you are dealing with when you are using HPLC. If we take a ratio of the above expressions, we get simple relationship between these parameters: S 2 = (3-2) V R K. K. Unger [6] found that in most cases, expression (3-2) shows a 15% discrepancy between measured and estimated adsorbent surface, which is very good when we take into account the above assumptions made in its derivation. The most commonly used base material is silica (SiO2), the most common substance on the Earth and thoroughly studied in the last two centuries. An excellent monograph on the properties of silica was published by Iler [7]. Development of modern HPLC techniques promoted advancement in porous silica technology. Almost all silica-based HPLC packings manufactured in the twenty-ﬁrst century are very uniform spherical porous particles with narrow particle and pore size distribution. Silica has one signiﬁcant drawback: It is soluble at high pH, although chem- ical modiﬁcation with high bonding density of attached alkylsilanes extends its stability range to over pH 10. Another porous base material suggested in the last decade as an alterna- tive to silica is zirconia. Zirconia is stable in a very wide pH range (pH 1–14), but zirconia surface has relatively low reactivity (more difﬁcult to bond dif- ferent functional groups to the surface), which signiﬁcantly limits a selection of available stationary phases. Polymer-based materials have been on the market for more than 30 years. Crosslinked styrene-divinylbenzene and methylmethacrylate copolymers are the most widely used. These materials show high pH stability and chemical inertness. Their rigidity and resistance to the swelling in different mobile phases is dependent on the degree of crosslinkage. Practical application of these materials for the separation of small molecules are somewhat limited due to the presence of microporosity. Gaps between cross-linked polymer chains are on the level of molecular size of low- molecular-weight analytes. These analytes could diffuse inside the body of a polymer-based packing material, which produce drastically different reten- tion of a small portion of injected sample than the rest of it. At the same time, polymers are the main packing material for size-exclusion chromatography.
80 STATIONARY PHASES 3.4 GEOMETRY 3.4.1 Shape (Spherical/Irregular) Recent technological advancements made spherical particles widely available and relatively inexpensive. Columns packed with spherical particles exhibit signiﬁcantly higher efﬁciency, and columns packed with irregular particles are seldom used and are becoming nonexistent for analytical scale separations. 3.4.2 Particle Size Distribution Packing materials are characterized by the average diameter of their particles and the distribution of the particle size around the average value. The particle size distributions shown in Figure 3-2 are for spherical pack- ing materials with nominal particle size of 10 µm. Distributions of different batches are symmetrical, with average width of approximately 50% of nominal diameter. Most critical for HPLC application is the presence of very small par- ticles (ﬁnes) less than 0.5 µm. These small particles are usually fragments of crushed particle (porous silica is a fragile material). These ﬁne particles will steadily migrate in the column toward the exit frit and clog it. These particles will eventually dramatically decrease the column efﬁciency, and peak distor- tion is usually observed for all peaks in the chromatogram. Particle size distribution itself does not affect chemical behavior of HPLC adsorbent, although it is known to inﬂuence the efﬁciency of packed column. Packings with wide particle size distribution contain a signiﬁcant amount of Figure 3-2. Example of cumulative particle size distribution of HPLC packing mate- rials (Waters µ-Bondapack). Average particle size is 10 µm (inﬂection point). (Reprinted from reference 8, with permission.)
GEOMETRY 81 ﬁne particles, which increases column backpressure; a big size difference between particles in the column decreases overall column efﬁciency. Halasz and Naefe [9] and Majors [10] suggested that if the distribution is not wider than 40% of the mean, then acceptable ﬂow resistance and column efﬁciency can be obtained. The narrower the particle size distribution, the better and the more reproducibly the columns could be packed. Generally accepted criteria is that 95% of all particles should be within 25% region around the mean particle diameter [11, 12]. 3.4.3 Surface Area Surface area of HPLC adsorbents is probably the most important parameter, although it is almost never used or accounted for in everyday practical chro- matographic work. As shown in the theory chapter (see Chapter 2), HPLC retention is proportional to the adsorbent surface area. The higher the surface area, the greater the analyte retention, although as we discuss later, depend- ing on the surface geometry, analytes of a different molecular size could effec- tively see different surface areas on the same adsorbent. The experimental methods for the measurement of the surface area of porous silica is fairly well established. Nitrogen or argon adsorption isotherms at the temperature of liquid nitrogen (77 K) are used in accordance with BET (Brunauer, Emmet, Teller) theory [13] for the calculation of the total surface area per unit of adsorbent weight.There are different variations of BET theory available as well as different instrumental approaches [14] for the measure- ment of nitrogen isotherms. For proper characterization of mesoporous (pore diameter is greater than 20 Å) adsorbents, the static measurement of adsorp- tion isotherm with proper equilibration at each measured point is preferable. Detailed discussion of all aspects of nitrogen adsorption isotherms and related theories could be found in the classic book Adsorption, Surface Area and Porosity by Gregg and Sing [15]. Full nitrogen adsorption isotherm (adsorp- tion and desorption branches) is shown in Figure 3-3. The region between 0.05 and 0.25 relative pressures is called the BET region, and it is used for the determination of the so-called monolayer capac- ity—the amount of nitrogen molecules adsorbed on the sample surface in a compact monolayer fashion. The BET equation represents the dependence of amount of adsorbed nitrogen as a function of the relative equilibrium pressure (p/p0): p p0 1 (C − 1) p = + (3-3) n 1 −  p nm C nm C p0  p0  where nm is the monolayer capacity, C is energetic constant of nitrogen inter- action with the surface; p/p0 is the relative equilibrium pressure, and n is the
82 STATIONARY PHASES Figure 3-3. Nitrogen adsorption isotherm. 1, Adsorption branch; 2, desorption branch. amount adsorbed. Figure 3-3 shows the experimental dependence of the amount of nitrogen adsorbed on the surface versus the relative pressure (pres- sure of nitrogen at the equilibrium in the gas phase over the adsorbent related to the saturation pressure at the temperature of the experiment). Equation (3- 3) is the linear form of the function shown in Figure 3-3, but only in the rela- tively low pressure region between 0.05 and 0.25 where the formation of adsorbed monolayer is complete and BET theory is valid. The plot of the experimental points in p/p0/(1 − p/p0) versus p/p0 allows for linear minimiza- tion and calculation of C and nm values [15]. It is generally assumed that a nitrogen molecule occupies 16.4 Å2 on the polar silica surface. The adsorbent surface area is then calculated as a product of the total amount of nitrogen in the monolayer (nm) and the nitrogen mol- ecular area (16.4 Å2). 3.4.4 Pore Volume At higher relative pressures, above 0.7 in Figure 3-3 a fast increase of the adsorbed amount of nitrogen is observed. This region is attributed to the process of capillary condensation of nitrogen inside the adsorbent pores. This increase is observed until the whole pore volume is ﬁlled with liquid nitrogen. When relative equilibrium pressure approaches the saturation pressure and all pores are already ﬁlled with liquid nitrogen, a small ﬂat section on the adsorption isotherm is usually observed (amax). This section indicates the com- pletion of the pore ﬁlling with condensed nitrogen, and it could be used for accurate determination of the adsorbent pore volume:
GEOMETRY 83 amax P Vpore = VL (3-4) RT where VL is the molar volume of liquid nitrogen (34.7 mL/mol); amax is the maxi- mum amount of nitrogen in the pores expressed in milliliters at 1 atm and L ⋅ atm 25°C; P is the pressure (1 atm); R is the gas constant ( 0.082 ); and T is K ⋅ mol the temperature, 298 K. The desorption branch of nitrogen isotherm is typically used for the deter- mination of the pore size distribution.The only important factor that should be carefully veriﬁed for each adsorbent is the presence of microporous structure. If the micropores (pores with diameter less than 20 Å) are present in base mate- rial, the actual surface to which HPLC analyte might be exposed will be differ- ent from the surface measured with nitrogen adsorption. This is due to the size difference of nitrogen molecule and practically any HPLC analyte molecule. Bigger molecules will have steric hindrance in micropores, and any interpreta- tion of the HPLC retention related to the surface area will be erroneous. In addition, proper chemical modiﬁcation of adsorbents with micropores is essen- tially not possible. Minimum pore diameter acceptable in HPLC adsorbents is approximately 50 Å. Adsorbent pore size provided by the manufacturer is the diameter corresponding to the maximum of the pore size distribution curve, obtained from the adsorption branch of nitrogen isotherm. The distribution of the pores could vary signiﬁcantly as it is shown in Figure 3-4. Figure 3-4. Pore size distribution of different HPLC materials. Allure Silica (Restek); Allure-PFPP (Restek), Prodigy-Silica (Phenomenex); Chromolith C18 (Merck KgaA, Germany); and research-type ordered silica with highly uniform pores of 50-Å pore diameter.
84 STATIONARY PHASES 3.4.5 Surface Geometry The roughness of the silica surface could introduce the steric hindrance of the surface accessibility similar to the effect of the micropores. In the discussion above, we assume the ideal tubular geometry of the silica surface. The use of different probe molecules for the BET measurement of silica surface area (such as N2, Ar, Kr, benzene, etc.) leads to signiﬁcant difference in the surface area values for the same silica sample. It was suggested that silica surfaces possess the property of fractals [16]; this essentially means that molecules of different size will see a different surface area. The surface constructed of ridges and valleys could be considered as an example of a fractal surface. The slopes of these ridges are also constructed by smaller ridges, and with the higher magniﬁcation even smaller ridges are visible. As an example, if a big ball is used to roll over this surface, it will see only the big ridges and thus a relatively small total surface. If, on the other hand, a smaller ball is rolling over the same surface, it will see much more of smaller ridges and a lot of them, resulting in a much higher effective surface area. The smaller the probing ball, the ﬁner the surface roughness it will see and correspondingly higher surface will be detected. Molecular nitrogen will see a signiﬁcant surface area due to its small size comparable to the dimensions of the surface roughness, while bigger mole- cules such as pyrene will not be able to see all ridges and valleys and will see a signiﬁcantly lower effective surface area. These factors have been studied extensively [17] for silica, and authors have found fractal factors to vary between 2 and 3, depending on the silica synthesis, treatment, and so on. Adsorbent surface area (S) is measured as a product of molecular area (s) of a probe substance and the number of the molecules (N) in complete adsorbed monolayer. On the fractal surface the total number of molecules in the monolayer is dependent on its roughness and could be expressed as D − N ~σ 2 (3-5) where D is a fractal number. Since S = Nσ, the adsorbent surface could be expressed as follows: S ~ s ⋅ N ~ s ( 2−D ) 2 (3-6) On the ﬂat surface, D is equal to 2 and only in this case the surface area is not dependent on the size of probe molecule. The higher the fractal number, the less accessible the surface (quasi-three- dimensional or rough surface). For silica with a pore diameter of 10 nm and higher, the fractal factor has a tendency to be between 2.05 and 2.3, which is close to the ﬂat surface. Figure 3-5 illustrates the apparent decrease of acces- sible silica surface in the form of a fraction of the total surface with the increase of the fractal number of this surface (roughness). For these types of
ADSORBENT SURFACE CHEMISTRY 85 Figure 3-5. Dependence of the surface accessible for the probe molecule on the surface fractal number. The probe molecule size is given as the ratio to the nitrogen (S/SN2), and the accessible surface is shown as the fraction of the surface measured by nitro- gen. (Reprinted from reference 18, with permission.) silica samples, all molecules with MW
86 STATIONARY PHASES 3.5.1.1 Mechanical Stability (Rigidity). Particles of packing material are subject to signiﬁcant mechanical stress under the column packing procedure and sometimes during column operation (pressure shock, or fast release of excessive pressure). The rigidity of material is, to a large extent, dependent on its surface tension (or surface energy), which is a function of material surface chemistry. Chemical modiﬁcation of base material signiﬁcantly alters this para- meter, and the rigidity of modiﬁed material usually is not the same as for orig- inal base silica [18]. 3.5.1.2 Chemical Stability. Hydrolytic stability of base material is the most important parameter because most reversed-phase HPLC separations are per- formed in water/organic eluents with controlled pH. Selection of the mobile phase pH is mainly dictated by the properties of the ionizable analytes to ensure that they are in one predominate ionization state. Chemical modiﬁcation of the adsorbent surface signiﬁcantly alters practi- cally all properties of the base material. Dense coverage of the adsorbent surface with inert ligands usually expands chemical stability of the packing material. 3.5.2 Silica Silica (SiO2) is the most widely used base material for HPLC adsorbents. The majority of HPLC packings are silica-based. The chemistry of silica, the methods of silica’s controlled synthesis, the surface structure of silica, and the properties of silica have been studied for over two centuries. It is possible to control a synthesis of ideally spherical particles with predeﬁned pore size and pore size distribution, as well as the synthesis of monolithic silica rods. Porous silica provides the high surface area necessary for successful separation; at the same time, silica particles are very hard (mechanically strong), which allow them to withstand harsh packing conditions and ﬂow of viscous liquids. Silica is not shrinking or swelling when exposed to the different solvents. Even though silica has an array of advantageous properties, it has some drawbacks. The main one is its solubility in water at high pH. The other is an extreme polarity of its surface. 3.5.2.1 Synthesis of Silica. Silica used in HPLC is an amorphous, porous solid, which could be obtained by different synthetic procedures. Colloidal Sol–Gel Procedure. Silica sol is passed through nonaqueous media, where it forms spherical droplets, which rapidly solidify into the hydrogel beads [19]. Solid beads are dried and calcinated at around 600–1000°C. This synthetic procedure usually gives spherical particles of silica with signiﬁcant amount of impurities (Na, Fe, B, etc.) in the body and on the surface of mate- rial. These impurities can increase the acidity of the surface silanols, thus low- ering the pKa of the respective silanol groups.
ADSORBENT SURFACE CHEMISTRY 87 Polycondensation Procedure. This method is essentially the controlled poly- merization of tetraethoxysilane (TES). In the ﬁrst step, TES is partially hydrolyzed in a viscous liquid, which then is emulsiﬁed in the mixture of ethanol and water and then undergoes further hydrolytic polycondensation when catalyzed. The formed solid beads of hydrogel are washed and dried into porous silica. This procedure allows synthesis of highly pure silica particles, which is essential for HPLC. Some recent modiﬁcations of the polycondensation process allow synthe- sis of silica with organic moieties embedded into the bulk material [20, 21], which manufacturers claim to give advantages over conventional silica in terms of higher pH and temperature stability. 3.5.2.2 Surface Silanoles. The surface of amorphous silica is constructed of several different terminal groups. The major portion of the silica surface is covered with single (isolated or free) silanols. Free silanols contain a silicon atom that has three bonds in the bulk structure, and the fourth bond is attached to a single hydroxyl group. The calcination process at high temperatures (over 800°C) often removes water molecules from adjacent silanols, leading to a formation of a siloxane bond. This process is known as dehydroxylation [18]. Dehydroxylated silica is very inert, but it can slowly absorb water and rehydroxylate. Some adjacent silanols can hydrogen-bond to each other, which requires rel- atively close position of silanols usually observed in the β-kristobalite form of silica. Typical chromatographic packing material is estimated to have not more than 15% of its surface with β-kristobalite-type silanols arrangement. In the chromatographic literature an additional type of surface silanols is often mentioned [8]; the geminal silanol shown below contains two hydroxyl groups attached to one silicon atom. Peri and Hensley [22] proposed the existence of these groups on silica surface, although their existence on the surface has not been conﬁrmed. These groups have only been experimentally observed on monomeric organosilicon com- pounds in solution.
88 STATIONARY PHASES The density of the silanole groups on the silica surface is the most impor- tant parameter deﬁning surface reactivity and polarity and is also claimed to have negative effects on chromatographic properties of modiﬁed adsorbents. Many attempts have been made to measure silanol surface density (αOH). Iler [7] estimated αOH to be equal to 8 groups/nm2 on the basis of the [100] face of β-cristobalite. However, most porous amorphous silicas show surface silanol concentration on the level of 4.6 to 5 groups/nm2 [6]. 3.5.3 Silica Hybrid In the last decade, several composite base packing materials were successfully introduced into the market [20, 21]. The primary driving force in developing these materials was the attempt to use all beneﬁts of well-known porous silica and suppress its drawbacks. The two main drawbacks are solubility at high pH and high surface density of silanols. Modiﬁcation of the base silica synthesis by addition of methylthrietoxysi- lane allows the introduction of methyl groups on the silica surface.A schematic representation of the expected surface is shown in Figure 3-6. Surface con- centration of methyl groups is dependent on the reagent ratio. The position of methyl groups within the silica body is essentially random, and their appearance on the surface may not be favorable during the poly- condensation process. The presence of terminating methyl groups within silica body may decrease the mechanical stability of base silica, and this is also dependent on the reagent ratio. Authors claim mechanical stability compara- ble to that of regular HPLC silica [23] while the amount of surface silanols is signiﬁcantly reduced [24, 25]. Another approach to manufacturing hybrid silica (Gemini) was introduced by Phenomenex [26]. They synthesize layered hybrid silica where the core of the particle is regular silica and the surface is covered by the layer of organic- embedded silica. This allows better control of the porous structure because Figure 3-6. Schematic of the synthesis of hybrid silica. (Reprinted with permission from reference 23.)
ADSORBENT SURFACE CHEMISTRY 89 Figure 3-7. Schematic of the formation of bridged hybrid silica. (Reprinted with per- mission from reference 28.) traditional synthesis of base silica is used and surface silanole concentration is decreased while maintaining the mechanical strength of silica. Recently, further development of hybrid materials allows the introduction of organic bridged silica (Figure 3-7). Embedded organic groups do not have terminating function any more but actually are forming an organic bridge between silicon atoms. According to authors, this hybrid particle, having an empirical formula SiO2(O1.5SiCH2CH2SiO1.5)0.25, is synthesized by the co- condensation of 1,2-bis(triethoxysilyl)ethane (BTEE, 1 equiv.) with TEOS (4 equiv.) [20]. The resulting hybrid material shows better pH stability because Si–C covalent bonds are much less prone to hydrolysis than Si–O–Si bonds. Surface energy is also signiﬁcantly reduced, as estimated by comparison of C- constants of the BET equation (BET C-constant represents the energy of nitrogen interaction with the surface [15]). Usual values for regular silica are between 100 and 150, while on hybrid silica a nitrogen interaction with the surface is signiﬁcantly weaker and the C-constant value drops to 49, which is comparable to that of phenyl-modiﬁed silicas [27]. A decrease in the surface energy is associated with the decrease in the surface silanols concentration, but this decrease is not reﬂected on the ability of this material to accept surface modiﬁcation. Chemical modiﬁcation with octadecilsilane ligands resulted in 3.2- to 3.3-µmol/m2 surface density, which is typical for most modern regular modiﬁed silicas [20]. 3.5.4 Polymeric Packings Variation of the mobile-phase pH is one of the most powerful tools in con- trolling the separation for ionizable analytes. The main drawback of silica- based HPLC packing materials is their narrow applicable pH range. The other limitations are surface activity (or polarity), which for speciﬁc applications (such as separation of proteins or biologically active compounds) could play a major role. All these factors are the driving force for the search in alterna- tive base materials for HPLC packings.
90 STATIONARY PHASES The majority of polymer-based packing materials are polystyrene- divinylbenzene crosslinked copolymers. While PS–DVB packings have the advantages of chemical stability at wide pH range, they suffer from the dis- advantage of yielding lower chromatographic efﬁciencies than silica-based bonded phase packings of the same particle size. Even a high degree of crosslinkage in three-dimensional polymer structure leave sufﬁcient space between polymer chains for small analyte molecules to diffuse into the body of the polymer. These “micropores” cause noticeable increase in the broad- ening of chromatographic zone. In another words, column efﬁciency is lower due to a slow intraparticle sorption rate and due to slow diffusion of solute molecules within the polymer matrix [29–36]. 3.5.5 Zirconia (Metal Oxides) Introduction of all these materials on the market is driven primarily by their superior stability at high mobile-phase pH and temperature range. There is a very limited selection of commercially available materials due to higher inertness of the metal oxide surface, and there are almost no repro- ducible methods of chemical surface modiﬁcation [37]. Most of the surface chemistry alteration is achieved by coating and not bonding. Control of the surface area and porosity is also limited. The commercial availability of zirconia-based HPLC packings are mainly related to the enormous extensive research of P. Carr and other workers [37, 38]. They applied zirconia as the starting material for a number of different polymer-coated RP phases. Carr and others have described the preparation and properties of polybu- tadiene (PBD) and polyethyleneimine (PEI), as well as aromatic polymer- coated and carbon-clad zirconia-based RP phases. The preparation of PBD-coated zirconia and the chromatographic evaluation of these phases have been described extensively by Carr, McNeff, and others [39–41]. From these studies, the authors conclude that at least for neutral analytes PBD zirconia-coated phases behave quite similar with respect to retention and efﬁ- ciency compared to silica-based RP phases [42]. For polar and ionic analytes, however, substantial differences with respect to retention, selectivity, and efﬁ- ciency have been reported [43]. 3.5.6 Porous Carbon (or Carbon-Coated Phases) For many years, different research groups attempted to create porous mater- ial with ideal graphite surface and strong enough to be used in HPLC. The advantage of this material would be that two main interactions can occur with analytes: hydrophobic and π–π interactions on an essentially planar surface of graphite (Figure 3-8). The use of graphitized carbon black in gas chromatog- raphy had shown signiﬁcant predictability of retention and speciﬁcity for the separation of conformational isomers, and similar advantages are expected for these adsorbents in HPLC.
SURFACE OF CHEMICALLY MODIFIED MATERIAL 91 Figure 3-8. Atomic structure of porous graphitic carbon. (Reprinted from reference 48, with permission.) The ﬁrst commercial HPLC packing material with graphitized carbon surface was made commercially available at the end of the 1980s under the name Hypercarb [44–46]. On the atomic level, porous graphitic carbon (PGC) is composed of ﬂat sheets of hexagonally arranged carbon atoms (about 105 atoms per sheet) [47]. Edges of graphite sheets are expected to be partially oxi- dized with the formation of hydroxyl, carbonyl, and carboxylate groups [49]. PGC-type packing material shows unique chromatographic properties, since it is more hydrophobic than conventional C18 phases and it shows sig- niﬁcantly higher methylene selectivity [50]. This material has high chemical stability in the wide pH range, and it has unique selectivity for the separation of polar compounds because of its high polarizability [51, 52]. This adsorbent is the primary choice if the separation of conformational isomers is required, because the planar nature of the main part of its surface provides the basis for isomeric separation (Figure 3-9). 3.6 SURFACE OF CHEMICALLY MODIFIED MATERIAL In the preparation of reversed-phase packing material the main purpose of chemical modiﬁcation is to convert polar surface of base material into the hydrophobic surface which will exert only dispersive interactions with any analyte. In porous packing materials with 10-nm average pore diameter, 99% of the available surface area is inside the pores. Conversion of highly polar silica with high silanol density (4.8 groups/nm2) [7] into the hydrophobic surface requires dense bonding of relatively thick organic layer which can effectively shield the surface of base silica material.
92 STATIONARY PHASES Figure 3-9. Selectivity of porous graphitic carbon for positional and conformational isomers under reversed-phase conditions. (Reprinted from reference 53, with permission.)
SURFACE OF CHEMICALLY MODIFIED MATERIAL 93 3.6.1 Limits of Surface Modiﬁcation A wide variety of different ligands have been bonded on silica surface [18] and used as HPLC packing materials. The most traditional and widely used are shown in Table 3-1. Molecular volumes and maximum ligand length shown in Table 3-1 allows for the estimation of theoretical maximum bonding density. The maximum possible thickness of bonded layer could not exceed the length of ligand in all- trans conformation. If we divide the molecular volume of a ligand (Å3/mole- cule) by its length (Å), we get the minimum possible molecular area (Å2) that the ligand can occupy on the surface with the densest bonding. Reciprocal value will be the number of ligands per unit of surface area, and this is only valid for a ﬂat surface. The majority of the surface of porous material is concave. Assuming a cylindrical pore model (standard assumption for silica surface), the maximum bonding density (db) corrected for the surface curva- ture is shown in equation (3-10) (and illustrated in Figure 3-10) as the func- tion of bonding density on the pore radius (R) and ligand length (l) and molecular volume (Vl) is 1  l2  db = l− (3-7) Vt  2R  In the literature the bonding density is often expressed in either number of moles (or micromoles) per square meter (µmole/m2) or in number of mole- cules (bonded ligands) per square nanometer. The relationship between these two units is the simple ratio 6.022 × 10 23 db( molecule nm 2 ) = db(µmol m 2 ) (3-8) 10 24 Calculated values are only a theoretical maximum; in reality, the average bonding density for C1–C18 alkyl ligands varies between 3 and 4 µmol/m2 or between 1.8 and 2.4 groups/nm2. Generally, less than half of available silanols (4.8 groups/nm2 or 8 µmol/m2) are reacted with bonded ligands; the other half is left on the surface. Because it is impossible to modify all available silanols, it is important to shield them, in order to make them inaccessible for analytes. 3.6.2 Chemical Modiﬁcation Surface silanols could react with many different functional groups to form the so-called bonded phase. The majority of the bonding agents used are chlorosi- lanes, although ethoxysilanes and sometimes alcohols are also used. Practically all commercially available chromatographic phases are made using silaniza- tion modiﬁcation process.
94 TABLE 3-1. Structures and Geometry Parameters of the Most Common Bonded Ligands Volumea # Structure Name Structural Formula (Å3) (Å3/molec) Lengthb (Å) 1 C1 150 3.7 2 C4 259 7.5 3 C8 365 13 4 C18 630 24 STATIONARY PHASES 5 Phenyl 303 9.2