Tài liệu HPLC for Pharmaceutical Scientists 2007 (Part 3) docx

Surface chemistry type of bonded ligands; bonding density; etc.4.. Surface area accessi-bility is dependent on the analyte molecular size, adsorbent pore diameter,and pore size distribut

chromatogra-Several thousands of different columns are commercially available, andwhen selecting a column for a particular separation the chromatographershould be able to decide whether a packed, capillary, or monolithic column isneeded and what the desired characteristics of the base material, bondedphase, and bonding density of selected column is needed Commercial columns

of the same general type (e.g., C18) could differ widely in their separationpower among different suppliers Basic information regarding the specificcolumn provided by the manufacturer, such as surface area, % carbon, andtype of bonded phase, usually does not allow prediction of the separation orfor 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 andshape; etc.)

75

HPLC for Pharmaceutical Scientists, Edited by Yuri Kazakevich and Rosario LoBrutto Copyright © 2007 by John Wiley & Sons, Inc.

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 includepore diameter can affect both the adsorbent surface area and the bondingdensity 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 thecolumn efficiency and flow resistance: particle size, particle shape, particle sizedistribution, 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 thesurface 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 rial defines the main type of chemical interactions of the surface with eluentand analyte molecules In essence, all C18-type columns should be similar withregard to their main interaction type, namely, hydrophobic interactions: Meth-ylene selectivity of all C18-type columns are virtually identical [1] Bondedphases of the same type differ in their ability to suppress (or shield) othertypes of interactions (ionic; dipole) exerted by the base material (e.g., silica).Energy of these unwanted interactions is about 10 times greater than theenergy of dispersive interactions [2] Due to the exponential nature of the rela-tionship between retention and interaction energy even the presence of 1%

mate-or less of these active centers in the packing material surface can significantlyaffect the analyte retention

Bonding density is the primary parameter in evaluation of the quality ofthe bonded material Usually the higher the bonding density, the better theshielding effect, although care should be taken in cross-evaluation of similarcolumns on the basis of their bonding density Surface geometry can also sig-nificantly affect bonding density Base material with smaller pores has highersurface area; however, bonding density is usually lower due to the smallerpores

All parameters of the packing material are interrelated in their influence

on the chromatographic performance of the column The quality of an HPLCcolumn is a subjective factor, which is dependent on the types of analytes andeven on the chromatographic conditions used for the evaluation of the overallquality

Long-term column stability (pH and temperature) and batch-to-batchreproducibility are probably the most important quality characteristics to beconsidered in column selection in the pharmaceutical industry Nevertheless,

these criteria should be evaluated with caution when selecting the columnevaluation parameters Long-term stability of retention and efficiency charac-teristics are usually different, depending on the testing conditions (mobilephase, temperature, and analyte probes) Efficiency is usually fairly stable atlow 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 ciency could be deteriorated

effi-3.2 TYPE OF PACKING MATERIAL

(POROUS, NONPOROUS, MONOLITHIC)

Majority of packing materials used in HPLC are porous particles with averagediameters between 3 and 10µm For most pharmaceutical applications, 3-µmparticle sizes are recommended Porosity provides the surface area necessaryfor the analyte retention (usually between 100 and 400 m2/g) Interparticlespace 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 Inthe column packed with porous particles, interparticle space is about 100-foldlarger than pores inside the particles, and liquid flow around the particles isalso 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(1.5µm) nonporous particles also require ultra-microinjection volumes and acorresponding increase of detector sensitivity

The introduction of monolithic columns in the 1990s was another and moresuccessful 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 pared to the conventional packed column with 5- or even 3-µm particles, thesilica skeleton in monolith is only approximately 1µm thick, which facilitatesaccessibility 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 inFigure 3-1

Com-3.3 BASE MATERIAL

(SILICA, ZIRCONIA, ALUMINA, POLYMERS)

In modern liquid chromatography, almost all reversed-phase separations areperformed on chemically modified adsorbents Analyte interactions with theBASE MATERIAL (SILICA, ZIRCONIA, ALUMINA, POLYMERS) 77

stationary phase surface are the primary factor for successful separations Mostcommercial adsorbents reflect their surface chemistry in their names (e.g., C18,C8, Phenyl, etc.) while the base material used usually is not specified, althoughits properties are very important.

Specific parameters of the base of packing material are:

• Density and distribution of the surface reactive centers

Surface area is directly related to the analyte retention [Equation (2-47) inChapter 2] Generally, the higher the surface area, the greater the retention.Pore size is a critical parameter for the surface accessibility Molecules ofdifferent 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 basematerial are cylindrical and neglecting the networked porous structure(assuming straight and not interconnected pores), it is possible to write thefollowing expressions for the surface area and pore volume [6]:

It may be interesting for the reader to estimate an approximate length of allpores 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-longHPLC column (4.6-mm I.D.) If you calculate the length of all pores in thiscolumn [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 betweenthese parameters:

(3-2)

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 itsderivation

The most commonly used base material is silica (SiO2), the most commonsubstance on the Earth and thoroughly studied in the last two centuries Anexcellent monograph on the properties of silica was published by Iler [7].Development of modern HPLC techniques promoted advancement in poroussilica technology Almost all silica-based HPLC packings manufactured in thetwenty-first century are very uniform spherical porous particles with narrowparticle and pore size distribution

Silica has one significant drawback: It is soluble at high pH, although ical modification with high bonding density of attached alkylsilanes extendsits stability range to over pH 10

chem-Another porous base material suggested in the last decade as an 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

alterna-of available stationary phases

Polymer-based materials have been on the market for more than 30 years.Crosslinked styrene-divinylbenzene and methylmethacrylate copolymers arethe most widely used These materials show high pH stability and chemicalinertness Their rigidity and resistance to the swelling in different mobilephases is dependent on the degree of crosslinkage

Practical application of these materials for the separation of small moleculesare somewhat limited due to the presence of microporosity Gaps betweencross-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 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

reten-S

V = R2

BASE MATERIAL (SILICA, ZIRCONIA, ALUMINA, POLYMERS) 79

3.4 GEOMETRY

3.4.1 Shape (Spherical/Irregular)

Recent technological advancements made spherical particles widely availableand relatively inexpensive Columns packed with spherical particles exhibitsignificantly higher efficiency, and columns packed with irregular particles areseldom 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 particlesand the distribution of the particle size around the average value

The particle size distributions shown in Figure 3-2 are for spherical ing materials with nominal particle size of 10µm Distributions of differentbatches are symmetrical, with average width of approximately 50% of nominaldiameter Most critical for HPLC application is the presence of very small par-ticles (fines) less than 0.5µm These small particles are usually fragments ofcrushed particle (porous silica is a fragile material) These fine particles willsteadily migrate in the column toward the exit frit and clog it These particleswill eventually dramatically decrease the column efficiency, and peak distor-tion is usually observed for all peaks in the chromatogram

pack-Particle size distribution itself does not affect chemical behavior of HPLCadsorbent, 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.)

fine particles, which increases column backpressure; a big size differencebetween particles in the column decreases overall column efficiency.

Halasz and Naefe [9] and Majors [10] suggested that if the distribution isnot wider than 40% of the mean, then acceptable flow resistance and columnefficiency can be obtained The narrower the particle size distribution, thebetter and the more reproducibly the columns could be packed Generallyaccepted criteria is that 95% of all particles should be within 25% regionaround 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), HPLCretention is proportional to the adsorbent surface area The higher the surfacearea, 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 ofporous 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 surfacearea per unit of adsorbent weight.There are different variations of BET theoryavailable as well as different instrumental approaches [14] for the measure-ment of nitrogen isotherms For proper characterization of mesoporous (porediameter 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

(adsorp-tion and desorp(adsorp-tion branches) is shown in Figure 3-3

The region between 0.05 and 0.25 relative pressures is called the BETregion, 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 acompact monolayer fashion The BET equation represents the dependence

of amount of adsorbed nitrogen as a function of the relative equilibrium

amount adsorbed Figure 3-3 shows the experimental dependence of theamount 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) 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 ofadsorbed monolayer is complete and BET theory is valid The plot of the

(3-experimental points in p/p0/(1− p/p0) versus p/p0allows for linear

minimiza-tion and calculaminimiza-tion of C and n mvalues [15]

It is generally assumed that a nitrogen molecule occupies 16.4 Å2 on thepolar silica surface The adsorbent surface area is then calculated as a product

of the total amount of nitrogen in the monolayer (n m) and the nitrogen ecular area (16.4 Å2)

mol-3.4.4 Pore Volume

At higher relative pressures, above 0.7 in Figure 3-3 a fast increase of theadsorbed amount of nitrogen is observed This region is attributed to theprocess of capillary condensation of nitrogen inside the adsorbent pores Thisincrease is observed until the whole pore volume is filled with liquid nitrogen.When relative equilibrium pressure approaches the saturation pressure andall pores are already filled with liquid nitrogen, a small flat section on the

adsorption isotherm is usually observed (amax) This section indicates the pletion of the pore filling with condensed nitrogen, and it could be used foraccurate determination of the adsorbent pore volume:

Figure 3-3 Nitrogen adsorption isotherm 1, Adsorption branch; 2, desorption branch.

deter-If the micropores (pores with diameter less than 20 Å) are present in base 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 sizedifference 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 Inaddition, proper chemical modification of adsorbents with micropores is essen-tially not possible Minimum pore diameter acceptable in HPLC adsorbents isapproximately 50 Å Adsorbent pore size provided by the manufacturer is thediameter corresponding to the maximum of the pore size distribution curve,obtained from the adsorption branch of nitrogen isotherm The distribution ofthe 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 porediameter

3.4.5 Surface Geometry

The roughness of the silica surface could introduce the steric hindrance of thesurface accessibility similar to the effect of the micropores In the discussionabove, we assume the ideal tubular geometry of the silica surface The use ofdifferent probe molecules for the BET measurement of silica surface area(such as N2, Ar, Kr, benzene, etc.) leads to significant difference in the surfacearea values for the same silica sample It was suggested that silica surfacespossess the property of fractals [16]; this essentially means that molecules ofdifferent size will see a different surface area

The surface constructed of ridges and valleys could be considered as anexample of a fractal surface The slopes of these ridges are also constructed bysmaller ridges, and with the higher magnification even smaller ridges arevisible As an example, if a big ball is used to roll over this surface, it will seeonly the big ridges and thus a relatively small total surface If, on the otherhand, a smaller ball is rolling over the same surface, it will see much more ofsmaller ridges and a lot of them, resulting in a much higher effective surfacearea The smaller the probing ball, the finer the surface roughness it will seeand correspondingly higher surface will be detected

Molecular nitrogen will see a significant surface area due to its small sizecomparable 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 studiedextensively [17] for silica, and authors have found fractal factors to varybetween 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 inthe monolayer is dependent on its roughness and could be expressed as

(3-5)

where D is a fractal number Since S = Nσ, the adsorbent surface could be

expressed as follows:

(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 dimensional or rough surface) For silica with a pore diameter of 10 nm andhigher, the fractal factor has a tendency to be between 2.05 and 2.3, which isclose 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 theincrease of the fractal number of this surface (roughness) For these types of

silica samples, all molecules with MW <5000 should see practically the samesurface [18].

3.5 ADSORBENT SURFACE CHEMISTRY

Interaction of the analyte molecules with the adsorbent surface is the drivingforce of HPLC separations.The surface of HPLC packing material should havespecific interactions with different analytes, and at the same time the packingmaterial itself should be mechanically and chemically stable Variation of theadsorbent surface chemistry is achieved via chemical modification of the basematerial surface—that is, chemical bonding of the specific ligands Chemicalmodification of the surface has two purposes: (a) shielding of the surface ofbase material and (b) introduction of the specific surface interactions

3.5.1 Surface Chemistry of the Base Material

Base material provides mechanically stable rigid porous particles (mostlyspherical) for reversed-phase HPLC adsorbents Particle porosity on themesoporous level (30 to 500-Å diameter) is necessary to provide high specificsurface area for the analyte retention Surface of the base material should havespecific chemical reactivity for further modification with selected ligands toform the reversed-phase bonded layer Base material determines the mechan-ical and chemical stability—the most important parameters of future (modi-fied) reversed-phase adsorbent

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/SN 2),and the accessible surface is shown as the fraction of the surface measured by nitro-gen (Reprinted from reference 18, with permission.)

3.5.1.1 Mechanical Stability (Rigidity). Particles of packing material aresubject to significant mechanical stress under the column packing procedureand sometimes during column operation (pressure shock, or fast release ofexcessive pressure) The rigidity of material is, to a large extent, dependent onits surface tension (or surface energy), which is a function of material surfacechemistry 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 mostimportant parameter because most reversed-phase HPLC separations are per-formed in water/organic eluents with controlled pH Selection of the mobilephase pH is mainly dictated by the properties of the ionizable analytes toensure that they are in one predominate ionization state

Chemical modification of the adsorbent surface significantly alters cally all properties of the base material Dense coverage of the adsorbentsurface with inert ligands usually expands chemical stability of the packingmaterial

practi-3.5.2 Silica

Silica (SiO2) is the most widely used base material for HPLC adsorbents Themajority of HPLC packings are silica-based The chemistry of silica, themethods of silica’s controlled synthesis, the surface structure of silica, and theproperties of silica have been studied for over two centuries It is possible tocontrol a synthesis of ideally spherical particles with predefined pore size andpore size distribution, as well as the synthesis of monolithic silica rods Poroussilica provides the high surface area necessary for successful separation; at thesame time, silica particles are very hard (mechanically strong), which allowthem to withstand harsh packing conditions and flow of viscous liquids Silica

is not shrinking or swelling when exposed to the different solvents Eventhough 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 extremepolarity of its surface

3.5.2.1 Synthesis of Silica. Silica used in HPLC is an amorphous, poroussolid, which could be obtained by different synthetic procedures

where it forms spherical droplets, which rapidly solidify into the hydrogelbeads [19] Solid beads are dried and calcinated at around 600–1000°C Thissynthetic procedure usually gives spherical particles of silica with significantamount 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 pK of the respective silanol groups

Polycondensation Procedure. This method is essentially the controlled merization of tetraethoxysilane (TES) In the first step, TES is partiallyhydrolyzed in a viscous liquid, which then is emulsified in the mixture ofethanol and water and then undergoes further hydrolytic polycondensationwhen catalyzed The formed solid beads of hydrogel are washed and dried intoporous silica This procedure allows synthesis of highly pure silica particles,which is essential for HPLC.

poly-Some recent modifications of the polycondensation process allow sis of silica with organic moieties embedded into the bulk material [20, 21],which manufacturers claim to give advantages over conventional silica interms of higher pH and temperature stability

synthe-3.5.2.2 Surface Silanoles. The surface of amorphous silica is constructed ofseveral different terminal groups The major portion of the silica surface iscovered with single (isolated or free) silanols Free silanols contain a siliconatom that has three bonds in the bulk structure, and the fourth bond isattached to a single hydroxyl group

The calcination process at high temperatures (over 800°C) often removeswater 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 atively close position of silanols usually observed in the β-kristobalite form ofsilica Typical chromatographic packing material is estimated to have not morethan 15% of its surface with β-kristobalite-type silanols arrangement

rel-In the chromatographic literature an additional type of surface silanols isoften mentioned [8]; the geminal silanol shown below contains two hydroxylgroups 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 groupshave only been experimentally observed on monomeric organosilicon com-pounds in solution

The density of the silanole groups on the silica surface is the most tant parameter defining surface reactivity and polarity and is also claimed tohave negative effects on chromatographic properties of modified adsorbents.Many attempts have been made to measure silanol surface density (αOH).Iler [7] estimated αOHto be equal to 8 groups/nm2 on the basis of the [100]face of β-cristobalite However, most porous amorphous silicas show surfacesilanol concentration on the level of 4.6 to 5 groups/nm2[6].

impor-3.5.3 Silica Hybrid

In the last decade, several composite base packing materials were successfullyintroduced into the market [20, 21] The primary driving force in developingthese materials was the attempt to use all benefits of well-known porous silicaand suppress its drawbacks The two main drawbacks are solubility at high pHand high surface density of silanols

Modification of the base silica synthesis by addition of lane allows the introduction of methyl groups on the silica surface.A schematicrepresentation of the expected surface is shown in Figure 3-6 Surface con-centration of methyl groups is dependent on the reagent ratio

methylthrietoxysi-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 silicabody may decrease the mechanical stability of base silica, and this is alsodependent on the reagent ratio Authors claim mechanical stability compara-ble to that of regular HPLC silica [23] while the amount of surface silanols issignificantly reduced [24, 25]

Another approach to manufacturing hybrid silica (Gemini) was introduced

by Phenomenex [26] They synthesize layered hybrid silica where the core ofthe 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.)

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 haveterminating function any more but actually are forming an organic bridgebetween silicon atoms According to authors, this hybrid particle, having anempirical 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 becauseSi–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 constants of the BET equation (BET C-constant represents the energy of

C-nitrogen interaction with the surface [15]) Usual values for regular silica arebetween 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 thesurface silanols concentration, but this decrease is not reflected on the ability

of this material to accept surface modification Chemical modification withoctadecilsilane ligands resulted in 3.2- to 3.3-µmol/m2surface density, which istypical 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 trolling the separation for ionizable analytes The main drawback of silica-based HPLC packing materials is their narrow applicable pH range The otherlimitations are surface activity (or polarity), which for specific applications(such as separation of proteins or biologically active compounds) could play

con-a mcon-ajor role All these fcon-actors con-are the driving force for the secon-arch in con-alterncon-a-tive base materials for HPLC packings

Figure 3-7 Schematic of the formation of bridged hybrid silica (Reprinted with

per-mission from reference 28.)

The majority of polymer-based packing materials are divinylbenzene crosslinked copolymers While PS–DVB packings have theadvantages of chemical stability at wide pH range, they suffer from the dis-advantage of yielding lower chromatographic efficiencies than silica-basedbonded phase packings of the same particle size Even a high degree ofcrosslinkage in three-dimensional polymer structure leave sufficient spacebetween polymer chains for small analyte molecules to diffuse into the body

polystyrene-of the polymer These “micropores” cause noticeable increase in the ening of chromatographic zone In another words, column efficiency is lowerdue to a slow intraparticle sorption rate and due to slow diffusion of solutemolecules within the polymer matrix [29–36]

broad-3.5.5 Zirconia (Metal Oxides)

Introduction of all these materials on the market is driven primarily by theirsuperior stability at high mobile-phase pH and temperature range

There is a very limited selection of commercially available materials due tohigher inertness of the metal oxide surface, and there are almost no repro-ducible methods of chemical surface modification [37] Most of the surfacechemistry alteration is achieved by coating and not bonding Control of thesurface area and porosity is also limited

The commercial availability of zirconia-based HPLC packings are mainlyrelated to the enormous extensive research of P Carr and other workers [37,38] They applied zirconia as the starting material for a number of differentpolymer-coated RP phases

Carr and others have described the preparation and properties of tadiene (PBD) and polyethyleneimine (PEI), as well as aromatic polymer-coated and carbon-clad zirconia-based RP phases The preparation ofPBD-coated zirconia and the chromatographic evaluation of these phaseshave been described extensively by Carr, McNeff, and others [39–41] Fromthese 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]

polybu-3.5.6 Porous Carbon (or Carbon-Coated Phases)

For many years, different research groups attempted to create porous ial with ideal graphite surface and strong enough to be used in HPLC Theadvantage of this material would be that two main interactions can occur withanalytes: hydrophobic and π–π interactions on an essentially planar surface ofgraphite (Figure 3-8) The use of graphitized carbon black in gas chromatog-raphy had shown significant predictability of retention and specificity for theseparation of conformational isomers, and similar advantages are expected forthese adsorbents in HPLC

The first commercial HPLC packing material with graphitized carbonsurface was made commercially available at the end of the 1980s under thename 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 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 chemicalstability in the wide pH range, and it has unique selectivity for the separation

oxi-of polar compounds because oxi-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 forisomeric separation (Figure 3-9)

3.6 SURFACE OF CHEMICALLY MODIFIED MATERIAL

In the preparation of reversed-phase packing material the main purpose ofchemical modification is to convert polar surface of base material into thehydrophobic surface which will exert only dispersive interactions with anyanalyte

In porous packing materials with 10-nm average pore diameter, 99% of theavailable surface area is inside the pores Conversion of highly polar silica withhigh silanol density (4.8 groups/nm2) [7] into the hydrophobic surface requiresdense bonding of relatively thick organic layer which can effectively shield thesurface of base silica material

SURFACE OF CHEMICALLY MODIFIED MATERIAL 91

Figure 3-8 Atomic structure of porous graphitic carbon (Reprinted from reference

48, with permission.)

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.)

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 usedare shown in Table 3-1

Molecular volumes and maximum ligand length shown in Table 3-1 allowsfor the estimation of theoretical maximum bonding density The maximumpossible 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) thatthe ligand can occupy on the surface with the densest bonding Reciprocalvalue will be the number of ligands per unit of surface area, and this is onlyvalid for a flat surface The majority of the surface of porous material isconcave Assuming a cylindrical pore model (standard assumption for silica

surface), the maximum bonding density (d b) corrected for the surface ture is shown in equation (3-10) (and illustrated in Figure 3-10) as the func-

curva-tion of bonding density on the pore radius (R) and ligand length (l) and molecular volume (V l) is

(3-7)

In the literature the bonding density is often expressed in either number ofmoles (or micromoles) per square meter (µmole/m2) or in number of mole-cules (bonded ligands) per square nanometer The relationship between thesetwo units is the simple ratio

(3-8)

Calculated values are only a theoretical maximum; in reality, the averagebonding density for C1–C18 alkyl ligands varies between 3 and 4µmol/m2orbetween 1.8 and 2.4 groups/nm2 Generally, less than half of available silanols(4.8 groups/nm2or 8µmol/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 theso-called bonded phase The majority of the bonding agents used are chlorosi-lanes, although ethoxysilanes and sometimes alcohols are also used Practicallyall commercially available chromatographic phases are made using silaniza-tion modification process

d b(molecule nm2)=6 022 10× d b( mol m2)

10

23 24

b t

aMolar volume evaluated using ACD software (version 9.3).

bLigand length calculated using ACD software for the distance between anchoring oxygen and foremost atom in all-trans conformation of corresponding ligand.

3.6.2.1 Types Modification Reactions. Chemical modification of lated surface of base material in more than 90% of commercially availableHPLC stationary phase is performed by its reaction with alkylchlorosilane-type modifiers, reaction (3-9) HCl is generated as a byproduct, and typically

hydroxy-a schydroxy-avenger for HCl is employed in the synthesis For most common bondedphases the two side groups are methyl; however, other side groups such as iso-

propyl and tert-butyl have been used.

(3-9)(3-10)

On the silica surface, silanols can react with alcohols forming Si–O–R bonds,reaction (3-10) This type of reaction allows for a very high bonding density,but hydrolytic cleavage of Si–O–R bonds is relatively high and this material

is not practically applicable for HPLC support

Chemical modification of the silica surface with hydridosilane-type fiers as shown in schematic (3-11) offers some advantages:

Figure 3-10 Schematic of the pore fragment with 100-Å diameter modified with C18

ligands in all-trans conformation illustrating the steric hindrance effect

3.6.2.2 Surface Modification with Chlorosilanes. Chlorosilanes are volatileand very sensitive to the presence of trace amounts of water Vapor-phasesilanization reactions with rigorous control of the absence of water show that

ClnSiMe4-n-type chlorosilanes react with aerosil (nonporous microparticulate

silica) as

(3-12)that is, only one surface silanol participates in this reaction [54] Unger [6], onthe other hand, advocates the possibility of the formation of bridging bonds:

Analysis of interatomic distances for these types of bonds shows that adjacentsilanols on the surface should be at a distance of approximately 2.5 Å, whichcorresponds to that distance in β-crystobalit and leads to the surface silanoldensity of 8 groups/nm2 The majority of porous silica have a silanol density

of 4.8 groups/nm2, and there is low probability of forming bridging bonds.Lisichkin and co-workers [18] discussed the interpretation of silica surface as

a mixture of different microcrystalline surfaces of quartz,tridimite, and crystobalite They estimate that approximately 15% of silica surfaces have aβ-crystobalite structure This means that this bridging reaction shown above ispossible but only 15% of available silanols could react in this manner, whiletrifunctional bonding is virtually impossible

β-The silanization reaction in liquid phase (usually in dry toluene) is logically more convenient, although it is practically impossible to ensure thecomplete absence of water in a reaction mixture that leads to hydrolysis ofchlorosilanes Using deuterium exchange, Roumeliotis and Unger [55] ana-lyzed surface silanol concentration before and after the modification with dif-ferent reagents (Table 3-2)

techno-All monofunctional modifiers decrease the surface concentration of silanols

by 50% (depending on the alkyl ligand) compared to original silica (7.6µmol/m2), while difunctional modifiers only slightly reduces the surfaceconcentration of silanols by 12% and trifunctional modifiers apparentlyincreases the amount of silanols This reflects actual hydrolysis of nonreactedchlorine groups of the alkyl di- and trifunctional modifiers and their conver-sion into silanols

When a difunctional modifier is used, it mainly reacts with only one silanole,and the second chlorine is converted into hydroxyl by the reaction with eitherresidual water in the solvent media or during the adsorbent washing processafter the main reaction is completed As the result, each surface silanole iseffectively substituted with another hydroxyl on the attached ligand and thetotal amount of silanols almost does not change

Si OH + Cl SiMe− n 4 −n→ −Si O + SiMe4–nCln− 1+HCl

SURFACE OF CHEMICALLY MODIFIED MATERIAL 97

When a trifunctional modifier is used each surface silanole is substituted withtwo hydroxyls, as shown in the following reaction:

Theoretically, the sum of surface concentrations of bonded ligands and ual silanols should be equal to the original silanols concentration for the reac-tion with monofunctional reagents In reality, there is an approximately1-µmol/m2difference; and the shorter the bonded alkyl chain, the smaller thedifference, at least in the range between C4 and C12 ligands The differenceitself could only be explained by the effectiveness of the surface shielding forthe deuteron exchange; and the longer the bonded chain, the more effectivethe shielding for comparable bonding density

resid-3.6.2.3 Monomeric and Polymeric Bonding. Difunctional and trifunctionalreagents are often used for the formation of so-called polymeric bonding Inthe previous section we discussed the controversy of the surface reactions ofmono-, di-, and trifunctional reagents At present, there is no definite conclu-sion regarding the possibility of the formation of bridging bonding, but most agree that it could not exceed 15% of the total amount bonded For the formation of the bond shown in Figure 3-11 the distance between surfacesilanols should be similar to that on β-kristobalite (approximately

TABLE 3-2 Surface Silanol Concentration Change after Chemical Modification [18]

Carbon Surface Concentration (umol/m

2)Modifier Content Ligands OH Groups (Lig + OH)-Silica

Cl3SiC8H17 7.36 2.35 11.70 6.42

Cl2Si(Me)C8H17 8.43 2.40 6.74 1.51ClSi(Me2)C8H17 9.24 2.35 3.73 −1.55

ClSi(Me2)C4H9 7.13 2.97 3.68 −0.98ClSi(Me2)C8H17 10.43 2.71 3.73 −1.19ClSi(Me2)C12H25 11.73 2.20 3.81 −1.62ClSi(Me2)C16H33 16.67 2.36 4.22 −1.05

2 Å), which corresponds to a very high surface concentration of silanols (4.8 groups/nm2) According to some estimations, thermally calcinated poroussilica may have small sections of its surface in the form of microcrystalline β-kristobalite (approximately 10–15%), and the formation of bridged (difunc-tional) bonding could be observed only on this type of surface.

The probability of the formation of trifunctional bonding is absolutely negligible

In most cases, di- and trifunctional reagents will polymerize in the presence

of water forming linear (for difunctional) and branched alkoxysilanes (for functional modifiers) Trace amounts of water are always present on thesurface of polar silica

tri-Figure 3-11 illustrates possible pathways of silica surface modification withmono-, di-, and trifunctional reagents

3.6.2.4 Mixed Bonding. In some cases, bonding of different ligands on thesilica surface is desirable On porous materials with small pore diameter(below 80 Å), steric hindrance restricts the achievement of high bondingdensity In this case, bonding of a mixture of randomly distributed short alkylligands and long chains allow for better shielding of polar silica surface.The practical synthesis of the uniformly distributed mixed bonded phases

is discussed in reference 18, where the authors indicate that reproducible thesis can be very challenging

syn-The authors in reference 18 also indicate that in most practically ble synthetic procedures for bonding of C18-type modifiers on a silica surface,the formation of the bonded layer proceeds according to the spot-formationprocess (in the presence of amine catalyst) In other words, octadecylsilanes

applica-SURFACE OF CHEMICALLY MODIFIED MATERIAL 99

Figure 3-11 Schematic of different types of bonding (Reprinted from reference 18,

with permission.)

are bonded close to each other, thereby forming clusters, and there is cant space between those clusters (schematically shown in Figure 3-12).

per-formed with the intention of covering accessible residual silanoles left afterthe main modification step Possible cleavage of the main ligands (substitu-tion) may result as a consequence [18]

Figure 3-12 shows the hypothetical structure of a bonded layer Cluster-typebonding of alkyl ligands leaves significant areas that are not covered due tothe concave nature of the inner silica surface and bulkiness of alkyl chains Wediscussed before that only about 50% of all available silanols actually reactwith a surface-modifying agent while the other half are left unreacted Some

of these so-called “residual” silanols are left underneath the closely bondedalkylsilanes, and others are between these bonded clusters The former areessentially inaccessible, whereas the latter are “accessible residual silanols.”These accessible residual silanols are believed to produce a lot of unwantedeffects in reversed-phase HPLC (peak tailing, bonded phase instability,fronting, etc.)

The remedy of these unwanted effects is the process called “end-capping,”which is essentially second-stage surface modification with small ligands thatcan squeeze between patches of C18 ligands and react with accessible resid-ual silanols, thus potentially deactivating them

Despite the widely accepted opinion that endcapping really covers ble residual silanols with relatively inert “mushrooms” of trimethylsilanes, wecan refer the reader to the old work of Berendsen et al [56], where they did

accessi-a rigorous accessi-anaccessi-alysis of accessi-alkyl-modified silicaccessi-a before accessi-and accessi-after end-caccessi-apping In accessi-all

Figure 3-12 (A) Cluster-type C18 bonding (B) Cluster bonding with end-capped

silanols

their samples, carbon content decreases after end-capping This can only meanthat long-chain bonded alkylsilanes are substituted with short trimetylsilanesthat actually have higher bonding energy If this process is indeed occurring,

it is impossible to estimate actual coverage with TMS and original ligands.Chromatographic testing usually shows the decrease of peak tailing and othereffects usually attributed to the influence of a reduced amount of a residualsilanols

It is possible that patches of accessible residual silanols are indeed coveredwith TMS and at the same time a fraction of bonded long-chain ligands(mainly at the borders of these patches) are substituted with TMS also Overallpacking material becomes more hydrophobic, since the amount of accessiblesilanols decreases with end-capping, but carbon content is decreased also

3.6.3 Types of Bonded Phases

3.6.3.1 Alkyl-Type Phases (C1–C18, C30). Probably 90% of all phase columns are alkyl-type bonded phases An enormous amount of publi-cations are devoted to the classification, standardization, and comparison of

reversed-these phases In their book Practical HPLC Method Development, Snyder and

Kirkland [57] indicate that reversed-phase retention for nonpolar and ionic compounds generally follows the retention pattern C1 < C4 < C8 ≈ C18

non-At the same time, they refer to the comparison of C18-type columns from ferent manufacturers and find dramatic variation in the retention of both polarand nonpolar compounds at the same conditions on different columns

dif-HPLC retention expressed either in absolute values (V R) or in relativeterms (retention factor) is a complex function of a multitude of parameters.Type of bonded phase is only one of them Surface area of base material, poresize, bonding density, end-capping, and even the column history all can signif-icantly affect analyte retention

The only valuable comparison of different columns found so far is so-calledmethylene selectivity Methylene selectivity is the slope of the dependence ofthe logarithm of retention factor of the members of homologous series on thenumber of carbon atoms in their alkyl chain (Figure 3-13) This parameterreflects the surface interaction energy of one CH2 group and is found to bevery similar on all C18-type columns (and even C8)

3.6.3.2 Phenyl-Type Phases. Phenyl-type phases have been studied for along time [58, 59] The presence of a phenyl ring on the surface of a bondedphase introduces so-called π–π interactions with some analytes that arecapable of these types of interactions This introduces an additional specificityfor HPLC separations on these stationary phases Compared to common alkyl-type phases, phenyl columns show lower methylene selectivity; in other words,the separation of members of homologous series will be less selective onphenyl columns than on alkyl-modified phases

SURFACE OF CHEMICALLY MODIFIED MATERIAL 101

3.6.3.3 Polar Embedded Stationary Phases. The introduction of polarembedded stationary phases was inspired by the need to develop reversed-phase methods in high aqueous mobile phases for separation of either highlypolar or ionic compounds At these conditions, the hydrophobic surface ofchemically bonded material has limited wettability In pure aqueous mobilephase the “phase collapse” effect was observed The consequence of this

“phase collapse” is that after some period of time the retention of any lytes on the column dramatically decreases, although it does not become equal

ana-to the void volume, but very close ana-to it The first proposed explanation wasthat bonded alkyl ligands are “collapsed” or do not allow analyte penetrationbetween bonded chains Recently, it was shown that alkyl chains are always

“collapsed” [60] They tend to minimize their energy by assuming such formations that allow the maximum number of contacts between these chains.The study of apparent viscosity of alkyl-bonded layers showed that it isapproximately three orders of magnitude higher than the viscosity of corre-

con-sponding free n-alkanes Recall that n-octadecyl is solid at room temperature;

this means that octadecyl chains immobilized on the surface form a rock solidmaterial

Recent studies of the “phase collapse” effect show that after steady removal

of a wetting agent (any previously used organic mobile-phase modifier), waterdoes not wet the hydrophobic inner surface of porous material, and the flow

of the mobile phase through the porous space and corresponding transport ofthe analyte molecules to the surface inside the pores is suppressed The effect

is essentially equivalent to an approximately 100-fold decrease of the bent surface area (the majority of the surface area is inside the pores ofpacking material) This effect is reversible, and pumping of an organic eluent

Figure 3-13 Slope of the logarithm of homologous series retention.

component restores the flow through the porous space, although, depending

on the adsorbent porosity, it may require up to several hours of organic flow.The smaller the pores, the longer it takes

Polar embedded stationary phases include a nonionizible polar groupembedded into the bonded chain Symmetry-Shield RP18 (Waters) and Supel-cosil ABZ (Supelco) are examples of this type of bonding Another example

is Synergi Fusion, (Phenomenex) which uses a polar embedded ligand and ahydrophobic ligand.The polar embedded ligands do not contain nitrogen func-tionality Other types of commercially available polar embedded phasesinclude XBridge Shield RP18 (Waters), Synergy Polar RP, Synergy Hydro-RP(Phenomenex), Zorbax SB-AQ (Agilent), YMC-Pack ODS-AQ (YMC),Dionex Acclaim PA (Dionex), and HiChrom ACE 3 AQ (ACE)

The second reason in the introduction of polar groups in the bonded ligands

is that these groups interact with residual silanols, which make the silanolseffectively inactive for the interaction with polar or basic analytes Sometimesthese phases are also end-capped with polar end-capping groups These phasesalso show a significant difference in selectivity compared to conventional C18-type phases In some cases they show improvement in the peak shape for basiccomponents

3.6.4 Structure of the Bonded Layer

Bonded alkyl ligands are flexible, although their flexibility is significantlyrestricted by immobilization on the surface; also, the higher the ligand bondingdensity, the less flexibility these chains have

The main goal of chemical modification of the surface is to create a ably uniform surface with a selected type of interactions Energetic uniformity

prefer-of the surface is dependent on the ligand’s bonding density, distribution, andconformations An additional desirable feature is hydrolytic stability, which inmost cases is achieved by proper shielding of anchoring bonds

Shielding of the base material, leading to the elimination of the influence

of residual silanols, is essentially the main focus in the development of mostrecent packing materials

SURFACE OF CHEMICALLY MODIFIED MATERIAL 103

The efficiency of the substrate shielding is dependent on the local density

of bonded ligands and their overall distribution on the surface Three ent types of surface ligand distribution could be distinguished: random,uniform, and island-like These distributions are illustrated in Figure 3-14.Type of bonding distribution is significantly dependent on the conditionsunder which the bonding reaction is performed Type of catalyst (usuallyamine), reagent concentration, temperature, and presence of residualadsorbed water on the substrate surface affect this process Detailed discus-sion of this could be found in reference 61

differ-Arrangement of the bonded ligands on the surface, along with tion of these ligands and their occupied volume, is also important for under-standing of the shielding effect Berendsen et al [62] studied the variation ofthe specific pore volume with surface modification Recently, this obviousdecrease of adsorbent pore volume was correlated with molecular volume ofbonded ligands [63] It was shown that molecular volume of bonded ligandscalculated from the volume of bonded layer and bonding density corresponds

conforma-to that for liquid normal alkanes of similar chain length (Figure 3-15)

A similar study was performed on packed columns at HPLC conditions, and

it was found that at any composition of the mobile phase, regardless of the type

of organic modifier (THF, methanol or acetonitrile), the volume of the bondedlayer corresponds to the densest arrangement of bonded ligands [60]

Molecular conformation of bonded ligands and their degree of freedom isdependent on bonding density The higher the bonding density, the lower thenumber of possible conformations and thus the less mobility the bondedchains have Immobilization of ligands on the surface already restrict their

mobility, so if we compare the state of free C18 molecules (n-octadecyl) with

immobilized octadecyl, we can expect more rigid (or solid-like) behavior ofimmobilized chain Indeed, the study of the viscosity of bonded layers [64]

Figure 3-14 Distribution of bonded ligands at low, average, and high bonding

density for island, random, and uniform bonding (Reprinted from reference 18, withpermission.)

showed that the apparent viscosity of alkyl chains in the bonded layerincreased by approximately two orders of magnitude compared to the viscos-

ity of corresponding n-alkanes Conformation of bonded ligands could be

interpreted as shown in Figure 3-16

3.6.5 Density of Bonded Ligands

Bonding density is a surface concentration of bonded ligands, and it isexpressed in either number of moles per square meter (µmol/m2) or in number

of groups per square nanometer (groups/nm2) Unit conversion is shown inequation (3-7)

The term bonding density is constantly used throughout this book This is

probably the most important characteristic of the bonded phase The higherthe bonding density, the more hydrophobic the adsorbent surface, the betterthe shielding of residual silanols, and the higher the hydrolytic stability.Above we briefly discussed the limitations on possible values of bondingdensity (Section 3.6.1) Molecular volume of bonded ligands is one of these

limitations If the most stretched conformation of a ligand has length l and its molar volume is v, then the minimum area,ω, it occupies on the surface will

bew= v/l and the maximum bonding density will be dmax= 1/w This maximumdensity is calculated for the flat surface, while on the concave internal surface

of the pore the maximum density is lower and could be expressed as

(3-13)

d

v l

l R

 

max

12

2

SURFACE OF CHEMICALLY MODIFIED MATERIAL 105

Figure 3-15 Correlation of molecular volume of bonded n-alkyl ligands with that for

liquid n-alkanes [63].

where v is molar volume of the ligand, l is maximum length of the ligand, and R is pore radius The molecular volume for a C18 ligand is estimated as

630 Å3with a maximum length in all-trans conformation is 24 Å

Maximum density means that the whole volume of a surface layer of

thick-ness l is actually filled with bonded ligands and is calculated for a uniform

cylindrical surface In reality, pores are interconnected and have a certain poresize distribution as well, and there are often gaps between bonded molecules(accessible silanols), so real values are usually lower as it could be seen inFigure 3-17

Bonding density cannot be measured directly; the measured value is either(a) carbon content on modified material, (b) weight loss after ashing in theoven, or (c) thermogravimetric weight loss between 200°C and 700°C Carbon

Figure 3-16 Hypothetical conformations of C18 chains on silica surface (Reprinted

from reference 63, with permission.)