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The CHROMacademy Essential Guide Webcast:
Critical Choices in HPLC – Selecting Column Stationary Phase and Dimensions

Thursday 20th March 2014

How do you currently select a column for an HPLC application? Do you just use the one that is on the instrument, root around in an old drawer and hope that you pull out one that works, use your favorite C18 or ODS column, or borrow one from a colleague who’s chromatography is currently working particularly well? These are probably not ideal situations but there will be many of us who are guilty of these exact scenarios. Instead, why not join us for the CHROMacademy Essential Guide webcast on HPLC column selection in which we will look at the available column stationary phases and support materials (including conventional fully porous and core-shell/superficially porous material), column dimensions and the effect that they can have on your chromatography, and how to optimize and improve your current chromatographic separations. This practical guide will give you the tools required to make an informed choice every time you develop a new HPLC method.

  • Available support materials –
    what are the relative advantages and disadvantages of fully porous and core-shell materials?
  • HPLC stationary phase classifications – what does it mean and how do they differ?
  • The effect of stationary phase chemistry on retention and selectivity
  • The effect of HPLC column dimensions on chromatographic parameters
  • How to choose the optimum HPLC column for your application

Who Should Attend:

  • Anyone working with HPLC who would like to better understand the common column stationary phases in use
  • Anyone who wants to improve their chromatographic separations
  • Anyone involved in developing HPLC methods
 

 

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The CHROMacademy Essential Guide Tutorial:
Critical Choices in HPLC – Selecting Column Stationary Phase and Dimensions

How do you currently select a column for an HPLC application? Do you just use the one that is on the instrument, root around in an old drawer and hope that you pull out one that works, use your favorite C18 or ODS column, or borrow one from a colleague who’s chromatography is currently working particularly well?
These are probably not ideal situations but there will be many of us who are guilty of these exact scenarios. Instead, why not join us for the CHROMacademy Essential Guide webcast on HPLC column selection in which we will look at the available column stationary phases and support materials (including conventional fully porous and core-shell/superficially porous material), column dimensions and the effect that they can have on your chromatography, and how to optimize and improve your current chromatographic separations.
This practical guide will give you the tools required to make an informed choice every time you develop a new HPLC method.

  • Available support materials – what are the relative advantages and disadvantages of fully porous and core-shell materials?
  • HPLC stationary phase classifications – what does it mean and how do they differ?
  • The effect of stationary phase chemistry on retention and selectivity
  • The effect of HPLC column dimensions on chromatographic parameters
  • How to choose the optimum HPLC column for your application

 

 

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Correct HPLC column choice is essential for successful chromatography.  This tutorial will examine the key column parameters, their effect on chromatography, and detail the important factors for successful column choice. 

The major column specifications and which chromatographic parameters they affect are shown in Figure 1. 

This is by no means an exhaustive diagram as there are many other column specifications that need to be considered and which will be detailed within the tutorial.  

 

Figure 1: HPLC column parameters and the chromatographic parameters which they affect.

 
 

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In order to obtain retention and separation of analytes there needs to be an interaction between the analyte and the column stationary phase.  In reversed phase chromatography the predominant interactions are hydrophobic.  Reversed phase chromatography has a mobile phase which is more polar than the stationary phase. 

The common stationary phases are hydrophobic in nature and will interact with analytes via hydrophobic interactions.  The retention of neutral analytes can be modified by altering the polarity of the mobile phase through addition of an organic modifier (i.e. methanol, acetonitrile etc.).  A higher percentage of water, which is polar, in the mobile phase will repel the analytes into the non-polar stationary phase where they will reside for a time until they partition into the mobile phase, ultimately, controlling their retention. 

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The chemical structure of the analytes will give a good indication of the retention order that can be expected; greater retention can be expected for analytes which are less water soluble (hydrophobic), have a greater number of carbon atoms compared to other analytes in a mixture, have straighter chains (opposed to branched, and are fully saturated (Figure 2).

 

The general order of elution exhibited in reversed phase chromatography will be:

Aliphatics > induced dipoles (e.g. CCl4) > permanent dipoles (e.g. CHCl3) > weak Lewis bases (esters, aldehydes, ketones) > strong Lewis bases (amines) > weak Lewis acids (alcohols, phenols) > strong Lewis acids (carboxylic acids)

 

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Figure 2: Analyte retention order in reversed phase chromatography.
 
 

Columns are required to separate analytes based upon small differences in molecular properties; for example difference in hydrophobicity, shape, or functional groups etc.  Within mixtures of analytes these parameters may be very similar.  Therefore, to achieve separation of analytes with very similar physicochemical properties the unique interactions between the analyte and stationary phase will need to be exploited (i.e. hydrophobic, dipole, π-π interactions etc.) along with careful design of the analytical conditions (mobile phase, isocratic or gradient elution etc.).

Finally, the column that is chosen for a particular application must be stable under the analytical conditions, including, pH, temperature, pressure, sample amount (overloading) in order to obtain good, robust chromatography.

The fundamental or Purnell resolution equation indicates that resolution is affected by three important parameters; efficiency (N), retention (k), and selectivity (α) (Figure 9).  It can be seen from the plot shown in Figure 8 that selectivity (α) has the largest impact on resolution.  Selectivity can be altered by modifying the mobile phase organic modifier, pH, solvent strength, and additives used in the mobile phase.  The choice of stationary phase will also have a significant impact on selectivity and, therefore, resolution.

 

Figure 3: The effect of efficiency (N), retention (k), and selectivity (α) on resolution.

 

The following sections will detail parameters associated with HPLC columns which should be considered when selecting a column for a particular application and how they will impact on the chromatographic parameters efficiency (N), retention (k), and selectivity (α) and ultimately resolution.

 
 

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There are many parameters quoted by column manufacturers which give additional information about the column stationary phase which need to be understood in order to select the correct column for a particular application. 

Not every manufacturer will report every parameter; however, it is worth being aware of the terminology in order to understand what they mean in relation to column choice and chromatography.  Some of these parameters will be covered more fully later within the tutorial, therefore, only a brief explanation is given below.

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Solid Support
The solid support refers to the solid particles within the HPLC column.  Solid supports can come in various different formats including fully porous, superficially porous, or non-porous (Figure 3).  The support material can be naked, coated, or have a chemically bonded phase. 

 

Figure 4: Representation of a superficially porous (left) and fully porous (right) silica particle.

 
 

Bonded Phase

A bonded phase is a stationary phase that has been chemically bonded to the surface of a support material (usually silica).  The range of bonded phase chemistries is prolific and will vary between manufacturers.  Some common stationary phase chemistries are based on alkyl, phenyl, cyano, and amino moieties.  It is the interaction between the analyte and the bonded phase which can be manipulated to alter retention and selectivity of a chromatographic separation.
Particle Size

The particle size or diameter (dp) is the average diameter of the column packing particles.
Particle Size Distribution

The particle size distribution will give a measure of the distribution of the size of particles used to pack the column.  It is desirable to have a narrow particle size distribution as this gives a more homogeneous packing and ultimately more reproducible chromatography.  A particle size distribution of dp ± 10% would indicate that the 90% of the particles are between 9-11 μm for a 10 μm averagedp packing.  A D10/D90 ratio will often be quoted and this will be discussed later.

Particle Shape

Two basic silica particle shapes are available, spherical and irregular (Figure 5).  Milling of silica particles followed by sieving to obtain the appropriate particle size and distribution produces irregular particles.  Although irregular particles are somewhat less expensive they are known to have poorer efficiency than spherical particles.  This is due to the way in which the particles pack into the HPLC column; with irregular particles packing homogeneity is much poorer leading to an exaggeration of eddy diffusion and mass transfer effects.
 For more information on the effects of eddy diffusion and mass transfer see:

CHROMacademy module - Band Broadening

The use of columns for high flow (pressure) applications and mechanical shock can cause the irregular particles to shear, forming smaller sub-particles known as fines which may migrate and eventually block the outlet frit of the column.  Therefore, spherical particles are generally favored for the reasons outlined above.

 
Figure 5: Silica particle shapes.
 
 

Pore Size

The pore diameter chosen is directly related to the hydrodynamic volume of the molecules in the sample.  Larger pores allow large molecules to access the bonded phase found within pores for maximum separating ability.  Select pore sizes of 150 Å or less for small molecules.  Large pores, 300 Å or greater, are used for samples having a molecular weight greater than 2000 Da.  As a rule of thumb the pore size should be at least three times the hydrodynamic diameter of the molecule.  Stationary phases with larger pores will have less surface area.

Surface Area

The surface area refers to the total area of the solid surface.  This is often determined using an accepted measurement technique such as the Brunauer-Emmet-Teller (BET) method, which uses the physical adsorption of nitrogen to measure the surface area.  The surface area can have an effect on several chromatographic parameters.  High surface area columns may exhibit greater retention, capacity, and resolution.  Low surface area packings will generally equilibrate more quickly which can be advantageous in gradient elution.

pH Range

The pH range gives the pH values at which the HPLC column can be used without degradation of the solid support and stationary phase which will ultimately result in deleterious chromatographic results.
Traditional silica has a working range of pH 2.5-7.5.  At low pH acidic hydrolysis of the silyl ether linkage between the bonded phase and silica surface will occur, resulting in column bleed (loss of stationary phase), poor peak shape, and loss of efficiency.  At high pH the silica surface itself is at risk of basic hydrolysis, sometimes referred to as silica dissolution, whereby, the solid silica support is cleaved apart and fines are created which block the support material pores, interstitial gaps between the particles, and the column outlet frit resulting in the system over pressurizing and shutting down.

Temperature Limit

Some column manufacturers will give an operating range or upper temperature limit at which the columns can be used without damaging the stationary phase.  Elevated temperatures will also promote basic hydrolysis of the silica support and acidic hydrolysis of the stationary phase.

Endcapping

Endcapping is used to remove surface silanol groups on the solid support which can cause unwanted analyte secondary interactions resulting in poor peak shapes; this is particularly evident when analyzing basic, ionizable, and ionic compounds.  A small silylating reagent (i.e. trimethylchlorosilane or dichlorodimethylsilane) is reacted with the surface silanol groups to produce, for example, a trimethylsilyl (TMS) capped group (Figure 6). 

 

Figure 6: Endcapped silica phase.

 
 

Carbon Load (%)

Carbon load is simply the elemental load of carbon on the support material (which is carbon free in its native form for traditional silica).  Hybrid particles such as BEH, XBridge, TriArt and Gemini contain a mixture of inorganic silica and organic groups and will always have a larger background carbon content present, even in its native, unmodified state. 

The carbon load is normally expressed as % carbon.  A higher carbon loading will result in greater analyte retention.  It should be noted that C18 columns will always have a higher % carbon and columns with different endcapping cannot be subjected to a like to like comparison as the % carbon for the different endcapping groups will differ.

Surface Coverage

Surface coverage gives a better measure of the retentivity/hydrophobicity of the column.  This refers to the concentration of stationary phase per unit area bonded to the support and is expressed as μmoles/m2 (Figure 7).

 
 

Figure 7: Representation of a silica surface with high and low coverage.

 
 

Silica Type

Acidic (lone) surface silanol groups give rise to the most pronounced secondary interactions with polar and ionizable analytes (Figure 8).  Modern silica is designated as being Type I or A and Type II or B, which primarily describes the nature of the silanol surface.  Type A or I silica is ‘high energy’ (non-homogenous) and contains a higher density of lone silanol groups, whereas Type B or II silica is much more homogenous (inter-hydrated) and, therefore, gives rise to much improved peak shapes. 

In order to create a more uniform (homogeneous) silica surface, manufacturers ensure the silica surface is fully hydroxylated prior to chemical modification.  The incorporation of an acid wash step and avoidance of treatments at elevated temperature renders the majority of the surface in the lower energy geminal and bridged (vicinal) confirmation, creating Type II silica.

 

Figure 8: Surface silanol groups present on the surface of silica particles.

 
 

This is clearly demonstrated with 5 basic analytes and the weakly acidic phenol as a control (Figure 9).  At pH 7.6 the acidic lone silanols will are be fully de-protonated and increased retention of the basic analytes 3 and 5 along with poorer peak shape on the Type A Hypersil type stationary phase are seen.  The stronger basic analytes 1, 2, and 4 are irreversibly bound to the column.  When chromatographed on the Hypersil BDS employing Type B silica, good retention times and peak shapes are observed for both the acidic and basic analytes.

 

Figure 9: Separation of basic analytes on Hypersil BDS C18 (Type B) and Hypersil C18 (Type A) columns.

 

The lone silanols acidity is further increased by the presence of metal ions.  The worst offenders being trivalent iron and aluminium.  Analytes which are capable of chelating can also interact with the column in this way leading to additional unwanted secondary interactions.  

By acid washing the silica to form Type B silica the metal ion impurities are also vastly reduced as is shown in the Table 1.  It can be seen that problematic iron and aluminium are present at sub 3 ppm levels.  Metal ion contamination these days is rarely resultant from the silica manufacturing process but from contact with the blades used for sub-sieve sizing and non-passivated stainless steel surfaces, i.e. column frits etc.

 

Silica

Na

K

Mg

Al

Ca

Ti

Fe

Zr

Cu

Cr

Zn

Type B

10

< 3

4

1.5

2

nd

3

nd

nd

nd

1

Type A

2900

n/a

40

300

38

65

230

n/a

n/a

n/a

n/a

Table 1: Metal ion content of Type A and Type B silica.  nd = none detected.

 
 

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Monolithic Silica Rods

Monolithic silica columns are formed from the dried rods of polymeric silica gel.  The columns are highly porous with a bi-modal pore structure containing mesopores with diameter of approximately 2-50 nm and macropores with diameters > 50 nm. 1  The surface area created by the mesopores is approximately 300 m2/g.  The total porosity of the monolithic silica matrix is greater than 80% which allows users to perform chromatography using much lower back pressures. 

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Monolithic phases exhibit better mass transfer properties in comparison to distinct particles (i.e. flatter Van Deemter curves); therefore, high speed separations are possible without a noticeable effect on resolution.  The low back pressures created also introduce the possibility of flow programming where the mobile phase flow rate is increased during the analysis to shorten analysis time.
Monolithic columns which carry a C18 surface modification and endcapping are comparable in selectivity to conventional reversed phase columns.  A comparison shows that although the selectivity of monolithic and traditional particle columns are the same the retention times achieved by monolithic columns are much shorter, therefore, resulting in a faster separation.

Fully porous (traditional silica)

Bare silica, chemically untreated, can be used as the support material for normal phase (adsorption) chromatography and after chemical modification is the primary support material for reversed phase (partition) chromatography (Figure 10).
Silica is a support material with exceptional assets including:

  • High mechanical strength - required to cope with back pressures of 1000+ bar
  • High surface area - required for high efficiency separations (300 m2/g is typical for a 5 μm particle with 100 Å pore diameter)
  • Available in a form pure enough to chromatograph polar and ionizable components when used in conjunction with bonded phases

Whilst we think of silica support materials as ‘spheres’, in reality there is less than 1% of the silica available surface on the outer surface of the sphere, the vast majority being found within the internal pore structure.
The major drawback to using silica is its susceptibility to hydrolysis at high pH (typically > pH 7.5) especially in highly aqueous environments.  Factors that influence the rate of silica hydrolysis include:

  • Aqueous content of the eluent (silica is more soluble in highly aqueous eluent systems)
  • Buffer type used
  • Temperature of the column/eluent
  • Pore volume (higher surface areas, achieved with smaller pore widths/volumes, will be more susceptible to faster hydrolysis especially in highly aqueous environments)
 

Figure 10: Fully porous (traditional silica) particle.

 
 

Fully porous (organo-silica hybrid)
Organic groups are grafted into the silica layers (Figure 11) making them more resistant to dissolution at high pH.  This makes them particularly appealing for high pH applications as they will have a longer lifetime.

 

Figure 11: Fully porous (organo-silica hybrid) particle.

 
 

Core-shell

Core-shell particles consist of a silica core and a porous outer layer (Figure 12).  The particle diameter will vary depending on manufacturer.  Core-shell particles will produce separations with very high efficiencies in comparison to conventional HPLC methods which use fully porous packing materials.  The size distribution of core-shell columns are narrower than those seen with more traditional solid support materials which again contribute to the increased efficiency of separations.  

 

Figure 12: Core-shell particle 4 μm, porous layer depth 0.6 μm (left) and comparison of peak efficiency generated using a column packed with core-shell particles and fully porous particles (right).

 

For further information on Core-shell particles watch the CHROMacademy webcast: Core-shell Particles - Present and Future

 
 

Porous graphitic carbon

Porous graphitic carbon is not necessarily a solid support as much as it is a stationary phase chemistry of its own.  It is composed of flat sheets of hexagonally arranged carbon atoms (Figure 13). 

There are no surface silanol groups; therefore, there are no possibilities for deleterious secondary interactions between analytes and the surface.  These columns are particularly amenable to separation of highly polar species, separations carried out at high pH, and they can be used for reversed phase, normal phase and LC-MS applications. 

The mechanism of interaction between the analyte and stationary phase is dependent upon the polarity and shape of the analyte.  These specific interaction mechanisms allow the resolution of analytes which may not be possible by typical reversed phase HPLC. 

Use of these types of phases may negate the need for complex buffering systems or ion pair reagents.  The use of increased organic modifier concentration for polar analytes makes porous graphitic carbon columns more amenable for use in LC-MS applications.

 
 

Figure 13: Porous graphitic carbon stationary phase.

 
 

Analyte retention on porous graphitic carbon phases is thought to occur via two mechanisms (Figure 14):

  1. Adsorption - the strength of the interaction between the analyte and stationary phase is dependent on the area of contact between the molecule and the surface and also on the type and position of functional groups which are in contact with the surface.  More planar molecules will exhibit greater retention than rigid molecules with a three-dimensional spatial arrangement.
  2. Charged induced interactions - this interaction accounts for the strong retention exhibited by polar analytes.  Polar groups have a permanent dipole which as it approaches the graphite surface induces a dipole on the surface resulting in an analyte/stationary phase interaction.
 

Figure 14: Representation of a polar molecule interacting with the graphite surface.

 
 

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Before selecting an HPLC column the physical and chemical properties of the analyte(s), the mode of analysis, and the analyte/stationary phase interactions need to be considered.  This process is summarized in the flow chart in Figure 15.

It should be noted that > 80% of all columns sold are reversed phase, therefore, when analyzing mixtures of analytes, which will have various physicochemical properties, reversed phase columns will often be the first choice of column and may produce the desired separation.  However, if separation is not satisfactory, mechanisms to further discriminate between functional group chemistry, shape (hydrodynamic volume), and dipole (induced dipole) capability will be important when selecting a column.  This may involve the use of columns with different chemistries and properties; for example PFP and phenyl phases, polar endcapping, Type II (III) silica, ligand density, polar embedded ligands etc.  Therefore, knowledge of the various column properties is imperative.

 

Figure 15: Flow chart for HPLC column selection.  RP = reversed phase, HILIC = hydrophilic interaction liquid chromatography.

 
 

Analyte Functional Groups, Solubility, and Interactions

Prior to commencing method development it is important to make some fundamental decisions based on analyte information, if this is available.  Listed below are just some of the important factors that need to be considered prior to any experimentation:

Analyte LogP – if this is known then the relative hydrophobicity of the analyte can be assessed. This gives an indication of the eluent strength required to elute the analyte (high positive values indicates greater hydrophobicity and require higher organic for elution).

Analyte pKa – gives an indication of the pH at which the analyte changes its state of ionization which affects its relative polarity and, therefore, it’s extent of retention under reversed phase HPLC conditions.  The analyte may have more than one ionizable functional group which may render the method development process more complex.  When dealing with ionizable analytes the first approach is usually to completely suppress or promote ionization in order to make optimization of the eluent organic composition more straightforward. 

A common modern approach is to employ 0.1% trifluoroacetic acid as the aqueous component of the eluent system which results in a pH of approximately 2.1.  At this pH, most organic acid functional groups will be fully ion suppressed and most basic functional groups will be fully ionized.  These two extremes can then be handled using a combination of column chemistry and mobile phase eluotropic strength.  Whilst this approach lacks finesse, it does simplify initial method development work and will give important pointers as to whether, for example, pH optimization is necessary to obtain a satisfactory separation.

Analyte assessment is important in deciding which mode of chromatography and stationary phase type are chosen.  The functional groups present in a molecule will affect its physicochemical properties (solubility, pKa etc.) and interactions with the column stationary phase.

Hydrophobic interactions are particularly important in reversed phase chromatography.  Hydrophobic (non-polar) functional groups are fundamentally hydrocarbon in nature and for the most part do not contain any heteroatoms (O, N, S etc.) (Figure 16).  Hydrophobic groups will interact with the stationary phase via dispersive forces which are relatively weak in comparison to other intermolecular forces.  Stationary phases which exhibit hydrophobic interactions include C18, C8, and phenyl etc.  Hydrophobic analytes prefer a non-aqueous environment and will be soluble in non-polar solvents which have similar functional groups characteristics, i.e. hydrocarbons such as hexane.

 

Figure 16: Cholesterol ester.  The pink area depicts the hydrophobic portion of the molecule.

 
 

Polar functional groups contain dipoles; molecular bonds where the electrons in the bond are shared unequally due to a greater electronegativity on one of the atoms in the bond, which results in one of the atoms in the bond having a negative charge, while the other has a positive charge.  The greater the electronegativity difference between the two atoms the stronger the dipole moment.  Most commonly strongly polar groups contain at least one heteroatom (O, N, S etc.) often bonded to a carbon atom or hydrogen atom (Figure 17).  Other weaker common dipoles include unsaturated carbon-carbon bonds, aromatic rings, and halogen carbon bonds. 

Analytes which contain polar functional groups will be soluble in highly aqueous or polar solvents, such as water, buffered solutions, or water miscible organic solvents.  The interaction of polar analytes with stationary phases will be via dipole-dipole and hydrogen bonding and will, therefore, be more strongly retained on stationary phases which contain functional groups which are capable of undergoing these types of interactions (diol, amino, cyano etc.).  Also, most silica based phases will exhibit polar interactions via surface silanol groups which can produce detrimental chromatographic results.

 

Figure 17: Norfenefrine.  The purple areas depict the polar functional groups.

 
 

Polar functional groups can be further divided into acidic and basic classes and some will also be termed ionic functional groups.  These functional groups are capable of donating a proton (acids) or accepting a proton (bases).  Ionic groups can exist in either a neutral or ionized form depending on the environment (in the case of HPLC the pH of the mobile phase will influence the ionization state of the analyte). 

The pH at which the functional group becomes ionized is dependent on the specific characteristics of that group.  Ionizable functional groups will have a pKa (Ka = acid dissociation constant) which indicates the pH at which the molecule will be 50% ionized; in fact this value is an equilibrium which means that the molecule is in equilibrium between its neutral and ionized form. 

When analyzing ionizable molecules it is incredibly important to carefully control the pH of the mobile phase as this will influence the extent of ionization of the analyte.  Analytes in their neutral form are less polar than their ionized counterparts and will, therefore, be more retained under reversed phase conditions.
1,4-Methyl-imidazoleacetic acid (Figure 18) contains both acidic (yellow) and basic (green) functional groups.

 
 

Figure 18: 1,4-Methyl-imidazoleacetic acid.  The yellow area depicts the acidic functional grous and the green areas depict the basic functional groups.

 
 

In reversed phase method development with ionizable compounds, plotting retention (k) against pH for the various analytes in the separation can be a very enlightening (Figure 19).  Plots of the retention factor (k) for each of the analytes at pH 3.5, 5.5, and 7.5 can often help to decide the optimum pH for the separation.  This can help even with fairly complex analytes as can be seen in Figure 19.

 

Figure 19: Dependence of retention factor on pH.

 
 

There are several observations from the pH vs. k plot that highlights the usefulness of this approach to optimizing separation selectivity.

  • Whilst we have no indication of peak efficiency, it can be assumed that as lines become closer together the separation (and hence the resolution) between analytes reduces
  • Crossing lines – indicate co-elution of the analytes, and therefore the pH would not suitable for the separation
  • At pH 2 all analytes are separated, although the peaks for some of the analytes are fairly close and good peak efficiency would be essential to ensure the required resolution between all peak pairs. More importantly, the response curves for all analytes are fairly flat – the importance of this will become evident in the next two points.
  • At pH 5 the peaks are all separated (in terms of selectivity).  However the response curves of lidocaine and ibuprofen are changing rapidly with pH which is of great concern for the robustness of the method.  Small changes in mobile phase pH, which can be expected, will lead to large changes in analyte retention and, therefore, potential loss of resolution between analytes – which is to be avoided at all costs in method development.
  • It should also be noted that when deciding on a pH at which to run the separation the column pH working range will need to be considered - even when inside the working range of the column, but close to either the upper or lower limit, column lifetime can be expected to be reduced.
  • Conclusion – analysis of these compounds (provided peak efficiency is good) at pH values 1-3 should produce a robust separation.
 
 

There are several online and print resources for obtaining physical and chemical data for known compounds including:

Websites

  • //www.chemicalize.org
  • //www.chemspider.com
  • //webbook.nist.gov/chemistry

Books

  • CRC Handbook of Chemistry and Physics 2
  • Lange’s Handbook of Chemistry 3

For more information on analyte functional groups see:

Sample Prep Channel/Solid Phase Extraction/Molecular Properties

 

For more information on optimizing separations with ionizable compounds see:

HPLC Channel/Theory of HPLC/Reversed Phase Chromatography

HPLC Channel/Webcasts & Tutorials/Developing Better HPLC (MS) Methods

HPLC Channel/Webcasts & Tutorials/Developing Better Methods for Reversed Phase HPLC

 
 

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In order for separation of analytes to occur they must be retained by the stationary phase.  The degree of retention will be dependent on the strength and type of analyte/stationary phase interactions. 

The type of interactions that a particular analyte will undergo is dependent on the functional groups, as has been discussed previously.  The main molecular interaction types are outline in Table 2.

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Molecular Interaction

Separation Principle

Dispersive (Van der Waals)

  • Exists in all molecules
  • Major retention mechanism for alkyl phases (C18, C8, C4 etc.)
  • Retention is proportional to the hydrophobicity of the molecule

Charge transfer (π-π interaction)

  • Seen for aromatic and unsaturated compounds and stationary phases
  • Enhanced with methanol mobile phase

Hydrogen bonding and dipole-dipole interactions

  • Analytes acts as a proton donor with a proton accepting group in the stationary phase
  • Evident with increased acidic analyte retention

Table 2: Primary molecular interactions.

 
 

Figure 20 details the polarity and the interaction types that the analyte/stationary phase can undergo for some common stationary phases.

 

Figure 20: Column polarity and interaction types.

 
 

The differences in interaction between the analyte and stationary phase can be exploited to achieve the desired separation.  In fact, selectivity is the method developer’s friend.  During method development phases should be screened for orthogonality (Figure 21). 
Even columns containing the same bonded phase i.e. C18, C8 etc., from the same manufacturer can show different selectivity.  The reproducibility of a column should also be investigated to determine if the separation will be susceptible to underlying manufacturing characteristics (silanol activity, metal ion content etc.). 
Method development should always be carried out with a new column.

Orthogonality refers to two separations in which the elution times are different; ideally the two separations will have been carried out using different retention mechanisms (i.e. reversed phase and normal phase, C18 column and amino column etc.)

 

Figure 21: Separation of a mixture of analytes carried out on different stationary phase types.

 
 

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Column Characterization (PQRI)

Stationary phase selectivity is critical for a successful (optimized) separation.  Separation on significant differences in LogP or LogD (hydrophobicity) using a C18 column is not always successful.  Mechanisms to further discriminate between functional group chemistry, shape (hydrodynamic volume), and dipole (induced dipole) capability are all very important in modern HPLC.  This may involve the use of differing ligand chemistry (PFP and phenyl phases are becoming increasingly important), polar endcapping, Type II and III silica, ligand density on the silica surface, polar embedded ligands etc. 

In modern HPLC an awareness of the various column properties which are described in modern databases and by manufacturers is a must.

Screening a column for ‘selectivity’ or selecting a column which is similar to or radically different (orthogonal) from one that has been used in the past can be a complex process.  Apart from experience and the literature, there are various in-silico column classification databases which use test results based on various chemical probes, which have been designed to highlight specific column characteristics. 

 

For example the USP use the PQRI method for characterizing columns which is based on the Hydrophobic Subtraction Models proposed by Snyder et al. 4  The PQRI system of column classification is a method of benchmarking key HPLC stationary phase criteria. This is achieved by testing each column with carefully selected probe compounds that assess six critical parameters that have been identified to account for the most significant aspects of stationary phase behavior and selectivity.  The compound mixtures are analyzed under the same chromatographic conditions (50% acetonitrile/buffer, pH 2.8 and 7.0), resulting in many reversed phase columns being characterized by six column-selectivity parameters.

H = Hydrophobicity - the ability to retain hydrophobic compounds
S = Steric or shape effect - ability to discriminate between molecules with similar size/LogP but with different 3D shape
A = Hydrogen bond acidity - secondary interaction via low activity silanol groups
B = Hydrogen bond basicity - gives significantly modified selectivity (large B = large retention of weak acids
C(2.8) = Silanol ionization at pH 2.8 - all residual silanols should be neutral
C(7.0) = Silanol ionization at pH 7.0 - all residual silanol species are fully ionized

Results of these test probes can be graphically represented (Figure 22); the more symmetrical the hexagon appears and the larger its area the more balanced the stationary phase is in the sum of its chromatographic properties.  This can be extremely useful when selecting a column or for easy comparison of different columns during method development.

 

Figure 22: Radar plot of PQRI parameters for Thermo Hypersil Beta Basic-18 HPLC column.

The USP column comparison database can be accessed via the following link:
//www.usp.org/app/USPNF/columnsDB.html

 
 

It should be noted, that when comparing columns the shape of the radar plot is a good indication to the similarity of the phase and, hence, if a similar or orthogonal separation will be obtained.  For example, Figure 23 shows the comparison of two C18 phases from Thermo Scientific, although these are both C18 phases from the same manufacturer there are differences in the plot shapes which may result in differences in chromatographic selectivity. 

 

Figure 23: Comparison of PQRI data for Thermo Hypersil ODS and Thermo Hypurity C18 phases.

 
 

However, the shape of the plots shown in Figure 24 are in good agreement which suggests that similar results would be obtained when using either of these columns for a particular separation under the same analytical conditions.

 

Figure 24: Comparison of PQRI data for Phenomenex Prodigy ODS(3) and YMC Pro C18 phases.

 

Therefore, knowledge of the differences in stationary phase types, interactions, and classifications are powerful tools for HPLC method development.  Table 3 details some common stationary phase types, the modes of chromatography under which they can be used, and some possible application areas.  However, each manufacturer will have a plethora of columns that can be used for a particular application and this is by no means an exhaustive list.

 
 

Name

Functional
Group

NP

RP

IE

HILIC

Application

Silica

−OH

 

 

Non-polar and moderately polar organic compounds

C1

−(CH3)3

 

 

 

Least retentive of all alkyl phases.  Moderately polar and multi-functional compounds

C4 (butyl)

−C4H9

 

 

 

Shorter retention than C8, C18.  Peptides and proteins.

C8

−C8H17

 

 

 

Less retentive than C18.  Small peptides/proteins, pharmaceuticals, steroids, environmental samples.

C18 (ODS)

−C18H37

 

 

 

Most retentive alkyl phase.  Pharmaceuticals, steroids, fatty acids, phthalates, environmental etc.

Cyano

−(CH2)3CN

 

 

Unique selectivity for polar compounds.  Better for NP gradient elution than silica.  Orthogonal selectivity to C18, C8 for RP.  Pharmaceuticals and mixtures with very different solutes.

Amino

−(CH2)3NH2

HILIC - Carbohydrates, polar compounds.
WAX - Anions, organic acids.
NP - Alternative selectivity to silica.  Good for aromatics.
Reversed phase.

Phenyl

−C6H5

 

 

 

Aromatic compounds, moderately polar compounds.

Pentafluorophenyl (PFP)

−C6F5

 

 

 

Extra selectivity and retention for halogenated, polar compounds, and positional isomers

Diol

−(CH2)20CH2
(CH2OH)2

 

RP - Proteins, peptides.
NP - Similar selectivity to silica, but less polar.
HILIC.

SCX

−RSO3H-

 

 

 

Organic bases.

SAX

−RN+(CH3)3

 

 

 

Organic acids, nucleotides, nucleosides.

AX
(anion exchange)

−(CH2CH2NH-)n

 

 

 

Organic acids, nucleotides, oligonucleotides.

Porous graphitic carbon

100% carbon

 

 

Particularly useful for highly polar compounds that are difficult to retain using conventional silica columns.  Structurally similar compounds (isomers, diastereomers)

Table 3: Summary of a range of HPLC stationary phase types. 
NP = normal phase chromatography, RP = reversed phase chromatography, IE = ion exchange, HILIC = hydrophilic interaction liquid chromatography.

 

There are some specialist HPLC column stationary phase types which are worth noting.  These stationary phase types are particularly amenable to separations that need to be carried out under conditions that would not be tolerated by traditional stationary phases, for example, at extremes of pH or with highly aqueous mobile phases.

 
 

Water Wettable Phases

Several modern applications in HPLC require the use of highly aqueous mobile phase compositions.  LC-MS techniques dictate that for optimum performance the analyte species should be in the ionized form, therefore, this will require the mobile phase to be highly aqueous in order to gain retention of the highly polar (ionized) analyte.  Traditional reversed phase columns are not suitable for use with highly aqueous mobile phases (> 95% H2O) due to a phenomenon known a phase collapse or self-association (Figure 25). 

Hydrocarbon bonded phases will tend to fold into themselves to escape the highly polar mobile phase resulting in gross losses of efficiency.  The columns may be restored but only after long and complex column washing procedures.  There are three possible ways of manufacturing water wettable phases for use in reversed phase HPLC.

 

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Figure 25: Phase collapse.
 
 

1. Aq Type Phases

One method of producing water wettable phases involves close control of the spacing between the bonded phase ligands on the silica surface (Figure 26).  Knowing the surface area of the silica and adjusting the carbon loading allows the inter ligand distance to be controlled.  This gives more (or less) access to the silica surface allowing the polar influence of low energy surface silanol groups to alter the selectivity of a separation. 

Stability in highly aqueous conditions is achieved via the adsorption of a layer of water at the silica surface (Figure 27).  The vicinal (low energy) silanol groups become hydrated and so the driving force towards self-association is lost as the layer of adsorbed water at the silica surface effectively repels the ligands and the remain ‘activated’.

 
Figure 26
 
 

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Figure 27: Adsorption of a water layer at the silica surface which prevents phase collapse.
 
 

2. Polar Endcapped Phases

The alternative method of introducing stability in highly aqueous environments is to use polar endcapping reagents.  These reagents are chosen to retain good peak shape with almost all applications whilst again introducing a polar characteristic to the separation and, hence, altering the column selectivity.  The mechanism of the high aqueous stability is the same as outlined previously where a layer of water is absorbed at the silica surface and prevents phase collapse.

 
 

3. Polar Embedded Phases

Polar embedded phases contain a modification to the alkyl chain which is usually an amide, carbamate, or other suitable polar functional group.
This phase has several distinct advantages including:

  • Use with 100% aqueous mobile phases
  • Polar group which gives an alternative selectivity
  • The ability to separate polar, ionizable, and especially highly basic compounds with excellent efficiency and peak shape
  • Enhanced robustness over shorter chain and polar bonded phases

The proposed mechanisms of interaction are shown (Figure 28).

  • Here the analyte polar functional group is shown interacting with the amide spacer in a manner analogous to the interaction with the silanol surface to produce alternative selectivity
  • A layer of water is attracted to the polar embedded moiety acts to stop phase collapse
  • The polar moiety interacts with a lone silanol group to shield the surface and reduce secondary interactions (poor peak shape)
 

Figure 28: All 100% aqueous stable phases can also be used for HILIC applications.

 
 

Extreme pH Applications

\The traditional working mobile phase pH range for silica based columns is 2.5-7.5.  Outside this range serious column damage would be inflicted with long-term use.  This is unfortunate as there are many advantages to working at high or low pH.
At low pH (1-2.5) the risk is due to hydrolysis of the silyl ether linkage between the bonded phase and the silica surface which will result in phase bleed.  Symptoms of phase bleed at low pH include deterioration of peak shape and loss of efficiency.  Most manufacturers offer bonded phases which are capable of operating at low pH.  These are based on one of two mechanisms (Figure 29):

  1. Bulky side groups on the ligand offer steric protection of the silyl ether linkage
  2. Difunctional binding to the silica surface to produce a more stable bond between the bonded phase and silica surface

It should be noted that tri-functional bonding is sterically impossible; however, some column manufacturers will use trifunctional ligands in order to increase the extent of difunctional bonding.
Working at low pH allows strong(er) acidic analytes to be analyzed without the need for ion pairing reagents.

 
Figure 29
 
 

At higher pH it is the silica surface itself that is most at risk from hydrolysis, primarily via the surface silanol species.  At mobile phase values above pH 7 traditional silica based packing materials may begin to hydrolyze leading eventually to the formation of small fines which migrate to and block the outlet frit of the column and the interstitial gaps between the particles; this will result in increased system back pressure.  Other earlier symptoms include loss of peak shape and efficiency.
Many approaches have been taken to protect the HPLC stationary phases at high pH including (Figure 30):

  1. Using hybrid silica which contains fewer surface silanol groups
  2. Using multiply bonded silica to reduce the instance of surface silanol species
  3. Using polymeric and non-silica based material

Working at high pH, especially with basic analytes, has the advantage of removing an experimental variable (the degree of analyte ionization and the necessity to very accurately adjust mobile phase pH) and increases method robustness and retention for highly basic analytes.

 

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Figure 30
 
 

Porous Graphitic Carbon

Porous graphitic carbon is composed of flat sheets of hexagonally arranged carbon atoms (Figure 31).  There are no surface silanol groups; therefore, there are no possibilities for deleterious secondary interactions between analytes and the surface.  These columns are particularly amenable to separation of highly polar species, separations carried out at high pH, and they can be used for reversed phase, normal phase and LC-MS applications. 

The mechanism of interaction between the analyte and stationary phase is dependent upon the polarity and shape of the analyte.  These specific interaction mechanisms allow the resolution of analytes which may not be separable by typical reversed phase HPLC.  Use of these types of phases may negate the need for complex buffering systems or ion pair reagents.  The use of increased organic modifier concentration for polar analytes makes porous graphitic carbon columns more amenable for use in LC-MS applications.

 

Figure 31: Porous graphitic carbon stationary phase.

 

Analyte retention on porous graphitic carbon phases is thought to occur via two mechanisms (Figure 32):

  1. Adsorption - the strength of the interaction between the analyte and stationary phase is dependent on the area of contact between the molecule and the surface and also on the type and position of functional groups which are in contact with the surface.  More planar molecules will exhibit greater retention than rigid molecules with a three-dimensional spatial arrangement.
  2. Charged induced interactions - this interaction accounts for the strong retention exhibited by polar analytes.  Polar groups have a permanent dipole which as it approaches the graphite surface induces a dipole on the surface resulting in an analyte/stationary phase interaction (Figure 31).
 

Figure 32: Representation of a polar molecule interacting with the graphite surface.

 

Further information on HPLC column stationary phase chemistry can be found here:

HPLC Channel/Classes/The Theory of HPLC/Column Chemistry

 
 

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Particle Size

The particle size of the support material is of primary importance when selecting a stationary phase.  By reducing the particle size diameter, not only is the efficiency of the column increased (plate height (H) is reduced), but the optimum linear velocity at which minimum plate height achieved is also increased. 

More efficient peaks can be achieved at elevated flow rates, leading to a reduction in analysis time accompanied, potentially, by an increase in resolution.  Shorter diffusion paths and, hence, increased mass transfer kinetics (C term from the Van Deemter equation) are one reason behind this reduction in plate height. 

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Figure 33 shows the relationship between particle size, flow rate (linear velocity), and plate height for columns packed with various silica particle diameters.

 

Figure 33: Van Deemter curves for various particle diameters.

 

It may seem that the smallest diameter particles will be the obvious choice for high efficiency separations; however, this benefit does come at a substantial price, that of increased back pressure.  The pressure increase is inversely proportional to the square of the particle diameter as shown in Equation 1.


Equation 1

Where any of the numerators increase, flow (F) or column length (L) for instance, a resultant increase in back pressure will be observed.  Where any of the denominators decrease, column radius (r) or particle size (dp) for instance, an increase in back pressure is also observed.  Note that the radius and particle size functions are raised to the second power, thus causing their impact on back pressure to be much more dramatic.
When selecting the appropriate particle size for an application some useful guidelines are as follows:

  • Smaller diameter particles - complex mixtures with similar components and high throughput separations
  • Large particles - routine analyses where analytes have greater structural differences
  • Particles 10+ μm - preparative HPLC

Particle size particularly affects the efficiency term of the fundamental resolution/Purnell equation (Equation 2). 
Efficiency is inversely proportional to particle size (Equation 3); hence, a decrease in particle size will result in an increase in efficiency. 
This increase in efficiency will allow the use of shorter columns and/or faster flow rates which will ultimately result in faster analyses without loss of resolution, as is demonstrated in Figure 34.  Note the increase in system pressure caused by reducing the particle size.


Equation 2
 
Equation 3
 
 
  :

Column:

Mobile phase:

Gradient:

Flow rate:

Temperature:

Detection:

Injection volume

 

Hypersil GOLD 200 x 2.1 mm

A - H2O; B - MeCN

65-95 %B in 1.5 min, hold for 1.5 min

600 μL/min

40 °C

247 nm

0.2 μL

1. Acetophenone, 2. propiophenone, 3. butyrophenone, 4. valerophenone, 5. hexanophenone, 6. heptanophenone, 7.  octanophenone.

Figure 34: Effect of particle size on efficiency and resolution. 5-6

 
 

Particle Size Distribution

The particle size distribution is another key parameter that should be considered; however, as this is not a user controlled variable, little practical attention is given to it (Figure 35).

Any particle size stated for an HPLC column (e.g. 5 μm) will actually be the mean value from a distribution of particle sizes, achieved practically by the manufacturer using automated sub-sieve sizing techniques.

 In reality a wide distribution of particles will lead to heterogeneity within the column and, as such, an increase in variable path lengths the analyte molecules can take through the column (Van Deemter A term, often called the column packing term) which will give rise to band broadening.

D10/D90 ratios are often quoted for packing materials as a relative measure of the particle diameter distribution.  D10 = particle diameter at 10% of the total size distribution and D90 = particle diameter at 90% of the total size distribution.  The closer this value is to unity, the more homogeneous the particle diameter distribution.
This phenomenon has contributed to the sudden upsurge in the commercial availability of Superficially Porous Silica (SPS) or core-shell particles with small pore volumes (80 Å - 120 Å) which exhibit increased efficiency/reduced plate height for the analysis of low molecular weight analytes. 

 

SPS particles have been available in their 300 Å guise for the analysis of high molecular weight analytes for a number of years because of the improved mass transfer kinetics (C term) which were debilitating for conventional porous microspheres.  These particles consist of a solid silica core with a layer of porous silica deposited around the outside, effectively shortening the path length for diffusion within the internal pore structure and improving mass transfer kinetics.

However, for lower molecular weight analytes (< 600 Da) the effects of increased mass transfer are less important and the increase in efficiency/reduction in plate height attributed, in the main part, to the very narrow particle size distribution that can be achieved from creating a particle with a solid core and the reduction of multiple path effects (A term).

 

Figure 35: Particle size distribution.

 
 

Length

Efficiency is directly proportional to column length (Equation 3).  Increasing column length will increase efficiency.  Doubling column length increases resolution but only by a factor of 1.4.  Short column lengths (30-50 mm) will give short run times and low backpressures and are ideal for gradient analyses.  Longer columns (250-300 mm) will give greater resolution but with longer analysis times and at a greater cost. 


Equation 3

The separation of four paraben compounds detailed in Figure 36 demonstrates the effect of column length.  As can be seen decreasing the column length reduces analysis time, however, resolution is also decreased.

 

Column:

Mobile phase:

Temperature:

Flow rate:

Detection:

Injection volume

 

Hypersil GOLD

H2O/MeCN (50:50) + 0.1% formic acid

40 °C

0.2 mL/min

UV 254 nm

1 μL

1. Methyl paraben, 2. ethyl paraben, 3. propyl paraben, and 4. butyl paraben.

Figure 36: Effect of column length. 7

 
 

Internal Diameter

As was shown previously (Equation 1) a decrease in column internal diameter will result in an increase in back pressure.


Equation 1
 

Larger diameter HPLC columns require higher flow rates; therefore, larger volumes of mobile phase will be used.  Changing from a 4.6 mm (1) to a 3.2 mm (2) ID column can reduce the flow rate and solvent volume required to reach the same optimum linear velocity without increasing the run time.  The new flow rate can be calculated using Equation 5.


Equation 5

Where:
F = flow rate (mL/min)
dc = column diameter (mm)
dp = particle diameter (μm)
Note: For columns with the same particle size the last term becomes obsolete.

 
 

When column internal diameter is decreased an increase in sensitivity (2-3 fold) can be expected when injecting the same analyte mass.  This is due to there being an increased analyte concentration in the mobile phase.

HPLC instrumentation may need to be adapted for columns with very narrow internal diameters to minimize band broadening effects which result from extra column effects i.e. mixing volumes out with the column; these can be reduced by reducing tubing length and diameter, using a microvolume detector flow cell etc.  Instrument manufacturers will be able to aid in recommendations for these types of alterations. Generally columns with an internal diameter of 2-5 mm are used for analytical applications.  For preparative applications the column internal diameter is much larger at 10-25.4 mm. 

A change in column diameter will affect the loading capacity, both the volume of sample and the mass of analyte which can be injected onto the column, along with the flow rates which can be used.  Some guidelines for these parameters are listed in Table 4.

 

Internal Diameter (mm)

Typical Injection Volume (μL)

Loading Capacity (mg)

Typical Flow Rates

4.6

15

1

0.5-2

10

100

4.7

4-15

21.2

400

19.5

10-50

30

1000

42.5

40-100

50

2000

118

100-300

100

10000

473

400-1000

Table 4: HPLC column dimensions, capacities, and flow rates.

 
 

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Sensitivity, or signal to noise ratio, is related to the concentration at the peak apex (Equation 6). 

Sensitivity can be increased by reducing column length, reducing internal diameter, and increasing efficiency (i.e. smaller particle size, minimize extra system/column volume, increase flow rate etc.). 

Ultimately the most effective way to maximize sensitivity is to reduce background noise.


Equation 6

Where:
N = efficiency
Vi = injection volume
L = column length
dc = column internal diameter
k = retention factor

 

Putting all of these factors together Figure 37 illustrates the effect of modifying each parameter to maximize the sensitivity of the chromatographic process.

Figure 37: Steps to maximize chromatographic sensitivity.

 
 

Resolution Capacity

A very practical measure of the relative separating power of columns of different dimension is the so-called Resolution Capacity (Table 5).  This can be calculated for a column by dividing the length by the particle size (both in μm).  Table 3 shows some values of resolution capacity for some common column geometries as well as some for core-shell columns.  When comparing, designing, or translating methods this can be a useful measure to determine whether the same, or increased, resolution can be achieved.  For example, to retain the amount of resolution from a 300 mm column with 10 μm particles (resolution capacity 30,000) a shorter 150 mm column with 5 μm particles could be used (Figure 38).  Or to increase resolution a 150 mm column with 1.7 μm particles could be used instead of a 100 mm column with the same particle size, this would result in an increase in resolution capacity from 58,820 to 88,230 respectively.  Core-shell columns produce highly efficient separations and exhibit increased resolution capacities.

 

Column Length (mm)

Particle Size (μm)

L/dp

300

10

30,000

150

5

30,000

100

3

33,000

50

1.7

29,500

 

 

 

100

1.7

58,820

150

1.7

88,230

Core-shell Columns

 

 

30

2.6

11,538

50

 

19,231

100

 

38,462

150

 

57,692

Table 5: Resolution capacity of standard and core-shell HPLC column geometries.

 
 

Figure 38: Separation of a mixture of 6 test compounds over a wide range of LogP values. 

Column:  Type II C8
Mobile phase: Water/Acetonitrile. 
Flow rate: 2 mL/min.

 
 

Mass Loading Considerations

The baseline width of a chromatographic peak (W) determines its separation from adjacent peaks.  For an overloaded separation this can be related to the sample weight and experimental conditions for isocratic elution (Equation 7). 8

The first term of Equation 7 relates to column effects while the second term relates to sample weight effects.  Therefore, for small samples peak width is determined by k, N, and t0, whereas, for large sample weights it is determined mainly by k, t0, and sample weight.  Rearrangement of Equation 7 to solve for the maximum column capacity (Ws) and plotting of these terms gives a graph with a slope with the value of Ws (Figure 39).  For different amounts of loaded sample, different values of (k0), (N0) and (W) will be found.


Equation 7

Where:
Ws = column capacity
Wx = weight of compound injected
W = peak width at base
t0 = column void volume
k0 = retention factor

 

Figure 39: Plot of the two terms from Equation 7.  Slope of the line gives a value for WS.

 
 

Even if the sample injection volume is small mass overload of the column can still occur resulting in fronting, tailing, or broadened peaks, a change in the retention time, or efficiency (Figure 40).  This is due to columns having a limited capacity (Table 6).  Mass overload will result in the stationary phase becoming saturated with sample.  Mass overload is likely to occur if the concentration of an analyte changes from sample to sample. 

The effect of changes in analyte concentration should be evaluated for an HPLC procedure after method development and a maximum analyte concentration should be established, Wmax (Equation 8). 9  If a sample exceeds this limit it should be diluted and re-run.  Equation 8 can be used to estimate values of Wmax, which will be in micrograms (μg) and is independent of column length.  The value of Wmax is for each analyte in the sample, not the total sample weight; therefore, this value can be multiplied by the number of analytes in the sample to give the maximum sample weight.


Equation 8

Where:
k = retention factor
dc = column diameter (cm)

 

Worked example:

For a separation performed on a 4.6 mm diameter column with an analyte k = 5.

 

Column Dimensions

150 mm

100 mm

50 mm

4.6 mm

20 μg

11.1 μg

1.2 μg

4 mm

15

8.3

0.9

3 mm

8.4

4.7

0.5

2.1 mm

4.2

2.3

0.3

1 mm

0.9

0.5

0.1

Table 6: HPLC column mass capacities per analyte.  μg Sample on column. 

 
 

Figure 40:  Peak fronting caused by column overload.

 
 

Effect of Column Dimensions on Isocratic and Gradient Elution

It’s interesting to draw a quick but effective analogy between isocratic and gradient HPLC.

In isocratic HPLC, the analytes enter the HPLC column and, depending upon the partition co-efficient of the analyte between the mobile and stationary phase material (governed by many factors including the hydrophobicity of the analyte and its shape), moves at a constant pace along the column, undergoing successive partitioning events into the stationary phase, which controls analyte retention.
Retention in isocratic HPLC is measured using the retention factor (k) which is defined as (Equation 9):

  sponsored by

Equation 9

Where:

t0 = retention time of a compound which does not interact with the stationary phase surface (typically estimated using the injection solvent peak or a compound such as Uracil in reversed phase HPLC or hexane for normal phase HPLC).

 
 

In gradient HPLC, things are somewhat different.  The gradient is formed by increasing the percentage of organic solvent.  Consequently; at the beginning of the analysis, when the mobile phase strength is low, the analyte will be partitioned wholly into the stationary phase (or ‘focused’) at the head of the column and will not be moving through the column at all.  As the mobile phase strength increases, the analyte will begin to partition into the mobile phase and move along the column.  As the mobile phase strength increases continuously, the rate at which the analyte moves along the column subsequently increases and the analyte ‘accelerates’ through the column. 

At some point within the column, the analyte may be wholly partitioned into the mobile phase, and will be moving with the same linear velocity as the mobile phase.  The point at which this occurs depends upon the nature of the analyte and its interaction with the stationary phase material.  As the rate at which the analyte elution changes during gradient HPLC, the retention factor (k) used above for isocratic separations is not applicable. Instead the use of an ‘average’ retention value, k*, or the retention factor of the analyte as it passes the mid-point of the column (Equation 10).  Most analytes will be moving at the same rate as they pass the mid-point of the column and hence the retention factors for all analytes in gradient HPLC are very similar.


Equation 10

Where:

tg = gradient time (min.)
F = flow rate
S = constant determined by strong solvent and sample compound (for small molecules < 500   Da the value is between 2 and 5; a value of 4 is used by convention when the value is not accurately known.  Proteins have much larger values; typically between 50 and 100 and need longer gradient times for separation).  S can be estimated from Equation 11:


Equation 11

Δ Φ = change in volume fraction of organic (final %B – initial %B)
VM = column void volume (Equation 12)


Equation 12

Increasing column length in isocratic separations can increase efficiency (N), increase retention, and therefore, affect resolution (Figure 41).  In gradient HPLC it is possible to encounter peak reversals depending upon the shape of the gradient profile and the length of the column used.  In the example shown (Figure 42), because of the relationship between %B and gradient retention factor, using a 10 cm column will result in Compound A eluting first.

However, when a 25 cm column is used, the retention factors dictate that compound B has enough distance to ‘overtake’ compound A and elutes first.  It is also important to note that although the column length is more than doubled, the retention times of the peaks has not increased by this factor as the analytes are travelling at the velocity of the mobile phase.

 

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Figure 41: Effect of column length on efficiency and resolution in isocratic separations.
 
 

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Figure 42: Effect of column length on analyte elution order in gradient separations.
 
 

Peak Capacity

The separation efficiency of columns under isocratic elution conditions is measured in theoretical plates.  Peak capacity is used to describe the separation efficiency for gradient elution.  Peak capacity describes the maximum theoretical number of components that can be successfully separated with a given column and set of analytical conditions with RS =1 (Figure 43 and Equation 13). 7  Equation 13 can be used to give an approximation of the number of components that can be separated under a specific set of conditions, if this number is lower than the number of components in a sample than the method will not produce a chromatogram with resolved peaks.


Equation 13

Where:

tg = gradient time (min.)
w = average peak width at 4σ (13.4% of peak height)

Note: This is an approximation but a good guide. 
The average peak width can be calculated by adding the peak widths of the first and last peaks and dividing by 2.
 

Figure 43: Calculation of peak capacity.

 

Peak capacity is a function of gradient time, flow rate, column length, and particle size.  Increasing column length while keeping particle size and gradient time constant results in a maximum value of peak capacity being reached, and in fact, for longer columns the value of peak capacity may decrease (Figure 44).  Improving peak capacity using particle size seems to give more promising results, with the decrease in particle size giving higher peak capacity values.  Increasing the gradient duration will increase the peak capacity; however, for longer gradients the increase in peak capacity with time becomes small as a maximum will be reached. 

Peak capacity can be optimized using the flow rate at a fixed gradient time (tg).  Peak capacity will increase proportionally to the square root of column efficiency (Equation 14); therefore, doubling column efficiency will increase peak capacity, but only by 40%.


Equation 14

Where:

N = efficiency
B = slope of the function ln k (k is the retention factor) versus solvent composition C
ΔC = change in solvent composition
t0 = breakthrough time
tg = gradient time

 

Figure 44: Plot of tg/ t0 vs. P giving the optimal peak capacity for a separation.

 
 

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  • Stationary phase selectivity is critical to a successful (optimized) separation
  • Separation on significant differences in LogP or LogD (hydrophobicity) using a C18 column is not always successful
  • Mechanisms to further discriminate between functional group chemistry, shape (hydrodynamic volume), and dipole (induced dipole) capability are all very important in modern HPLC
  • May involve differing ligand chemistry (PFP and Phenyl phases increasingly important), Polar End Capping, Type II (III) Silica, Ligand Density, Polar embedded ligands etc. etc.
  • In modern HPLC we need to be aware of these various column properties which are described in modern databases and by manufacturers
  • Knowledge of analytes is imperative for successful column selection
  • Appropriate column dimensions need to be chosen for the application (sample size, number of components etc.)
 
 

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  1. Klaus K. Unger (Editor), Nobuo Tanaka (Editor), Egidijus Machtejevas (Editor).  Monolithic Silicas in Separation Science: Concepts, Syntheses, Characterization, Modeling and Applications.  2011 Wiley-VCH Verlag GmbH & Co. KGaA. Chapter 4.
  2. Lide, D. R. ed., CRC Handbook of Chemistry and Physics, Internet Version 2005, <//www.hbcpnetbase.com>, CRC Press, Boca Raton, FL, 2005.
  3. Dean, J. A. ed., Lange’s Handbook of Chemistry, Fifteenth Edition, McGraw Hill Inc. 1999.
  4. Snyder, L. R.; Dolan, J. W.; Carr, P. W. J. Chromatogr. A, 2004, 1060, 77-116
  5. //www.analiticaweb.com.br/newsletter/04/Poster_Particulas-sub-2.pdf
  6. //www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma-Aldrich/Datasheet/t308183.pdf
  7. //www.analiticaweb.com.br/newsletter/13/HPLC_Columns.pdf
  8. Snyder, L. R.; Kirkland, J. J.; Glajch, J. L. Practical HPLC Method Development 2nd Ed. John Wiley & Sons Inc., 2011, p629.
  9. Snyder, L. R.; Kirkland, J. J.; Glajch, J. L. Practical HPLC Method Development 2nd Ed. John Wiley & Sons Inc., 2011, p56.
  10. Gilar, M.; Daly, A. E., Kele, M.; Neue, U. D.; Gebler, J. C., J. Chromatogr. A 2004, 1061, 183-192.
 
 

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How do you currently select a column for an HPLC application? Do you just use the one that is on the instrument, root around in an old drawer and hope that you pull out one that works, use your favorite C18 or ODS column, or borrow one from a colleague who’s chromatography is currently working particularly well? These are probably not ideal situations but there will be many of us who are guilty of these exact scenarios. Instead, why not join us for the CHROMacademy Essential Guide webcast on HPLC column selection in which we will look at the available column stationary phases and support materials (including conventional fully porous and core-shell/superficially porous material), column dimensions and the effect that they can have on your chromatography, and how to optimize and improve your current chromatographic separations. This practical guide will give you the tools required to make an informed choice every time you develop a new HPLC method.

Tony Edge
R&D Principal, Chromatography Consumables
Thermo Scientific

Dr. Dawn Watson
Technical Specialist
Crawford Scientific

Key Learning Objectives:

  • Understand available stationary phase types and chemistries
  • How to optimize HPLC separations by altering column geometry
  • Identify the correct HPLC column for a specific application
  • Understand column stationary phase classification