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The CHROMacademy Essential Guide Webcast:
Critical Evaluation of HPLC Methods

Thursday 19th September 2013, 16:00 GMT

There are numerous factors that affect the quality of your HPLC separation; including mobile phase composition, temperature, ionic strength, pH, column dimensions, and stationary phase and support material characteristics.  These can all be manipulated to change and improve your HPLC separations.  However, what are the changes we should expect?  Will retention improve if another solvent is used and would we expect selectivity to change?  Will certain analytes elute in the same order if the mobile phase pH, ionic strength, additives or temperature are changed? 

In this Essential Guide we will evaluate many of the aspects of the HPLC process giving you useful practical information on how to use these parameters to alter your HPLC separation resulting in better and more robust chromatography.  We will also discuss the use of in-line filters, guard columns, and the most appropriate detector for your application.

Scott Fletcher (Technical Manager, Crawford Scientific) and Dr Paul Ferguson (Associate Principal Scientist, Astra Zeneca) will draw on their vast HPLC knowledge to help you evaluate your HPLC process and highlight the important factors to consider when altering or developing HPLC methods.

 

Topics covered include:

  • The effect of mobile phase polarity on HPLC separations
  • The role of a buffer in an HPLC separation and how it should be chosen
  • The effect of temperature on analyte retention and selectivity
  • Additives for improved chromatography
  • Stationary phase and support material chemistry and important properties
  • The effect of column dimensions on gradient elution
  • Why and when you would use an guard column or in-line filter
  • Detectors – advantages, disadvantages, & applications

Who Should Attend:

  • Anyone who would like to improve their HPLC methods
  • Anyone who uses HPLC
  • Anyone involved in HPLC method development
  • Anyone who wants to better understand the factors that affect an HPLC separation
 


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The CHROMacademy Essential Guide Tutorial
Critical Evaluation of HPLC Methods

There are numerous factors that affect the quality of your HPLC separation; including mobile phase composition, temperature, ionic strength, pH, column dimensions, and stationary phase and support material characteristics.  These can all be manipulated to change and improve your HPLC separations.  However, what are the changes we should expect?  Will retention improve if another solvent is used and would we expect selectivity to change?  Will certain analytes elute in the same order if the mobile phase pH, ionic strength, additives or temperature are changed?  In this Essential Guide we will evaluate many of the aspects of the HPLC process giving you useful practical information on how to use these parameters to alter your HPLC separation resulting in better and more robust chromatography.  We will also discuss the use of in-line filters, guard columns, and the most appropriate detector for your application.
 
Scott Fletcher (Technical Manager, Crawford Scientific) and Dr Paul Ferguson (Associate Principal Scientist, Astra Zeneca) will draw on their vast HPLC knowledge to help you evaluate your HPLC process and highlight the important factors to consider when altering or developing HPLC methods.

Topics include:

  • The effect of mobile phase polarity on HPLC separations
  • The role of a buffer in an HPLC separation and how it should be chosen
  • The effect of temperature on analyte retention and selectivity
  • Additives for improved chromatography
  • Stationary phase and support material chemistry and important properties
  • The effect of column dimensions on gradient elution
  • Why and when you would use a guard column or in-line filter
  • Detectors – advantages, disadvantages, & applications

 

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Reversed phase HPLC is characterised by a non-polar stationary phase and a polar mobile phase.  Typical reversed phase stationary phases are hydrophobic and chemically bonded to the surface of a silica support particle.  The most commonly used stationary phases are shown (Table 1.).  As the hydrophobicity of the stationary phase decreases, its polarity increases, and the retention of non-polar analytes decreases.

Table 1: Commonly used reversed phase stationary phases.

 
 

The hydrophobicity of an analyte molecule is the primary indicator to its retentivity in reversed phase HPLC.  Hydrophobicity is often expressed as Log P which is a measure of the way an analyte (in its neutral form) partitions between two solvents (usually octanol and water, Eqn. 1) under standard conditions.  The higher the value of Log P the more hydrophobic the molecule.

 
 

The structure of the sample molecules will also give an indication as to their elution order (Figure 1 and 2).  Elution order is governed by the water solubility of the molecule and the carbon content of a particular homologous group.  Some general observations regarding sample elution order include:

  • The less water soluble a sample, the greater the retention
  • Charged species typically show the least retention followed by acid, then basic compounds all eluting early
  • Retention time increases as the number of carbon atoms increases, for a homologous group
  • Branched chain compounds elute more rapidly than normal, straight chain, isomers
  • Unsaturation, double bonds, decreases retention
 

The general elution order is:
Aliphatics > induced dipoles > permanent dipoles > weak bases > weak acids > strong acids

Ionic compounds generally elute with the column void volume.

 

Figure 1: Elution order for a series of homologous compounds.

 

Figure 2: Elution order for a series of benzologs.

 
 

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Composition
The mobile phase in reversed phase HPLC usually consists of water/aqueous solution (often a buffer) and an organic modifier.  Chromatographically in reversed phase HPLC water/aqueous is the weakest solvent as it is the most polar and repels hydrophobic analytes in to the stationary phase to a greater extent than any other solvent, resulting in long retention times.  A less polar organic modifier is added, resulting in faster elution of the hydrophobic analyte as it is no longer strongly repelled in to the stationary phase.  The organic modifier can, therefore, be considered as chromatographically strong – a strong eluter. 

 

Figure 3: Properties of common reversed phase HPLC solvents.

 
 

Choosing or modifying the mobile phase can alter the retention and selectivity of an HPLC separation; therefore, it is of utmost importance that the organic modifier, buffer, and additives are correctly chosen and prepared.  It is also important to understand how each of the parameters affects the chromatographic separation.

When deciding on the organic modifier that will be used for a separation the following points should be considered.  The chosen organic modifier must be miscible with water (MeOH, THF, MeCN are all water miscible).  The use of a low viscosity solvent is preferable (Table 2) due to the lower pressure drop produced at a specific flow rate.  It also allows for faster chromatography due to the increased rate of mass transfer.  It should be noted that the viscosity of mobile phase mixtures will be markedly higher, and also therefore instrument pressure, than the pure compounds.  Figure 4 illustrates the situation with different percentages of methanol, acetonitrile and tetrahydrofuran in aqueous.  The viscosity maximum for MeOH/Aq. mixtures is reached at 40% MeOH (1.62 mPa s at 25 oC) which is almost three times the value for MeOH alone.  Acetonitrile is often the solvent of choice due to its ability to solubilise many small molecules and its low viscosity.  As can be seen from Figure 4, the viscosity of MeCN/Aq. mixtures decreases with increasing amounts of MeCN, with the maximum value being ~0.95 mPa S (20 %B at 25 oC) which is almost half the maximum of MeOH/Aq.  Increased solvent viscosity will result in higher system pressures.  It is not uncommon for an instrument to shut down due to over pressuring when employing methanol as the organic solvent during the first injection of gradient method.

When mixing MeOH and aqueous mixtures, each solvent should be weighed or volumetrically measured due to the solvent contraction that occurs upon mixing i.e. 500 mL of water topped up to 1000 mL with MeOH will result in a solution with a MeOH content in excess of 50% by volume.  Care should also be taken if reactive analytes, (alcohols, aldehydes, carboxylic acids) are being analysed as in the presence of MeOH methyl esters can be formed giving rise to erroneous peaks in the chromatogram and quantification errors.

Tetrahydrofuran is an interesting solvent in that it is one of the strongest chromatographically and can produce separations in very short times, whilst still being fully miscible with water.  However, it does have a relatively high UV cut off.  Column equilibration can also be slower with THF than with MeOH or MeCN.  In the presence of air or oxidisers THF will also form hazardous, explosive peroxide species, which pose both a safety risk and can be reactive towards analytes.  Recently THF has also been upgraded to carcinogen status by some bodies.   

If a UV detector is being used it is important to consider the UV cut off of the mobile phase (organic modifier, buffers, additives etc. Table 2 and 6) to ensure that it does not interfere with the λmax of the analyte.

Once prepared, HPLC mobile phases will have a given shelf life (Table 3).  The values shown in table 3 are a conservative estimate of the usable time period for given mobile phases, therefore, it is important to monitor mobile phase stability periodically and local policies or procedures should take precedence.

 

Table 2: Solvent properties.

 
 

Figure 4: Viscosity of mixtures of aqueous and organic solvents at 25 oC. 2

 
 

Table 3: Estimated shelf-life for HPLC mobile phases.

 
 

Polarity, Retention, and Selectivity

Retention will be, and selectivity can be, affected by the mobile phase polarity.  The retention factor, k, is a means of measuring the retention of an analyte on the chromatographic column (Figure 5).  Ideal values of k are between 2 and 10 for conventional HPLC separations with values of 1 and 5 being more common for UHPLC, or high efficiency, separations.  If k < 1 early eluting peaks may not be properly resolved and may elute concurrently with unretained compounds and there will be a higher chance of chromatographic interferences at the beginning of the chromatogram.  The retention of analytes with these low k values will also be more susceptible to small changes in mobile phase composition. 

For complex mixtures large retention factors, k > 10 - 20 may be required; however, there may be no great gain in resolution by increasing retention factor at this point due to a decrease in efficiency (broader peaks) which is attributable to longitudinal molecular diffusion. 

 

Figure 5: Retention factor.

 
 

Selectivity factor, α, indicates the ability of the chromatographic system to chemically distinguish between sample components (Figure 6).  α is the ration of retention factors and must always be greater than 1, larger k divided by smaller k, as when it is equal to 1 the peaks co-elute.  Selectivity is dependent upon the analyte chemistry, stationary phase and a multitude of mobile phase parameters.  Selectivity is the main driver behind resolution and is therefore the most effective tool for optimisation.  The fundamental resolution equation shows that resolution is proportional to selectivity (α), efficiency (N), and retention (k).  Plotting these parameters against resolution (Figure 7) clearly demonstrates that selectivity has the greatest impact on resolution.

 

Figure 6: Selectivity factor.

 
 

Figure 7: Plot of α, N, and k vs. Rs.

 
 

Increasing the percentage of organic modifier in the mobile phase will reduce the polarity (increased strength) resulting in decreased retention times.  A 10% increase in %B will decrease retention by 2-3 times and as can be seen from Figure 8 resolution may also be altered.  For example, using the interactive tool (Figure 8) increasing %B from 45 – 65 % gives a k value of 6.9 (within the acceptable limits) for peak 6 and reduces the analysis time from 60 to 10 minutes.

 

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Figure 8: Effect of modifying %B on retention.  Column: C18 150 mm x 0.46 mm x 5 μm.  Flow: 2.00 mL/min.  t0 = 1.28 mins.

 
 

Changing the organic modifier can also have a large effect on retention and selectivity (Figure 9).  Retention is altered due to a change in the overall mobile phase polarity while selectivity is altered due to the solvents differing ability to undergo acid, base, and dipole interactions with analytes. 

 

Figure 9: Separation carried out using 40% MeOH, 40% MeCN, and 40% THF.

 
 

Each solvent will interact differently with differing analytes and can be classified by their solvochromatic parameters (Table 4).  Dipole character π*, is a measure of the ability of the solvent to interact with a solute via dipolar and polarisation forces and will be good for the retention of polarisable analytes.  Acidity α, is a measure of the ability of the solvent to act as a hydrogen bond donor towards basic (acceptor) solutes so will be good for the retention of bases.   Basicity β, is a measure of the ability of the solvent to act as a hydrogen bond acceptor towards an acidic (donor solute), therefore, it will retain acidic analytes well.  These characteristics, along with knowledge of the analyte chemistry, can be used to manipulate elution.

 

Table 4: Relative solvochromatic parameters of common HPLC solvents. The values for π*, α, and β are normalized so that their sum gives 1 and are therefore only relative numbers.

 
 

Eluotropic series


Eluotropic series – a listing of various compounds in order of eluting power for a given sorbent.
Isoeluotropic – mobile phases which possess the same eluting power or strength.

A nomogram (Figure 10) can be used for the selection of isoeluotropic mobile phases.  Isoeluotropic mobile phases produces separations in approximately the same time, however, they may show altered selectivity, due to the reasons as previously described.  Figure 11 illustrates how using an isoeluptropic mobile phase in each instance results in the final peak in each chromatogram eluting at the same approximate k, however, when the solvent is changed from MeCN or MeOH to THF the selectivity of peaks 1 and 2 is changed. 

 
Quick rules of thumb
  • THF reduces retention of straight chain analytes w.r.t. polarizable cyclic compounds
  • MeCN reduces retention of esters
 

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Figure 10: Nomogram for calculation of isoeluotropic mobile phases.

 
 
Figure 11: Separations carried out using different isoelutropic mobile phases.
 
 

Ionisable Analytes: pKa

When working with ionisable analytes, it is important to consider the pH of the mobile phase to optimise separation; therefore, knowledge of the analyte is of utmost importance.  Knowledge of the functional group chemistry and the associated pKa of an analyte will allow for tuning of the pH of the mobile phase.

When an acid or base is dissolved in water the equilibria shown are established (Eqn. 2 and 5).  The equilibrium constants (dissociation constants, Ka and Kb) are given by equations 3 and 6.  A strong base will have a large Kb and exists in equilibrium with its conjugate acid which is a weak acid with a small Ka.  The converse is true for acidic species.  All acid-base reactions in aqueous solutions can be viewed from the standpoint of the conjugate acid form losing a proton to form the conjugate base. When we do this we can always use the pKa in our calculations and do not need to deal with Kb or pKb making comparisons of acidic and basic molecules much more facile. 

 
 

For Acids

  Ka  
 
 

For Bases

  Kb  
 

Remember

The stronger the acid the smaller the pKa
The stronger the base the larger the pKa

 

As can be seen from Figure 12 the pKa gives an indication of the strength of the acid or the base, however, it cannot be decided from the pKa if the molecule is an acid or a base, the functional groups contained within the molecule must be known.  For example the pKa of aspirin and diazepam are very similar (3.5 and 3.3 respectively).  Aspirin is a weak acid as it contains carboxylic acid groups, while diazepam is a weak base and contains basic nitrogen functional groups.

 

Figure 12: Weak acid and base compounds.

 
 

As previously mentioned pH will affect the elution of ionisable analytes.  It will also affect the extent of analyte ionisation, which will in turn affect elution/retention properties.  For an acidic analyte, in a buffered solution, addition of an acid will lower the pH and the analyte will become less ionised i.e. the equilibrium will shift to the left.  For example the pKa of Ibuprofen is 4.9 (Figure 13 and table 5), at pH 6.9 it will be almost fully ionised.  Lowering the pH to 2.9 shifts the equilibrium left and ibuprofen will exists almost entirely in its non-ionised form.  At a pH equal to the pKa an ionisable molecule will be 50% ionised and 50% non-ionised, which can lead to poor peak shape.

 

Figure 13: Ibuprofen pKa.

 
 

Table 5:  Extent of ionization of Ibuprofen at selected pH values.

 

The change in degree of ionisation happens over a limited pH range.  Due to pH and pKa being logarithmic values it can be shown that 1 pH unit away from the pKa the extent of ionisation is approximately 90%.  At 2 pH units away from the pKa the extent of ionisation is approximately 99%, while at 3 pH units it is 99.9%. A good rule of thumb to predict the extent of analyte ionisation is the 2 pH rule (Figure 14).

 

Figure 14: 2 pH rule for determining the extent of ionisation of acidic and basic analytes.

 
 

The extent of ionization for salicylic acid and methamphetamine are shown in figures 15 and 16.

 

Figure 15: Plot of pH vs. concentration for salicylic acid. 3

 
 
Figure 16: Plot of pH vs. concentration for methamphetamine.
 

With regards to elution characteristics the ionized form of an acidic analyte is more polar, therefore, retention under reversed phase HPLC conditions is shorter (tR) and they will have smaller k values.  At a pH corresponding to the pKa the analyte is 50% ionized and it will exhibit poor peak shape (Figure 17).

 
 

Figure 17: Effect of ionization on elution of acidic species.

 

Unlike ionized acids, which when charged, elute rapidly from the column, protonated bases may have long retention times and poor peak shape due to interaction with residual silanol species on the silica surface.  Separations of basic compounds are not usually carried out under ion suppression conditions as the increase in pH to produce the neutral species would damage traditional silica columns (although hybrid columns can be used at extremes of pH).  Traditionally, analysis of weak bases have been carried out at low pH as the surface silanol species will be non-ionised ( pKa 3.5-4.5) resulting in improved peak tailing, as can be seen for the analysis of amphetamine at pH 7 and 2.5 (Figure 18).

 

Figure 18: Analysis of amphetamine at pH 7 and 2.5.

 
 

The pH of the mobile phase is controlled using a buffer (Table 6).  A buffer is a weak acid or base in co-solution with its conjugate acid/base.  It resists small changes in pH that may occur within the HPLC system.  A particular buffer is only reliable at 1 pH unit either side of its pKa.  Buffer concentration must be adequate but not excessive.  Below 10 mM buffers will have very little buffering capacity, whereas, above 50 mM there is a high risk of precipitation of the salt in the presence of high organic concentration (i.e. > 60% MeCN).  Buffers will usually be in the range 25 – 100 mM.  If LC-MS applications are being carried out the buffer must be volatile.  It is also good practice to prepare fresh buffers daily as pH can change on standing with the ingress of CO2.  Table 6 shows buffer pKa, working pH range and UV cut off values.

 

 Table 6: Properties of common buffers.

 
 

If pH cannot be used to improve peak shape and / or retention of basic analytes, sacrificial bases or ion pair reagents can be employed.  Sacrificial bases are sterically small, highly surface active species which interact with the surface silanol groups preferentially. Common sacrificial bases include triethylamine (TEA), piperazine, N,N,N’,N’-tetramethylethylenediamine (TMEDA), and dimethyloctylamine (DMOA).  They are added to the mobile phase in sufficient concentration to the mobile phase (10 – 100 mmol) to ensure the surface is fully covered (Figure 19).  Modern, high purity low silanol packing materials negate the need for sacrificial bases.

 

Figure 19: Effect of triethylamine (TEA) concentration on peak tailing.

 
 

Ion pair reagents can be used when other methods such as reversed phase and ion suppression techniques have not been successful.  Samples containing both anionic and cationic components have one type ‘masked’ by the ion pair reagent and the other suppressed by pH.  This is a useful technique if the pKa of sample analytes are not similar.
For example:

  • Tetrabutylammonium phosphate +N(C4H9)4 at pH 7.5 forms strong ion pair with acids, pH suppresses weak base ions
  • Alkylsulfonic acids -SO3(CH2)nCH3 (n = 4 - 7) at pH 3.5 forms ion pairs with bases and weak acids are suppressed by pH
  • TFA forms ion pairs with bases

The mechanisms by which ion pair reagents are thought to operate by are shown in figure 20. 

  1. The analyte is paired in solution with the ion pair reagent and the neutral complex undergoes partition interactions with the stationary phase or
  2. The ion pair reagent populates the hydrophobic surface and the analyte molecules undergo an ion exchange reaction.

Some disadvantages of ion pair reagents are the long equilibration times that are required (> 100 column volumes).  Ion pair reagents are notoriously difficult to remove from the column, therefore, it is recommended that a guard column or dedicated column is used for ion pair applications.  Ion pair reagents, along with irreversibly modifying the stationary phase, will drastically reduce the column lifetime.  Finally, ion pair reagents are not suitable for LC-MS work as they will suppress ion formation and reduce sensitivity.  There are, however, alternative approaches that can be used with LC-MS such as HILIC and mixed mode stationary phases (there is further information in CHROMacademy on these topics).

 

Figure 20: Mechanism of interaction of ion pair reagents.

 
 

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Temperature can affect retention and selectivity of a separation (Figure 21).  An increase in temperature will reduce retention times, this will allow the ability to work at higher flow rates (Figure 22).  Column performance may also increase due to reduced solvent viscosity which will improve mass transfer, reduce system backpressure, and allow faster flow rates.  The separation factor can either increase or decrease depending on the analyte and hence selectivity can be altered, this is particularly prevalent with ionisable analytes.

There are of course some disadvantages to working at higher temperatures including; solvent or sample decomposition, increased risk of bubbles in the detector due to increased solvent vapour pressure, this may manifest itself as a rising baseline, ghost peaks, or incomplete light adsorption.  Silica solubility increases with temperature (> 60oC for chemically bonded phases).  Poor reproducibility can also result if thermostating is inadequate.  Both pH and pKa are temperature dependant which may alter chromatographic parameters.  When working at elevated temperatures the flow rate may need to be increased to maintain the optimum linear velocity due to the decrease in solvent viscosity.  At increased pressure, the boiling point of a solvent will also increase (Figure 23).  Therefore, under HPLC conditions the boiling point of common solvents will be much higher than usual, allowing for column temperatures that exceed the normal boiling point of the solvent to be used.  However, to do this a backpressure regulator must be installed at the end of the system, i.e. after the UV detector, to suppress boiling of the mobile phase in the column. 7  The increase in solvent boiling point with pressure follows from the integrated Clausius-Clapeyron equation (Equation 1).8  A liquid boils when its vapour pressure exceeds the pressure acting on it, therefore, the column pressure must be kept above the vapour pressure of the mobile phase to prevent it from boiling.  A back-pressure regulator of approximately 30 bar is usually sufficient to keep typical mobile phases from boiling. 9

 

Figure 21: The effect of temperature on selectivity.

 

Figure 22: Van Deemter curves for various temperatures.

 

Figure 23: Effect of pressure on boiling point of MeCN and MeOH.

 
 



Where:



p* = vapour pressure of the liquid at a temperature of T*

p = vapour pressure at the temperature T

ΔHvap is the enthalpy of vapourization of the liquid

R = gas constant

 
 

Table 7: Enthalpy of vapourisation (ΔHvap) for various solvents.  Note: Enthalpies are generally fairly constant with temperature. 10

 
 

Analytical Chemists

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In-line filters are used to trap stray particles that could be injected with the sample which could block and damage the analytical column – particulate material in the sample or from wear and tear of pump seals or rotor injector.  In-line filters contain a frit that is most commonly 0.5 μm in porosity (0.2 μm are used for UHPLC applications).  The porosity should be similar to, or smaller than, the frit porosity of your analytical column so that the frit catches any particles that would be likely to block the column frit.  Two designs of in-line filter are available (Figure 24).

  1. Re-usable holder that can be disassembled to access the frit
  2. Disposable unit that screws directly into the end of the column

They should be mounted directly downstream from the autosampler.  The frit should be changed when the system backpressure increases significantly (+25%).

 

Figure 24: Re-usable in-line filter with replaceable frits (left) and disposable single piece in-line filters.

 
 

Guard columns are used to extend the life of an analytical column (Figure 25).  Compounds that would irreversibly bind to the column stationary phase will bind to the guard column instead.  They can also be used at extremes of pH, especially low.  The mobile phase will become saturated with stationary phase from the guard column and as such does not hydrolyse the column stationary phase.  The guard column should be chosen with a stationary phase that matches the analytical column so that it will interact with analytes in the same way as the analytical column. 

A guard column can also act as an in-line filter as it has frits at both ends.  It is fitted before the analytical column and should be changed after a pre-determined number of sample injections or a time period.  It is harder to judge when guard columns should be changed in comparison to in-line filters as there is no clearly designed physical manifestation of failure.  It is often recommended that an in-line filter is used in conjunction with a guard column.

 

Figure 25: Guard column.

 
 

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Silica Purity

Where the silica contains metal ions from the manufacturing and particle sizing production steps, in particular iron and aluminium, the acidity of the lone silanols is further increased leading to an increase in peak tailing with polar and ionisable compounds (Figure 26 and Table 8).  Further, analytes which are capable of chelating will also interact with the column in this way leading to additional unwanted secondary retention and more pronounced peak asymmetry (Figure 27).  Manufacturers use various silica washing processes to remove metal ions from silica and also to avoid the electrolytic decomposition of metal ions from the stainless steel column tubing and metal column end frits used to retain the packing within the column.

 

Figure 26: Chromatogram exhibiting peak tailing.

 

Figure 27: Interaction of phathalic acid with metal ion on the silica surface.

 

Table 8: Comparison of metal ion content in Type A silica vs. Type B silica.

 
 

Particle Shape

Two basic shapes of silica particle are available, ‘irregular’ and ‘spherical’ (Figure 28).  Milling silica xerogels 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 due to the way in which the particles pack into the HPLC column.  With irregular materials the packing homogeneity is much poorer leading to an exaggeration of eddy diffusion and mass transfer effects.  The use of columns for high flow (pressure) applications and mechanical shock can cause the irregular particles to sheer forming smaller sub-particles known as ‘fines’ which can migrate through the column eventually blocking the outlet frit.  Therefore, spherical particles are generally favoured.

 

Figure 28:  Silica particle shapes.

 
 

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 (Figure 29 and 30).  More efficient peaks can be achieved at elevated flow rates, leading to a reduction in analysis time accompanied, potentially, with 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.  Figure 29 shows the relationship between particle size, flow rate (linear velocity) and plate height for columns packed with various silica particle diameters.

It may seem that the smallest diameter particles will be the obvious choice for high efficiency separation; 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 8.

Where any of the numerators increase, flow (F) and Column length (L) for instance, a resultant increase in back pressure is observed.  Where any of the denominators decrease, column radius (r) and 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.

 

 

Figure 29: Van Deemter curves for various particle sizes.

 
 

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Figure 30: Effect of particle size on band broadening and resolution.

 
 

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 31).

Any particle size stated for an HPLC column (e.g. 5mm) 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.

 

Figure 31: Particle size distribution.

 
 

pH Stability

The chemical properties of silica are also of paramount importance and in particular are:

  • the stability of the silica support material to hydrolytic attack at elevated pH
  • the mode by which the surface has been chemically modified
  • the stability of the modifying ligand at low pH
  • secondary treatment of the remaining unreacted silica surface (endcapping)

The traditional working mobile phase pH range for silica based columns is 2.5 – 7.  At very low pH hydrolysis of the silyl ether linkage occurs.  This will be accompanied by a deterioration of peak shape and loss of efficiency.  Bonded phases with bulky side groups can be used to protect ether linkages (Figure 32).  The use of di-functional binding has also been utilised to manufacture materials with greater stability and extremes of pH.

 

Figure 32: Schematic of a silica surface showing the silyl ether linkage (left) and bulky side chains used to protect silyl ether linkages (right).

 
 

At high pH (> 7.0) the silica surface itself may hydrolyse resulting in the formation of fines.  Again a loss of peak shape and efficiency will be observed, along with increased system backpressure due to plugging of the interstitial gaps with the fines.  Hybrid silica, multiply bonded silica, or polymeric non-silica based materials can be utilised for applications that require high pH (Figure 33).

 

Figure 33: Examples of hybrid silica, multiply bonded silica, and polymeric non-silica based materials.

 
 

End-capped Silica

Fully hydroxylated silica will have a Silanol surface concentration of ≈8 μmol/m2.  Following chemical modification > 4 μmol/m2 of these silanols may remain even with optimum bonding conditions due to steric limitations of the modifying ligands.  This indicates that on a molar basis there are more residual silanols remaining than actual modified ligand.  In order to remove some of these residual silanols, an end-capping process may be undertaken.  Short chain, less sterically hindered hydrophobic ligands, (commonly trimethyl / tri-iodo chlorosilanes or similar), are chemically reacted with the remaining unbounded silanol species, leading to improved peak shape with polar and ionisable analytes (Figure 34).  This is only a partial solution, however, as not all of the surface silanol groups will be reacted even using sterically very small liagnds and optimised bonding conditions, also the end-capping ligand is prone to hydrolysis especially at low pH.

Acidic (lone) surface silanol groups give rise to the most pronounced secondary interactions with polar and ionisable analytes (Figure 35).  Modern silica is designated as being Type I or Type II, 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 34: End-capped silica phase.

 

Figure 35: Various surface silanol conformations.

 
 

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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:

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.  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.

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)

ΔΦ = change in volume fraction of organic (final %B – initial %B)

VM = column void volume (πr2L0.68)

 

Increasing column length in isocratic separations can increase efficiency (N), increase retention, and therefore, affect resolution (Figure 36).  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 37), 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 36: Effect of column length on efficiency and resolution in isocratic separations.

 

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Figure 37: Effect of column length on analyte elution order in gradient separations.

 
 

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Further in depth information on the operation of the detectors shown below can be found in CHROMacademy. 6

UV Visible

Advantages

  • Sensitive (0.5 - 1.0 ng detected)
  • Quantitative (Beer’s Law)
  • Suitable for gradient elution
  • Responds to most analytes

Disadvantages

  • Spectra are not highly specific
  • Absorption dependant on solution conditions
  • Relative response factors need calculated for impurity quantification

Applications

  • Small organic molecules
  • Biological macromolecules (peptides)
  • Any UV/visible active analyte - especially conjugated systems, analytes containing heteroatoms or metal ions
 

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Diode Array Detector

Advantages

  • Simultaneous multi-wavelength detection
  • Fast scan speeds
  • Interfering peaks can be eliminated
  • Peak purity can be measured

Disadvantages

  • Susceptible to lamp fluctuations
  • Peak purity is only indicative and needs to be carefully set up and operated
  • Sensitivity lower than single wavelength detector

Applications

  • Peak purity
  • Library searching for structural elucidation
 

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Fluorescence Detector

Advantages

  • Highly specific (only a minority of molecules fluoresce)
  • Very sensitive (10 - 100 pg)

Disadvantages

  • Most molecules do not naturally fluoresce
  • Affected by temperature fluctuations (increased molecular collisions, decrease in potential energy)
  • Excitation and emission wavelengths may differ from instrument to instrument

Applications

  • Biological applications (fluorescent labelled radionucleotides)
  • Food additives (tert-butylhydroquinone)
 

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Refractive Index Detector

Advantages

  • Good for the detection of non-ionic compounds, analytes which do not have a UV/visible chromophore, and molecules that do not fluoresce
  • The only universal detector in routine HPLC operation

Disadvantages

  • Not suitable for gradient analysis (eluent composition must remain constant throughout analysis)
  • Affected by changes in flow rate and temperature
  • Long equilibration times required

Applications

  • Food analysis (carbohydrates)
  • GPC
 

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Evaporative Light Scattering 4

Advantages

  • Analysis of non-volatile analytes
  • Universal
  • Not influenced by UV or refractive index properties or changing eluent composition - gradient compatible

Disadvantages

  • Mobile phase must be volatile
  • Detector response is a complex function of the injected amount of analyte
  • Small volatile molecules are difficult to analyse

Applications

  • Drug discovery
  • Natural product development
  • Food and beverage
 
 

Charged Aerosol Detector 5

Advantages

  • Compatible with gradient elution
  • Universal
  • Good sensitivity (ng quantities)
  • Wide dynamic range

Disadvantages

  • Analyte should be non-volatile
  • Volatile mobile phase is required

Applications

  • Lipid analysis
  • Formulation development
  • Stability analyses
 
 

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  1. //www.sanderkok.com/techniques/hplc/eluotropic_series_extended.html.

  2. Dolan, J.W., and Snyder, L.R.; Troubleshooting LC Systems, Humana Press, Clifton, 1989, p. 85.

  3. //www.chemicalize.org.

  4. Young, C. S.; Dolan, J. W.; LCGC Europe 2003, 2-5.

  5. Swartz, M.; Emanuele, M.; Awad, A.; Hartley, D.; LCGC 2009, ESA, Inc. Chelmsford, Massachusetts.

  6. //www.chromacademy.com/frameset-chromacademy.html?fChannel=0&fCourse=1&fSco=35&fPath=sco35/hplc_3_5_1.asp.

  7. Jensen, D.S.; Teutenberg, T.; Clark, J.; Linford, M.R.; LCGC North America 2012, 30, 850-863.

  8. P. Atkins and J. De Paula, in Atkins' Physical Chemistry, (W.H. Freeman and Company, New York, New York, 2006).

  9. C.V. McNeff, B. Yan, D.R. Stoll, and R.A. Henry, J. Sep. Sci. 30(11), 1672–1685 (2007).

  10. CRC Handbook of Chemistry and Physics, D.R. Lide, Ed. (CRC Press, Boca Raton, Florida, 2004).

 
 

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There are numerous factors that affect the quality of your HPLC separation; including mobile phase composition, temperature, ionic strength, pH, column dimensions, and stationary phase and support material characteristics.  These can all be manipulated to change and improve your HPLC separations.  However, what are the changes we should expect?  Will retention improve if another solvent is used and would we expect selectivity to change?  Will certain analytes elute in the same order if the mobile phase pH, ionic strength, additives or temperature are changed?  In this Essential Guide we will evaluate many of the aspects of the HPLC process giving you useful practical information on how to use these parameters to alter your HPLC separation resulting in better and more robust chromatography.  We will also discuss the use of in-line filters, guard columns, and the most appropriate detector for your application.

Dr. Paul Ferguson
Associate Principal Scientist
Astra Zeneca

Scott Fletcher
Technical Manager
Crawford Scientific

Key Learning Objectives:

  • Understand and evaluate each part of your HPLC method
  • Understand how each of the factors that affect your HPLC separation can be used to alter analyte retention and selectivity
  • Learn why and when you should use in-line filters or guard columns
  • Understand how impurities, free silanol groups, or end-capping on the silica support material  can impact on your HPLC separation – both positively and negatively
  • Understand the common HPLC detectors, their advantages and disadvantages, and their application areas