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The CHROMacademy Essential Guide to Troubleshooting your HPLC Chromatogram Part II
Selectivity, Resolution and Baseline Issues
Live Webcast - Thursday 19th July 2012, 11:00am EDT, 4.00pm BST.

This session examines the common causes of changes in selectivity, loss of chromatographic resolution and baseline issues – such as splitting, fronting, tailing and shouldering. Strategies for problem identification and calculation of critical chromatography performance indicators will also be discussed...

Topics Include

  • Identify common causes of problems within your chromatography
  • Learn logical strategies for rapid instrument and separation troubleshooting
  •  Explore common problems with
    •  Mobile phase composition
    • Temperature and pH
    • Column modification and age
    • Sample solvent strength
    • Sample preparation
    • System operating parameters
  • Understand issues with existing problematic methods
  • Instigate preventative steps in order to minimize problems in future methods

Find out more about this Month's Essential Guide Webcast »

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Troubleshooting your LC Chromatogram Part II
Selectivity, Resolution and Baseline Issues

This essential guide is the second and final installment in the ‘Troubleshooting your HPLC Chromatogram’ series and examines the common causes of changes in selectivity, resolution and baseline issues. Strategies for problem identification and calculation of critical chromatography performance indicators will be discussed. The degree to which these parameters are affected by mobile phase composition, temperature, sample solvent strength, and many other variables will be investigated alongside strategies for isolating the precise cause of the problem. Corrective and preventative actions will be described for the major causes of the symptoms observed.

 

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Step 1. Select your chromatographic symptoms.

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Step 3. We return a list of possible causes ranked by our industry experts.

The troubleshooter provides a concise summary of the problem and recommends solutions - supported by over 1000 references, feature articles and CHROMacademy content written by experts in HPLC.

 
 
 
 

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Overview

The concept of chromatographic parameters was introduced in Troubleshooting your HPLC Chromatogram – Part I where we focused on retention (capacity) factor, efficiency and asymmetry.

Just to re-cap, under ideal conditions, each peak should be narrow and symmetrical (Gaussian distribution). [1-8] A visual inspection of the chromatogram is often enough to highlight problems with a separation, however sometimes we need to be more quantitative to describe the extent of the issue.

To this end, a set of parameters, the most popular being retention time, retention factor, selectivity, efficiency, asymmetry and tailing factor, have been developed and in this Essential Guide we shall be focusing on selectivity (separation factor) and resolution.

 

Separation Factor or Selectivity (α)

The separation factor (IUPAC discourage the usage of selectivity [9]) refers to the ability of a chromatographic system to distinguish between two sample components. It is measured as a ratio of retention (capacity) factors (k’) of the two peaks in question and can be visualized as the distance between the two peak apices – i.e. how well they are separated.

 

Where: 

t0 is the time taken for an un-retained analyte to travel through the column and system and be detected
tr is the retention time of the analyte of interest
k is the retention factor of the analyte of interest

Figure 1.  Determination of Selectivity.

 

As separation factor is a ratio of retention factors, the largest divided by the smallest; by definition it must always by greater than one. An α value equal to one indicates that there is no separation of analytes and they are co-eluting (i.e. their retention times are identical). Larger values of alpha indicate that the apices of the two peaks are farther apart but does not guarantee they are well resolved – see below.

 
 

Resolution

Resolution is most important factor of any HPLC separation, whether it be for analyte identification (qualification) or for determining the amount of a specific analyte, or analytes, present (quantification). If you cannot accurately resolve your peak of interest from other contaminant peaks also present it is not possible to unequivocally assign a positive / or negative identification result. Likewise, it is also impossible to accurately determine the amount of a specific analyte present in this instance either.

The best description of chromatographic resolution is ‘optimum resolution in the minimum time’.[10] A resolution value of greater than or equal to 1.5 is generally desired as this confers ‘baseline resolution’ – following the complete elution of the first analyte of interest, the baseline is recreated before the second analyte of interest is initially detected.

In most instances the resolution between a ‘critical peak pair’ (closest eluting peaks in any separation) will decrease over time and it is therefore recommended that a cushion is built into the method and more typically resolution values of 1.7 or 1.8 or desired prior to validation and mainstream usage.

 

Where:           

tris the retention time of the analyte of interest
Wb is the width of the tangential lines where there dissect the baseline  

Figure 2.  Conventional Determination of Resolution.

 

The above equation requires the chromatographic peaks to be perfectly symmetrical and Gaussian, as this rarely the case in HPLC, due to secondary interactions, the alternative method for calculating selectivity is often advised – through experience and by regulatory agencies [11]

 

Where:           

b0.5 is the width of the analytes of interest at half their height

Figure 3.  Alternative Determination of Resolution.

 
 

The figure below demonstrates the resultant chromatogram at various resolution values.

Figure 4.  Visual representation of resolution.

 
 

Fundamental Resolution Equation

It is essential to briefly explore the fundamental resolution equation as it demonstrates the extent by which retention factor, efficiency and separation factor impact on and influence resolution.

Equation 1.  The Fundamental Resolution Equation.

 

As can be seen from the above equation each of the three chromatographic parameters we have discussed during Parts I and II of this ‘Troubleshooting Your HPLC Chromatogram’ series all influence resolution.  The extent to which they drive resolution is different and is highlighted in Figure 5 below.

 

Figure 5.  Individual Effect of Separate Chromatographic Parameters.

 

From the above graph it can be observed that separation factor has the largest impact on resolution, followed by efficiency with retention factor having the least influence.  It should be noted that whilst this is the case, all chromatographic parameters need to be explored individually when a loss in resolution is observed – a reduction in retention factor combined with a loss of efficiency can have a more profound affect that a change in selectivity.

 
 

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There are many factors that will influence separation factor and whilst most were initially discussed in part I, it is important for us to reexamine them but from the perspective of affecting separation, as appose to retention factor and / or efficiency.

 

Mobile phase Considerations

The role of the mobile phase in reversed phase chromatography was discussed during Troubleshooting your HPLC Chromatogram – Part I and the reader is directed to following the link below if they wish to revisit the topic.

Troubleshooting your HPLC Chromatogram – Part I

 

Amount of Organic Solvent (Strength)

The physico-chemical properties of the solvent, eluotropic series and isoeluotropism were again described during Part I.  In the following section we shall discuss how the strength of the mobile phase, amount of organic solvent, affects separation factor.  Not all analytes are affected to the same extent by changes in the strength of the mobile phase, albeit with the same solvent employed.  The degree by which analytes are affected by mobile phase strength is governed by its Shape Selectivity value, S.  An analytes S value is the slope of the line as generated by plotting the natural log of its retention time against the amount of organic – see later.  As a rule of thumb bigger analytes are affected to a much larger extent as compared to smaller analytes. [12]

Figure 6 demonstrates this phenomenon whereby the critical peak pair is 2 and 3 at 50% acetonitrile, however at 40% acetonitrile it is 4 and 5.

 
 

Figure 6.  Analytes affected to a differing extent by changes in solvent strength.

 
 

Type of Organic Solvent

The terms of eluotropic strength and isoeloutropism in relation to retention factor were introduced during Part I; we shall be examining how different solvents affect separation factor.  The degree by which analyte molecules can interact with the organic solvent are largely due to dipole interactions and hydrogen bonding.  This concludes that selective solvent-sample interactions are governed by solvent dipole moment, solvent basicity (proton acceptor) and solvent acidity (proton donor).   

For ease of visualizing how different solvents can affect separation factor, solvents are plotted based on their relative interactive strengths - this is traditionally referred to as the ‘solvent selectivity triangle’. [13]

 

Figure 7.  Solvent Selectivity Triangle.

 

Depending on analyte properties, altered retention, and therefore changes in separation factor, can be promoted even when changes in organic solvent are made in an isoeluotropic fashion.

 

Figure 8.  Isoeluotropic Separation Factor Changes.

 

For any readers wishing to investigate this phenomenon from a method development strategy they are directed to the following section within CHROMacademy:

LC/HPLC, The Theory of HPLC, Reversed Phase / Partition Chromatography, Changing the Organic Modifier

 
 

Mobile Phase pH

The pH is a key parameter of the mobile phase and is adjusted in order to ensure that all analyte molecules are in one ionic form. We have previously explored how pH affects the ionization state of an analyte, acidic or basic, and surface silanols, and the affect this can have on peaks shapes. We also discussed the 2 pH rule. [14]

Unadjusted mobile phase pH can also affect separation factor and this is demonstrated below whereby the changes in retention of a mixture of seven weakly acidic and neural analytes at differing pHs is plotted.  Please also note that not all acidic or basic analytes will be affected to the same extent due to their individual pKa’s and size etc.

 

Figure 9. pH changes influencing Separation Factor  

 
 

It should also be noted that measurements of the mobile phase should also be carried out using a suitable pH meter and a multipoint calibration on the aqueous portion of the mobile phase only.  The mobile phase should be changed and / or checked regularly as changes in pH can occur over time due to ingress of CO2 for instance. [15]

 

Figure 10.  Small Changes in pH Affecting Separation Factor

 
 

Mobile Phase Additives

The importance of accurately adjusting the pH of the aqueous portion of the mobile phase has been made above and the resultant changes in separation factor highlighted if inaccurately adjusted.  However, without the addition of a suitable buffer the pH will rapidly drift and fairly extreme changes in separation factor can be observed.  A buffer is the conjugate salt of the acid or base being used in order to adjust pH and a full list and appropriate buffers can be found in Part I. [14]

The concentration buffer is sometimes referred to as the ‘Goldilocks’ value – if it is too strong it can lead to precipitation which can cause hardware issue and if it is too weak then it can lead to unsatisfactory pH control. [15]

 

Figure 11.  Incorrect Buffer Concentration Affect.

 
 

Buffers are not the only component that is commonly added to mobile phases.  Although reducing in popularity there are still many applications that exist, and are being developed, that employ an ion-pair reagent.  Ion Pair Chromatography (IPC) is employed when ion-suppression is not an option – when analyzing strong acids or bases or mixtures of weak acids and bases. [16]

In a similar fashion to that described above, the concentration of the ion-pair reagent needs to be just right and should be empirically determined during method development.  The amount of ion-pair reagent bound to the stationary phase will reach a maximum value at its equilibrium.  Excess reagent added will lead to a LOSS in retention, and therefore separation factor, due to excess counter-ion present and too little will lead to equilibrium never fully being reached and changes in separation will follow.

 

Figure 12.  Incorrect Ion-Pair Reagent Concentration.

 
 

Mobile Phase Temperature

The main preface for the adjustment of the temperature of the separation is due to the associated reduction in operating back pressure – especially when long or reduced particle diameter columns are utilized. [17]

Care should be exercised when adjusting separation temperatures as it can lead to quite significant changes in selectivity.  There are two main instances where changes in separation factor can be observed. 

The first is only applicable to ionizable analytes when operating in ion suppression and ion pair modes.  We have previously discussed how the correct pH needs to set and suitable buffer and / or ion-pair reagent added.  Even quite small changes in temperature can affect the degree of ionization of an analyte and adjust the equilibrium in an ion-pair separation. [18]

The second instance where an analytes separation factor can change with temperature is when the analytes differ in shape and / or size – hydrodynamic volume. [19-20]

 

Figure 13.  Temperature Affecting Selectivity.

 
 

HPLC Column Considerations

Support Material

The physico-chemical properties of silica as the support material, in particular the nature of surface silanols and the degree of end-capping, were discussed in a Part I and the reader is directed to review that article for a deeper discussion. [14]

A point necessary to reinforce is that when changing to a different manufacturer of column, changes in separation factor can be observed even when the stationary phase is identical e.g. C18.  It is worth pointing out however that such changes are not always negative and the use of the most appropriate column available is encouraged as long as the impacts of changes in the support, and potential changes selectivity, are considered. 

 
Figure 14.  Basic and polar analytes analyzed using Type I (Hypersil C18) and Type II (Hypersil BDS C18) silica.
 
 

Stationary Phase

An explanation of the role of the stationary phase and common types available for reversed phase chromatography was given in the Part I. [14]

One of the most powerful tools a chromatographer has at their disposal in order illicit changes in separation factor is to change their stationary phase.  Wholesale changes in order of elution can be produced by differing the hydrophobicity / polarity of the stationary phase. 

 
Stationary Phase Affecting Separation Factor

Figure 15.  Stationary Phase Affecting Separation Factor.

 

This topic has been discussed in great detail previously - please see the links below for greater explanations;

Column Selection for Reversed Phase HPLC

Developing Better Methods for Reversed Phase HPLC Part 2

HPLC Column Characterization and Selection Essential Guide

LC/HPLC, The Theory of HPLC, Column Chemistry

 
 

Hardware Dimensions

We have already discussed how analytes are affected to different extents by changes in the sample solvent strength, this can also lead to changes in separation factor when the length of the column is changed when performing a gradient analysis.  This is due to the differing speeds at which different analytes travel and the distance over which they continue to interact with the column for and is highlighted in Figure 8 below.
 

Figure 16.  Analytes affected to a differing extent by changes in solvent strength and column length.

 

Under isocratic conditions, increasing (or decreasing) the column length will have a directly proportional affect in analyte separation factor and this phenomenon will not be observed.

 
 

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For ease of identifying an assignable cause and therefore identifying the necessary corrective actions for all associated baseline issues, all potential causes and associated solutions have been included in our interactive HPLC Troubleshooter.

 

Click the "Troubleshoot Now" button on the right to get started >>

 

If you're not familiar with our HPLC Troubleshooter - just watch the short movie below and you will be up and running (and solving your problems) in no time.

 
 
 
 

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  1. “Band Broadening” from “The Theory of HPLC” of CHROMacademy
  2. “Practical HPLC Troubleshooting & Maintenance” Crawford Scientific 2010
  3. “HPLC Troubleshooting & Maintenance” Crawford Scientific 2008
  4. G. Guiochon, in C. Horváth (Editor), High Performance Liquid Chromatography, Advances and Perspectives. Vol. 2, Academic Press, New York, 1980
  5. “Band Broadening” from the “HPLC Channel”
  6. Dao T.-T. Nguyen, Davy Guillarme, Sabine Heinisch, Marie-Pierre Barrioulet, Jean-Louis Rocca, Serge Rudaz, Jean-Luc Veuthey. “High throughput liquid chromatography with sub-2μm particles at high pressure and high temperature” Journal of Chromatography A, 1167 (2007) 76–84
  7. A.-M. Siouffi. “About the C Term in the Van Deemter’s Equation of Plate Height in Monoliths” Journal of Chromatography A, 1126 (2006) 86–94
  8. Alain Bertho and Alain Foucault. “Comments on Van Deemter Plot in High Speed Countercurrent Chromatography” Journal of Liquid Chromatography & Related Technologies. 2001; 24(13); Pp 1979 – 1985
  9. “UPAC. Compendium of Chemical Terminology, 2nd ed. (the "Gold Book"). Compiled by A. D. McNaught and A. Wilkinson. Blackwell Scientific Publications, Oxford (1997). XML on-line corrected version: http://goldbook.iupac.org (2006-) created by M. Nic, J. Jirat, B. Kosata; updates compiled by A. Jenkins. ISBN 0-9678550-9-8
  10. “Chromatographic Parameters” from “The Theory of HPLC” of CHROMacademy
  11. USP35-NF30, General Chapters, General Tests and Assays, Physical Tests and Determinations, <621> Chromatography, Definitions and Interpretations of Chromatograms
  12. “Understanding Gradient HPLC”, December 2010 Essential Guide, CHROMacademy
  13. “Reversed Phase / Partition Chromatography” from “The Theory of HPLC” of CHROMacademy
  14. “Troubleshooting your HPLC Chromatogram – Part I”, May 2012 Essential Guide, CHROMacademy
  15. “HPLC Troubleshooting – Eluents and Solvent Delivery Systems”, January 2012 Essential Guide, CHROMacademy
  16. “Ion-Par Chromatography” from “The Theory of HPLC” of CHROMacademy
  17. “High Efficiency HPLC separations - Sub 2µm (UHPLC) fully porous particles and Superficially Porous Silica particles (SPS)”, September 2010 Essential Guide, CHROMacademy
  18. Lloyd R Snyder, Joseph L Glajch and Joseph J Kirkland, Practical HPLC Method Development Chromatography, Wiley-Interscience Publication, 1988, p. 46
  19. L. R. Snyder, J. Chromatogr., 179 (1979) 167
  20. J. Chimieloweic and H. Sawatzky, J. Chromatogr. Sci., 19 (1979) 24
 
 

 

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In this session, Dr. Dafydd Milton  (Product Manager, Thermo Fisher Scientific) and Scott Fletcher (Technical Manager, Crawford Scientific), examine the common causes of changes in selectivity, loss of chromatographic resolution and baseline issues – such as splitting, fronting, tailing and shouldering.

Strategies for problem identification and calculation of critical chromatography performance indicators will be discussed. The degree to which these parameters are affected by mobile phase composition, temperature, column modification and age, sample solvent strength, sample preparation, system operation and many other variables will be investigated alongside strategies for isolating the precise cause of the problem.

Corrective chromatographic and / or HPLC system actions as well as key preventative maintenance operations will be described for the major causes of the symptoms observed.  Strategies for identifying the causes of problems with current methods and best practice for future method development are also included.

Scott Fletcher
Technical Manager
Crawford Scientific
Dafydd Milton, Ph.D
Product Manager – LC and LC/MS Columns
Thermo Fisher Scientific

Who Should Attend:

  • Anyone who uses HPLC equipment and who wants to improve their separation knowledge and troubleshooting and instrument maintenance skills

Key Learning Objectives:

  • Learn how to quickly and accurately identify the common causes of
    • Changes in selectivity
    • Loss of chromatographic resolution
    • Baseline issues
  • Understand when and how to employ the correct calculations in order to identify / quantify chromatographic issues
  • Appreciate how these parameters can be affected by
    • Mobile phase composition
    • Temperature and pH
    • Column modification and age
    • Sample solvent strength
    • Sample preparation
    • System operation
    • plus many other
  • Explain how to carry out chromatographic and / or system maintenance in order to alleviate the afore mentioned symptoms in existing methods
  • Propose an effective and accurate preventative approach to both chromatographic and system parameters in order to minimize the effects for all future methods