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The CHROMacademy Essential Guide
HPLC Troubleshooting Separations Retention Time, Efficiency and Peak Shape

This essential guide examines the common causes of retention time drift, loss of chromatographic efficiency and peak shape 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, 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. In Part II, July’s Essential Guide, we will focus on Selectivity, Resolution and Baseline issues.

 

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We developed the CHROMacademy HPLC Troubleshooter with busy chromatographers in mind. In 3 simple steps we can help you overcome your instrument, separation and quantitation issues.

Step 1. Select your chromatographic symptoms.

Step 2. Select your instrument symptoms.

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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Every separation in HPLC produces a chromatogram (we hope!).  Broadly speaking, every analyte is represented by a single peak eluting at a particular time, according to its various physicochemical properties.  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 retention time, retention factor and efficiency

Retention Time (tR)

The analyte retention time can be defined as the elapsed time between sample injection and the time of elution of the peak maximum of that analyte.  The non-retained mobile phase elutes at a time t0, which is known as the ‘hold up time’ or ‘dead time’.

 

 

Figure 1. Retention time interpretation.

 

There are several ways to determine t0 including:

  • The time at the baseline disturbance seen due to differences in absorbance or refractive index as the injection solvent passes through the detector
  • Retention time of Uracil (reverse phase)
  • Retention time of Hexane (normal phase)
 
 

Retention or Capacity Factor

Isocratic Operation

The retention factor (k’), also known as capacity factor, is a means of measuring the retention of an analyte on the chromatographic column.

A high k’ value indicates that the sample is highly retained and has spent a significant amount of time interacting with the stationary phase.
 
Figure 2. Determination of Retention Factor (k’).

The retention factor is equal to the ratio of retention time of the analyte on the column to the retention time of a non-retained compound. The non-retained compound has no affinity for the stationary phase and elutes with the solvent front at a time t0 .

Retention factor is independent of some key variable factors including small flow rate variations and column dimensions. Because of this, it is a useful parameter when comparing retention of chromatographic peaks obtained using different HPLC systems and when converting conventional HPLC methods to UHPLC systems (and vice versa!).  Chromatographers typically like to keep k’ values between 1 and 10 for good HPLC separations or 0.5 and 5 for methods developed on more modern UHPLC systems where highly efficient separations are the norm.  Please do note however that there is the risk of peak overlap (poor resolution) when separating a moderate number of analytes at low k’ values.

 
 

Gradient Operation

One cannot assign a fixed k’ value to a compound when gradient elution is applied; k’ is the retention coefficient and changes during gradient elution.

The equation for the gradient retention factor (k*) takes the form:

Gradient retention factor (k*) is difficult to visualize as it differs from its isocratic counterpart (k’) and resembles more the profile of the gradient elution. It is effectively defined as the retention factor for an analyte that has migrated half way down the HPLC column.

The Retention Factor as a Means to Evaluate the State of an HPLC Separation

The retention factor of peaks within a chromatogram can aid in the identification of many problems that are associated with HPLC system components.  Variable retention factors may result from changes in mobile phase flow rate, mobile phase composition, column stationary phase, and temperature.

 
 

Efficiency

The efficiency of a chromatographic peak is a measure of the dispersion of the analyte band as it travelled through the HPLC system and column. In an ideal world, chromatographic peaks would be pencil thin lines –however, due to dispersion effects the peaks take on their familiar ‘Gaussian’ shape.

The plate number (N) is a measure of the peak dispersion on the HPLC column, reflecting the column performance. N is derived from an analogy of Martyn and Synge who likened column efficiency to fractional distillation, where the column is divided into Theoretical Plates.

Each plate is the distance over which the sample components achieve one equilibration between the stationary and mobile phase in the column. Therefore, the more ‘theoretical’ plates available within a column, the more equilibrations possible and the better quality the separation.

Higher values for the Plate Number (N) are expected for subsequent peaks within a chromatogram.  Later eluting peaks that look broad in comparison to early eluters may have a higher plate count.  If this is not the case then your system contains a large extra-column dead volume which is dominating the diffusion process!

For a fractionating tower of a given length (L), the higher the number of plates, the lower will be the distance between each plate, shown as plate height in the diagram.  Therefore, for high efficiency separations, the plate number (N) will be high and the plate height (H) low.  Note that plate height is often called – ‘Height Equivalent to a Theoretical Plate (HETP).’  Modern columns which employ are reduced particle size, sub 2µm, enjoy an increased plate number (N) without increasing the length of the column (L) due to the reduction in distance between active sites, plate height (H)

These two terms are related through the expression:  H = L / N

 

Figure 3.  Factional distillation model of efficiency theory.

 

The number of theoretical plates is often used to establish the efficacy of a column for a given method.  The method developer may decide that a given method is no longer valid when the plate number falls below a predetermined value.  At that time, the column would be replaced with a new one.

 

Figure 4.  Comparison of two chromatograms with the same selectivity and different efficiency (and resolution).

 
 

Efficiency as a Means to Evaluate the State of an HPLC Separation

Peak efficiency within a chromatogram can aid in the identification of many problems that are associated with HPLC system components.  Reduced efficiency may result from incorrect or mobile phase deterioration, incorrect or deteriorated column, increased dead volume, addition of a disproportionate length of tubing or wider I.D. tubing, etc.

Peak Asymmetry

In the ideal world all chromatographic peaks would be symmetrical (or Gaussian).

However due to the effects of instrument dead-volume, adsorptive effects of the stationary phase and the quality of the column packing, peaks may often show a tailing behavior. Tailing describes a peak whose tail portion (distance ‘B’ in the diagram) is wider than the front portion (distance ‘A’ in the diagram). Also, if the sample concentration is too high or if the column is damaged and contains ‘channels’ then a fronting peak shape may occur.

There are different ways of calculating the amount of peak tailing (or fronting).  However, one of them, peak asymmetry (As), is probably the most commonly used, and is defined as:

Where A and B are measured at 10% of the peak height.

 

Figure 5.  Determination of Peak Asymmetry (AS) and examples of good and poor peak shape.

 
 

The US Pharmacopeia (USP) recommends the use of a different measurement, the tailing factor (Tf), which has been defined as:

Where A and B are measured at 5% of the peak height.

In general terms, it doesn’t matter which measurement parameter is used (either As or Tf), as long as we are consistent in our measurement and are aware of the ‘acceptable’ values for each measure.  The definition of ‘acceptable’ peak tailing or fronting for the two different measurements is  roughly the same  - see Table 1.

 

Table 1.  Asymmetry and tailing factor values comparison.

Parameter Acceptable Peak Unacceptable Peak
Peak Asymmetry (As) 1.0 1.1 1.3 1.7 2.5 3.8
Tailing Factor (Tf) 1.0 1.1 1.2 1.4 1.9 2.9
 
 
 

Peak Distortion as a Means to Evaluate the State of an HPLC Separation

Peak asymmetry and tailing factor can aid in the identification of many problems that are associated with HPLC system components. 

Peak shape problems may not adversely affect the resultant data, determine if the analytical results are truly compromised before taking actions.  Peak tailing and fronting are the most common forms of peak distortion in HPLC.

Basically, the primary cause of peak distortion is due to the occurrence of more than one mechanism of analyte retention; however, there are other reasons that account for this condition (mobile phase issues, column degradation, injection problems etc.).

 


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In order to effectively troubleshoot our separations for retention time, efficiency and peak shape issues, we need to understand the basics of retention and separation in HPLC.  This section presents a brief overview of the controlling variables and how they might be manipulated to change chromatographic behavior.

Reversed Phase solvents

Reversed phase mobile phases usually consist of water and an organic solvent often called a ‘modifier’. When ionisable compounds are analyzed, buffers and other additives may be present in the aqueous phase to control retention and peak shape.[9, 10, 11]

Chromatographically, in reversed phase HPLC, water is the ‘weakest’ solvent. As water is most polar, it repels the hydrophobic analytes into the stationary phase more than any other solvent, and hence retention times are long – this makes it chromatographically ‘weak’. The organic modifier is added (usually only one modifier type at a time for modern chromatography), and as these are less polar, the (hydrophobic) analyte is no longer as strongly repelled into the stationary phase, will spend less time in the stationary phase, and therefore elute earlier. This makes the modifier chromatographically ‘strong’ as it speeds up elution.

As progressively more organic modifier is added to the mobile phase, the analyte retention time will continue to decrease.

The values alongside the solvent chemical structures in Figure 6 represent the Snyder polarity index value.  The more polar a solvent, the ‘weaker’ it is in terms of eluotropic strength in reversed phase HPLC.  

 

 
 
Figure 6.  Solvents in reversed phase HPLC.   Figure 7.  Properties for selected HPLC solvents.
 

Use Figure 7 to assess the relative physic-chemical properties of the various solvents one might use as organic modifiers to decide upon their suitability for a particular application.

Changing the organic modifier within a mobile phase can alter the selectivity of the separation as well as the retention characteristics.

So how would one chose the most appropriate solvent – what are the major considerations?

First of all, the chosen organic solvent must be miscible with water. All of the solvents listed in Figure 6 are miscible with water. Second, a low viscosity mobile phase is favored to reduce dispersion and keep system back pressure low.

The solvent must be stable for long periods of time. This disfavors tetrahydrofuran which, after exposure to air, degrades rapidly, often forming explosive peroxides.

The two remaining mobile phase solvents are methanol and acetonitrile. The use of both solvents usually gives excellent retention characteristics.

Acetonitrile, however, has a lower viscosity and lower UV-cutoff (which is advantageous as the possibility detection interference is reduced). The UV-cutoff for methanol is 205 nm, while the UV cut-off for acetonitrile is 190 nm. For these reasons, most analysts begin reversed-phase method development with acetonitrile.

It is also important to note that, whilst the polarity index values of methanol and acetonitrile are similar, they have different Lewis acid / base characteristics (proton donator / acceptor) which allows them to act differently to alter separation characteristics.  We will study this in greater detail in Part II of this Essential Guide.

 
 

Mobile Phase Strength and Retention

Increasing the percentage of organic modifier in the mobile phase has a profound effect on analyte retention due to the change in polarity of the mobile phase.  Use the slider below to investigate the effect of altering the mobile phase composition.

 

Figure 8.  Solvent strength.

 
 

Eluotropic Series

Solvent strength in Reverse Phase HPLC depends upon the solvent type and amount used in the mobile phase (percentage organic modifier (%B)).

It is possible to interrelate the relative ‘strength’ of each of the common solvents using an Eluotropic Series. This is a table or listing of the various solvents and their relative strength on various media – for example Aluminium Oxide or Silica for Normal Phase HPLC and C18 for Reverse Phase HPLC.

Commonly – a nomograph might be used to find equivalent eluotropic strength between mobile phases that use different organic modifiers. The nomograph relates each solvent using a vertical line to indicate mobile phase composition (%B), which give equivalent elution strength – known as ‘isoelutropic’ mobile phase compositions.

Isoelutropic mobile phases produce separations in approximately the same time frame (measured using retention (capacity) factor of the last peak) – however they show altered selectivity. This can be very useful when developing or optimising separations.

You can use the slider bar below to find various isoelutropic compositions – note that the relationship yields results which are only approximately accurate to within about ±5% B.

 

Figure 9.  Solvent nomogram form reversed phase HPLC.

 

Table 2.  Eluotropic series of various reverse phase solvents.

Solvent εo(Al2O3) εo(SiOH) εo(C18) P’
Tetrahydrofuran 0.45-0.62 0.53 3.7 4.0
Acetonitrile 0.52-0.65 0.50-0.52 3.1 5.8
Methanol 0.95 0.70-0.73 1.0 5.1
Water - - - 10.2
 
 
 

Matching Injection Volume with Sample Solvent Strength

Under ideal conditions the strength of the sample solvent used would make no difference to the chromatographic separation, because the solvent would be diluted instantaneously to the same composition as the mobile phase. However, in practice it takes a finite period of time for the injected sample solvent to become diluted with the mobile phase.

The following table provides guidelines for sample injection volumes based on the sample solvent strength with respect to that of mobile phase.

 

Table 3.  Guidelines for sample injection volumes based on the sample solvent strength with respect to that of mobile phase.

Sample Solvent Strength Maximum Injection Volume
100% Strong Solvent ≤ 10μL
Stronger than Mobile Phase ≤ 25μL
Mobile Phase ≤ 15% of Peak Volume
Weaker than Mobile Phase Large
 

100% Strong Solvent

In this demanding situation it is necessary to keep the injection volume as small as possible, thereby minimising peak distortion. The recommendation is for no more than 10μL.

When the sample solvent is greater than 80% of the mobile phase it may be possible to inject larger volumes, as the compositional difference between the sample solvent and mobile phase is correspondingly less.

Stronger than Mobile Phase

When the sample solvent is no more than approximately 25% stronger than the mobile phase, then injection volumes as high as 25μL should be possible. However, always examine the chromatogram for possible peak distortion, given the inter-relationship of this and the preceding rule.

Generally, analyte solubility issues are the primary reason for increasing the sample solvent strength utilised. To minimise such solvent strength variations on injection dissolve the analyte at a high concentration in the strong solvent, then dilute with the weaker solvent to progressively correct for the difference in eluotropic strength. In a worst-case scenario dissolve in 100% strong solvent and inject as small a volume as possible.

Mobile Phase

The best injection scenario is when the sample solvent and the mobile phase are matched, both in terms of eluotropic strength and pH. In this situation, given the solvent homogeneity, you don’t have to be concerned with dilution effects.

However, the injection volume is limited. The contribution of the sample injection volume to the chromatographic peak width can be determined by the following equation:

 
 

Weaker than Mobile Phase

To assess the effect of the mobile phase strength consider the “Rule of Three”, which states that a 10% change in the strong solvent will result in an approximate threefold change in analyte retention time. Therefore, if a chromatographic peak had a retention time of 5min in a mobile phase of 40:60 water:acetonitrile, then in a mobile phase of 60:40 water:acetonitrile its retention time would increase to approximately 45min (3 × 3 × 5min = 45min).

Consequently, if the sample were to be injected in 60:40 water:acetonitrile instead, then the analyte molecules would travel much more slowly through the column, until the sample solvent was fully diluted with the mobile phase.

Chromatographic bands travelling more slowly than the mobile phase tend to compress, because as mobile phase reaches the analyte molecules at the peak “tail” they will begin to move faster down the column, catching those analyte molecules further down the column that are still effectively residing in a weaker mobile phase strength.

As the difference between the solvent strength of the sample solvent and the mobile phase increases, it is possible to use progressively larger and larger injection volumes. This effectively allows analyte on-column sample “focussing”, or concentration, and is often employed in environmental analysis to assist in the detection of trace quantities of pesticides.

Exceptions to the above are whenever the mobile phase contains trace additives, then it is always advisable to inject samples dissolved in the mobile phase. e.g. ion-pair separations rely on the equilibrium between the ion pair reagent in the mobile phase and the stationary phase. Injecting a sample in a solvent that doesn’t contain the ion pair shifts this equilibrium to the mobile phase, with resulting peak distortion.

Peak distortion can occur due to a mismatch between injection solvent and mobile phase strength.  In particular, when large amounts of sample are injected in a solvent that is much ‘stronger ‘than the mobile phase.  The diagram below shows typical peak distortion problems.

 

Figure 10. Peak shape abnormalities currently found when solvent strength is not considered when injecting.

 


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pH Considerations

The mobile phase pH can be used to influence the charge state of ionisable species in solution. The extent of analyte ionisation can be used to affect retention and selectivity.  The pH of a solution will influence the charge state of an acidic or basic analyte.

For example, addition of an acid to an aqueous solution of a basic analyte will increase the concentration of charged analyte in solution, as the hydrogen ion concentration increases. Conversely, raising the pH by addition of a base will increase the concentration of the neutral form of the basic analyte.

Take the example of homovanillic acid shown below – equilibrium between the ionised and non-ionised forms of the analyte will be established according to the solution pH.

 

 

Figure 11.  Homovanillic acid equilibrium.

 

Adding an acid to a buffered solution of homovanillic acid (i.e. lowering the mobile phase pH), will cause the equilibrium to shift to the left and the analyte will become less ionised (ion suppressed) as the analyte recombines to reduce the effect of the added hydrogen ions (protons) from the acidic species. Adding a base to a buffered solution of homovanillic acid (i.e. increasing the mobile phase pH), will cause the analyte to become more ionised as the solution attempts to regain equilibrium by producing more hydrogen ions to neutralise the added base. This principle was first described by Le Chetalier and the converse applies to acidic analyte species.

 
 

The 2 pH rule

It can be shown that at 1 pH unit away from the analyte pKa, the change in extent of ionisation is approximately 90%.  At 2 pH units away from the pKa the change in extent of ionisation is approximately 99%, at 3 pH units 99.9% etc.  Therefore – a rule a thumb known as the ‘2 pH rule’ is useful in predicting extent of ionisation.

 

Figure 12.  Schematic Representation of the 2pH Rule describing the effects of mobile phase pH on degree of analyte ionization in solution.

 
 

Basic Analytes & Ion Suppression

Unlike the dissociated (ionised) acids, which when charged, elute rapidly from the column, protonated bases often have long retention times and poor peak shape. This retention behaviour is due to the interaction with residual silanol species on the silica surface.

Separations of basic compounds, however, are not usually carried out under ion suppression conditions. The analyst would have to raise the pH to produce the neutral molecule. High pH mobile phases can damage traditional silica columns unless specifically designed to cope with high pH

Traditionally the analysis of weak bases has been carried out at low pH – essentially because the surface silanol species are non-ionised (pKa approx. 3.5 – 4.5) and peak tailing is improved somewhat.

The unwanted secondary silanol retention may be reduced (or even eliminated) by the addition of a small (sterically), highly surface active base such as Triethylamine (TEA), Piperazine, N,N,N’,N’-Tetramethylethylenediamine (TEMED), or Dimethyloctylamine (DMOA). These bases interact with the surface silanol species in preference to the analyte molecule and are called ‘sacrificial bases’. They are added to the mobile phase in sufficient concentration to ensure that the silica surface is fully deactivated at all times.

 

Figure 13.  Tailing factor versus concentration of TEA.

 
 

Buffers for Reverse Phase HPLC

In order to control the retention of weak acids and bases, the pH of the mobile phase must be strictly controlled. This usually involves meticulous preparation and adjustment of the mobile phase to the correct pH. Most workers will use a buffer to resist small changes in pH that may occur within the HPLC system (i.e. at the head of column when sample diluent and mobile phase mix, or via evaporation of the organic solvent in a pre-mixed mobile phase, ingress of CO2 into the mobile phase etc).  Some common HPLC buffers are listed in the table below.

Table 4.  Properties of some common Buffers used for Reverse Phase HPLC.

Buffer pKa pH range UV cutoff (nm)
TFA 0.3 1.1 - 3.1 210 (0.1%)
Methane Sulphonic Acid Ca – 2.0 > 1.0 n/a
Phosphate
pKa1
pKa2
pKa3

2.1
7.2
12.3

1.1 - 3.1
6.2 - 8.2
11.3-13.3
< 200
Citrate
pKa1
pKa2
pKa3

3.1
4.7
5.4

2.1 - 4.1
3.7 - 5.7
4.4 - 6.4
230
Carbonate
pKa1
pKa2

6.1
10.3

5.1 – 7.1
9.3 – 11.3
< 200
Formate* 3.8 2.8 - 4.8 210 (10 nM)
Acetate* 4.8 2.8 - 5.8 210 (10 nM)
Ammonia* 9.2 8.2 - 10.2 200 (10 nM)
Borate 9.2 8.2 - 10.2 n/a
Triethylamine 10.8 9.8 – 11.8 < 200
Tris (hydroxymethyl) aminomethane 8.3 7.3 - 9.3 205 (10 nM)
Pyrrolidine 10.5 9.5 - 11.5 n/a
* Users of LC-MS require volatile buffers to avoid fouling of the atmospheric pressure interface and reduced maintenance intervals.  The use of trifluoroacetic acid (TFA) is to be avoided for small molecule work due to its ion suppression effects.
 

A particular buffer is only reliable in the pH ranges given – usually around 1pH either side of the buffer pKa value (note some buffers have more than one ionisable functional group and therefore more than one pKa value).

The concentration of the buffer must be adequate, but not excessive. In general, HPLC buffers range in concentration from 25 to 100 mM. Buffers prepared at below 10mM can have very little impact on chromatography whilst those at high concentration (>50mM) risk precipitation of the salt in the presence of high organic concentration mobile phases (i.e. >60% MeCN), which may damage the internal components of the HPLC system.  The closer you are to the buffers pKa value then the more effectivey it can operate and the lower concentration required.

 


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The HPLC column lies at the heart of every chromatographic separation and the stationary phase is used to control retention.  The quality of the column packing, the physical attributes of the packing material and the quality of the column packing all have a direct influence over the efficiency of the analyte peaks produced. 

Problems with the column hardware and packed bed can result in poor peak shape.

For a more detailed discussion on HPLC column support materials please refer to the earlier Essential Guide -

Column Selection for Reversed Phase HPLC

 

 

Silica as a Packing Material

Silica is often used as a support material for adsorption (normal phase) chromatography and with chemical modification of the surface for partition (reverse phase), normal phase, ion-exchange, chiral and size exclusion chromatography.

Porous silica has a high surface area that leads to high efficiency columns (through a higher number of possible surface interactions – theoretical plates).

The surface of non-hybrid silica is covered with silanol groups that are used in adsorption (normal phase) chromatography to interact with polar molecules, or in reversed phase (as well as ion exchange, chiral etc.) chromatography to attach the bonded phase material.

Whilst most manufacturers are able to provide good bonded phase surface coverage, even the best manufacturing techniques will still leave a high number of silanol groups unreacted (due to steric effects).  The remaining silanol groups (depending upon their conformation) are able to interact with analytes containing ionic or polar functional groups to give rise to peak tailing, through unwanted secondary interactions.

Manufacturers may choose to end-cap the column surface, which involves reacting the silanol groups with a sterically small but highly reactive reagent that ‘caps’ the polar surface silanol with a non-polar (and much less reactive) trimethylsilyl group.

 
 

Reverse Phase Stationary Phases

Reverse phase separations are characterized by having a stationary phase that is less polar than the mobile phase.

Several popular reverse phase, bonded stationary phases are shown. Octadecylsilyl (or C18) is commonly used, as it is a highly robust hydrophobic phase, which produces good retention with hydrophobic (non-polar) analyte molecules. This phase can also be used for the separation of polar compounds when used with mobile phase additives, which will be discussed later.

 

Figure 14.  Selected reverse phase stationary phases.

 

In general, shortening the alkyl chain will shorten the retention time. There are only very slight selectivity differences between, for example, C18 and C8 columns.

The use of more polar phases such as cyano, phenyl or amino phases show altered selectivity compared to the alkyl phases. These phases are able to interact with polar analyte functional groups, via dipole-dipole interactions and the phenyl column can interact with analyte aromatic moieties via π-π electron interactions.

 
 

Silanols and Peak Tailing

Fully hydroxylated silica will have a Silanol surface concentration of ~8µmol/m2.  Following chemical modification > 4µmol/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 – as previously described. 

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 optimized bonding conditions, also the end-capping ligand is prone to hydrolysis especially at low pH.  Silanol groups are present in numerous conformations, with some being more active than others at causing analyte peak tailing and / or irreversible retention.

 

Figure 15.  Various Silanol conformations.

 
 

Acidic (lone) surface silanol groups give rise to the most pronounced secondary interactions with polar and ionisable analytes.  Modern silica is designated as being Type I (Type A) or Type II (Type B), which primarily describes the nature of the silanol surface.  Type I silica is ‘high energy’ (non-homogenous) and contains a higher density of lone silanol groups, whereas Type II silica is much more homogenous (bridged) 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.  The figure below shows how various basic and polar analytes are affected when analysed using Type I silica, Hypersil, as compared to Type II silica (Hypersil BDS).

 

Figure 16.  Basic, polar analytes analysed using Type I and Type II silica.

 
 

Mobile phase pH will affect the degree of silanol ionisation and therefore the degree with which they interact with polar and ionisable analytes, causing peak tailing.  Typically the pKa of surface silanol species lies in the range pH 3.8 – 4.5 and at eluent pH ≤3 all but the most acidic will be fully protonated and therefore peak tailing will be at a minimum (please refer to the figure below).

 

Figure 17.  Effect of pH on analyte retention.

 
 

As the eluent pH increases the degree of ionisation (through deprotonation) increases, and peak tailing becomes more pronounced as the silanol groups interact with charged and polar species in solution (please refer to the figure below).

 

Figure 18.  Silanol – Base interactions and effect on peak shape at different pH conditions (left hand side:  pH < 3.0.  right hand side pH > 3.0).

 
 

End Capping

The surface of non-hybrid silica is covered with silanol groups that are used in adsorption (normal phase) chromatography to interact with polar molecules, or in reversed phase (as well as ion exchange, chiral etc.) chromatography to attach the bonded phase material.

 

Figure 19.  Silanol end capping reaction scheme.

 
 

Figure 20.  Typical bonded phase silica surface.

 

The remaining silanol groups (depending upon their conformation) are able to interact with analytes containing ionic or polar functional groups to give rise to peak tailing, through unwanted secondary interactions.

 
 

Carbon Load

Carbon load is a measure of the amount of bonded phase bound to the surface of the packing.  In general terms:

  • high carbon loads: provide greater column capacities and resolution
  • low carbon loads: render less retentive packing and faster analysis

For more on stationary phases for HPLC, please follow the link below:

Column Chemistry    *** CHROMacademy Registered users only ***

 

Frits

A major cause of column deterioration and damage is the build-up of particulate and chemical contamination at the head of the packed stationary phase bed. This can lead to increased back pressure and poor analyte peak shape (usually tailing or in the worst cases split peaks).  HPLC Columns normally contain stainless steel inlet and outlet frits (acting as filters) which retain the column packing and allow the passage of the mobile phase.  The pore size of the frit must be smaller than the particle diameter of the packing, e.g., a 0.5 μm frit for 1.8 μm packing.  Sample depositing on the column inlet frit can lead to peak shouldering as demonstrated below.

 

Figure 21. Column inlet frits.

 

The simplest solution is to replace the frit, sometimes, however, you may be able to clean the frit in an ultrasonic bath.

 
 

Channelling

Channels occur when pockets of air under high pressure are forced through the packed bed – causing pathways with little or no packing material – creating a ‘path of least resistance’.  The presence of channels will cause peak fronting, as solvent (and solutes) will travel faster through them than in the rest of the column. Further – the available surface through these channels is limited, so overloading of this pathway quickly occurs and analytes undergo little (or reduced) retention in comparison with the majority of the sample band.

 

Figure 22.  The presence of channels will generate speed migration differences between different components of mobile phase, thus yielding peak shape problems.

 

In order to avoid channelling, you should not:

  • jar the column
  • shock the column through pressure changes or by jumping to immiscible solvents
  • reverse the column flow, or make sudden changes to flow rates

Remember to degas your mobile phase and prevent any particulate matter from entering the column (use an in-line filter where necessary).

 
 

Column Overload

This condition will cause different problems: poor peak shapes (broadening, tailing, fronting and asymmetry), variable retention times, selectivity issues, etc and occurs when the sample concentration or volume saturates the stationary phase surface in the area of the analyte band – causing unusual retention effects.  Column capacity depends upon many factors; however, the table below provides the means to quickly identify situations where the possibility of column overload exists.

 

Table 5.  HPLC column capacity (oversimplified).

Column Type Typical Dimensions Capacity (mg)
Analytical 25 cm × 4.6 mm < 0.5
Semi-Preparative 25 cm × 10 mm < 100
Preparative 25 cm × 21 mm < 500

Values per total amount of analyte.

 

Note that Table 5 is intended to indicate the possibility of overloading your column.  For more accurate information, for particular applications, consult with your column manufacturer.

There are two forms of column overloading: concentration and volume overloading. Both of them lead to a decrease in chromatographic resolution.  For more information on this topic, please follow the link below:

Troubleshooting the Autosampler, Column and Detector

 
 

Voids in the Stationary Phase

Working outside the ideal pH range of your column could compromise the integrity of the stationary phase (silica dissolution), so voids are likely to develop at the head of the column, due to bed compression.

Silica is soluble under high pH conditions (typically pH 7.5 and above for traditional silica based phases); therefore, silanol accessible functional groups (which are present in all silica based columns) can be attacked by strong bases while developing voids in the packing material, with the consequent loss of efficiency.

HPLC columns are designed to operate under high pressure conditions; however, columns can be damaged by sudden variations in pressure.  Reasons for pressure variation include:

  • Slow rotation of the sample injection valve
  • Fast start-up of the pump
  • Column switching operations

Voids are also formed when silica dissolution occurs and particles collapse causing bed compression when the column is pressurized.  Peak shouldering and broadening is one of the most common problems due to voids in the stationary phase.

 

Figure 23. Peak splitting, shouldering and broadening due to column voids.

 

Whereas in older style and cartridge type columns the inlet frit could be removed and loose stationary phase added as temporary fix, newer columns do not allow this with end fittings being permanently fixed in place.

Reasons for peak shouldering and splitting include:

  • Column void
  • Column contamination
  • Contaminated or partially blocked frit
  • Injection solvent stronger than mobile phase

Note that if peak shouldering affects only one peak within the chromatogram, then rather than stationary phase voids, co-elution should be suspected.

 


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Temperature

Temperature plays an important role in HPLC; this is because both the kinetics and thermodynamics of the chromatographic process are temperature dependent. 

In nearly all reversed phase separations, an increase in temperature will reduce analyte retention.

Additionally solvent viscosity is reduced at elevated temperature which in turn means lower backpressure.

 

 

Figure 24.  Effect of temperature on the HPLC separation. 
Column: 50 cm × 4.6 mm 1.8μm; solute: α-naphthol; mobile phase: 60% acetonitrile–40% water.

 
 

By increasing the temperature, the amount of organic solvent in the mobile phase can be reduced to maintain retention. In some cases a small increase in temperature (of only a few degrees Celsius) produces a similar effect on retention as changing mobile phase composition.

In the application below, a mixture of parabens were separated at three different temperatures (the optimum flow rate for each temperature was selected).

 

Figure 25.  HPLC analysis of a mixture of parabens (200 Bar).  Column: C18 (2.1mm I.D.×50 mm, 1.7μm); mobile phase: water + 30%acetonitrile.
Sample: a. Methylparaben; b. Ethylparaben; c. Propylparaben; d. Butylparaben.

Important:  due to lab temperature variations, some form of column temperature control, with mobile phase pre-heating is  strongly recommended.

 
 

Flow Rate

Keeping constant linear velocity is one of the key parameters when trying to reproduce a chromatographic separation on a different column.  Do not expect retention time similarities or same chromatographic efficiency when changing to a different column (dimensions, packing material, etc.).

As expected, there is a relationship between the column dimensions, more specifically column I.D., and its optimum flow rate.  Table 6 gives typical flow rates for selected HPLC columns.

 

Table 6.  Typical flow rates for HPLC columns.

Column I.D. (mm) Column Length (mm) Flow Rate (mL/min) for a Column Packed with
5.0 μm Particles 3.0 μm Particles
4.6 250 1.0 1.5
3.0 250 0.5 0.7
2.1 100 0.2 0.3
1.0 100 0.05 0.07
 
 

Interestingly, even if the same number of sample molecules is injected in each case, smaller diameter columns tend to provide higher sensitivity than larger diameter columns.  The main reason being that analytes are more concentrated in the mobile phase when using small diameter columns due to the reduction column volume.

For more on troubleshooting HPLC pumping systems, please follow the link below:

HPLC Solvent Pumping Systems    *** CHROMacademy Registered users only ***

Remember, the use of non pure solvents in HPLC causes irreversible adsorption of impurities at the column head.  Filters, guard columns and frits should be used to prevent this situation   For more information on HPLC mobile phase preparation and the use of in-line filters and guard columns, please follow the link below:

Mobile Phase Considerations    *** CHROMacademy Registered users only ***

 


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Introduction

The retention time of peaks within a chromatogram can aid in the identification of many problems that are associated with HPLC system components.  Variable retention time may result from changes in mobile phase flow rate, mobile phase composition, column stationary phase, and column temperature.  Analyte retention times can fluctuate, increase or decrease, and can be critical in the diagnosis and elimination of HPLC system problems.

Troubleshooting retention time variation requires that we first identify if the issue arises from the HPLC system or from the separation processes occurring within the HPLC column. From first principles it can be generally stated that:

  • If the void (hold-up) time (t0) and analyte retention time (tR) vary together, suspect a flow rate change. In this scenario the analyte capacity factor (k') will remain constant
  • If only the analyte retention time varies, with the void (hold-up) time remaining constant, then k' will change also. In this scenario suspect a change in the selectivity or retentivity of the separation system
 

Consider the following possibilities:

  • Mobile phase degradation
  • Selective evaporation of at least one mobile phase component
  • Incorrectly prepared mobile phase
  • Improper mobile phase mixing
  • Incorrect mobile phase
  • Absorbtion of sample or eluent components onto the stationary phase / selection and installation of the wrong column
 

Figure 26.  Retention time variation in all peaks except in t0.

 
 

In this case fresh mobile phase should be prepared, if the problem persists implement solvent degassing but care needs to be taken, sometimes mobile phase components evaporate when improperly degassed.  If only selected peaks change position then a change in the mobile phase pH or buffer concentration should be suspected.

 

Figure 27.  Retention time variation in selected peaks (t0 remains constant).

 

For more information on retention time related problems, please visit the links below: [12, 13, 14]

Retention Time Changes

Troubleshooting Basics, Part 3: Retention Problems

Variability — How to Control It.  –Why aren't Retention Times Constant?

 
 

Fluctuating Retention Times

Analyte retention time variation can be caused by changes in the mobile phase composition, and is typically the result of inconsistent on-line solvent mixing or insufficient column equilibration in gradient analysis. A 5−10% reduction in analyte retention by reversed phase is possible for every 1% increase in the proportion of mobile phase organic solvent. This variation is well recognized, and is often practically implemented to effect gross changes in retention (and sometimes selectivity) within a  separation during method development.

The figure below illustrates the retention time variation for consecutive injections of a four-component system suitability standard mix, using a low pressure mixing solvent delivery system. The initial part of the separation method uses a 12min isocratic hold at 62% B.

 

Figure 28.  Retention time variation.

 
 

Assuming that the solvent reservoir contents were correctly prepared, an incorrect (or inconsistent) mobile phase composition typically results from one of several possible issues as listed below;

1. A restriction in one or more of the solvent channel lines leading from the reservoir(s) to the gradient proportioning valve leading to incorrect mobile phase composition. Assess this by removing the fitting that connects the inlet line with the proportioning valve (you may need to do this for two sets of tubing if you have an in-line degasser installed in the system). Once disconnected, liquid should siphon freely; if it does not then there is a restriction. Loosen the solvent reservoir cap if sealed, to relieve any partial vacuum in the reservoir. If insufficient pressurization was the problem then the solvent will now siphon freely. If flow is still restricted, remove the inlet solvent frit (filter) and check for siphoning again. If the line siphons freely the frit is blocked. If the frit is of the sintered glass variety clean by soaking in 6M nitric acid, then rinse thoroughly first with H2O then MeOH, then finally with H2O again before reinstalling. Do not sonicate, as the glass may fracture due to the ultrasonic frequency applied. If a solvent inlet line were restricted then less solvent would be introduced, generating a slight vacuum in the gradient proportioning valve. Consequently, when the second valve opened there would be a surge of that solvent to relieve this partial vacuum, with the result that the proportion of solvent introduced would vary. An excess of solvent B in the mobile phase would elute the analytes earlier, the effect observed in the example chromatographic trace from figure 28 ?>?????

2. If solvent delivery is not restricted, then a problem with the controlling software or a mechanical problem with the proportioning valve might be suspected. Low pressure mixing systems operate by opening one proportioning valve for a given time, closing it, and then opening a second valve etc. according to the ‘duty cycle’ of the pumping or proportioning system. In the example shown in Figure 30, if the total valve cycle was 100ms, and an isocratic mobile phase of 38% A:62% B is required then proportioning valve A would remain open for 38ms and proportioning valve B for 62ms. To check for a gradient proportioning valve failure, prime all the channel lines with the same solvent, then with equal volumes in their reservoirs run a 1:1:1:1 (v/v) quaternary gradient for a fixed time at a fixed flow rate. The volume reduction in each solvent reservoir will be the same if the proportioning valves are operating correctly.

3. Mobile phase inconsistencies can also occur if improperly recycled solvent is used. Ensure the solvent reservoir contains a minimum of about three litres of mobile phase, and that it is constantly stirred. In this way sample contaminants or other small changes in the column eluent are effectively diluted out before passing through the system the next time.  In general it is better not to recycle mobile phase.

4. In gradient analysis, if the re-equilibration time is not sufficient, the initial mobile phase within the column will change from run to run.  This will cause variations (both earlier and later elution are possible on a run to run basis) in the retention time (and possibly the selectivity of the separation).  One should ensure that 10 column volumes of mobile phase, at the starting composition of the gradient, should pass through the column for proper re-equilibration of the whole packed bed (this may be shortened through experimentation).  Two important numbers are required to achieve this:  the internal volume of your column (sometimes called the ‘interstitial volume’) and the gradient dwell time (the time it takes for the system to change back from the final to the initial gradient composition and pump it to the inlet of your column).

Column volume (VM) is straightforward to estimate using the formula;

VM (ml) = πr2L x (0.6 x 10-3)

r = column radius (i.e. half of the column diameter) (mm)
L = column length (mm)

So for a 5cm x 2.1mm HPLC column this would represent a volume of:

VM (ml) = 3.14 x 1.052 x 50 x (0.6 x 10-3)

VM (ml) = 0.1

So, at a flow rate of 0.5mL/min., an equilibration of ten column volumes would mean allowing 2 minutes equilibration.

Now for that second important number – the gradient dwell volume.  We will show you how to calculate this is a future newsletter – however a well plumbed LC system will have a dwell volume below 0.5mL and some UPLC systems will be significantly lower.  However, if we err on the side of caution and assume 0.5mL – this will add a further 1 minute to our equilibration time at an eluent flow rate of 0.5 mL/min.

Therefore a total of 3 minutes will be required to re-equilibrate this particular HPLC column and avoid problems with irreproducible retention times and separation selectivity.

5. Improve the reliability of any LC analysis with respect to both analyte retention time variation and peak shape by ensuring that the buffering capacity of any mobile phase is sufficient to compensate for any changes in pH.

Generally a buffer will operate with greatest buffering efficiency when within ±1 pH unit of its pka. Importantly, phosphate buffers do not give such a desired buffering capacity in the pH range 3−6. This pH range could be filled by using either an acetate buffer (pka 4.8), or citrate as this buffer exhibits three pka’s in the pH range typical of reversed phase separations. However, citrate suffers from the fact that it is highly corrosive to stainless steel surfaces.

To maintain adequate buffering strength for analyte samples, start with a buffer concentration of between 25−50mM. Do not use less than 20mM, unless mass spectrometry is being used as the detection mechanism. Consider changing the buffer system used to one of a volatile nature i.e. ammonium acetate or ammonium formate. Volatile buffer systems will help prevent contamination and potential “fouling” of the mass spectrometer source, improving sensitivity and detection efficiency.

The figure below illustrates the detrimental effect varying pH has on both analyte retention time and peak shape. The two compounds being chromatographed are benzoic acid and sorbic acid respectively. At a buffered pH of 3.5 these weak acid compounds are fully protonated (ion suppressed), and consequently they exhibit long retention times on a reversed phase column (Trace A). At a buffered pH of 7.0 the acids are fully de-protonated (ionized). They now possess a much greater degree of polarity than at a pH of 3.5, and consequently their retention time decreases dramatically (Trace B). However, if an unbuffered pH of 7.0 is used then the ionisation state of these acid analytes are not controlled effectively, and as the dynamic equilibrium is constantly changing between their respective ion-suppressed and ionised forms then both their peak shape and retention times vary dramatically (Trace C).

 

Figure 29.  Effect of pH on peak shape and retention time.

 
 

Temperature Effects

Retention time variation may also be caused by fluctuations in temperature. A 1−2% change in retention time is possible for every 10oC change in column temperature. The simplest way to eliminate this potential problem is to ensure that both the column and mobile phase are thermostatically controlled. Check for potential temperature problems by using a calibrated thermometer, and determine if any observed temperature fluctuations correlate with changes in analyte retention time.

Column aging may also contribute to retention time variation.  The combined action of high temperatures and aggressive mobile phases (for example most HPLC silica based columns should be used with mobile phase pH between 2 and 8) will accelerate the natural column degradation process, that is responsible for many problems, such as reduced selectivity, reduced efficiency, peak shape problems, inconsistent retention time, etc. Using a guard column may extend the effective lifetime of a column. However, the use of a guard column is recommended for only one specific reason; to act as a chemical filter in removing any strongly retained material(s) that could potentially contaminate the analytical column.

Running check standards more frequently may allow a data capture system to effectively “keep up” with the observed retention time drift. Nominate a sample chromatographic peak as a reference peak. The use of internal standards may also help, especially if relative rather than absolute retention times are used.  The table below illustrates the observed effect of changing separation conditions on analyte tR.

 

Table 7.  Effect of Change in Separation Conditions on Analyte Retention.

Variable Method Variable Change Average Change in TR
Flow Rate All + 1% - 1%
Temperature All except SEC + 1oC - (1 – 2)%
Organic Solvent RP-HPLC + 1% (v/v) - (5 – 10)%
pH RP-HPLC + 0.01  unit ± (0 – 1)%
Strong Solvent Normal Phase + 1% - (1 – 2)%
Buffer: Organic Solvent SEC + 1% 0%
 
 
 

The table below helps you to identify the origin of and propose remedial actions for irreproducible retention time related problems.

Table 8.  Variable Retention Times.

Component Potential cause Potential Solution(s)
Mobile phase Air in mobile phase Degas mobile phase
Incorrect gradient operation parameters Ensure proper column equilibration
Mobile phase incompatible with sample solvent
  • Adjust solvent
  • Inject samples in mobile phase (if possible)
Change in mobile phase composition
  • Adjust mobile phase composition
  • If performing gradient elution ensure that the solvent components are being mixed and proportioned correctly
  • Increase re-equilibration time (gradient operation)
  • Cover solvent reservoirs
  • Prepare fresh mobile phase
Change in mobile phase pH 
  • Adjust mobile phase pH
  • Select correct buffer type and concentration (the 2 pH rule)
Mobile phase mixing problems
  • Ensure mobile phase system is delivered at a constant composition
  • When preparing mobile phases, remember to add the organic component to the buffer to avoid buffer precipitation
Pump Air in the pump
  • Test pump’s integrity (check valves, pistons, etc; look for leaks, salt buildup, unusual noises)
  • Degas mobile phase
  • Prime pump and degas solvent
  • Disconnect tubing and check for flow (look for leaks, and make sure the pump is not starved of mobile phase)
  • Purge pump at high flow rate
  • Allow air to escape.  Flush with methanol or isopropanol to remove air trapped in the pump
  • If necessary arrange for an expert visit
Cavitation
  • Degas solvent
  • Check for obstruction in line from solvent reservoir to pump
  • Decrease flow rate
Leak
  • Check system for loose fittings
  • Check pump for leaks
  • Change pump seals if necessary
Column Temperature fluctuation Implement temperature control
 
 

Decreasing Retention Times

If both the void time (t0) and retention time (tR) are decreasing, then the analyte’s capacity factor (k’) will remain constant. In this scenario suspect a progressive increase in the flow rate. However – let’s consider the situation in which analyte retention time tR is decreasing but the hold-up time t0 is not?

Typically this situation will occur when the analyte retention mechanism is affected.  This most often will be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule – usually due to an incorrect mobile phase pH (too high for acidic species or too low for bases).  Check that the mobile phase has been correctly prepared (check organic aqueous ratio / pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing.

Check the pH of any buffered mobile phase being used. Unless specifically stated by the column manufacturer, the operating pH should be kept within the pH range 2−8. Below approximately pH of 2 the siloxane bond attaching the stationary phase ligand to the silica support will be broken, and loss of bonded phase will result (phase bleed). Above a pH of approximately 8 dissolution of the silica support may occur. In both instances analyte retention times will commonly decrease, please do note that enhanced retention of basic and polar analytes can be observed when anlaysed on column that has experienced high phase bleed due to extra hydrogen bonding and cation exchange via the exposed silanols. One would also observe a reduction in analyte peak efficiency under these cricustances.  Consider switching to a column type specifically designed for use at extremes of pH.

Ensure the column has not been overloaded – the peak apex retention time can often be seen to move to earlier retention as the degree of column overload worsens.  Decrease the absolute amount of analyte being applied by diluting the sample or reducing the injection volume.

If performing gradient elution, ensure that the solvent components are being mixed and proportioned correctly. Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation, ensuring full pressurization and solvent line filling.

The table below helps you to identify the origin and propose remedial actions for decreasing retention time related problems.

 

Table 9.  Decreasing Retention Times.

Component Potential cause Potential Solution(s)
Mobile phase Change in mobile phase composition
  • If performing gradient elution ensure that the solvent components are being mixed and proportioned correctly
  • Cover solvent reservoirs
  • Prepare fresh mobile phase
Change in mobile phase pH Adjust mobile phase pH (check for ingress of CO2 lowering pH on standing)
Increased flow rate Locate and address the cause of the incorrect flow rate or adjust mobile phase flow rate if incorrectly set
Mobile phase contaminated or deteriorated
  • Prepare fresh mobile phase
  • Use HPLC-grade water and chemicals
  • Do not store pre-prepared mobile phase for very long periods before use
Pump Air in the pump Correct as appropriate – ensure that the eluent compressibility settings are correct
Column Temperature fluctuation Implement column temperature control which stabilizes column temperature and pre-heats the mobile phase prior to entering the column
Column overloading Retention times usually decrease as mass of solute injected exceeds column capacity. Inject smaller amounts of sample
Column degradation Correct column working conditions (for example: most HPLC columns use mobile phase pH between 2 and 8)
 
 
 

Increasing Retention Times

If both the void time (t0) and retention time (tR) are increasing, then suspect a progressive decrease in the flow rate. Check the method operating parameters and instrument flow rate calibration. Let’s consider the situation in which analyte retention time tR is increasing but the hold-up t0 isn’t?

A retention time increase may be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule – usually due to an incorrect mobile phase pH (too high for acidic species or too low for bases). 

When pre-mixed mobile phases are allowed to stand, the more volatile solvent component(s) can evaporate, thereby changing the overall retention characteristics, typically leading to increasing retention times. This problem is exacerbated with longer analytical campaigns as the gaseous headspace above the mobile phase increases as the level within the reservoir is depleted.   Ensure that the eluent reservoir is capped and ensure as little headspace as possible is maintained above the eluent liquid.

Check that the mobile phase has been correctly prepared (check organic aqueous ratio / pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing.

Check all pump-head components for leaks and if necessary perform a volumetric check of instrument flow rate using a calibrated electronic flow meter or by measuring the time take to deliver 10 mL of eluent into a measuring cylinder. For gradient elution, check that the gradient is being mixed and proportioned correctly. Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation, ensuring full pressurization and solvent line filling.

As the column gets older, secondary interactions are promoted by an increased number of exposed silanol groups, so retention time of polar and / or basic analytes may increase – this should be apparent in the first instance by an increase in the peak tailing of more pol;ar analytes.  Eliminate any column secondary interactions by using a mobile phase modifier or buffering the mobile phase appropriately.  Triethylamine can be added as a ‘competing base’ to eliminate polar analyte interactions with residual silanol groups, as well as increasing the effective carbon loading of the column or switching to an endcapped / type II silica column.

The table below helps you to identify the origin and propose remedial actions for increasing retention time related problems.

 

Table 10.  Increasing Retention Times.

Component Potential cause Potential Solution(s)
Mobile phase Change in mobile phase composition
  • Adjust mobile phase composition
  • If performing gradient elution ensure that the solvent components are being mixed and proportioned correctly
  • Cover solvent reservoirs
  • Prepare fresh mobile phase
Change in mobile phase pH Adjust mobile phase pH (check for ingress of CO2 lowering pH on standing)
Decreased flow rate
  • Locate and address the cause of the incorrect flow rate or adjust mobile phase flow rate if incorrectly set
  • Perform a volumetric flow rate check if necessary
Mobile phase contaminated or deteriorated
  • Prepare fresh mobile phase
  • Use HPLC-grade water and chemicals
  • Do not store pre-prepared mobile phase for very long periods before use
Pump Leaks/blockages
  • Identify the presenece of a leak via electronic or volumetric flow rate checks
  • Perform pump maintenance as required
  • If required arrange for an expert visit
Pump parameters wrongly selected Correct as appropriate
Column Temperature fluctuation Implement column temperature control in which the eluent is pre-heated prior to entering the column
Column degradation Correct column working conditions (for example: most HPLC columns use mobile phase pH between 2 and 8) or use a column designed to operate over a wide pH range
Other Leaks/blockages
  • Consider the entire HPLC system (including tubing, pump and accessories)
  • Check and fix as appropriate
 
 


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The shape of the chromatographic peaks can aid in the identification of many problems that are associated with the system components.

Peak shape problems may not adversely affect the resultant data, determine if the analytical results are truly compromised before taking actions.

For more information on peak shape related problems, please visit the links below:[15, 16, 17]

Peak Shape Problems

Split Peaks — A Case Study

Pinning Down Tailing Peaks

Peak Fronting, Column Life, and Column Conditioning

 

 
 

Peak Broadening

Correcting broadened chromatographic peaks requires information on whether the peak broadening is the result of a change in the HPLC system, column deterioration or a “late eluting” peak from an earlier injection.

If all of the separated peaks are broadened, with early peaks exhibiting more pronounced broadening, then the problem may have been simply caused by the introduction of a large extra-column volume in the system:

  • Addition of a disproportionate length of tubing or wider I.D. tubing
  • A poorly seated capillary or column connective fitting
  • Worn PEEK finger-tight nuts / poorly made (non-zero dead volume) connections
 

Figure 30.  Peak broadening.

 
 

If gradual peak broadening has been observed over a longer period of time, then it is indicative of a general deterioration in the column itself.

Column efficiency, or plate number (N), is used as a mathematical measure of the deterioration of a column over time and injection number. Measuring the plate number for a nominated sample compound, or evaluating the chromatographic peaks of a set of system suitability standards recommended by the column manufacturer, and comparing them to logbook records will indicate the extent of the deterioration. Providing the mobile phase and system settings have not been changed, analyte retention time should not vary significantly when efficiency drops.

Column efficiency can be calculated by applying the following equation:

Where:

N = Column plate number
tR = Analyte retention time
Wb = Width of peak where tangential lines intersect the baseline
W½ = Peak width at half height maximum (PWHH)

  • If N is seen to increase with increasing analyte retention time, then the column is the biggest contributor to the system dwell volume, and the HPLC is performing satisfactorily
  • If N is seen to decrease or is random with increasing analyte retention time, then the HPLC is the biggest contributor to the system dwell volume, and any contributor to extra column volume should be reduced (consider tubing length and i.d., number of connections and flow cell internal volume in the first instance)
 
 

Evaluate whether other system components may also have contributed to the broadening peaks -  including deteriorating guard columns for example, high molecular weight compounds, especially proteins, may have broad peaks on columns with pore sizes of < 80A; ensure > 300A pore size is used with such analyte species.

A late eluting peak from a previous sample injection should be suspected whenever a single broad band appears in a chromatogram with otherwise narrow bands (efficient peaks). Importantly this peak may not appear in all analyses, and therefore may not be noticed initially, especially in complex multi-component separations. Given the “late eluter” in question is suffering simply from an increased capacity factor (k’), and therefore much increased diffusion, then one simple approach to identifying its origin is to allow the chromatogram to run for several times the normal run time. The potential problem then arises of what happens if the “late-eluter” is not observed in every sample run?

A more efficient approach to identifying a late eluting peak is to calculate its true retention time first. This approach has the advantage that it allows you to identify with which particular sample injection the peak is actually associated.

First determine the plate number of a known peak in the chromatographic trace. As an example we will take a peak possessing a retention time of 12min, with a PWHH (W½) of 0.15min. Using the equation previously described this gives a plate number of:

N = 5.54 (tR / W½)2

N = 5.54 x (12 / 0.15)2 ≈ 35,456

 

Solving this equation for tR gives:

N = 5.54 (tR / W½)2 → tR = 0.425 W½ / N0.5

tR = 0.425 x (0.15) x (35,456)0.5 ≈ 12.0

 

By measuring the peak width at half height maximum of the late eluting peak it is possible to predict its retention time.

In this example suppose the peak width of the problem peak is 0.5min. Using this peak width, and the plate number for the column previously determined from the known chromatographic peak of 35,456, the calculated retention time is 40min. This means that the late eluting peak is associated with an injection made approximately 40min previously. Re-injecting the suspect sample, and altering the runtime accordingly can confirm this retention time value.

Often it is not possible to eliminate these late-eluting peaks. Therefore, instead of modifying the separation conditions, or waiting until the peak elutes before making the next injection, it is usually more convenient to modify the run time so that the late eluting peak falls in an unimportant region of a later chromatogram.

Table 11 helps you to identify the origin and propose remedial actions when peak broadening occurs.

 
 

Table 11.  Peak broadening.

Component Potential cause Potential Solution(s)
Mobile phase Buffer concentration too low
  • Increase buffer concentration
  • When preparing mobile phases, remember to add the organic component to the buffer and never otherwise, by so doing buffer precipitation is prevented
Mobile phase flow rate too low
  • Set the correct flow rate
  • Check for leaks and blockages
  • Inspect for blockages
Mobile-phase solvent viscosity too high
  • Increase column temperature
  • Change to lower viscosity solvent
Autosampler High dead volume Injector systems should have the smallest possible internal volumes – keep tubing internal diameter low and use the smallest loop (injection) size possible
Peak dispersion in injector valve Decrease sample loop size / inject a smaller volume of a more concentrated sample
Loading deteriorated sample Prepare and inject fresh sample
Column Column overloaded Inject smaller amount of sample
Large extra-column volume Use column uniformly packed with small diameter particles
Contamination
  • Flush column with strong solvent
  • Replace column
  • Replace guard column
  • Clean or replace frit
Void at column inlet Replace column and investigate reasons for void formation (silica dissolution at high pH and pressure shock on repeated injection or when changing flow rates are the most likely causes)
Two or more poorly resolved
compounds per peak
  • Improve separation selectivity
  • Change column
Elution of analytes retained
from previous injection
  • Flush column with strong solvent at end of run
  • End gradient at higher solvent concentration
Column degradation
  • A single fronting peak can result from a small peak eluting just before the chromatographic peak of interest
  • Use polymeric, sterically protected, or high-coverage reversed-phase column.
  • Reduce temperature
  • Correct column working conditions (for example: most HPLC columns use mobile phase pH between 2 and 8)
Retention time too long Use gradient elution or stronger isocratic mobile phase
Incorrect column temperature Correct temperature
Detector High dead volume Detector should be designed to give the smallest possible internal volume
HPLC tubing
and accessories
High dead volume
  • Minimize low dead volume
  • Use zero dead volume fittings and accessories
 
 
 

Peak Shouldering and Splitting

If all peaks in the chromatographic trace are doublets then the column has probably developed a void at the head of the packed bed or has been subjected to ‘channelling’. Substituting the column will quickly confirm this problem.

 

Figure 31.  Peak splitting.

 

If a void is suspected, reverse the column and wash in 100% strong solvent to remove any contamination from the column inlet filter and direct to waste, bypassing the detector. Check the manufacturer’s instructions as to whether the column should remain in the reversed flow orientation.

As a partial blockage of the column inlet frit is generally caused by deposition of particulates from the mobile phase, sample solvent, pump seals or rotor seal, ensure adequate filtering and include an inline filter between the injector and column head where required.

If only one peak in the chromatogram is a doublet, then a co-eluting or interfering peak should be suspected. Confirm the presence of an interferent by changing the detection wavelength, or improve the resolution of the separation by using a column offering greater efficiency i.e. a longer column or a column packed with smaller sized particles or an alternative selectivity (different stationary phase chemistry).

Peak shoulders usually indicate co-eluting compounds.  Adjust the selectivity shown to the co-eluting compounds by the separation system by altering the mobile phase composition or type, separation temperature or the column stationary phase. Use a sample clean-up procedure i.e. SPE to remove any interfering contaminants. Evaluate if any co-eluting peak may have come from a previous chromatographic run, by making a blank injection and evaluating the outcome.  If required flush your column (consult your column manufacturer).

 

Figure 32. Recommended flushing procedure for an HPLC column.

 
 

Routinely flush the column with a high elution strength solvent when performing isocratic separations, to wash any strongly retained non-polar compounds off the column. This is illustrated in the figure below, where the peak shape of a variety of basic drug compounds being separated isocratically in a 25mM Na2HPO4 (pH3.0):MeOH (60:40) mobile phase are significantly improved following a column wash with 100% acetonitrile.

 

Figure 33.  Injection effect on peak shape.

 
 

If too great a sample volume has been injected, and the sample solvent and mobile phase are not correctly matched, then two distribution equilibriums will occur, resulting in differential sample partitioning on-column. The consequence will be the formation of doublet peaks or peak shouldering.  Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows.  You can perform serial dilutions from a more concentrated sample, altering the eluent strength with each dilution to prevent precipitation in many instances.  Alternatively, one should restrict the injection volume to below 10μL.

 

Figure 34.  Peak distortion due to injection solvent and mobile phase mismatch.  The sample is a mixture of parabens. 
Column C18, 3.0 x 50 mm, 1.8 μm.  Temperature 45oC.  Mobile phase: 1.0 mL/min (30 - 65% CH3CN).

 

Table 12 helps you to identify the origin and propose remedial actions when peak shouldering or splitting problems occur.

 
 

Table 12.  Peak shouldering and splitting.

Component Potential cause Potential Solution(s)
Mobile phase Mobile phase wrongly prepared
  • Change buffer strength
  • Change pH – it is possible to observe ‘shouldering’ behavior when the mobile  phase pH is very close to the pKa of the analyte
  • Change mobile phase composition
  • If necessary, raise column temperature or change column type
  • When preparing mobile phases, remember to add the organic component to the buffer to avoid buffer precipitation
Mobile phase incompatible with sample solvent
  • Ensure mobile phase compatibility with buffer concentration
  • If possible inject sample in mobile phase
  • Decrease ionic strength or water organic solvent ratio
  • Select another mobile phase system
  • Inject the smallest sample volume possible to encourage mixing between the eluent and diluent
Mobile phase contaminated or deteriorated
  • Prepare fresh mobile phase
  • Use only HPLC grade water and chemicals
  • A single shouldering peak can result from a small peak co-eluting with the chromatographic peak of interest
Autosampler Sample solvent incompatible with mobile phase
  • Ensure mobile phase compatibility with buffer concentration
  • If possible inject sample in mobile phase
  • Decrease ionic strength or water organic solvent ratio
Loading contaminated sample
  • A single shouldering peak can result from a small peak co-eluting with the chromatographic peak of interest
  • Load fresh sample
Column Wrong column Change column type
Column degradation
  • Use polymeric, sterically protected, or high-coverage reversed-phase column
  • Reduce temperature
  • A single shouldering peak can result from a small peak co-eluting with the chromatographic peak of interest
  • Correct column working conditions (for example: most HPLC columns use mobile phase pH between 2 and 8)
Contamination
  • A single shouldering peak can result from a small peak coeluting with the chromatographic peak of interest
  • Flush column with strong solvent and reverse flush where necessary
  • Replace column
Channeling Replace the column
Other Low dead volume Minimize low dead volume
 
 

Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion.  A peak is classified as tailing if its asymmetry is greater than 1.2, although peaks with As values as high as 1.5 are acceptable for many assays.

 

Figure 35.  Peak tailing.

 

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention. In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase. However, polar interactions with any ionized residual silanol groups residing on the silica support surface are also common. Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups, producing tailing peaks. This is illustrated in the figure shown below at a mobile phase pH >3.0.

 

Figure 36.  Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing.

 
 

Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH, thereby ensuring the full protonation of such ionisable residual silanol groups.

 

Figure 37.  Reduction of secondary interactions through pH adjustment (low pH in this case).

 
 

Alternatively an end-capped column can be utilized as previously discussed.

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded. If necessary use a higher capacity stationary phase i.e. increased % carbon or pore size, use a column with an increased column diameter, or decrease the absolute sample amount or volume injected.

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit. Substituting the column will quickly confirm the problem.

If only one, or some, of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects.

 

Figure 38.  The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes.

 
 

Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5−10mM, will also reduce or possibly eliminate tailing of basic compounds. The TEA competes with basic polar analytes for binding to the residual silanol groups, performing a pseudo end-capping function. However, this approach is less favored, as the column’s stationary phase will be irreversibly altered by the addition of such a modifier, affecting subsequent selectivity shown by the separation system.

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes. An ion-exchange mode of chromatography is another option for ionic analyte species.

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest. The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored. As was described under peak splitting and shouldering, confirm the presence of an interferent by changing the detection wavelength, or improve the resolution of the separation by using a column offering greater efficiency i.e. a longer column or a column packed with smaller sized particles.

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type, separation temperature or the column stationary phase. Use a sample clean-up procedure i.e. SPE to remove any interfering contaminants. Evaluate if any co-eluting peak may have come from a previous chromatographic run, by making a blank injection and evaluating using the procedure described under "Peak Broadening".

The table below helps you to identify the origin and propose remedial actions when peak tailing occurs.

 
 

Table 13.  Peak tailing.

Component Potential cause Potential Solution(s)
Mobile phase Mobile phase wrongly prepared
  • pH and buffer strength of the eluent play a major part in the degree of interaction between surface silanol species and polar / ionised analytes – ensure the eluent buffer strength and pH are correct
  • Change mobile phase composition
  • If necessary, raise column temperature or change column type
  • When preparing mobile phases, remember to add the organic component to the buffer and never otherwise, by so doing buffer precipitation is prevented
Mobile phase incompatible with sample solvent
  • Ensure mobile phase compatibility with buffer concentration
  • If possible inject sample in mobile phase
  • Decrease ionic strength or water organic solvent ratio
  • Select another mobile phase system
Autosampler Sample solvent incompatible with mobile phase
  • Ensure mobile phase compatibility with buffer concentration
  • If possible inject sample in mobile phase
  • Decrease ionic strength or water organic solvent ratio
Column Wrong column Change column type
Analyte-stationary phase interaction
  • The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention
  • Change buffer strength
  • Change pH
  • Change mobile phase composition
  • If necessary, raise column temperature or change column type
  • Eliminate any column secondary interactions by using a mobile phase modifier or buffering the mobile phase appropriately
Column degradation
  • Use polymeric, sterically protected, or high-coverage reversed-phase column
  • Reduce temperature
  • Correct column working conditions (for example: most HPLC columns use mobile phase pH between 2 and 8)
Other Low dead volume Minimize low dead volume
 
 

Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded. If necessary use a higher capacity stationary phase i.e. increased % carbon or pore size, use a column with an increased column diameter, or decrease the absolute sample amount or volume injected.

 

Figure 39.  Peak fronting.

 

If too great a sample volume has been injected, and the sample solvent and mobile phase are not correctly matched, then two distribution equilibriums will occur, resulting in differential sample partitioning on-column.[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminating this phenomena.

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest. The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored. As was described under peak splitting and shouldering, confirm the presence of an interferent by changing the detection wavelength, or improve the resolution of the separation by using a column offering greater efficiency i.e. a longer column or a column packed with smaller sized particles.

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type, separation temperature or the column stationary phase. Use a sample clean-up procedure i.e. SPE to remove any interfering contaminants. Evaluate if any co-eluting peak may have come from a previous chromatographic run, by making a blank injection and evaluating.

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs.

 
 

Table 14.  Peak fronting.

Component Potential cause Potential Solution(s)
Mobile phase Mobile phase wrongly prepared
  • Change buffer strength
  • Change pH
  • Change mobile phase composition
  • If necessary, raise column temperature or change column type
  • When preparing mobile phases, remember to add the organic component to the buffer and never otherwise, by so doing buffer precipitation is prevented
Mobile phase incompatible with sample solvent
  • Ensure mobile phase compatibility with buffer concentration
  • If possible inject sample in mobile phase
  • Decrease ionic strength or water organic solvent ratio
  • Select another mobile phase system
Mobile phase contaminated or deteriorated
  • Prepare fresh mobile phase
  • Use only HPLC grade water and chemicals
  • A single fronting peak can result from a small peak eluting just before the chromatographic peak of interest
Autosampler Sample solvent incompatible with mobile phase
  • Ensure mobile phase compatibility with buffer concentration
  • If possible inject sample in mobile phase
  • Decrease ionic strength or water organic solvent ratio
Loading contaminated sample
  • A single fronting peak can result from a small peak eluting just before the chromatographic peak of interest
  • Load fresh sample
Column Wrong column Change column type
Column overloaded
  • Load smaller amount of sample
  • Use column with an increased diameter
  • If necessary use a higher capacity stationary phase
Wrong column temperature Adjust as appropriate
Analyte-stationary phase interaction
  • Change buffer strength
  • Change pH
  • Change mobile phase composition
  • If necessary, raise column temperature or change column type
  • Eliminate any column secondary interactions by using a mobile phase modifier or buffering the mobile phase appropriately
Silanol interactions with basic solutes
  • Use competing base such as triethylamine
  • Use a stronger mobile phase
  • Use base-deactivated silica-based reversed-phase column
  • Use polymeric column
Chelating solute interaction with trace metals in base silica
  • Use high purity silica-based column with low trace-metal content
  • Add EDTA or chelating compound to mobile phase
  • Use polymeric column
Column degradation
  • A single fronting peak can result from a small peak eluting just before the chromatographic peak of interest
  • Use polymeric, sterically protected, or high-coverage reversed-phase column
  • Reduce temperature
  • Correct column working conditions (for example: most HPLC columns use mobile phase pH between 2 and 8)
Contamination
  • A single fronting peak can result from a small peak eluting just before the chromatographic peak of interest
  • Flush column with strong solvent
  • Replace column
  • Restore column’s performance
Channeling Replace or repack column
Other Low dead volume Minimize low dead volume
 


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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. “Reverse Phase (Partition) Chromatography” from “The Theory of HPLC” of CHROMacademy
10. “Gradient HPLC” from “The Theory of HPLC” of CHROMacademy
11. “Mobile Phase Considerations” from “The Theory of HPLC” of CHROMacademy
12. Justin Chow, John W. Dolan.  “Retention Changes”.  January 2012
13. John W. Dolan.  “Troubleshooting Basics, Part 3: Retention Problems”  December 2011
14. John W. Dolan.  “Variability — How to Control It.  Why Aren't Retention Times Constant?”  December 2007
15. John Dolan.  “Peak Shape Problems”  July 2008
16. John Dolan.  “Split Peaks — A Case Study”  January 2005
17. Ryan D. Morrison and John W. Dolan.  “Peak Fronting, Column Life, and Column Conditioning”  April 2005
18. John Dolan.  “The Power of Mobile Phase Strength”  July 2006

 
 

Related Reference Materials from CHROMacademy:

Articles:

Peak Shape Problems – What causes fronting and tailing    *** CHROMacademy Registered users only ***

HPLC variability and how to control it    *** CHROMacademy Registered users only ***

Broad Peaks – HPLC Troubleshooting    *** CHROMacademy Registered users only ***

HPLC Retention Time Changes    *** CHROMacademy Registered users only ***

Troubleshooting Basics, Part I: Where to Start?    *** CHROMacademy Registered users only ***

 

HPLC E-Learning Modules:

HPLC Pump Systems    *** CHROMacademy Registered users only ***

Understanding HPLC Autosamplers    *** CHROMacademy Registered users only ***

Understanding HPLC Detectors    *** CHROMacademy Registered users only ***

 

Essential Guide Webcasts / Tools:

HPLC Troubleshooting - Eluents and Solvent Delivery Systems    *** CHROMacademy Registered users only ***

HPLC Troubleshooting – Autosamplers, Columns and Detectors    *** CHROMacademy Registered users only ***

Mobile Phase Optimization Strategies for Reversed Phase HPLC    *** CHROMacademy Registered users only ***

 


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The following subjects are covered in CHROMacademy.com

The Theory Of HPLC
Introduction (1.5hrs)
Chromatographic Parameters (3hrs)
Band Broadening (3hrs)
Column chemistry (4hrs)
Reverse phase (partition) chromatography (6hrs)
Ion-Pair Chromatography (3hrs)
Normal phase (absorption) chromatography (3hrs)
Gradient HPLC (3hrs)
Quantitative and Qualitative HPLC (3hrs)
FAST HPLC (4.5hrs)
HILIC (3hrs)
SFC (3hrs)
Ion Chromatography(3hrs)

Theory and Instrumentation of GC
Introduction (1.5hrs)
Chromatographic Parameters (3hrs)
Band Broadening (3hrs)
Gas Supply and Pressure Control (2hrs)
Sampling Techniques (4.5hrs)
Sample Introduction (5hrs)
GC Columns (5.5hrs)
GC Temperature Programming (3hrs)
GC Detectors (2.5hrs)
SFC (3hrs)

Instrumentation of HPLC
Mobile Phase Considerations (3.5hrs)
Solvent Pumping Systems (4hrs)
Autosamplers (4.5hrs)
Detectors (4.5hrs)

Solid Phase Extraction
Molecular Properties (4hrs)
SPE Overview (3.5hrs)
SPE Mechanisms (4.5hrs)
Method Development (5.0hrs)
Primary Sample Preparation Techniques (2hrs)
Liquid / Liquid Extraction Techniques (1.5hrs) Approaches to Automation for SPE (1.5hrs)

Fundamental GC-MS
Introduction (1.5hrs)
GC Considerations (4.5hrs)
GC -MS Interfaces (2.5hrs)

Fundamental LC-MS
Introduction (1.5hrs)
Electrospray Ionisation Theory (6hrs)
Electrospray Ionisation Instrumentation (4hrs)
Mass Analyzers (9.5hrs)
Atmospheric Pressure Chemical Ionisation (3.5hrs)
Atmospheric Pressure Photoionisation (3hrs)
Solvents, Buffers and Additives (3.5hrs)
Vacuum Systems (3hrs)
Flow Rates and Flow Splitting (3hrs)
Orbitrap Mass Analyzers (3hrs)

MS Interpretation
General Interpretation Strategies (11hrs)
Intro to MS Proteomics Research (3.5hrs)

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