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An Alternative Approach to Troubleshooting Variable Peak Height/Area in HPLC

When a problem with an HPLC separation occurs it is often best to take a step back and think about what may have caused the problem, before taking any drastic action such as dismantling the system and then finding that you don’t know how to put it back together, or that once you do, the problem still persists.  An Ishikawa diagram (also called a Fishbone diagram) is a useful tool that can be used to aid in troubleshooting.  They are used to show the causes of a specific problem.  The branches of the diagram that are more highly populated may have a greater influence on the final problem.

The problem of variable (irreproducible) peak height/area is a common and complex one, with many potential causes.  We have constructed an Ishikawa diagram to look at all the possible causes of variable peak height/area and to help prioritise the approach to fixing the problem.  Use the interactive diagram below to explore this more, or download the PDF as a useful reference tool for your lab. 

Download - Ishikawa Variable Peak/Height Area Diagram

Fishbone diagram

Inconsistent sample volume injected

Running an autosampler linearity test is a good way to identify this problem and get an idea of what may be going wrong.

    1. Sample too viscous
    2. Blocked sample needle
    3. Sample vial over/under filled
    4. Leaking sample valve
    5. Incorrect sample loop size

Loading air on injection
    1. Poorly made connections

    2. Leaking injection valve/sample loop
    3. Wrong needle depth
    4. Sample vial under filled
Leaking autosampler components

If the leak is not obvious check each component/connection using blue laboratory (absorbent) paper around each fitting and identify small leaks by looking for darkening of the paper.

  1. Poorly made connections
  2. Damaged rotor seal

    The operating conditions of the LC system can influene the choice of rotor seal material

Injector blockage

    1. Unfiltered mobile phase
      Here we need to consider the actual pore/frit size which is recommended for the various stationary phase particle size/system combinations.
      <3 µm particle size packing material – use a 0.2 µm porosity filter
      >3 µm particle size packing material – use a 0.45 µm porosity filter

    2. Insoluble sample components
Autosampler contamination
    1. Contaminated needle wash solvent
    2. Contamination from wash bottle cap
      When a needle wash bottle or vial is used, this should remain uncapped to prevent cross contamination from the cap itself

    3. Analyte or sample matrix absorption onto metal or plastic surfaces
      Autosampler contamination usually manifests itself as poor quantitative reproducibility, carryover (peaks in a blank injection which is made following a sample) or ghost peaks.

Faulty/ageing lamp

Leaking flow cell/cracked flow cell window

    1. Damaged gaskets

      Cross section of a UV flow cell

    2. Blockages in the flow cell fittings
    3. Flow cell pressure limit exceeded

Incorrect detector sampling rate

It is important to ensure that the sampling rate is correct, especially when working with Fast HPLC techniques to ensure that enough points are recorded across each peak for proper ‘modelling’ of the peak area.  Generally 20-25 points across a peak are required for accurate quantification.  Having an incorrect sampling rate can affect the peak shape, and if the rate is too low the peaks may look broad and one might interpret this effect as low column (system) efficiency.  A very high sampling rate can lead to the baseline appearing very noisy

Contaminated detector cell

Most flow cells can be cleaned with water, warm water, methanol or other solvent (0.1N nitric acid solution is sometines used in extreme circumstances) but be sure to follow manufacturer’s instructions.

Leaking inlet/outlet fitting
    1. Damaged fitting
    2. Incorrect fitting
      The wrong fitting will result in leaks i.e. a flat bottomed fitting in a coned port
    3. Poorly made connection
    4. Poorly cut tubing

In-line filter or guard column blocked/contaminated

This issue can lead to increased system backpressure, but also to degraded separation characteristics such as loss of efficiency or poor peak shape

    1. Strongly/irreversibly retained sample components
    2. Insoluble sample components
    3. Unfiltered mobile phase

Here we need to consider the actual pore/frit size which is recommended for the various stationary phase particle size/system combinations.

<3 µm particle size packing material – use a 0.2 µm porosity filter
>3 µm particle size packing material – use a 0.45 µm porosity filter

Contaminated water or solvents

Contaminated water or solvents can lead to increased background signal, system peaks and ghost peaks.

    1. Using recycled mobile phase
    2. Not using HPLC grade reagents (solvents, buffers etc.)
pH variation
    1. Using incorrect buffer salt

      Buffers are only effective within ±1 pH unit of their pKa.

      Buffer pKa pH range
      Phosphate 2.1, 7.2, 12.3 1. -3.1, 6.2-8.2,
      Citrate 3.1, 4.7, 6.4 2.1- 4.1, 3.7-5.7, 5.4-7.4
      Carbonate 6.1, 10.3 5.1-7.1, 9.3-11.3
      Formate 3.8 2.8-4.8
      Acetate 4..8 3.8-5.8
      Ammonia 9.3 8.3-10.3
      Borate 9.2 8.2-10.2

    2. Using old buffer

      Eluent lifetimes should be decided on a lab to lab basis.  For example,

      Mobile Phase Shelf-life
      H2O from purification system 3 days
      Aqueous solutions (without buffer) 3 days
      Buffer solutions 3 days
      Aqueous solutions with < 15% organic solvent 1 month
      Aqueous solutions with > 15% organic solvent 3 months
      Organic solvents 3 months

    3. Analyte(s) insoluble (partially insoluble) in mobile phase
Analyte(s) insoluble (partially insoluble) in mobile phase
Inconsistent flow rate
    1. Sticking check valve
    2. Air trapped in pump head

High pump dead volume

Incorrect tubing length/diameter

This issue mostly affects operation in gradient analysis mode.  Typically called the ‘gradient dwell volume’ the pump dead volume affects the time taken between the mixing of the solvents within the pump and the delivery of those solvents to the HPLC column (along with the internal volume of system tubing and the autosampler volume).

Column end frit(s) blocked/contaminated

Typically the problem can be solved by reverse flushing the column (disconnect from the detector first) in a solvent which will either flush away trapped particulate materials or dissolved absorbed contaminants, in which case one must choose a flushing solution in which the suspected contaminants are highly soluble.

    1. Unfiltered mobile phase
    2. Insufficient sample clean up
    3. No guard column or in-line filter

Analyte(s) or components strongly retained
    1. Insufficient sample clean up
    2. Incorrect stationary phase

Temperature variation

Oversimplified temperature profile in an HPLC column with temperature control

Sample too viscous

Aspiration rate may need to be altered

Sample deterioration
    1. Incorrect sample diluent
      Sample does not dissolve completely
      Immiscible solvents
      Sample diluent stronger than the mobile phase

    2. Incorrect sample storage
      Is the sample temperature sensitive?  Store in the fridge or freezer
      Is the sample light sensitive?  Use amber vials

Unstable derivative

Ensure the derivative being used forms a stable derivative under the analysis conditions being used

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