HPLC Troubleshooting


General Pattern:

  • Locate the problem by ranking possible causes.
  • Verify the presence of the most probable cause.
  • If present – fix the problem, otherwise verify the existence of the next possible cause.

First try to distinguish System problem or Method Problem

HPLC System Components

  • Pump
  • Injector/ Autosampler
  • Column
  • Detector
  • Data System/Integrator

Method vs. System Troubleshooting

System Parameters

  • Flow stability
  • Backpressure
  • Clogging
  • Detector problems
  • Injection suitability

Method Parameters

  • Flow rate
  • Eluent composition
  • pH &pH modifier (type)
  • Injection volume
  • Temperature
  • Gradient profile

System Parameters

Simple preliminary verification of system setup can save time

SolventDegasserPumpAuto samplercolumnDetector
Bottle fill-in Inlet filter dateFlush if solvent change >15 mLBack pressure Flow stability Check-valvesVial fill-in connections cross-contaminationColumn type connectionsWavelength

Categories of Column and System Problems

  • Pressure
  • Peak shape
  • Retention
  • Detection

I. Pressure Issues

Column observationsPotential Problems
High pressurePlugged frit
Column contamination
Plugged packing
Low pressureLeak
Flow Incorrect

Determining the Cause and Correcting High Back Pressure

Many pressure problems are due to blockages in the system.

If Column pressure is high:

  • Back flush column – Clear “dirty” frit surface
  • Wash column – Eliminate column contamination and plugged packing
  • – high molecular weight/adsorbed compounds
  • – precipitate from sample or buffer

Column Cleaning

Flush with stronger solvents than your mobile phase

Use at least 25 mL of each solvent for analytical columns

Reversed-Phase Solvent Choices in Order of Increasing Strength

  • Mobile phase without buffer salts
  • 100% Methanol
  • 100% Acetonitrile
  • 75% Acetonitrile:25% Isopropanol
  • 100% Isopropanol
  • 100% Methylene Chloride*
  • 100% Hexane*

* When using either Hexane or Methylene Chloride the column must be flushed with Isopropanol before returning to your reversed-phase mobile phase.

Prevention Techniques for column problems

Use column protection

  • In-line filters
  • Guard columns
  • Filter samples
  • Filter buffered mobile phases
  • Sample clean-up (i.e. SPE)
  • Appropriate column flushing

II.           Peak Shape Issue

What Are Common Peak Shape Issues?

  1. Split peaks
  2. Peak tailing
  3. Broad peaks
  • Many peak shape issues are also combinations – i.e. broad and tailing or tailing with increased retention
  • Symptoms do not necessarily affect all peaks in the chromatogram
  • Each of these problems can have multiple causes

Peak Splitting Caused By Disrupted Sample Path

  • Flow Path Disrupted by Void
  • Sample Allowed to Follow Different Paths through Column
  • Poorly Packed Bed Settles in Use
  • High pH Dissolves Silica

Split Peaks from Column Contamination

Column: Stable Bond SB-C8, 4.6 x 150 mm, 5 μm Mobile Phase: 60% 25 mM Na2HPO4, pH

3.0 : 40% MeOH Flow Rate: 1.0 mL/min

Temperature: 35°C Detection: UV 254 nm Sample: Filtered OTC Cold Medication:   

1.Pseudoephedrine 2. APAP 3. Chlorpheniramine

Peak Tailing, Broadening and Loss of Efficiency

May be caused by:

  • Column “secondary interactions”
  • Column contamination
  • Column aging
  • Column loading
  • Extra-column effects

Peak Tailing – Column Contamination

Trick: Reverse Column and Run Sample –If Improved, Possible Cleaning Will Help -No improvement-Column Damaged and Needs to be Replaced

Peak Shape: Fronting Peaks

Symmetry < 0.9

Causes: Column Overload

Peak Shape: Broad Peaks

All Peaks Broadened:

  • Loss of Column Efficiency.
  • Column Void.
  • Large Injection Volume

Some Peaks Broadened:

  • Late Elution from Previous Sample (Ghost Peak).
  • High Molecular Weight.
  • Sample – Protein or Polymer.

III.   Changes in Retention Time

Changes in Retention Can Be Chemical or physical May be caused by:

  • Column aging
  • Column contamination
  • Insufficient equilibration
  • Poor column/mobile phase combination
  • Change in mobile phase
  • Change in flow rate

Mobile Phase pH and pH Buffers Why Are These So Important in HPLC?

pH Effects Ionization

  • Silica Surface of Column
  • Sample Components of Interest

Buffers

  • Resist Changes in pH and Maintain Retention
  • Improve Peak Shape for Ionizable Compounds

Effects Column Life

  • Low pH strips Bonded Phase
  • High pH Dissolves Silica

Importance of pH and Buffers

  • pH is an effective tool for adjustment of selectivity and retention
  • pH can be used to optimize the resolution
  • Reversed phase packaging are most stable between pH’s 2 – 8.
  • Don’t Forget – Match Column to pH of mobile phase for maximum column lifetime.

IV. Detection Issues

Recognize Where the Problem Originates

  • Is it a consequence of technique?
  • Is It expected due to use of certain mobile phase components?
  • Can it be corrected by adjusting detector parameters?

Drifting Baselines

  • Detector (UV) not set at absorbance maximum but at slope of curve
  • Gradient Elution
  • Temperature Unstable (Refractive Index Detector)
  • Contamination in Mobile Phase
  • Mobile Phase Not in Equilibrium with Column

Baseline Noise

Ø  Mobile phase contaminated, deteriorated, or prepared from low-quality materials
Ø  Mobile phase solvents immiscible
 
Ø  Air trapped in system
 
Ø  Air bubbles in detector
 
Ø  Detector cell contaminated (even small amounts of contaminants can cause noise)
Ø  Weak detector lamp

Conclusions

HPLC column problems are evident as

  • High pressure (prevention better than the cure)
  • Undesirable peak shape
  • Changes in retention/selectivity

Often these problems are not associated with the column and may be caused by instrument and chemistry issues.

  • pH of mobile Phase
  • Instrument Connections
  • Detector Settings
  • Metal Contamination

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