HPLC Troubleshooting guide to visualize the posible solution !

HPLC Troubleshooting Guide

Although HPLC method development has been improved by advances in column technology and instrumentation, problems still arise. In this guide we offer you a systematic means of isolating, identifying, and correcting many typical HPLC problems.

The important segments of an HPLC system are the same, whether you use a modular system or a more sophisticated unit. Problems affecting overall system performance can arise in each component. Some common problems are discussed here. Solutions to these problems are presented in easy to use tables.

1: Isolating HPLC Problems

In an HPLC system, problems can arise from many sources. First define the problem, then isolate the source.

Use HPLC Problem Index to determine which component(s) may be causing the trouble. A process of elimination will usually enable you to pinpoint the specific cause and correct the problem.

HPLC troubleshooting
2: How to Prevent Mobile Phase Problems

Low sensitivity and rising baselines, noise, or spikes on the chromatogram can often be attributed to the mobile phase. Contaminants in the mobile phase are especially troublesome in gradient elution. The baseline may rise, and spurious peaks can appear as the level of the contaminated component increases.

Water is the most common source of contamination in reversed phase analyses. You should use only high purity distilled or deionized water when formulating mobile phases. However, several common deionizers introduce organic contaminants into the water. To remove these contaminants, pass the deionized water through activated charcoal or a preparative C18 column.

Use only HPLC grade solvents, salts, ion pair reagents, and base and acid modifiers. Cleaning lower quality solvents is time consuming, and trace levels of contaminants often remain. These trace contaminants can cause problems when you use a high sensitivity ultraviolet or fluorescence detector.

Because many aqueous buffers promote the growth of bacteria or algae, you should prepare these solutions fresh, and filter them (0.2 μm or 0.45 μm filter) before use. Filtering also will remove particles that could produce a noisy baseline, or plug the column. Prevent microorganism growth by adding about 100 ppm of sodium azide to aqueous buffers. Alternatively, these buffers may also be mixed with 20% or more of an organic solvent such as ethanol or acetonitrile.

To prevent bubbles in the system, degas the mobile phase. Generally an in-line degasser is a first choice, but sparging with helium can be an alternative if the mobile phase does not contain any volatile components.

Use ion pair reagents carefully. The optimum chain length and concentration of the reagent must be determined for each analysis. Concentrations can be as low as 0.2 mM, or as high as 150 mM, or more. In general, increasing the concentration or chain length increases retention times. High concentrations (>50%) of acetonitrile or some other organic solvents can precipitate ion pair reagents. Also, some salts of ion pair reagents are insoluble in water and will precipitate. Avoid this by using sodium-containing buffers in the presence of long chain sulfonic acids (e.g., sodium dodecyl sulfate), instead of potassium-containing buffers.

Volatile basic and acidic modifiers, such as triethylamine (TEA) and trifluoracetic acid (TFA), are useful when you wish to recover a compound for further analysis. These modifiers also let you avoid problems associated with ion pair reagents. They can be added to the buffer at concentrations of 0.1 to 1.0% TEA or 0.01 to 0.15% TFA. Increasing the concentration may improve peak shape for certain compounds, but can alter retention times.

Recycling the mobile phase used for isocratic separations has become more popular as a means of reducing the cost of solvents, their disposal, and mobile phase preparation time. A solvent recovery apparatus uses a microprocessor controlled switching valve to direct the solvent stream to waste when a peak is detected. When the baseline falls under the selected threshold, uncontaminated solvent is directed back to the solvent reservoir. [Index]

hplc intrumentation
Figure A. Components of an HPLC System
HPLC troubleshooting
3: Isolating Pump Problems

The pump must deliver a constant flow of solvent to the column over a wide range of conditions. HPLC pumps incorporate single or dual piston, syringe, or diaphragm pump designs.

Pumping system problems are usually easy to spot and correct. Some of the more common symptoms are erratic retention times, noisy baselines, or spikes in the chromatogram. Leaks at pump fittings or seals will result in poor chromatography. A sure sign of a leak is a buildup of salts at a pump connection. Buffer salts should be flushed from the system daily with fresh deionized water. To isolate and repair specific problems related to your apparatus, use the troubleshooting and maintenance sections of the operation manual. Pump seals require periodic replacement. You should perform regular maintenance rather than waiting for a problem to occur.[Index]

HPLC troubleshooting
4: Injector and Injection Solvents

The injector rapidly introduces the sample into the system with minimal disruption of the solvent flow. HPLC systems currently use variable loop, fixed loop, and syringe-type injectors. These are activated manually, pneumatically, or electrically.

Mechanical problems involving the injector (e.g., leaks, plugged capillary tubing, worn seals) are easy to spot and correct. Use a precolumn filter to prevent plugging of the column frit due to physical degradation of the injector seal. Other problems, such as irreproducible injections, are more difficult to solve.

Mechanical problems involving the injector (e.g., leaks, plugged capillary tubing, worn seals) are easy to spot and correct. Use a precolumn filter to prevent plugging of the column frit due to physical degradation of the injector seal. Other problems, such as irreproducible injections, are more difficult to solve.

Variable peak heights, split peaks, and broad peaks can be caused by incompletely filled sample loops, incompatibility of the injection solvent with the mobile phase, or poor sample solubility. Whenever possible, dissolve and inject samples in mobile phase. Otherwise, be sure the injection solvent is of lower eluting strength than the mobile phase. Be aware that some autosamplers use separate syringe washing solutions. Make sure that the wash solution is compatible with and weaker than the mobile phase[Table3]. This is especially important when switching between reversed and normal phase analyses.[Index]

HPLC troubleshooting
5: Column Protection

Although not an integral part of most equipment, mobile phase inlet filters, pre-injector and pre-column filters, and guard columns greatly reduce problems associated with complex separations. We recommend that all samples be filtered through 0.45 μm or 0.2 μm syringe filters. We strongly recommend the use of guard columns.

Filters and guard columns prevent particles and strongly retained compounds from accumulating on the analytical column. The useful life of these disposable products depends on mobile phase composition, sample purity, pH, etc. As these devices become contaminated or plugged with particles, pressure increases and peaks broaden or split. As an example, Figure B presents a clear case for the use of guard columns.[Index]

Figure B
HPLC troubleshooting
6: Getting the Most from Your Analytical Column

Regardless of whether the column contains a bonded reversed or normal phase, ion exchange, affinity, hydrophobic interaction, size exclusion, or resin/silica based packing, the most common problem associated with analytical columns is deterioration. Symptoms of deterioration are poor peak shape, split peaks, shoulders, loss of resolution, decreased retention times, and high back pressure. These symptoms indicate contaminants have accumulated on the frit or column inlet, or there are voids, channels, or a depression in the packing bed.

Deterioration is more evident in higher efficiency columns. For example, a 3 micron packing retained by 0.5 micron frits is more susceptible to plugging than a 5 or 10 micron packing retained by 2 micron or larger frits. Proper column protection and sample preparation are essential to getting the most from each column.[Index]

Overloading a column can cause poor peak shapes and other problems.

Typical Column Capacity

Column capacity depends on many factors, but typical values for total amounts of analytes on a column are:

  • Column dimensions
    • 25 cm x 4.6 mm
  • Column capacity
    • <500 μg

 

  • Column dimensions
    • 25 cm x 21.mm
  • Column capacity
    • <500 mg
  • Column dimensions
    • 25 cm x 4.6 mm
  • Column capacity
    • <500 μg

 

HPLC troubleshooting
7: Solving Detector Problems

Detector problems fall into two categories electrical and mechanical/optical. For electrical problems, you should contact the instrument manufacturer. Mechanical or optical problems usually can be traced to the flow cell. Detector related problems include leaks, air bubbles, and cell contamination. These usually produce spikes or baseline noise on the chromatograms or low sensitivity.

Some cells especially those used in refractive index detectors are sensitive to pressure. Flow rates or back pressures that exceed the manufacturer’s recommendation will break the cell window. Old or defective lamps as well as incorrect detector rise time, gain, or attenuation will reduce sensitivity and peak height. Faulty or reversed cable connections can also be the source of problems.[Index]

HPLC troubleshooting
8: Column Heater, Recorder

These components seldom cause problems with the system. They will be discussed in the troubleshooting [Table1].

HPLC troubleshooting
Most problems don’t occur overnight, but develop gradually. Accurate record keeping, then, is vital to detecting and solving many problems.
Evaluate every column you receive, when you receive it and at regular intervals thereafter. By keeping a written history of column efficiency, mobile phases used, lamp current, pump performance, etc., you can monitor your system’s performance.

Records also help prevent mistakes, such as introducing water into a silica column, or precipitating buffer in the system by adding too much organic solvent. Many analysts modify their HPLC systems in some way. Reliable records are the best way to ensure that a modification does not introduce problems. For problems relating to pumps, detectors, automatic samplers, and data systems, consult your instrument manual’s troubleshooting guide.[Index]

HPLC troubleshooting
HPLC troubleshooting
Table 1. HPLC Problems, Probable Causes, and Remedies

  1. Column temperature fluctuation. (Even small changes cause cyclic baseline rise and fall. Most often affects refractive index and conductivity detectors, UV detectors at high sensitivity or in indirect photometric mode.)

  2. Nonhomogeneous mobile phase. (Drift usually to higher absorbance, rather than cyclic pattern from temperature fluctuation.)

  3. Contaminant or air buildup in detector cell.

  4. Plugged outlet line after detector. (High pressure cracks cell window, producing noisy baseline.)

  5. Mobile phase mixing problem or change in flow rate.

  6. Slow column equilibration, especially when changing mobile phase.

  7. Mobile phase contaminated, deteriorated, or not prepared from high quality chemicals.

  8. Strongly retained materials in sample (high k’) can elute as very broad peaks and appear to be a rising baseline. (Gradient analyses can aggravate problem.)

  9. Detector (UV) not set at absorbance maximum but at slope of curve.

  1. Control column and mobile phase temperature, use heat exchanger before detector.

  2. Use HPLC grade solvents, high purity salts, and additives. Degas mobile phase before use, sparge with helium during use.

  3. Flush cell with methanol or other strong solvent. If necessary, clean cell with 1N HNO3 (never with HCl and never use nitric acid with PEEK tubing or fittings.)

  4. Unplug or replace line. Refer to detector manual to replace window.

  5. Correct composition/flow rate. To avoid problem, routinely monitor composition and flow rate.

  6. Flush column with intermediate strength solvent, run 10-20 column volumes of new mobile phase through column before analysis.

  7. Check make-up of mobile phase.

  8. Use guard column. If necessary, flush column with strong solvent between injections or periodically during analysis.

  9. Change wavelength to UV absorbance maximum.

HPLC troubleshooting

  1. Leak.

  2. Mobile phase contaminated, deteriorated, or prepared from low quality materials.

  3. Detector/recorder electronics.

  4. Air trapped in system.

  5. Air bubbles in detector.

  6. Detector cell contaminated. (Even small amounts of contaminants can cause noise.)

  7. Weak detector lamp.

  8. Column leaking silica or packing material.

  1. Check system for loose fittings. Check pump for leaks, salt buildup, unusual noises. Change pump seals if necessary.

  2. Check make-up of mobile phase.

  3. Isolate detector and recorder electronically. Refer to instruction manual to correct problem.

  4. Flush system with strong solvent.

  5. Purge detector. Install backpressure regulator after detector. Check the instrument manual, particularly for RI detectors (excessive backpressure can cause the flow cell to crack).

  6. Clean cell.

  7. Replace lamp.

  8. Replace column and clean system.

HPLC troubleshooting

  1. Air in mobile phase, detector cell, or pump.

  2. Pump pulsations.

  3. Incomplete mobile phase mixing.

  4. Temperature effect (column at high temperature, detector unheated).

  5. Other electronic equipment on same line.

  6. Leak.

  1. Degas mobile phase. Flush system to remove air from detector cell or pump.

  2. Incorporate pulse damper into system.

  3. Mix mobile phase by hand or use less viscous solvent.

  4. Reduce differential or add heat exchanger.

  5. Isolate LC, detector, recorder to determine if source of problem is external. Correct as necessary.

  6. Check system for loose fittings. Check pump for leaks, salt buildup, unusual noises. Change pump seals if necessary.

HPLC troubleshooting

  1. Problem in pump, injector, in-line filter, or tubing.

  2. Obstructed guard column or analytical column.

  1. Remove guard column and analytical column from system. Replace with unions and 0.010'' I.D. or larger tubing to reconnect injector to detector. Run pump at 2-5 mL/min. If pressure is minimal, see Cause 2. If not, isolate cause by systematically eliminating system components, starting with detector, then in-line filter, and working back to pump. Replace filter in pump if present.

  2. Remove guard column (if present) and check pressure. Replace guard column if necessary. If analytical column is obstructed, reverse and flush the column, while disconnected from the detector. If problem persists, column may be clogged with strongly retained contaminants. Use appropriate restoration procedure (Table 2). If problem still persists, change inlet frit or replace column.

HPLC troubleshooting

  1. Leak.

  2. Mobile phase flow interrupted/obstructed.

  3. Air trapped in pump head. (Revealed by pressure fluctuations.)

  4. Leak at column inlet end fitting.

  5. Air trapped elsewhere in system.

  6. Worn pump seal causing leaks around pump head.

  7. Faulty check valve.

  8. Faulty pump seals.

  1. Check system for loose fittings. Check pump for leaks, salt buildup, unusual noises. Change pump seals if necessary.

  2. Check mobile phase level in reservoir(s). Check flow throughout system. Examine sample loop for obstruction or air lock. Make sure mobile phase components are miscible and mobile phase is properly degassed.

  3. Disconnect tubing at guard column (if present) or analytical column inlet. Check for flow. Purge pump at high flow rate (e.g., 10 mL/min.), prime system if necessary. (Prime each pump head separately.) If system has check valve, loosen valve to allow air to escape.

  4. Reconnect column and pump solvent at double the flow rate. If pressure is still low, check for leaks at inlet fitting or column end fitting.

  5. Disconnect guard and analytical column and purge system. Reconnect column(s). If problem persists, flush system with 100% methanol or isopropanol.

  6. Replace seal. If problem persists, replace piston and seal.

  7. Rebuild or replace valve.

  8. Replace seals.

HPLC troubleshooting

  1. Mobile phase composition changed.

  2. Mobile phase flow rate too low.

  3. Leak (especially between column and detector).

  4. Detector settings incorrect.

  5. Extra-column effects:
    a. Column overloaded

    b. Detector response time or cell volume too large.

    c. Tubing between column and detector too long or I.D. too large.

    d. Recorder response time too
    high.

  6. Buffer concentration too low.

  7. Guard column contaminated/worn out.

  8. Column contaminated/worn out.

  9. Void at column inlet.

  10. Peak represents two or more poorly resolved compounds.

  11. Column temperature too low.

  1. Prepare new mobile phase.

  2. Adjust flow rate.

  3. Check system for loose fittings. Check pump for leaks, salt buildup, and unusual noises. Change pump seals if necessary.

  4. Adjust settings.


  5. a. Inject smaller volume (e.g., 10 μL vs. 100 μL) or 1:10 and 1:100 dilutions of sample.

    b. Reduce response time or use smaller cell.

    c. Use as short a piece of
    0.007-0.010" I.D. tubing as practical.

    d. Reduce response time.

  6. Increase concentration.

  7. Replace guard column.

  8. Replace column with new one of same type. If new column does not provide narrow peaks, flush old column (Table 2), then retest.

  9. Replace column or open inlet end and fill void.

  10. Change column type to improve separation.

  11. Increase temperature. Do not exceed 75 °C unless higher temperatures are acceptable to column manufacturer.

HPLC troubleshooting

  1. Column overloaded.

  2. Sample solvent incompatible with mobile phase.

  3. Shoulder or gradual baseline rise before a main peak may be another sample component.

  1. Inject smaller volume (e.g., 10 μL vs. 100 μL). Dilute the sample 1:10 or 1:100 fold in case of mass overload.

  2. Adjust solvent. Whenever possible, inject samples in mobile phase. Flush polar bonded phase column with 50 column volumes HPLC grade ethyl acetate at 2-3 times the standard flow rate, then with intermediate polarity solvent prior to analysis.

  3. Increase efficiency or change selectivity of system to improve resolution. Try another column type if necessary (e.g., switch from nonpolar C18 to polar cyano phase).

HPLC troubleshooting

  1. Detector operating outside linear dynamic range.

  2. Recorder gain set too low.

  3. Column overloaded.

  4. Sample-column interaction.

  5. Detector and/or recorder time constants are set too high.

  1. Reduce sample volume and/or concentration.

  2. Adjust gain.

  3. Inject smaller volume (e.g., 10 μL vs. 100 μL) or 1:10 or 1:100 dilution of sample.

  4. Change buffer strength, pH, or mobile phase composition. If necessary, raise column temperature or change column type. (Analysis of solute structure may help predict interaction.)

  5. Reduce settings to lowest values or values at which no further improvements are seen.

HPLC troubleshooting

  1. Contamination on guard or analytical column inlet.

  2. Partially blocked frit.

  3. Small (uneven) void at column inlet.

  4. Sample solvent incompatible with mobile phase.

  1. Remove guard column (if present) and attempt analysis. Replace guard column if necessary. If analytical column is obstructed, reverse and flush. If problem persists, column may be clogged with strongly retained contaminants. Use appropriate restoration procedure (Table 2). If problem still persists, inlet frit is probably (partially) plugged. Change frit or replace column.

  2. Replace frit (see above).

  3. Repack top of column with pellicular particles of same bonded phase functionality. Continue using the column in reverse flow direction.

  4. Adjust solvent. Whenever possible, inject samples in mobile phase.

HPLC troubleshooting

  1. Guard or analytical column contaminated/worn out.

  2. Mobile phase contaminated/deteriorated.

  3. Interfering components in sample

  1. Remove guard column (if present) and attempt analysis. Replace guard column if necessary. If analytical column is source of problem, use appropriate restoration procedure (Table 2). If problem persists, replace column.

  2. Check make-up of mobile phase.

  3. Check column performance with standards.

HPLC troubleshooting

  1. Sample reacting with active sites.

  2. Wrong mobile phase pH.

  3. Wrong column type.

  4. Small (uneven) void at column inlet.

  5. Wrong injection solvent.

  1. First check column performance with standard column test mixture. If results for test mix are good, add ion pair reagent or competing base or acid modifier.
  2. Adjust pH. For basic compounds, lower pH usually provides more symmetric peaks.
  3. Try another column type (e.g., deactivated column for basic compounds).
  4. See Split Peaks.
  5. Peaks can tail when sample is injected in stronger solvent than mobile phase. Dissolve sample in mobile phase.

HPLC troubleshooting

  1. One or more sample components deteriorated or column activity changed.

  2. Leak, especially between injection port and column inlet. (Retention also would change.)

  3. Inconsistent sample volume.

  4. Detector or recorder setting changed.

  5. Weak detector lamp.

  6. Contamination in detector cell.

  1. Use fresh sample or standard to confirm sample as source of problem. If some or all peaks are still smaller than expected, replace column. If new column improves analysis, try to restore the old column, following appropriate procedure (Table 2). If performance does not improve, discard old column.

  2. Check system for loose fittings. Check pump for leaks, salt buildup, unusual noises. Change pump seals if necessary.

  3. Be sure samples are consistent.
    For fixed volume sample loop, use 2-3 times loop volume to ensure loop is completely filled. Be sure automatic sampler vials contain sufficient sample and no air bubbles. Check syringe-type injectors for air. In systems with wash or flushing step, be sure wash solution does not precipitate sample components.

  4. Check settings.


  5. Replace lamp.

  6. Clean cell.

HPLC troubleshooting

  1. Pump off.

  2. Flow interrupted/obstructed.

  3. Leak.

  4. Air trapped in pump head. (Revealed by pressure fluctuations.)

  1. Start pump.

  2. Check mobile phase level in reservoir(s). Check flow throughout system. Examine sample loop for obstruction or air lock. Make sure mobile phase components are miscible and mobile phase is properly degassed.

  3. Check system for loose fittings. Check pump for leaks, salt buildup, unusual noises. Change pump seals if necessary.

  4. Disconnect tubing at guard column (if present) or analytical column inlet. Check for flow. Purge pump at high flow rate (e.g., 5-10 mL/min.), prime system if necessary. (Prime each pump head separately.) If system has check valve, loosen valve to allow air to escape. If problem persists, flush system with 100% methanol or isopropanol. If problem still persists, contact system manufacturer.

HPLC troubleshooting

  1. Recorder leads reversed.

  2. Refractive index of solute less than that of mobile phase (RI detector).

  3. Sample solvent and mobile phase differ greatly in composition (vacancy peaks).

  4. Mobile phase more absorptive than sample components to UV wavelength.

  1. Check polarity.

  2. Use mobile phase with lower refractive index, or reverse recorder leads.

  3. Adjust or change sample solvent. Dilute sample in mobile phase whenever possible.

  4. a. Change polarity when using indirect UV detection, or

    b. Change UV wavelength or use mobile phase that does not adsorb chosen wavelength.

HPLC troubleshooting

  1. Detector lamp off.

  2. Loose/broken wire between detector and integrator or recorder.

  3. No mobile phase flow.

  4. No sample/deteriorated sample/ wrong sample.

  5. Settings too high on detector or recorder.

  1. Turn lamp on.

  2. Check electrical connections and cables.

  3. See “No Flow” (Problem No. 2).

  4. Be sure automatic sampler vials have sufficient liquid and no air bubbles in the sample. Evaluate system performance with fresh standard to confirm sample as source of problem.

  5. Check attenuation or gain settings. Check lamp status. Auto-zero if necessary.

HPLC troubleshooting

  1. Contamination in injector or column.

  2. Late eluting peak (usually broad) present in sample.

  1. Flush injector between analyses (a good routine practice). If necessary, run strong solvent through column to remove late eluters. Include final wash step in gradient analyses, to remove strongly retained compounds.

  2. a. Check sample preparation.

    b. Include (step) gradient to quickly elute component.

HPLC troubleshooting

  1. Mobile phase contaminated/deteriorated (causing retention times and/or selectivity to change).

  2. Obstructed guard or analytical column.

  1. Prepare fresh mobile phase.

  2. Remove guard column (if present) and attempt analysis. Replace guard column if necessary. If analytical column is obstructed, reverse and flush. If problem persists, column may be clogged with strongly retained contaminants. Use appropriate restoration procedure (Table 2). If problem still persists, change inlet frit or replace column.

HPLC troubleshooting

  1. Leak.

  2. Change in mobile phase composition. (Small changes can lead to large changes in retention times.)

  3. Air trapped in pump. (Retention times increase and decrease at random times.)

  4. Column temperature fluctuations (especially evident in ion exchange systems).

  5. Column overloading. (Retention times usually decrease as mass of solute injected on column exceeds column capacity.)

  6. Sample solvent incompatible with mobile phase.

  7. Column problem. (Not a common cause of erratic retention. As a column ages, retention times gradually decrease.

  1. Check system for loose fittings. Check pump for leaks, salt buildup, unusual noises. Change pump seals if necessary.

  2. Check make-up of mobile phase. If mobile phase is machine mixed using proportioning values, hand mix and supply from one reservoir.

  3. Purge air from pump head or check valves. Change pump seals if necessary. Be sure mobile phase is degassed.

  4. Use reliable column oven. (Note: higher column temperatures increase column efficiency. For optimum results, heat eluant before introducing it onto column.)

  5. Inject smaller volume (e.g., 10 μL vs. 100 μL) or inject the same volume after 1:10 or 1:100 dilutions of sample.

  6. Adjust solvent. Whenever possible, inject samples in mobile phase.

  7. Substitute new column of same type to confirm column as cause. Discard old column if restoration procedures fail.

HPLC troubleshooting

  1. Increase or decrease solvent ionic strength, pH, or additive concentration (especially affects ionic solutes).

  2. Column changed, new column has different selectivity from that of old column.

  3. Sample injected in incorrect solvent or excessive amount (100-200 μL) of strong solvent.

  4. Column temperature change.

  1. Check make-up of mobile phase.

  2. Confirm identity of column packing. For reproducible analyses, use same column type. Establish whether change took place gradually. If so, bonded phase may have stripped. Column activity may have changed, or column may be contaminated.

  3. Adjust solvent. Whenever possible, inject sample in mobile phase.

  4. Adjust temperature. If needed, use column oven to maintain constant temperature.

HPLC troubleshooting

The following procedures should rejuvenate a column whose performance has deteriorated due to sample contamination.

Disconnect and reverse the column. Connect it to the pump, but not the detector. Follow the appropriate flushing procedure in this table, using a flow rate that results in column back pressure of 1500-4500 psi, but never higher than the maximum recommended pressure in the manufacturer’s instruction manual. Efficiency, symmetry, and capacity should be within 10-15% of the test sheet values. If not, repack the column inlet or replace the column.

Note: Volumes listed in Table 2 are for 25 cm x 4.6 mm I.D. columns, which have a column volume of 4.15 mL. When restoring a 4.6 mm I.D. column shorter or longer than 25 cm, multiply all volumes in Table 2 by the ratio of the column length to 25 (e.g., for a 15 cm column: 15/25, or 0.6 times the volumes in Table 2). When restoring a column of internal diameter other than 4.6 mm, multiply all volumes in Table 2 by the ratio of the square of the column I.D. to (4.6)2 (e.g., for a 3.2 mm I.D. column: (3.2)2/(4.6)2 = 10.24/21.16 = 0.48 times the values in Table 2).

HPLC troubleshooting
Table 2. Column Restoration Procedures

Silica Column
Flush with the following:

  1. 50 mL hexane
  2. 50 mL methylene chloride
  3. 50 mL 2-propanol
  4. 50 mL methanol
  5. 25 mL methylene chloride
  6. 25 mL mobile phase

Evaluate column performance according to conditions specified by the manufacturer.

A. To Remove Protein
Flush with 10 column volumes of acetonitrile:water, 50:50, containing 0.1% trifluoroacetic acid.

B. To Remove TCA
Flush with 10 column volumes of distilled water (adjust pH to 2.5 with H3PO4), then with 10 column volumes each of:

  1. water (to remove salts)
  2. methanol (to remove water)
  3. methanol/methylene chloride, 50:50 (a general clean-up solution)
  4. methanol

If column performance still is not acceptable, prepare the mobile phase buffer at 10X the concentration used for the analysis and recycle through the column overnight.*
Re-equilibrate the column with mobile phase at the normal buffer concentration and reevaluate.

*Use caution: with some buffer types and/or concentrations a 10-fold increase in concentration can cause precipitation.

Silica-Based Reversed Phase Column (alkyl (C8, C18, etc.), phenyl, or diphenyl column)

A. Water Soluble Samples Flush with the following:

  1. Flush with warm (60 °C) distilled water
  2. 50 mL methanol
  3. 50 mL acetonitrile
  4. 25 mL methanol
  5. 25 mL mobile phase

Evaluate column performance.

B. Samples Not Soluble in Water Flush with the following:

  1. 50 mL 2-propanol
  2. 50 mL methylene chloride
  3. 50 mL hexane
  4. 25 mL isopropanol
  5. 25 mL mobile phase

Evaluate column performance.

Silica-Based Ion Exchange Columns (strong or weak anion or cation exchange)

Most analyses involving ion exchange systems use ionic mobile phases. Compounds that may affect column performance are usually insoluble or only slightly soluble in water. The following procedure should be sufficient to remove these compounds.

Flush with the following:

  1. 50 mL hot (40-60 °C) distilled water
  2. 50 mL methanol
  3. 50 mL acetonitrile
  4. 25 mL methylene chloride
  5. 25 mL methanol
  6. 25 mL mobile phase

Evaluate column performance.

Polar-Bonded Phase Column (amino, cyano, or diol column or Pirkle-type chiral columns).

For a column used in the reversed phase mode (e.g., organic solvent/aqueous buffer mobile phase), follow the same cleanup procedure as for silica-based reversed phase columns. For a column used with nonaqueous mobile phases, use the following scheme:

Flush with the following:

  1. 50 mL chloroform
  2. 50 mL methanol
  3. 50 mL acetonitrile
  4. 25 mL methylene chloride
  5. 25 mL methanol
  6. 25 mL mobile phase

Evaluate column performance.

Silica-Based Columns for RPLC of Proteins and Peptides

Follow the protocol for silica-based reversed phase columns.

Alternatively, make one or more 100 μL injections of trifluoroethanol (determine the number of injections by evaluating column performance after each injection).

Evaluate column performance.

HPLC troubleshooting
Table 3. Properties of Organic Solvents Commonly Used in HPLC
HPLC troubleshooting
Nonbonded Silica Columns Exposed to Polar Solvent

Samples and mobile phases containing very strongly polar solvents, such as water or alcohols, can deactivate uncoated silica HPLC columns. This can drastically affect column performance, particularly solute retention and selectivity. (Figure C2). Even prolonged column flushing with a nonpolar solvent only partially restores column performance, while wasting chemicals. A silica regeneration solution quickly and inexpensively restores silica column performance by removing trapped polar material. Pump the solution through the affected column for 10 minutes at a rate of 4 mL/minute, then flush with mobile phase for 10 minutes at a rate of 2 mL/minute. Evaluate column performance by using the test mixture for evaluating silica columns. Performance should be virtually the same as before the polar solvent was introduced (Figure C3). [Index]

Figure C Regeneration Solution Restores Silica Column Performance
hplc troubleshooting
HPLC troubleshooting
Preventing Leaks
Leaks are a common problem in HPLC analyses. To minimize leaks in your system, avoid interchanging hardware and fittings from different manufacturers. Incompatible fittings can be forced to fit initially, but the separation may show problems and repeated connections may eventually cause the fitting to leak. If interchanging is absolutely necessary, use appropriate adapters and check all connections for leaks before proceeding.
Unclogging the Column Frit

A clogged column frit is another common HPLC problem. To minimize this problem from the start, use a precolumn filter and guard column.

To clean the inlet, first disconnect and reverse the column. Connect it to the pump (but not to the detector), and pump solvent through at twice the standard flow rate. About 5-10 column volumes of solvent should be sufficient to dislodge small amounts of particulate material on the inlet frit. Evaluate the performance of the cleaned column using a standard test mixture.

Replacing a Frit at the Column Inlet

Sometimes neither solvent flushing (see above) nor restoration procedures (see Table 2) restore a column’s performance. If you’ve isolated the column as the problem source, and other restorative procedures have failed, a void in the packing or a persistent obstruction on the inlet frit may exist.

As a last resort, open the inlet end of the column. Caution: opening the inlet end, and more so opening the outlet end, can permanently damage the packing bed. Before opening columns, consult the manufacturer’s literature. (Never open either end of a resin-filled column).[Index]

HPLC troubleshooting
Use the following procedure to open a column.
  1. Disconnect the column from the system. To prevent the packing from oozing out of the column, perform subsequent steps as quickly as possible.
  2. Using a vise and wrench, or two wrenches, carefully remove the inlet end fitting (see Figure D). If the frit remains in the fitting, dislodge it by tapping the fitting on a hard surface. If the frit stays on the column, slide it off rather than lift it off. This will help preserve the integrity of the packing bed.
    Modular columns may require a special tool, to remove the frit cap.
  3. Examine the old frit. Compression of the frit against the stainless steel tubing will leave a ring around the edge on the column side of a properly seated frit. No ring can mean the ferrule is seated too near the tubing end. The resulting loose connection can leak silica or act as a mixing chamber.
  4. Examine the packing bed. If it is depressed or fractured, you need a new column.
  5. Replace the frit.
  6. Replace the end fitting. Screw it down fingertight, then tighten 1/4 turn with a wrench.
hplc solutions

End Troubleshooting Guide

This page will continue to be updated and modified as manufacturers' guidelines and research are updated.