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.
In an HPLC system, problems can arise from many sources. First define the problem, then isolate the source.
Use Table 1 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.
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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.
Figure A.Components of an HPLC System
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.
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.
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 (Table 3). 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. This is especially important when switching between reversed and normal phase analyses.
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.
Figure B.Supelguard Columns Prolong the Lifespan of Your Analytical Columns
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.
Overloading a column can cause poor peak shapes and other problems.
Column capacity depends on many factors, but typical values for total amounts of analytes on a column are:
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.
These components seldom cause problems with the system. They will be discussed in the troubleshooting table (Table 1).
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.
|Column back pressure
| higher than usual
|lower than usual||3|
|Peak shapes, incorrect|
| broad peaks
| peak fronting
| rounded peaks
|tailing peaks, initial and later||9|
| height change
|Retention times, variable||5|
We also suggest referring to the maintenance and troubleshooting sections of your instrument manual. Modern HPLC systems often have self-diagnostic capabilities that help isolate the problem area within the instrument. For persistent problems relating to the column or your particular analysis, please contact Supelco’s Technical Service Department.
The remaining pages in this guide include procedures for restoring column performance following loss in resolution, retention, or selectivity, suggestions on how to prevent and solve column hardware problems, and a selection of column protection products from the Supelco catalog. Please refer to our catalog for our complete line of accessories that prolong column life and, in general, simplify or improve your HPLC or FPLC® analysis.
Finally, phone us to request additional literature about our HPLC and FPLC products, or use our ChromFax service for immediate access to all our free technical literature.
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. If you have a SUPELCOSIL column, analyze with the test mix and the conditions listed on the data sheet. 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).
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 (Cat. No. 58281). Performance should be virtually the same as before the polar solvent was introduced (Figure C3).
Silica Column Regeneration Solution, 200 mL (Cat. No. 33175)
Figure C.Regeneration Solution Restores Silica Column Performance
Performance evaluation mixes for HPLC columns.
Well defined test mixes enable you to troubleshoot chromatographic problems, optimize system efficiency, and evaluate columns under conditions where their performance is understood. We ship our test mixes in amber ampuls to prevent photodegradation, and we include instructions for proper use and interpretation of results.
Choose from column-specific or application-specific mixes. Refer to our catalog for our extensive selection of test mixes, or call our Technical Service Department.
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.
Highly concentrated salts (>0.2 M) and caustic mobile phases can reduce pump seal efficiency. The lifetime of injector rotor seals also depends on mobile phase conditions, particularly operation at high pH. In some cases, prolonged use of ion pair reagents has a lubricating effect on pump pistons that may produce small leaks at the seal. Some seals do not perform well with certain solvents. Before using a pump under adverse conditions, read the instrument manufacturer’s specifications. To replace seals, refer to the maintenance section of the pump manual.
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.
Sometimes neither solvent flushing (see above) nor restoration procedures (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).
Use the following procedure to open a column.
Figure D.Typical HPLC Column Designs
Protect your instrument and columns by removing particles and gases from solvents and other mobile phase components. Nylon 66 membrane filters are compatible with all solvents commonly used in HPLC.
Filtration Apparatus 1
(connects to 1000 mL sidearm flask)
Includes 250 mL glass reservoir, funnel base and stopper, clamp, stainless steel holder and screen, 10 Teflon gaskets, 50 Nylon 66 filters (47 mm, 0.45 μm pores).
Filtration Apparatus 2
(connects to aspiration line)
Includes 250 mL glass reservoir, 34/45 tapered funnel base, 34/45 tapered 1000 mL flask and glass cap, clamp, stainless steel holder and screen, 10 Teflon gaskets, 50 Nylon 66 filters (47 mm, 0.45 μm pores).
A precolumn filter is essential for protecting HPLC columns against particulate matter which can accumulate on the column frit, leading to split peaks and high back pressure. Sources of particles include mobile phases (especially when buffers are mixed with organic solvents), pump and injector seals, and samples. Use a 2.0 μm frit to protect columns containing 5 μm or larger particles, or a 0.5 μm frit for columns with particles smaller than 5 μm.
Direct-connect; protects analytical and guard columns.
Our precolumn filter can be connected directly, hand-tight, into any HPLC column or guard column listed in our current catalog, or with any other column that has Valco-compatible end fittings. PEEK cap and body, 2 μm stainless steel frit. For a metal-free system, order PEEK/Teflon replacement frits (Cat. No. 57430-U).
Valco Precolumn Frit and Screen Filters3
In-line installation. Efficient, low dead volume filters protect your columns from particles without reducing column performance. The replaceable 1/8" frit has 0.5 μm pores to protect 3 μm or 5 μm column packings, the replaceable screen has 2 μm pores. Choose the frit filter for higher filtration capacity (most applications) or the screen filter for less dead volume (e.g., with microbore columns). Use with 1/16" O.D. tubing; 1/16" fittings included.
In-line installation. High capacity inlet filter minimizes dead volume and band broadening, to prevent loss of column efficiency while protecting your column. Frit porosity: 0.5 μm. Complete as shown.
In-line installation. The 316 stainless steel filter disc (0.5 μm pores) is easily replaced without removing the column end fitting. Maximum operating pressure: 15,000 psi (1054 kg/cm²). For 1/16" tubing.
Place between the pump and injector to provide final filtration for the mobile phase. Easily replaced 316 stainless steel filter element (0.5 μm pores). Maximum operating pressure: 15,000 psi (105MPa). For 1/16" O.D. tubing, 10-32 threads2.
In-line installation. Stainless steel body with inert polyetheretherketone (PEEK) end fittings and a 0.5 μm or
2 μm PEEK frit in one end fitting.
The Rheodyne Model 7725 injector allows you to inject 1 μL-5 mL samples with accuracy and precision. The rugged, easily maintained design offers many advanced features:
Injector includes a 20 μL sample loop and is supplied with a VESPEL rotor seal that can be replaced with a Tefzel rotor seal for operation at pH 0-14. Factory set at 5000 psi (345 bar), adjustable to 7000 psi (483 bar). Model 7725i has an internal position sensing switch.
A Model 7725 Injector Reduces Wear and Tear on Your Columns
A conventional HPLC valve momentarily interrupts flow during sample injection, subjecting your column to repetitive pressure shocks. Rheodyne’s patented MBB (make-before-break) design makes the new connection before breaking the old one, providing uninterrupted flow.
A preventative maintenance program that includes routine replacement of pump parts that are subject to wear will help you avoid costly downtime. Our extensive selection of Optimize Technologies check valves, seals, and pistons meet or exceed pump manufacturers' specifications. For the most up-to-date selection of pump parts, refer to the current Supelco catalog, or call our Technical Service Department.
A pulse damper controls pump pulsations for a more stable baseline. The SSI LO-Pulse damper is a patented unit compatible with single piston reciprocating HPLC pumps (Altex 110A, Eldex pumps, LDC Mini-Pump VS, SSI Models 200 and 300, etc.). At pressures from 500 psi to 6000 psi (35-420 kg/cm²), it improves precision of quantitative analyses and detection limits for trace sample components. Fittings and instructions included.