HPLC Peak Splitting. Common Reasons For It

True "Split" HPLC peaks can be caused by a number of chromatography problems. Here are a few examples and their solutions:

  1. Sample overload. Sample overloading is one of the most common reasons for observing peak "splitting". Reduce the sample concentration by factors of ten to see if the peak shape improves.
  2.  A poor quality HPLC method. Poor quality methods which do not use mobile phase solutions which are at an appropriate pH, dissolve the sample in (are fully soluble) are unstable or show sample or mobile phase precipitation can cause this effect. Always check solubility before starting.
  3. A partially plugged or fouled column. A dirty or fouled column (from not washing down properly with a solution which is STRONGER than the mobile phase). Analysis methods should be followed by separate wash methods to remove all bound material and any late eluters,
  4. Wrong injection solution. If you dissolve and inject your sample in a solution that is stronger than your mobile phase, peak splitting can result. Dissolve and inject samples in the mobile phase or in a solution which is a slightly weaker solution (not stronger).
  5. A poorly packed column, void at column inlet or a dirty frit. These types of structural defects can each result in peak "splitting" (all of these are less common today than in the past using modern HPLC columns). When present, a dirty inlet frit can be replaced with a new one, or the column can sometimes be backflushed to remove any accumulated material.

How to Prevent Mobile Phase Problems in HPLC

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 in recent years as a means of reducing the cost of solvents, their disposal, and mobile phase preparation time. An apparatus such as the Supelco SRS-3000 or SRS-1000 Solvent Recovery System 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.

source:sigmaaldrich.com

Preventing Leaks In HPLC Analyses

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.

clogged column frit HPLC problem

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.

Troubleshooting HPLC- Loss in Response for all Analytes

Just as in GC applications, patterns in your HPLC chromatography can exhibit telltale signs that point toward probable sources of error.  In this brief series of posts, we will look at possible scenarios that you may encounter in the laboratory and how you might approach resolving those difficulties.

We occasionally get calls or emails from folks that are seeing lower than expected response for peaks of interest. This may be a matter of method development if you have not done this analysis previously. Suggestions in the post are provided assuming you are working with an established method and have had successful results over an extended period of time (until recently). These suggestions also assume that the response is low for quantitation standards as well as in prepared QC samples. If the problem appears only in your QC samples, then please review the earlier post on method troubleshooting. Otherwise, please see below, where we begin with a case where all peaks are equally low across the board for multiple analytes.

50 cent

This is often caused by:

1. Calculation/dilution error- This is one of the most common and easiest problems to resolve, although it is often the least suspected. A dead giveaway is an exact ratio of error, such as results that are close to exactly 50% low (2-fold).  Try having someone who has not been involved in your project prepare your standard or do the calculations starting from scratch to see if they obtain a different result. This can be a classic case of tunnel vision. Make sure the established method is being used for this and has not changed.

2. Injection volume is being measured incorrectly. This depends on how your specific autosampler and system are configured. This could be caused by:

a. Incorrect programming of injection volume

b. Incorrect size of syringe or sample loop, or inadequate volume of sample loaded onto the autosampler.

c. Malfunction of sample injection valve or metering device.

Depending on the situation, issues of this type might give results that are pretty consistent, similar to a calculation or dilution error.  If there is any doubt, try performing a manual injection to see if your response increases. (Consult your instrument manufacturer if you need help determining how to do this with your system.)

3. Injection volume is partially lost due to leaks. This could occur at either of the following locations:

a. Leakage in the autosampler from fittings or tubing or could be due to a malfunction/leakage from the sample injection valve.  An issue of this type may or may not be fairly consistent, although it would be surprising to see a numerical correlation such as in a dilution error. You may be able to see such an issue visibly, but if there is any doubt, try performing a manual injection to see if your response increases.

b. Leaks downstream from the injection valve- If you have a large enough leak between your valve and the detector, either before or after the column, it can result in low response. If the leak is before the column your system pressure will be lower also, thus, another good reason to keep a record of your operating pressures. If your leak is after the column or in the detector, you might see disturbances in the baseline, such as might be caused by air bubbles being present.

4. Peak broadening- When peaks become broader, the peak height is always lower. Try comparing your peak areas instead of heights and see if this explains what you are seeing. Broader peaks are also more difficult to get integrated properly, which makes matters worse. If this occurs for all of your analyte peaks, it is likely due to one of the following:

a. Excessively large injection volume. Check your operating parameters and see if you are using a higher volume that you did initially for this method. Try injecting a smaller volume of a more concentrated standard instead. (See the FAQ section on our website for suggested volumes.)

b. A change in system dead volume, resulting in increased extra-column band broadening. This effect is more pronounced for smaller ID columns. Try shortening those lengths of tubing and/or use smaller ID tubing. If you have changed detectors, see if the flow cell is smaller than the one you used previously and replace if needed.  Click here for more background information on the topic.

5. Detector maintenance needed- Detector lamp may be near the end of its life and need replaced. If baseline or background signal is high, the flow cell may be contaminated and should be cleaned. These issues are usually accompanied also by baseline drifts and/or noise and a steady decline in response over time.

6. If you have exhausted all of the above possibilities, check out the next post in this series, where we will discuss sources of loss for only certain analytes, but not always all of them. Of course, if you have only one analyte, or a handful of very similar analytes, the second post may be more applicable to your situation.

source:blog.restek.com

Troubleshooting HPLC- Loss in Response for Some, but Not All Analytes

Just as in GC applications, patterns in your HPLC chromatography can exhibit telltale signs that point toward probable sources of error.  In this brief series of posts, we will look at possible scenarios that you may encounter in the laboratory and how you might approach resolving those difficulties.

We occasionally get calls or emails from folks that are seeing lower than expected response for peaks of interest. This may be a matter of method development if you have not done this analysis previously. Suggestions in the post are provided assuming you are working with an established method and have had successful results over an extended period of time (until recently). These suggestions also assume that the response is low for quantitation standards as well as in prepared QC samples. If the problem appears only in your QC samples, then please review the earlier post on method troubleshooting. Otherwise, please see below, where we begin with a case where all peaks are equally low across the board for multiple analytes.

50 cent

This is often caused by:

1. Calculation/dilution error- This is one of the most common and easiest problems to resolve, although it is often the least suspected. A dead giveaway is an exact ratio of error, such as results that are close to exactly 50% low (2-fold).  Try having someone who has not been involved in your project prepare your standard or do the calculations starting from scratch to see if they obtain a different result. This can be a classic case of tunnel vision. Make sure the established method is being used for this and has not changed.

2. Injection volume is being measured incorrectly. This depends on how your specific autosampler and system are configured. This could be caused by:

a. Incorrect programming of injection volume

b. Incorrect size of syringe or sample loop, or inadequate volume of sample loaded onto the autosampler.

c. Malfunction of sample injection valve or metering device.

Depending on the situation, issues of this type might give results that are pretty consistent, similar to a calculation or dilution error.  If there is any doubt, try performing a manual injection to see if your response increases. (Consult your instrument manufacturer if you need help determining how to do this with your system.)

3. Injection volume is partially lost due to leaks. This could occur at either of the following locations:

a. Leakage in the autosampler from fittings or tubing or could be due to a malfunction/leakage from the sample injection valve.  An issue of this type may or may not be fairly consistent, although it would be surprising to see a numerical correlation such as in a dilution error. You may be able to see such an issue visibly, but if there is any doubt, try performing a manual injection to see if your response increases.

b. Leaks downstream from the injection valve- If you have a large enough leak between your valve and the detector, either before or after the column, it can result in low response. If the leak is before the column your system pressure will be lower also, thus, another good reason to keep a record of your operating pressures. If your leak is after the column or in the detector, you might see disturbances in the baseline, such as might be caused by air bubbles being present.

4. Peak broadening- When peaks become broader, the peak height is always lower. Try comparing your peak areas instead of heights and see if this explains what you are seeing. Broader peaks are also more difficult to get integrated properly, which makes matters worse. If this occurs for all of your analyte peaks, it is likely due to one of the following:

a. Excessively large injection volume. Check your operating parameters and see if you are using a higher volume that you did initially for this method. Try injecting a smaller volume of a more concentrated standard instead. (See the FAQ section on our website for suggested volumes.)

b. A change in system dead volume, resulting in increased extra-column band broadening. This effect is more pronounced for smaller ID columns. Try shortening those lengths of tubing and/or use smaller ID tubing. If you have changed detectors, see if the flow cell is smaller than the one you used previously and replace if needed.  Click here for more background information on the topic.

5. Detector maintenance needed- Detector lamp may be near the end of its life and need replaced. If baseline or background signal is high, the flow cell may be contaminated and should be cleaned. These issues are usually accompanied also by baseline drifts and/or noise and a steady decline in response over time.

6. If you have exhausted all of the above possibilities, check out the next post in this series, where we will discuss sources of loss for only certain analytes, but not always all of them. Of course, if you have only one analyte, or a handful of very similar analytes, the second post may be more applicable to your situation.

source:restek.com

Troubleshooting HPLC- Loss in Response for Some, but Not All Analytes

Patterns in your HPLC chromatography can exhibit telltale signs that point toward probable sources of error.  In this brief series of posts, we will look at possible scenarios that you may encounter in the laboratory and how you might approach resolving those difficulties.

When some, but not all of your peaks are low, and you are analyzing multiple analytes, the number one rule is to look for trends. As mentioned in the previous post for this series, when you have only one analyte, or a handful of very similar analytes, the following may also apply. Again in this post, the assumption is that you are using an established working method and that you see a loss in response for every sample or standard that is injected. If you are only seeing the problem with samples and not your quantitation, please the earlier post on Method Troubleshooting.

If you have a lower response for some but not all of your peaks, here are examples of some things that could account for this:response_cgram (2)

1. Incompatible solvent composition- Double-check the solvent composition of your prepared samples and standards and make sure nothing has changed. Also, if solubility is marginal, some of the other causes that follow may be pushing it over the edge. If your sample solvent is stronger than your starting mobile phase, you may see a “smearing” effect, which means some of your analyte is traveling through the column prematurely while the rest is retained somewhat normally. This will appear more pronounced for early eluting peaks. Of course, this type of peak distortion also creates a decreased overall peak height and apparent response. Also, check out previous blog posts such as the one entitled “Solvent mismatch isn’t only a GC issue:…” and the FAQ section on our website for further discussion of this phenomenon.

2. Incorrect detector settings- Make sure that a setting such as wavelength has not changed on your detector program.  If you started out with a good method, it is usually just a matter of making sure you’re still following it properly. Depending on the functional groups of various analytes, some chromophores may not be active at the wavelength you are monitoring, which would explain why response is still adequate for some of your analytes. This would be similar to monitoring an incorrect Mass/Charge ratio for LCMS.

3. Activity (pH-related)- Are the compounds that lack response characteristically acidic or basic? (Hint: Look up their pKa’s to see.) If they are, please see the earlier posts regarding buffers (Links shown below.) You might also notice poor peaks shape if buffer is needed or if your buffer isn’t prepared correctly.

When should you use a buffer for HPLC….

How much buffer to weigh…..

4. Matrix effects (short term)- Certain substances in sample matrices can affect analyte response in surprising ways and biological samples can vary significantly. These can suppress detector response, and/or ionization in mass spec. They can affect peak shape and, on rare occasions, they may form complexes with analytes of interest. This type of effects can usually be seen immediately and can be identified easily by comparison to injections made without matrix. Don’t forget that your quantitation standard might contain sample matrix, so you may need to make a new solution in clean solvent for troubleshooting purposes. To read more about these effects and their impact on LCMSMS analyses, check out the following links:

Matrix Effects in Multi-Residue Pesticide Analysis When Using Liquid Chromatography-Tandem Mass Spectrometry

Mitigating Matrix Effects: Examination of Dilution, QuEChERS, and Calibration Strategies for LC-MS/MS Analysis of Pesticide Residues in Diverse Food Types

Performance and Matrix Effect Observed in QuEChERS (Journal of Chromatographic Science, Vol. 49, Oct 2011)

5. Matrix effects (long term)- More insidious effects can occur over time, where the sample matrix can actually change the nature of the stationary phase inside the column, sometimes making it more prone to adsorption of analytes. This is harder to identify because once this happens, it tends to affect all subsequent injections, whether they contain matrix or not. While particulates can be filtered out prior to injection, it may take more effort to remove active components from the sample matrix in order to protect the column. Examples of these include salts and proteins from blood, plasma or urine samples.  This can also be caused by remnants of derivatization reagent that might be present. A guard column cartridge is the first line of defense in protection against this. However, it is more ideal to remove the offending components prior to injection by using sample/extract cleanup such as SPE. The earlier post Live Long and Prosper gives more suggestions for avoiding these issues. Sample matrix components can also coat the inside of tubing and valves and create similar symptoms. (Yes, I have seen this happen.) Usually the easiest fix is to replace tubing and/or fittings associated with that portion of the flowpath.  If you find that your column has been affected by repeated injections of sample, you might benefit from reading our technical note on LC Cleaning Recommendations.

6. Incompatibility with column phase- This is not encountered frequently, but should be mentioned, nonetheless. Please double check your Column Care Instructionsto make sure your analysis is compatible with the column phase. An example of an inappropriate solvent or sample would be an aldehyde or ketone solvent used with amino phase.

7. Column age- At some point a column needs to be replaced, although its life span depends on the application and frequency of usage. Matrix effects are usually the number one contributor to shortening of column lifetime, although changes back and forth between solvents, pH ranges, pressures, etc. contribute also. When in doubt, it is always best to keep a backup column on hand.

source:restek.com

Troubleshooting HPLC- Tailing Peaks

As in the previous posts for this series, these suggestions assume that you are working with an established method and had successful results in the past. If a method is rugged, it is developed to minimize tailing by using a stationary column phase that is base-deactivated as well as appropriate modifiers and pH adjustment in the mobile phase as needed.  Keep in mind that if the method is not as rugged as it should be, change(s) in the method might be advised if tailing continues to be an ongoing problem.

tailing

With troubleshooting, as always, it is critical to note when the change occurred and how it might correlate to changes in the system.  Equally important is to change one thing at a time to identify the source(s) of difficulty. The following are examples of things that could contribute to tailing. I suggest investigating these in this order:

  1. Issues with mobile phase- This could be a problem with preparation of new solution or degradation of an existing solution.  Particularly if you notice some retention time changes and/or interferences that weren’t present earlier, this could be the cause. Try preparing some fresh mobile phase and see if your tailing gets better.  Pay special attention to which peaks are tailing and whether the issue might be pH-related.  Lack of proper pH adjustment for the mobile phase could be a factor. For more on this topic, please see the earlier posts on Buffers, When should you use a buffer for HPLC, how does it work and which one to use? and How much buffer to weigh and what’s the best way to prepare my buffered mobile phase?. 
  2. Obstructed or dirty guard cartridge- Put simply, if you are using a guard cartridge, try replacing it with a new one. Replace the cap frit also if you are using a guard holder that accommodates the frit (such as catalog #25084). A buildup of sample material on the frit or adsorbed on the cartridge could create tailing that would most likely affect all of your analytes.
  3. Using an inappropriate guard cartridge- This is usually easy to correct and diagnose.  If using a guard cartridge, it should contain packing material that is the same as the analytical column or one that is very similar. Especially if you have just recently started using one of these, make sure that it contains the right packing material with the right bonded phase.  Use caution if you are using a Restek guard cartridge with an analytical column from another vendor.  This is addressed in the FAQ section of our website.  If there is a significant difference between the guard and analytical column packing, there will be two competing modes of separation, which could create split or distorted peaks or tailing.
  4. Increase in dead volume- This is most critical for columns with small ID and tailing is usually more evident with early eluting peaks. However, if it is bad enough, you might see an effect to some degree on all analytes.  If you have changed tubing lately, make sure you aren’t using a larger ID or longer length than previously.  Also make sure the tube fitting at the inlet of the column is fully seated and that you are using the proper fitting and ferrule. For instance, connection to a Waters column or Waters/Rheodyne parts sometimes requires alternate fitting parts due to deeper seated threads. Please see the LC FAQ pertaining to this for more information.  Our Universal PEEK connectors and new EXP fittings accommodate for all of the common fitting styles.  If you are using a guard holder that accommodates a cap frit, make sure that a cap frit is installed; otherwise, dead volume will result. If you are using a Trident Direct guard holder with an analytical column that is not from Restek, make sure you are using the appropriate PEEK tip. An alternate tip is offered for Waters-style column end fittings.
  5. Mass overloading- Injecting too much sample material, that is, a solution that is too concentrated in terms of sample material weight (including matrix), can create tailing, particularly for basic analytes. Try injecting a less concentrated solution to see if tailing decreases.
  6. Volume overloading-Injecting too large of a volume causes peak broadening, along with fronting and/or tailing. You can determine if this is the problem by injecting a smaller volume.   Some general guidelines as far as suggested volumes can be found in the FAQ section on our website.
  7. Interfering coelution- If you have tailing only for one or two of your peaks, there may be an interfering contaminant that coelutes.  If that is the case, you might see the tailing come and go, and a shoulder may appear at times. If you think this might be happening, try using a slower gradient or changing your organic/water ratio and see if the peak separates into two.
  8. Coating or obstructions on column frit or particles of column packing- This is due to sample matrix and very common.  This usually affects all of the analytes and is often accompanied by an increase in column backpressure.  It can best be prevented by filtering sample extracts prior to HPLC and by using sample cleanup techniques such as SPE. In some cases, flushing with various solvents may help. Please see our LC Cleaning Recommendations.  If cleaning does not help, your column probably has been damaged chemically and should be replaced.
  9. Physical damage to column- This does not happen frequently, but is a disturbance in terms of how the packing material is arranged inside the column. It could be related to a void, fracture, open channel, or non-uniform compression.  These can result from a physical shock in terms of pressure or flow.  The phenomenon may explain tailing on columns which have had no or very little sample matrix injected.  The tailing would occur for all analytes and is likely to be accompanied by a change in column pressure. A column with this type of damage should be replaced.
  10. Loss of inertness in tubing, injector parts or associated valves- If switching to a new column doesn’t help, it may actually be an issue with tubing or instrument parts.  This is a tough one to diagnose, but it can and has happened.  Symptoms of this tend to mimic a similar problem with the column, but may not be affected by efforts to clean the column.  This is caused by accumulation of certain components from sample matrix over time.  To remedy this, tubing should be replaced and valves physically cleaned or replaced. Please see your manufacturer’s instructions to do this.

Thank you for reading.  Don’t forget to check for the next post in this series on troubleshooting.

source:restek.com