HPLC Peak Splitting. Common Reasons For It

True "Split" HPLC peaks, not resulting from co-elution of another peak, 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 (*If the pH of the mobile phase is close to the pKa of the sample, then split peaks may result); which does not dissolve the sample in (should be fully soluble) or are unstable, 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. Peak splitting may be the result of dissolving and injecting your sample in a solution that is stronger than your mobile phase. 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, a dirty frit or poor mechanical connection (i.e. improperly swaged fitting). These types of structural or mechanical 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. Connections should always be double checked.
6. Detector data rate set too low. Too few peaks collected over time may result in integration errors and inaccurate peak symmetry problems. Read more about how to determine the best data collection rate at this link.

HPLC Mobile Phase Filtering & Solvent Inlet Filters

HPLC Mobile Phase Filtering: 



The tubing and valve passageways of the HPLC system are very narrow and clogs can result from using solutions which have not been properly filtered. Columns are expensive and will also clog up with particulate matter causing increased back pressure and/or changes in retention times. Running clean, particulate free HPLC grade solvents through your chromatograph is a basic maintenance requirement. High grade chromatography solvents (and ultra pure water) are often pre-filtered through 0.2 micron filters by the manufacturer to meet their grade for use in chromatographic systems. However, there are times when you also prepare (mix) your own mobile phases using theses solvents with or without chemical reagents and additives. When you prepare mobile phase using these reagent grade chemicals or additives you should also take the extra time to filter the final mixture through a 0.2 micron glass or steel filter prior to use. This helps to insure that you start with as clean a solution as possible. *This is a critical procedure to follow with buffer solutions. When using aqueous solutions, possible bacterial and algae growth can occur so remember to date the solutions and dispose of them after a suitable time period (Make up only what you will use in one week). Do not re-filter these solutions and then use them again.


HPLC Solvent Inlet Filters:

Most HPLC manufacturer's supply solvent inlet filters on the lines which draw solvent into the pump head. To protect the pump and components downstream, these lines often incorporate a filter. These solvent pre-filters are usually made from plastic (PEEK or PEAK), glass or stainless steel. Their porosity is typically ten or twenty microns. A smaller porosity could be used, but it would restrict the lines ability to draw up fresh solvent into the pump head at the required flow rate so a compromise in pore size is necessary. The filter is primarily designed to stop the pump from drawing up any large particles or debris which could cause damage to the system and is NOT used to filter the solution (as mentioned above, the solutions used should be pre-filtered). These filters can clog up over time and so should be monitored for restrictions. Stainless steel filters can be cleaned using sonication and heat. Plastic filters should usually be replaced with new ones. Glass filters, which are often made of sintered glass, can be washed, but should never be sonicated to clean them as this can cause the glass to fracture and plug them up even worse. When in doubt, replace them with new filters. Filters used with clean organic solvents often last for many years. Filters which are used with aqueous solutions last for shorter times due to build up of undesirable biological matter.

• Another way in which you can insure a clean source of liquid for your HPLC system is to make sure that your mobile phase reservoir bottles are clean and free of dirt and dust during use. Keep them covered. Always wipe off any dust and debris from the solvent bottles before you uncap them and pour them into another container (much of the dust in the mobile phase comes from dirt that falls into the bottles). Instead of 'topping-off' bottles, replace them with clean bottles containing new solution.