What causes peak tailing in reversed-phase HPLC and what can I do about it?

 

What causes peak tailing in reversed-phase HPLC and what can I do about it?

Some type of secondary interaction between an analyte and the column causes peak tailing. This interaction is in addition to the partitioning behavior seen for reversed-phase analyses. Peak tailing is most commonly seen with basic compounds and is usually a result of interactions between the residual silanols and positively charged basic compounds. The most common of these interactions is an ion exchange interaction between a positively charged basic compound and a negatively charged column surface silanol. Silanols on the surface of silica-based columns will have a negative charge when the pH of the mobile phase is above 4.5 – 5.0. Therefore the quickest way to reduce peak tailing is to operate with a buffered mobile phase at a pH below 4. Choosing newer columns with high purity, fully hydroxylated silica will also minimize peak tailing because silanol activity and ionization is reduced.         Some ZORBAX columns that use this type of silica are the StableBond columns, the Eclipse XDB columns, the Bonus-RP and the Extend-C18 column. Each of these columns can reduce peak tailing, but they are all a little different. The StableBond (SB) columns are ideal at low pH so they are often first choices to reduce peak tailing when using a low pH mobile phase. The Eclipse XDB columns are the first choice to reduce peak tailing if your mobile phase is pH 5 – 9. This column is double endcapped so it minimizes peak tailing by covering as many residual silanols on the column surface as possible and eliminating possible secondary interactions with silanols. The Bonus-RP column is also a good choice to reduce peak tailing in this intermediate pH region. The Bonus-RP column has a bonded-phase with an imbedded polar group. This group reduces interactions between basic compounds and residual silanols thereby improving the peak shape of basic compounds. This column can be used from pH 2 – 8. The Extend-C18 is designed as a high pH column and can be used up to pH 11.5. At high pH many basic compounds are no longer charged and interactions with silanols are minimized, reducing peak tailing.       Careful choice of a mobile phase can also reduce peak tailing. Buffered mobile phases (25 – 50 mM) will reduce peak tailing and low pH mobile phases are preferred (pH 2 –3). This should also result in more reproducible chromatography. Mobile phase additives such as triethylamine (TEA) can be added to reduce peak tailing of basic compounds, if needed. TEA acts as a competing base and ties up silanol sites, eliminating interactions between your analyte and residual silanols. But this type of additive is rarely needed at low pH and is only occasionally necessary at intermediate pH.        If you have peak tailing with an acidic compound the same process applies. Reduce the mobile phase pH to try to protonate the acids, then use a buffered mobile phase and try increasing the ionic strength of the mobile phase. Finally a competing organic acid can be added to the mobile phase and we have achieved excellent results with 0.1% trifluoroacetic acid (TFA), and this additive has a very low UV cutoff. Following these suggestions should reduce peak tailing of acids and bases.        Most columns now use spherical particles because columns packed with spherical particles will have higher efficiencies. Therefore start by choosing a column with spherical particles. The most common particle size choice for analytical separations is 5 um because it is easy to use, but more often today the better choice is 3.5 um particles. These smaller particles generate higher efficiencies in shorter column lengths and make it possible to do separations with shorter analysis times. If analysis time is important to you, consider choosing a ZORBAX Rapid Resolution (3.5 um) column to minimize analysis time. The 4.6 x 150 mm, 3.5 um Rapid Resolution column will have the same efficiency as a 4.6 x 250 mm, 5 um column and reduce analysis time by 40%. Other shorter Rapid Resolution columns (75 mm, 50 mm, 30 mm, and 15 mm) are available to further reduce analysis time.       Column pore size is selected based on the molecular weights of your analytes. A pore size of less than 100Å can be used for small molecules with molecular weights less than 4000. Larger molecules, such as proteins and peptides, should be analyzed on 300Å pore size columns. In addition, some smaller molecules with large, multi-ring, rigid structures can better be analyzed on 300Å pore size columns. Choosing the right pore size is important because most of the bonded-phase resides in the pores of the particles, therefore optimum retention and peak width are achieved only if the molecules can diffuse in and out of the pores rapidly and easily.-