When performing reversed phase LC separations, there is a natural tendency of method developers to reach for the most commonly used phase—a C18. However, there are so many different variations of C18 phases available to method developers that it often becomes difficult to choose a C18 column that one had originally thought.
This article explores the different C18 phases within the Kinetex core-shell family of HPLC/UHPLC columns and provides a general guideline for selecting the most appropriate C18 phase for a variety of applications.
To provide a streamlined method development approach, the 5 case studies listed below were used to determine the most appropriate C18 phase for each application:
– Acid, Base, Neutral Panel
– Highly Polar Analytes
– Highly Polar Bases (Catecholamines)
– Highly Polar Bases (Nucleotides)
– Hydrophobic Neutrals and Acids (Cannabinoids)
Three different Kinetex core-shell C18 phases were screened under the same conditions and optimized in some cases.
First Case: Acid, Base, Neutral Panel using a Formic Acid Gradient
In general, gradients will reduce selectivity differences between phases as the gradient rate itself will play a significant role in analyte elution. But, particularly when using formic acid and other weak buffers, differences in ion-exchange and polar interactions will still be apparent, giving subtle (or not so subtle) differences in selectivity. These differences will be less apparent when using stronger buffer salts like phosphate, or when ion-pairing agents (e.g. hexane sulfonic acid) or ion-masking agents (TEA; TFA).
Second Case: Highly Polar Analytes—Water- Soluble Vitamins and Third Case: Highly Polar Bases- Catecholamines
Our second and third case ended up with same results. As seen in the chromatograms below, you’ll want to take advantage of the aqueous stability of Kinetex Polar C18 to perform your analyses in 100% aqueous conditions to maximize retention of polar analytes. Traditional C18 phases like Kinetex C18 or XB-C18 should not be used below 2-3% organic to maintain phase stability.
Second Case Chromatograms
Third Case Chromatograms
Fourth Case: Highly Polar Bases- Nucleotides
To perform your analysis in 100% aqueous conditions to maximize retention of polar analytes you will want to take advantage of the aqueous stability of Kinetex Polar C18. The trade-off can be an increase in tailing, as can be seen below, but we include the data as a logical extreme; one would expect to optimize their method to find the right balance of retention and peak shape. In order to operate in highly aqueous conditions, one should use the Kinetex Polar C18 phase.
Fifth Case: Hydrophobic Neutrals and Acids- Cannabinoids
Even very hydrophobic molecules will display selectivity differences on different C18 phases due to structural or polar functional groups. In this case, although selectivity was similar overall, the traditional Kinetex C18 phase provided better selectivity for one critical pair (CBN and CBGA), which is interesting due to the fact that structurally, they are totally different. One would think they could be easily separated.
You will find differences in selectivity between the Kinetex C18, Kinetex XB-C18, and Kinetex Polar C18 stationary phases based upon the differences in surface chemistry. Under identical running conditions, the differences may be subtle, these are, after all, still all C18 phases, but subtle differences can make the difference between resolution and co-elution, or lead to reversals in elution order that can help to resolve a target analyte from matrix interferences. With the newest Kinetex Polar C18 phase, however, you can operate in 100% aqueous conditions, whereas the traditional C18 phases should not go below 2-3% organic. This open the potential to generate significant changes in retention for polar molecules.
A thorough method development approach should involve screening each of these phases, but if only one column could be screened, it is recommended that it be the Kinetex Polar C18, with its ability to operate in 100% aqueous conditions, followed by either the Kinetex C18 or Kinetex XB-C18 to complement it. The Kinetex Polar C18, with its aqueous stability and special surface modification, may provide a benefit for analyzing polar analytes, whereas the standard Kinetex C18 or XB-C18 phases, may be advantageous for separations based upon primarily hydrophobic differences.
Although it was not included in this study, our Kinetex EVO C18 is the obvious choice for operating in alkaline mobile phases or when you need improved peak shape for tailing basic analytes, especially when using weakly-buffered mobile phases like 0.1% formic acid.
For a deeper look into our Kinetex C18 portfolio, click here.