BIST™ Mechanism and properties

  • Doubly-charged anions of the buffer in the mobile phase form a bridge between the positive charges of the stationary phase surface and the positively-charged analyte
  • Bridge formation is possible when molecules of polar solvent (water) do not solvate ions sufficiently and, therefore, do not separate them
  • The more water the mobile phase has, the less retention of BIST™ type is observed
  • While BIST™ is based on the ionic electrostatic interaction, the concentration of ions in the mobile phase does not affect retention significantly (as opposed to the concentration of water
  • BIST™ is different from Ion-Exchange, HILIC, or any other common separation technique

But how exactly does BIST™ work, and how will it help your HPLC separations?

In order for BIST™ to perform, three conditions need to be met:

  1. A doubly charged buffer ion present in the mobile phase
  2. Buffer’s doubly charged ions should be opposite in charge to that of the stationary phase surface
  3. Reduced water in the mobile phase to minimize ion solvation

In traditional Ion Exchange Chromatography (IEC), analyte ions are usually separated by how they interact electrostatically with the column’s charged surface. If the ions and the surface are charged oppositely (i.e. a negatively-charged surface and positively charged ions), then the ions will be attracted to the surface and retained to a degree based on the number of charges, ion size, and other factors. However, if the ions and the surface share the same charge (i.e. if both are positively charged), the ions will be repelled by the surface charge to the point where they don’t even enter the pores of the stationary phase particles. This will result in extremely quick elution and is referred to as pre-void elution. This difference in same- and different- charges between ions and stationary phase is shown to the right, where a positively-charged ion was injected onto a positively-charged column (Anion Exchange) and onto a negatively charged column (Cation Exchange). The void time, or how long an analyte would take to travel through the column without any net interactions, is just under 1.5 minutes for the column dimensions given in the figure below. Notice that the red peak appears just a bit earlier, indicating the analyte eluted pre-void.

Column service charge effect on protonated Dopamine renetion in Ion-Exclusion and Ion-Exchange modes
Column service charge effect on protonated Dopamine renetion in Ion-Exclusion and Ion-Exchange modes

Doubly vs. Singly Charged Buffer Ions

Typically, when a positively-charged analyte is injected onto a column with a positively-charged surface, the analyte has very little interaction with the stationary phase. In fact, the analyte is repelled and does not even enter the particle pores, resulting in pre-void elution, as shown in blue below. However, when a buffer with double-charged ions (H2SO4) replaces a buffer with single-charged ions (TFA), retention occurs, as shown in red in Fig. 2. Conventional chromatographic wisdom tells us this shouldn’t be happening, yet positively-charged Dopamine is retained when a H2SO4 buffer and a high MeCN concentration are employed. Apparently, something unique and exciting is occurring!

Buffer effect on retention of protonated Dopamine
Buffer effect on retention of protonated Dopamine

BIST™ is not limited to just positively charged analytes and columns; it can work with negatively charged compounds and a negatively charged column surface. This can be seen below where negatively charged Tartrazine (with three acidic groups) was analyzed on a cation-exchange column. The key to BIST™ chromatography is a double charged cation of the buffer, such as magnesium acetate, calcium acetate, or N,N,N’,N’-tetramethyldiaminopropane (TMDAP) and high concentration of MeCN. If a buffer with a single-charged cation such as Sodium Acetate is used, no retention occurs for Tartrazine at the same high MeCN concentration.

Buffer type's effect on retention of negatively charged Tartrazine on a caution-exchange column
Buffer type’s effect on retention of negatively charged Tartrazine on a caution-exchange column

BIST™ provides not only great retention of charged analytes, but also offers good selectivity. When a bridge forms between the stationary phase and analyte, the structure of the analyte plays an important role in the bridge stability. Since this interaction happens close to the surface of the solid stationary phase, even small differences in the charge position within the solute molecule or presence of other functional groups may significantly influence analyte’s retention time and enhance column’s selectivity. It is clear that a single charged buffer can provide neither retention nor selectivity.

Selectivity of BIST™ separation of Paraquat and Diquat