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Primesep Mixed mode HPLC

Primesep for mixed-mode HPLC

 Primesep for mixed-mode HPLC stationary phases offer unprecedented selectivity in the separation of a broad array of chemical compounds and in multiple applications.

A wide range of HPLC columns with id’s from 0.5 mm to 22 mm is available. Corresponding Primesep guard columns are available with all stationary phases and do not require holders.

SIELC offers a method developmentservice is available for customers. Ask about special custom LC-phases tailored for specific separations.

Why to use Primesep for mixed-mode HPLC stationary phases?

For decades, liquid chromatography stationary phase design has been dominated by the idea of elimination of multiple, or “unwanted”, interactions that occur in mixed-mode separations. Reverse-phase chromatography is making the point of eliminating silanol interactions with amine-containing analytes by developing base-deactivated phases.

Within the same trend, adsorption interactions are considered undesirable as opposed to steric interactions in size-exclusion chromatography, and non-ionic interactions are generally viewed as complications in ion-exchange and ion-exclusion chromatography.

However, there are ways to benefit from multiple interactions on the stationary phase. SIELC Technologies introduces Primesep® HPLC columns, specifically designed for mixed-mode separations and capable of separating a tremendous range of compounds by different separation modes based only upon mobile phase selection.

The main benefit of having more than one type of interaction on a column is the ability to provide separation even when one separation mode has failed. For example, while a typical reverse-phase column is unable to retain polar compounds without the addition of ion-pairing reagent, Primesep® columns with their ion-exchange retention separate wide range of polar compounds without any difficulty. Primesep® columns are designed with the understanding that proper inclusion, not elimination, of secondary interaction is a powerful tool in selectivity control, column stability, and separation reproducibility. Primesep® multi-step manufacturing process guarantees good reproducibility of retention of neutral, acidic and basic compounds.

Primesep for mixed-mode HPLC columns efficiently separate organic and inorganic ions in ion-exchange and ion-exclusion modes.

They effectively work in normal and reverse phases, as well as in polar organic modes. Different modes of separation offer different selectivity. Working with Primesep® you won’t need an ion-pairing reagent in the mobile phase to separate ionizable polar compounds as these columns have an ion-pairing group embedded on a stationary phase. On the same column at the same time, an organic pharmaceutical can be quantified with its inorganic counter ion, or inorganic cations and anions can be run together without the ion-chromatography system. As opposed to reversed-phase columns, the selectivity of Primesep® columns can be altered not only by varying the concentration of the organic modifier, but also by changing the type and concentration of the acid modifier. These tools open a new realm of choices to alter the selectivity for the separation of various compounds previously unachievable.

Every Primesep® column has a dual chemistry stationary phase with a hydrophobic long alkyl chain and an ionizable cationic or anionic embedded group. When the polar group bears a charge, it effectively shields any other less polar groups of the stationary phase. As a result, silanol groups, which cause unwanted interaction in many reverse-phase columns, are completely undetectable and do not affect the peak shape and selectivity.

Product Range of Primesep for mixed-mode HPLC

The A Product and Number Group:

All these columns have  negatively charged functional groups and are for Cation Exchange.

We prvide several different columns with negatively charged functional groups due to the embedded anionic ion-paring reagent.

Primesep A is the strongest acidic column, while Primesep C is the weakest acidic column. Primesep 100 and Primesep 200 columns are in between.

Difference in the functional group acidity allows selecting the most appropriate column for a particular set of basic compounds that differ in their pKa value.
The embedded acidic functional group can be in an ionized form, or in a non-ionized form, depending on the pH of the mobile phase. In order to get retention by ion-exchange mechanism on Primesep® columns, the pH of the mobile phase should be selected close to, or above, the transition value of embedded acidic groups. Below the transition pH value, the column behaves as a regular reverse-phase column with an embedded non-ionized polar group.

Cation-exchange Column Type: About 50% of embedded acidic groups ionized at transition pH value Separates basic compounds

Primesep 500 : pH = 5.0 strong bases, dibases, polybases
Primesep C : pH = 3.5 strong bases, dibases
Primesep 200 : pH = 2 strong bases, dibases
Primesep P : pH = 1 weak, medium aromatic bases
Primesep 100 : pH = 1 weak, medium bases, AA, metals
Primesep A : Ionized in all working pH weak bases, AA, metals

Primesep 100 is a reverse-phase analytical column with embedded acidic ion-pairing groups. This is the most versatile column of the entire Primesep family for separation of a broad range of compounds.

  • Improves retention of basic compounds by cation-exchange mechanism.
  • Separates acids by ion-exclusion mechanism.
  • Retains neutral compounds by reverse-phase mechanism.
  • All mobile phases are LC-MS and preparative chromatography compatible

Primesep 200 : Is a reverse-phase analytical column with embedded weak acidic ion-pairing groups. Improves retention of strong basic compounds by cation-exchange mechanism.

  • Retains neutral compounds by reverse-phase mechanism.
  • Provides an additional interaction with polar molecules offering separation of isomers and structurally-related compounds.
  • All mobile phases are LC-MS and preparative chromatography compatible.

Primesep 500 :

Primesep A : Is a reverse-phase analytical column with embedded strong acidic ion-pairing groups.

  • Improves retention of weak basic compounds by cation-exchange mechanism.
  • Separates acids by ion-exclusion mechanism.
  • Retains neutral compounds by reverse-phase mechanism.
  • All mobile phases are LC-MS and preparative chromatography compatible

Primesep AB : Is a reverse-phase column with embedded acidic and basic ion-pairing groups.  This column offers also cation-exchange properties and is useful in separation of complex mixtures of polar anionic and cationic compounds.

  • Improves retention of strong acidic compounds and strong basic compounds by ion-exchange mechanism.
  • Retains neutral compounds by reverse-phase mechanism.
  • All mobile phases are LC-MS and preparative chromatography compatible.

Primesep AP  : 

The B-or Basic Group:

We provides 4 types of different B-type columns with positively charged functional groups.

Primesep B is a strong basic column. The recommended pH range is from 1.5 to 4, created by the addition of trifluoroacetic, phosphoric, perchloric, or formic acids to the mobile phase.

Primesep B2 is a weak basic column with acidic carboxyl functional groups. At pH of the mobile phase below 5, the acidic groups are not ionized and B2 column surface becomes positively charged. This dual chemistry offers extended pH range from 0.5 to 7, suitable for the ammonium acetate and ammonium formate buffered mobile phases.

Primesep D column, originally developed for direct plasma analysis, became very useful for other anion-exchange/reverse phase applications. It has extended pH range from 1.5 to 7 and offers similar properties as Primesep B2 column but the column has no carboxylic groups and remains positively charged throughout the working pH range. The fourth column, which offers anion-exchange properties, is Primesep AB column. This column also offers cation-exchange properties and is useful in separation of complex mixtures of polar anionic and cationic compounds.
All B-type columns provide at least two main interactions with analytes: the reverse-phase interaction and the anion-exchange interaction. Neutral analytes are retained by the reverse-phase interaction. The presence of the charged group in the alkyl chain provides additional selectivity uncommon for typical reverse-phase columns. Acidic analytes can be retained by both anion-exchange and reverse-phase mechanisms. To control retention and selectivity, there is a broad selection of the mobile phases with concentration of organic modifier in the 0-100% range. The concentration and type of acid used will also significantly affect the retention of anionic compounds. Basic compounds can be retained only by the reverse-phase mechanism but the presence of positively charged groups on both analyte and stationary phases produces the unique selectivity due to the ion-exclusion phenomena.

Primesep B columns retain acid residue in the stationary phase in equimolar amount. When a column switches to the mobile phase with another type of acid, it should be sufficiently equilibrated to replace all counter-ions from the previous mobile phase with new counter-ion.

Primesep B : Strong basic column for use with TFA, phosphoric or perchloric acid solutions in a mobile phase

Primesep B2 : Weak basic column for ammonium acetate buffered solution

Primesep B4 :

C Group of Column

Primesep C :Combined reverse-phase and complex formation properties for interaction with amines, sulfonium, phosphonium and metal ions

Primesep N :

P or Phenyl Group

Offer three interactions within one column: 1. reverse phase 2. pi-pi and 3.strong cation exchange

Primesep P
Choose for separation of structural isomers of aromatic compounds.

Improves retention of basic compounds by cation-exchange mechanism.
Separates aromatic compounds by difference in pi-pi interaction.
Retains neutral compounds by reverse-phase mechanism.
All mobile phases are LC-MS and preparative chromatography compatible.

Primesep P columns are available in all standard dimensions
Column IDs: 22 mm, 10 mm, 4.6 mm, 3.2 mm, 2.1 mm, 1 mm, 0.5 mm, 0.25 mm
Column Lengths: 250 mm, 150 mm, 100 mm, 50 mm, 25 mm, 10 mm
Particles: 10 um, 5 um
Pores: 100 A

Primesep P Application Examples
HPLC Separation of Aromatic Boronic Acids

Improves retention of basic compounds by cation-exchange mechanism.
Separates aromatic compounds by difference in pi-pi interaction.
Retains neutral compounds by reverse-phase mechanism.
All mobile phases are LC-MS and preparative chromatography compatible.

Primesep P columns are available in all standard dimensions
Column IDs: 22 mm, 10 mm, 4.6 mm, 3.2 mm, 2.1 mm, 1 mm, 0.5 mm, 0.25 mm
Column Lengths: 250 mm, 150 mm, 100 mm, 50 mm, 25 mm, 10 mm
Particles: 10 um, 5 um
Pores: 100 A

Primesep P Application Examples
HPLC Separation of Aromatic Boronic Acids

Improves retention of basic compounds by cation-exchange mechanism.
Separates aromatic compounds by difference in pi-pi interaction.
Retains neutral compounds by reverse-phase mechanism.
All mobile phases are LC-MS and preparative chromatography compatible.

Primesep P columns are available in all standard dimensions
Column IDs: 22 mm, 10 mm, 4.6 mm, 3.2 mm, 2.1 mm, 1 mm, 0.5 mm, 0.25 mm
Column Lengths: 250 mm, 150 mm, 100 mm, 50 mm, 25 mm, 10 mm
Particles: 10 um, 5 um
Pores: 100 A

Primesep P Application Examples
HPLC Separation of Aromatic Boronic Acids

Primesep P : Choose for separation of structural isomers of aromatic compounds.

Primesep PB :

Primesep S :

Primesep SB :

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