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SieLC Primesep

SieLC is a pioneer in mixed mode chromatographic materials and columns.

SieLC was formed by two highly innovative chemists Vlad Orlovsky and  Uri Seletionec in Prospect Hights, Il, north of Chicago.

Product range is structured as follows:

  • Coresep: Columns ombining mixed-mode and core-shell approaches
    • Coresep 100 (Reversed-phase cation exchange core shell column)
    • Coresep SB (Reversed-phase anion exchange core shell column)
    • Coresep S (HILIC/ion-exchange core shell column)
  • Primesep : Mixed Mode range of column
    • Primesep A
    • Primesep 500
    • Primesep 200
    • Primesep 100
    • Primesep A
    • Primesep C
    • Primesep P
    • Primesep SB
    • Primesep B2
    • Primesep D
    • Primesep AB
  • Primesep Specialities (Mixed Mode Specialities
    • Primesep N
    • Primesep S2
    • Primesep B4
    • Primesep PB
    • Primesep S
    • Primesep AP
    • Primesep X
  • Obelisc : Mixed Modes Columns based on Living Separation Cell Technology
    • Obelisc N
    • Obelisc R
  • Promix : A novel approach to separate proteins and peptide
    • Promix SP
    • Promix AP
    • Promix MP
  • Sharc : Separation primarily on hydrogen bonding

New

Coresep Columns

Introduction

SIELC Technologies has developed a new generation of unique  stationary phases combining mixed-mode and core-shell approaches -CoresepTM

In single single mode on a reversed-phase column compounds elute according to their hydrophobicity and analytes  with similar hydrophobicity may co-elute or show poor resolution. In Ion-Exchange columns compounds are retained based on their ionic  properties. Similarly, using ion-exchange chromatography, other  compounds may be closely eluting because they are identical in terms of  the strength of ionic interaction between stationary phase and analyte

In mixed-mode chromatography you have two  interactions,  reversed-phase and ion-exchang. You are exploring a  very small difference in reversed-phase and ionic properties of  compounds. These differences are enhanced due to synergy of two  mechanisms which results in much better separation (mixed-mode  example).

Sielc Coresep Comparison mixed mode vs RP-IEX

 

Core-shell particles are constructed with solid core and porous outer  layer. This porous layer is used as a stationary support for chemical  attachment of specific separation phases.

 

Sielc core-shell-small

The reduced pore length offers faster diffusion of molecules and as a result has the ability to do  chromatography faster compared to particles with through-pore structure. The same result can beachieved with smaller particles, but then the  pressure needs to be higher in order to produce the same flow rate and  speed of analysis.

Sielc porous-small particle

Core-shell technology offers the unique ability to achieve high efficiency at higher flow rates whilst generating back pressures which  can be achieved using traditional HPLC systems. This offers the end user the opportunity to improve separation speed and quality without the  requirement to¬  purchase UHPLC equipment. We have recognized the  exciting opportunities afforded by combining these two great  technologies in stationary phases which achieve unique selectivity and  high efficiency with traditional HPLC systems which are rated to 4000 or 6000 psi.

Mixed-mode columns offer much higher capacity and retention than  traditional reversed-phase columns. Since the surface area of core-shell particles is reduced, mixed-mode phases address potential loadability  issues whilst maintaining the selectivity advantages of multiple  interactions. Newly optimized ligand density and ionic / hydrophobic  ratios further increases the capacity of mixed-mode core-shell columns.

 

Coresep 100

Coresep 100 is a reverse-phase analytical column with embedded acidic ion-pairing groups embedded on core-shell particles.

  • 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.

Coresep 100 columns are available in all standard dimensions 
Column IDs: 4.6 mm, 3.2 mm, 2.1 mm, 1 mm, 0.5 mm, 0.25 mm
Column Lengths:150 mm, 100 mm, 50 mm, 25 mm, 10 mm
Particles: 2.7 um
Pores: 90 A

 

Coresep SB

Coresep SB is a reverse-phase analytical column with strong embedded basic ion-pairing groups embedded on care-shell particles.

  • Improves retention of acidic compounds by anion-exchange mechanism.
  • Separates bases by ion-exclusion mechanism.
  • Retains neutral compounds by reverse-phase mechanism.
  • All mobile phases are LC-MS and preparative chromatography compatible (working pH range from 1.5 to 5).

Coresep SB columns are available in all standard dimensions
Column IDs:
 4.6 mm, 3.2 mm, 2.1 mm, 1 mm, 0.5 mm, 0.25 mm
Column Lengths:¬ 150 mm, 100 mm, 50 mm, 25 mm, 10 mm
Particles: 2.7 um
Pores: 90 A

Coresep S

Coresep S is a normal-phase analytical column with embedded acidic ion-pairing groups embedded on core-shell particles.

  • Most suitable for separation of common sugars and other neutral or very polar molecules.
  • Retains and separates amino acids with different buffers at low concentration.
  • Separates weak bases by ion-exchange mechanism.
  • Separate acids by ion-exclusion mechanism.
  • All mobile phases are LC-MS and preparative chromatography compatible (working pH range from 2.0 to 7.0).

Coresep S columns are available in all standard dimensions
Column IDs:
4.6 mm, 3.2 mm, 2.1 mm, 1 mm, 0.5 mm, 0.25 mm
Column Lengths: 150 mm, 100 mm, 50 mm, 25 mm, 10 mm
Particles: 2.7 um
Pores: 90 A

 

Primesep Columns

Introduction

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.

Primesep 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 development service is available for customers. Ask about special custom LC-phases tailored for specific separations.

Literature

Primesep Literature for download

  • Primesep® Columns, Methods, Applications
  • Primesep® Separation of Ions, New Alternative to Ion Chromatography
  • Primesep® Solving Problems of Pharmaceutical HPLC Analysis

Pharmaproblems

Primesep Catalogue

 

Acidic Primesep Phases for cation exchange

SIELC Technologies provides  several different columns with negatively charged functional groups due  to the embedded anionic ion-paring reagent.

SieLC_acidic_pic

Primesep A

Is the strongest acidic column, while Primesep C is the weakest acidic column. The 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.

The column name corresponds to pH value of the mobile phase where acid residue on the column switches from ionize form to neutral one.

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 500

Is a reverse-phase analytical column with embedded acidic ion-pairing groups.

  • Primesep 500 has an embedded carboxilic acid with a pKa of about 5.
  • Improves retention of basic compounds by cation-exchange mechanism at pH > 5.
  • Separates acids by ion-exclusion mechanism at a mobile phase pH > 5.
  • Retains basic compounds, as any RP column, at pH < 5
  • Retains neutral compounds by reverse-phase mechanism.
  • All mobile phases are LC-MS and preparative chromatography compatible.

Primesep 500 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 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 200 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 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 100 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

SieLC_Versatility of Primesep Stationary Phases

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 A 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

 

Complex Interaction with Primesep C

SIELC Technologies has developed a new class of columns with combined reverse-phase, cation  exchange, and complex-formation properties in interaction with amines,  sulfonium, phosphonium and metal ions.

SieLC_complex_pic

This column has a unique unparallel selectivity for complex mixtures separation.

The pH working range  for these columns is from 1 to 7, but their complex formation and  cation-exchange properties are substantially suppressed at the pH below  3. In order to facilitate the complex formation, the pH of the mobile  phase should remain in the range of 3-7. The degree of complex formation can be adjusted by selecting the pH of the mobile phase.

The unique complex properties of the  column can be observed in the separation of alkali metals ions. The ions elute on the Primesep C column in reverse order compared to  ion-exchange elution. The unusual elution order is found in primary,  secondary, and tertiary amines. Secondary and tertiary amines have  higher pKa values than primary amines, which makes them retain longer by ion-exchange mechanism.

With the complex forming  properties of Primesep C columns, the primary amines are more retainable than secondary and tertiary amines. This extraordinary elution pattern  allows tailoring the separation of amines to provide the most  appropriate peak order for quantitation and preparative chromatography.

Primesep C

Is a reverse-phase analytical column with embedded complex-forming groups.

  • Improves retention of positively charged compounds by forming a host-guest complex.
  • Retains neutral compounds by reverse-phase mechanism.
  • All mobile phases are LC-MS and preparative chromatography compatible.

Primesep C 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

HPLC separation of aromatic compounds on Primesep P columns.

SieLC_phenyl_pic

SIELC Technologies has developed a new class of stationary phases, which provides three interactions with analytes:

    reverse-phase interaction
    pi-pi interaction
    strong cation exchange interaction
Depending on analyte properties, one, two, or all three interactions can be applied to the separation

This stationary phase facilitates the separation of structural isomers of aromatic compounds.

Additional pi-pi interaction creates a difference in the bind state for structural isomers and often resolves critical pairs of compounds. The degree of pi-pi interaction can be adjusted by varying the amount of acetonitrile in the mobile phase

When methanol is used as an organic modifier, the highest degree of aromatic interaction can be achieved.

Primesep P

Is a reverse-phase analytical column with embedded aromatic and acidic ion-pairing groups.

  • 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

 

Anion Exchange with Primesep B Type of columns

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

Primesep SB

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

Primesep B2

Is a weak basic column. It also has carboxilic acidic 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

Was 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 carboxilic groups and remains positively charged throughout the working pH range.

SieLC_Direct Plasma Analysis with Primesep D

The fourth column, which offers anion-exchange properties is Primesep AB column. This column offers also cation-exchange properties and useful in separation of complex mixtures of polar anionic and cationic compounds.

SieLC_basic_pic

All B-type columns provide at least two main interactions with analytes: the reverse-phase interaction and the anion-exchange interaction. Neutral analytes retained by 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 the type of an acid 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.

SieLC_Primesep B_S_5_2

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 SB

Is a reverse-phase analytical column with strong embedded basic ion-pairing groups.

  • Improves retention of acidic compounds by anion-exchange mechanism.
  • Separates bases by ion-exclusion mechanism.
  • Retains neutral compounds by reverse-phase mechanism.
  • All mobile phases are LC-MS and preparative chromatography compatible (working pH range from 1.5 to 5).

Primesep SB 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 B2

Is a reverse-phase analytical column with embedded basic ion-pairing groups.

  • Improves retention of acidic compounds by anion-exchange mechanism.
  • Separates bases by ion-exclusion mechanism.
  • Retains neutral compounds by reverse-phase mechanism.
  • All mobile phases are LC-MS and preparative chromatography compatible (working pH range from 1.5 to 7).

Primesep B2 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 D

Is a  reverse-phase analytical column with embedded basic ion-pairing groups  specifically design for direct plasma analysis.

Does not retain high-molecular weight plasma components.

  • Retains hydrophobic and acidic components.
  • All mobile phases are LC-MS and preparative chromatography compatible (working pH range from 1.5 to 4).

Primesep D 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 AB

Is a reverse-phase column with embedded acidic and basic ion-pairing groups.

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

 


Primesep Specialty Products

Primesep N

Is a normal-phase analytical column with embedded acidic groups.

  • Primesep N has embedded acidic groups with a pKa of about 5.
  • Improves retention of basic compounds by cation-exchange mechanism at pH > 5.
  • Separates polar compounds by HILIC mechanism.
  • All mobile phases are LC-MS and preparative chromatography compatible.

Primesep N 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: 300 A, 100 A

 

Primesep S2

Is a silica-based acidic-column with hydrophilic properties.

  • Separates basic compounds by cation-exchange mechanism.
  • Separates basic and neutral polar compounds by HILIC mechanism.
  • All mobile phases are LC-MS and preparative chromatography compatible.

Primesep S2 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 B4

HPLC Columns for Separating inorganic anions

Primesep B4 is a reverse-phase (short carbon chain) analytical column with embedded basic ion-pairing groups.

  • Improves retention of acidic compounds by anion-exchange mechanism.
  • Retains and separates basic and acidic surfactants based on multi-mode mechanism.
  • Separates hydrophobic bases by ion-exclusion mechanism.
  • Retains hydrophobic neutral compounds by reverse-phase mechanism.
  • All mobile phases are LC-MS and preparative chromatography compatible (working pH range from 2.0 to 4.5).

Primesep B4 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 PB HPLC Columns

Primesep PB is a reverse-phase analytical column with embedded aromatic and basic ion-pairing groups.

  • HILIC Retention of Polar Compounds
  • Improves retention of acidic compounds by anion-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 (working pH range from 2.0 to 5).

Primesep PB 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 S HPLC Columns

Primesep S is a normal-phase analytical column with embedded acidic ion-pairing groups

  • Most suitable for separation of common sugars and other neutral or very polar molecules.
  • Retains and separates amino acids with different buffers at low concentration.
  • Separates weak bases by ion-exchange mechanism.
  • Separate acids by ion-exclusion mechanism.
  • All mobile phases are LC-MS and preparative chromatography compatible (working pH range from 2.0 to 7.0).

Primesep S 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 AP HPLC Columns

Primesep AP is a silica-based amino-column with hydrophilic properties.

  • Separates acidic compounds by anion-exchange mechanism.
  • Separates basic and neutral polar compounds by HILIC mechanism.
  • All mobile phases are LC-MS and preparative chromatography compatible.

Primesep AP 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 X HPLC Columns

Primesep X is a silica-based amino-column with weak hydrophobic properties.

  • Separates acidic compounds by anion-exchange mechanism.
  • Separates basic and neutral compounds by RP mechanism.
  • All mobile phases are LC-MS and preparative chromatography compatible.

Primesep X 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


 

Obelisc Columns

With the increasing demand for the analysis of polar and ionisable  compounds, SIELC Technologies have developed Obelisc - a new  generation of mixed mode columns. Obelisc R and Obelisc N are complimentary phases for the separation  of polar and non-polar compounds using multiple separation  mechanisms (RP, ion-exchange, NP, ion exclusion). Both phases have  positive and negative charges separated by a long organic chain,  allowing both charges to simultaneously participate in electrostatic

SIELC_Obelisc_Intro_Brochure

Compound / Phase Interaction Mechanisms

SIELC_Obelisc_N_Interactions

SieLC_Obelisc_R_Interactions

Two complimentary columns, Obelisc R and Obelisc N, based on Liquid Separation Cell ( LiSC ) technology, offer a new approach to separate a variety of small molecules. Buffer concentration, buffer pH, and organic modifier concentration are three orthogonal parameters of the mobile phase that allow adjustment of column properties to separate complex mixtures.

SieLC_LIving_Separation_Cell

Obelisc N

Is a column which has very polar characteristics and works well for polar and charged analytes.

  • Obelisc N offers both positively and negatively charged groups to interact with positively or negatively charged analytes.
  • Multiple separation modes (NP, HILIC, IE).
  • In HILIC mode, Obelisc N offers different selectivity form other HILIC or bare silica columns.
  • Mass spec, ELSD, preparative and low UV (<220 nm) compatible, low concentration buffers.

Obelisc R

Is a reverse-phase analytical column and can be used in traditional, reversed-phase type applications.

  • Due to the presence of ionic groups and a long hydrophobic chain,  Obelisc R offers additional retention and tuning that is not available  with traditional reversed-phase columns.
  • Multiple separation modes (RP, NP, IE).
  • Mass spec, ELSD, preparative and low UV (<220 nm) compatible, low concentration buffers.

Obelisc R & N  HPLC Columns

SIELC Product

 

Particle diameter um

Pore size A

Column Id mm

Column Length mm

Obelisc R

 

5, 10

100

22

 250, 150, 100, 50, 25, 10

Obelisc R

 

5, 10

100

10

 250, 150, 100, 50, 25, 10

Obelisc R

 

5, 10

100

4.6

 250, 150, 100, 50, 25, 10

Obelisc R

 

5, 10

100

3.2

 250, 150, 100, 50, 25, 10

Obelisc R

 

5, 10

100

2.1

 250, 150, 100, 50, 25, 10

Obelisc R

 

5, 10

100

1

 250, 150, 100, 50, 25, 10

Obelisc R

 

5, 10

100

0.5

 250, 150, 100, 50, 25, 10

Obelisc R

 

5, 10

100

0.25

 250, 150, 100, 50, 25, 10

Obelisc N

 

5, 10

100

22

 250, 150, 100, 50, 25, 10

Obelisc N

 

5, 10

100

10

 250, 150, 100, 50, 25, 10

Obelisc N

 

5, 10

100

4.6

 250, 150, 100, 50, 25, 10

Obelisc N

 

5, 10

100

3.2

 250, 150, 100, 50, 25, 10

Obelisc N

 

5, 10

100

2.1

 250, 150, 100, 50, 25, 10

Obelisc N

 

5, 10

100

1

 250, 150, 100, 50, 25, 10

Obelisc N

 

5, 10

100

0.5

 250, 150, 100, 50, 25, 10

Obelisc N

 

5, 10

100

0.25

 250, 150, 100, 50, 25, 10

 

 

Promix  for biomolecule separation

Introduction

Promix is an alternative chromatography technology for efficient resolution of peptides and proteins. The technology is based on a combination of two interactions - hydrophobic and ionic. This approach is possible due to a new type of separation media: a chemical combination of hydrophobic and ionic functional groups on a ligand bonded to a silica support. With this phase, unparalleled selectivity and peak capacity can be achieved. Independent adjustment of the amount of buffer and organic modifier creates an infinite number of separation conditions that are suitable for many types of biomolecules.

Literature

Promix® Enter a New Era in Biomolecule Analysis with Promix Columns Unsurpassed Selectivity and Peak Capacity for Peptides and Proteins

SieLC_Peptides Analytic with Promix

SieLC_Alternative Selectivity of Promix

SieLC_Peptide Digest Separation with Promix MP

SieLC_Peptides Different Gradient Types with Promix MP

SieLC_Proteins and Peptides on Promix Columns

SieLC_Separation of Insulin Analogues With Promix MP

 

There are four different Promix Columns to be selected as follows:

Use the following chart to select a column based on the analyte's properties

SieLC_Promix_Selection_Chart

 Select the column length based on sample complexity

    • 150-250 mm for proteomics, protein digests, and complex samples
    • 50-150 mm for synthetic analysis and purification

Select the column i.d. based on sample loading and sensitivity needs

Mobile Phase should be pH 2-4 with:

  • TFA (0.05-0.3%)
  • Formic Acid (0.1-0.9%)
  • Ammonium Acetate or Ammonium Formate
  • Organic Modifier should be Acetonitrile
  • Ionic strength, pH, and organic modifier gradients can be used to optimize the separation

Promix SP HPLC Columns

Promix SP is a stationary phase for small peptides under 1 kDa with PI value below 6.0.

  • Reverse phase and ion-exchange mechanism of interaction.
  • Use acidic mobile phase pH from 2 to 5.5.
  • Use ion strength at least 20 mM.
  • 100√… pore size is available.
  • Promix SP is a column for very short and/or very hydrophilic peptides. Typically it is difficult to retain such peptides on RP column. The Promix SP column offers an improved retention profile and alternative selectivity if compared with RP separation.

Promix SP 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: 5 um
Pores: 100 A

Promix AP HPLC Columns

Promix AP is a stationary phase for small and medium size peptides under 3 kDa with PI value below 7.0.

  • Reverse phase and ion-exchange mechanism of interaction.
  • Use acidic mobile phase pH from 2 to 5.5.
  • Use ion strength at least 20 mM.
  • 100√… and 300√… pore sizes are available.
  • Promix AP is a column designed for short basic peptides. This phase allows the retention of peptides with several histidine and lysine residues. A pH gradient is the most convenient way to control retention of basic peptides on this column. Use a buffer with a neutral or slightly acidic pH (4.5 - 7) in the beginning of the gradient, and use a more acidic buffer (pH 2 - 3.5) in the end of the gradient elution.

Promix AP 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: 5 um
Pores: 300 A, 100 A

Promix MP HPLC Columns

Promix MP is a stationary phase for medium size proteins and peptides from 1 to 10 kDa.

  • Broad pI value.
  • Reverse phase and ion-exclusion mechanism of interaction.
  • Use acidic mobile phase pH from 2 to 5.5.
  • Use ion strength at least 1 mM.
  • 300√… and 800√… pore sizes are available.
  • Promix MP is a column designed for medium size hydrophobic peptides. Typical applications include protein digest which increases peak capacity of the digest by 30 to 50%. This column offers alternative selectivity if compared with simple RP columns. Typically difficult to separate pairs of peptides can be resolved on this stationary phase.

Promix MP 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: 5 um
Pores: 800 A, 300 A

 

 

Sharc ™

 

Introduction

SHARC™ HPLC columns are the latest innovation from SIELC Technologies. SHARC columns are the first commercially available columns with separation based primarily on hydrogen bonding. SHARC stands for  Specific Hydrogen-bond Adsorption Resolution Chromatography.

Hydrogen bonding is an interaction between hydrogen atom bound to electronegative atoms in a molecule, such as oxygen, nitrogen, fluorine. This is typically a weak interaction, especially when separation is performed in aqueous solutions. Liquid chromatography techniques evolved as tool for separation of different molecules based on their physico-chemical properties. Most common techniques of the separation are:

  1. Hydrophobic separation based on degree of hydrophobicity of the molecule
  2. Ion-exchange separation based on number, nature, and distribution of charges in the molecule.
  3. Normal phase separation based on hydrophilic properties of the molecules including molecule dipoles value and dipoles position
  4. Size exclusion separation (SEC) based on molecule size and shape

 

Columns and stationary phases based on these techniques never perform purely with one type of interaction. Hydrogen bonding is omnipresent in every one of these techniques with minor contribution to retention and selectivity. However, in some cases, especially in normal phase chromatography, the contribution of hydrogen bonding can be significant. SHARC 1 is the first column specifically design to perform a separation based entirely on the interaction of the molecules capable providing hydrogen atom (donor) or attract hydrogen atom (acceptor) to the stationary phase with special properties.

Technology

SHARC 1 column operation conditions are unique. A mixture of acetonitrile (MeCN), a weak solvent, and  methanol (MeOH), a strong solvent, are used as the mobile phase. Pure MeCN  has very insignificant amount of hydrogen bonding with the SHARC stationary phase, while MeOH interacts strongly with SHARC stationary phase, which reduces the retention of analytes based on it’s capacity to hydrogen bond. By changing ratio of MeCN/MeOH the optimum retention profile can be obtained for many types of molecules with high selectivity, peak shape, efficiency, and speed.

Sharc_Hydrogen_bonding_interactio
                                                    Sharc_H_bonding_Chromatography

Surface interaction within the column with hydrogen bonding                                                            Surface interaction within the column with hydrogen bonding

 capable analytes with acetonitrile as the mobile phase                                                                      capable analytes with methanol as the mobile phase

Hydrogen bonding energy is usually in range of 30 kJ/mol or less and depends strongly on the nature of the functional groups and their orientation within the molecule. This energy difference is the basis for selectivity of the interaction between the stationary phase and the molecule of different structure and chemical characteristics.

SieLC_Bonding_energies

A given molecule can retain on the stationary phase with more than one hydrogen bond, while also performing as a donor or acceptor of a hydrogen atom.

Presence of a polarized hydrogen atom is not always enough to observe retention by hydrogen bond mechanism. Sometimes a compound can form intramolecular interactions, as oppose to intermolecular hydrogen bonding, and does not participate in stationary phase interaction.

 Advantages

SPEED.
MeCN/MeOH mixture has 2-3 times lower viscosity than water/MeOH or water/MeCN mixture (fig. 5). As result smaller particles for column packing can be used without increasing the working pressure. UPLC-like conditions can be easily obtained on regular HPLC instrument with 2-3 times higher velocity with a shorter column with small particles. This can increase the speed of analysis up to 5 times. Using UPLC (RRLC, UHPLC) equipment allows to increase speed of analysis by another  factor  of 5 or 6.

SOLUBILITY.
MeOH is one of the most universal solvent for organic compounds. Combination of MeOH with MeCN allows to dissolve almost any molecules with high or low polarity. Hydrophobic molecules such as surfactants, lipids, and oil soluble vitamins, are soluble in this solvent combination. Very polar molecules such as sugars, di-ols, salts of amino compounds, carboxylic acids also dissolve in this solvent system.

SELECTIVITY.
Since hydrogen bonds formation is very specific in the interaction energy and strongly depends on molecule geometry, number of functional groups and the position of the functional groups, the selectivity of resolution of molecule of similar nature such as isomers, related impurities, product of oxidation or reduction can be achieved with SHARC separation very efficiently.

PREPARATIVE SEPARATION.
MeCN/MeOH mixture has low boiling point and  much easier to evaporate than water. As result this solvent system is much friendlier for preparative type chromatography. The additional benefit of low viscosity allows SHARC to perform prep separation with higher throughput.  This mobile system is MS friendly and allows implementation of MS-driven sample collection.  In most cases isocratic method can be used due to high selectivity of the column which allows to recycle  most of the mobile phase minimizing solvent consumption.

WIDE RANGE OF COMPOUNDS.
Wide range of molecules can be analyzed with SHARC technique. Practically any molecule with functional group which contains oxygen and  nitrogen can be retained and separate from similar compounds in this technology. Hydrocarbons is one class of compounds that will be retained poorly in this system, but they are typically well separated in reverse phase HPLC or GC.

Sharc_solvent_viscosity-profile

Method Development

Hydrogen-bonding interaction offers unique selectivity based on number of “interaction points” available for hydrogen bonding. One of the useful characteristics to determine retention patterns in hydrogen-bonding mode is the molecular polar surface area (PSA). This calculated parameter is usually used for prediction of drug transport properties, but we successfully applied it to hydrogen-bonding interactions. Polar surface area is defined as a sum of surfaces of polar atoms (usually oxygens, nitrogens and attached hydrogens) in a molecule.  Since those polar  atoms can participate in hydrogen-bonding interaction, estimation of elution order can often be made based on PSA. While PSA is a good indicator of elution time, it must be noted that polar surface area does not account for the accessibility of hydrogen-interaction sites. Not every polar surface participates in intermolecular hydrogen interactions with the stationary phase.

Proximity of “interaction points” to each other within one molecule also needs to be considered since molecules can form an intramolecular hydrogen-bonding, which competes with intermolecular interaction between analyte and stationary phase. This reduces retention time in hydrogen-bonding mode.  Such structural factors provides unique selectivity among similarly structural (isomers, homologs, degradation products, precursors) molecules.

Since SHARC 1 column is a mixed-mode column, pKa is another useful parameter in method development for these columns. SHARC columns operate in non-aqueous mobile phase, but some effect of charge interaction of stationary phase and ionizable molecules still exists and contributes to the retention profile.

Literature:

SHARC®  LC Column with Hydrogen Bonding Based Separation

 

 

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