Kromasil technical evidence

• Introduction
• Mechanical stability
• Chemical stability
• Surface properties
• Loadability
• Scalability

Surface properties influence your separations

Chromatographic purifications are influenced by the surface properties of the stationary phase. Purification of difficult types of compounds like amines, basic peptides and chelating compounds is influenced by the following parameters:

1. Hydrophobicity

Hydrophobicity determines the separation power of HPLC materials when hydrophobic interactions are dominating. High surface coverage and type of ligand influence this parameter. How selectivity is influenced by the stationary phase hydrophobicity, is shown in fig. 10.

Fig. 1 - Increase in separation time of a mixture of substances due to increased hydrophobicity of the stationary phase (Kromasil 100 Å, 5 µm,  C4, C8 and C18).
Sample:
Uracil, benzamide, methyl benzoate, toluene, propyl benzene, butyl benzene.
Conditions:
Column size: 4.6 x 250 mm
Mobile phase: Acetonitrile/Water(70/30)
Flow rate: 2 ml/min
Detection: UV 254 nm

2. Undesired Ion Exchange Interactions

The interaction of basic samples with acidic silanols is mainly an ion exchange process:

These strong interactions may cause undesired tailing when separating basic compounds, such as tricyclic antidepressants.

To illustrate this, the separation of a mixture of tricyclic antidepressants, and toluene as a hydrophobic reference, is shown in figure 2 for Kromasil and other commercial materials.

Fig. 2 - Separation of a mixture of antidepressants and toluene as hydrophobic reference with Kromasil 100 Å, 5 µm, C18, a comparable, high quality commercial material and a low quality commercial one.
Sample:
1. Uracil, 2. Phenylpropanol amine (3.0 µg), 3. Nortriptyline (1.75 µg), 4. Toluene (3.0 µg), 5. Imipramine (3.9 µg) and 6. Amitriptyline (3.0 µg).
Conditions:
Column size: 4.6 x 250 mm
Mobile phase: Acetonitrile/10 mM sodium phosphate pH 7.0 (60/40)
Flow rate: 1.5 ml/min
Temperature: 40°C
Detection: UV 215 nm

3. Metal impurities in the stationary phase

Metal impurities in the silica structure affect the acidity of silanols, creating non-homogeneous structures which strongly complex with chelating compounds.

For this reason, metal impurities must be carefully monitored in silica-based materials. In figure 3, the content of metal impurities of Kromasil and other commercial materials is shown.

Fig. 3 - Content of metal impurities of Kromasil and some other commercial materials.

The chromatographic purification of the chelating compound 2,2'-bipyridyl with Kromasil C8 and another commercial material is shown in figure 4. Note that for Silica A, only the non-chelating reference, 4,4'-bipyridyl is eluted and the chelating compound is irreversible adsorbed.

Fig. 4 - Chromatogram of a chelating compound 2,2'-bipyridyl (Rt=5.44) and 4,4'-bipyridyl (Rt=3.93) for Kromasil and another commercial material. The test is performed in metal-free system and column (Peek).
Conditions:
Column size: 250 x 4.6 mm
Eluent: MeOH/H ₂O (60/40)
Samples: 2,2'-bipyridyl (2 µg) and 4,4'-bipyridyl (0.5 µg)
Flow rate: 1 ml/min.
Detection: UV 254 nm