The Scherzo SM-C18 column is an ODS column modified with weak cation and anion ligands. It can retain highly polar ionic compounds that conventional ODS columns cannot retain. It is also capable of reversed-phase separation of general compounds. By optimizing the mobile phase, it has the potential to analyze a wider variety of compounds than traditional methods.
Exsample Initial Conditions for General-Purpose Analysis with SM-C18 [LC-MS Compatible]
| Column: | Scherzo SM-C18, 150 x 3 mm (SM035) |
| Mobile phase A: | 10mM Ammonium Acetate / Acetonitrile = 90 / 10 |
| Mobile phase B: | 100mM Ammonium Acetate / Acetonitrile = 50 / 50 |
| Gradient: | 0-100% B (0-15 min) |
| Flow rate: | 0.4 mL/min |
| Column temperature: | 37°C |
| Detection: | UV, ELSD, MS, etc. |
| Injection Volume: | 1-10 μL |
| Sample solvent: | 0.1% Formic Acid, 0.1% Acetic Acid,50mM Ammonium Acetate, etc. |
Explanation of Analytical Condition Settings
Adsorption
The stationary phase surface of Scherzo C18 column has both anion and cation ligands. At neutral pH, both ions are in a dissociated state.
Using a mobile phase with "neutral pH" and "low ionic strength" like 10mM Ammonium Acetate as the initial gradient condition allows both cationic and anionic compounds to be adsorbed "ionically."
Of course, being an ODS column, "hydrophobic" adsorption is also possible.
Elution
Since the solutes are adsorbed to the stationary phase "ionically," increasing the "ionic strength (salt concentration)" in the mobile phase will cause the ions in the mobile phase to interfere with the ionic interactions between the solutes and the stationary phase, resulting in elution from the stationary phase (principle of ion-exchange chromatography).
Additionally, increasing the concentration of the organic solvent will disrupt "hydrophobic interactions," leading to elution.
The strength of ionic and hydrophobic interactions with the stationary phase varies depending on the compound being analyzed. Therefore, to achieve precise separation, it is necessary to optimize the "salt concentration" and "organic solvent concentration" of the B solution.
Gradient
Ionic interactions are much stronger than hydrophobic interactions. Therefore, for better peak shape and reproducibility, isocratic analysis with this column is generally not recommended. A gradient elution with changes in ionic strength is preferred.
Depending on the elution and separation behavior, it may be necessary to optimize the initial and final concentrations of the gradient and the gradient slope.
Detection
Using ammonium acetate under these conditions makes it difficult to use UV detection at short wavelengths like 220 nm. In such cases, an ionic strength gradient with a neutral phosphate buffer is required, for example, a gradient of 5mM -> 30mM phosphate buffer (pH 7) + acetonitrile.
Sample Solvent
For ionic substances, it is essential to control the dissociation state in the sample solution by adjusting the pH of the sample solvent. Generally, the sample is dissolved in solution A, but depending on the properties of the compound, acid or organic solvent may be necessary.
The peak shape and retention time can be influenced by the sample solvent. In such cases, it is necessary to examine the sample solvent.
Examples of General-Purpose Analysis Using Scherzo SM-C18
Comparison with SS-C18, SW-C18
Scherzo SM-C18 demonstrates good overall responsiveness and versatility under the above conditions.
- Pinaverium Bromide
A spasmolytic used to treat gastrointestinal disorders. The basic nature of pinaverium makes it difficult to elute with SS-C18, while SM-C18 shows appropriate elution and good peak shape.
- Amlodipine Besylate
A comparison of Scherzo columns for this antihypertensive drug.
SM-C18 shows good results in terms of peak shape for besylate and amlodipine elution. - Nelfinavir Mesylate (MS)
An anti-HIV drug expected to be effective against COVID-19.
SM-C18 shows good elution balance for the highly polar, ionic mesylate and the highly hydrophobic nelfinavir. - Chlorpheniramine Maleate (MS)
An antihistamine drug. SS-C18 and SM-C18 show good retention and peak shape under these conditions.
- Rizatriptan Benzoate
Rizatriptan, a migraine medication, forms a benzoate salt (with cationic rizatriptan and anionic benzoate).
The above standard method is convenient for simultaneous analysis of both components.
Additionally, Scherzo SM-C18, with its moderate ionic properties, offers better retention balance compared to other Scherzo columns. - Vancomycin
A glycopeptide antibiotic. SM-C18 (and SW-C18) is effective for impurity separation.
- Tizanidine
A muscle relaxant with basic properties, showing good retention and peak shape with SM-C18's weak ionic nature.
- Tadalafil
A phosphodiesterase 5 inhibitor. Comparison of the three Scherzo columns shows similar retention, indicating weak ionic nature and hydrophobic retention behavior.
This suggests that the comparative evaluation of Scherzo columns is useful for understanding the ionic nature of solutes. - Eperisone
A spasmolytic with basic properties.
SM-C18 shows better elution behavior compared to SS-C18 and SW-C18 with strong ionic nature. - Etodolac
A nonsteroidal anti-inflammatory drug.
SW-C18 shows good peak shape under these conditions, with SM-C18 also showing appropriate retention and shape.