Silica reversed phase chromatography (RPC) is a popular purification tool for peptide-based therapeutics such as insulin and GLP-1 compounds. Conventional silica media commonly have a short lifetime in these chromatographic processes that require high pH conditions, such as column regeneration by 0.1 M NaOH solution. Amerigo Scientific offers UniSil® Revo reversed phase media that are produced on the Precisely Controlled Silica (PCS) technology for the formation of monodispersed particles. A further revolutionary process is utilized to eliminate the micropores, making the pores of UniSil® Revo particles more interconnected and making the silica skeleton thicker. Thus, UniSil® Revo particles have pores that are more easily accessible to analytes, superior mechanical strength, and higher pH stability compared to other conventional silica particles.
UniSil® Revo Chromatography Media
Characteristics
| Particle Size |
Pore Size |
Surface Area |
Pore Volume |
Max. Pressure |
pH Range |
| 10 µm |
120 Å |
320 m²/ g |
1.1 ml/g |
600 bar |
2-9 |
Features of UniSil® Revo
- Next generation of particles with optimized pore structure and thicker pore walls for improved lifetime
- Good selectivity and resolution of impurity peaks
- High yield and high purity for insulin and GLP-1 purification
Mechanical Strength Test
UniSil® Revo particles have thicker silica skeleton and therefore have better mechanical strength. As shown in Figure 1, the linear relationship of back pressure of a 2.1 x 150 mm column with flow rate indicates that the particles are strong enough up to 9000 psi (600 bar).
High pH stability Test
After 0.1 M NaOH (pH 13) flash for 9 hours, UniSil® Revo 10-120 C8 still maintained a good peak shape compared to other silica C8 media.
Figure 3. Comparison of UniSil® Revo 10-120 C8 and the competitor's 10-100 C8 media in pH 13 condition
Applications of UniSil® Revo Chromatography Media
UniSil® Revo for Insulin Purification
Figure 4. Comparison of UniSil® Reva and the competitor's silica media
Compared to the competitor's silica media, UniSil® Revo showed a narrower peak width and better resolution for front and back impurities.
Figure 5. Analytical chromatogram of human insulin puried using UniSil® Reva and the competitor's silica media
UniSil® Revo for the Purification of Synthetic Semaglutide
Purification of Semaglutide is a challenge in GLP-1 drug development. Amerigo Scientific offers UniSil® Revo and UniHybrid® Eterne media for Semaglutide purification using a two-step strategy.
1st Step: Purification of Semaglutide under basic condition using UniHybrid® Eterne C8
- The sample to be purified for the first time usually contains a lot of impurities which requires more 0.1 M NaOH washings/regenerations. UniHybrid® Eterne C8 provides a high yield under basic condition and excellent chemical lifetime for 0.1 M NaOH.
2nd Step: Purification under acidic pH 3.5 condition using
UniSil® Revo 10-120 C8
- The impurities in the second purification sample are usually less, which requires less 0.1 M NaOH regenerations. The use of UniSil® Revo 10-120 C8 can achieve a higher purity of Semaglutide.
Figure 6. Purification flow chart of synthetic Semaglutide
Figure 1. Pressure vs. flow of 
Figure 2. 
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