Evolution not Revolution A History of Silica [Top] Irregular shaped silica particles of wide pore size distribution were the original support used to manufacture HPLC columns. At the time these supports were developed they were the best technology available. Due to inherent deficiencies and inconsistencies these columns limited chromatographers to the use of organic solvents and Normal Phase Chromatography and were not very reproducible. To achieve reverse phase separations, silica had to be bonded with low polarity organo-silanes using siloxane bonding technology. These siloxane (Si-O-Si-C) bonds used in this process can be hydrolytically susceptible to attachment failure and during the lifetime of column can result in separation problems when working at extreme pH or with strong buffers or ion pair reagents. The development of spherical shaped silica particles of 10µm and later 5µm particle size proved to be a great advancement in HPLC supports. The uniformity of shape and size allowed for better packed columns which resulted in an increase in precision and ruggedness. These tightly controlled particles did not create “fines” as irregular shaped particles do resulting in columns that lasted longer and were more stable. Even with this improvement, HPLC columns still suffered from frequent tailing problems when basic compounds were separated at desired pH. Today, Reverse Phase Separations are predominantly being performed on silica bonded with hydrocarbons mostly C8 and C18. The next evolution in HPLC support development was the High Purity Silica which was specifically produced to minimize the amount of trace metals in the silica lattice. Spherical silica manufactured with low metal content (especially aluminum) minimizes the effect that the free, residual silanols would have on the chromatography when they are ionized (high pH). This step forward was termed Type-B silica to differentiate it from the lower purity, more acidic, higher metal content, spherical silica which preceded it. This major advancement in chromatography produced improved peak shape and increased pH tolerance and packed bed stability. Columns not only lasted longer, but produced better chromatography for many compounds of interest and were more stable.

...to perform Organic-Normal Phase HPLC you were limited to un-bonded silica or specialty bonded phases such as Cyano or Amino. Silica supports were hydroscopic in nature and quite strongly retained water, the most polar common solvent used and adsorbed by organic solvents variably depending on atmospheric conditions and the type of solvent used. The stationary phase (Type B silica) then adsorbs the water from the mobile phase due to free silanols and as the water content on the support begins to increase, the retention of the analytes begin to change. Long tedious steps had to be taken to tightly control the water content in the mobile phase solvents.

Some of the inherent limitations of Type-B silicas associated with free silanols have been overcome with TYPE-C Silica™ products. These include but are not limited to;

• surface acidity
• improved pH stability
• less hydroscopic

The silica surface and bonded phase of HPLC columns are responsible for adsorption. Partitioning takes place on the “quasi liquid phase layer” that forms around the surface of the Stationary Phase. The “Liquid Stationary Phase” is mostly formed by solvating it with water. This “hydration shell” and the subsequent organic phase that forms around the Stationary Phase contributes to the separation mechanism in conjunction with the underlying solid phase which can have different functionalities at work. Depending on the bonded phase and liquid phase that forms, the amount of partitioning will vary.

With Type-B silica, water is readily adsorbed onto the silica’s surface, which contains active silanol groups; this strong adsorption of water forms a durable “Hydration Shell” which is responsible for longer equilibration times and lack of reproducibility in Normal Phase separations as well as other chromatographic difficulties and is often the main cause of pH hysteresis and lack of reproducibility. This is often the case in RP when using competitive, polar embedded phases.

Strong adsorption of water to type-B silica (shown above) makes using normal phase chromatography very difficult. The silicon-hydride groups (Si-H) found on the surface of TYPE-C Silica™ are not prone to such strong water retention (shown above) as type-B silica making it an excellent choice for Organic-Normal Phase with improvements in speed and range of solvent possibilities. The weaker water adsorption also accounts for the little to no hysteresis observed when changing from Organic-Normal to Aqueous-Normal/Reverse Phase with TYPE-C™ products or when changing pH with TYPE-C Silica™ based HPLC columns such as Cogent UDC-Cholesterol™. This feature makes the column preferred over polar-embedded phases which often exhibit long term memory effects. TYPE-C Silica™ products extend the useful range of Normal phase from Hexane/Ethyl Acetate all the way to Water/Acetonitrile with excellent precision.

Initial products using the above bonding include Cogent TYPE-C Silica™, Cogent UDC-Cholesterol™ (10 carbon straight chain with an ester linkage to cholesterol) on TYPE-C Silica™, and Cogent Bidentate C18™ on TYPE-C Silica™. These products will be sequentially released starting March 2003. The particle size is 4µm, the pore size is 100A with 300A phases to be released during the year.

An example of this mechanism is the work done by MicroSolv on Metformin and Glyburide; two anti-diabetic drugs of vastly different partition coefficients. When separating these two compounds as a single mixture, the Cogent TYPE-C™ Silica column produces good separation of the compounds with good peak shape for Glyburide and alightly tailing peak shape for Metformin. All other features of the TYPE-C™ series are evident in the separation. When the Cogent UDC-Cholesterol™ column is used instead of the Silica column the peak shape is excellent for both compounds. This suggests that the bonded phase interacts differently from the silica surface and that the silica surface is acting on the compounds similarly in both columns. For a complete review of this work and how to apply it, please contact us. Click here for a detailed description of how this works.