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Unique Type-C Silica based HPLC columns
Evolution not Revolution; A History of Silica
Before the Introduction of Cogent TYPE-C Silica™
Introducing TYPE-C Silica™
Unique Surface Chemistry of TYPE-C Silica™ Products
Hydration Shell, Liquid Stationary Phase type-B v. TYPE-C™
Unique Bonding Capabilities Produce Unique Selectivities with Problem Solving Benefits
Separate Mechanisms for Separations
Features and Benefits to Chromatographers


Evolution not Revolution
A History of Silica
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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 10m and later 5m 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.

 





Before the Introduction of Cogent TYPE-C Silica™
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... all silica based HPLC stationary phases had polar, acidic, silanol (Si-OH) functional groups on the surface. Even with end-capping technology after exhaustive bonding, as much as 30%-50% of the silanols remain un-bonded and can contribute to unwanted separation results due to electrostatic interaction with solutes. To minimize type-B silica silanols it may be desirous to use small end capping groups such as C1 to cover these sites. The disadvantage of this concept is that these small groups are readily hydrolyzed away in reverse phase solvents below pH 3. Therefore this column technology is suited to a higher pH range; 6 to 9. Since many of the silanols sites are not fully ionized in the mid pH range, using this it will cause a lack of precision in the method. Silanols may be fully ionized (therefore do not contribute to lack of precision) at the higher pH but higher pH causes dissolution of silica; end capping is effective at retarding this dissolution (which begins to occur at and above pH 8). Most loss of retention at high pH is not due to loss of bonded phase and end capping groups (lost to hydrolysis) as it is at low pH but is due to dissolution of the underlying silica bed that results in the production of newly formed silanol sites causing a change of carbon load and instability of the packed column.
Before the Introduction of Cogent TYPE-C Silica™...


...all other silica based HPLC stationary phases had mixed separation mechanisms involving reverse phase type interactions with the bonded ligands and normal phase type interactions with the silanols groups on the silica support. High carbon loadings of HPLC columns (achieved through organic bonding schemes) were developed to minimize this effect. The disadvantage of high carbon loading is that it reduces the ability to separate highly polar compounds and limits the number of analytes successfully separated with these phases although they can perform well for protonated compounds.

...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.




Introducing TYPE-C Silica™
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Cogent TYPE-C Silica

Think Inside the Box... Solve difficult separation problems with your current skill set and experience.

TYPE-C™ silica based HPLC columns have opened a new chapter in the book that describes chromatographic supports and phases. Based on high purity-low metal content Type-B silica, the manufacturing process for TYPE-C Silica™ continues where others leave off. Our patented, proprietary process produces a surface populated with silicon-hydride groups (SI-H) which are very stable and non polar. The TYPE-C Silica™ support still has all the advantages of Type-B silica such as spherical shape, low metal content, high purity, high mechanical strength, narrow pore size distribution, wide range of pore sizes, easily chemically modified, no swelling in the presence of solvents for stable packed beds and does not produce fines as irregular silica. TYPE-C Silica™ products also have...



Unique Surface Chemistry of TYPE-C Silica™ Products
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Cogent TYPE-C Silica

The surface of TYPE-C Silica™ is predominantly populated with non-polar, silicon-hydride (Si-H) groups instead of the polar, silanol groups (Si-OH) that dominate the surface of previous varieties of silica. This feature of TYPE-C Silica™ particles, gives it many unique, useful and helpful chromatographic qualities.

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
as well as a unique separation character that allows for improved aqueous based reverse phase, organic solvent based normal phase and aqueous based normal phase without an organic bonded stationary phase.



Hydration Shell, Liquid Stationary Phase type-B v. TYPE-C™
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Cogent TYPE-C Silica

There are two basic mechanisms that create retention of solutes in HPLC: partitioning and adsorption. Partitioning can be defined as changes between two or more phases. Adsorption can be defined as “attractive forces” between solutes and a solid surface. HPLC columns have different retention phenomena taking place to varying degrees on the Stationary and within the Mobile Phases.

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.



Unique Bonding Capabilities Produce Unique Selectivities with Problem Solving Benefits
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Structures
Click here to view NMR Spectra

Because of the unique silica surface, Cogent TYPE-C Silica can bond with any chemical moiety which possesses either a terminal double or triple bond. Due the resulting strong chemical bond between silicon and carbon, these bonded phases are much more stable and resistant to conditions that can cause hydrolysis.

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 4m, the pore size is 100A with 300A phases to be released during the year.



Separate Mechanisms for Separations
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Another unique feature of the Cogent TYPE-C Silica™ based products is that the silica surface, with silicon-hydrides, can act independently of the bonded phase giving it the ability to interact differently with non-polar to mid-polar compounds.

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.




Features and Benefits to Chromatographers
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Some Features of TYPE-C™ Columns:
Chromatographer’s Benefits
Silicon-Carbon Bonds instead of Siloxane
More stable and durable.
UDC-Cholesterol: pH 2.0 – 8.2
Bidentate C18: pH 1.5 – 10.0+ Bidentate C8: pH 1.5 – 10.0+

Si-H replaces Si-OH
Rapid equilibration between gradients
Temperature Stability Increased
Use Temperature as a selectivity Tool
Can be used with Hard Lewis Acids
Use Lewis Acids as Mobile Phase Additives
Free of Salts
Contaminant Free Surface
Bonded to a Silicon-Hydride Surface
Will not strongly bind Carboxylic Acids
or Sulfonic Acids
Minimized Silanophilic Activity
Stable at high flow rates (up to 3ml/minute)
Weakly Associated Hydration Shell
Water Friendly Columns, easy to use.
Very rapidly remove water from the stationary phase and silica.
Use 100% water on C18
Without loss of retention with time
Lack of pH hysteresis
Quickly change mobile phases and pH buffers.
Perform ANP & RP at the same time
Separate Polar & Non-Polar compounds in the same run
Retain Polar compounds at extremely high organic content
Increases sensitivity of mass detectors using ESI
User Non-Polar solvents
Retain & separate compounds which are non-soluble in water
Low affinity for water
Run normal phase separations on these "Bonded" columns without the hassles of moisture in solvents
Use high organic % content in mobile phases
Shorten sample prep time. No need to dry down samples after SPE. Inject sample diluent (high organic) right on the column
Bonded phase that performs ANP & RP
Get the performance of HILIC columns on a stable, robust bonded phase
High efficiency & stable
Great peak shapes & columns that last a long time




 

 
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