Cogent TYPE-C™ Silica HPLC columns are made with silica hydride and make the silica slightly hydrophobic. This very unique feature is why only TYPE-C silica can produce the Aqueous Normal Phase (ANP), Reversed Phase and Normal Phase modes of chromatography. Background information, technical information and a link to our information rich Knowledge Base (KB) are listed below. These columns, based on a silica hydride surface, have been an Industry leading column for polar compounds including amines, acids, based, peptides and sugars since 2003. The only columns that can deliver Aqueous Normal Phase HPLC are a best choice when you are interested in different selectivity, method precision and ease of use. Often used in metabolomics to separate unknown unknowns that may be polar and non polar.
Every column is individually tested for reliability and is supplied with a test certificate. Choose from analytical, prep or guard columns. Our onsite laboratory is equipped and eager to help you develop new methods, troubleshoot or answer questions that you might have.
- Application Notes (Knowledge Base)
- Bibliography of References (Knowledge Base)
- Frequently Asked Questions (FAQ) (Knowledge Base)
- How to Use Cogent TYPE-C™ Columns
- Listing of TYPE-C™ Stationary Phases
- Method Development Quick Start for ANP (PDF)
- Specifications of TYPE-C Phases
- Storing Cogent TYPE-C™ Columns (Knowledge Base)
- YouTube© Video - What are TYPE-C™ HPLC Columns
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Background & Technical Information: Quick Links.
- A Brief History of Silica
- Bonding Uniqueness Produce Great Selectivities
- Cogent TYPE-C™ Silica an Introduction
- Features and Benefits to Chromatographers
- Unique Surface Chemistry of TYPE-C Silica™ Products
- Hydration Shell, Liquid Stationary Phase type-B v. TYPE-C™
- Hydration Shell, No Adsorbed Water
- Separate Mechanisms for Separations
Irregular shaped silica particles with a wide pore size distribution were the original support used to manufacture HPLC columns. At the time that 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 using Normal Phase Chromatography.
To achieve "Reversed Phase" separations with water soluble samples, the silica had to be modified with low polarity, organo-silanes using siloxane bonding technology. Stationary phases commonly used today such as C18 and C8 were created. The siloxane (Si-O-Si) bonds used in this process suffer from being hydrolytically susceptible to attachment failure and during the lifetime of column, result in separation problems when working at low pH or with strong buffers or ion pair reagents often resulted.
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 of the columns. 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 tailing problems when basic compounds were separated at desired pH. Today, reversed phase separations are predominantly being performed on silica bonded with hydrocarbons.
Before the introduction of Cogent TYPE-C™ Silica, 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. These surface silanols also have a large participation in the partitioning mechanism. To minimize tailing and column reactivity, type-B silica silanols it became desirous to use small end capping groups such as C1 to "cover" or tie up 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.
After irregular particles were replaced with spherical silica particles, it became necessary to develop low metal, ultra high purity particles. This was given the name of Type-B silica and ordinary spherical silica was retrospectively named Type-A. Most development after this was different bonded phases, end capping and particle size. The mechanisms of separation remained the same. Partitioning and Adsorption are the major mechanisms and others such as hydrogen bonding remain the same from phase to phase. Until TYPE-C™ Silica.
Cogent TYPE-C™ Silica based HPLC columns had opened a new chapter in the book of chromatographic supports and phases for polar compounds in 2001. Based on high purity-low metal content Type-B silica, the manufacturing process for TYPE-C Silica™ continues where others leave off. Our process produces a surface populated with silicon-hydride groups (Si-H) which are very stable. This change in the surface of the silica particles makes the particle slightly hydrophobic where all other silica is extremely hydrophilic. This differences allows, when in the presence of at least 5% water in the mobile phase, polar compounds to be retained on the charged, non-polar surface by adsorption of H+ from the auto-dissociation of water. A proposed explanation is similar to how hydrophobic oil droplets in water adsorb H+ and become soluble in the ocean.
The TYPE-C™ Silica support still has all the physical 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. Among the differences between TYPE-C™ and Type-B silica is that the same, one column can be used for 3 different modes of HPLC. This produces an new selectivity power no other column has. On one column you can separate polar and non polar compounds at the same time or in different runs with different mobile phases. One column can be used for classic reversed phase, normal phase with non polar solvents such as hexane and can be used for normal phase elution with aqueous solvents. This is called Aqueous Normal Phase (ANP).
ANP has many advantages over HILIC including but limited to precision, speed to equilibrate, much lower salt to retain polar compounds and flexibility with bonded phases such as C18.
Other benefits are the very fast speed to equilibrate between gradient runs or to clean your column from matrix effects which now is feasible do to between runs. In typical runs less than one minute is needed or 3-5 column volumes. TYPE-C columns are especially beneficial for polar & water labile compounds and peptides.
The surface chemistry or structure of the surface is unique with TYPE-C™ Silica. The image above depicts the differences. This difference is responsible for all the uniqueness of Cogent TYPE-C Silica.
TYPE-C Silica has a weak association with water due to the hydrophobic nature of the particle. Even once H+ ions are adsorbed to the surface, TYPE-C Silica does not adsorb any water layer similar to Type-B silica. This water shell on Type-B silica is semi-permanent and becomes an integral part of every day HPLC mechanisms (Partitioning cannot exist with out it). Since TYPE-C Silica does not adsorb Water, there is no partitioning mechanism and the retention is simplified. Mostly adsorption and ion interaction (Hydrogen Bonding) are the dominant mechanisms for the Silica Particle.
The surface of Cogent TYPE-C™ Silica is predominantly populated with non-polar, silicon-hydride (Si-H) groups instead of the polar, silanol groups (Si-OH) that are prevalent on the surface of ordinary varieties of silica. This feature gives the Cogen TYPE-C™ columns many unique, useful and helpful chromatographic qualities. Especially for polar & water labile compounds and peptides.
The Cogent TYPE-C columns overcomes some of the inherent limitations of ordinary silicas associated with free silanols.
- Surface acidity is reduced
- Improved low pH stability
- Less hydroscopic - no water shell
Less than 1/2 monolayer of water forms.
Strong adsorption of water to type-B silica is well known and well documented. However, the silicon-hydride groups (Si-H) found on the surface of Cogent TYPE-C Silica™ are not prone to such strong water retention (shown above) making it an excellent choice for polar and non-polar compounds. The weaker water adsorption also accounts for the little to no hysteresis observed when changing from Organic-Normal to Aqueous-Normal to Reversed Phase when changing pH of your sample and mobile phase.
This feature makes the column preferred over HILIC phases which often exhibit long term memory effects and equilibration 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.
Examples of the unique bonds formed on Cogent TYPE-C™ Silica, the only direct Silicon to Carbon bonds which are extremely stable.
Because of the unique silica hydride surface, Cogent TYPE-C™ silica are bonded with ligands to create unique stationary phases but also have unique points of attachment. Due to the strong bonds formed directly between silicon and the ligand without the ordinary intermediary functional group, the bonded phases are stable and resistant to conditions that can cause hydrolysis which results in much longer, useful column lifetimes.
Creating different stationary phases offers changes in selectivity which works in addition to the basic adsorption mechanism of the silica hydride for excellent separation of closely related compounds. The overall mechanism of the bonded stationary phase is largely dominated by the TYPE-C Silica particle and the bonded phase often acts as a secondary selectivity and retention mechanism but can be extremely important.
Click HERE for a listing of all Cogent TYPE-C™ Stationary Phases.
Another unique feature of the Cogent TYPE-C Silica™ based columns is that the silica surface, silica hydride, 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-C™ (unmodified TYPE-C silicas) column produces good separation of the compounds with good peak shape for Glyburide and a lightly tailing peak shape for Metformin. All other features of the TYPE-C™ columns are evident in the separation.
When the Cogent UDC-Cholesterol™ column is used instead of the Silica-C™ 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.
|Some Features of TYPE-C™ Columns:||Chromatographer’s Benefits|
Silicon-Carbon Bonds instead of Siloxane
More stable and durable.
|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 & ONP||Get the performance of HILIC columns on a stable, robust bonded phase|
|High efficiency & stable||Great peak shapes & columns that last a long time|