Introducing an evolution in HPLC silica support

Introducing TYPE-C Silica, A selling Guide

MicroSolv Technology Corporation Long Branch, NJ USA

Technical Background: TYPE-C SILICA PRODUCTS

WHAT ARE THEY? HOW CAN THEY HELP CHROMATOGRAPHERS? Evolution not Revolution A History:

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 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.

Cogent 4µm Spherical Silica 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 (low 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... ... 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. With changes in temperature in the lab, mobile phase solvents used in Normal Phase Chromatography adsorb varying amounts of water from the atmosphere. 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 Type-C Silica based HPLC products are opening a new chapter in the book that describes chromatographic supports and phases. Based on high purity-low metal content manufacturing technology of Type-B Silica, the manufacturing process for Type-C Silica™ continues where others leave off. Our patented, proprietary process produces a surface populated with Silica Hydrides (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 .

Unique Characteristics of TYPE-C Silica™ Products 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 forms of silica. This unique property of Type-C Silica particles, gives it many unique, useful and helpful chromatographic qualities as well as overcoming some inherent limitations.

Some of the inherent limitations of Type-B silica 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 and a unique separation character that allows for improved Aqueous-Reverse Phase, Organic-Normal Phase and Aqueous-Normal Phase without the organic bonded phase.

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

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.

Type-B Silica with Strongly Associated Water Shell

TYPE-C Silica with Weakly Associated Water Shell

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 OrganicNormal 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.

Reverse Phase Silica without a bonded phase Type-C Silica columns (no carbon bonded phase or end capping), can work in Aqueous-Reverse Phase mode unlike Type-B silica which cannot be used in Reverse Phase.

Carbon Free: LCMS and Prep Market.

As there is no bonded carbon phase or end-capping, the Type-C Silica phase offers “phase stability” and “bleed-free” chromatography from at least pH 2 to pH 8. This is a major requirement for many laboratory/ pilot/process preparative applications and a great feature for LC-MS. In addition to the unique selectivities provided by TYPE-C Silica™ products which are detailed in the MicroSolv Website, www. MicroSolvTech.com with relevant pharmaceutical applications, etc.

Dual Mode, RP & NP Type-C Silica columns, can work in Aqueous-Reverse Phase mode or Normal Phase mode with a unique feature. Methods that utilize a mobile phase consisting of water (solvent A) and Acetonitrile (solvent B which is less polar) that varies the concentration of B from 0% and up to 70% will produce retention of the analytes decreasing as the less polar solvent (B) is increased. With 100% Aqueous (A the more polar solvent) retaining the most for neutral compounds. What is unique is that when analyzing ionizable (acids or bases) compounds, if in an appropriately charged state (pH) is used and when the Acetonitrile (solvent B) concetration is above 70%, then a different maximum retention of 100% Acetonitrile (solvent B) occurs. From this maximum retention which occurs at 100% Acetonitrile (solvent B) the retention time will now decrease as the more polar solvent A (aqueous) increases. This Normal Phase Separation using an Aqueous mobile phase. What this means is that for the same compounds, you can change elution order and retention times by changing either pH (removing the charged state) or by changing the organic concentration of the mobile phase. In both “Aqueous” (using water) and or “Organic” (using non polar solvents) Normal Phase mode as the more polar solvent is increased, the retention time of the analyte decreases. The elution order is based on functionality/ionic state of the analytes. The maximum retention of the analytes is at 100% concentration of the least polar solvent. This is a definition of Normal Phase.

Unique Bonding Capabilities for Unique Selectivities and Problem Solving.

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. Typical Mono-functional Bonded Phase Type B Hydrolysis

Initial products using the above bonding include Cogent TYPE-C Silica™, Cogent UDCCholesterol™ (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.

Selling the Cogent TYPE-C Silica Products to First Time Users. Separate Mechanisms for Separations: 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 Dr. Les Brown, Technical Director of 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 bad peak shape for Metformin. All other features of the TYPE-C series are evident in the separation. When the Cogent UDCCholesterol™ 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.

Some Features of TYPE-C Silica products include: Silicon-Carbon Bonds instead of Siloxane.

Free of Salts Bonded to a Silicon-Hydride Surface

Customer Benefits More stable and durable. Silica: pH 2.0 – 8.2 UDC-C: pH 2.0 – 8.2 Bident C18: pH 1.5 – 10.0+ Closer Attachment to the surface Unique Adsorption Qualities Temperature Stability Increased Can be used with Hard Lewis Acids at high pH Contaminant Free Will not strongly bind Carboxylic Acids or Sulfonic Acids Minimized Silanophilic Activity Stable at high flow rates (up to 3ml/minute) No Bleed of organic materials. Water Friendly Columns Very rapidly remove water from the stationary phase and silica. Easy to pack, highly efficient columns Use 100% water on C18 without loss of retention with time. Lack of pH hysteresis. Can quickly change mobile phases and pH buffers.

Target Markets for this evolutionary HPLC Product Line.

MicroSolv Technology Corporation has identified the early adopters and the thought leaders of innovator companies as the initial target market for these products. It is imperative that the end user get complete training and hands on Customer Care and Customer Service to assure that these columns are not used as traditional Type-B columns. Although they will most likely out perform Type-B Columns on a level playing field, the real advantages to this product line’s uniqueness are the novel separations that can be achieved. MicroSolv is committed to personal, one on one attention through all our sales reps and dealers for every Cogent TYPE-C HPLC column sold. Specific target segments include but are not limited to: LCMS where phase and end capping bleed is an issue. Method Development and Trouble shooting with highly polar, non retentive compounds. Scientists working with normal phase separations. Scientists working with semi-prep applications (up to 21.2mm ID) that are looking for increased pH range, solvent range or struggling with water content issues. Potential markets are endless as these columns are high purity silica products that offer stability, range and efficiency. Due to the lack of silanols and silanophilic activity, protein, peptide and other biological separations for bio molecules with hydrodynamic spheres that do not exceed 25% of the pore diameter are possible with TYPE-C Silica 100A. 300A is in R&D now and will be available in the future.

Available Column Configurations. Not listed: mini-prep, semi-prep columns and custom sizes. Silica

UDC-Cholesterol Bidentate C18 Description

Dimensions

40000-01

69069-01

40018-01

Guard Column Kit, 5e 4x20mm with 1 SLS Integral Holder 4x20

40000-01-R

69069-01-R

40018-01-R

Mini Column Replacement Kit, 5e 4x20mm Columns

40000-02

69069-02

40018-02

Mini Column Kit, 5e 4x20mm Columns with 1 Stand Alone 4x20 Holder

40000-02-2R

69069-02-2R

40018-02-2R

Mini Column Replacement Kit, 5e 2x20mm Columns

2x20

40000-05-2

69069-05-2

40018-05-2

Super Link System Column, 50 x 2.1mm Cmplt w/End Fittings

50x2.1

40000-05R-2

69069-05R-2

40018-05R-2

Super Link System Replacement Column Only 50 x 2.1mm

50x2.1

40000-10-2

69069-10-2

40018-10-2

Super Link System Column, 100 x 2.1mm Cmplt w/End Fittings

100x2.1

40000-10R-2

69069-10R-2

40018-10R-2

Super Link System Replacement Column Only 100 x 2.1mm

100x2.1

40000-15

69069-15

40018-15

Super Link System Column, 150 x 4.6 mm Cmplt w/End Fittings

150x4.6

40000-15-2

69069-15-2

40018-15-2

Super Link System Column, 150 x 2.1 mm Cmplt w/End Fittings

150x2.1

40000-15R

69069-15R

40018-15R

Super Link System Replacement Column Only 150 x 4.6mm

150x4.6

40000-15R-2

69069-15R-2

40018-15R-2

Super Link System Replacement Column Only 150 x 2.1mm

150x2.1

40000-25

69069-25

40018-25

Super Link System Column, 250 x 2.1 mm Cmplt w/End Fittings

250x4.6

40000-25R

69069-25R

40018-25R

Super Link System Replacement Column Only 250 x 4.6mm

250x4.6

40000-7.5

69069-7.5

40018-75

Super Link System Column, 7.5 x 4.6 mm Cmplt w/End Fittings

75x4.6

40000-7.5R

69069-7.5R

40018-75R

Super Link System Replacement Column Only 75 x 4.6mm

75x4.6

4x20

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MicroSolv Technology Corporation 103 Brighton Avenue Long Branch, NJ 07740 USA Phone: 1-732-229-3400 Fax: 1-732-229-2403 email: [email protected] Visit our website at: www.MicroSolvTech.com