TYPE-C Column Information – MicroSolv Technology Corporation

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AppNotes Cogent RP C8
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Prep Chromatography

“Normal Conditions” for Cogent TYPE-C Silica columns

The Cogent TYPE-C Silica™ columns have been observed to last 20-50% longer than those based on ordinary silica under normal conditions. What this means is following the specifications of the columns in terms of temperature, pH, and so on. In addition, please be sure to follow proper storage protocols to make sure the material is not damaged due to […]

Amide vs. Amine/Amino HPLC columns.

The amide functional group should not be confused with the amine or amino moiety. The presence of the carbonyl group imparts the amide functionality with significant physical and chemical differences compared to an amine. Most notably, amines are ionizable (positively charged when ionized) while amides are not. Amines are more reactive than amides, such as with aldehydes, which can present some […]

ANP Replaces HILIC in HPLC and LCMS – With Benefits.

Over the years, many chromatographers have been given the task to develop HPLC and LCMS methods for polar compounds. Those who have chosen HILIC columns often report their gradient methods are problematic causing them frustration, more method development time and additional costs. Mostly the complaints are the “downtime” between runs that HILIC and all ordinary […]

Are the Cogent TYPE-C Silica based stationary phases available as bulk materials?

No, the Cogent 4um and 2.o TYPE-C Silica™ based stationary phases are not available as unpacked bulk material. TYPE-C Silica™ is a very unique material and to properly pack columns or capillaries requires proprietary technology. For this reason, it is only sold in a pre-packed column format. However, 10um particles can be made available upon […]

Assuring retention of polar compounds in an ANP gradient in positive mode LCMS.

When using a Cogent Diamond Hydride™ column prepare your solvents for positive ionization in LC-MS: Solvent A: 50% methanol/50% DI water + 0.1% formic acid and Solvent B: 90% acetonitrile/10% DI water/0.1% formic acid. ANP gradients need to start between 100% – 90% of Solvent B (to retain polar compound) and then reduce the percent of Solvent B […]

Can I use acetone as a mobile phase solvent in ANP?

If you are using LCMS, the simple answer is yes but pH is also something to consider. Acetone has a high UV cutoff and therefore can be a problem for UV detection. However, this is not a problem when using LCMS or ELS detectors such as the Corona CAD. You can use acetone to replace acetonitrile with the Cogent TYPE-C™ […]

Can I use phosphoric acid or sodium phosphate with the Cogent Diamond Hydride HPLC column?

Yes, but there is some evidence that the use of phosphoric acid may semi-permanently change the surface chemistry, and therefore the chromatography of the column. For example, if you get a certain separation using formic acid and then introduce phosphoric acid to the column, you may not get the same separation when you try the […]

Cleaning Cogent TYPE-C silica based HPLC columns

The Cogent TYPE-C™ HPLC columns are very stable and can be washed between runs with 100% Acetonitrile, DMSO THF and DMF among many other solvents. We use these solvents when typical washing procedures are not adequate. A precaution I would give you is the stay between pH 2 and pH 7.5 when using acids and bases. […]

Cogent Diamond Hydride 2.o HPLC Column Retention Time Precision – AppNote

This Graph shows a Study Investigating the Day to Day Retention Time Precision with two test solutes on the Cogent Diamond Hydride 2.o™ HPLC Column. Peaks: Blue Trace: Phenylalanine Red Trace: Phenylglycine Many factors can affect Column Robustness and lifetime, so it is not possible to define specific values for those parameters. Mobile Phase: —A: DI […]

Cogent UDA HPLC Column Use – Extended AppNote

Ion exchange chromatography can be a useful tool available to the analytical chemist. In this separation mode, an ionized moiety on the stationary phase interacts electrostatically with an oppositely charged analyte. There are several types of ion exchange that can be used, such as strong anion exchange, weak anion exchange, strong cation exchange, and weak […]

Eliminate Broad Split Peaks in ANP Methods with Acetonitrile / Methanol – Tips & Suggestions

I am analyzing Pyrazinoic Acid by Aqueous Normal Phase HPLC (ANP) using the Cogent Diamond Hydride™ Column. I observe two broad, Split Peaks for the Analyte. I am using an Acetonitrile / DI Water / Formic Acid based Mobile Phase and my diluent is 50:50 Methanol / Acetonitrile. Retention is also low. What can I […]

Glucose, Pyruvate, Taurocholic Acid, Gamma Glutamylcysteine etal Analysis – Tips & Suggestions

What suggestion can you offer for Analysis of Metabolites such as Glucose, Pyruvate, Taurocholic Acid, Gamma Glutamylcysteine, Glycochenodeoxycholate, Glycerol-3-phosphate, Spermidine, Taurine, and S-Adenosylmethionine? Suggestion: We would recommend starting by using the Cogent Diamond Hydride™ HPLC Column for this Analysis. Many of these compounds are quite Polar and would be very difficult to Separate by Conventional […]

How does efficiency in ANP compare to HILIC?

Aqueous Normal Phase (ANP) is a distinctly different retention mode than Hydrophilic Interaction Liquid Chromatography (HILIC). In HILIC, evidence has shown that retention is achieved by partitioning in and out of a water layer surrounding the stationary phase surface. With a much less polar surface, TYPE-C Silica columns do not exhibit this same partitioning retention behavior. Speculated local […]

How to analyze phosphorylated compounds with the Cogent Diamond Hydride™ column with HPLC.

Phosphorylated compounds can be difficult to analyze with HPLC. However, a wide variety of compounds with the phosphate functional group have been able to be retained with the Cogent Diamond Hydride™ column in ANP mode. Examples include phosphorylated sugars, herbicides, and nucleotides. The strategy can also be applied to other compounds of this type such […]

Literature References for Cogent TYPE-C HPLC Columns

The following is a list of Articles published in peer-reviewed Journals and books that discuss Cogent TYPE-C™ Silica based materials and their Applications: Authors Title Publication Date Volume Pages Marisa C. May, David C. Pavone, Dr. Ira S. Lurie The separation and identification of synthetic cathinones by portable low micro-flow liquid chromatography with dual capillary […]

Literature References for Cogent UDC-Cholesterol™ HPLC Columns

The following is a list of articles published in peer-reviewed journals and books that discuss Cogent UDC-Cholesterol™ based materials and their applications: Authors Title Publication Date Volume Pages H. Grajek, Z. Witkiewicz, M. Purchała, W. Drzewiński Liquid Crystals as Stationary Phases in Chromatography Chromatographia 2016 1-29 J. E. Young, M.T. Matyska, A.K. Azad, […]

NMR Structure confirmation data for Cogent Bidentate C18™ phases

The following solid state CP-MAS 13C NMR spectrum was recorded for a Cogent Bidentate C18™ stationary phase in order to elucidate the structure. The assignments for the different carbon groups are shown below. The spectrum highlights the unique bonding of these phases, which is not possible with conventional silica based stationary phases. For example, the bidentate attachment, […]

NMR Structure confirmation data for Cogent Bidentate C8™ HPLC phases.

The following solid state CP-MAS 13C NMR spectrum was recorded for a Cogent Bidentate C8™ stationary phase in order to elucidate the structure. The assignments for the different carbon groups are shown below. The spectrum highlights the unique bonding of these phases, which is not possible with conventional silica based stationary phases. For example, the bidentate attachment, […]

NMR Structure confirmation data for Cogent UDC-Cholesterol™ phases

The following solid state CP-MAS 13C NMR spectrum was recorded for a Cogent UDC-Cholesterol™ stationary phase in order to elucidate the structure. The assignments for the different carbon groups are shown below. The spectrum highlights the unique bonding of these phases, which is not possible with conventional silica based stationary phases. Cogent UDC-Cholesterol HPLC Column Ordering […]

On-column hydrolysis in HPLC columns and what causes it? What will be the effect on Cogent TYPE-C™ Silica columns.

Hydrolysis is a common degradation mechanism, which is facilitated by the presence of acids or bases in solution or on the surface of solids the liquid is in contact with. With solids in contact with liquids, the larger the surface area the greater the potential for hydrolysis. Irregular (Type A) & spherical (Type-B) HPLC phases […]

Retention and/or peak shape for citric acid has changed compared to data from a previous HPLC run. What happened?

Citric acid analysis using LCMS can be compromised due to the presence of iron in the system. When iron is present, it can cause peak distortion for compounds like this. A possible solution to this problem is to use a small concentration of a chelating agent (EDTA) in the mobile phase/sample diluent to sequester the […]

Retention time vary from instrument to instrument

To obtain the same retention times for analyzed compounds as in application note gradient conditions have to be adjusted according to the instrument used. Each instrument has different plumbing (dimensions of the tubing), so obtaining exactly the same retention times is virtually impossible. This can be especially significant when attempting to duplicate application notes from […]

Sample Diluents should match your mobile phase.

For best results, it is always best to try to dissolve your target in as close as possible to your isocratic eluent or the start of your chosen gradient. If this is not possible gradually increase the percentage of the solvent in your eluent that the target is most soluble in.

Silicon carbon bonds for Cogent TYPE-C HPLC columns.

Typical HPLC bonded phases (C8, C18, NH2 etc) are made with siloxane (Si-O-Si-C) bonds which are prone to hydrolysis. The oxygen in the this link is the weak point where the bonded phase is cleaved off the particle. Silicon-Carbon bonds (available only on Cogent TYPE-C™ columns) are much more stable and resistant to hydrolysis and is […]

Tandem LCMS approach to Metabolite Profiling – Poster

Hydrophilic and Hydrophobic Molecules In a Single Line Injection Presented at the Metabolomics Society meeting in Amsterdam, June 2010; below is the introduction from the poster or you can download and view the entire poster by clicking on the link below. INTRODUCTION: A tandem liquid chromatography (LC) setup has been developed to analyze a larger […]

Tips on solving baseline problems in ANP with the Cogent Diamond Hydride

When using a gradient method with the Cogent Diamond Hydride™ column, an inconsistent baseline may be observed in some instances. The following are some possible causes of the issue along with suggestions to fix it. 1. Is the mobile phase filtered before use on the instrument? A 0.45µm nylon membrane filter with vacuum filtration is […]

Using temperature changes for separations of terpenoids with the Cogent Bidentate C18 and Cogent UDC-Cholesterol HPLC columns

Temperature is a useful variable to consider in HPLC separations. Control of temperature can reduce run times, alter selectivity, and change analyte efficiency. In this study of three terpenoids (bexarotene, tretinoin, and tazarotene), we compared the Cogent Bidentate C18™ and Cogent UDC-Cholesterol™ columns at different temperatures in an attempt to gain insight into their retentive behavior. The […]

Using the Cogent Diamond Hydride and Cogent Bidentate C18 columns together in a tandem LC separation.

If you connect these two columns in parallel but use the same mobile phase program on both columns, it will be difficult to have both reversed phase and aqueous normal phase mechanisms operating simultaneously and therefore not get optimal results; a gradient starting at high organic may work well for the Cogent Diamond Hydride™ column but only very hydrophobic compounds will retain on […]

What are the main differences between HILIC and Aqueous Normal Phase?

There are many important differences between HILIC columns and Cogent TYPE-C Silica™ columns but for the sake of brevity, we will only address the main differences. The Cogent TYPE-C™ silica columns (silica hydride) perform similarly to HILIC columns as far as polar compound retention in a “Normal Phase” elution order is concerned. With both types of columns using higher than 70% organic composition of the […]

What is Aqueous Normal Phase (ANP) and how can it help you? Wikipedia Article.

Aqueous normal phase chromatography From Wikipedia, the free encyclopedia Aqueous normal phase chromatography (ANP) is an HPLC technique which encompasses the mobile phase region between reversed-phase chromatography (RP) and organic normal phase chromatography (ONP) and is used mainly for polar compounds. In normal phase chromatography, the stationary phase is polar and the mobile phase is nonpolar. In […]

What is the chemical structure of the Cogent DIOL HPLC stationary phase?

The Cogent Diol stationary phase structure has a single point attachment as shown below. Note this bonded phase is attached to the silica hydride surface and offers very unique retention mechanisms for both RP and ANP HPLC.

What is the metal content of the silica used in Cogent TYPE-C HPLC columns?

Metal impurities testing via ICP (Inductively Coupled Plasma) analysis is peformed for the following, along with acceptance specifications: Al: Not more than 5.0 ppm Fe: Not more than 20.0 ppm Ti: Not more than 0.5 ppm Mg: Not more than 5.0 ppm Ca: Not more than 15.0 ppm Na: Not more than 20.0 ppm It is important to note that the actual […]

What is the most effective way to clean HPLC columns after or before use?

Many commonly used cleaning methods feature a strong wash solvent, run isocratically in order to elute strongly retained compounds or residual solvents from the column. After some time of washing in this manner, the stationary phase is essentially in equilibrium with the wash solvent, so it may still take a long time to remove contaminants from […]

What is the purpose of using isopropanol (IPA) as part of the aqueous mobile mobile phase solvent in ANP methods?

Some of our Cogent HPLC column application notes that feature Aqueous Normal Phase (ANP) use 50% isopropanol (IPA) as part of the aqueous component of the mobile phase. The main reason for this use is to help keep the column free of undesired adsorbed contaminants from a sample or sample matrix. Contaminants can change the retention behavior of […]

What is the structure of the Cogent Phenyl Hydride bonded ligand? What is a “direct silicon-carbon bond”?

The Cogent Phenyl Hydride phase is a 4 carbon chain with a phenyl group at the end attached directly to the silica hydride surface with a single, direct silicon-carbon bond. With bonds in traditional silica B based materials, the organo-silane starting material is Si-O-Si-C4-Phenyl and this reagent is reacted with the silica to form the […]

What Temperature Should I Use with The Cogent TYPE-C 2.o HPLC Columns?

We recommend a starting temperature of 25 °C for most applications. Generally you will see higher retention at higher temperatures and lower retention at lower ones with Aqueous Normal Phase (ANP) chromatography. This can sometimes be a useful tool for fine-tuning the retention/selectivity of the peaks. Although temperature is a variable to consider during method development, we […]

When is the acid group of the Cogent UDA HPLC column ionized?

The pKa of most simple carboxylic acids is in the range of ~pH 4.7-5.0. The carboxylic acid group of the Cogent UDA™ HPLC column can be either ionized or non-ionized depending on the pH of the mobile phase used in your method. The pH at which the group is ionized or non-ionized is dictated by […]

Why ANP HPLC Methods are Better to use than HILIC.

Why ANP is better than HILIC for Polar Compound Methods Compared to ANP, HILIC will require more time to Equilibrate, takes more salt to retain some compounds, lacks Retention Time (RT) Precision and Lifetime of the Columns can be very short. With Cogent TYPE-C Columns using ANP, Equilibration is extremely fast, Retention Time (RT) Precision […]

Why Doesn’t Triphenylamine Tetain in ANP on a Diamond Hydride HPLC Column?

There can be many reasons why any compound does not retain in any given HPLC Method, however in this case, Triphenylamine has three bulky Phenyl Groups (see structure below), which can create steric hindrance from interaction of the Stationary Phase with the Tertiary Amine. NOTE: Tertiary Amines are the least Polar Amine and will have […]

Why is it important not to exceed a mobile phase pH of 7 when using the Cogent Diamond Hydride?

Selection of too high a pH could lead to voids developing in your column. Data for the dissolution rate of silica in pure aqueous solvents as a function of pH shows significantly increasing solubility above pH 7. If a void has developed in your column due to dissolution, you will observe significant peak shape distortion for every […]

Working with plasma samples and Cogent TYPE-C HPLC columns

When working with plasma extracts, we recommend using 50% methanol/50% DI water for washing between injections. Flow rates of 0.5ml/min for 20-30 minutes for a 4.6mm x 250mm column should be sufficient. When working with LC-MS and biological samples we recommend using 50% methanol/50% Di water as the A solvent. It helps clean the column […]