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 […]

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EDTA plasma samples for creatine & creatinine – Blog.

Since we have done work with creatine and creatinine in urine, see appliction note we been asked about measuring levels of these compounds in EDTA plasma samples. It  should be possible unless there is some interference from the EDTA; we have not measured EDTA retention under these conditions (where we determined creatine and creatinine). Obviously plasma is a different matrix […]

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Fosetyl-Aluminum Analysis by HPLC. Optimizing Tips.

You can use the Cogent Bidentate C18™ column in reversed phase (RP) with a high water content method. According to a 2014 third-party research article, if the analysis is done by reversed phase an ion pair agent is recommended to increase retention and reduce peak tailing. Here, 8 mM sodium sulfate was used in a […]

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How can I separate methacrylic acid and dodecyl benzene sulfonic acid?

Good retention and peak shapes have been observed for similar compounds. Methacrylic acid is similar in structure to acrylic acid, which can be well-retained with a mobile phase of DI water + 0.1% formic acid and a Bidentate C18 column. Dodecyl benzene sulfonic acid has some of the same functional groups as docusate sodium, which also has […]

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How does a Pulse from an HPLC Pump Affect the Column and its Performance?

If your HPLC pump produces a “pulse” (i.e. cyclic fluctuation in flow/pressure), this can cause problems for your Method and Column. The pump will not be delivering optimally Precise/Accurate Flow and this can cause your system to not meet System Suitability Criteria as well as other issues. Sudden changes in the Pressure can also cause […]

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How To Choose a Cogent HPLC Column. Order of Hydrophobicity.

The first thing to consider is the chemical structures of the Analytes in your Sample and the corresponding most suitable Retention mode for them. For Reversed Phase, the general order of Stationary Phase Hydrophobicity (and therefore retentiveness in RP) is as follows (left to right) and for hydrophilicity (and therefore retentiveness in ANP) is shown […]

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How To Condition Cogent RP, HPS & Axis HPLC Columns for Storage.

For  Cogent RP™, Cogent HPS™ & Cogent Axis™ HPLC Columns Long Term Storage: Rinse the Column with 8 Column Volumes with pure Solvent that you use as your mobile phase. It is important to use non-buffered Mobile Phase. Then purge the Column with 8 Column Volumes of 100% HPLC Grade, Filtered Isopropyl Alcohol. Then purge […]

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Hydrophilic & Hydrophobic peptides analyzed by LCMS in ANP

The Cogent Diamond Hydride™ HPLC columns using aqueous normal phase (ANP) chromatography are very good choices for LCMS or HPLC analysis of most peptides. We have published an article on peptides demonstrating the ability of the column to retain a very broad range of peptides.  This paper uses experimental, capillary ID columns but it works just as well with standard […]

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I heard that there is Not a Good Alternative to HILIC to Achieve Sufficient Retention for some compounds. Is this true?

This is an untrue statement that can lead chromatographers to frustration as THERE IS an alternative mode of chromatography that surpasses HILIC in reproducibility, precision and the ability to separate both hydrophobic and hydrophilic compounds. It is well documented that Aqueous Normal Phase HPLC (ANP) is a very viable alternative to HILIC for polar compounds and in many cases superior […]

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Types of isomers and suitable HPLC columns for separation

There are many types of chemical isomers, and each may require different approaches to obtain chromatographic separation. Some examples are as follows: Empirical Isomers: These compounds have the same empirical formula but the atoms could be arranged in any possible configuration. This is the most general type of isomer. Knowing all of the possible isomers […]

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Using different Cogent™ HPLC stationary phases for different selectivity.

Each of the Cogent™ stationary phase materials has a different bonded ligand and therefore potential for different selectivity of given analytes. Consider the example, featuring the Cogent Bidentate C18™ and Cogent UDC-Cholesterol™ columns in another Knowledge Base article:  https://kb.mtc-usa.com/article/AA-00781 Note the two columns give substantially different retention for the same analytes under the same conditions. As […]

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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 […]

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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 […]

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What is the most polar Cogent TYPE-C HPLC column?

The most polar HPLC stationary phase manufactured by MicroSolv Technology Corporation is the Cogent Amide™. It is very robust compared to most other “amide” columns and can be very effective for compounds such as sugars and other carbohydrates but also for compounds with amines. The Cogent Amide™ column is made with TYPE-C™ silica so it […]

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When to use PEEK over stainless steel for HPLC column Harware.

Data in certain types of analyses may be adversely affected by use of stainless steel HPLC columns. In these cases, you may want to consider a column housed in PEEK hardware instead of traditional stainless steel. Mainly used in Ion Chromatography and for some bioactive compounds, PEEK columns may circumvent the issues associated with stainless […]

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Which Column would you Recommend for a Bromhexine HPLC Assay Method?

I am currently using the following Method for analysis of Bromhexine. What Column and/or alternative Method Conditions can you Suggest? Mobile phase: Potassium di-hydrogen phosphate buffer(take Potassium di-hydrogen phosphate 1.0g, add 900ml DI Water, then change pH to 7.0 with 0.5mol/L NaOH solution, and dilution it to 1000ml by DI Water )-acetonitrile(20:80), Column temperature: 40C, Wavelength:245nm […]

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Which HPLC column diameter should I use for LC-MS?

For most standard HPLC applications such as UV detection methods, a 4.6mm or 3.0mm I.D. is suitable. Lower diameters will present unnecessary problems for standard UV methods. For instance, the system tubing and especially the flow cell may cause band broadening with a 2.1mm column due to extra-column dispersion effects. With most LC-MS (as well as some other […]

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Which HPLC column do you suggest for analysis of carotene or lycopene?

With these types of compounds, the main challenge will be separating the different isomers. There are numerous double bonds in the molecules, and many combinations of cis/trans isomers are likely to be encountered. Because the isomers differ little in hydrophobicity but significantly in shape, a column with shape-selective capabilities would be required for an adequate separation. Reversed […]

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