How to Avoid Microbial Growth in HPLC Mobile Phases.

Bacteria can be a problem in the case of Aqueous Solvents. If present, they can create problems for an instrument’s operation. There are a few things you can do to avoid their formation in your mobile phase. One is to add an acid such as 0.1% Formic Acid. Many HPLC methods may use an acidified […]

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How to Back Flush an HPLC Column.

1. Reverse the Flow Direction in the Column. Attach the Column fitting that is normally at the outlet to the tubing that is normally connected to the inlet. Direct the Column Outlet to Waste, not to the tubing leading to the detector. The reason for this is so any contaminants coming off the Column don’t end […]

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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 Clean Sparging Stones and Inline SS Filters.

Inlet Stainless Steel Filters also known as Sparging Stones, can be cleaned by ultra-sonication in 30% Phosphoric Acid, clean afterwards with DI Water (ultra-sonication) and Methanol (ultra-sonication). Same would be true for all Metal Filters, like for Column Filters, Inlet Filters and more. But be aware, only the metal part! No PEEK Rings, Connectors or […]

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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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How to Condition Controlled & Zero EOF Capillaries.

Capillary Conditioning Procedure: Never Use Strong Bases on these Capillaries Carefully remove the capillary from the shipping/storage container. Remove protective polyimide coating by using 98% fuming sulfuric acid heated to 100ºC. One drop should be sufficient for complete removal. The MicroSolv Window Maker be used as well as long as you do not allow the […]

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How To Condition Diamond Hydride Columns for use with Serum Samples.

For Serum and other biological samples, we Recommend using: Solvent A: 50% DI Water/50% Methanol (or 2-propanol) + 0.1% Formic Acid (for positive mode MS) or +10 mM ammonium acetate or formate (for negative mode MS). Solvent B: The same as you would ordinarily use. Cleaning Tip: Methanol or 2-Propanol (IPA) helps to clean the […]

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How To Convert HPLC Columns Between Reversed Phase & Normal Phase.

To start work in normal phase mode, a simple switching procedure is recommended. A simple 30 minute procedure allows switching from one mode to another. Procedure: A – Moving from Reversed Phase to Normal Phase HPLC; pump 100% Methanol for 15 minutes at 1 mL/min. Flow Rate, followed by 15 minutes 100% Methylene Chloride. The […]

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How to Cut PEEK Tubing, Use a Clean Cut90 Cutter.

It is Extremely Important to have a Proper Cut for all HPLC Tubing Including PEEK Tubing. The Cutting Blade of the Clean-Cut90™ PEEK Tubing Cutter (Shown Below) has 2 sides. One side will be marked on the tool with an Arrow, and this produces the Perpendicular Cut (90° angle) that you want to use. Be […]

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How To Cut Stainless Steel Tubing for HPLC.

Cutting Stainless Steel Tubing for HPLC can be challenging. Here are some tips and instructions on cutting tubing using the MicroSolv tubing cutter, Catalog number 46001-01. Introduce the Tubing into the Cutter and lightly Tighten the Screw. If you tighten too much you will deform the Tubing walls. Turn the Cutter a few times around […]

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How To Determine Pore Volume of an HPLC Column by HPLC Injections.

The Interior of an HPLC Column is comprised of Spherical Particles. There are two types of empty space in the column, i.e. Volume not occupied by these particles. The first Type is the Interstitial Space between adjacent Particles. This actually comprises a small percentage of the total Void Volume. The majority of the empty space […]

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How to Fix a Saw Tooth Pattern in the UV Baseline. Immiscible Solvents.

This Saw Tooth Pattern may be due to the presence of an Immiscible Solvent remaining in the Column. Even trace amounts of these Solvents can cause issues. The Immiscibility manifests as an unstable Baseline, such as the Example shown below. Suggestion: In order to fix this issue, try flushing the Column overnight with a mutually […]

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How To Integrate a Main Peak with a Shoulder.

It is always better to have baseline separation between a Main Peak and any nearby Minor Peaks. However, this is not always possible and you may have to deal with integration of a Peak with a Minor Shoulder or Peak present. It is less accurate to integrate from the base to the point where the […]

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How to Keep HPLC Columns Free of Adsorbed Contaminants.

Depending on the analytes you use the column with, there are a number of strategies you can try: If your HPLC System can have more than two Solvents, you could include a Third Line with a Cleaning Solvent. Then in the Injection Sequence you could insert a Wash Step which uses only the Third solvent. […]

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How to Minimize HPLC Column Blockage. Most Common Causes.

A Column Blockage can be a problematic Issue, resulting in undesirable phenomena such as High Backpressure. The most important thing you can do is to prevent the problem from occurring in the first place. To do so, one must consider the Potential Causes: 1. Samples. When dealing with Samples such as Plasma, there can be Particulate […]

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How To Perform an Ohms Law Plot.

Ohm’s Law Plot to Optimize your Separation can bring many benefits and is essential for good CE Methods. Select the optimal Voltage Setting for your Separation. Maximize the Efficiency of your Method. Learn the Upper Voltage Limits of your Method. Use to Validate your Capillaries and Columns in your Method. Performing an Ohm’s Law Plot […]

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How To Prepare a 0.05% Solution from a Tablet for HPLC Injection.

Preparation of a 0.05% solution for HPLC injections depends on the Pharmaceutical Tablet Strength (dosage) and how much Volume of Diluent you use. In most cases we would recommend using your intended starting HPLC Mobile Phase as the Diluent. The Drug Tablet should first be ground using a mortar and pestle and then dispersed in […]

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How To Properly Cut a Fused Silica Capillary for CE.

A Recommended Cleaving or Cutting Procedure for Simplus Brand or any Bare Fused Silica Capillaries is as follows: Cleaving Procedure: Place the Capillary over a large Diameter Radius Surface such as large pipe or tube. Keep the Capillary under slight Tension. Hold a fresh, Clean Cleaving Stone at an approximately 30º angle to the Capillary. […]

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How to Purge Metals from HPLC System using EDTA.

In LC-MS, metal ions can be responsible for distorted analyte peak shapes via chelating. If you determine that this is the cause of a peak issue, here is what you can do to eliminate metals from the system and see that they don’t cause further problems. 1. Add 5-10 micro Molar EDTA to both A and B […]

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How To Search an Article for Exact Text Matches – Ctrl-F.

On a PC, you can instantly find any specific text that is found in a document using Ctrl-F (Command-F on a Mac). This will work for webpages, Word files, pdfs, and any other document with text (note: will not work for image files such as jpg, png, etc.). This step by step tutorial will show […]

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How To Separate Maleic Hydrazide.

Maleic Hydrazide has a pKa of 5.62 and a logP of -1.96. This logP value means it is very Hydrophilic and will not retain well in Reversed Phase HPLC. For this reason, we would suggest you try the Cogent Diamond Hydride™ column in Aqueous Normal Phase (ANP) mode in order to analyze and retain it. The pKa of […]

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How to Separate Phenylephrine HCl, Acetaminophen, and Dextromethorphan.

The Cogent Phenyl Hydride™ would be recommended for these compounds because of Dextromethorphan. It has a strong tendency to tail on most columns but the Phenyl Hydride™ Column can produce a very Symmetrical Peak; a Mobile Phase Additive of 0.1% TFA would be beneficial as well for reducing Dextromethorphan Tailing. Phenylephrine HCL does not normally […]

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How to Separate Phospholipids by HPLC – AppNote

Phospholipids present a challenge for Chromatographers for various reasons. Here some points to consider. They have negligible UV Absorption and generally require other Detection Methods. Subtle Structural Differences among Phospholipids often lead to Poor Selectivity or co-elution. For detection, Refractive Index (RI), Charged Aerosol Detection (CAD), ELSD (Evaporative Light Scattering Detection), or other “universal detectors” […]

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How to Store & Condition Cogent TYPE-C Silica™ HPLC Columns.

This article describes how best to prepare Cogent TYPE-C Silica™ HPLC Columns for Initial Use when you first receive them as well as How To Store them, for either Overnight or Long-Term Storage, to prevent Column degradation. Storage Solvents for Reverse Phase (RP) or Aqueous Normal Phase (ANP): If RP and ANP are your desired next use […]

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Validation Protocol for the PQ Kit Template.

The validation protocol in the attachment is intended to document and validate the MicroSolv HPLC PQ Kit Excel Template used for HPLC Qualifications. The Excel file given for the latest version of the Performance Qualification is for 97-2003 Microsoft Excel Worksheet, but this is also compatible with Excel 2010.  For operating in Excel 2010, you […]

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