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Trouble Shooting HPLC Methods

1 Analyte retention decreases when I increase water content in my mobile phase using the Cogent Bidentate C18 HPLC column. Has anything happened to the column?

There are several explanations to this phenomenon. 1. This could be due to Aqueous Normal Phase (ANP) HPLC and is a unique property of Cogent HPLC columns with Cogent TYPE-C™ silica. In this case the…

2 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%…

3 Bubble formation when using inlet filters for HPLC mobile phases.

When an HPLC pump is active, a vacuum is present in the tubing and any filter that is connected to the pump. If the liquid cannot flow fast enough, an air bubble will result which is commonly called "cavitation".…

4 Can I use methanol instead of acetonitrile in my HPLC method?

Methanol is a very common choice for the HPLC mobile phase organic solvent component in reversed phase HPLC even though acetonitrile is often superior in several aspects. On the other hand, methanol is…

5 Can my HPLC stationary phase be responsible for a blocked frit?

Under certain circumstances, it is possible to damage the stationary phase particles (dropping the column on a hard surface, pressure shock etc) such that they are comparable in size to the frit pore…

6 Changing retention times of amino acids in biological extracts using ammonium acetate in HPLC methods.

If you observe changing retention times of amino acids using a Cogent Diamond Hydride™ HPLCL column, consider the following . Suggestions, tips and tricks 1. Acid in the Mobile Phase; Formic or acidic…

7 Do I need to use a high mobile phase pH for compounds with a high pKa?

No, while some types of analyses for compounds have traditionally required a high mobile phase pH (e.g. up to 10, 11 or 12) in order to get acceptable chromatographic retention due to a high pKa, it is…

8 Fused insert glass vials (cat# 9532S-WCV) break during sample injection

Question: When injecting samples using fused insert glass vials, sometimes the needle hits the insert wall, causing it to break. Are the inserts not aligned vertically? Answer: This is more likely due…

9 High background noise using new Cogent column

If you are seeing significant background noise on your detector using a new Cogent HPLC column, it may simply need an initial conditioning to remove any trace residual packing solvents. Flushing the column…

10 High molecular weight compounds that won't retain on Cogent Diamond Hydride column note.

If you are working with an analyte of MW ~10,000 or greater, you may observe no retention when using the 100A columns such as the Cogent Diamond Hydride™. This is because the pores of an HPLC column…

11 High pressure develops after flushing Cogent Diamond Hydride with acetonirile. Cause and solution.

Question: I was running a method using an ammonium acetate-based mobile phase and decided to clean the column. I flushed it with acetonitrile but noticed the column pressure was higher than before when…

12 High pressure traced to the pump module

Excessive backpressure is a common problem encountered in HPLC. However, there are many things that can cause a high pressure, and therefore troubleshooting may be quite puzzling. The best approach is…

13 How can I purge metals from my 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…

14 How can I reduce tailing for atomoxetine, reboxetine, and maprotiline in a reversed phase C18 method? I am using a pH 4 phosphate/acetic acid buffer.

Atomoxetine, reboxetine, and maprotiline alll have amine groups. Ordinary C18 phases may have residual silanols on the surface which can interact electrostatically with the amines from these analytes.…

15 How can I separate phenylephrine HCl, acetaminophen, and dextromethorphan?

The Cogent Phenyl Hydride™ would be recommended for these compounds because of the dextromethorphan. It has a strong tendency to tail on most columns but the Phenyl Hydride™ column and can produce…

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