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Trouble Shooting HPLC Methods

1 A case to eliminate retention time drift in ANP using the Cogent Diamond Hydride in LCMS.

We had a significant run to run RT drift ( up to 1 min for 10 repeated runs) using the Cogent Diamond Hydride™ HPLC column we ordered two weeks ago. After conditioning the column with 90% IPA and 10X…

2 Add 10 micro molar EDTA to mobile phase for better peak shapes.

Metals from glass bottles and vials or your instrument can be one cause of poor peak shapes for the polyprotic acids and nucleotides in LCMS. This varies greatly from instrument to instrument so you should…

3 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…

4 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%…

5 Blocked column from proteins in the sample matrix can be recovered

It is of crucial importance to always filter your samples prior to introduction to an HPLC instrument. Most samples will contain particulates and other matrix components that can cause blockages in the…

6 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".…

7 Buffer concentration can affect ANP retention of acids in HPLC Analyses.

If you are using ammonium acetate or formate in an ANP method, the concentration used may play a significant role in the retention of organic acid analytes. Although buffer concentration may affect a…

8 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…

9 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…

10 Can sample concentration have an effect on peak shape when using an HPLC column?

Injecting a very high concentration can result in lower retention and distortion in peak shape for your analyte in Aqueous Normal Phase (ANP) or Reversed Phase (RP) HPLC. The following study examined…

11 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…

12 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…

13 Dwell volume's effect on HPLC analyte retention in gradient methods

Dwell volume is the volume between the point where the solvents are mixed and the HPLC column. Suppose we have a method with a hypothetical gradient as follows: Time (min) % B Solvent 0 20 9 60 10 20…

14 FAQ: I am observing broad, split peaks in Aqueous Normal Phase (ANP) when using acetonitrile/methanol diluent, What can I do to improve peak shape?

Question: 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…

15 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…

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