Add 10 micro molar EDTA to Mobile Phase for Better Peak Shapes.

Metals from glass Bottles and Autosampler 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 identify the source of your poor shapes. One possible solution in this case would be to add 10 micro-molar […]

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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 column is behaving normally. 2. If you know the mechanism is most likely to be reversed phase, then there is potentially a […]

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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 instrument and the column. In addition, some macromolecules such as proteins may need to be precipitated and removed during sample preparation so that they do not […]

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Bubbles Forming When Using Inlet Filters for HPLC Mobile Phases.

Bubbles Can Form in an HPLC Mobile Phase. 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”. This cavitation can occur for a variety of […]

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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 chromatographic environment in numerous ways, one particularly significant effect (in the case of acids) is thought to involve adsorbed acetate or formate on the […]

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Eliminate Retention Time Drift in ANP in LCMS.

A significant  run to run RT drift could be possible. A case study. Up to 1 min for 10 repeated runs using the Cogent Diamond Hydride™ HPLC column showed Retention Time Drift.  After conditioning the column with  90% IPA and 10X  1% phosphoric acid injections as suggested for our specific method, we were able to […]

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Fosetyl-Aluminum Analysis by HPLC. Optimizing Tips – AppNote

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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High background noise using new Cogent HPLC Column can be eliminated.

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 at a low flow rate overnight with isopropanol, 50/50 isopropanol/DI water, or 50/50 methanol/DI water may be effective.  Another strategy you can […]

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High pressure traced to the HPLC 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 to try to isolate where in the flow path the high pressure originates. For example, if the column is suspected, try replacing it with a […]

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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 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 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 Remove a Ferrule that is Stuck in an HPLC System or Column.

Sometimes a Ferrule will become jammed inside a Column or elsewhere in the HPLC System. When this happens, don’t worry! The Ferrule can be Removed using our Specially Designed Ferrule Removal Tool. The Tool works due to the direction of the threads, which go the opposite way from that of a standard Drill Bit. This […]

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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 Store and Maintain Cogent Diamond Hydride HPLC Columns.

Before the First use of the Diamond Hydride Column: 1. Condition it with 50:50 MeOH/Water for 30 minutes. Conditioning Steps – Depends on Your Samples: 1. When using urine, plasma, or other biological samples etc: A. Conditioning the Column with 80% Acetonitrile/ 20% DI water/ 0.2% Acetic Acid (or 0.1% Formic Acid) is recommended for […]

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HPLC Retention time shift troubleshooting tip; purge solvent lines.

As one possible solution to unexpected retention times is to ensure that the mobile phase solvent lines currently contain the mobile phase solvents you want to use. A previous user may have used a different mobile phase on the instrument and there can be some residual solvent left in the lines. In this case, simply purge […]

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HPLC Stationary Phase can 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 size. This would then produce a high Column pressure due to the partially blocked flow area. Some of our Cogent TYPE-C™ Silica […]

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Ideas on pressure increases after each HPLC injection. FAQ.

Question: I am injecting cell extract samples on my HPLC instrument and I find that the back pressure increases after each run. Any ideas on what the problem might be? Answer: There are a number of possible causes. First, be sure to always sonicate and filter your mobile phase before introducing to the LC system especially […]

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Inconsistent peak heights obtained for glucosamine help in HPLC.

Issue: I am running an HPLC method for glucosamine. However, my peak heights are inconsistent from run to run. What is causing this? Solution: One possibility that could account for this behavior is chelation of glucosamine with trace metals in the HPLC system. A prominent source of metals is the mobile phase reservoir bottle, which […]

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Issues with a steep UV trace in gradient elution in HPLC methods.

A common problem encountered in gradient elution chromatography can be the angle or slope of the UV trace. This reason for this phenomenon can be understood by considering how each of the solvents (A) and (B) have their own UV absorption profile. Because of these differences, the UV readout (trace) will change continuously over the course of the gradient, producing the undesirable slope that is observed. […]

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Lower ANP retention is observed when group in compound is ionized

Question: I am analyzing S-adenosyl methionine (SAMe) with the Diamond Hydride column in ANP mode. I used a gradient with a formic acid additive and then tried an ammonium acetate additive. With ammonium acetate, I got lower retention than with formic acid using otherwise identical method conditions. I was expecting greater retention with ammonium acetate since the carboxyl […]

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Missing Peaks using UV Detection in HPLC.

If you do not see a Peak for your Analyte with UV Detection in HPLC, it could mean any number of things. The compound could be poorly retained and hence obscured by the Solvent Front Peak. Conversely, it could be strongly retained and not eluted during the run. A Sample Prep Issue could also be […]

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Random shifting of retention times in HPLC chromatograms.

If you see retention times that are getting either higher or lower with each injection, this often points to a column equilibration issue. However, if the retention time shifts are more random in nature from run to run, this may be due to the solvent proportioning valve instead. What this means is that the solvents are not […]

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Sample Concentration can Have an Effect on Peak Shape in HPLC.

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 three concentrations (3mg/mL, 0.3mg/mL, and 0.06mg/mL) of venlafaxine using the same ANP method. The effects of “overloading” the Column are clearly observed in the 3mg/mL injection. The […]

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Sensitivity slowly diminishes in HPLC.

Problems with the noise floor and sensitivity can be due to factors like the mobile phase purity, poor analyte recovery, and various other factors.  However, if the UV lamp is at fault, the phenomenon is usually gradual; the baseline will slowly becomes more noisy and/or the peak sensitivity will slowly attenuate. If this is the case, the […]

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Shoulder Peaks in HPLC method using RP C18 columns.

With regards to your question about the shoulder peaks observed using the Cogent C18 HPLC column. First I would like to ask you about the shoulder peaks. Have you determined whether these are due to distortion of the peaks of interest or elution of contaminants? There are several ways you can tell the difference. One is to spike […]

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