Trouble Shooting Suggestions – MicroSolv Technology Corporation

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A White Film Developed on my MS Source. Is this Column Packing Material? -FAQ

I see a white film developing on my MS source after doing LCMS. How do I know I’m not losing Packing Material? Suggestion: If the film was from Dissolved Stationary Phase Material, you would see a significant Distortion in Peak Shape for all your analytes due to the Void that would be developing in the […]

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 […]

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 […]

Analyzing Metformin in Plasma Samples, how do I Obtain Reproducible Results and Peak Shapes? – FAQ

Suggestion: Try substituting Formic Acid in DI Water and adding Ammonium Acetate (10mM) and Acetic Acid (1%) to the Aqueous Phase. This should improve the Reproducibility of Peak Areas and Retention Times. In addition Extensive Reconditioning of the Analytical Column with pure Acetonitrile in between Separations is Essential for good Reproducibility when working with Plasma […]

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% formic acid. ANP gradients need to start between 100% – 90% of Solvent B (to retain polar compound) and then reduce the percent of Solvent B […]

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 […]

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 […]

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 […]

Changing Retention Times and a peak Showing up in my Blank with a Silica-C Column. What can be the issue? – FAQ

Suggestion: It is most likely a Matrix effect. You should wash the Column with a reasonable concentration of the Strong Solvent of your Mobile Phase, with it being higher than at the end of the Gradient. Then you should Inject the blank again. Also, check to make sure the Peak you are getting in the […]

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 acid is generally used when analyzing amino acids, so the use of ammonium acetate (or formate) might be a problem. 2. Contamination from the […]

Does the HSQ Kit Qualify Quaternary HPLC Pumps? – FAQ

Yes, the HSQ Kit™ will work on many types of pumps; from single, binary, and also quaternary pumps. For Gradient Qualification Tests, the base method is called “GRDAB” for “Gradient, pump A/B*”, where the “*” designates that the B fluid circuit is “visualized” with the addition of the Gradient Visualization Solution (GVS). For ternary or […]

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 From the table, we can see that the gradient begins immediately, but in practice this is […]

Eliminate Broad Split Peaks in ANP Methods with Acetonitrile / Methanol – Tips & Suggestions

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 Water / Formic Acid based Mobile Phase and my diluent is 50:50 Methanol / Acetonitrile. Retention is also low. What can I […]

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 […]

Fosetyl-Aluminum Analysis with 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 […]

Ghost peaks appearing results using a gradient method with TFA in the mobile phase – TFA degrades with time

Question: I am running a gradient HPLC method and see a number of ghost peaks towards the end of the HPLC run. I see them in both samples and blanks. I have 0.1% TFA (tri-fluoro acetic acid) in the A and B solvents. Do you have any suggestions or a solution? Answer: The problem may be due […]

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 […]

High molecular weight compounds that won’t retain on Cogent Diamond Hydride column, try Cogent Bidentate C8 300.

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 account for most of the surface area of the phase and therefore interactions involved in retention. If an analyte is too large to fit […]

High Pressure Develops after Flushing a Cogent Diamond Hydride Column with Acetonitrile, Cause and Solution. FAQ.

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 I returned to my method. What happened? Answer: The problem is most likely the Solubility of Ammonium Acetate in pure Acetonitrile. We […]

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 […]

How can I reduce tailing for Atomoxetine, Reboxetine, and Maprotiline in a Reversed Phase C18 HPLC method? – FAQ

How can I reduce Tailing for Atomoxetine, Reboxetine, and Maprotiline in a Reversed Phase C18 HPLC method? I am using a pH 4 Phosphate/Acetic Acid Buffer. Atomoxetine, Reboxetine, and Maprotiline all have Amine Groups. Standard C18 phases may have Residual Silanols on the surface which can interact Electrostatically with the Amines from these analytes. These […]

How can Sample Concentration Have an Effect on Peak Shape in HPLC – FAQ

Injecting a very high concentration sample can result in lower Retention and Distortion in Peak Shape 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 0.3mg/mL […]

How do I Avoid Distorted Peak Shapes when using a Basic Mobile Phase pH for the Analysis of a Lipophilic Amine? – FAQ

You may not require a Basic Mobile Phase pH for HPLC analysis of these Types of Analytes. Use of the Cogent Diamond Hydride™ Column in ANP Mode with 0.1% Formic Acid instead of a Basic Mobile Phase may be a better approach. There are a number of possible reasons that you may have observed a […]

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

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 […]

How to Back Flush and Wash an HPLC Column for Reuse – Tips & Suggestions

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 […]

How to Fix a Saw Tooth Pattern in the UV Baseline. Immiscible Solvents- Tips & Suggestions

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 miscible […]

How to improve poor peak shapes, retention & carryover for polar compounds using Cogent Diamond Hydride™ HPLC column with LC-MS

Peak shape for polar vitamins in LCMS has been shown to be improved when using a 0.1% formic acid additive instead of ammonium acetate. The sample diluent should be 50% DI water/ 50% acetonitrile/ 0.1% formic acid. If you are using 50% isopropanol or 50% methanol in the aqueous solvent, the carryover could be due […]

How to Increase HPLC UV Detector Response with a Peak Height of 300 mAU – Tips & Suggestions

In accordance with Beer’s Law, there are only three things you can do to obtain increased Analyte Response in UV Detection: The first is Increasing the Analyte Concentration. The second is Increasing the Path Length of the UV Flow Cell. —–To do this, you could use an Instrument with or allows a Longer Path Length. […]

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 […]

How to Remove a Ferrule that is Stuck in an HPLC System or Column- Tips & Suggestions

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 […]

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 […]

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 […]

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 […]

HPLC Stationary Phase can be Responsible for a Blocked Frit- Tips & Suggestions

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 would be 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™ […]

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, always be sure to sonicate and filter your mobile phase before introducing to the LC system especially […]

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 […]

Injection volume suggestions for Cogent TYPE-C HPLC columns

When using an analytical column it is recommended to inject 1 uL to 10 uL of the biological sample which is prepared either in DI water or 50% solvent A/50% solvent B of the mobile phase. When a 100 uL injection is performed the column is overloaded.

Issues arising from changes in mobile phase with acids to one with ammonium salts: Cogent Diamond Hydride.

If you need to use both acid (e.g. formic) and ammonium salts (acetate or formate) with a Cogent Diamond Hydride™ HPLC column for your samples we recommend dedicating one column for use with mobile phases containing ammonium and another for use with mobile phases containing acids. The rationale in this is that once you introduce ammonium acetate or formate to […]

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. […]

Low quantitative results in HPLC: Influence of autosampler vial caps and other factors.

Issue: I am running HPLC methods and observe low analyte quantitation. Other observations: – The problem appears to be random in nature – Three different autosamplers are involved – Mobile phases and samples are the same on other instruments that do not exhibit this behavior – Replacing the needle does not change the low result – Use of […]

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 […]

Lower than Normal Back Pressure seen using Cogent Diamond Hydride HPLC Columns. – FAQ

I have recently been using the 2.1 x 150mm 4um 100A Cogent Diamond Hydride™ HPLC Column. I am using a 400uL / minute flow rate with Acetonitrile and an Aqueous Buffer (Column Temperature = 30 °C). I’m seeing a very low back pressure (around 20 bar at the beginning of the gradient while acetonitrile level is […]

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 […]

No Need to Use a pH for High Mobile Phases for Compounds with a High pKa.

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 not necessary to use high pH to get retention. These extreme pH conditions can be damaging to any HPLC […]

Peaks are broad and non-symmetrical in LC-MS method using Reversed Phase HPLC Columns. Any Suggestions?

There are various possible solutions using Cogent™ HPLC columns: 1. Diluent: Is your diluent too strong for the mobile phase? You should not have a 90% acetonitrile diluent with a 10% acetonitrile reversed phase method. You may observe peak distortion for this reason. 2. Mobile phase/diluent pH: Do you have any basic analytes? If so, perhaps they are […]

Poor HPLC peak shape can be due to diluent mismatch: Example using Resveratrol.

In this example, resveratrol samples were found to produce poor peak shape when a diluent of 100% acetonitrile was used. This is caused by a mismatch of the diluent compared to the mobile phase, which is 80% aqueous. Your diluent should be the same as your mobile phase if possible. If this cannot be achieved, […]

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 […]

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 […]