Cysteine Analysis by LC-MS.

Cysteine is difficult to analyze by LC-MS because it is hard to ionize and also very sensitive to any metals in the instrument such as tubing, injectors, seals, joints etc.  We do have a method for cystine, which is a disulfide bonded cysteine dimer and may be helpful in your scouting and method development.

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Determining Efficiency of HPLC Methods, 4um v. 2um. FAQ.

Question: In Application Note A307, it may appear that the 4um column produces a narrower peak width than the 2.o column. Does this mean it is more efficient than the 2.2um column? Answer: Efficiency is a function of two variables: peak width and retention time. Hence, a compound may have a higher efficiency on one […]

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Determining HPLC Method Accuracy. A Primer.

The accuracy of an HPLC method is the closeness of the measured value to the true value for the sample. To determine the accuracy of a proposed method, different levels of the analyte concentrations: lower concentration (LC, 80%), intermediate concentration (IC, 100%) and higher concentration (HC, 120%) must be prepared from independent stock solutions and analyzed (n=10). Accuracy is assessed as the percentage relative error […]

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Determining the Asymmetry Factor in HPLC. Primer.

Measure the left half of the peak width at 10% peak height (A) and then measure the right half of the peak width at 10% peak height (B) (see figure below).  The ratio of B over A is the asymmetry factor.  A value of 1.0 is perfect symmetry and 0.9 – 1.2 is acceptable.  Value less […]

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Difference between adsorption and absorption.

Although these two terms sound similar, they are actually quite distinct and should not be confused. • Adsorption is the adhesion of a substance to a surface. This process creates a film of the adsorbate on the surface. In HPLC, an example might be the adsorption of water molecules on the surface of a hydrophilic stationary […]

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Difference between adsorption and absorption. For Chromatographers.

Although these two terms sound similar, they are actually quite distinct and should not be confused. • Adsorption is the adhesion of a substance to a surface. This process creates a film of the adsorbate on the surface. In HPLC, an example might be the adsorption of water molecules on the surface of a hydrophilic stationary […]

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Different Bonded Phases when using Cogent TYPE-C Silica: Retention of Polar and non Polar analytes using the different HPLC columns – Tips & Suggestions

The degree of analyte Retention in Reversed Phase (RP) and Aqueous Normal Phase (ANP) on Cogent TYPE-C (silica hydride ) Columns depends on the degree and type of modification. For no or minimal modification the ANP mode is stronger than RP mode.   As the degree of modification becomes greater, i.e. larger groups and/or higher surface coverage, […]

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Do Cogent TYPE-C Silica™ stationary phases for HPLC have a partial positive charge? If so, is retention for basic compounds reduced because of this?

No, studies using zeta potential experiments have shown that the Cogent TYPE-C Silica™ stationary phases have a negative charge due to the presence of hydroxide ions on the surface.  Retention for basic compounds is therefore not reduced. With ordinary silica, silanols on the surface which can have a negative charge adversely affect the peak shape […]

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Does Cogent TYPE-C Silica™ HPLC columns have a partial positive charge? If so, is retention for basic compounds reduced because of this?

No, studies using zeta potential experiments have shown that the Cogent TYPE-C Silica™ material has a negative charge due to the presence of hydroxide ions on the surface. Retention for basic compounds is therefore not reduced. With ordinary silica, silanols on the surface which can have a negative charge adversely affect the peak shape of […]

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Drawbacks in terms of robustness for HILIC columns

It is reported that HILIC columns can be less robust than traditional reversed-phase (RP) columns. Partly this is due to the fact that the surface can be easily contaminated and is hard to clean. In other cases, the bonded group is not very robust and is susceptible to being cleaved from the surface in mobile […]

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Dursan Coated HPLC Columns Comparison to PEEK Column Performance – Tips

Dursan is Excellent for any Compound that will Interact with Stainless Steel PEEK is a highly inert material which has been used in HPLC applications for years. However PEEK has some limitations: Mechanical strength: PEEK begins to flex at 3000 to 5000 PSI, making it unsuitable for UHPLC and other applications where high pressure is […]

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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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Factors that affect sensitivity in HPLC.

Sensitivity of an HPLC chromatogram can be said as the ability to observe all analytes in sample, even a very small quantity. One of the most important factors in sensitivity is the “Signal to Noise Ratio” of the detector you are using which can be defined as the ratio of analyte signal to background noise, […]

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Factors that affect Sensitivity in HPLC.

Sensitivity of an HPLC chromatogram can be said as the ability to observe all analytes in sample, even a very small quantity. One of the most important factors in sensitivity is the “Signal to Noise Ratio” of the detector you are using which can be defined as the ratio of analyte signal to background noise, which derives from the method of detection and […]

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For sugars analyzed by LCMS, sodium acetate is added to obtain greater sensitivity of Na adducts. Could you do this using ammonia instead, observing the NH4 adducts?

In LCMS, you will get much better sensitivity of Na-sugar adducts in the EICs compared to sugars themselves. The same principle will work for NH4 adducts as well, but there are notable drawbacks. The problems with NH4 is that it will change the pH of the mobile phase and its concentration is hard to control in the sample.  […]

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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 […]

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Fosetyl-aluminum HPLC Analysis Tips – Tips & Suggestions

Reversed Phase & Aqueous Normal Phase Method 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 pair agent is recommended to increase retention and reduce peak tailing. Here, […]

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Glucose, Pyruvate, Taurocholic Acid, Gamma Glutamylcysteine etal Analysis – Tips & Suggestions

What suggestion can you offer for Analysis of Metabolites such as Glucose, Pyruvate, Taurocholic Acid, Gamma Glutamylcysteine, Glycochenodeoxycholate, Glycerol-3-phosphate, Spermidine, Taurine, and S-Adenosylmethionine? Suggestion: We would recommend starting by using the Cogent Diamond Hydride™ HPLC Column for this Analysis. Many of these compounds are quite Polar and would be very difficult to Separate by Conventional […]

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High baseline noise with Cogent Diamond Hydride column

One reason background would be high is if something was injected that was stuck on the column and it is bleeding off or if harsh pH conditions were used. Either of these can occur sometimes without the user knowing it if the instrument is shared and it was not flushed properly between uses. Cogent Diamond […]

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High baseline noise with Cogent Diamond Hydride HPLC column.

One reason background would be high is if something was injected that was stuck on the column and it is bleeding off or if harsh pH conditions were used. Either of these can occur sometimes without the user knowing it if the instrument is shared and it was not flushed properly between uses. Click HERE […]

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How can I calculate the ligand density of a bonded phase?

The ligand density can be calculated using the Berendsen-de Galan equation: α = 106 %C/(102 MW carbon nC – %C MW ligand) SBET where α is the ligand density (µmol/m2), %C is the percent carbon (%), MW carbon is the molecular weight of carbon (g/mol), nC is the number of carbon atoms per bonded ligand, MW ligand is the molecular weight of the organic bonded ligand (g/mol), and SBET is the specific surface area of silica material (m2/g). For […]

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How can I get separation of tartaric and malic acid in a complex mixture?

Suppose you have a sample containing lactic acid, glycolic acid, formic acid, L-tartaric acid, DL-malic acid, and succinic acid. The Cogent Diamond Hydride™ HPLC column will be an excellent column choice for this separation because all these organic acids are very polar. In the mobile phase you want to keep the acids ionized so that […]

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How can I get separation of tartaric and malic acid in a complex mixture?

Suppose you have a sample containing lactic acid, glycolic acid, formic acid, L-tartaric acid, DL-malic acid, and succinic acid. The Cogent Diamond Hydride™ HPLC column will be an excellent column choice for this separation because all these organic acids are very polar. In the mobile phase you want to keep the acids ionized so that […]

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How can I separate capsaicinoids in hot sauce with HPLC?

If you have a hot sauce extract containing nordihydrocapsaicin, capsaicin, dihydrocapsaicin, and homodihydrocapsaicin, the Cogent Bidentate C18 2.o™ HPLC column is a good choice for sufficient separation when the compounds are analyzed by LC-MS; look for the [M+H]+ ions. The following gradient should be a good starting point: You may get EICs which look like […]

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How can I separate capsaicinoids in hot sauce with HPLC?

If you have a hot sauce extract containing nordihydrocapsaicin, capsaicin, dihydrocapsaicin, and homodihydrocapsaicin, the Cogent Bidentate C18 2.o™ HPLC column is a good choice for sufficient separation when the compounds are analyzed by LC-MS; look for the  [M+H]+ ions. The following gradient should be a good starting point: You may get EICs which look like this: 1. nordihydrocapsaicin, 294.2064 […]

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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 […]

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How do autosampler vials cause missed injections in HPLC and LCMS?

HPLC instrument manufacturers require “Fit and Form” specifications for vials used in their autosamplers. These include minimum and maximum vial dimensions for height, neck, shoulder and outer dimensional width to prevent jamming the autosampler. If vials are within these specifications jamming problems due to the vials shouldn’t occur. However, most modern autosamplers have an injection […]

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How do autosampler vials cause missed injections?

HPLC instrument manufacturers require “Fit and Form” specifications for vials used in their autosamplers. These include minimum and maximum vial dimensions for height, neck, shoulder and outer dimensional width to prevent jamming the autosampler. If vials are within these specifications jamming problems due to the vials shouldn’t occur. However, most modern autosamplers have an injection sequence […]

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How do I condition new Cogent TYPE-C™ Columns for use?

For all new Cogent TYPE-C™ HPLC columns with the exception of new Diamond Hydride™ columns, all one needs to do is run 7-10 column bed volumes of your mobile phase through the column at your normal flow rate. Then it is best to inject a known standard and repeat the injections until duplicate chromatograms are […]

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How does ammonium fluoride affect HPLC & LCMS chromatography using Cogent TYPE-C Columns?

Ammonium fluoride functions beneficially in several ways in a chromatographic method:      • As a buffer (i.e. pH control) • Improvement of peak shape • Improvement of sensitivity in MS for certain compounds. For further reading, please refer to the following third-party journal article reference: J. Pesek, M. Matyska, “Ammonium fluoride as a mobile phase additive in aqueous normal phase chromatography,” J. […]

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How is the Cogent UDA HPLC ligand bonded to the surface?

The Cogent UDA™ HPLC column ligand is a C11 chain with a carboxylic acid on one end. On the other end, there are two orientations for how the ligand could be bonded to the Cogent TYPE-C™ Silica support. The first orientation is shown in A. In this case, only one hydro-silation occurs from the starting […]

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