Increasing selectivity and resolution for xylose and ribose method

When our team created the method for our application note of D-ribose and D-xylose, we had achieved a good level of separation which was a key feature for this particular set of analytes. We recently revisited this work to see if any additional improvements could be made. By increasing the length of the column dimension […]

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Using Cogent TYPE-C Silica columns for analysis of different amphiphilic compounds: Separation when both reversed phase and ANP mechanisms operate.

It is relatively unlikely that Reversed Phase (RP) and Aqueous Normal Phase (ANP) HPLC retention contributions would be equal for different amphiphilic compounds because it would require that both mechanisms would be operating at equal efficiency for those particular compounds.  In most cases one mechanism would be more predominant than the other and retention would be different.  However, even in […]

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When I run gradients on my HPLC and switch from one gradient to another, my baseline shows as “negative”. If I auto zero, it becomes positive for the remainder of the run but when I start again, it is negative. What can you suggest?

Negative baselines in gradients are not that unusual. If the method is using even moderately UV absorbing components at the wavelength of interest, it is very difficult to exactly balance the absorbance signals of both channels. Acetic acid is very bad in this respect. The lower the wavelength, the more difficult it is to manage. […]

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Since Cogent TYPE-C™ columns can be used in Reversed Phase (RP) or Normal Phase (ONP) modes, what do you suggest to “switch” them with since these solvent systems are not miscible?

Suggested Procedure: A – moving from Reverse Phase to Normal Phase HPLC; pump 100% methanol for 15 minutes at 1 mL/min. flow rate, followed by 15 minutes 100% methylene chloride. The column is ready to be equilibrated with mobile phase for NP-HPLC. B – moving from Normal Phase to Reverse Phase HPLC; pump 100% methylene chloride for […]

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Adjusted retention time in HPLC. A Primer.

Adjusted retention time (tR‘) is the retention time adjusted for the hold-up time: tR‘ = tR – tM where tR is the retention time and tM  is the hold-up time. The hold-up time is the time of an analyte (small molecule) which completely penetrates the pores and which is not retained at all by the stationary phase.

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The pH range of common buffers for HPLC.

The following table shows a list of common buffers and their working buffer ranges:   Buffer pH Range Trifluoroacetic acid (TFA)  1.5-2.5 Phosphoric acid/monobasic phosphate (pKa 1)  1.1-3.1 Formic acid/formate  2.8-4.8 Acetic acid/acetate  3.8-5.8 Mono/dibasic phosphate (pKa 2)  6.2-8.2 Ammonia  8.2-10.2 1-methylpiperidine  9.1-11.1 Triethylamine  10.0- 12.0 As for concentration, about 5mM is suitable for Cogent TYPE-C Silica™ […]

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HPLC Columns that could be used to separate both polar and nonpolar compounds.

The most versatile columns for this type of separation are the Cogent TYPE-C Silica™ columns because the amount of retention in both modes can be adjusted by the type of modification and the mobile phase composition.  Polar end-capped and polar embedded columns have this same capability since they are also classified as mixed-mode stationary phases.  The degree of […]

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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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The best way to deal with ion suppression issues in LC-MS if you need to use an ion-pair agent. Simple and easy technique.

In many cases, you will not need ion-pair agents when using Cogent TYPE-C Silica™ columns. Ion pair agents are generally used to either increase retention of a poorly retained compound in reversed phase (RP) or to improve peak shape due to silanolic tailing. The Cogent TYPE-C™ Silica columns can retain analytes by aqueous normal phase (ANP) which is […]

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Typical validation parameters such as accuracy and precision of HILIC vs. Reversed Phase HPLC methods.

In most cases the average level of accuracy and precision in a HILIC analysis is less than typical values obtained by reversed-phase (RP).  The accuracy and precision of a HILIC analysis is strongly influenced by the equilibration time if a gradient method is used.  In addition, Aqueous Normal Phase (ANP) is more suitable than HILIC in terms of accuracy and precision. The reason for this is that in […]

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Which membrane is best for aqueous buffers when using Syringe Filters?

The best membrane is a difficult question to answer as it depends on your sample that you are filtering and the solvent is it dissolved in. For most HPLC and Dissolution testing, hydrophilic Nylon membranes are generally used because they are durable, compatible with most aqueous solvents including water and have the lowest extractables with […]

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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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Theoretical plate N: Estimate for real samples.

One can estimate a reasonable plate number for any column as:                          N ≈ 300 L/dp L is the column length (in millimeters) and dp is the particle diameter (in micrometers). For example for 250 mm x 4.6 mm column packed with 5 micron particles, the predicted value is                                        N ≈ 15,000    If the measured value of N is about […]

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What is “Post Time” in an LCMS or HPLC gradient?

In HPLC application notes that use gradients, you will see a value listed for “post time” at the end of the gradient table. This parameter specifies the time to hold at these mobile phase conditions until the next injection. Generally, the conditions at the end of the gradient and the beginning of the next run […]

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What can cause excessive back pressure in HPLC runs?

There can be many causes of excessive back pressure but one very common cause is build up of foreign materials on the top of the HPLC Column. This situation can result when sample precipitates (retains or accumulates) or dust and other particles from the degradation of the pump seals. This situation can be avoided by […]

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HPLC Method Linearity tip.

To establish linearity of the proposed method, ten point calibration curves should be generated with appropriate concentrations of calibration standard solutions. The calibration range can be for example: 10-100 microg/mL. The linearity should be evaluated by the least squares regression method using unweight data.

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Can I make the PTFE Syringe Filter hydrophilic?

Yes, if you are wiling to introduce Methanol into your method. For a 25mm device, if you use 15-20mls of methanol as a blank filtrate, the membrane will give you less back pressure after that for aqueous samples. This is due to “wetting” the membrane with Methanol allowing more aqueous solvents to pass through it […]

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System suitability requirements for a USP HPLC method.

USP (United States Pharmacopoeia) existing methods for analysis of drugs or active compounds are very frequently used in the pharmaceutical industry because they do not need to be validated if they can be set up and demonstrated that they pass system suitability requirements, which include: A minimum resolution between the active ingredient and a related […]

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Activity in HPLC. Simple and easy definition.

It is the relative strength of the surface of the stationary phase in adsorption chromatography.  In the case of silica gel, the more silanol groups are available on the surface, the more active it is.  One can modifty activity by adding water or other polar solvent that will bond to active sites, reducing activity.   Cogent TYPE-C HPLC Columns […]

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If I use one syringe filter for many samples will I get sample carry over?

You will most likely get some kind of carry over. It is not recommended to use one filter for more than one sample. Reusing the same syringe filters for multiple samples is never recommended due to carry over problems. Click HERE for AQ Brand Syringe Filter Ordering Information. Attachments: MicroSolvFiltersEquivalency.pdf   26.9 Kb   Download File

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Poor peak shape for Ethylenediamine in LCMS Analyses.

Ethylenediamine is known to associate with metals, forming organometallic complexes. In terms of HPLC runs, this can present problems for analyte peak shape. Most commercial HPLC systems will most likely have some trace metals present that can be problematic. These metals can come from many sources such as the glass bottles, the pump components, columns etc. What you can […]

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Should the pre-column filters replace my HPLC guard columns?

No, a guard column is both a particulate filter (non dissolved chemicals) and a chemical filter (removes dissolved chemicals). The pre-column filter is a particulate filter and adds extra protection of your expensive analytical and guard columns.   Click HERE for Column Protection Ordering Information including HPLC Pre-Column Filters.  

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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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What is the difference between weak and strong cation exchange for IEX Columns?

An HPLC stationary phase is considered a weak cation exchanger when it has a functional group such as a carboxylic acid which proton does not come off (ionize) until around pH 4 or so.  Columns with sulfonic acid groups for example, are considered strong cation exchangers because they are essentially un-protonated (ionized) at any reasonable mobile phase pH for […]

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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 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 to fix poor peak shape for phosphorylated compounds in HPLC analysis.

Question: I am analyzing phosphorylated sugars and obtain poor HPLC peak shapes with very broad tailing. What are some possible solutions? Answer: We have a number of strategies for fixing problems with peak shape for phosphorylated compounds: • Ensure the column is not overloaded. We recommend an injection volume of 1 µL or less with […]

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Precision and suitability of the Cogent TYPE-C Silica HPLC columns in LC-MS.

The Cogent TYPE-C™ Silica HPLC Columns produce lower background noise in LC-MS than other HPLC columns. For column precision, typically %RSD for retention time of replicate injections is less than 1%. As for detection precision, we highly recommend using an appropriate internal standard in LC-MS.  This will help with reliable quantitation of your analytes.

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Compatibility of Cogent TYPE-C Silica HPLC Columns with UHPLC instruments.

Cogent™ HPLC columns are compatible with ultra high performance liquid chromatography (UHPLC) instrumentation. However, it is important to note the low pressure produced in ANP and the pressure limits of these columns. The Cogent 2.o™ is considered “Near UHPLC” with pressure limits near 9,000 psi. If you are using Aqueous Normal Phase (ANP) HPLC it is unlikely […]

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Quaternary Amines Analysis by LC-MS: Finding the right ion: Tip.

When analyzing quaternary amines in LC-MS, you should remember that they are permanently charged species. A tertiary amine, for example, may be observed in the positive ion mode as the [M + H]+ ion, but because quaternary amines are already ionized, you should look for the [M]+ ion in the Extracted Ion Chromatograms (EICs) instead.  If you look […]

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Limits of Detection LOD and Limits of Quantification LOQ definitions.

The limits of detection (LOD) and quantification (LOQ) are evaluated using the following equations [1-4]: LOD=3.3 S0/b LOQ=10 S0/b    where S0 is the standard deviation of the calibration line’s y-intercept where b is the slope of the linear regression line of best fit. The limit should be subsequently validated by the analysis of a suitable […]

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