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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Best Practices for Crimping a Cap on a Vial

It is important to understand that crimping a cap could result in an inadequate seal if not done properly. Follow the steps shown below to achieve the best seal. You need a calibrated crimping tool (https://mtc-usa.com/Crimping-Tool) This means that the chuck has to be adjusted every 10 -15 crimps with the adjusting tool it comes with. […]

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What type of autosampler vial can be used with crimp caps? – FAQ

We sell 3 types of vial closures and vials; Screw thread, Snap Top and Crimp Top. Our Snap Top Vials will accept any of our Snap Caps as well as our Crimp Caps and are often called Snap/Crimp Tops in the market place. However, to ensure the best long term seal we recommend using our  Crimp Top (Serum Finish) Vials. When a quality […]

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Adsorption can Happen Quickly in an Autosampler Vial.

We have shown from previous experiments of how concentration of certain analytes can be adsorbed to glass surfaces over time which is an indicator of potential problems for the laboratory for many other analytes as well. In this experiment, a sample was taken in small timed increments to display the magnitude of potential sample loss […]

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Can I use acetone as a mobile phase solvent in ANP?

If you are using LCMS, the simple answer is yes but pH is also something to consider. Acetone has a high UV cutoff and therefore can be a problem for UV detection. However, this is not a problem when using LCMS or ELS detectors such as the Corona CAD. You can use acetone to replace acetonitrile with the Cogent TYPE-C™ […]

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Observing broad, split peaks in Aqueous Normal Phase (ANP) when using acetonitrile/methanol diluent

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 water/formic acid based mobile phase and my diluent is 50/50 methanol/acetonitrile. Retention is also low. What can I do to improve peak shape and/or retention? Answer: The absence […]

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Fosetyl-aluminum analysis tips with HPLC

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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Using the Cogent Diamond Hydride and Cogent Bidentate C18 columns together in a tandem LC separation.

If you connect these two columns in parallel but use the same mobile phase program on both columns, it will be difficult to have both reversed phase and aqueous normal phase mechanisms operating simultaneously and therefore not get optimal results; a gradient starting at high organic may work well for the Cogent Diamond Hydride™ column but only very hydrophobic compounds will […]

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Qualitative and Quantitative method for milk caseins

I am looking to quantify and qualify milk samples with HPLC. In terms of quantification I am would like a column that will resolve the common caseins i.e. alpha, beta and kappa as well as the common whey proteins alpha lactalbumin, beta lactoglobulins and bovine serum albumin (BSA). We need a rapid and robust column as we […]

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Tips on solving baseline problems in ANP with the Cogent Diamond Hydride

When using a gradient method with the Cogent Diamond Hydride™ column, an inconsistent baseline may be observed in some instances. The following are some possible causes of the issue along with suggestions to fix it.   1. Is the mobile phase filtered before use on the instrument? A 0.45µm nylon membrane filter with vacuum filtration […]

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Can I use primary amines with acetone as the mobile phase component in LCMS?

Yes, you can. However, some labs react acetone with primary amines (+heat) to form acetone adducts (imines) in GC and GCMS. This reaction while using acetone as a mobile phase component in LCMS along with primary amines as part of the sample is something to consider. This reaction, according the University of Liverpool requires a primary amine, a ketone or aldehyde (in this […]

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Improving peak shape in an HPLC method in Aqueous Normal Phase (ANP).

Try the following tips: 1. It is generally better to have the sample solvent strength stronger that the mobile phase solvent, especially at the beginning of a gradient.  In addition, Cogent TYPE-C™ silica hydride phases are less susceptible to peak distortions than HILIC.  With the proper gradient, most compounds will give good peak shape. 2. […]

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What is the difference between potency and purity in drug products? A simple and easy definition.

Please refer to the International Conference on Harmonization (ICH) guidelines for more detail. A quick definition would be: –Potency is a measure of drug activity expressed in terms of the amount required to produce an effect of given intensity. –Purity is a measure of the amount of API present in a sample compared to those of related substances, impurities, residual solvents, etc.

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I want to use the post column photochemical reactor for Aflotxins analysis. Which knitted reactor coil (KRC) do you recommend? What other accessories would I need?

The recommended KRC for Aflatoxin analysis is the 49900-KRC2525, 1.25ml void volume. The uv lamp supplied in the Photochemical Reactor is a low pressure mercury with 254nm light and is purchased with one polished support plate 8″ (49900-55) .The KRC requires 2 polished support plates. Click HERE for Post Column Reactor Ordering Information including knitted […]

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Caution when using the term 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 phases […]

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Repairing a blocked frit in an HPLC column. A simple technique.

The frit assembly in a Cogent HPLC Column is a possible cause of blockage and resulting high pressures and other possible issues. For non UHPLC, the frit pore size is a distribution of sizes with an average of only 2um wide and therefore can be easily blocked by small un-dissolved sample. matrix or other particulate matter that was not […]

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Purpose of different bonded phases using Cogent TYPE-C Silica: Retention of polar and nonpolar analytes using the different HPLC columns.

The degree of retention in reversed-phase and aqueous normal phase on silica hydride columns depends on the degree and type of modification.   For no or minimal modification the aqueous normal phase mode is stronger than reversed-phase.   As the degree of modification becomes greater, i.e. larger groups and/or higher surface coverage, the reversed-phase properties increase.

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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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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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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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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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