How to Avoid Microbial Growth in HPLC Mobile Phases.

Bacteria can be a problem in the case of Aqueous Solvents. If present, they can create problems for an instrument’s operation. There are a few things you can do to avoid their formation in your mobile phase. One is to add an acid such as 0.1% Formic Acid. Many HPLC methods may use an acidified […]

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How to Calculate the Internal Volume of Tubing.

To calculate the internal volume (V), you need to know the length of tubing (L) and the inner diameter (ID). You then use the formula for volume of a cylinder. Convert L and ID into cm first. (1 inch = 2.54 cm). This gives V in cm3. 1 cm3 = 1 mL. You can then […]

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How To Convert HPLC Columns Between Reversed Phase & Normal Phase.

To start work in normal phase mode, a simple switching procedure is recommended. A simple 30 minute procedure allows switching from one mode to another. Procedure: A – Moving from Reversed 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 […]

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How To Determine Pore Volume of an HPLC Column with HPLC Injections.

The Interior of an HPLC Column is comprised of Spherical Particles. There are two types of empty space in the column, i.e. Volume not occupied by these particles. The first Type is the Interstitial Space between adjacent Particles. This actually comprises a small percentage of the total Void Volume. The majority of the empty space […]

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

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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How To Integrate a Main Peak with a Shoulder.

It is always better to have baseline separation between a Main Peak and any nearby Minor Peaks. However, this is not always possible and you may have to deal with integration of a Peak with a Minor Shoulder or Peak present. It is less accurate to integrate from the base to the point where the […]

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How To Prepare a 0.05% Solution from a Tablet for HPLC Injection.

Preparation of a 0.05% solution for HPLC injections depends on the Pharmaceutical Tablet Strength (dosage) and how much Volume of Diluent you use. In most cases we would recommend using your intended starting HPLC Mobile Phase as the Diluent. The Drug Tablet should first be ground using a mortar and pestle and then dispersed in […]

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How to Purge Metals from HPLC System using EDTA.

In LC-MS, metal ions can be responsible for distorted analyte peak shapes via chelating. If you determine that this is the cause of a peak issue, here is what you can do to eliminate metals from the system and see that they don’t cause further problems. 1. Add 5-10 micro Molar EDTA to both A and B […]

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How to Repair a Blocked Frit in an HPLC Column – Tips & Suggestions

A simple technique. The frit assembly in all HPLC Columns is a possible site of blockage and resulting high pressures and other possible issues. Rationale: 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 […]

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How To Separate Maleic Hydrazide.

Maleic Hydrazide has a pKa of 5.62 and a logP of -1.96. This logP value means it is very Hydrophilic and will not retain well in Reversed Phase HPLC. For this reason, we would suggest you try the Cogent Diamond Hydride™ column in Aqueous Normal Phase (ANP) mode in order to analyze and retain it. The pKa of […]

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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 Separate Phospholipids with HPLC – Tips

Phospholipids present a challenge for Chromatographers for various reasons. Here some points to consider. They have negligible UV Absorption and generally require other detection methods. Subtle structural differences among Phospholipids often lead to poor Selectivity or co-elution. For detection, Refractive Index (RI), Charged Aerosol Detection (CAD), ELSD (Evaporative Light Scattering Detection), or other “universal detectors” […]

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How to Store & Condition Cogent TYPE-C Silica™ HPLC Columns.

This article describes how best to prepare Cogent TYPE-C Silica™ HPLC Columns for Initial Use when you first receive them as well as How To Store them, for either Overnight or Long-Term Storage, to prevent Column degradation. Storage Solvents for Reverse Phase (RP) or Aqueous Normal Phase (ANP): If RP and ANP are your desired next use […]

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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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HPLC Method Accuracy: Determination

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 […]

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HPLC Method Linearity

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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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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I am using a Cogent TYPE-C™column with LC-MS and solvents containing 10 mM ammonium acetate. I noticed that my source was getting white film on it. At the same time the background noise was very low. Is my column bleeding the packing material?

Cogent TYPE-C™ columns including the Cogent Diamond Hydride have a very low background noise even when used with LC-MS and the white film most likely comes from ammonium acetate build up from your mobile phase. These columns have been proven to be extremely durable and because of the direct silicon-carbon bonds of these phases, bonded phase bleed is almost […]

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I heard that there is Not a Good Alternative to HILIC to Achieve Sufficient Retention for some compounds. Is this true? – FAQ

This is an untrue statement that can lead chromatographers to frustration as THERE IS an alternative mode of chromatography that surpasses HILIC in reproducibility, precision and the ability to separate both hydrophobic and hydrophilic compounds. It is well documented that Aqueous Normal Phase HPLC (ANP) is a very viable alternative to HILIC for polar compounds and in many cases superior […]

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I use normal phase HPLC, which syringe filter do you recommend?

PTFE is the best for use with non polar, organic solvents required for normal phase chromatography. You should also consider using Aqueous Normal Phase chromatography if possible. See link below. AQ Syringe Filter Ordering Information Attachments MicroSolvFiltersEquivalency.pdf   26.9 Kb   Download File

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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 Syringe Filter Ordering Information Attachments MicroSolvFiltersEquivalency.pdf   26.9 Kb   Download File

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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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Improve or Obtain Flat Baselines in HPLC Methods with a Gradient – Tips

Due to differences in UV absorbance between two solvents at a particular monitored wavelength, you will generally observe a sloped baseline when performing Gradient Methods with HPLC-UV. As the Solvent composition changes, the UV “background” changes and results in a sloped baseline. This affect is generally less pronounced at higher wavelengths due to the less […]

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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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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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