How To… – MicroSolv Technology Corporation

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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 […]

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 Calculate Assay of Lisinopril Dihydrate Based on the Anhydrous Form.

When you weigh your reference standard and use the mass in the assay calculations of your formulation, you must correct for the water since the reference standard is the di-hydrate form. You know the molecular weight of Lisinopril and of the two H2O molecules so you would apply a mass correction factor of M.W. anhydrous […]

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 […]

How to Calibrate UV-vis and HPLC Detectors with NIST Traceable Solutions Below 230nm

Holmium Oxide is an internationally recognized wavelength calibration primary standard, supported by NIST and other standards organizations. It provides 14 official absorbance bands ranging from 241 nm to 641 nm. Caffeine is considered a secondary standard, with two absorbance bands at 205 nm and 273 nm. Caffeine is also widely used as a wavelength and […]

How To Choose a Cogent HPLC Column. Order of Hydrophobicity.

The first thing to consider is the chemical structures of the Analytes in your Sample and the corresponding most suitable Retention mode for them. For Reversed Phase, the general order of Stationary Phase Hydrophobicity (and therefore retentiveness in RP) is as follows (left to right) and for hydrophilicity (and therefore retentiveness in ANP) is shown […]

How To Choose a Suitable Mobile Phase Additive for Negative Ionization Mode LC-MS.

For negative ionization we usually use: Solvent A: 10 mM Ammonium Formate or Acetate in DI Water Solvent B: 90% Acetonitrile/10% DI Water (v/v) /10 mM Ammonium Formate or Acetate Solvent B has to have Water in order to dissolve Ammonium Formate or Acetate. Usually a 200 mM solution of Ammonium Formate or Acetate is […]

How To Choose an Appropriate Guard Column when using a Cogent TYPE-C HPLC Column.

Two sizes of Guard Columns are available: 2.0 and 4.0 mm Internal Diameter (ID). Both sizes are 20mm in length and are used with the same Guard Column Holder. The 2.0mm ID guard columns are for use with the 2.1mm ID Analytical Columns and the 4.0mm ID Guard Columns should be used with the 3.0mm […]

How To Choose an Appropriate re-Equilibration or Post Time for Gradient HPLC.

When using Gradient Methods in HPLC or LCMS, the Mobile Phase and Column must be allowed to Equilibrate back to its initial Conditions before starting the next run in order to ensure consistent Chromatography. This portion of the gradient is called the “Post Time“. It is best to use as low a Post Time as […]

How To Clean Sparging Stones and Inline SS Filters.

Inlet Stainless Steel Filters also known as Sparging Stones, can be cleaned by ultra-sonication in 30% Phosphoric Acid, clean afterwards with DI Water (ultra-sonication) and Methanol (ultra-sonication). Same would be true for all Metal Filters, like for Column Filters, Inlet Filters and more. But be aware, only the metal part! No PEEK Rings, Connectors or […]

How To Condition Cogent RP, HPS & Axis HPLC Columns for Storage.

For Cogent RP™, Cogent HPS™ & Cogent Axis™ HPLC Columns Long Term Storage: Rinse the Column with 8 Column Volumes with pure Solvent that you use as your mobile phase. It is important to use non-buffered Mobile Phase. Then purge the Column with 8 Column Volumes of 100% HPLC Grade, Filtered Isopropyl Alcohol. Then purge […]

How to Condition Controlled & Zero EOF Capillaries.

Capillary Conditioning Procedure: Never Use Strong Bases on these Capillaries Carefully remove the capillary from the shipping/storage container. Remove protective polyimide coating by using 98% fuming sulfuric acid heated to 100ºC. One drop should be sufficient for complete removal. The MicroSolv Window Maker be used as well as long as you do not allow the […]

How To Condition Diamond Hydride Columns for use with Serum Samples.

For Serum and other biological samples, we Recommend using: Solvent A: 50% DI Water/50% Methanol (or 2-propanol) + 0.1% Formic Acid (for positive mode MS) or +10 mM ammonium acetate or formate (for negative mode MS). Solvent B: The same as you would ordinarily use. Cleaning Tip: Methanol or 2-Propanol (IPA) helps to clean the […]

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 […]

How to Cut PEEK Tubing, Use a Clean Cut90 Cutter.

It is Extremely Important to have a Proper Cut for all HPLC Tubing Including PEEK Tubing. The Cutting Blade of the Clean-Cut90™ PEEK Tubing Cutter (shown below) has 2 sides. One side will be marked on the tool with an Arrow, and this produces the Perpendicular Cut (90° angle) that you want to use. Be […]

How To Cut Stainless Steel Tubing for HPLC.

Cutting Stainless Steel Tubing for HPLC can be challenging. Here are some tips and instructions on cutting tubing using the MicroSolv tubing cutter, Catalog number 46001-01. Introduce the Tubing into the Cutter and lightly Tighten the Screw. If you tighten too much you will deform the Tubing walls. Turn the Cutter a few times around […]

How To Determine Concentration of an Impurity in HPLC with the Relative Response Factor – RRF.

The concentration can be calculated once you have values for the following two equations: Response Factor (RF) = Peak Area / Concentration Relative Response Factor (RRF) = RFimpurity / RFAPI You can use RRF and RFAPI to solve for RFimpurity. Then you can use the measured Peak Area of the Impurity to solve for its Concentration.

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 […]

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 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 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 […]

How to Keep HPLC Columns Free of Adsorbed Contaminants – Tips & Suggestions

Depending on the analytes you use the Column with, there are a number of Strategies you can try: If your HPLC System can have more than two Solvents, you could include a Third Line with a Cleaning Solvent. Then in the Injection Sequence you could insert a Wash Step which uses only the Third solvent. […]

How To Measure Signal-to-Noise Ratio in an HPLC Chromatogram.

The best way is to zoom in on the peak of interest using your data analysis software so that the baseline near the peak is also visible as well. Depending on how sharp or narrow your peak is, you’ll want to zoom in so that the time axis covers a range of about 0.5 –1.0 […]

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 Perform an Ohms Law Plot.

Ohm’s Law Plot to Optimize your Separation can bring many benefits and is essential for good CE Methods. Select the optimal Voltage Setting for your Separation. Maximize the Efficiency of your Method. Learn the Upper Voltage Limits of your Method. Use to Validate your Capillaries and Columns in your Method. Performing an Ohm’s Law Plot […]

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 […]

How To Properly Cut a Fused Silica Capillary for CE.

A Recommended Cleaving or Cutting Procedure for Simplus Brand or any Bare Fused Silica Capillaries is as follows: Cleaving Procedure: Place the Capillary over a large Diameter Radius Surface such as large pipe or tube. Keep the Capillary under slight Tension. Hold a fresh, Clean Cleaving Stone at an approximately 30º angle to the Capillary. […]

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 […]

How To Recover an HPLC Column that has had Ammonium Acetate precipitated in it.

If you are using a method with a buffer such as 16 mM ammonium acetate in the mobile phase and then stored the column in pure Acetonitrile immediately afterwards. You may observe peak splitting when using your Ammonium Acetate Method. It is possible that Ammonium Acetate has precipitated in the Column since it is insoluble […]

How To Search an Article for Exact Text Matches – Ctrl-F.

On a PC, you can instantly find any specific text that is found in a document using Ctrl-F (Command-F on a Mac). This will work for webpages, Word files, pdfs, and any other document with text (note: will not work for image files such as jpg, png, etc.). This step by step tutorial will show […]

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 […]

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 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” […]

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 […]

How to Use Bubble Point to Filter Water through Hydrophobic Membranes.

Bubble Point pressure is the differential pressure required for a liquid to flow through a dry filter membrane. The differential pressure is either a positive pressure on the liquid layer on top of the membrane or a vacuum applied below the membrane or a combination of the two. The bubble point pressure is determined by […]

Validation Protocol for the PQ Kit Template- Tips & Suggestions

The Validation Protocol in the attachment is intended to Document and Validate the MicroSolv HPLC PQ Kit Excel Template used for HPLC Qualifications. The Excel file given for the latest version of the Performance Qualification is for 97-2003 Microsoft Excel Worksheet, but this is also compatible with Excel 2010. For operating in Excel 2010, you […]

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