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1 Aqueous Normal phase (ANP) defined & compared to Reversed Phase & Normal Phase in HPLC

Aqueous Normal Phase (ANP) is probably the newest HPLC technique or at least the latest to be investigated well. Often we are asked what is ANP. Below is a link with more explanation than this article…

2 Assuring retention of polar compounds in an ANP gradient in positive mode LCMS.

When using a Cogent Diamond Hydride™ column prepare your solvents for positive ionization in LC-MS: Solvent A: 50% methanol/50% DI water + 0.1% formic acid and Solvent B: 90% acetonitrile/10% DI water/0.1%…

3 Determining dwell volume of gradient HPLC systems

With some instruments the gradient dwell volume can be quite significant, even a few milliliters. At the start of the gradient, the volume in the instrument is filled with the mobile phase corresponding…

4 Formulas for chromatography

Efficiency (Theoretical Plates): N = 5.54 ( tR / w1/2 ) 2 5.54 is a constant when using the "Half Height" method. tR is the retention time of the peak and w1/2 is the width at half height. Capacity Factor…

5 How can my finger tight fitting be producing band broadening?

Most chromatographers may not realize it but if fittings are not properly set and a perfect connection is accomplished, a dead volume can occur that does not get swept. This extra volume can cause band…

6 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…

7 I am analyzing metformin in plasma samples using acetonitrile + DI water with 0.5% formic acid. How I can obtain reproducible results and peak shape?

You can try substitute formic acid in DI water by adding ammonium acetate (10 mM) and acetic acid (1%) to the aqueous phase. This should improved the reproducibility of peak areas and retention times.…

8 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…

9 I was analyzing maleic and fumaric acids in biological extract using a Cogent Diamond Hydride column. After 10 perfect injections (%RSD of retention times around 0.5), I noticed that the peaks intensity diminished about 10 times, however the peaks still have the same retention time. What happened to the column?

Most likely, nothing happened to the column. Your mass spectrometer probably needs some attention. I would clean the ion source and check the nebulizer. © Copyright MicroSolv Technology Corporation,…

10 Injection volume suggestions for Cogent TYPE-C HPLC columns

When using an analytical column it is recommended to inject 1 uL to 10 uL of the biological sample which is prepared either in DI water or 50% solvent A/50% solvent B of the mobile phase. When a 100 uL…

11 Restoring the initial results with a Diamond Hydride™ method used for analyzing AMP, ADP, ATP, UDP and GPT in biological extracts?

"My results deteriorated after two days of using the Diamond Hydride column for AMP, ADP, ATP, UDP and GPT in biological extracts". Phosphate containing metabolites that you are using are very sensitive…

12 Sample Diluents should match your mobile phase.

For best results, it is always best to try to dissolve your target in as close as possible to your isocratic eluent or the start of your chosen gradient. If this is not possible gradually increase the…

13 Use a linear flow rate test to check pressure on an HPLC column.

If you are suspecting high pressure on an HPLC column, a semi-prep column for example, you can check whether it is operating properly by testing it against an analytical column of the same stationary…

14 What are the maximum changes I can make to a method to increase the chances of meeting system suitability in HPLC?

This is a question that is always asked and the following are only guidelines. For exact details, consult the FDA or the USP. Change of Detector Wavelength Not Allowed (you can vary up to 3nm in accuracy…

15 What is the effect of extra column volume for different HPLC instruments on the same method?

The difference can be very significant. Extra column volume that is “hidden” in your HPLC system, the fittings, the tubing and the flow cell can greatly impact the efficiency of your method. Below…

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