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Seven common mistakes in the use of HPLC

Time:2018/09/21   Pageviews:0    Share:
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Since there are no alternatives to use, there are still seven traditional practices still in the technical application of HPLC, which are actually not conducive to the efficiency of HPLC technology, the reduction of operating costs and the reduction of running time. In this article, some alternatives to feasible traditional practices will be proposed, and some may be controversial about these alternatives, but if applied properly, these alternatives are bound to have a significant effect. Because analysts should work smarter than mechanical work.

One of the common mistakes - the column is filled with particles of 5 μm particle size

Recall that standard HPLC analytical columns (250mm × 4.6mm) have been packed with 5μm particle size for many years. However, in fact, the 3μm particle size packed column (150mm × 4.6mm) has better separation. Effect and shorter analysis time. At the same time, many of the lab's standard chromatographs are UHPLC and LC-MS, so the column should be replaced with a 3μm or sub-2μm packed column.

It is worth noting that the 3μm particle size packed column has a smaller inlet porosity and is more susceptible to clogging by “dirty” samples.

Common mistake 2 - use 4.6mm inner diameter column (1mL / min)

Since the early 1970s, the "standard" internal diameter of HPLC analytical columns has been 4.6mm. In recent years, many laboratories have begun to use 3.0mm id columns in an effort to reduce solvent consumption and save samples. As a better alternative, instead of using a 4.6mm id column. If a modern intermediate dispersion HPLC instrument is used, the effects of column rate and extra-column band broadening are generally not adversely affected. In summary, it is advantageous to use a column with a smaller inner diameter to achieve higher resolution under gradient elution conditions.

Common mistakes three - filtering the mobile phase of HPLC

The HPLC grade solvent and water normally obtained from the purification system are already clean enough. If we give it an extra filtration, it will only be counterproductive and introduce unnecessary chemical contamination. In fact, many laboratories have their own set of chromatographic instrument maintenance programs, and the filters in the HPLC system are replaced every year, so there is no need to further filter the mobile phase.

The exception is: If you are using ion-pairing reagents, low-purity buffers or high-salt mobile phases, it is highly recommended that you filter the mobile phase.

There is also a case where additional filtration is necessary, that is, if the water flowing out of the purification system is not clean enough due to poor quality of the water source or other reasons.

Common mistakes four - using buffer mobile phase

When analyzing acidic or basic analytes, it is necessary to acidify or alkalinize the mobile phase. A simple mobile phase such as an aqueous solution containing 0.1% by volume of formic acid can be prepared quickly by simply diluting 1 mL of formic acid into 1 liter of water. 1.5ml snap HPLC vials Similarly, an aqueous solution containing 0.1% by volume of ammonia can be used in a variety of high pH compatible columns. When using a low silicon hydroxyl active column, buffers are rarely used, whether running with low or high pH mobile phases, including when using mobile phase A as a diluent to prepare a sample solution.

The exception is: when analyzing complex molecules, complex mixtures, it may still be necessary to use a buffer mobile phase to maintain a high selectivity and a particularly moderate pH mobile phase.
Common Mistakes 5 - Reconstitute a new reference standard solution for each test

In the quality control laboratory of a drug, each test is often reconstituted with a new reference standard solution, which is actually unnecessary. Many drugs have sufficient stability at low temperatures and under appropriate storage conditions. So you can prepare hundreds of qualified reference standard solutions in one time in an HPLC vial and then refrigerate or freeze store for later analysis. For the long-term stability study of the test results, the standard solution prepared from the same original solution should be used. Of course, for the usual tests, the stability (shelf life) of the standard solution under storage conditions should be verified and the first preparation time recorded. What deserves our attention here is that all operations need to be recorded.

Common mistakes six - shaking the vial 1.5ml snap HPLC vials

Shaking (or reversing) the vial creates a liquid film underneath the vial cap, which can interfere with the HPLC autosampler to give a confusing test result. So always be wary of this even if the fault is eliminated.

The exception is: if the sample is frozen, after thawing, it needs to be vortexed or shaken to ensure the sample is even.

Common mistakes seven - the use of stainless steel ferrules as a joint of the column 

HPLC systems are usually fitted with stainless steel fittings and ferrules, and in most cases they work well, but they are not suitable for connections that require frequent column changes.

First, stainless steel ferrules can only be matched to their tailored end fittings, which can only be counterproductive for different columns.

Second, prefabricated stainless steel joints can only be resealed 5-10 times. This problem becomes especially noticeable when some manufacturers' stainless steel fittings are used on other brands of columns because of the different end joints and depth of insertion.1.5ml snap HPLC vials

One possible solution is to use finger-tightened polyetheretherketone (PEEK) fittings, which are inexpensive and have a good seal at 5000 psi (34.47 MPa). Many newer UHPLC fittings are rated at 20,000 psi (137.9 MPa) and can be tightened with a finger and then turned a quarter turn with a wrench.

This is the end of the introduction of Seven common mistakes in the use of HPLC. I hope it can help you.


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