Encountering preventable technical issues when running high-performance liquid chromatography analysis can wreak havoc on your entire analysis. Want to learn how to avoid some common pitfalls? These seven tips will teach you how to make your run flow smoother.
The Pressure of Your System
The proper pressure in your HPLC system is one of the most critical parameters for a successful run. The continuous flow of the mobile phase through the instrument drives the movement of compounds from the injection site to the detector. A suboptimal amount of pressure compromises how well your compounds move through the instrument and separate on the column.
Low-pressure issues may yield chromatograms with zero peaks meaning your sample did not even reach the column and stuck somewhere in the instrument. A low-pressure problem could mean your pump is breaking down or needs maintenance, or you may have a leak in your tubing or fittings from wear and tear. Volatile organic solvents and higher temperature runs will inevitably breakdown your lines and fittings over time, so monitor the function of these parts regularly and replace them with quality parts, like the ones supplied by PEEK Fits.
On the other hand, if the pressure of the system is too high, the cause may arise from a clog in your tubing, fittings, or column. You should individually diagnose each of these potential failures, starting with changing the column. If the pressure in your system is still too high with the new column, then check for clogs in your tubes and the appropriate connection points. Pressures that exceed the manufacturer’s recommended value may damage your column permanently, sending you on an unwanted column shopping spree.
The Cleanliness of Your Column
Often HPLC instruments in academic and industrial facilities are used multiple times a day with various operators, running a variety of chemical solutions. If there is no established operating produce in your facility on how to clean and flush the column after use, then you should clean the column before use. The last thing you want to happen when you run an HPLC is to have a dirty column contaminated with compound(s) from a prior run that elute with your sample.
An excellent way to wash the column and prevent contamination of your sample is by running a solvent flush on your system for a minimum of ten column volumes before injecting your sample. You should wash the column with the exact eluent mixture you plan to use in your run. If you happen to need a gradient solvent system, then you want to make sure and run the gradient as part of your wash. If your chromatogram is free of any peaks after the flush, then you are free to inject your sample into the instrument.
In the unfortunate event, cleaning the column elutes compounds, then you need to take a few steps back and flush the column with the exact solvent mixture from the previous run. Keep cleaning your system until the instruments no longer detect compounds eluting off the column, then repeat the ten-column volume flush with your solvent system. Proper maintenance and regular cleaning of the HPLC instrument are imperative to a long-lasting and functional column.
Solvent Mixture in the Preceding Run
In HPLC, the polarity of the column and solvents mixture dictate the separation of compounds. A modification in the chemical composition of either factor affects separation and retention time.
Let’s say, for example, the eluent system used in the prior run was a mixture of hexanes-to-acetonitrile, while the solvent mixture for your procedure is a mixture of acetonitrile-to-methanol. This solvent difference between two unique runs could pose a problem during the run since hexanes and methanol have opposite polarities. The presence of any amount of methanol remaining in the instrument can skew your retention times and may prevent a clean separation.
Since compounds are identified and reported in HPLC by the retention time, you don’t want to collect inaccurate data as a result of poor instrument practice. Be sure to find out the solvents used in the last run and if the device flushed with this solvent system or a different mixture. Just like we recommend above, be sure to wash your instrument and run your solvent system through the column with the ten-column volume.
Leaks in Your Lines
Have you ever heard the term leaky lines sink loose ships? Neither have we, but taking the time to inspect all the attachment points for tubing and fittings helps identify leaks.
Leaks in your lines happen in many ways, like the breakdown of your fittings and tubing over time from exposure to volatile organic solvents, high temperature, or damage from over tightening the fit, to name a few. Loose fittings and leaky lines and can stir up a whole number of problems like a noisy baseline, broadening or splitting of peaks, and a shift in retention times.
When your lines leak, salts, and solid compounds, especially ones soluble in volatile organics like hexanes, build up around the leaky site. Perform a visual inspection at all the attachment points and look for the presence of solid or crystalline substances. If you see any solid material outside your lines, then you need to identify and fix the leaky site immediately. You do not have time to waste on this easily preventable mishap, so save yourself from an HPLC nightmare.
The Freshness of Your Solvents
The separation of your sample on the column relies majorly on the chemical composition of your solvent system. Make sure you run HPLC grade solvents in your experiments and prepare your solvent mixtures fresh.
Over time, solutions like alcohol will pick up water, buffers dissolve oxygen and carbon dioxide, and aromatic solvents oxidize.
All of these chemical changes can give rise to a change in the pH of your buffers solutions, or increase the polarity of your solvents, in the case of picking up water or oxidation.
While these reactions may seem subtle, these chemical modifications will throw off the separation of molecules, reproducibility of your retention times, and the sharpness of your peaks. A good rule of thumb is to replace your aqueous and buffer solutions after forty-eight hours and your organic solvents every seven days.
Presence of Particulates in Your Samples
This topic might seem like a no brainer, but we like to emphasize the importance of solubility in HPLC analyses, especially if your runs require a gradient method for separation. Make sure to test how soluble your sample is in each solvent mixture and filter your samples through a 0.2 um syringe filter before injection!
Injecting samples with solid particles or the precipitation of compounds from the sample while in the instrument resulting from a change in the solvent mix is the last thing you want. Insoluble particles can easily ruin your experiment by clogging tubes, connection points, and even worse your column.
The Purity of Your Standard Solutions
As a component of good lab practice, all HPLC protocols should incorporate the use of a standard solution to improve the precision of the quantitative analysis.
In most cases, standard chemicals are purchased from a supplier and not manufactured and purified by you. Depending on the process controls put in place at your facility, incoming material specifications may or may not require certificates of analysis with HPLC or NMR traces or some form of analysis that shows proof of purity. Either way, you should carry out a run on just your standard sample to validate the purity before you inject your sample into the instrument. You will thank yourself for the time saved in the unfortunate event your standard sample is not pure.
Be Good to Your HPLC Instrument
We cannot emphasize enough that good lab and manufacturing practice always pays off. Regularly cleaning and checking the components of your HPLC instrument can pay off by saving you on time, material costs, instrument maintenance, solvent use, and your sanity.