With that said, one of the important and often overlooked tips you can do to increase the performance of your system is to understand how to use and prepare your buffers and solvents. Even the simplest element such as buffer preparation, if not done well, could cause inconsistent results and loss of precious sample. Accurate preparation and correct selection of buffers is essential to obtain reproducible results in chromatography. Here we present some fundamental tips and tricks of buffer preparation to help you get the best separation of your compounds.
Understand your compound
This might seem obvious, but there are times when we just want results and rush into an experiment without taking the time to do some background research. If you are looking for a particular compound within a mixture, you should use a buffer/solvent combination that best separates your analyte from others. For example, understanding the polarity and solubility (polar or non-polar), ionization, the UV absorbance of what you are looking for will help guide you to a particular column and solvent set. There are several resources that help guide you based on the properties of your analyte. ChromaBLOGraphy and CHROMacademy are two that help explain buffer choice for HPLC separations. ZirChrom offers a tool designed to help you with the calculations needed to prepare a pH buffer for use in liquid chromatography after a simple registration.
It’s tempting to use lower grade and less costly reagents to make your buffers to save some money but in the long run, it ends up being more expensive. Compared to HPLC grade reagents, which contain minimal to no impurities, less pure reagents can cause unwanted peaks and noisy baselines. They also wreak havoc on your system causing blockages that can lead to system failures and more expensive maintenance. All reagents and solvents, including the water you use, should be of the highest quality, HPLC grade, to lessen unwanted particulates in your buffers. The high grade reagents may cost slightly more than lower grade reagents, but the difference in purity is worth the price. HPLC grade reagents can also contribute to more consistent results and keep your system running smoothly. HPLC grade reagents are available from many sources, so test what works the best for your analysis and stick with that reagent.
Filter the bad stuff out
Although the buffer may be chemically pure by using HPLC grade reagents, dust, particulate matter from a bottle-cap, or precipitation of salts may be present by the time the buffer is made. To remove these unwanted particulates, filter the buffer after it has been prepared. For best results, use a 0.45 µm filter both buffers and solvents prior to use. After filtration, the solvents should be stored in a covered reservoir to prevent contamination with dust, or other unwanted materials.
Get rid of the bubbles
Degassing and/or vacuum filtering buffers prior to use with your system minimizes air and particulates in your mobile phase. Recent improvements to many instrumentation offerings now offer in-line degassers to address some issues but not all issues related to dissolved air in the mobile-phase. If mobile-phase outgassing occurs within your liquid chromatography system, it will mostly affect the pumps and detector. To help with this, degassing before the freshly prepared mobile phase is pumped around the HPLC system, along with the in-line degasser, should be thoroughly degassed to remove all dissolved gasses. The most efficient form of degassing is bubbling with helium or another low solubility gas. If this method is available, it is recommend that the mobile phase be continually degassed at very low levels throughout the analysis. This will inhibit the re-adsorption of gases over the analysis time.
Although it seems unlikely that bacteria could grow in your solvent buffer, they can. Bacteria can adapt and grow in almost any solution, even organic solvents depending on the concentrations. To prevent bacterial growth from blocking your column frits, replace buffer reservoirs each time a new buffer batch is prepared and visually inspect buffer bottles/bags for signs of bacterial growth. Solutions that appear cloudy upon shaking or stirring should be discarded. Treatment with a bacteriostatic agent such as 0.02% sodium azide will prolong solution storage although these agents might affect your chromatogram.
Fresh is often best
For best results, make your solutions fresh, as needed. Set an expiration date for discarding unused buffer. Although you may be able to get longer stability, many laboratories specify one-week expiration dating on dilute buffers. This practice ensures that the buffer pH is unaffected by prolonged storage and that there is no microbial growth present. Both pH change and microbial growth will affect your chromatography run and cause inconsistencies between runs. Although you can add stabilizing agents such as sodium metabisuphite, these agents can affect the optical and chromatographic results.
Summing it up
Chromatography can be a challenging technique to master. Following the tips above on how to prepare and use buffers for your purification, will help to make each run consistent and reproducible. Vendor websites such as Bio-Rad offer technical guidance on the latest systems, reagents and protocols to help make your journey in protein purification a successful one.