HomeSmall Molecule HPLCHPLC Tips & Tricks – Mobile Phase Preparation

HPLC Tips & Tricks – Mobile Phase Preparation

Dr. Egidijus Machtejevas, Lead Expert, Analytical Science Liaison

Article from Analytix Reporter - Issue 10


The most important part of a chromatographic set up is the column. But even though it provides retention, the final separation also depends strongly on the mobile phase (MP). The various effects offered by the mobile phase influence the retention and differential migration (selectivity) of the solutes through the column. Therefore, during method development and optimization, the separation might have to be tuned by changing mobile phase parameters, such as solvent type, additives (different buffers, ion pair reagents), or operating conditions (gradient time/ steepness, temperature, flow rate).

Often, problems with chromatographic separation are related to an incorrectly/inconsistently prepared mobile phase. Hence, an inclination to use simpler mobile phases can be observed for practical reasons of increased method robustness, easier method transfer, and ease of use (e.g. in clinical diagnostics or in process control).

Solvent/Mobile Phase Additive Purity

Most of you are familiar with the rule: “garbage in, garbage out.” This adage is especially true when selecting the correct purity of mobile phase components. For example, it is an absolute must to use gradient grade solvents & reagents for gradient grade separations to get an accurate, reproducible and clean baseline (free of ghost peaks), and sensitive chromatographic separation (see also HPLC Tips and Tricks in Analytix Reporter 9 "Increase Your HPLC/UHPLC Method Sensitivity“). Using the correct and suitable grade of solvents based on the application (e.g. hypergrade for LC-MS for LC-MS methods, see more at also minimizes the chances of contaminations and extends the longevity of a chromatographic column.

For cases requiring addition of any reagent like buffer, it is to be ensured that the reagent meets the required quality and has not passed its expiry date. The degradants from expired additives lead to ghost peaks in sample chromatograms. Certain additives degrade quicker, depending on their nature (for example 20 mM, pH 7 phosphate buffer). Improper/careless handling (for example left over solvent put back into bottle, bottle left on the lab bench without the cap closed, lost pipette tips floating inside the bottle and so on) of these reagents spoil chemicals quickly.

Mixing Mobile Phases

For isocratic separations with premixed mobile phases, solvent volumetric contractions in commonly used mixtures (water/acetonitrile, methanol or tetrahydrofuran) should be taken into account during their preparation. The only correct way to prepare such mobile phase mixtures is to separately take precisely measured volumes of the components and mix them. For example, to get a 70% organic mobile phase, 300 mL of water and 700 mL of organic solvent should be precisely measured separately and then combined together in a flask. But if only the water is measured precisely and the organic solvent is then added to make up the required final volume, due to the solvent mixture contraction, the resulting solvent strength will be a little higher (or weaker in case organic solvent was added first and water was added later). For premixing of MPs, attention should be taken to the toxic solvent fumes that might be emitted, under a fume hood.

Nowadays, gradients are generally correctly formulated using gradient pumps; however, some minor differences in retention behavior might be observed during comparison of the instruments with low- pressure and high-pressure gradient systems due to their mixing mechanisms.

Personally, in Reversed-Phase chromatography, I prefer HPLC methods with premixed mobile phases such as 5% acetonitrile in water and/or 5% water in acetonitrile. The rationale behind such preference is to increase degassing effectiveness, avoid mixture heating (e.g. methanol in water) or cooling (e.g. acetonitrile in water) upon mixing, and also to improve mixing efficiency by making the two mobile phases more similar in viscosity and surface tension. The limitations of such premixed solvents are that solvent strength of mobile phase B cannot be 100% and an extra step in mobile phase preparation – which is an additional, potential source of error. Typically, it is recommended to use mobile phase solvents directly out of their delivery containers to prevent additional chances of contamination.

The final step in the mobile phase preparation is filtration. There are many different types of filters that could be chosen based on the solvents being filtered. Recommended are membrane-type filters with pore sizes of at least 0.45 µm for HPLC systems, and 0.22 µm for UHPLC systems. Filtering removes particles from the prepared mobile phase and prevents clogging of the system and column.

See more on mobile phase preparation in HPLC Tips & Tricks: Mobile Phase Preparation Part 2 - Buffers.

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