What HPLC Operators Need to Know, Part I: Sample Preparation and Column Selection

August 1, 2016
Dawn Wallace Watson
LCGC North America

Volume 34, Issue 8

Page Number: 574

How to optimize the key variables in HPLC analysis-sample preparation and column selection

Sample Preparation

Sample preparation is required for a number of reasons, including rendering the sample in the correct format for the analysis type (for example, when the sample is too dilute, too dirty, or has an incompatible sample matrix), reducing spurious peaks by removing interferences or matrix components, improving analyte peak shape, generating robust analytical methods, and increasing column lifetime and instrument up-time.

The type of sample preparation selected depends on the sample type, requirements of the analytical method, time available to run the sample preparation method, time investment available for method development, and ultimately cost per sample. Sample preparation can be performed off- or on-line, with many techniques being amenable to automation, which increases sample throughput. Modern approaches use miniaturization to reduce solvent use and make the extraction process “greener.”
Even the simplest of sample preparation techniques will require some optimization. For example, when using syringe filters the type of membrane, filter diameter, and porosity all need to be considered. One must be aware of solvent compatibility of both the membrane and housing materials and the possibility for analyte–membrane binding.

The ideal filter diameter balances filtration performance with the risk of extractables and analyte binding, and depends on the sample volume (that is, with <1 mL of sample use a 4-mm diameter filter, with 1–10 mL of sample use a 13-mm diameter filter, and with 10–150 mL of sample use a 25- or 33-mm diameter filter).

The porosity of the syringe filter should be considered in conjunction with the porosity of the column inlet frit or packing material diameter so that any particulates that would be large enough to block the column inlet frit or column itself are removed before they reach the high performance liquid chromatography (HPLC) system. For columns packed with sub-2-µm particles, a 0.2-µm ultrahigh-pressure liquid chromatography (UHPLC) filter should be used; 0.2- or 0.45-µm porosity filters are suitable for columns packed with particles >2 µm.

Liquid–liquid extraction (LLE) is still commonplace in many laboratories. Selecting the correct solvent to perform LLE is key to the success of this technique. Normally a water–water immiscible organic solvent mixture is used because there is a polarity difference between the phases, which allows for analyte extraction and phase separation. Addition of an inorganic salt (salting out) allows LLE to be extended to the extraction of analytes using water–water miscible solvent systems. Analytes must have preferential solubility in one solvent over the other to facilitate optimum recovery. The solubility and phase preference of ionizable analytes can be altered by controlling the extent of ionization via adjustment of the pH of the aqueous portion.


Solid-phase extraction (SPE) is one of the most complex, but specific sample preparation methods. With applicability in numerous research areas it provides ultraclean samples, can be used to concentrate target analytes, and is amenable to automation. SPE optimization is a multistep process that includes selection of the correct sorbent chemistry and development of condition, load, wash, and elution steps.

Column Selection

There are several approaches to selecting an HPLC column for a new method. Consulting the literature may provide a method for your compounds. If no literature method exists, using a column already in use in the laboratory may be a viable approach based on the fact that the molecules being analyzed are possibly similar to other molecules already being studied in the laboratory.

Using an existing column or method may require some rudimentary changes to the analytical conditions, such as adjusting the mobile-phase composition (organic solvent type, percentage of organic solvent, and pH) or gradient. Developing methods from scratch requires knowledge of the physicochemical properties of the molecule and how analytes interact with the stationary phase. However, there may be an alternative to the traditional method of selecting a column and iteratively altering the eluent conditions.

The ultimate goal of any chromatographic analysis is to gain optimum resolution in the minimum amount of time. Resolution is related to the chromatographic parameters of retention, efficiency, and selectivity by the Purnell equation; with selectivity having the largest impact on resolution.

In turn, column chemistry has the greatest impact on selectivity; therefore, there has been an increased interest in screening different column chemistries (which can be automated) to select the most suitable column for an application. Conceivably, by using only four column phases, a polar embedded C18, high-pH-stable C18, biphenyl, and pentafluorophenyl propyl phase in conjunction with a single scouting gradient (5–98% B), analysis conditions for almost any type of molecule can be achieved (1).

Optimization of any method through changing the organic modifier or pH after determining an initial column-mobile phase choice may of course be required.


  • D.K. Appulage, E.H. Wang, F. Carroll, and K.A. Schug, J. Sep. Sci.39, 1638–1647 (2016).

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