Analytical Methods Committee, AMCTB No. 111
First published on 2nd August 2022
The increasing trend for non-expert users to undertake analytical measurements using an expanding range of chromatographic approaches can lead to the use of unsuitable separation methods and the generation of poor-quality data. Technical Brief AMCTB No. 107 introduced liquid chromatography and guidance on selection of the appropriate separation modality. This publication aims to provide a quick and easy to use educational tool for optimizing the mode of liquid chromatographic separation. It will be of value to both expert and non-expert users across the natural, life and physical sciences.
Fig. 1 A periodic table of liquid (fluid) phase chromatography detailing common separation modes in terms of partitioning (HILIC), adsorption (NPLC, SFC, RPLC, IPC and IEX) and size exclusion (gel permeation chromatography (GPC) and gel filtration chromatography (GFC)). This includes information regarding the suitability of the modality based on the physicochemical characteristics of the compound (i.e., logD) and potential detectors (see Table 1). |
In addition, to better meet the breadth of the separation range required for many complex matrices, stationary phase chemistries were developed that spanned non-polar hydrophobic interactions (e.g., reversed phase LC, RPLC), polar hydrophilic interactions (e.g., normal phase LC, NPLC) and ionic interactions (e.g., ion-exchange chromatography, IEX). However, these were often limited in their ability to separate on the basis of more subtle differences in analyte chemistries (resulting from the range of logD and pKa values encountered). This has led to the introduction of multi-modal stationary phases (e.g., ion-pair chromatography (IPC) or mixed-mode (MM) IEX), alternative mobile phases (e.g., supercritical fluid chromatography (SFC)) and additional mechanisms of separation (e.g., partition through the use of hydrophilic interaction chromatography (HILIC)) within the chromatographic platforms.
The plethora of techniques has, therefore, often become a source of confusion in selecting the most appropriate modality for an application, particularly when new users of chromatographic technology are attempting method development and where more than one separation option may apply. AMCTB No. 1078) provides a decision tree to help select an appropriate modality. Building on these principles, we have prepared a periodic table for liquid chromatography separation modes (Fig. 1) that provides a guide as to the general applicability of LC modes and stationary phases in a recognisable scientific format, helping users to refine the method options for the range of analyte chemistries detailed above. To help explain the detector terms, a glossary has been included (Table 1). However, users should note that the application of the optional separation mode will be dependent on available laboratory instrumentation and should be considered as part of method selection.
Abbreviation | Term | Description |
---|---|---|
a These descriptions have been abbreviated or adapted from the text in the cited reference. | ||
AAS | Atomic Absorption Spectroscopy | Measures the amount of a chemical element based on the measurement of the absorption of characteristic electromagnetic radiation by atoms in the vapour phasea.9 |
AES | Atomic Emission Spectroscopy | Measures the amount of a chemical element based on the measurement of the intensity of characteristic electromagnetic radiation emitted by atoms or moleculesa.9 |
ICP-MS | Inductively Coupled Plasma-Mass Spectrometry | Mass spectrometry technique based on coupling a mass spectrometer with an inductively coupled plasma as an ion source that both atomizes samples into their constituent atoms and ionizes them to form atomic cationsa.10 |
UV | Ultraviolet Spectroscopy | Molecular absorption spectroscopy in the ultraviolet (UV) and visible (VIS) is concerned with the measured absorption of radiation in its passage through a gas, a liquid or a solida.11 |
DAD | Diode Array Detector | A UV spectrophotometry detector that uses an arrangement of a number of photodiodes on a single chipa.12 |
FLD | Fluorescence Detector | Measures the fluorescence of a molecule/sample.13 |
ELSD | Evaporative Light Scattering Detector | Measures the light scattered by non-volatile molecules/particles following desolvation.14 |
FID | Flame Ionization Detection | Technique that uses a hydrogen flame to ionize gaseous molecules to measure the resulting change in electrical current.15 |
APCI-MS | Atmospheric Pressure Chemical Ionization-Mass Spectrometry | Chemical ionization of a sample that is a gas or nebulized liquid, using an atmospheric pressure corona discharge or beta emitter such as 63Nia.10 |
RI | Refractive Index | Measures the change in the direction of light when passing through media that have a different refractive index based on alternative (bio)chemical compositions.14 |
ESI-MS | Electrospray Ionization-Mass Spectrometry | Spray ionization process in which either cations or anions in solution are transferred to the gas phase via formation and desolvation at atmospheric pressure of a stream of highly charged droplets that result from applying a potential difference between the tip of the electrospray needle containing the solution and a counter electrodea.10 |
— | Conductivity Detection | Measures the electrolyte concentration of a solution via its conductivity.14 |
ECD | Electrochemical Detection | Methods in which either current or potential is measured during an electrochemical reaction. The gas or liquid containing the trace impurity to be analysed is sent through an electrochemical cell containing a liquid or solid electrolyte and in which an electrochemical reaction specific to the impurity takes placea.15 |
Fig. 2 Chemical structure of nifedipine with relevant physicochemical properties for the separation. |
From the decision tree in AMCTB No. 107,8 the analyte is in a liquid sample and could require LC for separating nifedipine from the remaining matrix components. Using the logD for nifedipine it is clear from the periodic table (Fig. 1), that RP-LC or IPC could be used to successfully retain the drug via the use of a non-polar stationary phase (providing the sample is relatively polar). Furthermore, by using the chemical structure information (e.g., weakly acidic, negatively charged, functional groups), IEX-LC may also be used to retain the drug providing a low ionic strength (IS) eluent is employed to ensure retention where the analyte is expected to elute when an appropriate shift in pH exceeds the drug pKa of 3.93 (see AMCTB No. 1078) for further explanation of pKa). Given nifedipine is a small molecule, a possible eight detection options may be explored, each with their own degree of selectivity and sensitivity, and should be chosen according to the needs of the analysis.
From the decision tree in AMCTB No. 107,8 the analyte is in a liquid sample and could require LC for separating the drug from the remaining matrix components. Given captopril is strongly polar as indicated by the logD, the use of RP would likely result in weak retention (see Fig. 1). Hence, NP-LC, or even a weak IEX sorbent given the ionizable functional groups, may be preferable to retain captopril, however, the latter would require a high to moderate IS eluent via an appropriate counter ion or shift in pH to exceed the drug pKa (see AMCTB No. 107 8 for further explanation of pKa) to ensure the release of captopril from the retention mechanism. Similarly, IPC could be employed using a relevant ion-pair that is first loaded onto the RP stationary phase prior to the sample matrix that contains captopril. As with nifedipine, a possible eight detection options for this small molecule may be explored, each with their own degree of selectivity and sensitivity, and should be chosen according to the needs of the analysis.
Ruth Godfrey 0000-0002-8830-3625 (Swansea University), and Scott Fletcher and Robert Boughtflower (RSC Separation Sciences Group (SSG)).
This Technical Brief was prepared for the Analytical Methods Committee (AMC), with contributions from members of the AMC Instrumental Analysis Expert Working Group and the RSC Separation Sciences Group (SSG) and approved by the AMC on 28 May 2022.
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