Analysis of Small Organic Acids by Capillary Electrophoresis - - Chromatography Online
Analysis of Small Organic Acids by Capillary Electrophoresis

LCGC Europe
Volume 25, Issue 12, pp. 682-687

Small organic acids such as acetic, citric and lactic are ideal candidates for analysis by capillary electrophoresis (CE) because they are small and highly charged. Methods have been developed and validated for a range of applications and are in routine use in a number of industries. This article covers the applications, separation conditions and reasons for using CE.

What Are These Small Organic Acids?

They are simple low-molecular-weight non-aromatic carboxylic acids. Examples include citric, maleic, succinic, lactic, acetic and tartaric acid. These species are ideal candidates for analysis by capillary electrophoresis (CE) because they are small and highly charged and a number of applications have been developed which are in routine use in many industries. A comprehensive review of the subject area is available in reference (1).

Why Use CE?

The methods are relatively fast with typical analysis times of 5 min. Kits are available from a number of suppliers and contain pre-prepared reagents and standards which provide reproducible separations. The separations are performed on standard CE equipment and capillaries which may prevent the need to purchase specific equipment and consumables such as ion-exchange chromatography systems and columns. The capillaries can be rinsed between samples which allows direct injection of liquid samples and can reduce the need for sample clean-up prior to analysis.

How Are They Detected?

There are a number of detection approaches used (1).

Indirect UV Detection: This is the most frequently used detection mode. An additive is included in the electrolyte which migrates at the same speed as the acids and provides the background signal for indirect detection. It is important that the additive moves at a similar rate to obtain good peak symmetry and improved detection limits. Examples that are frequently used are phthalate (2) and PDC (2,6-pyridinedicarboxylic) (3). Additive concentrations are typically in the order of 5–10 mM to give optimum sensitivity. Zwitterionic buffers such as MES (morpholinoethanesulphonic acid) are used (2) in indirect ultraviolet (UV) detection methods because they provide good buffering capacity with low operating currents. High currents lead to high temperatures within the capillary which generates refractive index changes and poor baseline noise.

Direct UV Detection: The organic acids have a limited amount of conjugation which allows them to be directly detected at low wavelengths. This is achieved using inorganic buffers such as phosphate or borate that have no residual UV absorbance. Enhanced detection is possible (4) using wavelengths as low as 185 nm. In some cases, diode array detection (DAD) is used (5) to enhance sensitivity and eliminate interference from co-migrating species, for example 200 nm is used as the primary wavelength with 260 nm as the reference wavelength.

Pre-separation derivatization of the organic acids is also possible to enhance sensitivity. For example, acids were derivatized with 2-nitrophenylhydrazine and determined with UV detection at 230 nm (6).

Mass-Spectrometry: Capillary electrophoresis-mass spectrometry (CE–MS) has been a routine combination because robust and reliable interfaces are available. Negative ion mode detection was used to determine a range of organic acids and amino acids in metabolic studies (7). This was applied specifically to pineapple leaves to study acid metabolism. CE–MS has also been used for analysis of organic acids in biological samples (8).

Conductivity Detection: Contactless conductivity detectors are commercially available and these have been applied to the analysis of organic acids in biological samples (9). This is more of a niche detection mode.

What Separation Conditions Are Used?

Often cationic surfactants are added to the electrolyte to reverse the electroendosmotic flow (EOF) by forming a double layer and a positive charge on the capillary surface. The EOF moves in the same direction as the negatively charged acids and gives good peak shape. Typically, the cationic surfactant used (2) is tetradecyltrimethylammonium bromide (TTAB) with concentrations in the region of 0.5 mM.

Selectivity can be optimized through pH adjustment of the electrolyte because 0the acids have multiple pKa values. For example, an optimal separation of tartaric, citric, succinic and acetic acids was obtained using a pH of 5.2 (2).

Selectivity additives can also be used. For example, 0.24 mM CaCl2 was used to optimise selectivity (10). The organic acids selectively chelate/interact with the organic acids which alters the separation and can be used to enhance resolution.


blog comments powered by Disqus
LCGC E-mail Newsletters
Global E-newsletters subscribe here:



Column Watch: Ron Majors, established authority on new column technologies, keeps readers up-to-date with new sample preparation trends in all branches of chromatography and reviews developments. LATEST: When Bad Things Happen to Good Food: Applications of HPLC to Detect Food Adulteration

Perspectives in Modern HPLC: Michael W. Dong is a senior scientist in Small Molecule Drug Discovery at Genentech in South San Francisco, California. He is responsible for new technologies, automation, and supporting late-stage research projects in small molecule analytical chemistry and QC of small molecule pharmaceutical sciences. LATEST: HPLC for Characterization and Quality Control of Therapeutic Monoclonal Antibodies

MS — The Practical Art: Kate Yu brings her expertise in the field of mass spectrometry and hyphenated techniques to the pages of LCGC. In this column she examines the mass spectrometric side of coupled liquid and gas-phase systems. Troubleshooting-style articles provide readers with invaluable advice for getting the most from their mass spectrometers. LATEST: Radical Mass Spectrometry as a New Frontier for Bioanalysis

LC Troubleshooting: LC Troubleshooting sets about making HPLC methods easier to master. By covering the basics of liquid chromatography separations and instrumentation, John Dolan is able to highlight common problems and provide remedies for them. LATEST: How Much Can I Inject? Part I: Injecting in Mobile Phase

More LCGC Columnists>>

LCGC North America Editorial Advisory Board>>

LCGC Europe Editorial Advisory Board>>

LCGC Editorial Team Contacts>>

Source: LCGC Europe,
Click here