Most chemists vaguely remember a portion of their instrumental analysis course that covered electroanalytical chemistry. The section covered topics such as potentiometry, coulometry, amperometry, and polarography. The section usually ended up with a graduate student operating the polarograph because of the toxicity of the mercury. After this class, most chemists may use a pH meter or run a coulometric Karl Fischer water determination, but never really have the chance to explore these areas again. However, these methods can be quite accurate and specific. This article addresses amperometric detectors for chromatography and, more specifically, analysis of sugars and aminoglycosides.
Amperometric detectors have been around for some time but have never become popular. This is mostly due to the lack of knowledge regarding function, as well as design problems with early detectors that have since been rectified. The good news is that the field is maturing and the newer equipment is rugged and easy to use.
Historically, amperometry was known as a technique that could be accurate, precise, and specific. The specificity comes from the fact that only certain compounds will undergo a redox reaction under conditions such as pH and specific voltage. Other compounds either will not respond to the detector under these conditions or will respond so weakly that they do not interfere. For example, when the proper waveform is used, carbohydrates will respond, but most other compounds will not. When amperometry is coupled with chromatographic techniques, the carbohydrates are separated before they reach the detector; thus the specificity is increased significantly.
The problem in the past was that the surface of the working electrode had to be cleaned repeatedly. As compounds were oxidized or reduced, the surface of the working electrode became fouled. Cleaning was normally performed by using a pencil eraser to remove the surface layer. The resulting inconsistent electrode surface created variations from run to run. Additionally, as the surface fouled during long runs, the area response of the analyte decreased, causing low results.
Today, the gold working electrodes that are used with carbohydrates are cleaned by a voltage waveform. A waveform is a series of voltage steps that work to charge the electrode, detect the analyte, clean the electrode, and then restore the surface.1 The entire process takes milliseconds and is ongoing throughout the run. This repeated cleaning and detecting is called pulsing; the process is known as pulsed amperometric detection, or PAD. The pulsing waveform ensures the surface is always clean and the results are reproducible. Because the cleaning takes off the top layer of atoms, the disposable electrode must be replaced about every two weeks. Overall, the current technology has improved dramatically from just a few years ago.
Table 1 - Working electrodes for specific analytes
The product of just about any redox reaction can be determined with the correct electrode and chromatography column. Table 1 gives a much-abbreviated list of what can be done with pulsed amperometric detection.
These examples are from the Dionex (Sunnyvale, CA) product manual for disposable electrodes,2 but there are many more. A boron-doped diamond disposable electrode is also available. There are many applications, and the published literature is expanding rapidly.
The author’s laboratory has more recently focused on simple carbohydrates. Carbohydrates such as sugars are often a problem because they are not detected very well by UV or refractive index. The carbohydrates absorb at low wavelengths by UV and often have interferences that lead to a loss of sensitivity and specificity. The refractive index detector is much less sensitive, requiring larger amounts of sample and relatively large amounts of costly reference standards. Pulsed amperometric detection allows quantitation down to the picomole levels.
Alcohols and amines can be oxidized electrolytically. Carbohydrates get their name from carbon + hydrate. Most carbohydrates have alcohol functional groups that can be oxidized. The pulsed amperometric detector can be selective for this reaction and very sensitive, and therefore is the ideal method for carbohydrate analysis. However, the electrode will see all carbohydrates. This problem is overcome by an initial chromatographic separation to give a rapid, sensitive, and selective determination of carbohydrates.
Table 2 - pKa of common carbohydrates2
Carbohydrates and most alcohols may be ionized at high pH levels. Once the alcoholic functional group on the carbohydrate is ionized, the carbohydrate may be separated from other carbohydrates by ion chromatography. The high pH would destroy most silica-based columns; therefore, a polymeric-based backbone is needed. The CarboPac® series of columns (Dionex) were designed with this kind of separation in mind. An example of the pH levels needed for the ionization of the carbohydrates is given in Table 2.
Notice in the table that there is a significant difference between the pKa’s of the sugars and the sugar alcohols. The pKa’s are used in the separation scheme to give further sensitivity. In the same manner, neutral or cationic analytes are not retained on the column and elute from the column in the solvent front.
Figure 1 - Glucose, fructose, and sucrose at 20 μg/mL.
Sugar analysis by amperometry is a simple and effective way to demonstrate the power of PAD. The example presented in Figure 1 used a CarboPac PA1 column to separate glucose, fructose, and sucrose. The run was isocratic and the eluent was KOH. The samples were prepared from 12 to 28 μg/mL. Five injections of the 20-μg/mL sample were used for system suitability. Figure 1 shows a chromatogram of the 20-μg/mL sample. Table 3 gives the results for the analysis.
Table 3 - Results for simple sugars
The most interesting features of the analysis were simple sample preparation, no eluent to prepare, and an entire process that took less than 4 hr without trying to optimize the chromatography.
The simple process can be applied to many applications. Many biologically active compounds have a sugar molecule attached. These molecules are called glycosides. Glycosides are found in many classes of molecules such as sweeteners, alkaloids, flavonoids, hormones, and antibiotics. The sugar portion of the molecule can be vital for the activity of a drug. The sugar modifies the hydrophilic character of a drug and influences the membrane transport activity of the drug in the cell. A significant amount of research is ongoing in this area.3
If a glycosidic molecule is stable under the high pH of the hydroxide mobile phase, it can be quantitated using the separation techniques described above. If the molecule is not stable under basic conditions, it can still be quantitated by amperometry, but the initial chromatographic separation must be different. However, it is surprising how many molecules are stable enough to withstand attack from the mobile phase over the analysis time, which is usually 15 min or less.
Figure 2 - Kanamycin and amikacin by electrochemical detection.
The USP contains several drugs within the mycin class. Many of these drugs are aminoglycosides, which can be analyzed using the procedure above. For this study, amikacin and kanamycin were used. They share the same assay method in the USP. The method was modified by adopting the waveform appropriate for the gold working electrode. The results are shown in Figure 2. Five replicates were used to establish the precision of the method. The %RSD for kanamycin was 1.1% and the %RSD for the amikacin was 0.9%.
There can also be other advantages. Many of the biologically active compounds have more than one sugar molecule attached. Often the degradation products can be separated and determined by the same method. Dionex has shown this in its paper on streptomycin.4
This overview only presents a brief summary of the power of pulsed amperometric detection coupled with chromatography. Those who have been involved in amperometry in the past will really appreciate the new innovations. Published literature on new methods are on the rise, and smaller systems are being developed that will help with precious samples and standards. The breadth of the applications should make any analytical chemist take notice.
- Dionex Technical Note 21. Optimal Settings for Pulsed Amperometric Detection of Carbohydrates Using the Dionex ED40 Electrochemical Detector, 1998.
- Dionex Technical Note 20. Analysis of Carbohydrates by High Performance Anion Exchange Chromatography Using the Dionex ED40 Electrochemical Detector; http://www.dionex.com/en-us/webdocs/5023-TN20.pdf, 1993. Revised 2004.
- Kren, V.; Martinková, L. Glycosides in medicine: the role of glycosidic residue in biological activity. Curr. Med. Chem. 2001, 8, 1313–38.
- Dionex Application Note 181. Determination of Streptomycin and Impurities Using HPAE-PAD, 2007.
Mr. Limpert is Advisory Scientist, Celsis Analytical Services, 6200 S. Lindbergh Blvd., St. Louis, MO 63123, U.S.A.; tel.: 314-487-6776; fax: 314-487-8991; e-mail: GLimpert@Celsis.com.