Complex Carbohydrate Analysis by HPAE-PAD Made Easier: 'Just Add Water'

Carbohydrates are ubiquitous in nature and in our lives. Various forms of carbohydrates (aka glycans) play major metabolic roles as sugars in our foods that enter our bodies. Carbohydrates are also present in certain therapeutic drugs used for the treatment of various diseases. Therefore, for food safety and therapeutic drug development, carbohydrates need to be characterized and measured reliably.

Analysis of complex carbohydrates with HPAE-PAD

Carbohydrates do not carry any chromophores, or charge, making it a challenge to analyze carbohydrates via liquid chromatography. Luckily, the hydroxyl groups in carbohydrate molecules can be ionized at high alkaline conditions allowing carbohydrates to be retained and separated in anion exchange columns. These hydroxyl groups can be oxidized on gold electrode in alkaline solution making electrochemical detection of carbohydrates possible.

High-performance anion exchange chromatography with pulsed amperometric detection (HPAE-PAD) offers superior resolution of carbohydrates with high sensitivity without the need for derivatization, thereby reducing overall cost of analysis and labor as well as eliminating the errors associated with analyte derivatization.

Eluent preparation for HPAE-PAD

The most common barrier to success for complex carbohydrate analysis via HPAE-PAD is the proper preparation and use of eluents. The carbonate contamination of the eluents can significantly affect the retention time and resolution of carbohydrates.

To minimize the carbon dioxide intrusion into sodium hydroxide (NaOH)/sodium acetate (NaOAc) eluents, meticulous care is required in the eluent preparation during multiple steps to ensure consistency in the eluent composition and minimize carbonate intrusion throughout the eluent preparation and use.

The prepared solution must be thoroughly degassed and promptly transferred to a plastic eluent bottle, and the solution must be protected from the ambient air via the introduction of helium or nitrogen gas. Fresh eluent must be prepared every week or at any time helium or nitrogen headspace is lost for more than 15 minutes.

Dual EG for complex carbohydrates analysis: just add water

Electrolytic eluent generation is a process that allows the production of high-purity eluents for ion chromatography and is a part of reagent-free ion chromatography (RFIC). The entire process requires only deionized water, eliminating the need to handle any acids or bases, thereby offering an easy-to-use platform with excellent reproducibility.

Dual electrolytic eluent generation (Dual EG mode) employs two eluent generator cartridges to produce high-purity potassium hydroxide (KOH) and potassium methanesulfonate acid (KMSA) eluents for complex carbohydrate analysis. It significantly reduces labor, time, and human error associated with the manual preparation of the NaOH/NaOAc eluent solutions.

Users only need to set the eluent concentration in the Chromeleon chromatography data system (CDS), then be rest assured that the eluent generators will deliver a highly accurate and precise eluent concentration for their applications. Time and effort can then be focused on method development and other innovation.

Method development for Dual EG applications

One of the frequently asked questions is, “How do I convert my existing methods using the NaOH/NaOAc eluents to the Dual EG system?”

The general recommendation is to start with a KMSA concentration of 1/2 or 1/3 of NaOAc concentration. Within the total concentration limit (maximum total combined concentration of KOH and KMSA of 200 mM at 0.063 mL/min), a higher KOH concentration can help achieve sharper peaks and better resolution (and better sensitivity).

For strongly retained analytes, if total concentration reaches the limit, lower KOH concentration can help decrease the retention time, therefore achieving a shorter run time. Figure 1 shows an example of the gradient methods developed for Dual EG 1-mm column application compared to the original NaOH/NaOAc method.