HPLC and GC Articles

This month's installment of "Column Watch" is the first of a two-part series in which columnist Ron Majors examines the trends and highlights in columns and consumables at Pittcon 2008. Here, he discusses new high performance liquid chromatography (HPLC) columns and packings, including reversed-phase, ion-exchange, ion chromatography, size-exclusion, and specialty columns.

Ronald E. Majors
Pittcon 2008 — the 59th Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy — was held in New Orleans, Louisiana, March 1–7, 2008, the first time the Conference has returned there since the Katrina disaster. This year's event hosted more than 1150 instrument manufacturers and laboratory suppliers in more than 2500 booths. In addition to attending the exposition, the conferees listened to 1250 oral presentations, viewed more than 1000 posters, checked numerous company seminar rooms, or attended one of 114 short courses.

As I write this, I have just finished a week of teaching a liquid chromatography (LC) training class in Chongqing, China. On the last day, we spent several hours looking at problems that the students brought to class, and it reminded me how chromatographers worldwide encounter the same type of problems. One problem that came up was related to the transfer of a method from one instrument to another. This kind of problem is one that all of us encounter at one time or another, so I would like to dedicate this month's "LC Troubleshooting" installment to method transfer problems.

The Rule of One

It is strange how we automatically apply the scientific method to most of our work in the laboratory, but somehow we discard it when it comes time to troubleshoot an LC problem. For troubleshooting purposes, I summarize the recommended technique as the of One." This reminds us to change just one variable at a time when investigating a problem.

Bacillus anthracis, the etiological agent of anthrax, is a Gram-positive, rod-shaped bacterium that forms spores that are highly resistant to heat, ultraviolet light, radiation, pressure, and chemical agents. The durability of these spores make this bacterium a potential bioweapon (1).

Gum arabic (A. senegal) is a highly heterogeneous complex polysaccharide that consists of three main fractions. These fractions are arabinogalactan protein complex (AGP), arabinogalactan (AG), and glycoprotein (GP). Each fraction contains a range of different molecular weight components with different protein contents (1). The AGP fraction is composed of hydrophilic carbohydrate blocks linked to a protein chain and has been reported to have a wattle blossom-type structure due to being degraded readily by proteolytic enzyme (Figure 1) (2–4).

Figure 1
The carbohydrate blocks are made up of galactose, L-arabinose, D-glucuronic acid and 4-O-methyl glucuronic acid.

Gum arabic is used mainly in food, pharmaceutical, and cosmetic industries due to its unique physiochemical and functional properties (5,6). These functional properties are related directly to the molecular structure.

Choosing and effectively employing analytical technology is the broader discussion that underlies the diversity of topics that appear in this installment of "MS—The Practical Art." Over the last five years, we have presented the reasoning of the practitioners we consult, to disseminate their wisdom and further our own knowledge in a number of areas such as accurate mass measurement, sample preparation, modern small-particle ultrahigh-pressure liquid chromatography (UHPLC) technology, and evaluating the viability of emerging non-LC ionization prospects.

In almost every instance, I relied on the expertise of well-recognized practitioners, not only for their "nuts-and-bolts" explanations but for the unique insight their experience affords. Insights developed from their well-earned knowledge we cannot glean from reading peer-reviewed work.

In 2001, the second glossary of common and not-so-common terms and "buzz words" for reference to high performance liquid chromatography (HPLC) columns and column technology was published (1). It is time for an update as new terms have arisen or, in some cases, their original meanings have expanded or changed. We have also decided to expand the terms dealing with HPLC and LC to cover some of the common terms that we neglected to include in the earlier glossary. To make room for this expansion, we have decided to remove most of the terms referring to capillary electrophoresis (CE) because this technique is rather specialized and not all liquid chromatographers are also performing the various forms of CE. We will also stick to the conventions of the International Union of Pure and Applied Chemistry (IUPAC) in their "Nomenclature for Chromatography" that provides guidance and changes in some of the more commonly accepted terms (2).

The dissolution performance test is a required test for all solid oral dosage forms for product release testing. It also is used commonly as a predictor of a drug product's in-vivo performance. To help satisfy dissolution requirements, the USP provides information in the way of a general chapter on dissolution, as well as related chapters on disintegration and drug release (1–3). The USP and the FDA also provide guidelines on development and validation of dissolution procedures (4–9), and while this month's column will draw from this information and will discuss the available guidance in some detail, we encourage readers to consult the references for additional details.

Reversed-phase liquid chromatography (LC) is a simple and easy-to-use technique that will give satisfactory separations for the samples that most of us encounter. Based upon column sales, somewhere around 70–80% of samples can be separated by reversed-phase techniques. The high-purity silica-based columns available today are quite robust in the 2 < pH < 8 range and there are several choices for columns that will work well up to pH 10. The first choice for most workers is to work at low pH, in the pH 2–3 range. Under these conditions, most acidic samples will have suppressed ionization and be well retained, as will be neutral molecules. Basic analytes will be ionized, but if the bulk of the sample molecule is nonpolar, adequate retention often is obtained. At pH < 10, many basic compounds will not be ionized, so they can be chromatographed successfully, but other bases will still be ionized, so retention can be poor.

Analysis of the solvents involved in the manufacturing process is a longstanding regulatory requirement in the pharmaceutical industry. According to the initial guidelines proposed by the International Conference on Harmonization (ICH) and by the United States Pharmacopeia (USP, General Chapter <467>) and European Pharmacopeia (EP), manufacturing solvents must be carefully regulated because they have varying levels of toxicity or environmental hazard and they have no therapeutic benefit (1–3). The solvents are grouped into three classes:

Class 1: Solvents to be avoided because they are highly toxic or are especially hazardous to the environment. Required daily exposure limits are 2–1500 ppm.

Class 2: Solvents with moderate toxicity, including most common solvents used in synthetic processes. Required daily exposure limits are 20–4800 ppm.

Analysis of the solvents involved in the manufacturing process is a longstanding regulatory requirement in the pharmaceutical industry. According to the initial guidelines proposed by the International Conference on Harmonization (ICH) and by the United States Pharmacopeia (USP, General Chapter <467>) and European Pharmacopeia (EP), manufacturing solvents must be carefully regulated because they have varying levels of toxicity or environmental hazard and they have no therapeutic benefit (1–3). The solvents are grouped into three classes:

Class 1: Solvents to be avoided because they are highly toxic or are especially hazardous to the environment. Required daily exposure limits are 2–1500 ppm.

Class 2: Solvents with moderate toxicity, including most common solvents used in synthetic processes. Required daily exposure limits are 20–4800 ppm.