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The laboratory notebook: answering important questions about quality.

This month we discuss a critical aspect of HPLC analysis; the effect the elapsed time of an HPLC run has on the analysis. For you fiber fans we also describe the differences between soluble fiber, insoluble fiber and dietary fiber.

Q. I bought a significant amount of green tea and had an independent laboratory test it by HPLC. I got the chromatogram, which had six labeled peaks, with the amount of EGCG being rather low. The seller then provided a chromatogram that had only one peak, labeled epigallocatechin gallate (EGCG), and its level was quite high. I did notice that the time period reported on my laboratory's chromatogram ran for 40 minutes and the one from the seller ran for only 10 minutes. Can you explain the reasons for these widely different results?

A. This is a great question describing a situation we see from time to time. If you read this column you know we (and most analytical chemists) favor HPLC analyses over all others where it is applicable. This does not mean, however, that all HPLC analyses are created equal. To understand the issue let's quickly review the basic principles of HPLC analysis.

HPLC stands for "high pressure liquid chromatography" (also called "high performance liquid chromatography"). Simply stated, the material of interest is put into solution (if it is not already a liquid) and injected into the HPLC. HPLC utilizes additional liquid (called the "mobile phase") to move the material through the system and to assist in the separation of the components of a mixture. These components (or analytes) are then forced to flow through a chromatographic column under a high pressure.

When the solvent composition of the mobile phase remains constant throughout the analysis the elution is called isocratic. While this works for simple materials, it is insufficient for more complex ones. For the tough stuff, the solvent composition is gradually changed during the analysis. This type of analysis is called gradient elution. Generally, this is done by progressively increasing the proportion of organic solvent or salt buffer with time.

The column (referred to as the "stationary phase") consists of immobile packing material, generally silica. This material actually retains analytes. In the column, the mixture is separated (technically called "resolved") into its components. The degree of resolution is critical. A greater degree of resolution will mean greater specificity, that is, a more exacting separation of the various components. If resolution is great, closely related but not identical components will be separated from one another. If resolution is poor, closely related but not identical components will not be separated and instead may be lumped together, appearing to be one uniform component with the combined amount of the similar but not identical components. The analyst's mission and challenge is to separate each analyte present, no matter how minor the differences may be.

As you can probably tell by now, the degree of resolution is dependant upon many factors, such as the type of mobile phase used and the packing material placed in the column. Different materials require different combinations of each. The degree of resolution is also dependant upon time. All analyses must run for enough time for adequate resolution. Obviously, the faster you can run a sample through the system, the more samples you can run within a given time. This is good for busy analytical labs. However, you have to be sure you do not compromise your analytical goals. Running a gradient elution too fast compromises resolution and can lead to incorrect analyses. The proper analysis of green tea requires differentiation among the six primary catechins: catechin, gallocatechin, epicatechin, epigallocatechin, epicatechin gallate and epigallocatechin gallate (also known as EGCG). EGCG is considered to be the most active component in green tea and the one most people look for. The proper HPLC analytical method employs a gradient elution. Sufficient time must be allowed for adequate resolution of the six closely related catechins. No matter who is running the system and what instrumentation is used, the analysis cannot be completed in 10 minutes--20 to 40 minutes is more like it. What we suspect has happened is that the gradient elution went way too fast resulting in poor resolution. Some or all of the catechins were not separated from each other and instead were lumped together, giving the appearance of a high level of one of them, EGCG.

Unfortunately, we see situations like this more often than we like to. Perhaps some labs speed up HPLC run time to meet volume demand for which they do not have sufficient capacity. Or worse, perhaps some labs speed up run time because this can have the effect of improving the appearance of what is actually an inferior product. There is always a desire for an analytical lab (the seller of services) to provide the client (the consumer of services) with the answer they would like. But reputable firms cannot let business get in the way of good science.

The moral of the story is do not accept anything at face value. Know the lab you work with and investigate the results of all other labs. The actual run time of an HPLC analysis is easy to determine; it should be clearly indicated at the bottom of each chromatogram. Ask for a copy and see for yourself. While there are exceptions, any HPLC run of complex material that is 10 minutes or less warrants close scrutiny.

Q. Is there a difference between soluble and insoluble fiber, on the one hand, and total dietary fiber, on the other?

A. Yes there is. Simply stated, the term fiber refers to carbohydrates that cannot be digested. Fiber is present in all plants that are eaten for food, including grains, vegetables, fruits and legumes. However, not all fiber is the same. One way to categorize fiber is by the ease in which it dissolves in water. Soluble fiber dissolves in water, while insoluble fiber does not. Insoluble fiber can, however, be partially digested by enzymes. As such, it can be utilized by the body and contribute to caloric value. Dietary fiber consists of soluble fiber and the part of the insoluble that can be digested by enzymes.

Total dietary fiber is determined by AOAC methodology. In short, the material to be tested undergoes enzymatic digestion with amylase, protease and amylogy-cosidase. The resultant material is then treated with alcohol to precipitate the soluble dietary fiber and then filtered, washed, dried and weighed.

Robert S. Green is the president of Integrated Biomolecule Corporation. The IBC Labs division is a leader in the analytical testing of nutritional supplement raw materials and finished products and provides research, development, production and technical marketing services. The IBC Organics division conducts pharmaceutical research and development. Every other month he answers questions on quality issues in an easy-to-understand manner. He can be reached at 520-219-2900; Fax: 520-219-6090; E-mail:; Website:
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Title Annotation:QUALITY FOCUS
Author:Green, Robert
Publication:Nutraceuticals World
Geographic Code:1USA
Date:Nov 1, 2005
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