Liquid Chromatography – Sample Preparation and Quantitative Errors

Part 1. Introduction

The process of performing analysis using HPLC can be divided into few stages:

• Sample Preparation
• Injection
• Separation & Detection
• Data Processing

The causes of quantitative errors may originate from any of the above stages, but this month we’ll look at those related to standards and sample preparation.

Part 2. Preparation of Standard Solutions

There is a tendency to omit the sample/standard preparation steps when evaluating the causes of quantitative errors. During sample / standard preparation there are number of points which require your total attention to ensure preparation is carried with the consistent and accurate approach; refer to Table 1 below. Each of the points listed in Table 1 should be carefully considered when preparing standard/sample solutions.

Out of the listed, particularly important is the “adsorption of target constituent to container” (Table 1 – point I). To help you avoid these interactions, we listed chemical entities and container types which can potentially interact including preventative steps in Table 2. In some cases, adsorption can be inhibited by selecting an appropriate solvent, on the other hand you may need to change the container/flask type to a different material. Should you investigate if the adsorption occurs, it is possible to detected it based on the lack of linearity in the calibration curve or repetitive calibration curve failure to pass through the origin with the diluted standard solutions.

Table 1. Standard/sample solution preparation – potential errors in preparation

Another factor to look at is the “oxidation or decomposition of target constituent” (Table 1 – point K). Let’s look at the ascorbic acid which is easily oxidized by dissolved oxygen and iron (III) ions present in aqueous solutions, causing the ascorbic acid concentration to drop over time. In this case the control of the solution / diluent pH (lowering pH) and elimination or binding of the iron (III) ions with EDTA-2Na are recommended. It is also recommended that injection is made immediately after preparation, if possible.

Table 2. Container adsorption and ways to prevent it.

In general, oxidation and decomposition are inhibited by, for example, adding a reducing agent, performing nitrogen substitution, using a nonaqueous solvent, using a brown bottle, or storing in a dark, cold places. Oxidation and decomposition can also be detected from a reduction in area value that occurs when the sample solution is injected several times and commonly accompanied by increase in peak area of one of the impurities or formation of a new peak in your chromatograms.

Part 3. Pretreatment of Actual Samples

When preparing sample solution, you always have to consider the extraction recovery and efficiency. In solid-liquid extraction and liquid-liquid extraction systems, it may not be possible to realise a high recovery, or the recovery rate may be unpredictable. In protein removal, the target constituent may adsorb to degenerated protein, causing a reduction in the recovery rate. Typically, the recovery rate is evaluated by adding the target constituent to the sample and extracting it. The change in the relationship between the amount of target constituent added and the increase in the peak area in the chromatogram compared to when the target-constituent solution is injected without extraction is investigated. If there is a problem in the recovery rate you have two options:

• Option 1 – change the extraction method
• Option 2 – performed extraction after adding an internal standard

While going with Option 2 it is critical that the internal standard used has a similar chemical structure to the target constituent and the extraction efficiency must be approximately the same. Usually, sample pretreatment is often performed with the Option 2 in cases where the recovery rate is not 100% but a relatively stable rate is observed. This way you will be able to apply a correction factor to appropriately calculate your results.

Part 4. Summary

Looking at the simple task of sample or standard solution preparation we typically neglect the fact that there are multiple, complex steps included which may potentially impact the accuracy of our results. Beyond obvious such as weighing and dilution errors, the unforeseen like adsorption to surface, compound stability, if not considered, will influence results reporting, which we all want to avoid. Hope this month’s read leaves you with some thoughts and considerations when preparing your next sample or standard solution. Always strive for a high level of accuracy in daily analysis work by considering the potential causes of errors that require particular attention.

Liquid Chromatography – Master the Basics

This article is part of our “Liquid Chromatography – Master the Basics” series, your go-to resource for comprehensive and insightful updates on the world of liquid chromatography. Each month in 2024 we will dive into a Liquid Chromatography topic, offering content that is both accessible to beginners and beneficial for experienced scientists.

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