What is an internal standard and why is it used in analytical chemistry?

An internal standard is a substance added in a known amount to standards, samples, and blanks during analysis. It compensates for both systematic and random errors that occur during sample handling and measurement. By calculating the ratio of signals between the internal standard and analyte, called the response factor, accurate concentration calculations are possible even when sample loss occurs.

How does an internal standard help account for sample loss?

Any sample losses occurring after the internal standard is added should be similar for both the internal standard and the analyte. Since both substances experience proportional losses, their signal ratio remains constant. This allows the response factor to accurately reflect concentration ratios, making it possible to calculate analyte concentration despite sample loss during preparation and transfer steps.

What are the key requirements for choosing a suitable internal standard?

An internal standard must have a signal similar but not identical to the analyte, allowing the measurement method to distinguish between them. It cannot affect the analyte measurement and must not be natively present in the sample. The concentration must be well known, ensuring the only source in solution is the known amount deliberately added during analysis.

What is a response factor and how is it calculated?

The response factor is the ratio of analytical signals for the internal standard and analyte, which is proportional to their concentration ratio. It is calculated by plotting the ratios of peak areas versus concentration ratios from known standards. The slope of the resulting calibration curve equals the response factor, which can then be used to determine unknown analyte concentrations.

How do internal standards correct for systematic errors like matrix effects?

Matrix effects interfere with analyte measurement by affecting the signal in solution. When an internal standard experiences the same matrix effect as the analyte, the ratio of their signals remains unaffected. Performing calibrations in the same matrix as the sample ensures both substances are equally influenced, allowing the response factor ratio to accurately reflect true concentration differences.

What analytical techniques commonly use internal standards?

Internal standards are widely used in chromatography, spectroscopy, and electrochemistry. In gas chromatography, the ratio of peak areas for the internal standard and analyte is calculated. In spectroscopy, internal standards correct for random errors from variable light source intensity. These techniques rely on internal standards to minimize errors and ensure accurate quantitative analysis across diverse scientific disciplines.

How is sample preparation optimized to minimize sample loss before using an internal standard?

Sample loss is minimized by reducing the number of handling and transfer steps. Massing solids directly into the final flask eliminates unnecessary transfers. When transfers are necessary, triple rinsing glassware ensures complete sample transfer. For samples that adsorb to glass, using polypropylene tubes prevents losses. These preparation strategies work alongside internal standards to ensure accurate concentration determination.

内标物

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1. 正确的样品处理：配制溶液

取一个洁净的烧杯，称取适量样品放入其中，记录实际使用质量。本示例中，在容量瓶中配制腺嘌呤溶液，作为后续分析的内标。腺嘌呤的质量为 100 mg。切勿直接在容量瓶中称量，因其瓶颈较长，腺嘌呤难以加入或取出。
向烧杯中加入约 25 mL 溶剂（本例中为二甲基亚砜（DMSO）），搅拌至溶解。本示例中最终溶液在 50 mL 容量瓶中定容，因此仅加入约 25 mL 溶剂，以便后续冲洗烧杯并将溶液转移至最终体积。
待固体完全溶解后，将溶液倒入容量瓶中。
用少量溶剂（约 10 mL）冲洗烧杯和搅拌子，并将洗涤液倒入容量瓶中，重复此操作两次。这有助于确保溶液完全转移。

2. 内标校准曲线的制备

为气相色谱分析准备所需的标准样品。本示例中，使用乙腈从咖啡中提取咖啡因，然后使用腺嘌呤作为内标物进行测量。
对于咖啡因样品，称取配制浓度为 1 mg/mL 所需量的样品。若使用 10 mL 容量瓶，则称取 10 mg。
将分析物称入烧杯中，加入几毫升溶剂（此处为甲醇）溶解，然后用溶剂冲洗 3 次，定量转移至容量瓶中。
以相同方法制备另外 3 个浓度分别为 0.2、0.5 和 2 mg/mL 的咖啡因标准样品。
将每种咖啡因标准溶液各取 1 mL 加入样品瓶中。
向每个样品瓶中加入 0.2 mL 浓度为 2 mg/mL 的腺嘌呤内标溶液。
对每种咖啡因标准溶液进行气相色谱实验。对每张色谱图，计算咖啡因与内标物的峰面积比值。
绘制峰面积比值与浓度比值的关系图。该图的斜率即为响应因子。

3. 气相色谱内标法实际样品的制备

准备用于气相色谱分析的所需样品。在此示例中，使用乙腈从咖啡中提取咖啡因。
对于咖啡样品，称取 2 g 咖啡至 100-mL 烧杯中。记录咖啡的精确质量。
向烧杯中加入 20 mL 乙腈。
静置 20 分钟，期间频繁搅拌。
使用漏斗中的滤纸过滤除去咖啡渣。
用少量乙腈（5 mL）冲洗滤纸 3 次。
测量滤液的最终体积，应约为 35 mL。

4. 运行样品并计算浓度

取1 mL咖啡提取物样品，加入0.2 mL内标物至样品瓶中。将样品瓶放入自动进样器样品架。
运行样品的气相色谱分析。确保气相色谱条件能使咖啡因和腺嘌呤分离。本示例中，在200 °C下进行等温分离。
分析完成后，计算内标物峰和分析物峰的峰面积。利用峰面积比值，计算样品中咖啡因的含量。

5. 结果：采用内标法对咖啡因进行气相色谱分析

咖啡因的气相色谱分析结果如图1所示。腺嘌呤用作内标物。可通过测量峰面积比值并对其浓度比值作图。该图的斜率即为响应因子（本例中为1.8）。
图2显示了含有腺嘌呤内标物的咖啡样品的色谱图。峰面积比值为1.78。利用响应因子和已知的腺嘌呤浓度（0.33 mg/mL），计算得出未知样品中咖啡因的浓度为0.33 mg/mL。

$线性校准曲线，咖啡因分析，方程 $A_{\text{caff}}/A_{\text{IS}}$ 对 $C_{\text{caff}}/C_{\text{IS}}$。$
图1. 使用内标法的校准曲线图。 三种咖啡因标准样品（1、0.5 和 0.2 mg/mL）与每种样品中添加的 0.33 mg/mL 腺嘌呤内标物的峰面积比相对于浓度比的图示。直线的斜率为 1.8，即为响应因子。

气相色谱图，FID 响应值随时间变化；溶剂、咖啡因、腺嘌呤、棕榈酸。
图 2. 含腺嘌呤内标物的咖啡样品色谱图。 FID 检测器对样品响应值的图谱。三个主要峰分别为腺嘌呤（内标物）、咖啡因和棕榈酸。

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来源：弗吉尼亚大学 B. 茉莉·文顿博士实验室

许多化学分析的目标是定量分析，即确定样品中某种物质的含量。为了准确计算样品中未知物质的浓度，精细的样品前处理至关重要。每次处理或转移样品时，都可能造成部分样品损失。然而，存在多种策略可将样品损失降至最低，同时也有一些方法可在发生样品损失的情况下，仍实现…