📚 Experimental Design in Biological Molecules | 生物分子实验设计
Understanding how to design experiments to identify and quantify biological molecules is a fundamental skill in A-level Biology. This article explores the key tests for carbohydrates, proteins, and lipids, and provides guidance on planning reliable investigations, controlling variables, and interpreting results.
了解如何设计实验来鉴定和定量生物分子是A-level生物学的基本技能。本文探讨了碳水化合物、蛋白质和脂质的关键测试，并指导如何计划可靠的调查、控制变量和解释结果。
1. Introduction to Biomolecule Tests | 生物分子检测简介
Biological molecules can be identified by specific chemical tests that produce visible colour changes or precipitates.
生物分子可以通过特定的化学测试来鉴定，这些测试会产生可见的颜色变化或沉淀。
These tests exploit the unique chemical properties of functional groups, such as aldehyde groups in reducing sugars or peptide bonds in proteins.
这些测试利用了官能团的独特化学性质，例如还原糖中的醛基或蛋白质中的肽键。
2. Test for Reducing Sugars | 还原糖测试
Reducing sugars, including all monosaccharides and some disaccharides like maltose, reduce Benedict's reagent upon heating.
还原糖，包括所有单糖和一些二糖如麦芽糖，在加热时能还原本尼迪克特试剂。
Benedict's reagent contains copper(II) sulfate; Cu²⁺ ions are reduced to Cu⁺, forming a brick-red precipitate of copper(I) oxide (Cu₂O).
本尼迪克特试剂含有硫酸铜；Cu²⁺ 离子被还原为 Cu⁺，形成砖红色的氧化亚铜(Cu₂O)沉淀。
Reducing sugar + Benedict's solution (Cu²⁺) → heat → Cu₂O (brick-red ppt)
实验时，将样品与本尼迪克特试剂混合，在沸水浴中加热2-5分钟，颜色从蓝色经绿色、黄色、橙色最终变为砖红色，表明还原糖存在。
To improve reliability, use a water bath at a fixed temperature (e.g., 80°C) and standardise the heating time.
为了提高可靠性，应使用固定温度的水浴（例如80°C）并标准化加热时间。
Always run a negative control using distilled water and a positive control with a known reducing sugar solution.
始终使用蒸馏水进行阴性对照，使用已知的还原糖溶液进行阳性对照。
3. Test for Non-reducing Sugars | 非还原糖测试
Non-reducing sugars like sucrose must be hydrolysed into reducing sugars before testing.
诸如蔗糖等非还原糖必须在测试前水解成还原糖。
Add dilute hydrochloric acid to the sample, heat in a water bath, then neutralise with sodium hydrogencarbonate solution before adding Benedict's reagent.
向样品中加入稀盐酸，在水浴中加热，然后用碳酸氢钠溶液中和，再加入本尼迪克特试剂。
A positive result after hydrolysis confirms the presence of a non-reducing sugar, provided the initial test for reducing sugar was negative.
水解后出现阳性结果则证实存在非还原糖，前提是初始的还原糖测试为阴性。
4. Starch Detection with Iodine | 碘液检测淀粉
Iodine solution (iodine in potassium iodide) turns blue-black in the presence of starch.
碘液（碘溶于碘化钾）在淀粉存在时呈蓝黑色。
The colour change results from iodine molecules becoming trapped within the helical amylose structure of starch.
颜色变化是由于碘分子被包埋在淀粉的螺旋直链淀粉结构中。
This test is highly sensitive and works best at room temperature; heating may break the starch-iodine complex and cause the colour to fade.
该测试非常灵敏，在室温下效果最佳；加热可能会破坏淀粉-碘复合物，导致颜色褪去。
5. Biuret Test for Proteins | 蛋白质双缩脲测试
The Biuret test detects peptide bonds in proteins and peptides.
双缩脲测试检测蛋白质和多肽中的肽键。
Add sodium hydroxide solution followed by a few drops of copper(II) sulfate solution; a violet or purple colour indicates proteins.
加入氢氧化钠溶液，然后滴加几滴硫酸铜溶液；出现紫色表明含有蛋白质。
The reaction requires at least two peptide bonds; thus, individual amino acids give a negative result.
该反应至少需要两个肽键；因此，单个氨基酸会产生阴性结果。
Be careful not to add excess copper sulfate, as a blue precipitate of Cu(OH)₂ can mask the purple colour.
注意不要加入过量的硫酸铜，因为蓝色的Cu(OH)₂沉淀会掩盖紫色。
6. Emulsion Test for Lipids | 脂质乳液测试
Lipids can be identified by the emulsion test: mix the sample with ethanol, then pour into water.
脂质可通过乳液测试进行鉴定：将样品与乙醇混合，然后倒入水中。
A milky-white emulsion indicates the presence of lipids because the lipid droplets disperse light.
出现乳白色乳状液表明存在脂质，因为脂质小滴会使光发生散射。
If a solid sample contains lipids, crush it first and shake with ethanol to dissolve the lipid components.
如果固体样品含有脂质，应先将其研碎，再与乙醇一起摇晃以溶解脂质成分。
Use a control tube with water instead of ethanol to ensure the cloudiness is not caused by other substances.
使用水代替乙醇的对照管，确保浑浊不是由其他物质引起的。
7. Quantitative Analysis Using Colorimetry | 使用比色法定量分析
Colorimetry allows the concentration of reducing sugars or proteins to be estimated by measuring absorbance.
比色法可通过测量吸光度来估算还原糖或蛋白质的浓度。
Prepare a series of known concentrations, treat each with the appropriate reagent, and measure the absorbance at a specific wavelength using a colorimeter.
准备一系列已知浓度，用相应的试剂处理，并使用比色计在特定波长下测量吸光度。
A calibration curve of absorbance versus concentration can then be used to determine the concentration of an unknown sample.
然后可以使用吸光度-浓度标准曲线来确定未知样品的浓度。
For reducing sugars, the supernatant after centrifugation can be compared; for proteins, the Biuret colour intensity is proportional to peptide bond concentration.
对于还原糖，可以比较离心后的上清液；对于蛋白质，双缩脲颜色强度与肽键浓度成正比。
8. Designing a Controlled Experiment | 设计对照实验
A well-designed experiment must include appropriate controls, standardised conditions, and repeated measurements.
精心设计的实验必须包含适当的对照、标准化的条件和重复测量。
Controls: a negative control (replacing the sample with water or buffer) confirms no false positives; a positive control (known macromolecule) verifies the test is working.
对照：阴性对照（用水或缓冲液代替样品）确认无假阳性；阳性对照（已知大分子）验证测试是否正常工作。
Identify the independent variable (e.g., concentration of sugar), the dependent variable (e.g., absorbance), and control variables (temperature, pH, reagent volume).
确定自变量（例如糖的浓度）、因变量（例如吸光度）和控制变量（温度、pH、试剂体积）。
Always state how variables are controlled, such as using a thermostatically controlled water bath for temperature.
始终说明如何控制变量，例如使用恒温水浴来控制温度。
9. Interpreting Results and Unknown Identification | 结果解读与未知物鉴定
When given an unknown mixture, perform a logical sequence of tests to identify its components.
遇到未知混合物时，按逻辑顺序进行测试以鉴定其成分。
Start with iodine for starch, then emulsion test for lipids, Biuret for proteins, and finally Benedict's test for reducing and non-reducing sugars.
先用碘液检测淀粉，然后进行脂质乳液测试，接着用双缩脲检测蛋白质，最后用本尼迪克特测试还原糖和非还原糖。
Record observations in a table, noting initial and final colours, and clearly state whether each macromolecule is present or absent.
用表格记录观察结果，注明初始和最终颜色，并明确说明每种大分子是否存在。
10. Common Pitfalls and Improvements | 常见错误与改进
Inadequate mixing of reagents can lead to
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