Stop Being a Lab Robot: Mastering National Standards in HPLC Analysis
Author: Chromatography Mound
Introduction
As chromatographers, we owe our laboratories more than merely executing prescribed procedures. Rigidly following national standards without appreciating their underlying rationale perpetuates a “machine-like” mindset—one that falters when confronted with unexpected sample matrices or urgent troubleshooting.
This mindset is common, but avoidable. To break free from passive execution, we must adopt a different attitude—one that seeks to understand the why behind the how of standard methods.
How to Read a National Standard Method the Right Way
National standard methods may vary across countries, but their cores and principles share commonality. When approaching a new method, these core aspects should always be focused on:
- Scope of application
- Extraction procedure
- Cleanup steps
- Derivatization requirements
- Instrumentation
- Quantification approach
- Solvents and reagents involved
Let’s now analyze GB 5009.28-2016, the standard used in China for determining benzoic acid, sorbic acid, and saccharin sodium in food. (GB means “national standard” in Chinese, by the way.)
LC vs GC: Method Applicability
This method offers two options:
- Method 1: HPLC, suitable for a wide range of foods
- Method 2: GC, limited to soy sauce, juice, and jam
Clearly, Method 1 is more versatile. Method 2 targets mainly liquid matrices, suggesting limitations in its extraction compatibility. Understanding method scope helps you select the most appropriate approach for your sample type.
Why Hexane, Ammonia, and Ethanol for High-Fat Samples?
In this method, potassium ferrocyanide (K4[Fe(CN)6]·3H2O) and zinc acetate (Zn(CH3CO2)2·2H2O) are used to remove proteins after ultrasonicate extraction with water, and methanol, ammonium acetate (CH3COONH4), and formic acid are used as the mobile phase.
Why then, are hexane (n-hexane), ammonia, and ethanol specified for high-fat food samples in Section 5.2.3 (quoted below)?
To understand this, first recall that benzoic acid and sorbic acid, despite being acids, often exist as water-soluble salts in food. This helps them become more stable. However, their extractability varies with pH and matrix composition. In high-fat matrices, even the salts may resist extraction.
- Hexane disperses and removes lipids, breaking down the matrix for better analyte release.
- Ammonia raises the pH, helping dissociate target compounds from the matrix, especially under heat.
- Why Ethanol? It acts as a bridge, since ammonia is highly polar and doesn’t mix well with fatty matrices, which are low in polarity. Ethanol’s intermediate polarity promotes mixing of ammonia and matrices, improving extraction efficiency.
Why Include Ammonium Acetate and Formic Acid in the Mobile Phase?
Another question worth discussion in this method is, why not just use water and methanol as the mobile phase, but include ammonium acetate, with formic acid in it?
The answer lies in analyte chemistry. Benzoic acid and sorbic acid contain carboxylic groups, making them highly water-soluble. This causes them to elute too early without buffer salts, and may suffer from diffusion in the mobile phase, resulting in peak broadening and poor retention.
- Ammonium acetate increases ionic strength, improving retention and peak shape.
- Formic acid stabilizes the mobile phase pH, keeping the acids predominantly in their protonated form, enhancing detector response and method reproducibility.
Furthermore, the use of UV detection is due to both benzoic acid and sorbic acid having conjugated systems that strongly absorb in the UV range—thus making them ideal candidates for UV detection.
Real Method Mastery Starts with the Chemistry
Every solvent, and every step in a national standard exist to reflect specific chemical reasoning. Without understanding their mechanisms, it's easy to overlook critical details that compromise accuracy and reproducibility.
To truly master a national standard method, start with the chemistry of your analytes. Understand their structure, polarity, solubility, and behavior under different pH conditions and matrices.
The key isn’t just technical skill—it’s curiosity. If you cultivate a habit of asking why at each step, you’ll move beyond routine execution. When problems arise, you’ll have the insight to resolve them—not as a “lab robot”, but as a competent analytical scientist.