Team:TUST China/HPLC




Current and Commonly Used Tetracycline Detection Methods

Antibiotics as antibacterials and growth promoters are widely used in various fields, resulting in a lot of residual hazards that are difficult to eradicate. At present, there are many methods to detect antibiotic residues in water, among which chromatographic and spectral methods are the most widely used. In order to assess the advantages of our project over traditional methods of testing, we chose a representative traditional method for comparison. This page mainly compares the characteristics of these two methods, and on this basis summarizes a set of methods suitable for our project.

1. Chromatography

Chromatography is used to separate various components in the mixture. The chromatographic system consists of two phases, stationary phase and mobile phase. When a moving phase flows through a stationary phase with a sample, each component moves at different speeds and separates from each other because of the different concentration ratios between the two phases. The stationary phase can be solid or liquid supported by solid or gel. [1]

1.1 High Performance Liquid Chromatography

High performance liquid chromatography (HPLC) uses liquid as mobile phase, pumping a single solvent with different polarity or mixing solvent with different proportion into chromatographic column. The components in the column are separated and then enter the detector (such as ultraviolet, fluorescence, diode array detector) for detection. [2] As the most commonly used detection method, HPLC is very suitable for high molecular weight, difficult to gasify, volatile or heat-sensitive substances, and has the following advantages: 1) Resolution higher than other chromatography. 2) Fast, 10 minutes to dozens of minutes to complete. 3) Repeatability, high-performance chromatographic column can be used repeatedly. 4) Automatic operation with high accuracy.[3]

1.2 Chromatography-mass Spectrometry

Chromatography-mass spectrometry (GC-MS) combines the powerful separation function of chromatography with the accurate identification function of mass spectrometry (MS). It can well analyze trace levels of antibiotic residues and simultaneously determine various antibiotic components. Among them, gas chromatography, liquid chromatography and mass spectrometry (LC-MS) are the most widely used. This method has high detection accuracy. Pretreatment operation is relatively simple, combined with solid phase extraction pretreatment method, not only can make the separation speed and separation degree higher, but also reduce the consumption of liquid, reduce the cost of analysis. However, the detection limit achieved by this method is not high, and the pretreatment method is tedious, inconvenient and time-consuming. [2]

2. Spectral Method

Spectrometry, as a new nondestructive and rapid detection technology, is also used to detect antibiotic residues in water. Spectral data usually need to be analyzed by chemometrics, including spectral pretreatment and modeling. The multivariate calibration model of sample composition data and spectral data was established by chemometrics. Finally, the model was used to predict unknown samples. Although spectroscopy does not require complex pretreatment, simple operation and less sample loss, and can be used for in situ detection of samples, its detection limit is inferior to chromatographic method. [2]

2.1 Infrared Spectroscopy

Infrared spectrophotometry works in the infrared light. And different chemical bonds or functional groups in the molecule can occur vibration absorption when the infrared spectrum in different positions, so it can qualitatively analyze of substances. Because of the miniaturization of dopants in water and the complexity of water, conventional one-dimensional spectroscopy cannot effectively extract the characteristic information of dopants, thus two-dimensional correlation spectroscopy must be used to analyze the information source. [2]

2.2 Fluorescence Spectrometry

Fluorescence spectroscopy is based on the mechanism of molecular absorption spectrum and fluorescence spectrum energy level transition. Substances with the ability to absorb photons can emit fluorescence longer than the excitation wavelength under a certain wavelength of light. The fluorescence spectra of materials are used for quantitative analysis. The fluorescence properties of different kinds of antibiotics are quite different, and the environmental conditions (such as solvent, temperature, pH value, surfactant, derivative reagent, etc.) also have great influence on the fluorescence characteristics. [2]

Considering the advantages and disadvantages of the above methods and the available resources in the laboratory, TUST_China choses high performance liquid chromatography to complete the experiment, and summarized a set of best scheme in the experiment.

Experimental results

Standard sample: 0.1mg/ml Standard sample: 0.05 mg/ml Standard sample: 0.01 mg/ml Standard sample: 0.001mg/ml Rivers nearby the fish pond sample: Fish pond sample: Chicken feces sample: Pig feces sample:

All of the images show the peak at around 8.4min.

Because the machine is not enough sensitive to the samples(the concentration of TC is too low), so there are no peaks in the images. And the altitude of 0.0001mg/ml is very low, so we predict that it can only detect the concentration above 1ug/ml.


[1] Zhang Shuzheng. Fundamentals of Liquid Chromatography. Bulletin of [J]. Microbiology, 1978 (02): 29-37.

[2] Wu Nan, Haixia, Yang Renjie, Wang Jian, Xu Chen-shan, Wang Jun, Zeng Ming. Application of Chromatography and Spectroscopy in the Detection of Milk Antibiotics [J].Heilongjiang Animal Husbandry Veterinary, 2018 (17): 194-197.

[3] Chapter 1. The Advantages and Application of High Performance Liquid Chromatography in Practical Problems [J]. Chemical Management, 2014 (24): 127.