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Detecting and Removing Mycotoxins in the Food Chain
Exploring Chromatographic Techniques
No matter whether it is for purification, identification, or extraction of specific compounds from a mixture, we need effective ways to separate individual compounds from a mixture. Chromatography is a useful technique to separate compounds, such as amino acids, pesticides and drug molecules for qualitative and quantitative analysis.^1
In chromatography, the properties of a stationary phase and a mobile phase are utilized to separate molecules in a mixture. The molecules are carried by the mobile phase to move along the stationary phase. Due to the different properties of different molecules, they will elute at different times.
The continuous development of stationary phase and mobile phase chemistry, as well as the use of different combinations of the two phases bring new advancement to our modern chromatography methods. Chromatography can be classified into two types, planar and column chromatography. The stationary phase in planar chromatography is in the form of a flat thin layer while that of column chromatography is packed into a column.^2 Thin-layer chromatography (TLC) and paper chromatography are the common planar chromatography techniques. On the other hand, gas chromatography (GC) and liquid chromatography (LC) are popular examples of column chromatography.
Nowadays, column chromatography has become a common tool for analytical laboratories to quantify and identify compounds. With the development of different lengths and materials in the packed column, column chromatography can now detect and analyze multiple compounds in a mixture in a single injection. In particular, GC and LC are two of the most popular techniques widely used in testing laboratories and academic institutions across the globe.
1. What is gas chromatography and how does it work?
In a nutshell, GC uses gas as the mobile phase to separate compounds in a mixture. The stationary phase is a long, coiled column. GC takes advantage of the differences in boiling point or vapor pressure of different compounds in a mixture to separate different compounds. It was developed in 1952 by James and Martin who successfully separated volatile fatty acids using a stream of gas.^3
By Elizabeth Suk-Hang LAM
Figure 1. Schematic diagram showing different parts of a GC.
Figure 1 shows a schematic diagram of a GC. The sample mixture containing volatile compounds of interest is injected through the injection port into the GC. The mixture is then heated by the oven and the compounds in the mixture are vaporized. The carrier gas supply (such as helium) provides a continuous gas flow (mobile phase), which carries the gaseous sample compounds into the column (stationary phase). Differences in mass and chemical properties of the component gaseous molecules result in the difference in elution points, enabling separation. The detector then generates an electronic signal which is proportional to the amount of compounds hitting the detector. By recording the time when a compound is detected and its signal intensity, each compound can be identified and quantified.^4
2. How is gas chromatography useful in reality?
Case 1 – Applications in the pharmaceutical industry
GC is an important safeguard in drug production. During the synthesis of drugs, some solvents used in production may remain with the drug compounds. These organic solvents include acetone, ethyl acetate, isopropyl alcohol, methanol, tetrahydrofuran and toluene which are popular in the manufacture of active pharmaceutical ingredients. As these compounds are volatile and have low boiling points, GC is useful to monitor the residual solvents in drug formulations to make sure they do not exceed the safety threshold. Even at low oven temperatures, GC is able to retain the solvent molecules. Therefore, by adjusting the oven temperatures, the solvent molecules can be separated. Due to the associated health concerns, the quantity of residual organic solvents in the pharmaceutical industry is routinely measured by GC.^5
Case 2 – Pesticides residue analysis
When GC is coupled with mass spectrometry (MS), it is extremely useful in pesticide residue analysis. For instance, organochlorine pesticides (OCPs) are highly toxic organic compounds. This class of pesticides includes the notorious DDT (dichlorodiphenyltrichloroethane), chlordane, aldrin, dieldrin, endrin, heptachlor, and hexachlorobenzene.^6 They are commonly used as pesticides in agriculture, causing contamination of soil and groundwater. Some OCPs are very persistent and can accumulate in the food
Detector
Carrier Gas Supply
Flow Direction
Oven
Column
Injector
signal which is proportional to the amount of each compound reaching the detector. By recording the time when a compound is detected and its signal intensity, each compound can be identified and quantified.
Figure 2 - Schematic diagram of the LC system.
There are four common types of LC according to the different types of column (stationary phase).^12
a. Normal phase LC – the stationary phase is polar while the mobile phase is non-polar. For example, the column is made up of silica, while hexane, chloroform and diethyl ether could be used as the mobile phase.
b. Reverse phase LC – the mobile phase is polar while the stationary phase is non-polar or hydrophobic, which is the reverse of the normal phase LC. This enables the column to retain more organic compounds as they are often non-polar.
c. Size-exclusion LC – the column is composed of precisely controlled substrate molecules. The separation of compounds is based on the difference in molecular sizes.
d. Ion-exchange LC – the stationary phase has a charged surface opposite to that of the compounds of interest. An aqueous buffer is used as the mobile phase to control pH and ionic strength.
6. What’s the difference between LC and HPLC?
Nowadays, high-performance liquid chromatography (HPLC) may be a synonym of LC for laboratories. In traditional LC, solvents travel in a natural flow under the force of gravity. In HPLC, solvents travel under high pressure generated by a pump to overcome densely packed columns. This reduces the time of separation and saves instrumentation time.
7. How is liquid chromatography useful in reality?
LC is widely used in the areas of pharmacy, environmental, forensic, food and clinical analysis where it helps in the separation and purification of compounds of interest.
Pump Column
Mobile Phase
Detector
Injector
Column
- Pharmaceutical applications: By analyzing the components in drug samples, LC can be used to control drug stability, study dissolution characteristics as well as quality control. - Environmental applications: Water pollution can be monitored by LC as it can identify and quantify pollutants and potentially harmful substances in water samples, such as drinking water and groundwater. - Forensic applications: The molecules in textile dyes can be analyzed by LC to check whether the compounds included are safe to be in contact with humans. The presence of drug and steroid components in biological samples can also be determined. - Food and flavor applications: The sugar content in fruit juices and products can be quantified, and preservatives in vegetables or dried fruits can be analyzed by LC. - Clinical applications: LC can detect endogenous neuropeptides in biological samples like blood and urine, which is helpful to monitor people’s health.
8. Does liquid chromatography have limitations?
Of course! Although LC can detect non-volatile compounds, it has its own limitations. The elution of LC depends strongly upon the polarity of the compounds of interests as well as the mobile phase. If there are multiple compounds with similar polarity and affinity towards the stationary phase, it is hard to separate them in the LC system. This could result in a broad peak or a merged peak. In addition, the LC column is delicate and requires good care. For example, if the sample mixture contains hydrophobic contaminants, they can stick to the column and reduce the retention power of the column to the compounds of interests. Similarly, if the mobile phase solvents are not pure, impurities could also bind to the column and interfere with the results.
9. Summary
Although GC and LC have their own drawbacks, they still dominate in separation techniques. Their robustness and the continuous development of many spectral libraries have made chromatography the dominant technique for quantification and identification in the food, pharmaceutical and manufacturing industries.
