FOOD ANALYSIS: CHROMATOGRAPHY
Diagram
Two ways to separate a mixture
Gas chromatography (GC)
A gas carries the sample through a long coiled column in an oven. Only substances that can vaporise will travel through it.
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High performance liquid chromatography (HPLC)
A liquid pumped at high pressure carries the sample through a steel column packed with fine particles. Almost anything that dissolves will travel through it.
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The choice
GC suits volatile substances such as fatty acids and flavours. HPLC suits dissolved substances such as sugars, vitamins and preservatives.
Figure 1: GC uses a gas as the moving fluid and HPLC uses a liquid. Drawn in our own style from the chromatography unit of the FTN Instrumental Methods of Food Analysis notes, Unit 3, and from Holcapek and Byrdwell, Handbook of Advanced Chromatography / Mass Spectrometry Techniques.
Introduction
Gas chromatography, written GC, and high performance liquid chromatography, written HPLC, are the two main chromatographic methods of food analysis, and both separate a mixture by carrying it in a moving fluid past a stationary material. The difference is the moving fluid: GC uses a gas, while HPLC uses a liquid pumped at high pressure. That one difference decides what each method can measure: GC needs substances that can vaporise, while HPLC needs substances that dissolve.
The FTN Instrumental Methods of Food Analysis notes, Unit 3, walk through both methods side by side, from paper chromatography to HPLC and gas chromatography, and use the same worked example, the sugars in honey, to show where each method fits. This article follows that unit, with the two books below.
Quick comparison
Table 1: GC compared with HPLC
| Aspect | GC | HPLC |
|---|---|---|
| Mobile phase | A gas, often helium or nitrogen, called the carrier gas | A liquid, pumped through at high pressure |
| Stationary phase | A liquid held on a solid support inside the column | Fine particles packed into a steel column |
| Sample form | Must vaporise in a heated inlet before it can travel | Must dissolve in the mobile phase |
| How separation happens | Substances divide between the gas and the liquid, and by their volatility: a substance that likes the liquid stays longer, and one that vaporises easily moves faster | Substances divide between the moving liquid and the stationary particles: how much a substance likes each one decides how long it stays |
| Column | A long coiled column held in an oven, a thin tube tens of metres long | A short steel column packed with very fine particles |
| Detector | Often a flame ionisation detector, which burns the substances and measures the ions formed | Ultraviolet absorption, fluorescence, refractive index or mass, chosen for the substances being measured |
| Best for | Volatile substances: fatty acids, flavours, aromas, alcohols and pesticide residues that can be vaporised without breaking down | Almost anything that dissolves: sugars, organic acids, vitamins, amino acids, colours, preservatives, mycotoxins and pesticide residues |
| Elution control | Temperature of the oven is raised through the run | The mobile phase can stay the same (isocratic elution) or change gradually (gradient elution) |
| Reading the result | The detector records each substance as a peak; peak area gives the amount and retention time gives the identity, both compared with standards | The same: peak area gives the amount and retention time gives the identity, both compared with standards |
| Limit | A sample that cannot be vaporised, such as a protein or a large sugar, cannot be run unless it is first changed chemically into a volatile form | Cost is in the instrument, the columns and the solvents, and skill is needed to choose the column, the mobile phase and the detector |
Scientific explanation
Definition
Gas chromatography is chromatography with a gas as the mobile phase and a liquid held on a solid support as the stationary phase: the sample is vaporised in a heated inlet and swept through a long coiled column in an oven by a carrier gas, often helium or nitrogen. High performance liquid chromatography is chromatography with a liquid mobile phase pumped at high pressure through a steel column packed with very fine particles. Both separate a mixture into its parts, and both record each part as a peak, but GC works only for substances that can be vaporised, while HPLC works for almost anything that dissolves.
Working principle
In GC, the sample is injected into a heated inlet, where it vaporises, and the carrier gas sweeps it through the column. Substances separate by how they divide between the gas and the liquid, and by their volatility: a substance that likes the liquid stays longer, and a substance that vaporises easily moves faster. The detector at the end, often a flame ionisation detector that burns the substances and measures the ions, records each one as a peak. Fatty acids are first turned into methyl esters, which vaporise easily, and the pattern of peaks gives the fatty acid profile of an oil, which identifies the oil and detects adulteration. In HPLC, the sample is injected, the substances separate in the column, and a detector at the end records each one as a peak. The mobile phase can be kept the same through the run, called isocratic elution, or it can be changed gradually, called gradient elution, so that substances held strongly are pushed out in a reasonable time. The method is quantitative: the peak area gives the amount, and the retention time gives the identity, both compared with standards.
Apparatus
GC needs a heated inlet, a supply of carrier gas, a long coiled column held in an oven, and a detector, often a flame ionisation detector. HPLC needs a high pressure pump, a steel column packed with fine particles, an injector, and a detector, which may measure ultraviolet absorption, fluorescence, refractive index or mass.
Measurement procedure
Both methods follow the same pattern: separate, detect, compare with standards, and correct for dilution. A honey sample shows it: paper chromatography would show its three sugars as spots, but the measurement would be rough, and GC would need the sugars to be changed into volatile forms, which is possible but slow. HPLC with a refractive index detector separates glucose, fructose and sucrose directly, without changing them, and measures each one from its peak area. The worked example is given under Formulas and calculations below.
Factors affecting readings
For GC, the temperature of the oven, the choice of carrier gas, and whether the sample can be vaporised without breaking down decide whether the run works: a substance that breaks down on heating cannot be run. For HPLC, the choice of column, mobile phase and detector decides the separation, and gradient elution is used when some substances are held much more strongly than others. In both methods the result rests on standards run beside the samples, because the peak area is only meaningful when compared with a known amount.
Formulas and calculations
The books give the worked example for HPLC in the honey test. Standards of glucose at 10, 20 and 30 g per litre give peak areas of 100, 200 and 300 units, a straight line with a slope of 10 units per gram per litre. A honey sample, diluted tenfold, gives a glucose peak area of 150 units. The concentration in the diluted sample is 150 divided by 10, which is 15 g per litre. The original honey holds ten times that, which is 150 g per litre. The derivation shows the pattern that runs through all chromatography: separate, detect, compare with standards, and correct for dilution.
For GC the books give no worked numerical example, so none is given here: the same pattern applies, with peak areas compared against standards of the fatty acids or residues being measured.
Industrial applications
- GC: measuring the fatty acid profile of an oil, which identifies the oil and detects adulteration.
- GC: measuring flavours, aromas and alcohols that vaporise easily.
- GC: measuring pesticide residues that can be vaporised without breaking down.
- HPLC: measuring the sugars in honey, glucose, fructose and sucrose, directly and without changing them.
- HPLC: measuring a vitamin in a juice, a preservative in a sauce, or a mycotoxin in a grain, all with the same instrument and different columns.
- HPLC: measuring organic acids, amino acids and colours in foods that dissolve in the mobile phase.
Advantages and limitations
- GC, advantage: its columns give separations that no liquid method can match for volatile mixtures, and the method is fast, sensitive and precise.
- GC, limitation: the sample must vaporise, so proteins and large sugars cannot be run unless they are first changed chemically into a volatile form.
- HPLC, advantage: it separates almost anything that dissolves directly, without changing the sample, and one instrument covers sugars, vitamins, preservatives and mycotoxins.
- HPLC, limitation: the cost is in the instrument, the columns and the solvents, and the skill is in choosing the column, the mobile phase and the detector for the substances at hand.
FAQ
What is the main difference between GC and HPLC?
The mobile phase: GC uses a gas, usually helium or nitrogen, while HPLC uses a liquid pumped at high pressure.
Why is HPLC used for sugars in honey, while GC is used for fatty acids in oil?
Sugars do not vaporise, so GC would need them changed into volatile forms, which is slow; HPLC separates them directly. Fatty acids are turned into methyl esters that vaporise easily, and GC separates them with a sharpness no liquid method can match.
What does the peak area tell the analyst?
The amount: peak area gives the amount of the substance, and retention time gives its identity, both compared with standards.
What is gradient elution in HPLC?
Changing the mobile phase gradually through the run, so that substances held strongly in the column are pushed out in a reasonable time.
Can GC measure a protein?
No, not directly: a protein cannot be vaporised without breaking down, so it must first be changed chemically into a volatile form, or another method such as HPLC must be used.
References and further reading
- FTN subject: Instrumental Methods of Food Analysis, Unit 3: Chromatographic Techniques.
- Michal Holcapek and Wm. Craig Byrdwell (eds.), Handbook of Advanced Chromatography / Mass Spectrometry Techniques, Elsevier, 2017 (Google Books page read)
- Leslie S. Ettre, Chapters in the Evolution of Chromatography, World Scientific, 2008 (Google Books page read)
