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Food, Flavors, & Fragrances Article

Evaluating Undiluted Essential Oils

By Julie Kowalski, Ph.D., Innovations Chemist
  • Improve accuracy and speed by eliminating dilution.
  • Prevent downtime due to plugged equipment.
  • Optimize your method to ensure a clean baseline.

Gas chromatography-mass spectrometry (GC/MS) is used extensively by the essential oil industry. Essential oils are complex natural products consisting of many components that span a wide concentration range. This complexity makes the analysis of essential oils challenging. Many methods involve sample dilution to prevent syringes from clogging but this is not always necessary and can compromise analytical quality, especially for less abundant compounds near the solvent peak. The analytical method used will dictate the quality of the data collected; therefore it is important to optimize your GC/MS method to obtain the most accurate data.

Part of method development is determining the best way to inject your sample. In most cases, for GC analysis, the sample is diluted into an organic solvent like methylene chloride or methanol. This dilution helps to adjust the concentration of the sample so that the column and detector will not be overloaded and to decrease the viscosity of thicker oils. Whenever possible, methods are optimized to avoid compounds of interest eluting near the solvent peak. However, with natural products like essential oils, the analyst does not control the components or their concentrations. Injection of a diluted essential oil may cause components to be obstructed by the solvent peak—and this can be considered an adulteration of the sample.

Injecting essential oils without dilution is an easy solution to this problem, but care must be taken to avoid sample carryover. This can be accomplished by extensively rinsing the syringe. Determining the best solvents and number of times your syringe is rinsed is an easy, yet important, part of method development. Most autosamplers are equipped to allow a syringe rinse program to consist of two solvents. Most essential oils will be soluble in alcohols like methanol and ethanol. Methylene chloride is an excellent general solvent. The following example illustrates the process used to determine the number of syringe rinses required so that no sample memory or syringe plugging occurred.

Figure 1 shows typical neat essential oil chromatography — many peaks at very different signal intensities. Note the syringe was rinsed ten times with ethanol and ten times with methylene chloride directly after this injection. It is important to rinse immediately after the injection so that the essential oil sample does not sit in the syringe during the runtime and plug the syringe. Immediately before the next injection, the syringe is rinsed an additional two times with each solvent. During method development, you should increase the number of times the syringe is rinsed until no sample component peaks are present in a blank injection. Insufficient rinsing is characterized by a few peaks which correspond to the most abundant compounds in the sample (Figure 2). The number of rinses should be increased until the solvent blank injection results in a clean baseline, free of any sample carryover (Figure 3). The best solvents and the number of rinses required may vary with specific essential oils but investing the time in optimizing this part of your method allows you to analyze essential oils without dilution and is critical to obtaining accurate results, particularly for less abundant or early eluting compounds.


Figure 1  Undiluted peppermint oil—many peaks with variable signal intensity.

  1. menthone
  2. menthol
  3. menthone
  4. menthol
  5. menthyl acetate
(A) Representative chromatogram from commercial peppermint sample B


(B) Separation of major component isomers using the Rt-γDEXsa™ phase for four commercial samples which differ in harvest location and year.

Column:
Rt-γDEXsm™ 30m x 0.25mm ID, 0.25µm (cat.# 13113)
Sample:
commercial peppermint oil, sample B (Mentha piperita, Mountain Rose Herbs, certified organic by Oregon Tilth, therapeutic grade according to international standards)
Inj.:
1.0µL neat, split (split ratio 1:150)
Inj. temp.:
230°C
Carrier gas:
helium, constant pressure
Flow rate:
35cm/sec. at 100°C
Oven temp.:
40°C to 120°C @ 5°C/min. to 135°C @ 3°C/min. to 200°C @ 5°C/min.
Det:
MS
Transfer line temp.:
200°C
Scan range:
40-300amu
Ionization:
EI
Mode:
scan

GC_FF00802

Figure 2  Inadequate rinsing—sample carryover is still apparent in the solvent blank even after five ethanol and five methylene chloride rinses.


Column:
Rt-γDEXsa™, 30m, 0.25mm ID, 0.25µm (cat.# 13113)
Sample:
Solvent blank following injection of commercial peppermint oil sample B, five ethanol rinses, and five methylene chloride rinses
Inj.:
1µL, split (split ratio 1:150)
Inj. temp.:
230°C
Carrier gas:
helium, constant pressure
Flow rate:
35cm/sec. at 100°C
Oven temp.:
40°C to 120°C @ 5°C/min., to 135°C @ 3°C/min., to 200°C @ 5°C/min.
Det:
MS
Transfer line temp.:
200°C
Scan range:
40-300amu
Ionization:
EI
Mode:
scan

GC_FF00913

Figure 3  Sufficient rinsing—flat baseline with no sample carryover following ten ethanol and ten methylene chloride rinses.


Column:
Rt-γDEXsa™, 30m, 0.25mm ID, 0.25µm (cat.# 13113)
Sample:
Solvent blank following injection of commercial peppermint oil sample B, ten ethanol rinses, and ten methylene chloride rinses
Inj.:
1µL, split (split ratio 1:150)
Inj. temp.:
230°C
Carrier gas:
helium, constant pressure
Flow rate:
35cm/sec. at 100°C
Oven temp.:
40°C to 120°C @ 5°C/min., to 135°C @ 3°C/min., to 200°C @ 5°C/min.
Det:
MS
Transfer line temp.:
200°C
Scan range:
40-300amu
Ionization:
EI
Mode:
scan

GC_FF00914


How to optimize a syringe rinse program for injecting undiluted essential oils:

  1. Determine the best solvents for your essential oil by literature review or experimentation. Many common essential oils are soluble in ethanol and methylene chloride.
  2. Perform a blank injection to determine the baseline.
  3. Inject your essential oil. Typically, you will not need to inject more than one microliter with the GC set to run at a 100-300:1 split ratio.
  4. The method should be programmed to rinse at least five times with at least one solvent immediately after sample injection.
  5. After the sample run is complete, rinse the syringe at least one more time immediately before injection of a blank.
  6. Examine the chromatogram for chromatographic peaks. Be sure to use the zoom feature so that you can investigate at low levels. The baseline should be flat.
  7. If peaks are observed, increase the number of rinses and/or add the second solvent.
  8. Repeat until the baseline is flat.