Application Note

Food Testing and Agriculture

AuthorVanessa Abercrombie

AbstractThe analysis of cold-pressed citrus essential oils, such as pink grapefruit, can be challenging due to the presence of higher molecular weight aromatic compounds, which can cause carryover or long run times. The Agilent J&W DB-HeavyWAX column, with a maximum operating temperature of 280 °C isothermal and 290 °C programmed, can operate at temperatures greater than traditional WAX-type columns. The increased temperature range allows faster and more reproducible analysis of compounds in cold-pressed pink grapefruit oil over extended periods of time. The DB-HeavyWAX showed very similar selectivity to the Agilent J&W DB-WAX GC columns.

Reduced carryover and increased reproducibility in the analysis of cold-pressed pink grapefruit essential oil

Using an Agilent J&W DB-HeavyWAX GC column

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IntroductionEssential oils are used in many household items, from cleaning products to perfumes, and contain a great deal of aromatic compounds. The two primary extraction methods for these essential oils are distillation and cold-pressing. Steam distillation applies heat to the organic material, while cold-pressing, also referred to as expressed, is a mechanical process where no heat is applied1. An advantage of the cold-pressed extraction process is that it contains higher boiling and higher molecular weight compounds that are lost in the steam distillation process. The higher molecular weight compounds produce a fresher and more flavorful aroma, and are widely used to extract citrus oils, such as pink grapefruit essential oil2.

Analyzing compounds in cold-pressed essential oils is commonly done with a 100 % polyethylene glycol phase column, also known as a WAX column. Due to issues such as decreased thermal stability, traditional WAX columns have a limited maximum operating temperature of 250 °C isothermal and 260 °C programmed. This limit can cause difficulty when analyzing the higher molecular weight compounds found in cold-pressed citrus essential oils. To work around the issue of analyzing all the compounds in cold-pressed citrus essential oils, several options are available:

• Hold the final oven temperature at the maximum for an extended period

• Risk taking the column above the stated Maximum Allowed Operating Temperature (MAOT)

• Select a different column with a phase that allows the use of higher temperatures, but may not have the optimum selectivity

Increasing the final hold time on a GC run may facilitate the elution of some later-eluting compounds, but it will also increase the overall run time. Taking a column above the stated MAOT, to elute all of its volatile compounds, risks damage to the phase. This damage can be seen in shifting retention times, increased column bleed, and sometimes a change in the retention order of certain compounds over extended periods of time3.

The Agilent J&W DB-HeavyWAX has an extended temperature limit of up to 280/290 °C. This permits the analysis of the higher boiling compounds in cold-pressed citrus oil without risking the integrity of the column phase. This Application Note examined the benefits of the extended temperature limit of the DB-HeavyWAX column. This increased temperature limit can increase the

reproducibility and decrease carryover in the analysis of cold-pressed pink grapefruit essential oil. The thermal stability of a traditional WAX column versus the DB-HeavyWAX was examined, with the application of cold-pressed pink grapefruit essential oil. This was done to demonstrate the improved thermal stability of the DB-HeavyWAX.

ExperimentalAn Agilent 7890 GC/FID equipped with a split/splitless inlet, and an Agilent 7693 sampler with Agilent MassHunter control software was used for GC/FID experiments.

Sample preparationA sample of cold-pressed pink grapefruit essential oil was purchased from Emily’s Oils & Essentials (Fair Oaks, CA), and injected neat.

GC Conditions

ColumnAgilent J&W DB-HeavyWAX, 30 m × 0.25 mm, 0.25 µm (p/n 122-7132) Agilent J&W DB-WAX, 30 m × 0.25 mm, 0.25 µm (p/n 122-7032) Commercially available WAX column, 30 m × 0.25 mm, 0.25 µm

Carrier Helium, constant flow, 1 mL/min

Oven Program 1: 60 °C (2.0 minutes), ramp 5 °C/min to 250 °C (30 minutes) Program 2: 60 °C (2.0 minutes), ramp 5 °C/min to 280 °C (30 minutes)

Inlet Split mode, 250 °C, split ratio 200:1

Inlet liner Ultra Inert, low pressure drop (p/n 5190-2295)

GC/FID Agilent 7890B GC equipped with FID/MSD

Sampler Agilent 7693 autosampler

FID conditions

Temperature 280 °C

Hydrogen 30 mL/min

Air 400 mL/min

Col + make up 25 mL/min

Flowpath supplies

Septum Bleed and temperature optimized, BTO, 11 mm septa (p/n 5183-4757, 50/pk)

Gold seal Ultra Inert gold seals (p/n 5190-6145, 10/pk)

Vials 2 mL, screw top, amber, write-on spot, certified (p/n 5182-0716, 100/pk)

Vial inserts 250 µL glass inserts, deactivated (p/n 5181-8872, 100/pk)

Vial caps 9 mm blue screw cap, PTFE/red silicone septa (p/n 5185-5820, 500/pk)

Inlet/FID 85:15 Vespel: graphite ferrules (p/n 5062-3508, 10/pk)

Instrument conditions

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Results and discussionA sample of cold-pressed pink grapefruit essential oil was injected onto a DB-HeavyWAX column. An initial oven temperature of 60 °C was held for two minutes, then ramped to a final temperature at a rate of 5 °C/min,

and held for 30 minutes. Figure 1 demonstrates the effect of achieving a final temperature of 280 °C versus 250 °C on the analysis of pink grapefruit essential oil. By increasing the final temperature, the peak shape is better for the later-eluting compounds, and it is also possible to elute more compounds.

Figure 1. A sample of pink grapefruit essential oil injected on an Agilent J&W DB-HeavyWAX column run to a final oven temperature of 250 and 280 °C.

×102

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×102

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Final temperature: 250 °C

A

B

Final temperature: 280 °C

Acquisition time (min)

Coun

ts (%

)Co

unts

(%)

3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 55 57 59 61 63 65 67 69

The largest of the two final eluting compounds, Meranzin and Isomeranzin, did not elute with a final temperature of 250 °C and a hold time of 30 minutes. Therefore, these compounds are present in subsequent injections, as seen in Figure 2, as carryover. This carryover causes irreproducible and incorrect results if the column is not operated at a final temperature of 280 °C.

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Figure 3 illustrates five of the aromatic compounds identified by mass spectrometry in cold-pressed pink grapefruit essential oil that benefit from the increased MAOT of the DB-HeavyWAX column.

Figures 4A and 4B demonstrate the shortcomings of a traditional WAX column with maximum operating temperatures of 250/260 °C, when taken above its MAOT. In Figure 4A, a sample of cold-pressed pink grapefruit essential oil was injected onto a commercially

available traditional WAX column, and run to a final temperature of 280 °C. To examine what happens to the resolution and thermal stability of a traditional WAX column taken above the MAOT over an extended period, four well resolved peaks were chosen and identified.

42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Initial injection

Repeat injection

Meranzin

Isomeranzin

Osthole

Figure 2. Carryover in repeated injections of pink grapefruit essential oil run to a final oven temperature of 250 °C, held for 30 minutes.

Figure 3. Higher molecular weight compounds in pink grapefruit essential oil (cold-pressed) that elute with a final oven temperature of 280 °C. These compounds were identified using mass spectrometry.

58.996

51.390

59.466

55.967

68.174

Auraptenol260.289 g/mol

Meranzin260.289 g/mol

Bergapten216.192 g/mol

Osthole244.29 g/mol

Acquisition time (min)50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72

Isomeranzin260.289 g/mol

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Figure 4a. A sample of cold-pressed pink grapefruit essential oil injected onto a commercially available WAX column, run to a final oven temperature of 280 °C

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Acquisition time (min)

Resp

onse

uni

ts (%

)

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54

1

2

Peak ID1. Linalool2. γ-Amorphene

Figure 4b. A sample of pink grapefruit essential oil (cold-pressed) run on a commercially available WAX column, operated for 100 hours at 280 °C.

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0 Hours spent at 280 °C

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50 Hours spent at 280 °C

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13.5 13.7 13.9 14.0 14.2 14.4 14.6 14.8 15.0 15.2 15.4 15.6 15.8 16.0 16.2 16.4 16.6 16.8 17.0

1

1

1, 2

2

2

Acquisition time (min)

Resp

onse

uni

ts (%

)Re

spon

se u

nits

(%)

Resp

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uni

ts (%

)

Peak ID1. Linalool2. γ-Amorphene

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Figures 4A and 4B demonstrate that the two peaks, linalool and γ-amorphene, confirmed with a mass spectrometer, are fully resolved upon their initial injection of pink grapefruit essential oil, and zero operating hours at 280 °C. After 50 hours of operation at 280 °C, the retention times of the peaks have not only shifted earlier, but linalool and γ-amorphene coelute. Operation of this column at 280 °C for 100 hours demonstrates greater retention time shifting, coelution, and a change in position of the linalool and γ-amorphene peaks.

The shift in retention time, seen in Figure 4B, indicates that the phase in the commercially available WAX column has been significantly degraded after repeated use at 280 °C. This loss of phase has caused the four compounds to elute at different parts of the temperature programs, and has changed their elution order.

Figure 5 demonstrates the improved thermal stability of the DB-HeavyWAX column when operating at high temperatures for extended periods of time. Even after 100 hours of use at 280 °C, the elution order on the DB-HeavyWAX is still the same, which is not the case with the competitor WAX column. The stability of the elution order over long periods of time at high temperatures demonstrates the improved thermal stability of the DB-HeavyWAX.

0 Hours at 280 °C

50 Hours at 280 °C

100 Hours at 280 °C

Agilent J&W DB-HeavyWAX

1

2

1

2

1

2

Competitor WAX column

1

2

1

2

1,2

Figure 5. Comparison of stationary phase thermal stability over 100 hours operating at 280 °C for the Agilent J&W DB-HeavyWAX and a traditional commercially available WAX column.

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Figure 6 shows the same sample of cold-pressed pink grapefruit essential oil run on a DB-WAX and a DB-HeavyWAX column to a final oven temperature of 250 °C. This temperature was held until the final peaks eluted after 60 minutes. Both the DB-WAX and the DB-HeavyWAX show similar selectivity for the analysis of cold-pressed pink grapefruit essential oil.

ConclusionThe Agilent DB-HeavyWAX column provides an increased maximum temperature range. This range allows for more reproducible analysis of higher boiling compounds in cold-pressed citrus essential oils, without fear of carryover from sample to sample. Traditional WAX columns have a lower upper temperature limit, and cannot maintain retention time stability at high temperatures, such as 280 °C, for extended periods of

Figure 6. Agilent J&W DB-WAX has a similar selectivity to Agilent J&W DB-HeavyWAX, as demonstrated in the analysis of pink grapefruit essential oil.

Agilent DB-HeavyWAX

Agilent DB-WAX

time. The increased temperature limit of the DB-HeavyWAX provides a more thermally stable column, even after 100 hours operating at 280 °C. The DB-HeavyWAX has a similar selectivity to an Agilent DB-WAX, allowing for easy method translation when replacing a DB-WAX column with a DB-HeavyWAX. The DB-HeavyWAX has the added benefit of the extended temperature limit, up to 280 °C isothermal and 290 °C programmed.

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This information is subject to change without notice.

© Agilent Technologies, Inc. 2018 Printed in the USA, March 19, 2018 5991-9078EN

References1. https://naha.org/explore-

aromatherapy/about-aromatherapy/how-are-essential-oils-extracted, Accessed 8 January 2018.

2. Lin, J.; Rouseff, R. L. Characterization of aroma-impact compounds in cold-pressed grapefruit oil using time–intensity GC–olfactometry and GC–MS. Flavor and Fragrance Journal 2001, 16, 457–463.

3. Abercrombie, V.; Provoost, L. Increased Thermal Stability and Maximum Temperature of the Agilent J&W DB-HeavyWAX Column. Agilent Technologies Application Note, publication number 5991-9035EN, 2018.