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Method quickly sniffs out fragrance molecules: [UPC.sup.2] technology, teamed with mass spectrometry, enables simple, quick chiral analyses of fragrance compounds.

Perception of aroma occurs at the olfactory membrane. This membrane is comprised in part of proteins and carbohydrates, which are chiral in nature. This makes it possible for the olfactory receptors to distinguish between enantiomers. Many enantiomers of fragrance molecules are perceived differently by our sense of smell. For example, carvone is a chiral terpenoid where the R enantiomer smells like spearmint while the S enantiomer has the distinct odor of caraway seed.

Chiral composition of fragrance molecules is important for many industries, including food, cosmetics and consumer products, in order to control the olfactory perception of their products. In addition, chiral analyses are routinely performed to authenticate the natural sources of essential oils. Since naturally chiral sources of essential oils are generally more costly and have a greater perceived health benefit than their synthetic counterparts, rapid chiral analysis allows manufacturers to quickly exclude adulterated products containing inexpensive racemic synthetic materials at the time of purchase.

Historically, chiral separations of fragrance compounds have primarily been carried out using chiral stationary phases (CSPs) in capillary gas chromatography (GC). The analysis time often ranges from 15 to 50 min. More recently, supercritical fluid chromatography (SFC) with CSPs has been applied to these separations, often resulting in comparable resolution and reduced run time. Despite the great success in chiral separation by SFC, the associated instrumentation and CSPs have been slow to tap into the technology advancements that have taken place in the HPLC field. For example, one of most significant breakthroughs in HPLC in the past decade is the advent of the ACQUITY UPLC system that capitalizes on sub-2 pm particles. The ACQUITY UPLC system retains the practicality and principles of HPLC while increasing the overall interlaced attributes of speed, sensitivity and resolution. Until very recently, the standard particle size for commercially available CSPs has remained 5 pm.

Convergence chromatography is the next evolution in SFC. UltraPerformance Convergence Chromatography ([UPC.sup.2]) is a holistically designed system that has similar selectivity to normal phase chromatography. The [UPC.sup.2] system offers minimized system and dwell volume, enabling users to leverage the superior separation power inherent to smaller particle sizes.

Presented here is the enantiomeric and diastereomeric separations of tour fragrance compounds using ACQUITY [UPC.sup.2] Trefoil AMY 1 and CEL1 columns on a Waters ACQUITY [UPC.sup.2] System. Compared to the traditional method of analysis by GC, the [UPC.sup.2] chromatography offered similarly high resolution with significantly shorter run times, resulting in improved productivity.

Experimental

Instrumentation: All experiments were performed on a Waters ACQUITY [UPC.sup.2] System equipped with an ACQUITY [UPC.sup.2] PDA detector and an ACQUITY TQD mass detector. The system is controlled by MassLynx software.

Samples: The standard samples used in this study were purchased from TCI Americas. Essential oils were purchased from various commercial sources. All samples were dissolved in tert-butyl methyl ether (TBME) for the analyses.

[UPC.sup.2] conditions: The chiral separations were performed on either a Waters ACQUITY [UPC.sup.2] Trefoil AMY1 or CEL1 column (3.0 x 150 mm, 2.5 pm). For all experiments, the backpressure was 1740 psi and the temperature was 40 C. The mobile phases were A: C[O.sup.2] and B: isopropanol. The MS was operated under APCI positive mode.

Results and discussion

S(+) carvone, R(-) carvone and racemic carvone were all separated on an [UPC.sup.2] Trefoil CEL1 column. The chromatograms (not pictured) revealed that S(+) carvone peaked at 1.85; R(-) carvone peaked at 1.76; and racemic carvone peaked at 1.75 and 1.86. The enantiomeric pair was baseline resolved in less than 2.5 min. The peak widths at half-height were 2 to 3 sec. It is also interesting to note that there were detectable antipode present in both single enantiomer standards (S(+) carvone and R(-) carvone). In both cases, the minor peaks account for approximately 1% of the main peaks, resulting in a 98% enantiomeric excess (e.e.). This example clearly demonstrates a high efficiency chiral separation, resulting in short analysis time, sharp peaks and improved detection sensitivity.

Linalool is a terpene alcohol with a soft floral odor. Linalool can be found in different plant extracts. Figure 3A shows the enantiomeric resolution of the linalool standard on an ACQUITY [UPC.sup.2] Trefoil AMY1 column. It is noted that the linalool standard was non-racemic (Figure 1A), suggesting the standard was derived from a natural source. The e. e. was estimated to be 40% in favor of the late eluting isomer. Figure IB is the [UPC.sup.2]-UV chromatogram of a commercially available lavender essential oil obtained under the same condition. The two linalool enantiomers were identified by both retention time and corresponding mass spectra (results not shown). It is noted that MS plays a critical role for the positive identification of the target analytes in a complex matrix. While bearing a similar selectivity to normal phase LC, [UPC.sup.2] is inherently advantageous in incorporating MS detection because of its MS-friendly mobile phase. The linalool in this lavender essential oil exhibited a 92% e. e. in favor of the later eluting peak at 2.07 min.

Similarly, terpinen-4-ol is a terpene with a pleasant conifer odor. It is a major constituent of tea tree oil. The researchers ran an enantiomeric resolution of the two isomers of a terpinen-4-ol standard on an ACQUITY [UPC.sup.2] Trefoil AMY1 column. The results indicated that the terpinen-4-ol standard was nearly racemic, suggesting its synthetic origin. Examination of a tea tree essential oil revealed that the terpinen-4-ol exhibited a 37% e. e. in favor of the earlier eluting isomer at 1.95 min.

Nerolidol is a sesquiterpene with a pleasant woody odor reminiscent of fresh bark. It can be found in the neroli essential oil derived from the bitter orange plant. The nerolidol molecule contains a chiral center and a double bond-generating cis/trans isomerism, resulting in four possible stereoisomers in a mixture. Figure 2 shows the simultaneous separation of all four nerolidol isomers on an ACQUITY [UPC.sup.2] Trefoil AMY 1 column in less than 3 min. Figure 2B is the selected ion recording (SIR) for the isomeric mixture at m/z 205.2, corresponding to the [(M+H)-H20]+ of nerolidol. The observation of the base peak of nerolidol resulting from the loss of water is consistent with other reports.

This study has demonstrated the successful chiral separations of some fragrance compounds on the Waters ACQUITY [UPC.sup.2] Trefoil AMY 1 and CEL1 columns using [UPC.sup.2]. The low system volume and extra-column volume of the [UPC.sup.2] system, combined with the reduced particle size of the Trefoil AMY1 and CEL1 columns, enable superior, faster and more efficient separations compared with traditional SFC and GC. The demonstrated analysis times range from 2 to 3 min, significantly shorter than the 15 to 50 minute analysis time typical for chiral GC, which allows for a fast authentication of the natural sources of essential oils. In all cases, the closely eluting isomers were baseline resolved. For the essential oil analysis, the oil samples were diluted and directly injected onto a [UPC.sup.2] system without tedious sample preparation. Furthermore, the inherent compatibility between [UPC.sup.2] and MS offered an unambiguous identification of the target analytes in a complex sample matrix. The high efficiency, short analysis time and simple sample workup demonstrated in this study would be beneficial to industries where chiral analyses of fragrance compounds are routinely performed.

by John McCauley and Rui Chen, Waters Corp., Milford, Mass.
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Author:McCauley, John; Chen, Rui
Publication:Chromatography Techniques
Geographic Code:1USA
Date:Feb 1, 2014
Words:1263
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