Inside:
Definitions ofChirality and ChiralChromatography
Chiral ColumnsOffer UniqueSelectivity
Optimizationof ChiralSeparations
Chiral SpecificApplications ofEssential Oils,Flavors, andPharmaceuticals
A Guide to theAnalysis of Chiral
Compounds by GC
Technical Guide
2
The Rt-βDEXsm and Rt-βDEXsechiral capillary columns offer
extensive enantiomeric separationof monoterpenes, monoterpene
alcohols, and monoterpene ketonesthat cannot be matched by
permethylated β-cyclodextrincolumns. Rt-βDEXsp and Rt-
βDEXsa are secondary columnsthat best resolve specific flavor and
fragrance chiral components. TheRt-βDEXcst provides excellent
resolution of some complex flavorcompounds and has demonstrated
great potential with pharmaceuticalsubstances as well.
Index:
Definitions of Chirality andChiral Chromatography ......3
Chiral Columns OfferUnique Selectivity ...............5
Optimization of ChiralSeparations ..................... 10
Chiral Specific Applicationsof Essential Oils, Flavors, andPharmaceuticals .............. 14
A team of researchers at the
University of Neuchâtel
developed βββββ-cyclodextrins with
superb enantiomeric
selectivity. They joined forces
with Restek, a manufacturer of
top quality columns, to provide
a unique line of commercially
available βββββ-cyclodextrin
stationary phases with
enhanced capabilities for
chiral capillary gas
chromatography.
Dr. Raphael TabacchiBorn in Ticino, Switzerland,Dr. Tabacchi has been aprofessor for Analytical andOrganic Structure at theUniversity of Neuchâtel,Switzerland, since 1978. Hisresearch interests focusedupon natural productchemistry, and developmentof HPLC and GC stationaryphases. He has developed β-cyclodextrins with uniquesubstitutions to create novelchiral phases for capillaryGC.
Dr. Georges Claude SaturninBorn in St. Joseph,Martinique, Dr. Saturninbecame a Senior Assistantand Assistant Professor in1990 at the University ofNeuchâtel. He is involved inthe development of HPLCphases. His focus is thesynthesis of these newcyclodextrin materials thatcharacterize the new chiralcolumns.
Lori BitzerAfter completing herChemistry degree at WestVirginia University in 1995,Lori joined Restek as a FusedSilica ManufacturingChemist. She is involved withthe design and production ofnew products includingcapillary chiral columns. Loriensures product quality andconsistency that arecharacteristic of all Restekproducts.
Sherry SponslerSherry is an ApplicationsChemist and has been withRestek since 1990. Sheconducts method and productdevelopment for analysis offoods, flavors and fragrances,as well as some pharmaceuti-cal samples. Frequentcommunication withcustomers has helped Sherryto identify many importantchiral applications in theseindustries. She has demon-strated many of these keyseparations, especially forfragrances and amphet-amines, using the newcyclodextrin capillarycolumns.
Maurus BiedermannMaurus has been with Dr.Konrad Grob’s GC/LCGCgroup at the KantonalesLaboratory Zurich (OfficialFood Control Authority inSwitzerland) since 1990 andhas participated in thedevelopment of severalLCGC methods for foodanalysis. During hissabbatical at Restek, hedemonstrated the ability ofthese new and unique chiralphases for many applicationssuch as the authenticity ofessential oils, “natural”flavor extracts, and theanalysis of drugs forenantiomeric composition.
Claire-Lise PorretBorn in Neuchâtel, Switzer-land, Ms. Porret waspreviously a technician atNestlé and joined Dr.Tabacchi’s team in 1991. Sheis also involved in thesynthesis of organiccompounds and with thedevelopment of GC andHPLC stationary phases.
○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○
3
Linalool is a chiral compound because it contains an assymmetric carboncenter. The mirror images are not superimposable and so they are enanti-omers.
S configuration
Enantiomers can be distinguished by configuration. Following groups fromhigh to low priority in the clockwise direction is denoted R, and S for thecounterclockwise direction.
R configuration
Figure 1A
Linalool oxides have two chiral centers at carbon numbers 2 and 5 andexist as four enantiomers.
Figure 1B
2S, 5R(+)-cis-linalool oxide
2R, 5S(-)-cis-linalool oxide
2S, 5S(-)-trans-linalool oxide
2R, 5R(+)-trans-linalool oxide
WHAT ARE CHIRALCOMPOUNDS?○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○
Any carbon atom that is bonded tofour different functional groups istermed a chiral or an assymetric car-bon. Molecules containing one ormore of these carbon centers are con-sidered chiral molecules. Chiral cen-ters can exist in two forms calledenantiomers. These two forms arenon-superimposable mirror images ofeach other, but both have similarproperties. For example, both enan-tiomers will have the same boilingpoint, densities, and reaction rates asachiral molecules. They do, however,generally possess different aroma andflavor characteristics; more impor-
tantly, they possess differences intoxicity and biological activity.
Enantiomers are also known as opti-cal isomers because they rotate planepolarized light in different directions.Optical isomers that rotate plane po-larized light to the right, or clockwise,are termed dextrorotary {denoted as(d) or (+)}, Optical isomers that rotatein the left direction are termed levoro-tary {denoted (l) or (-)}.
Enantiomers can be denoted by thespecific configuration around thechiral center. Groups on the carboncenter are assigned a “priority” basedon atomic number of the first bondedatom (Cahn-Ingold-Prelog rules). Thegroup with the highest atomic num-
ber is rated first. If priority cannot beestablished with the first atom, workoutward until priority differences canbe determined. Once priorities havebeen established for all four groups,specific configuration can be deter-mined. An R configuration is desig-nated when the priority around theassymmetric carbon is in a clockwisedirection, whereas a counterclockwisedirection is denoted as S. (Figure 1A)1
A chiral compound can possess mul-tiple chiral centers and many combi-nations of configurations. Linalooloxides possess two chiral centers, re-sulting in four enantiomers. (Figure1B) Note that configuration (R or S) isindependent from optical activity (+ or-) or interaction with plane-polarizedlight.
CH3(CH2)3CH2 CH2(CH2)3CH3CHCH2 CH2CH
OH HO
WHAT ARE CHIRALCOMPOUNDS?
Any carbon atom that is
bonded to four different groups
is termed a chiral or an
assymetric carbon. Molecules
containing one or more of
these carbon centers are con-
sidered chiral molecules.
Chiral centers can exist in two
forms called enantiomers.
These two forms are non-
superimposable mirror images
of each other, but both have
similar properties.
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4
Figure 2Restek’s chiral columns demonstrate exceptional lifetime and stability for
more than 250 injections without loss of resolution.
min. 10 20 30
min. 10 20 30
1,2
3
4
89
5
67
10 11
12
13
14
15
1,2
3
4
8
5
67
10 11
12
13
14
15
9
Chiral chromatography is the
separation of enantiomeric
compounds. Common liquid
stationary phases do not
possess adequate selectivity
for enantiomeric separation.
Addition of derivatized
cyclodextrin macromolecules
to common stationary phases
creates capillary columns with
the ability to separate
many enantiomers.
1. (-) α-pinene2. (+) α-pinene3. decane4. (-) 2,3-butanediol5. (+) 2,3-butanediol
30m, 0.32mm ID, 0.25µm Rt-βDEXsa (cat.# 13108)Oven temp.: 40°C (hold 1 min.) to 230°C @ 2°C/min.;
Carrier gas: hydrogen; 80cm/sec. set @ 40°C; Detector: FID set @ 220°C
WHAT IS CHIRALCHROMATOGRAPHY?○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○
Chiral chromatography is the separa-tion of enantiomeric compounds.Common liquid stationary phasesused in gas chromatography resolvecomponents from one another, butthey do not possess adequate selec-tivity for enantiomeric separation.Addition of derivatized cyclodextrinmacromolecules to common station-ary phases creates capillary columnswith the ability to separate enanti-omers as well.
The permethylated derivative of beta-cyclodextrin in cyanopropyl-dim-ethylpolysiloxane liquid stationaryphase is commonly used for such ste-reochemical separations, but it exhib-its limited applications. Beta-cyclodextrins derivatized with alkylsubstituents can enhance the enan-tiomeric resolution of various com-pound classes. Restek’s five capillarycolumns incorporate various combi-nations of alkylated beta-cyclodex-trins into a cyanopropyl-dimethylpolysiloxane liquid stationary phaseto achieve significant separation.
These columns also exhibit stabilityand extended lifetime. From the firstinjection to the 250th injection on achiral column, enantiomeric separa-tion is maintained with almost no lossin resolution (Figures 2A and B).
A: 1st injection
B: 250th injection
6. undecane7. nonanal8. 1-octanol9. 2,6-dimethylphenol
10. (+) phenylethanol
11. (-) phenylethanol12. methyl decanoate13. dicyclohexylamine14. methyl undecanoate15. methyl dodecanoate
○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○
5
Figure 3The Rt-βDEXsm column provides
the best chiral separation ofisoborneol and α-terpineol.
Figure 4The Rt-βDEXsm column offers
complete resolution of α-ionone.
30m, 0.32mm ID, 0.25µm Rt-βDEXsm (cat.# 13104)Oven temp.: 40°C (hold 1 min.) to 230°C @ 2°C/min. (hold 3 min.);
Carrier gas: hydrogen; 80cm/sec. set @ 40°C; Detector: FID set @ 220°C
min. 30 40
(+/-) isoborneol
(+/-) terpineol
min. 30 40
α-ionone
Figure 6The Rt-βDEXse column resolves
limonene enantiomers.
min. 18 20
limonene
Figure 5The Rt-βDEXse column resolves optical isomers of
ethyl-2-methylbutyrate, styrene oxide, and camphorwith some overlap.
min. 10 20 30
ethyl-2-methylbutyrate
styrene oxide
camphor
R S
S (-)
R (+)
30m, 0.32mm ID, 0.25µm Rt-βDEXse(cat.# 13106)
Oven temp.: 40°C (hold 1 min.) to 230°C @2°C/min. (hold 3 min.); Carrier gas: hydrogen;
80cm/sec. set @ 40°C; Detector: FID set @ 220°C
RESTEK’S CHIRAL COLUMNS OFFER UNIQUESELECTIVITY○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○
Each of the five chiral columnsposesses a specific combination ofalkyl substituents on the derivatizedβ-cyclodextrins. These unique combi-nations provide a wide range ofutilitization for each type of chiralcolumn. Table I, on page 8, indicatesthat certain columns provide betterresolution of specific compounds.
Rt-βββββDEXsm○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○
Of the chiral columns evaluated, onlythe Rt-βDEXsm separates all of the 25tested compounds, with 19 beingbaseline resolved. This column pro-vides the best enantiomeric separa-tion of α-pinene, isoborneol, α-ionone,linalool oxides, hexobarbital, and me-phobarbital (Figures 3 and 4).
Rt-βββββDEXse○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○
The Rt-βDEXse is similar in perfor-mance to the Rt-βDEXsm, but it pro-vides better resolution for limonene,linalool, linalyl acetate, ethyl-2-methylbutyrate, 2,3-butane-diol, andstyrene oxides. Sometimes extensiveseparation results in overlap of enan-tiomeric pairs, as shown in Figures 5and 6.
SR
○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○
6
Rt-βββββDEXsm andRt-βββββDEXse○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○
Cis and trans linalool oxides, linalool,and linalyl acetate are commonlyfound together in lavender oils, andresolution of all enantiomers is desir-able. The Rt-βDEXsm separates thelinalool oxides, but it does not resolvelinalyl acetate. Conversely, Rt-βDEXse separates linalool and linalylacetate, but does not resolve all of thelinalool oxides. Combining both to-gether in a dual column system willprovide resolution for all of theseenantiomers (Figure 7).
Rt-βββββDEXsp○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○
Rt-βDEXsp is a specialized columnthat best resolves menthol. It wouldbe useful in addition to the Rt-βDEXsm or Rt-βDEXse column foranalyzing complete profiles of mintoils (Figure 8).
Rt-βββββDEXsa○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○
The Rt-βDEXsa has a significantly dif-ferent selectivity than the other chiralcolumns. It provides the best separa-tion of 1-octen-3-ol, carvone, cam-phor, 1-phenylethanol, β-citronellol,and rose oxides (Figure 9).
Figure 7ACis and trans linalool oxide
enantiomers separated on anRt-βDEXsm column.
Figure 7BLinalool and linalyl acetateenantiomers are resolved on
the Rt-βDEXse column.
Figure 8Menthol enantiomers are best resolved on the
Rt-βDEXsp column.
min. 20 30
linalool oxides
30 min. 20
linalool
linalyl acetate
menthol
min. 30
Figure 9A1-octen-3-ol and carvone
enantiomers are best resolvedon an Rt-βDEXsa column.
Figure 9BAn Rt-βDEXsa column provides
baseline chiral resolution forβ-citronellol.
Figure 9CAn Rt-βDEXsa column
separates racemic cis andtrans rose oxides.
1-octen-3-ol
carvone
(-)(S
)
(+)-
cis
(+)(R
)
(+)-
tran
s
30 40min. 20 52 53min. 51 25 26min. 24 27
(+)-
tran
s
(+)-
cis
(-)-
cis
R
S
RS
(+)(-)
(+/-)
(-)
(+)
(-)-
cis
(-)-
tran
s
30m, 0.32mm ID, 0.25µm Rt-βDEXsp (cat.# 13110)Oven temp.: 60°C (hold 1 min.) to 200°C @
2°C/min.; Carrier gas: hydrogen; 80cm/sec. set @40°C; Detector: FID set @ 220°C
(-)-
tran
s
30m, 0.32mm ID, 0.25µm7A: Rt-βDEXsm (cat.# 13104)7B: Rt-βDEXse (cat.# 13106)
Oven temp.: 40°C (hold 1 min.)to 230°C @ 2°C/min. (hold 3min.); Carrier gas: hydrogen;
80cm/sec. set @ 40°C;Detector: FID set @ 220°C
30m, 0.32mm ID, 0.25µmRt-βDEXsa (cat.# 13108)
Oven temp.: 40°C (hold 1 min.)to 230°C @ 2°C/min. (hold 3min.); Carrier gas: hydrogen;
80cm/sec. set @ 40°C;Detector: FID set @ 220°C
7
1. (+/-)δ-pentalactones2. (+/-)δ-hexalactones3. (+/-)δ-heptalactones4. (+/-)δ-octalactones5. (+/-)δ-nonalactones6. (+/-)δ-decalactones7. (+/-)δ-dodecalactones
Figure 10All of the irone isomers are resolved
without overlapping of the enantiomericpairs on the Rt-βDEXcst column.
Figure 11The Rt-βDEXcst column provides maximumresolution of the γ-lactones and δ-lactones.
A. γ-lactones on the Rt-βDEXcstcolumn
B. δ-lactones on the Rt-βDEXcstcolumn
min. 40
1. (-)-(2R,6R)-trans-α-irone2. (+)-(2S,6S)-trans-α-irone3. (+)-(2R,6R)-trans-γ-irone4. (-)-(2S,6S)-trans-γ-irone5. (+)-(2R,6S)-cis-α-irone6. (+)-(2R,6S)-cis-γ-irone7. (-)-(2S,6R)-cis-γ-irone8. (-)-(2S,6R)-cis-α-irone9. (+)-(2R)-β-irone
10. (-)-(2R)-β-irone
1
3
4
85
6
7
10
2
9
min. 100 120
1
3
4
5
2
140
1. (+/-)γ-heptalactones2. (+/-)γ-octalactones3. (+/-)γ-nonalactones4. (+/-)γ-decalactones5. (+/-)γ-dodecalactones
min. 100 120
1
34 5
2
140
67
30m, 0.32mm ID, 0.25µmRt-βDEXcst (cat.# 13102)
Oven temp.: 60°C (hold 1 min.) to 200°C@ 1°C/min.; Carrier gas: hydrogen;
40cm/sec. set @ 60°C;Detector: FID set @ 220°C
30m, 0.32mm ID, 0.25µmRt-βDEXcst (cat.# 13102)
Oven temp.: 40°C (hold 1 min.) to230°C @ 2°C/min. (hold 3 min.);
Carrier gas: hydrogen; 80cm/sec. set @ 40°C;Detector: FID set @ 220°C
Rt-βββββDEXcst
This column is optimum for semi-volatile chiral compounds becauselower boiling components show peakbroadening (discussed in the “Optimi-zation of Chiral Separations” section).All of the irone isomers (found in irisflowers) are resolved without overlap-ping of the enantiomeric pairs (Figure10). This is also the best column forresolution of the γ- and δ-lactones(Figures 11A and B).
The Rt-βDEXcst column is good forseparating the enantiomers of somebarbiturates and TFA-derivatives ofamphetamines as well. This is dis-cussed in further detail on pages 22and 23.
Visit Restek on-line at
www.restek.com,
or call 800-356-1688,
ext. 4, for technical
assistance.
30m, 0.32mm ID, 0.25µmRt-βDEXcst (cat.# 13102)
Oven temp.: 60°C (hold 1 min.) to 200°C@ 1°C/min.; Carrier gas: hydrogen;
40cm/sec. set @ 60°C;Detector: FID set @ 220°C
○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○
8
Table IResolution of common chiral compounds on Restek’s cyclodextrin columns.
ns = no separation of enantiomerst = peak tailingb = peak broadening
ad = adsorption of nonderivatized drug compound* = cis and trans linalool oxides analyzed @ 9.0 psi head pressure
To demonstrate the abilities of the fivedifferent types of chiral columns, weanalyzed twenty-five chiral com-pounds commonly found in flavors,fragrances, and pharmaceuticalanalyses. The extent to which twoenantiomers are resolved (or any twopeaks) can be determined by the reso-lution equation and are known asresolution factors, sometimes de-
noted R. An R value of 1.5 indicatesbaseline resolution. Resolution fac-tors for all chiral compounds on allthe β-cyclodextrin columns were com-pared to those obtained on the exist-ing Rt-βDEXm (permethylatedcyclodextrin) column. Table I showsthe degree of enantiomeric separationby resolution factor for all twenty-fivecomponents on each column. The col-
umn that has the largest resolutionfactor provides the best separation ofa particular compound. These valuescan easily be compared to help deter-mine which column is optimum forspecific chiral components. Charts 1–6 illustrate the degree of enantiomericseparation of each compound on eachchiral column.
Compounds Formula m.w.
Terpenes 1. α-pinene C10H16 136
2. limonene C10H16 136
Alcohols 3. 1-octen-3-ol C8H16O 128
4. linalool C10H18O 154
5. α-terpineol C10H18O 154
6. terpinen-4-ol C10H18O 154
7. isoborneol C10H18O 154
8. β-citronellol C10H20O 156
9. menthol C10H20O 156
10. 2,3-butanediol C4H10O2 90
11. 1-phenylethanol C8H10O 122
Ketones 12. carvone C10H14O 150
13. camphor C10H16O 152
14. α-ionone C13H20O 192
Lactones 15. γ-nonalactone C9H16O2 156
16. γ-undecalactone C11H20O2 184
17. δ-decalactone C10H18O2 170
Esters 18. ethyl-2-methylbutyrate C7H14O2 130
19. linalylacetate C12H20O2 196
Epoxides 20. styreneoxide C8H8O 120
21. trans-linalooloxides C10H18O2 170
21. cis-linalooloxides
Drugs 22. hexobarbital C12H16O3N2 236
23. mephobarbital C13H14O3N2 246
24. fenfluramine C12H16F3N 231
25. fenfluramine TFAA der. C14H15F6NO 327
Column Resolution FactorsRt-βDEXsm Rt-βDEXse Rt-βDEXsp Rt-βDEXsa Rt-βDEXcst Rt-βDEXm
3.50 0.80 ns ns 0.90b 3.30
5.10 7.30 3.20 ns 2.70b 1.40
1.10 ns 0.50 2.30 0.60b 0.50t
3.30 6.00 4.30b 3.70 2.60t 1.00
5.30 5.50 2.20 4.00 4.30t 1.70
2.40 2.20 ns ns 1.20t 1.80t
4.00 3.30t ns ns 1.90t 2.00
0.90 0.80 ns 1.00t ns ns
1.10 1.10 2.20 ns 1.10 1.60t
7.50 8.10 2.20 4.60 4.00 2.60
7.30 6.40 1.10 7.80 6.30 6.60
1.30 ns ns 2.50 1.10 ns
1.80 2.10t 1.30b 4.30 2.50t ns
6.40 3.20 1.30 4.40 ns 3.20
4.70 5.00 3.40 4.40 5.90 1.00
2.90 2.90 1.70 3.70 4.50 ns
0.90 ns ns 2.10 2.70 ns
1.30 3.50 1.10b ns ns ns
0.60 2.20 1.20 0.10 ns ns
4.80 10.20 6.50 1.00 1.40t 2.50t
10.10 2.40 ns 1.50 4.30b 6.80*
6.20 3.60 ns 1.00 3.50b 4.20*
11.30 4.70 0.90 ns 8.90 6.30
8.40 3.80 0.60 ns 6.20 6.10
1.80 2.90t ad ad ns ad
1.20 ns ns 2.40 3.40 ns
○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○
9
Charts 1-6 illustrate the unique separation capabilities of each cyclodextrin column.
Rt-βββββDEXsm
12
10.5
9
7.5
6
3
1.5
0
4.5
1
252 3 4 5 6 7 8 9
10
11
12
13
14
15
16
17
18
19
20
21
21
22
23
24
Rt-βββββDEXse
Rt-βββββDEXsp Rt-βββββDEXsa
Rt-βββββDEXcst Rt-βββββDEXm
Compound Number*
Res
olu
tion F
acto
r
Compound Number*
Res
olu
tion F
acto
r
Compound Number*
Res
olu
tion F
acto
r
Compound Number*
Res
olu
tion F
acto
r
Compound Number*
Res
olu
tion F
acto
r
Compound Number*
Res
olu
tion F
acto
r
*Refer to Table I for compound identification.
12
10.5
9
7.5
6
3
1.5
0
4.5
1
252 3 4 5 6 7 8 9
10
11
12
13
14
15
16
17
18
19
20
21
21
22
23
24
12
10.5
9
7.5
6
3
1.5
0
4.5
1
252 3 4 5 6 7 8 9
10
11
12
13
14
15
16
17
18
19
20
21
21
22
23
24
12
10.5
9
7.5
6
3
1.5
0
4.5
1
252 3 4 5 6 7 8 9
10
11
12
13
14
15
16
17
18
19
20
21
21
22
23
24
12
10.5
9
7.5
6
3
1.5
0
4.5
1
252 3 4 5 6 7 8 9
10
11
12
13
14
15
16
17
18
19
20
21
21
22
23
24
12
10.5
9
7.5
6
3
1.5
0
4.5
1
252 3 4 5 6 7 8 9
10
11
12
13
14
15
16
17
18
19
20
21
21
22
23
24
○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○
10
Figure 12BFaster linear velocities provide maximum
resolution of chiral pairs.
Figure 12AHigher Trennzahl values are obtained at 40 cm/sec
with hydrogen carrier gas.
Rt-βββββDEXsa: 40°C (80°C for lactones) to 200°C @2°C/min. (hold 1 min.). Hydrogen carrier gas.
Rt-βββββDEXsa: 40°C to 200°C @ 2°C/min. (hold 1 min.).Hydrogen carrier gas.
OPTIMIZATION OFCHIRAL SEPARATIONS○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○
Although the new β-cyclodextrin col-umns can resolve a variety of chiralcompounds, certain parameters mustbe optimized to obtain maximumseparation and column performance.Variation in linear velocity and tem-perature ramp rate can greatly affectthe resolution of enantiomers. De-pending on the type of chiral column,initial GC oven temperature can affectpeak width. Column sample capacityvaries with different compounds, andoverloading results in broad tailingpeaks and reduced enantiomericseparation.
Linear Velocity (Column Flow)○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○
The resolution between the enantio-meric pairs can be improved by in-creasing the linear velocity. This is es-pecially important if the resolutionfactor is below two for optical isomers(see Table I). Trennzahl values aremeasurements of column separationefficiency, which are often optimumat a linear velocity of 40 cm/sec withhydrogen carrier gas. This is illus-trated in Figure 12A. Although opti-mal linear velocity can be different foreach chiral compound and column,the typical optimum linear velocity formaximum enantiomeric separation isaround 80 cm/sec with hydrogencarrier gas, as illustrated with sixchiral compounds on the Rt-βDEXsacolumn in Figure 12B. This is twicethe linear velocity required to achievemaximum efficiency as indicated bythe Trennzahl values of 1-octen-3-olenantiomers in Figure 12A.
1-octen-3-ollinaloolα-iononeγ-nonalactoneγ-undecalactoneδ-decalactone
30 40 80 120
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
30 40 80 120
30
25
20
15
10
5
0
Linear Velocity (cm/sec)
Tre
nnza
hl
Val
ue
Linear Velocity (cm/sec)
Res
olu
tion F
acto
r
11
Figure 13BLower temperature ramp rates provide maximum
resolution of chiral pairs.
Figure 13ATrennzahl values increase with enantiomeric resolution factors as
temperature ramp rates decrease.
Rt-βββββDEXsa: 40°C (80°C for lactones) to 200°C @2°C/min. (hold 1 min.). Hydrogen carrier gas.
Rt-βββββDEXsa: 40°C to 200°C @ 2°C/min. (hold 1 min.).Hydrogen carrier gas.
1-octen-3-ollinaloolα-iononeγ-nonalactoneγ-undecalactoneδ-decalactone
1.5°C/min.
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
5.0
5.5
2.0°C/min. 2.5°C/min. 3.0°C/min.
30
25
20
15
10
5
01.5°C/min. 2.0°C/min. 2.5°C/min. 3.0°C/min.
Temperature Program
The resolution between the enantio-meric pairs can be improved by usingslow temperature ramp rates. Thebest temperature ramp rates are 1-2°C/min. Trennzahl values improvealong with enantiomeric resolution asthe temperature ramp rate is de-creased (Figures 13A and B).
Temperature Program Rate
Tre
nnza
hl
Val
ue
Temperature Program Rate
Res
olu
tion F
acto
r
Remember, to optimize
chiral separation use:
1) Faster linear velocities(80 cm/sec.) with hydrogencarrier gas.
2) Slower temperature ramprates (1-2°C/min.).
3) Appropriate minimumoperating temperature(40 or 60°C).
4) On-column concentrationsof 50ng or less.
○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○
12
Minimum Temperature○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○
For maximum resolution of chiralcompounds with low boiling points(below 100°C), initial temperatures of35-40°C are recommended for the Rt-βDEXsm, Rt-βDEXse, and Rt-βDEXsacolumns. In contrast, the same vola-tile compounds exhibit a very broadpeak shape on the Rt-βDEXsp and Rt-βDEXcst columns at these initial oventemperatures. Linalool oxides arevolatile compounds that exhibit peakbroadening and almost no resolutionon the Rt-βDEXcst column with aninitial oven temperature of 40°C (Fig-ure 14A). The peak shapes and over-all resolution of the linalool oxides im-prove when initial temperature isincreased to 70°C, even though the in-dividual enantiomers of both the cisand trans isomers are not separated(Figure 14B). Higher initial tempera-tures do not always completely elimi-nate peak broadening for some com-ponents. However, the improvementin solvent peak shape indicates aphysical transition of the cyclodextrinmacromolecules from a crystallinestructure to a liquid at this highertemperature. Thus the recommendedminimum operating temperature ofRt-βDEXsp and Rt-βDEXcst columnsis 60°C.
Figure 14ALinalool oxides exhibit extreme peak broadening and poor resolution on the
Rt-βDEXcst column with an initial oven temperature of 40°C.
Figure 14BLinalool oxides exhibit improved peak shape and resolution on the
Rt-βDEXcst column when initial oven temperature is increased to 70°C.
cis & trans linalool oxides
15min. 20 25
cis & trans linalool oxides
15min. 20 25
30m, 0.32mm ID, 0.25µm Rt-βDEXcst (cat.# 13102)Oven temp.: 70°C (hold 1 min.) to 200°C @
2°C/min.; Carrier gas: hydrogen; 80cm/sec. set @ 40°C;Detector: FID set @ 220°C
30m, 0.32mm ID, 0.25µm Rt-βDEXcst (cat.# 13102)Oven temp.: 40°C (hold 1 min.) to 200°C @
2°C/min.; Carrier gas: hydrogen; 80cm/sec. set @ 40°C;Detector: FID set @ 220°C
Higher initial
temperatures do not
always completely elimi-
nate peak broadening
for some components.
However, the improve-
ment in solvent peak
shape indicates a physi-
cal transition of the
cyclodextrin macromol-
ecules from a crystalline
structure to a liquid at
this higher temperature.
○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○
13
Figure 15ALinalool and linalyl acetate havesymmetrical peak shapes and
excellent chiral separation on theRt-βDEXse column at 25ng per
component on-column.
linalool
min. 30 35 40
linalyl acetate
linalool
min. 30 35 40
linalyl acetate
linalool
min. 30 35 40
linalyl acetate
Figure 15CExcessive overload of chiral com-pounds on cyclodextrin columns
results in extreme peak tailing andcomplete loss in resolution.
Figure 15BLinalool shows signs of overload
with slight peak tailing, and linalylacetate has a small loss in
resolution when sample load isincreased to160ng.
R S
R S
RS
R
S
R S
R,S
30m, 0.32mm ID, 0.25µmRt-βDEXse (cat.# 13106)
Oven temp.: 40°C (hold 1 min.) to 200°C@ 2°C/min.; Carrier gas: hydrogen;
80cm/sec. set @ 40°C;Detector: FID set @ 220°C
30m, 0.32mm ID, 0.25µmRt-βDEXse (cat.# 13106)
Oven temp.: 40°C (hold 1 min.) to 200°C@ 2°C/min.; Carrier gas: hydrogen;
80cm/sec. set @ 40°C;Detector: FID set @ 220°C
30m, 0.32mm ID, 0.25µmRt-βDEXse (cat.# 13106)
Oven temp.: 40°C (hold 1 min.)to 200°C @ 2°C/min.;Carrier gas: hydrogen;80cm/sec. set @ 40°C;
Detector: FID set @ 220°C
Overloading and Tailing○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○
Some chiral compounds show over-loading at lower concentrations thanachiral compounds. One reason is thedifferent amounts of cyclodextrin (5-50%) dissolved in the stationaryphase. Unlike the classical frontingpeaks of normal stationary phases,the characteristic of an overloadedpeak on cyclodextrin stationaryphases is indicated by a tailing peak.Overloading chiral compounds re-sults in loss of resolution, even whencolumn capacity has not been ex-ceeded. Figure 15A shows the enan-tiomeric separations of linalool andlinalyl acetate on the Rt-βDEXse. Theamount for each component in thecolumn is about 25 ng. Figure 15Bshows the same components at ahigher concentration of 160ng on-col-umn. Note that the linalool enanti-omers are beginning to tail, and thereis a small loss in chiral resolution forlinalyl acetate. Even though the maxi-mum sample capacity for 0.32mm IDcapillary columns is normally 400-500ng per component, the peakshapes of chiral compounds indicateoverload at one-third of the sampleamount. Again, there is much lesscyclodextrin for which a chiral com-pound can interact. Figure 15Cshows pronounced overloading ofthese compounds at 5µg on-column.Extreme tailing and complete loss inresolution are the result.
○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○
14
CHIRAL SPECIFIC APPLICATIONS OF ESSENTIALOILS, FLAVORS, AND PHARMACEUTICALS○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○
ESSENTIAL OILS○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○
Chiral capillary GC has proven to be a convenient method for characterizingessential oils and differentiating natural flavors from those of synthetic origin.Chiral compounds from natural origins usually exist as one predominant op-tical isomer. Also, the inspection of enantiomeric ratios can characterize re-gional differences between oils. Although sometimes a result of processing, thepresence of racemic pairs (one-to-one ratios of each enantiomer) most oftenindicates adulteration or unnatural origin.
Since most chiral compounds natu-rally exist as one predominant isomer,resolution is more challenging, espe-cially for components in higher con-centrations. For primary constituentsin essential oils, select a chiral col-umn that provides a resolution factorvalue greater than two to overcomepossible loss of resolution.
Since essential oils are mixtures ofmany compounds, coelution of peaksand overlapping of certain opticalpairs are sometimes hard to avoid.Not all of the chiral compounds foundin an essential oil or flavor extract mayseparate on the same column. Con-necting two different columns to-gether is possible, but the elution or-der of some enantiomers may reversewith this combination, resulting inloss of separation. Dual columnanalysis is a logical alternative to ob-tain a more complete enantiomericprofile and to provide confirmationalidentification of individual constitu-ents. To reduce analysis time, bothcolumns can be installed into thesame injection port for simultaneousconfirmation. (Consult Restek’s Chro-matography Products Guide for moreinformation about dual columnanalysis.)
For primary constituents
in essential oils, select
a chiral column that
provides a resolution
factor value greater than
two to overcome possible
loss of resolution.
15
Lemon Oil
The Rt-βDEXsm is the optimum col-umn for obtaining chiral profiles ofLemon and other citrus oils since itprovides enantiomeric separation forthe main terpene constituents like α-and β-pinenes, sabinene (these enan-tiomers overlap with those of β-pinene) and limonene (Figure 16).
Rosemary Oil
Chiral constituents in this oil includeα-pinene, β-pinene, camphene, li-monene, linalool, camphor, terpinen-4-ol, α-terpineol, borneol, andisoborneol. Baseline enantiomericseparation is easily achieved for all ofthese compounds on the new Rt-βDEXsm column. The commonpermethylated β-cyclodextrin columncannot completely resolve the opticalisomers of limonene, linalool, andcamphor (Figure 17).
Figure 17The Rt-βDEXsm, the most versatile β-cyclodextrin column for essential oil
analysis, resolves enantiomers of α-pinene, β-pinene, camphene, limonene,linalool, camphor, terpinen-4-ol, α-terpineol, borneol, and isoborneol in
rosemary oil.
10 020 .nim
1
30 40
2
3
3
4
4 1. (-/+)α-pinene2. (+/-)camphene3. (+/-)β-pinene4. (-/+)limonene5. eucalyptol (cineole)6. linalool7. camphor8. (-/+)terpinen-4-ol9. isoborneol
10. borneol11. α-terpineol
66
7
7
8
899 10
10
1111
5
30m, 0.32mm ID, 0.25µm Rt-βDEXsm (cat.# 13104);Oven temp.: 40°C (hold 1 min.) to 200°C @ 2°C/min. (hold 3 min.);
Carrier gas: hydrogen; 80cm/sec.; Detector: FID set @ 220°C
Figure 16The Rt-βDEXsm column provides chiral resolution of the primary
terpenes in Artificial Lemon oil.
15min. 10 20 25
1
30 35 40 45
1
22
3
3
4
4
1. (-/+)α-pinene2. sabinene3. (+/-)β-pinene4. (-/+)limonene
30m, 0.32mm ID, 0.25µm Rt-βDEXsm (cat.# 13104);Oven temp.: 40°C (hold 1 min.) to 200°C @ 2°C/min. (hold 3 min.);
Carrier gas: hydrogen; 80cm/sec.; Detector: FID set @ 220°C
The Rt-βββββDEXsm is the
optimum column for
obtaining chiral profiles
of Lemon and other
citrus oils.
Visit Restek on-line at
www.restek.com,
or call 800-356-1688,
ext. 4, for technical
assistance.
○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○
16
Figure 18The Rt-βDEXsm column is optimum
for the separation of (+/-) α- andβ-pinene, limonene, and menthone
in peppermint oil.
Figure 19AAnalyze spearmint oil on theRt-βDEXsm column to obtain
maximum separation of (+/-) α-and β-pinene, limonene, and
menthone enantiomers.
15min. 10 20 25 30 35 40 45
1
2
3
3
4
1. (-/+) α-pinene2. (+/-) β-pinene3. (-/+) limonene4. (+/-) menthone5. (+/-) menthol (no sep.)
5
{
{
{
15min. 10 20 25 30 35 40 45
1. α-pinene2. β-pinene3. limonene4. menthone5. menthol (no sep.)6. carvone (no sep.)
1
2
33
4
5
{
{
{
6
Figure 19BThe optical isomers of carvone bestseparate on the Rt-βDEXsa column.
30m, 0.32mm ID, 0.25µm Rt-βDEXsm (cat.# 13104); Oven temp.: 40°C (hold 1 min.) to200°C @ 2°C/min. (hold 3 min.); Carrier gas: helium; 60cm/sec.; Detector: MSD set @ 220°C
30m, 0.32mm ID, 0.25µm Rt-βDEXsm (cat.# 13104); Oven temp.: 40°C (hold 1 min.) to200°C @ 2°C/min. (hold 3 min.); Carrier gas: helium; 60cm/sec.; Detector: MSD set @ 220°C
MidwesternSpearmint
(d)
(l)
RacemicCarvoneStandard
(d)(l)
min. 38 39 40
30m, 0.32mm ID, 0.25µm Rt-βDEXsa(cat.# 13108); Oven temp.: 40°C
(hold 1 min.) to 200°C @ 2°C/min.;Carrier gas: helium; 60cm/sec. set @ 40°C;
Detector: MSD set @ 220°C
Peppermint Oil providedby AM Todd.
Spearmint Oil providedby AM Todd.
Peppermint Oil○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○
The Rt-βDEXsm column is optimum for the separation of (+/-) α- and β-pinene,limonene and menthone. Since menthone and menthol enantiomers are ma-jor constituents of peppermint oil, reducing the sample size to prevent over-loading of these components and provide better enantiomeric resolution maybe necessary.
An alternative solution is to use an Rt-βDEXsp as a secondary column since itprovides better resolution of menthol. However, do not use this column in adual column system with an Rt-βDEXsm. The minimum temperatures of thesephases differ by 20°C, which would minimize volatile terpene separation onthe Rt-βDEXsm (Figure 18).
Spearmint Oil○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○
The Rt-βDEXsm column yields maxi-mum separation of (+/-) α- and β-pinene, limonene and menthoneenantiomers in spearmint oil. Theenantiomeric ratios of the primarychiral constituents in an artificialspearmint oil can be seen in the chro-matogram shown in Figure 19A.
Although the optical isomers ofcarvone best separate on the Rt-βDEXsa column, α-pinene and li-monene do not. For the separation ofcarvone, use a dual column systemcomprised of 30-meter Rt-βDEXsmand Rt-βDEXsa columns, since bothhave a minimum operating tempera-ture of 40°C. Figure 19B comparescarvone in natural sources of spear-mint oil to a racemic standard on theRt-βDEXsa column.
min. 38 39 40
17
Figure 20ALinalool and linalool oxides in lavender oils are stereochemically
separated on the Rt-βDEXsm column.
Figure 20BThe Rt-βDEXse column resolves enantiomers of
linalyl acetate in lavender oils.
20 05040301 .nim
20 05040301 .nim
1a. (-)-limonene1b. (+)-limonene2. (+)-trans-linalool oxide3. (-)-cis-linalool oxide
4a. (-)- (R)-linalool4b. (+)-(S)-linalool5. (+/-)camphor6. R,S linalyl acetate
1b
2 3
4a
5{
6
1a
Lavender oils provided by Belmay.
Lavender oils provided by Belmay.
30m, 0.32mm ID, 0.25µm Rt-βDEXse (cat.# 13106);Oven temp.: 40°C (hold 1 min.) to 200°C @ 2°C/min. (hold 3 min.);
Carrier gas: hydrogen; 80cm/sec.; Detector: FID set @ 220°C
30m, 0.32mm ID, 0.25µm Rt-βDEXsm (cat.# 13104);Oven temp.: 40°C (hold 1 min.) to 200°C @ 2°C/min. (hold 3 min.);
Carrier gas: hydrogen; 80cm/sec.; Detector: FID set @ 220°C
1a. (-)-limonene1b. (+)-limonene2. (+)-trans-linalool oxide3. (-)-cis-linalool oxide
4a. (-)- (R)-linalool4b. (+)-(S)-linalool5. (+/-)camphor
6a. (-)-(R)-linalyl acetate6b. (+)-(S)-linalyl acetate
1a
1b +
eu
caly
pto
l
2 3
4a
{5
6a6b
Visit Restek on-line at
www.restek.com,
or call 800-356-1688,
ext. 4, for technical
assistance.
Lavender Oil
The Rt-βDEXsm column separatesthe enantiomers of the primary chiralcompounds found in lavender oil, in-cluding linalool. Both the cis andtrans enantiomeric pairs of thefuranoid linalool oxides, which con-tribute characteristic odors to laven-der oils and Clary sage oil, are sepa-rated on this column. (R)-Linalool ispresent in at least 85% enantiomericexcess. (2R)-Configured linalool ox-ides are present in about 77% enan-tiomeric excess in authentic Lavenderoils.2 Both the cis and trans (R)-lina-lool oxides are essentially enantio-merically pure in this oil, as shown inFigure 20A (peaks 2 and 3).
Linalyl acetate is another primaryconstituent in lavender oils. The (R)-(-) enantiomer is predominant in au-thentic lavender oils.3 A dual columnsystem comprised of both the Rt-βDEXsm and Rt-βDEXse columnscan be used to resolve the enanti-omers of linalyl acetate as well (peak6 in Figure 20B). Note that the (-)-(R)-enantiomer constitutes >92% oflinalyl acetate in this lavender oil.
4b
4b
18
6b
7
1a. (+)-(2R,4S)-cis-rose oxide1b. (-)-(2S,4R)-cis-rose oxide2a. (-)-(2R,4R)-trans-rose oxide2b. (+)-(2S,4S)-trans-rose oxide3. isomenthone4. menthone
5a. (-)-(R)-linalool5b. (+)-(S)-linalool6a. (-)-(S)-β-citronellol6b. (+)-(R)-β-citronellol7. geraniol8. citronellyl formate
Figure 21AThe Rt-βDEXsa column provides chiral resolution for cis and trans rose
oxides, linalool, and β-citronellol in Chinese Geranium oil.
Figure 21BThe Rt-βDEXsa column reveals authenticity of Rose oil Maroc.
1a
40 060503 .nim
40 060503 .nim
1b
2a2b
3
4
5a 5b
6a
8
1. (-)-(2S,4R)-cis-rose oxide2. (-)-(2R,4R)-trans-rose oxide
3a. (-)-(R)-linalool3b. (+)-(S)-linalool4. phenylethyl alcohol
5a. (-)-(S)-β-citronellol5b. (+)-(R)-β-citronellol6. geraniol
6
1
2
3a
4 5a
5b
3b
Geranium oils provided by Belmay.
30m, 0.25mm ID, 0.25µm Rt-βDEXsa (cat.# 13109);Oven temp.: 60°C to 110°C @ 1°C/min. (hold 30 min.);
Carrier gas: hydrogen; 40cm/sec. set @ 60°C; Detector: FID set @ 220°C
30m, 0.25mm ID, 0.25µm Rt-βDEXsa (cat.# 13109);Oven temp.: 60°C to 110°C @ 1°C/min. (hold 30 min.);
Carrier gas: hydrogen; 40cm/sec. set @ 60°C; Detector: FID set @ 220°C
Geranium Oil
Chiral constituents in geranium oilsinclude cis and trans rose oxides,linalool, and β-citronellol. The Rt-βDEXsa column provides chiral reso-lution for all of these compounds. Inauthentic samples of geranium oil,(-)-(4R)-configured diastereomers ofcis- and trans-rose oxides predomi-nate over their (+)-enantiomers.3 The(-)-(S) form of β-citronellol is 74-80%of the enantiomeric ratio.4 Note thatcis- and trans-rose oxides and β-cit-ronellol are racemic in this particu-lar commercial geranium oil, asshown in Figure 21A. These racemiccompounds indicate that this oil isnot authentic.
Rose Oil
As with geranium oils, (-)-(2S,4R)-cisand (-)-(2R,4R)-trans rose oxides and(-)-(S)-β-citronellol are specific indica-tors of genuine rose oils.5 Note theenantiomeric purity of these com-pounds in Rose Oil Maroc, as shownin Figure 21B.
Visit Restek on-line at
www.restek.com,
or call 800-356-1688,
ext. 4, for technical
assistance.
○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○
19
Figure 22The Rt-βDEXse column can differentiate Bergamot extract
from Bergamot flavor.
(R)-
lim
onen
e
13min. 8 18
(R)-
lin
aloo
l
(R)-
lin
alyl
ace
tate
(R)-
lin
aloo
l
(R)-
lim
onen
e
(R)-
lin
alyl
ace
tate
13min. 8 18
(S)-
lin
aloo
l
(S)-
lim
onen
e
(S)-
lin
alyl
ace
tate
30m, 0.32mm ID, 0.25µm Rt-βDEXse (cat.# 13106);Oven temp.: 40°C (hold 1 min.) to 200°C @ 4°C/min.;
Carrier gas: helium; 60cm/sec. set @ 40°C; Detector: MSD set @ 220°C
FLAVORS○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○
Bergamot oil and a few of the popularfruit flavorings such as raspberry,strawberry, and peach were exam-ined. The composition of extracts fromnatural sources were compared tothose from commercially available fla-vored teas and drinks. Some targetchiral compounds examined werelinalool and linalyl acetate in berga-mot oil, α-ionone and δ-decalactone inraspberry, and γ-lactones in peachextracts.
Bergamot Flavor○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○
A genuine cold-pressed bergamot oilshould contain only the (R)-isomers oflinalool and linalyl acetate.6 The enan-tiomeric purity of (R) limonene shouldalso be considered.7 Chromatogram Ain Figure 22 is a natural source ofbergamot oil. Only the (R)-enanti-omers of limonene, linalool and linalylacetate were present. ChromatogramB illustrates an extract from an arti-ficially flavored tea. Both sampleswere analyzed on an Rt-βDEXse col-umn. The presence of racemic linalooland linalyl acetate indicates bergamotflavor of unnatural origin.
A: Bergamot Extract
B: Bergamot Flavor
(S)-
lim
onen
e
○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○
20
Figure 23The γ-lactones are analyzed for the adulteration of
peach flavor on the Rt-βDEXsa column.
190 min. 38
γ-dec
alac
ton
e
γ-dod
ecal
acto
ne
R
R
S+
30m, 0.32mm ID, 0.25µm Rt-βDEXsa (cat.# 13108); Oven temp.: 60°C (hold 2 min.)to 100°C @ 15°C/min., then to 220°C @ 3°C/min.;
Carrier gas: helium; 60cm/sec. set @ 60°C; Detector: MSD set @ 220°C.
γ-u
ndec
alac
ton
e
R
γ-u
ndec
alac
ton
e
R S
R
γ-dec
alac
ton
e
γ-oc
tala
cton
e
R S
Peach Flavor○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○
Both δ- and γ-lactones are present inpeaches, but only the γ-lactones areanalyzed for the adulteration of peachflavor. Gamma-decalactone occurs inthe 89% (R) : 11%(S)- enantiomers innatural Peach flavor.8 In Figure 23,Chromatogram A is a peach extract.A significant amount of the (R)-γ-decalactone was present, along withthe (S)-enantiomer, which coelutedwith an unknown. A small amount of(R)-γ-dodecalactone was also de-tected. Chromatogram B is an extractfrom a beverage with “all natural”peach flavor. Gamma-decalactonewas not present, but racemic γ-undecalactone was found in a 1:1 ra-tio. This was the same result with an-other peach-flavored beverage.Chromatogram C is from an “all natu-ral-flavored beverage,” with peachand vanilla flavors. Although only the(R)-enantiomer of γ-decalactone waspresent, the amount is very small.Both γ-octalactone and γ-undecalac-tone were found to be racemic, indi-cating adulteration.
A: Peach Extract
B: Peach Flavor
C: Peach/Vanilla Flavor
The γγγγγ-lactones are
inspected for the
adulteration of
peach flavor.
S
○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○
21
Raspberry Flavor○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○
Alpha-ionone from raspberries occursas an enantiopure (R)(+)-enantiomer,illustrated on an Rt-βDEXsa columnin Figure 24A. Chromatogram B rep-resents a “naturally flavored” rasp-berry iced tea. A racemic mixture ofα-ionone was present, indicating thatit is not a completely natural rasp-berry flavor. Thus, α-ionone serves asa good marker compound for deter-mining raspberry authenticity.
Figure 24The Rt-βDEXsa column resolves isomers of α-ionone to
determine raspberry authenticity.
α-io
non
e
20min. 10 30
β-io
non
e
δ-dec
alac
ton
e
R
R
S
S
40
20min. 10 30 40
30m, 0.32mm ID, 0.25µm Rt-βDEXsa (cat.# 13108);Oven temp.: 60°C (hold 2 min.) to 200°C @ 3°C/min.;
Carrier gas: helium; 60cm/sec. set @ 60°C; Detector: MSD set @ 220°C.
α-io
non
e
A: Raspberry Extract
B: Raspberry Flavor
Alpha-ionone serves as a
good marker compound
for determining
raspberry authenticity.
30m, 0.32mm ID, 0.25µm Rt-βDEXsa (cat.# 13108);Oven temp.: 60°C (hold 2 min.) to 200°C @ 3°C/min.;
Carrier gas: helium; 60cm/sec. set @ 60°C; Detector: MSD set @ 220°C.
○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○
22
Figure 25The optical isomers of TFA-fenfluramine and its metabolite are
well resolved on the Rt-βDEXcst column.
Figure 26 The Rt-βDEXcst column can resolve the enantiomers of
common barbiturates.
20min. 15 25 30 35 40
20min. 10 30 5040
Figure 27The Rt-βDEXcst column simultaneously resolves the enantiomers of
TFA-amphetamine and TFA-methamphetamine.
12min. 10 14 16 18 20 22 24
30m, 0.32mm ID, 0.25µmRt-βDEXcst (cat.# 13102);
Oven temp.: 60°C (hold 1 min.) to220°C @ 3°C/min.; Carrier gas:hydrogen; 80cm/sec. set @ 60°C;
Detector: FID set @ 220°C.
30m, 0.32mm ID, 0.25µmRt-βDEXcst (cat.# 13102);
Oven temp.: 90°C (hold 1 min.) to200°C @ 2°C/min. (hold 3 min.);
Carrier gas: hydrogen; 80cm/sec. set@ 60°C; Detector: FID set @ 220°C.
(+)-
fen
flu
ram
ine
(-)-
fen
flu
ram
ine
(+)-
nor
fen
flu
ram
ine
(-)-
nor
fen
flu
ram
ine
(+/-)
-hex
obar
bit
al
(+/-)
-mep
hob
arbit
al
30m, 0.25mm ID, 0.25µm Rt-βDEXcst (cat.# 13103); Oventemp.: 120°C (hold 1 min.) to
175°C @ 1.5°C/min.; Carrier gas:helium; 25cm/sec. set @ 120°C;
Detector: MSD set @ 220°C.
met
ham
ph
etam
ine
amph
etam
ine
DRUGS○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○
Stereochemical properties of chiraldrugs have been found in many in-stances to be the controlling factorconcerning activity. One enantiomermay provide a biological function. Theother may be inactive or exhibit an-other functionality, which could re-sult in side effects. In some cases, oneoptical isomer may be harmful. TheFDA requires drug manufacturers totest the individual enantiomers ofnew drugs for toxicity.
Fenfluramine○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○
Fenfluramine is an appetite suppres-sant to promote weight loss withobese patients.9 Although it is struc-turally similar to amphetamines, itdiffers somewhat pharmacologically.Norfenfluramine is a metabolite thatis found in urine and serum of pa-tients. The purpose of the chiral iso-lations, like many analyses in thepharmaceutical industry, is of propri-etary nature. The TFA derivatives ofboth fenfluramine and norfenflur-amine are separated into their enan-tiomers on the Rt-βDEXcst column(Figure 25).
Barbiturates○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○
Mephobarbital and Hexobarbital arebarbiturates with sedative, hypnoticand anticonvulsant properties.10 Be-cause psychological and physical de-pendence may occur with continuinguse, they are controlled substances inthe U.S. Code of Federal Regulations.The optical isomers of these barbitu-rates can be simultaneously resolvedon an Rt-βDEXcst column (Figure26).
Amphetamines○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○
Dextroamphetamine (d-amphet-amine), d,l-amphetamine, and d-methamphetamine are sympathomi-metic amines with central nervoussystem stimulant activity.11 They aresignificant drugs of abuse in theUnited States and are includedamong the drugs to be tested under
(+)
(-)
(+) (-)
23
1. John McMurray, Organic Chemistry, Brooks/Cole Pub., Monterey, CA, 1984.2. C. Askari and A. Mosandl, Phytochem. Anal., Vol.2 p.211 (1991).3. Uzi Ravid, Eli Putievsky, and Irena Katzir, “Chiral Analysis of Enantiomerically pure (R)-(-) Linalyl
Acetate in some Lamiaceae, Myrtle, and petigrain Essential Oils,” Flavour Fragrance J.Vol.9,Pp. 275-276 (1994).
4. Peter Kreis and Armin Mosandl, “Chiral Compounds of Essential Oils. Part XIII. SimultaneousChirality Evaluation of Geranium Oil Constituents.” Flavour and Fragrance Journal, Vol. 8. Pp.161-168. (1993).
5. Peter Kreis and Armin Mosandl, “Chiral Compounds of Essential Oils. PartXII. AuthenticityControl of Rose Oils, Using Enantioselective Multidimensional Gas Chromatography.” Flavourand Fragrance Journal, Vol. 7, Pp. 199-203 (1992).
6. W.A. Konig, C. Fricke, and Y. Saritas, “Adulteration or Natural Variability? Enantiomeric GC inPurity Control of Essential Oils.” University of Hamburg, Institute of Organic Chemistry.
7. H.P Neukum and D.J. Meier, “Detection of Natural and Reconstituted Bergamot Oil in Earl GreyTeas by separation of the Enantiomers of Linalool and Dihydrolinalool,” Mitt. Gebiete Lebensm.Hyg., Vol. 84, Pp. 537-544 (1993).
8. Anna Artho and Konrad Grob, “Determination of γ-Lactones Added to Foods as Flavors. How FarMust “Nature-Identical” Flavors be Identical with Nature ?,” Mitte. Gebiete Lebensm. Hyg. Vol. 81,Pp. 544-558 (1990).
9. Physician’s Desk Reference, 46th ed., Pp. 1867-1868 (1992).10. Physicians’ Desk Reference, 46th ed., Pp. 2061-2062 (1992).11. Physicians’ Desk Reference, 46th ed., Pp. 514-515 (1992).12. W. Haefely, E. Kyburz, M. Gerecke, and H. Mohler. “Recent Advances in the molecular
pharmacology of bezodiazepine receptors and in the structure-activity relationships of theiragonists and antagonists.” Adv. Drug Res.Vol. 14, Pp. 165-322 (1985).
13. J.T. Cody and R. Schwartzhoff, “Interpretation of Methamphetamine and AmphetamineEnantiomer Data” J. Anal Toxicol. Vol. 17, Pp. 321-326 (Oct. 1993).
PRODUCT LIST
Rt-βββββDEXsm (30m, 0.25µm) Rt-βββββDEXse (30m, 0.25µm)
mm ID cat.# min. temp. max. temp. mm ID cat.# min. temp. max. temp.
0.25 13105 40°C 230°C 0.25 13107 40°C 230°C
0.32 13104 40°C 230°C 0.32 13106 40°C 230°C
Rt-βββββDEXsa (30m, 0.25µm) Rt-βββββDEXsp (30m, 0.25µm)
0.25 13109 40°C 230°C 0.25 13111 60°C 230°C
0.32 13108 40°C 230°C 0.32 13110 60°C 230°C
Rt-βββββDEXcst (30m, 0.25µm) Rt-βββββDEXm (30m, 0.25µm)
0.25 13103 60°C 230°C 0.25 13100 40°C 230°C
0.32 13102 60°C 230°C 0.32 13101 40°C 230°C
Stereochemical proper-
ties of chiral drugs have
been found in many in-
stances to be the control-
ling factor concerning
activity. One enantiomer
may provide a biological
function and the other
may be inactive or may
exhibit another function-
ality, which could result
in side effects.
the federal guidelines for workplacedrug testing.12 However, l-metham-phetamine (deoxyephedrine) is foundin over-the-counter decongestantsand is not a controlled substance.13
Enantiomeric separation of these
compounds, which is necessary foraccurate interpretation of drug tests,is easily achieved on the Rt-βDEXcstchiral capillary GC column (Figure27).
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