A Guide to the Analysis of Chiral Compounds by GC (PDF) - Restek

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.

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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

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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.);


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

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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;


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;



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.




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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

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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

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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

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



The Rt-βββββDEXsm is the

optimum column for

obtaining chiral profiles

of Lemon and other

citrus oils.


www.restek.com,

or call 800-356-1688,


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.);




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


www.restek.com,

or call 800-356-1688,


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.);


30m, 0.25mm ID, 0.25µm Rt-βDEXsa (cat.# 13109);Oven temp.: 60°C to 110°C @ 1°C/min. (hold 30 min.);


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.


www.restek.com,

or call 800-356-1688,


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.;


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.;


○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○

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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A Guide to the Analysis of Chiral Compounds by GC (PDF) - Restek

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