Chiral Separations in Gas Chromatography

Olga Inozemtseva – Dai ThaiDr. Dixon

Chem. 230 Dec. 9, 2014

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Chiral Separations in GC Outline

• Introduction

• Theory

• Advantages and Disadvantages

• Applications

• Conclusions

• References

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Chiral Separations in GC Introduction:

• One of the limitations of conventional GC– Cannot be used to separate enantiomers.– Enantiomers are nonsuperimposable mirror-image molecules. – Since they have identical properties, they elute at the same time in

non-chiral GC.

• However diastereomers can be separated using chromatography:– Different chromatographic properties – Different retention times

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Chiral Separations in GC Introduction:

• Chiral separations in GC can be achieved via:– Using a derivatization procedure with a chiral auxiliary

– Using mobile phase additive to form diastereomer complexes which then separated on an achiral

stationary phase.

– Using a non-racemic chiral stationary phase (CSP)

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Chiral Separations in GC Introduction:

• In the late 1960s, chiral separation in GC was a challenging problem among scientists.

• In 1967, Prof. E. Gil-Av was successfully demonstrated chiral separation for L- and D-amino acid esters with lauryl ester of N-trifluoroacetyl-L-isoleucine as CSP.

• In 1977, the first polysiloxane bonded CSP, Chirasil-VAL (thermal stability > 200⁰C) was developed by H. Frank, G. Nicholson and E. Bayer.

• In 1987, the first capillary column coated with cyclodextrins was invented by Prof. W. A. Konig in cooperation with Macherey-Nagel.

• In the early of 1980s, the successful separations of o-, m-, p-xylenes and ethylbenzene on a cyclodextrins CSP were published.

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Chiral Separations in GC Theory:

The main purpose of chiral separations is to determine the precise enantiomeric compositions or enantiomeric excess:

% ee = 100(R-S)/(R+S)

with R= major enantiomer

S= minor enantiomer of chiral analytes

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Chiral Separations in GC Theory:

• Based on their unique chiral selectors, many of CSP in GC are classified into three main groups:

• chiral separation on non-racemic chiral amino acid derivatives via hydrogen bonding

• chiral separation on non-racemic chiral metal coordination compounds via complexation

• chiral separation on biogenic cyclodextrin derivatives via inclusion Subsequently, all of the chiral selectors are chemically linked to

polysiloxanes.

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Chiral Separations in GC Theory:

• Non-racemic amino acid derivatives as CSP

polysiloxane

Chiral center

Hydrogen bonding site

http://www.spectroscopyonline.com/spectroscopy/data/articlestandard//lcgc/162007/420842/i8.gif

Non-racemic AA derivatives

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Chiral Separations in GC Theory:

• Non-racemic amino acid derivatives as CSP• Hydrogen bonding interaction • Enantiomeric separation of polar analytes such as alcohols, diketones, amines and

2- and 3-hydroxyl carboxylic acids. • Peak switching principle For example: on Chiral-L-Val, the D-AAs elute before the L-AAs.• Used for trace analysis and purity studies of amino acids (AAs).• The enantiomer with a significantly lower concentration must be eluted first for

the quantification to be accurate. • Upper temperature limit of 200⁰ CDrawback: racemization of the stationary phase.

derivatization of analytes in order to increase volatility and interaction sites

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Chiral Separations in GC Theory:

• Chiral metal coordination compounds as CSP

http://www.sciencedirect.com.proxy.lib.csus.edu/science/article/pii/S0021967300005057?np=y

polysiloxane

Complexation site

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Chiral Separations in GC Theory:

• Chiral metal coordination compounds as CSP• Metal coordination principles

• Using various chiral 1,3-diketonate bis chelates of Mn(II), Co(II) and Ni(II)

• Chiral analysis of volatile non-hydrogen-bonding compounds

• Chiral analysis of chiral oxygen-, nitrogen- and sulfur-containing compounds.

• Used for mechanistic investigations and for understanding inherent principles of chiral recognition (entropy/enthalpy compensation)

• Not widely used

Drawback: low temperature range of operation (25-120 ⁰C)

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Chiral Separations in GC Theory:

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Chiral Separations in GC Theory:

• Cyclodextrins (CDs) as CSP Unfunctionalized CDs• Lower possibility of formation of the

opposite chirality

• Lower chance of reversal elution

Drawback: Low solubilites from the

hydroxyl groups at positions 2, 3, and 6.

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Chiral Separations in GC Theory:

• Cyclodextrins (CDs) as CSP Functionalized CDs

• Derivatization of the hydroxyl groups

• Improving the solubility of the CD

• Distort the shape of the molecule making inclusion more difficult, increasing interactions at the modified surface.

• May applied to column neat or dissolved in polysiloxanes at different percentage.

• Wide operating temperatures and low column bleed.

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Chiral Separations in GC Theory:

• Mixed Stationary Phases– Using favorable interactions of each class

–Widening the range of analytes

– Often consist of two different selector classes, primarily amino acid derivatives and inclusion-type structures, such as CDs.

– Efficiencies of separation may decrease if the separations are dependent on one type of interaction.

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Chiral Separations in GC Advantages and Disadvantages:

• Advantages:– No sample derivatization is required (except for very polar analytes)– High efficiency at high speed– Sensitive– Temperature-programming tools– The CSPs need not be enantiomerically pure– In contrast with liquid chromatographic and electrophoretic methods, the

optimization of mobile phases with respect to solvents, pH of buffers, modifiers and gradients is absent.

– Development time and optimization is faster than for HPLC.– Lower LODs– No waste solvent

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Chiral Separations in GC Advantages and Disadvantages:

• Disadvantages:- Volatility

- Thermal stability

- Stereochemical integrity (not prone to racemization)

- Derivatization is required, if compound is polar

- Optically pure chiral reagent (ideally 99% optically pure)

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Chiral Separations in GC Hyphenated Approaches in Chiral GC

• Chiral analysis by GC-MS (SIM)– When high sensitivity is required, the MS system in the SIM mode is set

to monitor only selected ions• 2D approaches in chiral GC– Used for sequential analysis of enantiomers with two or more columns

connected in series by flow-controlled connectors.– 1st column is non-enantioselective (pre-separates the compounds of

interest)– 2nd column is enantioselective

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Chiral Separations in GC Applications:

• Essential oils• Environmental science– General pollutants– Pharmaceuticals in the environment

• Pheromones• Pesticides• Amino acids• Drugs• Markers in aged books• Geochemical biomarkers

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Chiral Separations in GC Applications: Essential Oils

– EOs – compounds obtained from plants– Chiral GC is applied to EO research with particular interest in the origin,

authenticity and characterization of the product.– Recently developed CDs (next slide) have been shown to be useful for

single-run analysis of EOs.

– Bergamot oil – a cold-pressed essential oil produced by cells inside the rind of a bergamot orange fruit.

– The chiral components: α-pinene, β-pinene, sabinene, limonene, linalool, linalyl acetate, and α-terpineol.

– The new CD derivatives separated the enantiomers of all seven components with resolution above 1.5.

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MeMe-CD

Es-GC-MS profile of bergamot essential oil (5) α–pinene, (6) β–pinene, (7) sabinene, (3) limonene, (57) linalool, (20) linalyl acetate, (73) α–terpineol.a: (R) enantiomer b: (S) enantiomer

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EtMe-CD

Es-GC-MS profile of bergamot essential oil (5) α–pinene, (6) β–pinene, (7) sabinene, (3) limonene, (57) linalool, (20) linalyl acetate, (73) α–terpineol.a: (R) enantiomer b: (S) enantiomer

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Chiral Separations in GC Applications: Amino Acids

– Due to nonvolatile and polar nature of AAs, derivatization is normally required before analysis.

– Both D- and L-AAs exist in the natural world.– AAs, under certain circumstances, convert from an L-form to a D-

form over a period of time, which has been related to age since death in human and animal remains and archaeological specimens.

– Aspartic acid (Asp) has the most rapid racemization reaction speed of all the amino acids.

– Asp racemization rates in dentin (D-aspartic acid/L-aspartic acid) are used as an index for age estimation in forensic dentistry.

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Chiral Separations in GC Applications: Amino Acids

– N-trifluoroacetyl isopropyl ester derivatives were applied for the analysis of Asp in human teeth, using a non-racemic chiral AA-type phase.

– Successful separation within approximately 4 min– Favorable elution order: D- and then L- (minor,

followed by a major enantiomer)– The application of chiral GC stationary phases to

AAs and other small organic molecules allows the potential, in combination with MS, for chiral analysis in extra terrestrial environments such as the planet Mars.

Figure —Gas chromatogram of N-trifluoroacetyl isopropyl esters of amino acids in dentin. 25

Chiral Separations in GC Applications: Markers in Aged Books

– Volatile organic compounds (VOCs) are produced as breakdown products from the paper found in aged books.

– Chiral molecules (e.g. 2-ethyl-1-hexanol) are detected, but only in the (S)-isomer.

– Chiral separations of (R)- and (S)- 2-ethyl-1-hexanol were carried out on a Cyclosil-B stationary phase.

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Chiral Separations in GC Conclusions:

• Chiral separations using GC represent a popular and advanced technique.

• Chiral separations using GC employ two methods: (1) direct approach utilizing chiral stationary phases; (2) indirect approach requiring derivatization with a chiral reagent.

• The selection of a CSP still remains a matter of trial and error (no universal CSP for GC is yet available).– Permethylated β-cyclodextrin-derived CSPs appear to be leading

• New types of stationary phases are constantly being developed.• The wide range of applications demonstrates that scientists from

widely differing disciplines find the technology useful.

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References1. Schurig, V., Separation of enantiomers by gas chromatography. J. Chromatogr. A 2001, 906 (1-2),

275-299.2. Schurig, V., Chiral separations using gas chromatography. TrAC, Trends Anal. Chem. 2002, 21

(9/10), 647-661.3. Morrison, C. In Chromatographic separations and analysis: chiral gas chromatography, Elsevier

B.V.: 2012; pp 333-353.4. Bicchi, C.; Cagliero, C.; Liberto, E.; Sgorbini, B.; Martina, K.; Cravotto, G.; Rubiolo, P., New

asymmetrical per-substituted cyclodextrins (2-O-methyl-3-O-ethyl- and 2-O-ethyl-3-O-methyl-6-O-t-butyldimethylsilyl-β-derivatives) as chiral selectors for enantioselective gas chromatography in the flavour and fragrance field. J. Chromatogr. A 2010, 1217 (7), 1106-1113.

5. Ohtani, S.; Yamamoto, T., Age estimation by amino acid racemization in human teeth. J Forensic Sci 2010, 55 (6), 1630-3.

6. Gaspar, E. M.; Santana, J. C.; Lopes, J. F.; Diniz, M. B., Volatile organic compounds in paper-an approach for identification of markers in aged books. Anal. Bioanal. Chem. 2010, 397 (1), 369-380.

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Questions:

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