Transcript Chiral Separations in Gas Chromatography

Chiral Separations in Gas
Chromatography
Olga Inozemtseva – Dai Thai
Dr. Dixon
Chem. 230
Dec. 9, 2014
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Chiral Separations in GC
Outline
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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
Non-racemic AA
derivatives
Hydrogen bonding
site
http://www.spectroscopyonline.com/spectroscopy/data/articlestandard//lcgc/162007/420842/i8.gif
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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 C
Drawback: 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
polysiloxane
Complexation
site
http://www.sciencedirect.com.proxy.lib.csus.edu/science/article/pii/S0021967300005057?np=y
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Chiral Separations in GC
Theory:
• Chiral metal coordination compounds as CSP
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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
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Drawback: low temperature range of operation (25-120 ⁰C)
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Chiral Separations in GC
Theory:
• Cyclodextrins (CDs) as CSP
• Cyclic molecules consisting of six (α- CD), seven (β- CD) or eight (γ-CD)
D-glucopyranose units bonded through α- 1,4 linkages.
• Formed as enzymatic degradation products of starch.
• CDs can interact with an analytes via:
Functionalized CDs
Hydrogen bonds (functional groups)
» Dipole/dipole interactions (functional groups)
» Hydrophobic interactions (carbon content)
» Steric interactions
Unfunctionalized CDs
» Inclusion (size of the molecule)
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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:
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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
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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 Dform 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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References
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Schurig, V., Separation of enantiomers by gas chromatography. J. Chromatogr. A 2001, 906 (1-2),
275-299.
Schurig, V., Chiral separations using gas chromatography. TrAC, Trends Anal. Chem. 2002, 21 (9/10),
647-661.
Morrison, C. In Chromatographic separations and analysis: chiral gas chromatography, Elsevier B.V.:
2012; pp 333-353.
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-Ot-butyldimethylsilyl-β-derivatives) as chiral selectors for enantioselective gas chromatography in the
flavour and fragrance field. J. Chromatogr. A 2010, 1217 (7), 1106-1113.
Ohtani, S.; Yamamoto, T., Age estimation by amino acid racemization in human teeth. J Forensic Sci
2010, 55 (6), 1630-3.
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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