BY Yogesh K. Chaudhari M Pharm 1st year (Pharmacology University of Mumbai

Chiral: A molecule is chiral if it is not superimposable on its mirror image.

Most chiral molecules can be identified by their lack of a plane of symmetry or center of symmetry.

Enantiomers or Optical isomers: Two mirror images of chiral molecules.

Chiral chromatography refers to the separation of enantiomers using a chiral HPLC column, an HPLC column packed with chiral stationary phases.

Approximately 60% of Pharmaceutical Drugs are chiral.

Isomers: Compounds with the same molecular formula

Constitutional (or structural)isomers

Stereoisomers

Same atomconnectivity

Different atomconnectivity

Interconvert through rotation about asingle bond

Conformationalisomers or rotamers

Configurationalisomers

Not readily Interconvertible

EnantiomersDiastereomers

Chiral

w/ chiral centers (optically active)

Geometric isomers

Achiral

Configurational isomersConstitutional (structural)isomers

mirror images Enantiomers

Chiral MoleculeChiral Molecule: : • Has one stereogenic center Has one stereogenic center

(typically C, but can be N, P, (typically C, but can be N, P, etc.), which is attached to 4 etc.), which is attached to 4 different substituents different substituents asymmetricasymmetric

• one that is one that is notnot superi superi mposable on its mirror image mposable on its mirror image

(the (the two are not identical)two are not identical)

– i.e. hands, keys, shoesi.e. hands, keys, shoes• the two mirror image forms the two mirror image forms

are called are called enantiomersenantiomers • Optically activeOptically active

Achiral MoleculeAchiral Molecule: : • Has no stereogenic center; Has no stereogenic center;

the carbon atom has less than the carbon atom has less than 4 non-equivalent substituents 4 non-equivalent substituents attachedattached

• has a plane of symmetryhas a plane of symmetry• one that one that isis superimposable on superimposable on

its mirror image (the two are its mirror image (the two are identical)identical)– i.e. nail, ball, a baseball bat i.e. nail, ball, a baseball bat

• Not optically activeNot optically active

• Each enantiomer has an equal but opposite optical rotation; can be measured using optical rotation polarimeter• One enantiomer rotates polarized light in a clockwise direction and is then designed as (+), or dextrorotatory• The other enantiomer rotates polarized light in counter-clockwise direction and is the (-) enantiomer, or levorotatory• Racemates (1:1 mixture of enantiomers) have no observable optical rotation; they cancel each other outSpecific Rotation =

[]D

l * c

where = observed rotation, l = cell length in dm, c = concentration in g/mL, and D is the 589nm light from a sodium lamp

Isomers : Compounds with the different chemical structures and the same molecular formula

Stereoisomers: compounds made up of the same atoms but have different arrangement of atoms in space

Enantiomers are the 2 mirror image forms of a chiral molecule can contain any number of chiral centers, as long as each center is the

exact mirror image of the corresponding center in the other molecule Identical physical and chemical properties, but may have

different biological profiles. Need chiral recognition to be separated.

Different optical rotations (One enantiomer is (+) or dextrorotatory (clockwise), while the other is (-) or levorotatory (counter clockwise))

Racemate: a 1:1 mixture of enantiomers. Separation of enantiomers occurs when mixture is reacted with a chiral

stationary phase to form 2 diastereomeric complexes that can be separated by chromatographic techniques

Diastereomers: stereoisomers that are not enantiomers Have different chemical and physical characteristics, and can be

separated by non-chiral methods. Has at least 2 chiral centers; the number of potential diastereomers for

each chiral center is determined by the equation 2n, where n=the number of chiral centers

• Single enantiomers of chiral active pharmaceutical ingredients (APIs) may have different:– Pharmacokinetic properties in animal models

• Absorption, distribution, metabolism and excretion– Pharmacological or toxicological effects

• Biologically “active” isomer may have desirable effects• Biologically “inactive” isomer may have undesirable side effects (i.e.

increased toxicity)• Increased pressures by regulatory authorities to

switch from racemic to single enantiomer APIs• Development of chiral APIs raises issues regarding:

– acceptable manufacturing control of synthesis and impurities– pharmacological and toxicological assessment of both

enantiomers– proper assessment of metabolism and distribution– proper clinical evaluation of these drugs

Albuterol (anti-asthmatic inhalant) D-albuterol may actually cause airway constriction Levalbuterol (L-albuterol) avoids side effects

Allegra (allergy medication) Single enantiomer of Seldane that avoids life-threatening

heart disorders of Seldane Fluoxetine (generic name for Prozac,

depression medication) R-Fluoxetine – improved efficacy; minimizes side effects, i.e.

anxiety and sexual dysfunction. Other indications (eating disorders)

S-Fluoxetine – use for treatment of migraines

Chiral Recognition: Ability of chiral stationary phase, CSP, to interact differently with each enantiomer to form transient-diastereomeric complexes; requires a minimum of 3 interactions through:

H-bonding π-π interactions Dipole stacking Inclusion complexing Steric bulk

Five general types of CSPs used in chromatography:1. Polymer-based carbohydrates2. Pirkle or brush-type phases3. Cyclodextrins4. Chirobiotic phases 5. Protein-based

CSP Biphenyl derivative

1) Polymer-based Carbohydrates Chiral polysaccharide derivatives, i.e. amylose and cellulose,

coated on a silica support Enantiomers form H-bonds with carbamate links between

side chains and polysaccharide backbone Steric restrictions at polysaccharide backbone may prevent

access of one of enantiomers to H-bonding site Can be used with normal phase HPLC, SFC, RP-HPLC Limitations: Not compatible with a wide range of solvents

other than alcohols

• Available columns:– i.e. Chiralpak AD, AD-RH, AS, AS-RH, and Chiralcel OD, OD-RH, OJ, OJ-RH,

etc. from Chiral Technologies, Inc.– Chiralpak IA and IB…same chiral selectors as AD and OD, respectively, but

these are immobilized on the silica; more robust and has much greater solvent compatibilities

CH3

O

OH

MeO

Conditions:Chiralpak AD-HHexane/IPA/TFA, 80:20:0.1Flow: 1.0 mL/min

Conditions:Chiralpak AS-RHaq. H3PO4 (pH2)/ACN, 60:40Flow: 0.7mL/min

Conditions:Chiralpak AD-H, 100x4.6mmCO2/MeOH, 80:20Flow: 5.0 mL/min

Conditions:Chiralpak AD-H, 100x4.6mmCO2/MeOH, 90/10Flow: 2.0 mL/min

2) Pirkle or Brush-type Phases: (Donor-Acceptor)– Small chiral molecules bonded to silica– More specific applications; strong 3-point interactions through 3 classes:

• π-donor phases• π-acceptor phases• Mixed donor-acceptor phases

– Binding sites are π-basic or π-acidic aromatic rings (π-π interactions), acidic and basic sites (H-bonding), and steric interaction

– Separation occurs through preferential binding of one enantiomer to CSP– Mostly used with normal phase HPLC, SFC. May get less resolution with RP-

HPLC; compatible with a broad range of solvents– Limitations: only works with aromatic compounds

• Available columns:• Whelk-O 1, Whelk-O 2, ULMO, DACH-DNB (mixed phases), -Burke 2, β-

Gem 1 (π-acceptor phases), Naphthylleucine (π-donor phases), from Regis Technologies, Inc.

• Phenomenex Chirex phases

(Normal phase)(Reversed phase)

3) Cyclodextrin CSPs Alpha, beta and gamma-cyclodextrins bond to silica and

form chiral cavities 3-point interactions by:

Opening of cyclodextrin cavity contains hydroxyls for H-bonding with polar groups of analyte

Hydrophobic portion of analyte fits into non-polar cavity (inclusion complexes)

One enantiomer will be able to better fit in the cavity than the other

Used in RP-HPLC and polar organic mode Limitations: analyte must have hydrophobic or aromatic

group to “fit” into cavity

• Available columns:– Cyclobond (-, -, and -cyclodextrins) from Astec, Inc.– ORpak CDA (), ORpak CDB (), ORpak CDC () from JM

Sciences

Conditions Results

Column: CYCLOBOND I 2000

Dimensions (mm): 250x4.6mm

Catalog Number: 20024

Mobile Phase: 10/90: CH3CN/1% TEAA, pH 4.1

Flow Rate (mL/min): 1.0 mL/min.

Temp (oC): 23°C

Chart Speed (cm/min): 0.4cm/min.

Detection (nm): 254nm

Injection Volume (µL): 2.0µL

Sample Concentration (mg/mL): 5.0mg/mL

Peak1 16.1 Peak2 18.1

Chlorpheniramine example using

Cyclodextrin-type CSPchlorpheniramine

4) Chirobiotic Phases Macrocyclic glycopeptides linked to silica Contain a large number of chiral centers

together with cavities for analytes to enter and interact

Potential interactions: π-π complexes, H-bonding, ionic interactions Inclusion complexation, steric interactions

Capable of running in RP-HPLC, normal phase, polar organic, and polar ionic modes

• Available columns:– Chirobiotic V and V2 (Vancomycin), Chirobiotic T and T2

(Teicoplanin), Chirobiotic R (Ristocetin A) from Astec

Peak1 8.78 Peak2 10.48

Conditions Results

Column: CHIROBIOTIC V

Dimensions (mm): 250x4.6

Catalog Number: 11024

Mobile Phase: 10/90:THF/0.1% TEAA, pH7

Flow Rate (mL/min): 1.0 mL/min.

Temp (oC): 25°C

Chart Speed (cm/min): 0.5

Detection (nm): 254

Injection Volume (µL): 2

Sample Concentration (mg/mL): 5

Naproxen example using Chirobiotic-type CSP

Naproxen

5) Protein-based CSPs Natural proteins bonded to a silica matrix Proteins contain large numbers of chiral centers and interact

strongly with small chiral analytes through: Hydrophobic and electrostatic interactions, H-bonding

Limitations: Requires aqueous based conditions in RP-HPLC Analyte must have ionizable groups such as amine or acid. Not suited for preparative applications due to low sample capacity

• Available columns:– Chiral AGP (-glycoprotein) from ChromTech– HSA (human serum albumin) from ChromTech– BSA (bovine serum albumin) from Regis Technologies

Naproxen examples using Protein-based type CSP

Human Serum Albumin CSP Acid glycoprotein CSP

1.0 It is possible to effect an enantiomeric separation using conventional HPLC stationary phases by adding a chiral selector to the mobile phase.

2.0 Chiral selector additives generally interact via ion pair ligand exchange or inclusion interactions with enatiomeric analytes, forming diastermeric complexes that free separable to conventional normal phase or reversed phase columns.

3.0 When free cyclodexrins are added to the mobile phase, inclusion complexes are formed and separation can approach those obtained on cyclodextrins based chiral stationary phases.Chiral separation of Ketoprofen on an achiral C8 column by HPLC using norvancomycin as chiral mobile phase additives.S (+) Analgesic and antiinflammatoryR (-) slows periodontal bone loss

Chiral derivatising agent (chiral resolving agent) react with enantiomers to gives diasteromers. Since diasteromers have different physical properties, that they further analyzed by HPLC and NMR spectroscopy.Two compounds that are enatiomers have same NMR spectral properties.e.g. Analysis of enantiomers of chiral phenylethylamine i.e. Amphetamine vy capillary GC/MS/FID and precolumn chiral derivatisation from biological fluids.

Following derivatising agent widely usedN-alpha-(2,4-dinitro-5-fluorophenyl)-L-alaninamide (FDAA), 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl isothiocyanate (GITC), (S)-N-(4-nitrophenoxycarbonyl)phenylalanine methoxyethyl ester (S-NIFE), or o-phthalaldehyde/isobutyryl-L-cysteine (OPA-IBLC),

One enantiomers exhibits desired biological activity and other enantiomers may exhibit undesired sideffects thereby making chiral purity an important part.Circular Dichiorism (CD) detector: Optically active compounds with chromophore close to a chiral center may be absorb circularly polarized light , which can be detected with excellent sensitivity and selectivity.

When chiral compounds are measured using UV-visible detectors , d and l enantiomers cannot distinguished eventhough separated by chiral column. Chiral detectors measures the angle of rotation of plane polarised light caused by optically active isomers and useful for the chiral compounds with no absorption.

ULTRA PERFORMANCE LIQUID CHROMATOGRAPHY (UPLC)

UPLC brings dramatic improvements in sensitivity, resolution and speed of analysis can be calculated. It has instrumentation that operates at high pressure than that used in HPLC & in this system uses fine particles (less than 2.5 µm) and mobile phases at high linear velocities decreases the length of column, reduces solvent consumption & saves time. Therefore, by using smaller particles, speed and peak capacity (number of peaks resolved per unit time in gradient separations) can be extended to new limits, termed ultra performance liquid chromatography.

UPLC system allows shortening analysis time up to nine times comparing to the conventional system.

ADVANCES IN HPLC SYSTEMHigh temperature liquid chromatographyIncreased temperature of liquid mobile phase is correspondingly lowers mobile phase viscosity, which allows increased mobile phase flow rate through liquid chromatography system while maintaining desired chromatographic analysis attributes. Zironica a packing material for stationary phase, is thermally stable and provides relatively stable analytical separations at temperature even in excess of 200 °C. In fact, recent tests have demonstrated that packing materials utilizing Zironica as the substrate material are chemically and thermally stable at temperature approaching the critical point of water (375 °C). Water increasingly resembles an organic solvent as temperature increasingly towards critical temperature of water. In fact, recent tests and calculations indicate that at 250 °C, water exhibits solvent properties approaching those of the pure organic solvent, such as methanol and acetonitrile. Thus, in reversed phase applications, the use of only water as a mobile phase is environmentally and economically highly desirable.

ADVANCES IN HPLC SYSTEMMonolithic reversed phase silica columnA monolithic HPLC column is a special type of column used in HPLC with porous channels rather than beads. In these, tiny beads of an inert substance, typically a modified silica, are packed tightly into a tube. Monolithic columns possess a different structure from traditional columns. Their construction is more akin to a rod with lots of random channeling and outcroppings. Monoliths support high flow rates without sacrificing resolution as they exhibit no void volume and can withstand flow rates up to 9.0 mL/minute.

ADVANCES IN HPLC SYSTEMMicrochip HPLC system: Recent microchip HPLC focused on control of pumping pressure and sample injections in polymer and glass microsystems. Rapid microchip RP-HPLC of peptides and proteins at pressure gradients of 180 psi/cm has been performed using a microdevice that integrates on-chip injection, separation, and detection with a miniaturized LIF detector. Separation was achieved via definition of a C18 side-chain porous polymer monolith using contact lithography, and injection was achieved via definition of a pressure switchable fluoropolymer valve using projection lithography. Preliminary separations of peptide standards and protein mixtures were performed in 40-200 s, and switching between samples with no detectible sample carryover has been performed at 72 injections/h. Sample volumes ranging from 220 to 800 pL could be linearly metered by controlling the pressure injection pulse duration with conventional timing and valving.

ADVANCES IN HPLC SYSTEM Development of new high-capacity, high-selective and high

efficient stationary phases for separation of chiral molecules, i.e. molecules that exist as different mirror image forms.

The stationary phase particles become even smaller to increase performance further; at present particles with 1.7 μm in diameter are commercially available. LC columns packed with such materials require ultra-high pressure pumps to provide sufficient flow rates this technique also called ultra performance liquid chromatography (UPLC).

Column miniaturization to decrease sample and mobile phase consumption.

The development of monolithic stationary phases. Instead of packing the column with spherical particles, a single-piece stationary phase is synthesized by in situ polymerization.

Operation at very high temperatures to decrease mobile phase viscosity, increase solute solubility and to enable the use of nontoxic eluents such as water. Stationary phases are developed to withstand extreme conditions, such as very high or very low pH.