CYCLOBOND™ Handbook - Sigma-Aldrich

C Y C L O B O N DH A N D B O O K

A G U I D E

T O U S I N G

C Y C L O D E X T R I N

B O N D E D P H A S E S

F O R C H I R A L L C

S E P A R A T I O N S

TM

CYCLOBOND HANDBOOKTable of Contents

Introduction to Cyclodextrins......................................................................................... 01Chemical Structure............................................................................................... 01Physical Properties.............................................................................................. 01

Bonded Native Cyclodextrins: CYCLOBOND..................................................................... 01Reversed Phase Separations on CYCLOBOND.......................................................... 02

Introduction................................................................................................. 02General Retention Mechanism: Inclusion Complexation........................................ 02Inclusion Selectivity...................................................................................... 02Solvent Strength.......................................................................................... 03Functional Group Interaction........................................................................... 03Rules for Obtaining a Chiral Separation in the RP Mode......................................... 04Controlling Parameters.................................................................................. 04

Proper Choice of Cyclodextrin Phase....................................................... 04pH Effects.......................................................................................... 04Buffer Effects..................................................................................... 04Flow Rate Effects................................................................................ 05Temperature Effects............................................................................ 05

Reversed Phase Mode Protocol....................................................................... 06Polar Organic Mode Separations on CYCLOBOND...................................................... 07

Introduction................................................................................................. 07General Retention Mechanism......................................................................... 07Controlling Parameters.................................................................................. 08

Proper Choice of Cyclodextrin Phase....................................................... 08Mobile Phase Composition..................................................................... 08Flow Rate Effects................................................................................ 09Temperature Effects............................................................................ 09

Polar Organic Mode Protocol........................................................................... 12Bonded Derivatized Cyclodextrins................................................................................... 13

CYCLOBOND I 2000 DM........................................................................................ 13CYCLOBOND I 2000 AC......................................................................................... 15CYCLOBOND I 2000 SP and RSP............................................................................ 15CYCLOBOND I 2000 SN, RN and DMP...................................................................... 16

Introduction................................................................................................. 17Normal Phase Operation................................................................................ 17

Preferred Solvent Conditions................................................................. 17Optimum Flow Rates............................................................................. 17Temperature Effects............................................................................ 17Normal Phase Mode Protocol................................................................. 18Normal Phase Mode Separations............................................................ 18Reversal of Elution Order...................................................................... 20

Polar Organic Mode Operation......................................................................... 20Reversed Phase Operation............................................................................. 21Fluorescent Tagged Primary Amines on CYCLOBOND I 2000 Carbamates................ 21

Trace Analysis on CYCLOBOND Phases.......................................................................... 22SFC Using CYCLOBOND Phases.................................................................................... 23Preparative Separations on CYCLOBOND......................................................................... 26General Operating Conditions......................................................................................... 29

Determining Void Volume (Vo)................................................................................. 29Temperature........................................................................................................ 29Injection Volume and Concentrations....................................................................... 29Stability.............................................................................................................. 29Column Evaluation................................................................................................ 29Flow Rate............................................................................................................ 30Pressure............................................................................................................ 30Regeneration....................................................................................................... 30Storage.............................................................................................................. 30Column Assessment Parameters............................................................................. 30

Compounds Separated on CYCLOBOND Columns.............................................................. 31Bibliography................................................................................................................ 46Ordering Information..................................................................................................... 56

1

CYCLOBOND™Bonded Cyclodextrin Stationary Phases for Liquid Chromatography

Introduction to Cyclodextrins

Chemical Structure

Cyclodextrins are produced by the partial degradation ofstarch and enzymatic coupling of cleaved glucose unitsinto crystalline, homogenous toroidal structures ofdifferent molecular size. Three of the most widely charac-terized are alpha, beta and gamma cyclodextrin. Theycontain 6, 7 and 8 glucose units, respectively.Cyclodextrins are, therefore, chiral structures. Forexample, beta-cyclodextrin has 35 stereogenic centers.The toroidal structure has a hydrophilic surface resultingfrom the 2, 3 and 6 position hydroxyl groups making themwater soluble. The cavity is composed of the glucosideoxygens and methylene hydrogens giving it an apolarcharacter. As a consequence, cyclodextrins can includeother apolar molecules of appropriate dimensions andbind them through dipole-dipole interactions, hydrogenbonding or London dispersion forces.

Based on the evidence of x-ray crystallographic data, thebeta and gamma structures appear to have quite rigid andinflexible structures and are stable to a wide variety ofaqueous and organic solvents. The bonds of alpha-cyclodextrin appear weaker and capable of stretching.These facts can be used to induce the fit of largermolecules into the alpha cavity by deformation of itsstructure under high aqueous buffer conditions.Cyclodextrins are pH stable from 3 to 14.

One or two of the primary hydroxyl groups are used to linkthe cyclodextrin to the surface of a media. The secondaryhydroxyl groups can be derivatized selectively, generallythose in position 2 first and then position 3. Assubstitution increases reaction rates decrease. Severalderivatives of the native cyclodextrins have been made tochange the physical and chemical properties of thecyclodextrin and extend the separation capabilities.

Physical Properties

CyclodextrinNo. ofGlucoseUnits

MW CavityDiameter

WaterSolubility(g/100mL)

Alpha 6 972 0.57nm 14.5Beta 7 1135 0.78nm 1.85*Gamma 8 1297 0.95nm 23.2

*Note: If position 2 or 3 hydroxyl groups of theβ-cyclodextrin are ethoxylated or propoxylated thesolubility increases to 45g/100mL.

Alpha, Beta and Gamma Cyclodextrin Structures

α-cyclodextrin

β-cyclodextrin

γ-cyclodextrin

Bonded Native CyclodextrinsCYCLOBOND™The three bonded native cyclodextrins are the basis ofthe entire line of CYCLOBOND products. They areCYCLOBOND I (β-cyclodextrin bonded), CYCLOBOND II(γ-cyclodextrin bonded) and CYCLOBOND III (α-cyclo-dextrin bonded). The cyclodextrin with the broadestapplicability has been the beta form. It has demonstratedthe greatest suitability for small analytes of generalinterest in the pharmaceutical, chemical and environ-mental areas. As a consequence of this diversity,CYCLOBOND I has been used as the basis for severalderivatives. These derivatives were designed for HPLC to

2

accommodate various spacial requirements and providespecific interactions with certain functional groups toproduce highly selective separations for a versatile arrayof analytes.

Since the introduction of the native cyclodextrin bondedphases as CYCLOBOND at the 1984 Pittsburgh Confer-ence, there have been several advances in the technol-ogy that have increased performance and applicability ofthis technology. The first of these advances was theintroduction of cyclodextrin derivatives. Of the originalseven derivatives, the most significant today have beenthe CYCLOBOND I 2000 RSP (R,S,-hydroxypropyl ether)and the CYCLOBOND I 2000 SN (S-naphthylethylcarbamate) and RN (R-naphthylethyl carbamate). Thenext major advance was the introduction of the PolarOrganic Mode and, finally, the introduction of theCYCLOBOND I 2000 series, the second generationCYCLOBOND I phases giving improved selectivity andresolution.

Reversed Phase Separationson CYCLOBOND

Introduction

All CYCLOBOND columns can be run in the reversedphase mode. The following principles govern its use andare applicable to all native and derivatized forms ofCYCLOBOND, the difference being in the position andtype of functional group or groups near or on thestereogenic center. A study of the examples will aid inchoosing the proper column.

General Retention Mechanism:Inclusion Complexation

+

C YX

Z

Ks

X

C Y

Z

When cyclodextrin stationary phases (CYCLOBOND) areused with aqueous mobile phases, the basic mechanismthat retains solutes is referred to as inclusioncomplexation. This mechanism represents the attractionof the apolar molecule or segment to the apolar cavity.When an aromatic group is present, the orientation in thecavity is selective due to the electron sharing of thearomatic methylene groups with those of the glucosideoxygens. Linear or acyclic hydrocarbons occupy morerandom positions in the cavity. It is essential, therefore,if a chiral separation is attempted in the reversed phasethat the analyte have at least one aromatic ring. The onlyexception observed so far has been the separation ofcertain heterocyclic analytes and t-boc amino acids onthe CYCLOBOND I 2000 RSP.

The high density of secondary hydroxyl groups at thelarger opening of the toroid acts as an energy barrier for

polar molecules attempting to complex, and preferentialhydrogen bonding occurs. Amines and carboxyl groupsinteract strongly with these hydroxyl groups as a functionof the pK of the analyte and pH of the aqueous system.This relationship is important in understanding how todesign mobile phases for chiral separations on bondednative cyclodextrins.

The first important consideration for proper retention andchiral recognition is proper fit of the molecules to thecyclodextrin cavity. This fit is a function of both size andshape of the analyte relative to the cyclodextrin cavity.

Retention of Polyaromatic Hydrocarbons

CH3

2

4

6

8

10

12

14

16

18

20

22

24

26

CY

CLO

BO

ND

III (

alph

a)

CY

CLO

BO

ND

I (b

eta)

CY

CLO

BO

ND

II (g

amm

a)

Rt

Chrysene

Pyrene

PhenanthreneAnthracene

Naphthalene

Toluene

It is easy to recognize that for cyclodextrin inclusion, themolecular weight of a polyaromatic ring structure is not ascritical as its bulk. The enantiomers of an analyte likeNorgestrel (a 5 ring steroid structure) are better separatedon the gamma-cyclodextrin (CYCLOBOND (II), whileenantiomers of a naphthylene like structure or singlesubstituted aromatic ring structures would better fitbeta-cyclodextrin (CYCLOBOND I).

As a general rule, substituted phenyl, naphthyl andbiphenyl rings can be separated on the beta-cyclodextrin(CYCLOBOND I) while smaller molecules separate on thealpha (CYCLOBOND III). Molecules with three to five ringsin their structure, i.e. steroids, can best be separated onthe gamma (CYCLOBOND II).

Inclusion Selectivity

Inclusion experiments with pyrene have demonstratedboth the apolar nature of the cyclodextrin cavity as wellas the relationship to the fit of the analyte. The better thefit of pyrene, the more complete the inclusion and thegreater its fluorescence output indicating a more nonpolarenvironment. Using this data, the general polarity of the

3

cavity is comparable to that of the oxygenated solventssuch as n-octanoic, n-octanol, isopropyl ether and t-amylalcohol. This correlation is applicable to free or bondedcyclodextrins and is independent of cavity size.33

The response to mobile phase polarity for the bondedcyclodextrins would be equivalent to a reversed phaseC8. The correlation is not exact in that smaller watersoluble aromatics show a greater affinity to thecyclodextrin structure than to C8. This is due to the lowcapacity of standard reversed phase in high aqueousmobile phase compositions resulting from the interactionof the hydrocarbon moieties to the exclusion of otheranalytes. For the purpose of mobile phase design, thecorrelation is adequate.

Solvent Strength

The weakest solvent to interact with the cyclodextrincavity is water. Alcohols are next in displacementstrength increasing with the number of methylene groups,i.e., CH3OH<C2H5OH<C3H7OH, etc. Generally, acetoni-trile is stronger than methanol and closer in its polarity topropanol. Halogenated solvents have a very strongdisplacement effect and are generally not recommended.

In many cases, solvent strength is independent of chiralrecognition as it effects only the displacement of theanalyte from the cavity. Methanol is often used as thestarting solvent because it is the weakest displacerafter CO2.

Acetonitrile, however, as in standard reversed phase,generally gives the most efficient peaks but exhibits astronger displacement effect. In a number of cited cases,i.e., Ibuprofen, methanol overwhelms the weak hydrogenbonding forces of the secondary hydroxyl groups,diminishing separation. Acetonitrile produces the bestseparation in this case.

A balance generally exists between buffer concentrationand the percent of organic modifier. Lower concentrationsof buffer (0.1 to 0.5%) allow for higher concentrations oforganic modifier.

Functional Group Interaction

Certain functional groups have a strong affinity for thecyclodextrin cavity. Other polar groups strongly hydrogenbond to the high density diol surface. For chiralrecognition to occur in the reversed phase mode, theorientation of the analyte in the cyclodextrin cavity isimportant. Knowing the attraction of certain groups to thecavity helps in this determination.

Preferred Inclusion Functional Groups

■ Iodide>Bromide>Chloride>Fluoride

■ Nitrate, Sulfate, Phosphate

■ Hydroxyl (as a function of pH)

Preferred Hydrogen Bonding Groups

■ Carboxyl, Carbonyl

■ Amines, primary, secondary, tertiary, free or in ring structures

Retention of an aromatic structure can be increased bysubstitution with a preferred inclusion functional group aswell as by the presence of a preferred hydrogen bondinggroup.

In the following example, chiral recognition is favorable forbeta-cyclodextrin (CYCLOBOND I), however, the nitrogroup pulls the analyte toward the cavity and thehydrogen acceptor group away from interaction with thecyclodextrin hydroxyl group at 2 position. CYCLOBOND I2000 SP (hydroxypropyl) is a better choice since theflexible hydrogen bonding group can extend the hydrogenbonding to the carbonyl to satisfy the second rule forchiral recognition.

Example of Preferred Inclusion Groups

Calcium Channel Blocker

N OC

O

NO2

C OCH

O

Changes plane of inclusion

Hydrogen acceptor site

Weaker cyclodextrin inclusion

Preferred cyclodextrin inclusion

*

Chiral center

CH3 (CH3)3S

H

N

N

CYCLOBOND I 2000 SP15/85: CH3CN/1% TEAA (pH 4.1)

1.0 mL/min.

Note: For CYCLOBOND I 2000 SP structure seeSummary of Derivatives, page 13.

4

Rules for Obtaining a Chiral Separationin the Revered Phase Mode

Rule 1:

The analyte must have a proper structural fit to thecyclodextrin. This generally dictates that at least onearomatic ring structure must be present in the solutemolecule.

Rule 2:

A substituent on or near the stereogenic center mustinteract (attract or repulse) with the 2 or 3 positionhydroxyl groups at the mouth of the cyclodextrin cavity.Steric effects due to large bulky groups near thestereogenic center can have a similar effect. Remember,these hydroxyl groups are sterically fixed and the positionof the interacting groups in relation to the aromatic ringmay indicate the better use of a derivatized cyclodextrin.When a stereogenic center lies between a single aromaticring and a carbonyl group, the separation of enantiomerscan be easily achieved on a CYCLOBOND I 2000 column.In summary, successful separations can be achieved inthe reversed phase mode on CYCLOBOND stationaryphases with the proper use of controlling parameters.

Controlling Parameters

■ Proper Choice of Cyclodextrin Phase■ pH■ Buffer■ Flow Rate■ Temperature

Proper Choice of Cyclodextrin Phase

This decision is based on ring size, substitution positionand distance/bonding strength of the functional group onor near the stereogenic center. Refer to polyaromatichydrocarbon chart page 2 and preferred inclusion/hydro-gen bonding group list on page 3.

pH Effects

The type of buffer, pH and buffer concentration all effectthe degree of resolution. To isolate the effects of each ofthese factors, an aqueous solution of 0.01M acetic acid isused and pH adjusted with dilute sodium hydroxide. TestpH at 4, 5 and 6 to observe effect on resolution. Theoptimum pH is then used with a select type of buffer andthe buffer concentration is studied. It is possible tocontrol the hydrogen bonding of polar groups on or nearthe stereogenic center with the use of small amounts ofweak acid and base (see Polar Organic Mode).

Simplified Study of pH Effect on Resolution

Analyte: Dansyl D,L-PhenylalanineColumn: CYCLOBOND I 2000Mobile Phase: MeOH/Buffer: 80/20Flow Rate: 1.5 mL/min.

Buffer 1pH 6.0

Buffer 1pH 5.5

Buffer 1pH 5.0

Buffer 1pH 4.0

Buffer 2pH 4.0

Buffer 1 = 0.1M HOAc, adjusted with NaOHBuffer 2 =10mM TEAadjustedwith HOAc

Plot Rt/Rs versus pH3 3.5 4 4.5 5 5.5 6

pH

45

30

15

Rt (min)

(Rs)

(1.0)

(0.5)

(0.0)

Plotting resolution as a function of pH will identify theproper pH for maximum resolution. The correspondingretention time minimum is also obtained. The higher Rtdata could be used for the solid phase extraction versionof CYCLOBOND which would give a higher capacity at thelower pH for this example.

Buffer Effects

Buffers are known to include into the cyclodextrin cavity.As the buffer concentration is increased, solute peaksbecome sharper and Rt decreases. For Dansyl amino

5

acids, buffer concentrations up to 1% can be used. ForIbuprofen (single aromatic ring) more dilute solutions arerequired. Ammonium nitrate strongly hydrogen bonds to

the primary hydroxyl groups and can be used to reducesolute retention (naphthyl ring) for strongly retainedanalytes as shown in the following chromatograms.

Separation of Dansyl D,L-Phenylalanine

#1 #2 #3 #4 #5 #6*B = No Buffer 0.01% TEAA 0.1M NH4NO3 0.05M NH4NO3 0.025M NH4NO3 0.025M NH4NO3Peak 1 - 25.0 min. Peak 1 - 24.6 min. Peak 1 - 6.70 min. Peak 1 - 9.25 min. Peak 1 - 16.8 min. Peak 1 - 14.7 min.Peak 2 - 38.0 min. Peak 2 - 27.9 min. Peak 2 - 7.34 min. Peak 2 - 10.0 min. Peak 2 - 18.6 min. Peak 2 - 16.6 min.

#1-5 Mobile phase is 80/20: CH3OH/Buffer*#6 Mobile phase is 80/20: CH3CN/Buffer.

Flow Rate Effects

When the separation mechanism involves onlydisplacement from the cyclodextrin cavity the relationshipof flow rate to efficiency is identical to reversed phase.However, for enantiomers the response follows a curvewith a slope increase generally most dramatic between1.0 and 0.2 mL/min. This is observed for a wide variety ofenantiomers. The shape of the curve is always the samehaving a midpoint where the rate of change above orbelow increases by some multiple.

Effect of Flow Rate on theResolution of Enantiomers

Flow Rate(mL/min) α Rs

1.00 1.22 0.850.80 1.24 0.920.60 1.24 0.970.40 1.25 1.120.30 1.26 1.220.20 1.24 1.20

25cm β-CD (CYCLOBOND I) column using 50:50(v/v) methanol/water7

Temperature Effects

For chiral separations on CYCLOBOND, temperaturedependence has to be evaluated. Lower temperaturesgenerally lead to increased retention and increasedresolution. The degree to which temperature effectsresolution is dependent on the analyte. In order toevaluate the effect of temperature, it is necessary to

determine both alpha and efficiency at threetemperatures, i.e., 5°C, 15°C and 25°C. A plot of thesevalues versus 1/T will be helpful in optimizing the chiralseparation. In mobile phase compositions of 40 to 60%aqueous methanol, the higher viscosity at lowertemperature negates any beneficial effect. Always tryto use CH3CN as the organic modifier. For accuratetemperature control (0-80°C), we recommend theJetstream Plus Column Thermostat. See AstecChromatography Product Guide or visit our websiteat www.astecusa.com.

Reversed Phase Separation of Ibuprofen

0.98

1.06

1.14

1.22

1.3

Rs

°C

5 15 2 5

CYCLOBOND I 2000CH3CN/0.1% TEAA, pH 41 mL/min.

6

Reversed Phase Mode Protocol

35/65: CH3CN/BUFFERSEE PREFERRED pH’s

PEAKS ELUTE NO ELUTION

75/25: CH3CN/BUFFER

NO SEPARATION SEPARATION PEAKS ELUTE NO ELUTION

1. CHANGE pH

2. CHANGE ORGANIC MODIFIER

1. ADJUST CONC. ORGANIC MODIFIER TO OPTIMUM k’

2. VARY pH, BUFFER STRENGTH, BUFFER TYPE AND ON OCCASION, NATURE OF MODIFIER

1. CHANGE pH

2. CHANGE ORGANIC MODIFIER

NO SEPARATIONOPTIMIZE BY VARYING

PARAMETERS IN SMALLER INCREMENTS

NO SEPARATION

GO TO NORMAL PHASE MODE

NO SEPARATION

SEPARATION

GO TO NORMAL PHASE MODE

CYCLOBOND I 2000

GO TOCYCLOBOND I 2000 RN

SEPARATION

SEPARATION

Preferred BuffersConcentration

Range

TEAA*/TEAPO4 (0.01 to 2.0%)

NH4NO3 (10 to 500mM)

CITRATE (10 to 200mM)

NH4Ac (10 to 200mM)

HOAc, TEA, DEA (10 to 25µL/250mL)

As the concentration of buffer increases, the effect offlow rates decreases. Also, Rt decreases with increasedbuffer concentration.

*Use good grade triethylamine. Dilute to properconcentration and adjust pH with 5% acid. For solutions1% and greater, use glacial HOAc. Different counter-ionslead to different selectivities and efficiencies andultimately in peak resolution. In order of success Ac,PO4, NO3, SO4.

7

Polar Organic Mode Separations onCYCLOBOND

Introduction

The term polar organic mode was applied to this mobilephase design because the composition (a) contains thepolar constituents anhydrous triethylamine and glacialacetic acid; and, (b) does not include any water.

In the past, chiral separations on CYCLOBOND bondedcyclodextrins and cyclodextrin derivatives have been runin basically one of two solvent modes: normal phase, withtypical solvents like Hexane/IPA, CH3CN/CH3OH, pureCH3OH, EtOH; or, reversed phase utilizing CH3OH orCH3CN/Buffer. Inclusion complexation was the drivingforce to obtain enantioselectivity in the reversed phasemode and π-π/hydrogen bonding forces in the normalphase mode. Recently, however, it was discovered that itis possible to override inclusion complexation in favor ofinteracting directly with the secondary hydroxyl groupsacross the larger opening of the cyclodextrin toroid or theappendant carbamate, acetate or hydroxypropylfunctional groups. To accomplish this, the polar organicmode was developed which produces very efficientseparations not previously possible on these phases.This development has been one of the most powerfuladvances in chiral separation on cyclodextrin phases inthe last fifteen years.

Compounds like warfarin, all the various beta-blockers,i.e., propranolol, atenolol, timolol, etc., and an increasingnumber of molecules with particular structural featureshave been separated. The method has also worked formolecules that do not contain an aromatic group. Inaddition to producing many separations that were notpossible in the reversed phase or normal phase modes,the polar organic mode often yields separations that aremore efficient, less retentive and more reproducible, andgenerally, higher sample loads are possible.

General Retention Mechanism

Since the secondary alcohols of the native cyclodextrinsradiate from stereogenic centers, the surface of thecyclodextrin is chiral and can form selective chiralinteractions (diastereomers) with the hydrogen bondinggroups of chiral analytes. CYCLOBOND derivatives likeCYCLOBOND I AC (acetate), CYCLOBOND I RN or SN(naphthylethyl carbamate), CYCLOBOND I SP or RSP(hydroxypropyl), offer this same potential with the RN, SNand SP derivatives adding additional stereogenic centers.The use of acid/base under anhydrous solvent conditionsappears to affect the interactions of compounds withfunctional groups like amines, alcohols, acids andcarbonyls. Since at least three points of interaction arenecessary for chiral recognition, the solvent pool in the

cyclodextrin cavity is seen as one of the interactions. Atleast two functional groups must be present in the analytefor this technique to work and one of the interactivegroups must be on or near the stereogenic center of theanalyte.

The major advantages of the polar organic mode aresimilar to those for normal phase, i.e.:

� Column stability is greater

� Sample capacity is higher

� Retention times are shorter

� Resolution is generally higher

� Solvent is easier to remove

� Better overall preparative scale recoveries

As has been noted, the polar organic mode has worked asa replacement for both reversed phase and normal phasesolvents if certain structural requirements are met.Compare the following separation of ciprofibrate in thenormal phase and propranolol in the reversed phase to theseparation in the polar organic mode.

Note: Severe peak deformation can occur if the sample isdissolved in the wrong solvent. Preference is to use themobile phase but CH3CN can also be used.

Separation of Ciprofibrate (Underivatized)

ClO

Cl

C COOH

CH3

CH3

*

Normal Phase Polar Organic PhaseCYCLOBOND I SNCH3CN/1% HOAc in EtOH:80/201.0 mL/min.

CYCLOBOND I 2000 SNCH3CN/CH3OH/HOAc/TEA:95/5/0.5/0.41.0 mL/min.

Peak 1 - 43.9 min.Peak 2 - 49.1 min.

Peak 1 - 12.84 min.Peak 2 - 14.45 min.

8

Separation of Propranolol

O CH2CHCH2NHCH(CH3)2

OH

Reversed Phase Polar Organic PhaseCYCLOBOND I(2 x 250x4.6mm)CH3OH/1% TEAApH 4.1: 25/75,1.0 mL/min.

CYCLOBOND I 2000(1 x 250x4.6mm)CH3CN/CH3OH/HOAc/TEA:95/5/0.3/0.21.0 mL/min.

Peak 1 - 29.79 min.Peak 2 - 31.46 min.

Peak 1 - 17.1 min.Peak 2 - 18.5 min.

Note: Some chromatograms show the CYCLOBOND I2000 as opposed to CYCLOBOND I. The 2000 series is anew, second generation product designed primarily toguarantee batch to batch reproducibility. The selectivityand retention characteristics of the CYCLOBOND I 2000are very close to the original CYCLOBOND product.

Controlling Parameters

� Proper Choice of Cyclodextrin Phase� Mobile Phase� Flow Rate� Temperature

Proper Choice of Cyclodextrin Phase

All cyclodextrin phases have been used in the polarorganic mode. The most successful have been:

CYCLOBOND I 2000CYCLOBOND I 2000 ACCYCLOBOND I 2000 RNCYCLOBOND I 2000 SNCYCLOBOND I 2000 RSPCYCLOBOND IICYCLOBOND III

For more details on types of structures see theCYCLOBOND bibliography reference #119.

Mobile Phase Composition

There are four components to this mobile phase whenused with cyclodextrin phases. They are mixed byvolume and are used in an anhydrous and pure form. Thefirst two components, acetonitrile and methanol, controlretention. As the methanol concentration is increased,retention decreases. There are cases where methanolmay be eliminated from the mixture. The second twocomponents, glacial acetic acid and anhydroustriethylamine, control selectivity. These two componentsare in low concentration.

Best starting mobile phase composition :CH3CN/CH3OH/HOAc/TEA: 95/5/0.3/0.2.

Separation of Arotinolol (Underivatized)

SH2NC

S

N

SCH2CHCH2NHC(CH3)3O

OH

CYCLOBOND ICH3CN/CH3OH/HOAc/TEA: 95/5/0.3/0.22.0 mL/min.

Peak 1 - 14.89 min.Peak 2 - 16.74 min.

To obtain or optimize resolution in the polar organic mode,it is only necessary to adjust the ratio of acid to base.For the native as well as derivatized cyclodextrins, aneffective ratio of acid to base has been found in the range1:1 to 4:1, the typical average being 1.5:1.0. Formolecules containing carboxyl groups, the ratio has fallento 1.0:1.2. The combined acid/base concentration rangehas been from 0.002% to 2.5% with the average being0.5%. For carboxyl groups, sometimes DEA has beensubstituted for TEA to increase the efficiency of thepeaks. No other substitutions have been found for theother mobile phase components.

9

Effect of Changes in Polar Organic Mode

CYCLOBOND I 2000 SN (250x4.6mm)Mobile Phase: CH3CN/CH3OH/HOAc/TEAFlow Rate: 0.6 mL/min

Analyte: R,S-Coumachlor

A. 95/5/0.3/0.2

Peak 1 - 8.2 min.Peak 2 - 8.6 min.

B. 100/0.3/0.2

Peak 1 - 9.7min.Peak 2 - 10.4 min.

C. 100/0.25/0.05

Peak 1 - 10.7 min.Peak 2 -12.0 min.

To decrease retention:

1) Increase methanol concentration.

2) Increase acid/base concentration at same ratio.

To increase retention:

1) Reduce or eliminate methanol.

2) Decrease acid/base concentration at same ratio.

Separation of Warfarin

O O

OHCHCH2COCH3

C6H5

CYCLOBOND I CYCLOBOND I 2000CH3CN/CH3OH/HOAc/TEA:95/5/0.3/0.2

CH3CN/HOAc/TEA:100/0.3/0.2

1.0 mL/min. 1.0 mL/min.

Peak 1 - 5.91 min.Peak 2 - 6.57 min.

Peak 1 - 6.89 min.Peak 2 - 7.85 min.

Note: Eliminating methanol increased retention with aslight improvement in resolution. This increase is primarilydue to initial low retention of analytes. At higher retentiontimes the methanol effect on Rs is not observed. In thosecases, to improve resolution, work on altering acid-baseconcentration and ratio.

Flow Rate Effects

Unlike the reversed phase mode (inclusion), there is verylittle effect of flow rate on resolution. However, this is yetanother parameter to reduce retention time withoutaffecting resolution. Optimal flow rates: 1.0-2.0 mL/min.

Temperature Effects

Below ambient conditions, resolution increases dramati-cally with a decrease in temperature. At temperatureconditions up to 40°C little effect is seen on resolutionbut retention times can be reduced effectively.

10

Polar Organic Separation of Propranolol

0

0.5

1

1.5

2

2.5

3

3.5R

s

°C

5 15 25

For accurate temperature control (0-80°C), we recommendthe Jetstream Plus Column Thermostat. See AstecChromatography Product Guide or visit our website atwww.astecusa.com.

The response to temperature effects is analytedependent and covers a broad range of responses.

Comparison of Separation Characteristics of Structural Analogues on Cyclodextrin Phases

CompoundCYCLOBOND

Column1 k 2 α3 Rs4 Mobile PhaseComposition5

Oxazepam

N

NCl

O

OH

HSNRN

0.640.66

1.281.35

1.81.9

99/1/0.02/0.0299/1/0.0064/0.0064

MiscellaneousCO2H

O O

S

Ac 1.92 1.12 1.7 90/10/0.45/0.3

Cateolol

OOH

HN O N

H

β-CD 2.24 1.09 1.6 98/2/1.5/1.0

Labetalol

H2N

HO

O OH

N

β-CD k1 = 8.80k2 = 9.17k3 = 9.53k4 = 10.34

98/2/0.8/0.6

Nadolol

O N

HO

OH

OH H

β-CD k1 = 9.83k2 = 10.03k3 = 11.50k4 = 12.08

98/2/0.8/0.6

CH3CN/MeOH: 95/5 + 0.3 ml HOAc +0.25 ml TEA1 mL/min.

11

Metoprolol

OH

O

O N

H

β-CDAc

7.523.33

1.211.15

3.22.9

95/5/0.2/0.295/5/0.3/0.2

Pindolol

OH H

HN O N

γ-CD 8.21 1.10 1.7 99/1/0.2/0.1

Propranolol

OH H

O N

β-CD 4.36 1.12 2.1 95/5/0.3/0.2

Timolol

NS

N

NO

H

O N

OH

β-CDAc

4.522.48

1.151.15

3.02.8

95/5/0.3/0.295/5/0.3/0.2

Suprofen

SO

OH

O

RN 3.28 1.16 1.9 95//5/0.2/0.2

CoumachlorO O

OH

Cl

O

β-CDSNRN

1.271.710.46

1.131.231.25

1.31.31.5

90/10/0.004/0.00495/5/0.005/0.00398/2/0.8/0.6

Warfarin

OOH

OOβ-CDSN

1.241.51

1.171.23

1.61.5

90/10/0.004/0.00495/5/0.005/0.003

2-(2-Chlorophenoxy)propionic acid

Cl

O

O

OH

RN 0.80 1.05 0.6 95/5/0.6/0.4

2-(4-Chlorophenoxy)propionic acid

OH

O

O

Cl

RN 1.00 1.27 2.6 95/5/0.6/0.4

2-Phenoxypropionic acid

OH

O

O

RN 0.92 1.11 1.3 95/5/0.6/0.4

12

1 Columns (250x4.6mm):β-CD CYCLOBOND IAC CYCLOBOND I AC (acetylated β-CD)SN CYCLOBOND I SN (S-naphtylethyl carbamated β-CD)RN CYCLOBOND I RN (R-naphtylethyl carbamated β-CD)

2 Capacity factor, k, of the first eluted enantiomer; configuration indicated as superscript, when known.

3 Selectivity factor, α = k 2

k 1

4 Resolution (Rs),. Rs = 2 ( t r 2 - t r 1 ) W 1 + W 2

5 Mobile phase conditions are given as volume amounts of acetonitrile/methanol/acetic acid/triethylamine. The UV detector wavelength was 254 nm. The flow rate was 1.0 mL/min.

Note: If published literature shows a separation of any racemate in the reversed phase mode and the analyte possessesat least two functional groups, then the polar organic mode should be tried. The chiral stationary phase on which theoriginal separation was obtained should be used for the polar organic mode test.

For additional information about the CYCLOBOND polar organic mode, see the following references which correspond toour CYCLOBOND bibliography on page 41:

Ref. 108 - "A New Approach for the Direct Resolution of Racemic Beta Adrenergic Blocking agents by HPLC", Armstrong,D.W., Chen, S., Chang, C. and Chang, S., Journal of Liquid Chromatography, 15(3), 545-556 (1992).Ref. 119 - "Evaluation of a New Polar-Organic HPLC Mobile Phase for Cyclodextrin Bonded Chiral Stationary Phases",Chang, S.C., Reid III, G.L., Chen, S., Chang, C.D., Armstrong, D.W., TRACS, 12(4),144-153 (April 1993).

Polar Organic Mode Protocol

CH3CN/CH3OH/HOAc/TEA:95/5/0.3/0.2


SEPARATION NO SEPARATION

1. OPTIMIZE BY ALTERING RATIO HOAc/TEA

2. ADJUST Rt BY INCREASING CONC. HOAc/TEA IN SAME RATIO

1. DECREASE CH3OH to 1%*

2. ALTER RATIO AND DECREASE CONC. HOAc/TEA

1. INCREASE CH3OH TO 10%

2. INCREASE CONC. HOAc + TEA

CYCLOBOND I 2000

SEPARATIONPEAKS ELUTE

NO SEPARATION

Notes:1. Compounds with primary amines on the stereogenic center have not been resolved as yet, however, secondary

amines work.

2. Organic salts may be used directly.

3. A reversal of elution order has been reported in running separations in the polar organic mode from the reversed phase mode.

*Can go to pure CH3CN.

13

Bonded DerivatizedCyclodextrins

The following list of derivatives are those currentlyavailable. They are based predominantly on the beta-cyclodextrin and offer additional opportunities for chiralseparation in a variety of mobile phase modes.

Summary of Derivatives of CYCLOBOND I 2000

Silica Gel

R R

R R

R RRR

R=CYCLOBOND I 2000

SUFFIX

OCH3

COCH3

CH2CHCH3

OH

*

CONHCH

CH3

*

CH3

CH3

CONH

DM(dimethylated)

AC**(acetylated)

SP or RSP(hydroxypropyl ether)

RN or SN(naphthylethyl

carbamate)

DMP(3,5-dimethylphenyl

carbamate)

*Stereogenic Center.

**Note: Acetylated versions also available in gamma(CYCLOBOND II AC) and alpha (CYCLOBOND III AC).

CYCLOBOND I 2000 DM

The CYCLOBOND I 2000 DM separates a variety ofstructural and geometric isomers as well as a group ofenantiomers not resolved on CYCLOBOND I 2000. In alimited number of cases, analytes could be resolved onboth while in a few cases resolution was improved with theDM phase.

This phase operates only in the reversed phase mode.Strong interactive groups like carbonyls off thestereogenic center are better resolved because theinclusion and diastereomeric complexation energies arebalanced. Steric interactions at the mouth of the cavityare more important to the chiral recognition than hydrogenbonding, therefore, multiple ring structures show greaterselectivity over native CYCLOBOND.

Separation of Warfarin Separation ofOxybutynin

OO

OH O

CH3

OH

O

O

CC

CH3

N CH3

k′1 - 2.62k′2 - 3.90

CYCLOBOND I 2000 DM30/70: MeOH/1% TEAA, pH

4.11.0 mL/min.

k′1 - 1.57k′2 - 1.98

CYCLOBOND I 2000 DM25/75: MeOH/1% TEAA, pH

4.11.0 mL/min.

14

Compounds Resolved Only by CYCLOBOND I 2000 DM in the Reversed Phase Mode

Compound and Strucure k′a α b Rsc Mobile Phase

3-(α-Acetonyl-4-chlorobenzyl)-4-hydroxycoumarin

OH

O

O

Cl

O

3.06 1.37 3.38 70/30:MeOH/1% TEAA, pH 4.1

2-Amino-9-hydroxyfluorene

OHNH2

2.40 1.29 2.36 15/85:MeOH/1% TEAA, pH 7.1

BAY COOH

N

CF3

COOH

MeMe

O2N

H

1.06 1.18 1.0 15/85:CH3CN/1% TEAA, pH 4.1

Coumafuryl O

CHCH2CCH3O

OH

O3.08 1.20 1.25 20/80:

MeOH/1% TEAA, pH 4.1

Crown ether analogue #19

(CH2)8

O

O

1.25 1.30 2.06 40/60:CH3CN/1% TEAA, pH 4.1

Fenoxaprop-ethyl

ClO

N O

OO

O

13.1 1.13 1.00 20/80:MeOH/1% TEAA, pH 7.1

Idazoxan

O

O

N

N

H

1.11 1.42 1.93 100%:1% TEAA, pH 4.1

1-IndanolOH

1.34 1.18 1.25 3/97:MeOH/1% TEAA, pH 7.1

Methylidazoxan

O

O

N

N

H

1.31 1.26 1.03 10/90:MeOH/1% TEAA, pH 7.1

a k′ = capacity factor of the first eluted enantiomer, b The selectivity factor, α, is equal to k1′, k2′, c The resolution is equal to 2(tr2-tr1)/(w1-w2).

15

CYCLOBOND I 2000 AC

CYCLOBOND I 2000 AC has sterically fixed hydroxylgroups in position 2 and 3 of the beta-cyclodextrinacetylated to change the nature of the interactions thatform the diastereometric complex from simple hydrogenbonding to hydrogen donor and acceptor sites. This is amuch stronger type of interaction. This chiral stationaryphase is especially beneficial when the includablearomatic portion of an analyte has a stereogenic centerwith a hydroxyl or amine group in the alpha or betaposition. Under these circumstances the acetylated betawill enhance the complex formation leading to higherselectivity and shorter retention than experienced withthe native CYCLOBOND. The differences can bedramatic. For example, the separation of phenylephrineon the CYCLOBOND I 2000 requires a retention time of30-35 minutes to achieve near baseline resolution. TheCYCLOBOND I 2000 AC will separate this racemate tobaseline in under 15 minutes (see chromatograms below).

Phenylephrine Norphenylephrine

OH

HOCH CH2NH CH3

NH2CH2HOCH

Peak 1 - 10.9 min.Peak 2 - 13.3 min.


CYCLOBOND I 2000 AC10/90: CH3OH/0.5% NaOAc, pH 5.5

0.5 mL/min.

In addition to the situation described above, in thereversed phase mode this stationary phase has beenused in the polar organic mode for those racemates thathave more than one functional group capable of donatinga hydrogen. One of those groups has to be on or near thestereogenic center. Some examples of the potential ofthis chiral stationary phase in this mobile phase systemare compounds like homoatropine, trihexylphenidyl,rulene and other chiral phosphorus and sulfur pesticides.For more information on applications in this mobile phasesee reference 119 in the CYCLOBOND bibliography.

CYCLOBOND I 2000 SP and RSP

The S and R hydroxypropyl derivatives of CYCLOBONDhave been very successful. There are two reasons forthis:

1. Marginal separations obtained on CYCLOBOND I2000 are often enhanced on the CYCLOBOND I 2000RSP and SP columns in the reversed phase mode withgreater long term stability.

2. The RSP and SP separate, for the first time, non-aromatic racemates. It has been one of the best methodsfor t-boc amino acids and cyclic hydrocarbons.

The appendant hydroxyl groups of this derivativeincrease hydrogen bonding flexibility and provideadditional sites for hydrogen bonding thereby assistingin immobilization of the solute and steric interactionsespecially for enantiomers which have bulky substituentsbeta to the stereogenic center. Molecular structures thatfit this derivative would have hydrogen bonding groups oracceptor sites that are within 2-4 carbons of an aromaticor heterocyclic ring. This chiral stationary phasefunctions in both reversed phase and polar organicphase. Protocols for the native CYCLOBOND areapplicable here. See pages 6 and 12.

Selectivity EnhancementSeparation of 2 Enantiomers/Meso Compound

CYCLOBOND I CYCLOBOND I RSP0.05% TEAPO4 pH 7

0.3 mL/min.0.25% TEAPO4 pH 7

0.5 mL/min.

Peak 1 - 26.73 min. Peak 1 - 11.91 min.Peak 2 - 27.61 min. Peak 2 - 13.90 min.Peak 3 - 33.26 min. Peak 3 - 14.04 min.

16

Separation of Methadoneon CYCLOBOND I 2000 RSP

15/85: CH3CN/1% TEAA, pH 4.1 @ 1.0 mL/min.

Separation of t-BOC Amino Acids:(1) Leucine and (2) Alanine

on CYCLOBOND I 2000 RSP7/93: CH3CN/1% TEAA, pH 6.9 @ 0.8 mL/min.

C CH2CHN(CH3)2C2H5 C

O CH3

(1)

H3C

CH3 NH2

O

OH

(2)

OH

O

NH2

CH3

Note: All t-BOC amino acids can be separated by altering the mobile phase composition, pH and the eluent flow rate. TheL form usually elutes first. See CYCLOBOND bibliography reference #113.

See CYCLOBOND bibliography reference #88 for additional applications with CYCLOBOND I SP and RSP. Allseparations cited for the "S" configuration (CYCLOBOND I SP) in that reference can be successfully achieved on theracemic form, CYCLOBOND I 2000 RSP.

CYCLOBOND 2000 Carbamates SN, RN and DMP

Broad based, multi-modal chiral selectors - Cyclodextrin carbamate derivatives:

CYCLOBOND I 2000 SN S-Naphthylethyl carbamate, beta-cyclodextrinCYCLOBOND I 2000 RN R-Naphthylethyl carbamate, beta-cyclodextrinCYCLOBOND I 2000 DMP 3,5-Dimethylphenyl carbamate, beta-cyclodextrin

Interactive Region InteractionMobile Phase to

Promote Interaction

*

O

CH3CH

NH

C

O

OH

OH OH

OH

Silica Gel

Chiral R or S NEC

Hydrogen bondingdonor/acceptor sites

Chiral D glucose

= π acid/π-π

= chiral complex

= inclusion complex

Normal Phase:Hex/IPA, EtOH

Polar Organic Phase:CH3CN/CH3OH/HOAc/TEA

Reversed Phase:CH3CN/Buffer

Peak 1 - 5.70 min.Peak 2 - 6.38 min.

Peak 1 - 8.21 min.Peak 2 - 11.34 min.

Peak 1 - 10.49 min.Peak 2 - 11.62 min.

17

Introduction

The carbamate coupling of the π base, 1-naphthylethyl toa bonded cyclodextrin creates a complex chiralenvironment that has demonstrated diverse chiralseparations. It has been labeled a multi-modal chiralstationary phase because it has been operatedsuccessfully in three distinctly different mobile phasesthat function by three distinctly different mechanisms. Ithas, therefore, produced a range of separations notpreviously possible and has also enhanced certainseparations due to more favorable diastereomericcomplexation.

To utilize these chiral stationary phases to their fullest, itis necessary to understand all three solvent modes and tomake choices based on that understanding and therequirements of the analysis, i.e., clinical (low detectionlevels), analytical (routine purity checks), or preparative(high capacity).

The three mobile phases are:

1. Normal phase2. Polar organic phase3. Reversed phase

The best starting point for choosing the proper mobilephase is dictated by the analyte structure, solubility andstability. For example, if the analyte is π acidic, animmediate test in a normal phase solvent is essential. Ifthe analyte is not π acidic but contains 2 hydrogenbonding groups, one on or near the stereogenic center,then a first test should be the polar organic mode.Reversed phase is the last choice because (a) Rs isgenerally lower, (b) column life is shorter, and (c) samplecapacity is lower. Of course, these are generalizationsand there are many exceptions.

The most desirable mobile phases for overall performanceare:

Normal phase > Polar organic phase > Reversed phase

Of the three carbamates available, the S-naphthylethylcarbamate (SN) has shown statistically, so far, thegreatest selectivity and versatility especially in the polarorganic phase. However, since the naphthylethylcarbamate configuration does play a role inenantioselectivity, the R form can be useful if separationdoes not occur on the S form.

Normal Phase Operation

If the analyte is π acidic or can be made so by derivatiza-tion to the 3,5-dinitrobenzoyl derivative, often a simplemanipulation of Hexane/IPA on the CYCLOBOND I 2000SN will allow for the rapid development of a separation. Ina publication by Berthod, Chang and Armstrong, Anal.

Chem. , 64, p. 395-404 (1992), a total of 121 racemiccompounds were reported separated in the normal phaseon the CYCLOBOND I SN and 74 on the CYCLOBOND IRN. From their data, they predicted the separationpotential for over 1.6 million chiral compounds in thenormal phase. This publication offers a possible methodfor acquiring a database that will estimate separationfactors (α) in the normal phase. Under normal phaseconditions, a CYCLOBOND I 2000 SN and RN can providea reversal of the elution order using the oppositeconfiguration of the chiral stationary phase, i.e. RN, mayrequire additional optimization. A useful feature of thesephases is that once IPA concentration exceeds 50%,neat alcohols like methanol or ethanol have been useddirectly with excellent results.

Preferred Solvent Conditions

Solvent/Range

Hexane/IPA 98-50% Hexane/2-50% IPA (>50% use neat EtOH)

Acetonitrile 100% (for acids or bases + 0.1-0.2%HOAc or TEA)

Methanol or Ethanol 100% (for acids or bases + 0.10.2%HOAc or TEA)

Acetonitrile/Methanol 90-99% Acetonitrile/10-1% Methanol

Optimal Flow Rates

Optimal flow rate is 1.0 - 2.0 mL/min

Temperature Effects

Normal Phase Separation of Phensuximide

0

0.5

1

1.5

2

2.5

3

3.5

Rs

°C

5 15 25

CYCLOBOND I 2000 DMPHexane/IPA: 20/80,1 mL/min.

18

Normal Phase Mode Protocol

90/10: HEXANE/IPA

NO ELUTION

INCREASE IPA TO 50%

NO SEPARATION

SEPARATION


GO TO1. POLAR ORGANIC MODE2. REVERSED PHASE MODE

OPTIMIZE SEPARATION BY VARYING % IPA. OPTION TO ALTER MODIFIER (e,g. BuOH,

PENTANOL, 2-BuOH).

USE NEAT CH3CN, EtOH OR MeOH. ADDITION OF 0.1 TO 1.0% HOAc AND TEA FOR

ACIDS AND BASES.

OPTIMIZE BY SEPARATION BY VARYING AMOUNTS OF HOAc AND TEA OR SEEING EFFECT OF DEA OR MEA.

CYCLOBOND I 2000 SN

NO SEPARATION

SEPARATION

Normal Phase Mode Separations

3,5-DNB-2-Aminoheptane3,5-DNB-D,L-Tryptophan

Methyl Ester3,5-DNB-R,S-1(α-

methylbenzylamine) Homophenylalanine

CONHCHCH2(CH2)3CH3

CH3O2N

O2N

1 - 12.3 min.2 - 13.7 min.

N

H

CH2CHNHCO

COOCH3 NO2

NO2

1 - 5.1 min.2 - 5.7 min.

H3C NHCO

NO2

NO2

1 - 6.0 min.2 - 8.8 min.

CH2CH2CHCOOH

NH2

1 - 25.1 min.2 - 33.3 min.

CYCLOBOND I 2000 SNHexane/IPA: 90/10

1.0 mL/min.

CYCLOBOND I 2000 SN100% Ethanol1.0 mL/min.

CYCLOBOND I 2000 SNIPA/Hexane: 30/70

1.0 mL/min.

CYCLOBOND I 2000 RNMeOH/HOAc: 99/1

1.0 mL/min.

19

Normal Phase (Pirkle-type) SeparationsCYCLOBOND I 2000 SN/RN

Acidic Compounds

NO2

NO2

R= NH

3,5-Dinitrobenzylamide Derivatization Method

To a 1 dram vial with rubber lined screw cap add:

Step 1: 100mg (0.65mM) free acid. Add dropwise (in ahood) 0.2 mL (~ 3mM) SOCl2. Warm reaction in a 60°Cwater bath. Reaction is complete when bubbles cease toevolve. Evaporate off SOCl2.. Dissolve residue in 0.2 mLmethylene chloride and place in an ice bath.

Step 2: Dissolve 119mg (0.65mM) 3,5-dinitrobenzylaminein 0.2 mL methylene chloride and add carefully to the acidchloride prepared in Step 1. Let reaction come to roomtemperature and then warm to 60°C in the water bath for10 minutes or until all solvent has evaporated. Dissolveresidue in ethanol for HPLC.

Compound k Rs Type Mobile Phase

2-bromopropionic acidCH3 CH CO2R

Br

4.14 3.8 SN 20% IPA/HEX

2-bromobutyric acidCH3 CH CO2R

Br

CH2 3.31 3.1 SN 20% IPA/HEX

2-phenylpropionic acid

CHCO2R

CH3 4.12 8.1 SN 20% IPA/HEX

2-phenylbutyric acid

CHCO2R

CH2CH3 3.54 10.7 SN 20% IPA/HEX

Normal Phase (Pirkle-type) SeparationsCYCLOBOND I 2000 SN/RN

Alcohols/Amines

NO2

NO2

CR=

O

3,5-Dinitrobenzoyl Derivatization Method

To a 1 dram vial with rubber lined screw on cap add:1.3mg 3,5-dinitrobenzoyl chloride1mg sample0.2 mL methylene chloride1 drop triethylamine

Heat in a boiling water bath for 45 minutes. Let vial cool toroom temperature, remove cap and gently evaporate thesample to dryness. Reconstitute sample in EtOH.

Compound k Rs Type Mobile Phase

sec-butylamine

CH3 CH2 CH CH3

NHR 11.45 1.5 SN 10% IPA/HEX

2-aminoheptane

CH3 CH

NHR

(CH2)4CH3

8.54 1.5 SN 10% IPA/HEX

3-aminoheptane

CH3 CH2 CH

NHR

(CH2)3CH3

8.32 2.6 SN 10% IPA/HEX

α-methylbenzylamine

CHCH3

NHR 4.56 6.4 SN 30% IPA/HEX

2-pentanol

CH3 (CH2)2 CH CH3

OR 3.76 1.2 SN 10% IPA/HEX

2-hexanol

CH3 CH CH3

OR

(CH2)3

3.29 1.4 SN 10% IPA/HEX

4-methyl-2-pentanol

CH3 CH CH3

OR

CH2 CH

CH3

3.24 2.3 SN 10% IPA/HEX

2-octanol

CH3 CH3

OR

CH(CH2)5

2.82 1.3 SN 10% IPA/HEX

α-methylbenzylalcohol

CHCH3

OR 6.82 4.5 SN 10% IPA/HEX

20

Reversal of Elution Order

In normal phase operation it is possible to reverse theorder of elution by substituting a CYCLOBOND I 2000 RNfor the SN.

Reversed Elution Order - CYCLOBOND I 2000 SNversus CYCLOBOND I 2000 RN

3,5-DNB-α-Methylbenzylamine, 20/80: Hexane/IPA,Sample Conc. 2:1; S:R (2:1 mg/mL)

(A)CYCLOBOND I 2000 RN1.0 mL/min.

t1 - 9.80 (S)t2 - 11.62 (R)

(B) CYCLOBOND I 2000 SN1.0 mL/min.

t1 - 6.42 (R)t2 - 9.42 (S)

(C)CYCLOBOND I 2000 RN0.5 mL/min.

t1 - 11.31 (S)t2 - 14.13 (R)

(D)CYCLOBOND I 2000 SN0.5 mL/min.

t1 - 13.15 (R)t2 - 19.57 (S)

For the separation potential of normal phase operation,see Ref. 110 in the CYCLOBOND bibliography. Over 1.6million potential separations are predicted using thismethodology.

Reasons normal phase operation may not work:

� Analyte is π basic; inadequate interaction of thearomatic structure of the analyte with thenaphthylethyl structure.

� Poor solubility of analyte in mobile phasesolvents.

� Hydrogen donor or acceptor site interactions notof appropriate distance.

Conclusions:

� Go to polar organic mode if structuralrequirements are met.

� Go to reversed phase mode if structuralrequirements are met.

Polar Organic Phase Operation

The complex chiral environment of these carbamates canbe taken advantage of when using the polar organicmode. All the benefits of fast elution, high NTP and highsample capacity are maintained with these phases. Thisis believed to be a surface interaction and no inclusioncomplexation is thought to take place. This has been oneof the broadest applicable mobile phases for pharmaceutical analysis and the most important advance forchiral separations . See pages 7-12.

Preferred Solvent Conditions

SolventTypical

Composition v/v Range

CH3CN 95 85-100%CH3OH 5 15-0%HOAc 0.3 0.001-1.2%TEA 0.2 0.001-1.2%

Separations in the Polar Organic Mode

Oxazepam

N

N

O

OH

H

Cl

Peak 1 - 13.3 min.Peak 2 - 15.4 min.

CYCLBOND I 2000 SN100/0.001/0.001:CH3CN/HOAc/TEA

1.0 mL/min.

AtenololO

CH2CNH2(CH3)2CHNHCH2CH(OH)CH2O

Peak 1 - 28.3 min.Peak 2 - 33.5 min.

CYCLOBOND I 2000 SN95/5/0.3/0.25:

CH3CN/CH3OH/HOAc/TEA1.0 mL/min.

21

PyridogluthemideNO O

C2H5

N


CYCLOBOND I 2000 RN95/5/0.3/0.4:


Timolol

O N OCH2CCH2NHC

OH CH3

CH3

H CH3

N NS

Peak 1 - 11.3 min.Peak 2 - 12.8 min.

CYCLOBOND I 2000 RN95/5/0.6/0.4:


Reversed Phase Operation

As with the native cyclodextrins, a number of parametersmust be investigated for developing and optimizing aseparation in the reversed phase mode. The mostsignificant parameter is pH. If the pK of the molecule isknown, then operating 0.5 pH unit above and below thisnumber will demonstrate the influence of pH on selectiv-ity. If it is not known, then a buffer run at pH 4 and pH 6.9will demonstrate the influence of pH and appropriateincremental steps can subsequently be taken. Thestability range for this derivative is pH 4.0 - pH 6.9.

Preferred Reversed Phase Conditions

Conditions are identical to those covered on pages 4-6.

Reversed Phase Mode Separations

Indapamide Bendroflumethiazide

N

NH

H

CH3

CO Cl

SO2NH2

Peak 1 - 21.0 min.Peak 2 - 24.2 min.

CYCLOBOND I 2000 SN20/80: CH3CN/1% TEAA

pH 7.1, 1.0 mL/min.

SN

N

H2NO2S

F3C

H

H

CH2

O O

Peak 1 - 5.85 min.Peak 2 - 7.31 min.

CYCLOBOND I 2000 SN30/70: CH3CN/1% TEAA

pH 4.5, 1.2 mL/min.

Fluorescent Tagged Primary Amines onCYCLOBOND I 2000 Carbamates

Reversed phase separation of a number of primaryamines while showing some selectivity on CYCLOBONDphases had poor detectability. A search for the bestmethodology to improve this situation revealed that thereagent Accu-Tag, available from Waters Corporation,had simple chemistry, was easy to automate, no sampleclean-up, good long term stability and excellent selectiv-ity on CYCLOBOND phases. Use of CYCLOBOND I 2000SN indicated some steric effects since even though alarge alpha was obtained, the separation would not go tobaseline. The smaller ring structure of the CYCLOBOND I2000 DMP, however, gave excellent results as seenbelow.

Separation of D,L-Amphetamine

Column:Size:Mobile Phase:Derivative:Flow Rate:Observation:

CYCLOBOND I 2000 SN250x4.6mm35/65 CH3CN/0.1% TEAA pH 4.1AQC0.75 mL/min.Not baseline, Rt long, D>L

Peak 1 - 23.99Peak 2 - 26.42

L D

Separation of D,L-Amphetamine

Column:Size:Mobile Phase:Derivative:Flow Rate:Observation:

CYCLOBOND I 2000 DMP250x4.6mm45/55 CH3CN/0.1% TEAA pH 4.1AQC1.0 mL/min.Rs-2.4, L elutes before D

Peak 1 - 12.70Peak 2 - 14.50

22

AQC.Amphetamine Linearity

0

500000010000000

1500000020000000

2500000030000000

3500000040000000

Are

a C

ount

0 2 4 6 8 10

Injection Volume

Inj. Vol(µL)

AreaL-Amphetamine

AreaD-Amphetamine

2 7293472 72210435 18872928 1838899210 37209408 36719776

L-Amphetamine: Y=3.732x106X - 23412 R=0.99991D-Amphetamine: Y=3.685x106X - 1.061x106 R=0.99999

Trace Analysis on CYCLOBOND Phases

The development of pure enantiomers has been a majoreffort since the first discovery of the ill effects of the firstdrug racemate. It has accelerated dramatically as morecost effective means were sought to produce pure singleenantiomers. Traditionally, molecules from naturalsources have been used in chiral synthesis since theywere often available at reasonable cost. More recently anincreasing number and variety of new chiral agents havebecome available. Many of these are not from naturalsources and need to be analyzed for their chiral impuritiesin order for the final product to meet the industryrequirements of >99% purity.

As can be seen from the work of D. W. Armstrong and J.T. Lee, enantiomeric impurities can range from a low of0.01% to a high of 10%. Chiral contaminants of 1-10% arenot uncommon. The surprising fact is that suppliers andconsumers alike were not able to verify the chiral purity oftheir starting materials. Only the non-chiral impuritieswere evaluated.

In the first published work of Armstrong and Lee, 83 chiralreagents were studied. For these analyses, 4 chiral GCphases (42.9% on the CHIRALDEX G-TA), 5 chiral HPLCphases (33.3% on the CYCLOBOND I 2000 RSP) and 3types of CE chiral additives were used. In all 39 differentsets of conditions were employed to evaluate the 83 chiralreagents.

In the second study, 109 chiral reagents were evaluated.This was accomplished with 5 chiral GC phases (39.3% onthe CHIRALDEX G-TA, 33.7% on the CHIRALDEX B-DM)and 6 chiral HPLC phases, the largest percentage beingon the CHIROBIOTIC V (23.5%).

Approximately 64% of these latter reagents had moderateto high levels of enantiomeric impurities. The range wasfrom >0.1% to <16%.

See references 156 and 157 in the CYCLOBONDbibliography.

2-Phenylbutyric AcidCYCLOBOND I 2000 RSP

24.9

8

37.9

2

33.3

228

.17

30/70:MeOH/1% TEAA (pH 4.1) @ 1.0 mL/min.R - 98.18%, S - 1.82% R - 2.16%, S - 97.84%

R

SR

S

CH C OH

O

C2H5

23

[3aR-[2(3′aR*,8′aS*),3′aβ,8′aβ]]-2,2′-methyl-ene-bis[3a,8a-dihydro-8H-indenol[1,2-d]]oxazole

CYCLOBOND I 2000 SN28

.65

38.9

24.6

5

34.0

15/85:ACN/1% TEAA (pH 4.1) @ 1.0 mL/min.R - 99.97%, S - 0.03% R - 0.05%, S - 99.95%

2-Amino-1,2-diphenylethanolCYCLOBOND I 2000 AC

5/95:ACN/1% TEAA (pH 4.1) @ 1.0 mL/min.1S,2R - 3.60%1R,2S - 96.40%

1R,2S - 0.10%1S,2R - 99.90%

cis-4,5-Diphenyl-2-oxazolidinoneCYCLOBOND I 2000 AC

23.5

5

30.0

9

24.2

1

33.6

2

20/80:ACN/1% TEAA (pH 4.1) @ 1.0 mL/min.4S,5R - 0.01%

4R,5S - 99.99%4R,5S - 0.01%4S,5R - 99.99%

SFC Using CYCLOBOND PhasesComparisons between LC and SFC demonstrated that,although selectivity was sometimes lower in SFC than inLC, the improved efficiency in SFC resulted in higherresolution. Identification of optimum chromatographicparameters was facilitated in SFC, and most of thecompounds investigated were resolved with a carbondioxide/methanol eluent. However, the alcohol modifierplayed an important role in enantioselectivity in SFC, andthe nature of this role was not the same for all analytes.Only when run in the polar organic mode was a substantialsavings in time noted for LC. Resolution was also betterfor LC in this mode.

Reference:

Williams, K.L., Sander, L.C., Wise, S.A., Comparison of Liquid andSupercritical Fluid Chromatography Using Naphthylethyl-carbamoylated-β-cyclodextrin Chiral Stationary Phases. SeeCYCLOBOND bibliography reference 145.

S

S

R

R

O

N

O

N

R

S

R

S

CH

NH2

CH

OH

R

SR

S

O

N

O

24

Comparison of Chromatographic Results for N-(3,5-dinitrobenzoyl) Derivatized Analytes in Normal Phase LCand SFC on the CYCLOBOND I 2000 SN

Compound ka α Rs Mobileb,c

Phase4-Chlorophenylalanine ethyl ester LC 3.13 1.25 2.0 60:40

SFC 5.08 1.14 2.3 85:15

2-Aminoheptane LC 6.55 1.17 1.2 90:10SFC 9.27 1.14 2.6 95:5

1-Cyclohexylethylamine LC 3.60 (R) 1.23 1.7 80:20SFC 6.73 (S) 1.45 5.9 90:10

α-Methylbenzylamine LC 3.29 (R) 2.10 6.8 70:30SFC 3.55 (R) 1.56 7.8 80:20

1,2,3,4-Tetrahydro-1-naphthylamine LC 2.15 1.92 5.0 70:30SFC 3.51 1.46 6.4 80:20

Conclusion: SFC gave consistently higher resolution with retention times only slightly longer.aConfiguration of the first eluting enantiomers is shown in parentheses, when known.

bMobile phases for LC are volume ratios of hexane to 2-propanol.

cMobile phases for SFC are volume ratios of carbon dioxide to methanol.

Comparison of SFC and Polar Organic LC on the CYCLOBOND I 2000 RN

Compound k α Rs Mobilea,b

Phase2-(4-Chlorophenoxy)- LC 0.87 1.18 2.1 95:5:0.6:0.4propionic acid SFC 30.90 1.14 2.0 80:20

Coumachlor LC 0.33 1.27 1.5 98:2:0.8:0.6SFC 19.99 1.06 1.1 85:15

Proglumide LC 1.07 1.19 1.8 95:5:0.8:0.6SFC 15.44 1.10 1.9 92:8

Suprofenc LC 3.23 1.10 1.0 95:5:0.2:0.2SFC 21.50 1.05 0.6 80:20

aMobile phases for LC are volume ratios of acetonitrile/methanol/acetic acid/triethylamine.

bMobile phases for SFC are volume ratios of carbon dioxide to methanol.

cEthanol was used as the modifier.

Conclusion: SFC separations appear to mimic the separations obtained by LC in this solvent mode but a substantialincrease in retention time was noted.

25

Comparison of Chromatographic Results for SFC and Reversed Phase LCon the CYCLOBOND I 2000 SN and RN

Compound CSP k α Rs MobilePhase

Ancymidol SN LC 4.72 1.14 1.3 80:20 (7.0)a

N

N

HO

OCH3

SN SFC 6.32 1.08 1.3 90:10b

Bendroflumethiazide SN LC 6.36 1.22 1.9 70:30(4.5)

S

N

NHS

H2N

F3C H

O O O O SN SFC 9.95 1.11 1.9 70:30

Cromakalim SN LC 2.19 1.00 0.0 80:20(4.5)

O

OH

CH3

CH3

N

O

NC

SN SFC 10.25 1.08 1.5 96:4

5-(4-Hydroxyphenyl)-5-phenylhydantoin

HOC6H4

C6H5

O

H

O

N NH

RNRN

LCSFC

8.5136.24

1.101.15

0.71.5

80:20(4.5)85:15

Mephenytoin SN LC 1.29 1.22 1.3 70:30(4.1)

O

H3C

HN

NCH3O

SN SFC 3.15 1.25 3.0 95:5

Tropicamide SN LC 1.56 1.22 1.1 70:30(4.5) N

N

CH3

O

OH SN SFC 12.48 1.15 2.1 90:10

aMobile phases for LC are volume ratios of triethylammonium acetate buffer to acetonitrile; pH is given in parentheses.

bMobile phases for SFC are volume ratios of carbon dioxide to methanol.

Conclusion: Compounds resolved in the reversed phase mode generally were more highly retained in the SFC mode buthigher resolution was also observed. This differs dramatically from the conditions observed in the polar organic mode.

26

Preparative Separationson CYCLOBOND

Defining the Need

It is important when trying to select a purification schemeto identify the following:

� Amount needed� Time frame� Starting purity� Final purity� Acceptable yield� Available resources

Equipment Manpower Money

As an example, we will limit the information to:

� Milligram to multigrams� One day� 50% (Racemic)� 98+% Pure� 90%+ Yield� Prep system with flow rates capability for up to

2" diameter columns with and without recycle

It should be noted that chromatography offers thefastest, most reliable approach to isolating gramquantities of pure enantiomers especially if analyticalHPLC methods have already been developed.

Practical Considerations

There are significant numbers of texts and publicationsthat discuss the theoretical models and appliedtechniques for preparative and process HPLC. However,few seem to discuss the total purification process.

Over the years, we have conducted informal surveysabout how time is spent in the prep lab. Here is what wehave found.

� 40% converting analytical methods to prep andscaling up the method

� 20% actually running the prep HPLC

� 40% analysis of the collected fractions andrecovery of the sample from the mobile phase

While the above percentages were not rigorouslydeveloped, they do stimulate a discussion that can helpreduce costs and increase sample throughput.

Method Development

If an analytical method has been developed and can beused directly for the preparative separation, thenconsiderable amounts of time can be saved.

For analytical HPLC, baseline resolution is desiredbecause we are collecting quantitative information.However, for preparative HPLC our need is to collect puresample and even if we do not have baseline resolution, wecan still collect pure sample. Therefore, when convertingan analytical method to prep we might want to considerthe following:

A. Sample solubility in the mobile phase

B. Inexpensive and safe solvents

If recycling is not available on your prep system, then youmay need to improve the resolution between theenantiomers in order to increase throughput.

If only one enantiomer is desired then you may want todevelop the method so that the desired enantiomer elutesfirst. As you will see later, displacement mechanisms canallow for collection of high purity and good yields of thefirst eluting enantiomer. If both enantiomers are neededand recycle is not available, then improved selectivity ormore injections at lower load may be required.

If the analytical method requires the use of buffers ormodifiers, you should determine if they are necessary forthe selectivity. Often these are used to sharpen thepeaks but do not affect selectivity. If this is the case, tryremoving the modifiers. However, if they are critical to theselectivity at least try using volatile modifiers such asammonium acetate or triethylamine acetate. The reasonfor the above is that you need to remove the mobile phaseafter the purification. Recovery of the separatedenantiomer from an aqueous mobile phase can beaccomplished on a reversed phase column. (See methodin Astec CHIROBIOTIC HANDBOOK).

Sample Solubility

Often analytical methods are developed with little or nothought to sample solubility. In order to minimize runproblems (precipitation on column) and improve through-put, you should pick a mobile phase in which the samplehas good solubility. Target - approximately 20-50mg/mL.

Scaling Up

The method that has been developed should be testedusing the same packing that is in your preparative columnfor a loading study on the analytical size column.Because the analytical system detector's path length isusually 10mm, we will quickly saturate the electronics butyou will at least have an idea about how much to startloading on the prep column.

27

The Prep Run

Because you have developed the prep method on ananalytical column that contains the identical packing thatis in the prep column, you can scale the flow rate andsample load by the proportional (cross sectional areabetween the analytical and prep column). You will mostlikely find that you will want to continue your loading studyin order to get the best throughput.

Scale-Up Guide

This guide was developed from a range of racemates oncyclodextrin phases.

As a general rule, sample capacity of a column can besquared when the column diameter is doubled, i.e., 2 x ID= (Cp)2 @ 4 x mL/min.

The usefulness of column switching and recycle willbecome apparent in the following examples. It should benoted that these techniques reduce the time spent inmethod development.

ColumnDiameter

Av.Sample

Capacity

Typical FlowRates

InjectionVolume

10.0mm ID 2-20mg 4-10 mL/min. 0.2-2.0 mL

22.1mm ID 20-200mg 15-50 mL/min. 1.0-10.0 mL

50.0mm ID 200mg-5g 60-250 mL/min. 5.0-50.0 mL

After the Prep Run

If you did not use recycle in the prep run you will haveprobably collected more than two fractions. These haveto be analyzed before removing the mobile phase. Usingrecycle will reduce the number of fractions taken and willsignificantly improve yield.

Preparative Process

The highest capacity for working with cyclodextrin phasesis in the polar organic mode. This is followed by normalphase separations on the CYCLOBOND I 2000 SN and RNseries. A wide variation exists for reversed phase modesthat appears to be dictated by the strength of theinteractions, i.e. presence of strong amines or carbonylgroups in the analyte.

Structures of the Thienopyran Enantiomers

O

SCH3

CH3

OHN O

N+O-

O

(-)

O

SCH3

CH3

OHN O

N+O-

O

(+)

CYCLOBOND I, 250x10.0mmInjection Volume and concentration: 2.0ml (15mg/mL)

Loading studies indicated that the maximum amount ofracemate the 10mm ID column could tolerate withoutcompromise to resolution was 30mg. A single pass wasmade for each injection and two fractions were collectedfor each peak. This method provided 100mg of the (-)enantiomer in 99.8% enantiomeric excess and 100mg ofthe (+) enantiomer in 99.4% enantiomeric excess.

Note: Sample Recovery from Mobile Phase - To recoversamples from reversed phase mode, adsorption onto ashort C18 cartridge and elution with methanol works well.

Automatic Purification of Enantiomers Using RepetitiveInjection, Recycling and Peak Detection

In an effort to purify gram quantities of single enantiomersfor use as standards we automated the repetitive injectionof the racemate mixture. This mixture was separated on aCYCLOBOND I 2000 (Beta) semi-preparative column, 250x 22.1mm.

In order to reduce solvent consumption and improvepurity and yield recycling is the best technique.Throughput then is limited only by sample solubility. Thepeaks were collected using a slope-threshold fractioncollector.

This method can be fully automated and run unattended.

28

Warfarin 1000µL

The above chromatogram is an example of the improvedselectivity using recycle. No additional solvent is usedduring the recycle process.

1. Overload Method, Direct ElutionWarfarin, 76mg, 1.0mL, 1mm path

Fractions below were collect as marked. The analysis ofthese fractions is seen below.

Fraction1

Fraction2

Fraction3

Fraction4

Fraction5

7.15 min. 7.12 min.8.32 min.

7.12 min.8.01 min.

7.12 min.8.09 min.

7.20 min.8.21 min.

It can be seen above that nearly pure (98%+) enantiomerA can be collected by collecting up to the valley.However, enantiomer A tails into enantiomer B andcontaminates all of the subsequent fractions.

2. Overload with Recycle MethodWarfarin, 152mg, 2.0mL

3. Overload with Shave and Recycle Method60mL/min, Warfarin, 150mg, 2.0mL

In order to collect the highest possible purity of bothenantiomers we decided on the following strategy. Werecycled until a valley appeared, then we collectedenantiomer A on each successive recycle up to thevalley. Enantiomer B is recycled until most of enantiomerA has been separated out, then we collect enantiomer B.

Recycle Reproducibility, Warfarin, 152mg, 2.0mL

29

The previous method was programmed into the VersaPrepsoftware and automated, repetitive injections andcollections were made. This shows the run to runreproducibility. Approximately 500mg of each enantiomerwas collected using this automated method.

Throughput = 152mg each enantiomer per hour on a250x22.1mm column.

All of the preparative work was done on the VersaPrepmanufactured by AmeriChrom Global Technologies. TheVersaPrep was designed and optimized for recycle/shavechromatography.

General Operating Conditions

Determining Void Volume (Vo)

Since the cyclodextrin cavity is capable of including awide variety of ions and molecules including the usualvoid volume indicators, the determination cannot be madedirectly. A graphical method has been proposed in whichthe retention volume of several low molecular weightalcohols (C1-C2) were determined and plotted against theirbinding constant to the cyclodextrin. The extrapolatedretention volume for a binding constant equal to zero wastaken as column void volume.7

Retention Volume versus Binding Constant

2.8

2.9

3

3.1

3.2

3.3

3.4

3.5

3.6

VR (m

l)

Kf

0 0.3 MeOH 0.9 EtOH

ll

ll

(2)

(1)

Conditions: (1) MeOH/H20: 50/50 @ 1.0 mL/min.(2) CH3CN/H20: 50/50 @ 1.0 mL/min.

Temperature

Lower temperatures enhance the weaker bonding forces.The net result is that the chromatographer has anadditional powerful means to control selectivity andretention. This capability is not as pronounced with moretraditional bonded phases. For a more comprehensivereview see Ref. 9. Column reproducibility can beestablished by maintaining a constant temperaturecondition within 1°C. Typically 18°C is used as a base.See the Jetstream Column Thermostat in the AstecChromatography Product Guide or visit our website atwww.astecusa.com.

Injection Volumes and Concentrations

Two distinct examples of column capacity have beenobserved for CYCLOBOND I 2000 phases whether run inthe reversed phase, normal phase or polar organic phasemode. The first example relates to a predominantinclusion effect. In this case the separation is dependentupon sample volume and concentration. Typical examplesof 1-5µL of 1 mg/mL concentration are required for goodresolution. In other cases, volumes up to 20µL may beused. It is advisable to begin the separation study at thelowest volumes and concentrations until a properdetermination can be made of its effect. Always usemobile phase to dissolve sample. Peak deformation canoccur especially in the normal phase or polar organicphase systems.

Stability

The stability of CYCLOBOND I 2000 to pH is in the rangeof 3.0 to 7.0. Strong acids attack the cyclodextrinstructure while strong alkaline conditions attack the silicabase. Pure water can cause column deterioration overlong periods of time. Use of 90% aqueous mobile phasesshowed no change over many injections as long as thecolumn is washed with water and re-equilibrated withacetonitrile subsequent to an analysis. A precolumn(before injector) of silica should be used when operatingwith buffers greater than 6.0. Successful runs of pH 3.0buffer have been run for short periods immediatelywashing the column with water, then acetonitrile.

Column Evaluation

To establish column performance on CYCLOBOND I 2000,it is recommended that the separation of ortho, meta andpara nitroaniline be run in 40/60: MeOH/H2O. Theselectivity between the ortho and para establishes theintegrity of the bonded cyclodextrin. When p-nitroaniline(Peak 3) falls below a retention of 17 minutes, the columnwill no longer separate most chiral analytes.

30

CYCLOBOND I 2000 Selectivityfor m-, o-, p-Nitroanaline

Flow Rate

Initial flow rates may be set at 1.0 mL/min unless theeffects of mass transfer are clearly visible. Generally,lower flow rates (0.25 to 1.0mL) give higher efficiency andgreater resolution for enantiomers. Non-chiralseparations based on inclusion complexing behavesimilarly to reversed phase columns.

Astec columns may be operated from either directlywithout loss of performance due to the uniform packingsystem that produces uniform packing density. This istrue for all Astec columns.

Pressure

Operating pressure for CYCLOBOND I 2000 columns hasbeen recorded in the range of 700 to 800 psi (100mm) and2000 to 2500 (250mm) at 1.0 mL/minute for 40/60: MeOH/H2O. As with standard reversed phase columns, thehigher the water content, the higher the back pressurewith a maximum at 50/50. Care should always beexercised in prefiltering and degassing the water andsolvent used with these columns. In general, pressureshould not exceed 3500 psi (238 bars).

Regeneration

Columns showing decreased resolution can sometimes beregenerated by passing several column volumes of pureethanol followed by pure HPLC water and then acetonitrilethrough the column at 0.5 mL/min. The ethanol is twotimes more efficient for displacing substances from thecavity than methanol. Acetonitrile may be used for finaldisplacement and storage. Long term storage (>24 hours)is best done in IPA.

Storage

NEVER STORE COLUMNS, EVEN FOR SHORT PERIODSOF TIME IN ANY BUFFER. WASH THE COLUMN WITHWATER.

Subsequent to the quality control test, the column isconditioned with IPA for storage and shipment. Whenanalysis is complete the column should be returned to thissolvent to ensure long life.

Column Assessment Parameters

Column Assessment Parameters provide useful data asto the reproducibility of the column performance and canbe used to evaluate the column for signs of deterioration.Each column is individually tested and assigned a serialnumber for tracability of all column components.

40/60: MeOH/H2O1.0 mL/min.

Peak 1: meta-nitroanilinePeak 2: ortho-nitroanilinePeak 3: para-nitroaniline

7.08

7.85

8.81

17.93

31

Compounds Separated on CYCLOBOND ColumnsA comment on the compound list:

We report here information obtained from published papers referencing CYCLOBOND. The conditions chosen or theexperience of the author does not always reflect the best state of conditions. For instance, a preponderance of earlypublications focused on reversed phase type separations on the native CYCLOBOND phases. The polar organic modeoften offers superior results and should be considered as a viable alternative. Second, often a CYCLOBOND derivativeenhances selectivity and resolution over the native. All of these papers should be viewed in light of the contents of thisHandbook.

In an effort to simplify finding a compound in the CYCLOBOND bibliography, the following is a partial list of compoundsthat have been separated on CYCLOBOND HPLC phases and the corresponding number in the bibliography or in theAstec Chromatogram Library.

Note: Some applications from our bibliography show the CYCLOBOND I as opposed to the CYCLOBOND I 2000 columnsdescribed in this Handbook. The 2000 series was introduced in 1993 as a second generation product designed primarilyto guarantee batch to batch reproducibility. The selectivity and retention characteristics are very close to the originalCYCLOBOND product.

Compounds

Compound ReferenceNumber

CYCLOBOND ColumnUsed in Separation

Acenaphthalene 52 CYCLOBOND IAcenaphthenal 74 CYCLOBOND II(D,L)-3-(α-Acetonyl-4-chlorobenzyl)-4-hydroxycoumarin(See also (D,L)-Coumachlor)

828895

CYCLOBOND I Ac/RNCYCLOBOND I SPCYCLOBOND I SN

(D,L)-Adonitol (See also Ribitol) 61 CYCLOBOND I(D,L)-Allose 61, 62 CYCLOBOND IAlprenolol 108,119 CYCLOBOND IAlthiazide 95

119CYCLOBOND I SNCYCLOBOND I/II

2-Amino-3,3-dimethylbutane 97 CYCLOBOND I RNAminobenzoic acid (m-,o-,p-) 64 CYCLOBOND I2-Amino-1,2-diphenyl ethanol 157 CYCLOBOND I 2000 AC(D,L)-Aminoglutethimide 85 CYCLOBOND I2-Aminoheptane 97 CYCLOBOND I SN3-Aminoheptane 97 CYCLOBOND I SN2-Amino-9-hydroxyfluorene 154 CYCLOBOND I 2000 DMAmoxycillin 159 CYCLOBOND I 2000Amphetamine, AQC 148

148see page 42

CYCLOBOND I 2000 DMPCYCLOBOND I 2000 RSPCYCLOBOND I 2000 DMP

Amylase (α−, β−, γ−) 66 CYCLOBOND I

(±)-Anabasine 72 CYCLOBOND IAnacymidol 127 CYCLOBOND I SN(R,S)-Anatabine 72 CYCLOBOND IAncymidol 95, 145 CYCLOBOND I SNAniline 36 CYCLOBOND IIAnisidine (m-,o-,p-) 36 CYCLOBOND IIAnthracene 103, 105

107CYCLOBOND ICYCLOBOND I/III

(D,L)-Arabinose 61, 62 CYCLOBOND I(D,L)-Arabitol 61 CYCLOBOND I

32

Arogenate 38 CYCLOBOND I(R,S)-Arotinolol See page 42 CYCLOBOND I 2000(R,S)-Atenolol 108

119CYCLOBOND I ACCYCLOBOND I

Atropine 44, 98, 119 CYCLOBOND I3'-Azido-3'-deoxythymidine (AZT) 57 CYCLOBOND IBAY CNET 88 CYCLOBOND I SPBAY COOH 88 CYCLOBOND I SPBendroflumethiazide 95

145, seepage 42

CYCLOBOND I SNCYCLOBOND I 2000 SN

Benzene 64, 87 CYCLOBOND I1-Benzocyclobutene carbonitrile 154 CYCLOBOND I 2000 DM(±)-1-Benzocyclobutenecarboxylic acid 88 CYCLOBOND I SP1,4-Benzodiazepin-2-ones 77 CYCLOBOND I(R,S)-(±)-Benzoin 45

95156

CYCLOBOND ICYCLOBOND I SNCYCLOBOND I 2000

Benzoin Ethyl Ether 154 CYCLOBOND I 2000(±)-Benzoin methyl ether 95 CYCLOBOND I SNBenzo{ghi}-perylene 107 CYCLOBOND I/IIIBenzo [a] pyrene in aviation fuel 70 CYCLOBOND IIBenzo {a} pyrene 89, 105

107CYCLOBOND ICYCLOBOND I/III

Benzo {b} pyrene 105107

CYCLOBOND ICYCLOBOND I/III

Benzo {k} pyrene 105107

CYCLOBOND ICYCLOBOND I/III

N'-Benzoylnornicotine 53 CYCLOBOND I4-Benzyl-5,5′-dimethyl-2-oxazolidinone 156 CYCLOBOND I 2000 RN

(R,S)-4-Benzyl-2-oxazolidinone 49 CYCLOBOND IN'-Benzyl-nornicotine 53, 69

82CYCLOBOND ICYCLOBOND I RN

3-Benzylphthalide 95 CYCLOBOND I SN4-Benzyl-3-propionyl-2-oxazolidinone 167 CYCLOBOND I 2000 SNBicyclic 1,3-amino alcohols 166 CYCLOBOND I 2000 SN(R,S)-2,2'-Bi-2-naphthol 82 CYCLOBOND I DMP(R,S)-(±)-1,1'-Bi-2-naphthol 88 CYCLOBOND I SP1,1′-Binaphthalene-2,2-diyl hydrogen phosphate 156 CYCLOBOND I 20002,2′-Binaphthyldiyl-8-crown-2 154 CYCLOBOND I 2000/DM2,2′-Binaphthyldiyl-11-crown-3 154 CYCLOBOND I 2000/DM(R,S)-2,2'-Bi-naphthyldiyl-17-thiacrown-5 82 CYCLOBOND I PT(R,S)-2,2'-Bi-naphthyldiylcrown-4 82 CYCLOBOND I RNBiphenyl 39, 47, 88 CYCLOBOND IBiphenyldiols 92, 93, 94 CYCLOBOND IBiphenylols 92, 93, 94 CYCLOBOND IBromobenzoic acid (m-,o-,p-) 64 CYCLOBOND I2-Bromobutyric acid 97 CYCLOBOND I SN(±)-2-Bromoproprionic acid 97 CYCLOBOND I SNp-Bromotetramisole oxalate 154 CYCLOBOND I 2000Brompheniramine 49 CYCLOBOND IBulan 127 CYCLOBOND I AC(R,S)-2-Butanol 97 CYCLOBOND I(R,S)-sec-Butylamine 97 CYCLOBOND I SN/RNButylbenzene (n-, sec-, tert-) 87, 103 CYCLOBOND ICarboxyibuprofen 65 CYCLOBOND I

33

ß-Carotene 71 CYCLOBOND Iß-Carotene-7,8,7',8'-tetrahydro-15 cis 71 CYCLOBOND Iß-Carotene-7,8,7',8'-tetrahydro 71 CYCLOBOND I15,15'-cis-ß-Carotene 71 CYCLOBOND ICateolol 108, 119 CYCLOBOND IN-CBZ-(D,L)-proline 82 CYCLOBOND I RN/PT(D,L)-Cellobiose 61, 62 CYCLOBOND ICephaloridin 159 CYCLOBOND I 2000Cephalosporins 158 CYCLOBOND I 2000(D,L)-4-Chloro-2-(a-methylbenzyl)-phenol 49 CYCLOBOND I(D,L)-1-(5-Chloro-2-(methylamino)-phenyl)-1,2,3,4-tetrahydroisoquinoline

4974

CYCLOBOND ICYCLOBOND II

Chloroaniline (o-,p-) 3679

CYCLOBOND IICYCLOBOND I

Chlorobiphenols 92, 93, 94 CYCLOBOND IChlorophenols (monoaromatic, diaromatic) 92, 93, 94 CYCLOBOND I2-(4-Chlorophenoxy) propionic acid 145 CYCLOBOND I 2000 RN4-(2-Chlorophenyl)-2-hydroxy-5,5-dimethyl-1,3,2-dioxaphosphorinane-2-oxide

156 CYCLOBOND I 2000 RSPCYCLOBOND I 2000

p-Chlorowarfarin 144 CYCLOBOND I 2000 RNChlorpheniramine 85

See page 42CYCLOBOND ICYCLOBOND I 2000

Chlorthalidone 8588See page 42

CYCLOBOND ICYCLOBOND I RSPCYCLOBOND I 2000 RSP

5-Cholesta-7-ene-2α ,3α ,5α ,6ß,9α ,1α ,19-heptol sterol 96 CYCLOBOND I

Chorismate 38 CYCLOBOND I(R,S)-Ciprofibrate 82

119See page 42

CYCLOBOND I SNCYCLOBOND I/SN/RNCYCLOBOND I 2000

Clavulanic acid 159 CYCLOBOND I 2000Cocaine 44 CYCLOBOND I(S)-(-)-Continine 72 CYCLOBOND ICoproporphyrin 78, 91 CYCLOBOND I(D,L)-Coumachlor(See also (D,L)-3-(α-Acetonyl-4-chlorobenzyl)-4-hydroxycoumarin)

8282/1458895,119154

CYCLOBOND I ACCYCLOBOND I RNCYCLOBOND I SPCYCLOBOND I SNCYCLOBOND I 2000 DM

Coumafuryl 154 CYCLOBOND I 2000 DMCresol (m-,o-,p-) 64

See page 42CYCLOBOND ICYCLOBOND I 2000

Cromakalim 145 CYCLOBOND I 2000 SNCyclohexane (cis-, trans-) 35 CYCLOBOND I5,6-Cyclohexenonicotine 53 CYCLOBOND I(R,S)-1-Cyclohexylethylamine 97 CYCLOBOND I RN3-{(Cyclopentylhydroxyphenyl-acetyl)oxy}-1,1-dimethylpyrrolidiniumbromide

83 CYCLOBOND I

Decahydro-2,6-naphthalenedimethanol (cis-,trans-) 35 CYCLOBOND I2-Decanol 97 CYCLOBOND I RN/SNDeguelin 40 CYCLOBOND Icis-17,18- Dehydro-PGB1 43 CYCLOBOND I

19,20-Dehydro-PGB2 43 CYCLOBOND I

2-Deoxy-3′-thiacytidine 149, 150 CYCLOBOND I 2000 ACDeoxy-D-galactose 61, 62 CYCLOBOND IDeoxy-D-glucose 61, 62 CYCLOBOND I

34

Deprenyl 148 CYCLOBOND I 2000 SNN-Desmethyl-tamoxifen 32 CYCLOBOND I2′,1′:1,2;1″,2″:3,4-Dinaphthcyclohepta-1,3-diene-6-aminomethyl-6-carboxylic acid

160 CYCLOBOND I 2000 RSP

(R,S)-N-(3,5-Dinitrobenzoyl)-α-methylbenzylamine 6982

CYCLOBOND ICYCLOBOND I DMP

(R,S)-N-(3,5-Di-nitrobenzoyl)-1-(1-naphthyl)-ethylamine 97 CYCLOBOND I SN/RNN,N'-Dibenzyl-(D,L)-tartramide 49 CYCLOBOND I15,15'-Didehydro-11-cis-ß-carotene 71 CYCLOBOND I15,15'-Didehydro-ß-carotene 71 CYCLOBOND I2',3'-Dideoxycytidine (DDC) 57 CYCLOBOND I2',3'-Dideoxy-3'-thiacytidine 150 CYCLOBOND I ACDiethybenzene (m-,o-,p-) 64 CYCLOBOND IN'-(2,2-Difluoroethyl)-nornicotine 53 CYCLOBOND I15,15'-Dihydro-11-cis-ß-carotene 71 CYCLOBOND I15,15'-Dihydro-ß-carotene 71 CYCLOBOND I2,3-Dihydro-7a-methyl-3-phenylpyrrolo[2,1-b]oxazol-5(7aH)-one 157 CYCLOBOND I 2000 RSPDihydrorotenone 40 CYCLOBOND I3,5 -Diiodo-(D,L)-thyronine 88 CYCLOBOND I SPN,α-Dimethyl-N-(2-naphthylmethyl)-3-pyridinemethanamine 53 CYCLOBOND I

2,5-Dimethylaniline 36 CYCLOBOND IIN,N-Dimethylaniline 36 CYCLOBOND II2,6-Dimethylaniline 36 CYCLOBOND IIN,N-Dimethyl-1-ferrocenyl-ethylamine 156 CYCLOBOND I 2000 SN1,5-Dimethylhexylamine 97 CYCLOBOND I SN1,3-Dimethylnaphthalene 53 CYCLOBOND I1,4-Dimethylnaphthalene 53 CYCLOBOND Iα,α-Di(2-naphthyl)-2-pyrrolidine methanol 157

See page 42CYCLOBOND I 2000 SN

Diniconazole 117 CYCLOBOND I3,5-Dinitrobenzoic acid 81 CYCLOBOND I3,5-DNB bicyclic 1,3-amino alcohols 166 CYCLOBOND I 2000 SNN-3,5-DNB alanine methyl ester 145 CYCLOBOND I 2000 SNN-3,5-DNB 2-aminoheptane 145 CYCLOBOND I 2000 SNN-3,5-DNB chlorophenylalanine methyl ester 145 CYCLOBOND I 2000 SNN-3,5-DNB 1-cyclohexylethylamine 145 CYCLOBOND I 2000 SNN-(3,5-Dinitrobenzoyl)-(D,L)-leucine 45 CYCLOBOND IN-3,5-DNB α-methylbenzylamine 145

See page 43CYCLOBOND I 2000 SNCYCLOBOND I 2000 DMP

N-3,5-DNB norleucine methyl ester 145 CYCLOBOND I 2000 SNN-3,5-DNB phenylalanine 145 CYCLOBOND I 2000 SN3,5-Dinitrobenzoyl-phenylglycine 95 CYCLOBOND I SNN-3,5-DNB 1,2,3,4-Tetrahydro-1-naphthylamine 145 CYCLOBOND I 2000 SNN-3,5-DNB valine methyl ester 145 CYCLOBOND I 2000 SNDinitrocresols ( 2,6-; 4,6-) 63 CYCLOBOND IDinitrophenols (2,6-) 63 CYCLOBOND I2,4-Dinitrophenyl 81 CYCLOBOND IDiphenidol 164 CYCLOBOND I 2000Diphenylamine 87 CYCLOBOND Icis-4,5-diphenyl-2-oxazolidinone 157 CYCLOBOND I 2000 ACα,α-Diphenyl-2-pyrrolidine methanol 156 CYCLOBOND I 2000 AC2,3'-Dipyridyl 72 CYCLOBOND IDNP-(D,L)-α-n-caprylic acid 100 CYCLOBOND I

DNP-(D,L)-citrulline 100 CYCLOBOND IDNP-(D,L)-ethionine 100 CYCLOBOND I

35

DNP-(D,L)-glutamic acid 100 CYCLOBOND IDNP-(D,L)-methionine 100 CYCLOBOND IDNP-(D,L)-methionine sulphone 100 CYCLOBOND IDNP-(D,L)-methionine sulphoxide 100 CYCLOBOND IDNP-(D,L)-α-amino-n-butyric acid 100 CYCLOBOND I

Dothiepin See page 43 CYCLOBOND I 2000 ACDyfonate 95 CYCLOBOND I RNEfaroxan 85 CYCLOBOND IElliptone 40 CYCLOBOND IEnilconazole See page 43 CYCLOBOND I 2000Erythritol 61 CYCLOBOND I(D,L)-Erythrose 61 CYCLOBOND IEstradiol 138 CYCLOBOND IEthoxyaniline (m-,o-,p-) (see also Phenetidine) 79 CYCLOBOND IN-Ethyl-3-phenyl-2-norboranamine 49 CYCLOBOND I(±)-Ethyl-3-phenylglycidate 45 CYCLOBOND I5-Ethyl-5-(p-tolyl)-2-thiobarbituric acid 49 CYCLOBOND Io-Ethylaniline 36 CYCLOBOND IIEthylanthraquinone 39 CYCLOBOND IEthylbenzene 87, 103 CYCLOBOND IEthylidazoxan 85 CYCLOBOND I6-Ethylnicotine 69 CYCLOBOND IEtomidate 85 CYCLOBOND IFenofibric Acid See page 43 CYCLOBOND I 2000Ferrocenylalkyl benzimidazoles 162 CYCLOBOND IIFlavanone See page 43 CYCLOBOND I 2000 DMPFluoranthene 105 CYCLOBOND IFluorene 87 CYCLOBOND IFluoxetine 85 CYCLOBOND IFlurbiprofen 95 CYCLOBOND I SNFonofos 127 CYCLOBOND I RN(D,L)-Fructose 61, CYCLOBOND IGalactol 61 CYCLOBOND I(D,L)-Galactose 61, 62 CYCLOBOND IGentiobiose 61, 62 CYCLOBOND I(D,L)-Glucose 62 CYCLOBOND I(D,L)-Glutamic acid 84, 100 CYCLOBOND IGlutethimide 82

95CYCLOBOND I DMPCYCLOBOND I SN

(D,L)-Glyceraldehyde 61 CYCLOBOND IGlycopyrrolate 83 CYCLOBOND IGycerine 61 CYCLOBOND I1-Heptanol 80 CYCLOBOND III(R,S)-2-Heptanol 97 CYCLOBOND I SNHeptaporphyrin 78, 91 CYCLOBOND ID-gluco-D-gulo-Heptose 61 CYCLOBOND Itrans- 6,6a,7,10,10a,11-Hexahydro-8,9 dimethyl-11-oxodibenz{b,e}oxepin-3-acidic acid

48 CYCLOBOND I

1-Hexanol 80 CYCLOBOND III(±)-2-Hexanol 97 CYCLOBOND I SN3-Hexanol 97 CYCLOBOND I SNHexaporphyrin 78, 91 CYCLOBOND IHexobarbital 46, 85 CYCLOBOND IHippuric and methyl hippuric acid (m-,o -and p-) 68

137CYCLOBOND ICYCLOBOND I AC

36

(D,L)-Homatropine 44119

CYCLOBOND ICYCLOBOND I AC

(D,L)-Homophenylalanine 9597

CYCLOBOND I SNCYCLOBOND I RN

Huperzine A 85 CYCLOBOND IHydrobenzoin 95

156CYCLOBOND I SNCYCLOBOND I 2000 RSP

9519-Hydroxy PGB1 43 CYCLOBOND I



3 -Hydroxy-(D,L)-kynurenine 45 CYCLOBOND I4-Hydroxy-tamoxifen 32 CYCLOBOND I2-Hydroxybenzyl alcohol 47 CYCLOBOND I3-Hydroxybenzyl alcohol 47 CYCLOBOND I4-Hydroxybenzyl alcohol 47 CYCLOBOND I2-Hydroxy-5,5-dimethyl-4-phenyl-1,3,2-dioxaphosphorinane-2-oxide 156 CYCLOBOND I 2000 RSPHydroxyelliptone 40 CYCLOBOND IHydroxyibuprofen 65 CYCLOBOND I2-Hydroxy-4-(2-methoxyphenyl)5,5-dimethyl-1,3,2-dioxaphorinane-2-oxide

156 CYCLOBOND I 2000

(D,L)-5-(4-Hydroxyphenyl)-5-phenylhydantoin 49145

CYCLOBOND ICYCLOBOND I 2000 RN

4-Hydroxypyrazole 55 CYCLOBOND IIbuprofen 65,85

95,123, 145CYCLOBOND ICYCLOBOND I SN/RN

Idazoxan 88154

CYCLOBOND I RSPCYCLOBOND I 2000 DM

Idazoxan derivatives 85 CYCLOBOND I(R,S)-1-Indanol 88 CYCLOBOND I RSP1-Indanol 154 CYCLOBOND I 2000 DMIndapamide 95 CYCLOBOND I SNIndeno-{1,2,3-cd}-pyrene 107 CYCLOBOND I/IIImyo-Inositol 61 CYCLOBOND I4-Isomyosmine 69 CYCLOBOND I2-Isonicotine 69 CYCLOBOND I4-Isonicotine 69 CYCLOBOND I2-Isonornicotine 69 CYCLOBOND I4-Isonornicotine 69 CYCLOBOND IIsorotenolone 40 CYCLOBOND IIsorotenone 40 CYCLOBOND IJacobsen’s Catalyst 156 CYCLOBOND I 2000 RSPKetoprofen 85 CYCLOBOND I(D,L)-Lactose 61 CYCLOBOND I(D,L)-Lactulose 61 CYCLOBOND ILeucine, AQC 165 CYCLOBOND I 2000 SNLeucine, FMOC-Gly 142, 143 CYCLOBOND ILorazepam 95 CYCLOBOND I SNLutein 71 CYCLOBOND ILycopene 71 CYCLOBOND I(D,L)-Lyxose 61 CYCLOBOND IIIMaltitol 61 CYCLOBOND I(D,L)-Maltose 61, 62 CYCLOBOND I(D,L)-Maltotriose 61 CYCLOBOND I(D,L)-Mandelic acid 45 CYCLOBOND I(D,L)-Mandelic acid benzyl ester 49 CYCLOBOND I

37

(D,L)-Mandelic acid methyl ester 45 CYCLOBOND I(D,L)-Mannitol 61 CYCLOBOND I(D,L)-Mannose 62 CYCLOBOND IMelezitose 61 CYCLOBOND III(D,L)-Melibiose 61, 62 CYCLOBOND IMephenytoin 85

145CYCLOBOND ICYCLOBOND I 2000 SN

Mephobarbital 85 CYCLOBOND IMesoporphyrin 78, 91 CYCLOBOND IMethadone 85

See page 43See page 43151

CYCLOBOND ICYCLOBOND I 2000 SPCYCLOBOND I 2000 RSPCYCLOBOND I 2000 RSP

Methadone Major Metabolite - 2-Ethylidene-1,5-dimethyl-3,3-diphenyl-pyrrolidine

151 CYCLOBOND I 2000 RSP

Methamphetamine, AQC 148, Seepage43

CYCLOBOND I 2000 DMP

(R,S)-2-Methoxy-2-phenylethanol 8297

CYCLOBOND I DMPCYCLOBOND I SN

Methoxyaniline (m-,o-,p-)(See also Anisidine (m-,o-,p-) 36 CYCLOBOND II(R,S)-α-Methoxyphenylacetic acid 82 CYCLOBOND I PT

5-Methoxypsoralen 51 CYCLOBOND I8-Methoxypsoralen 51 CYCLOBOND Iα-Methoxy-α-(trifluoromethyl)phenyacetic acid 156 CYCLOBOND I 20003,5-Dinitrobenzoyl-α-Methylbenzylamine LC1014 CYCLOBOND I 2000 DMPN'-(2-Methyl benzyl)-nornicotine 53 CYCLOBOND I1-Methyl-2-(3-pyridyl)-azetidine 69 CYCLOBOND I4-Methyl-2-Pentanol 97 CYCLOBOND I SN1-Methyl-2-phenylpyrrolidine 69 CYCLOBOND IMethyl-5-formyl-2,4 pentadienoate iron tricarbonyl 99 CYCLOBOND I5-Methyl-5-phenylhydantoin 49 CYCLOBOND I(4S,5R;4R,5S)-4-Methyl-5-phenyl-2-oxazolidinone 156 CYCLOBOND I 2000 SN

α-Methyl-α-phenyl-succinimide 49 CYCLOBOND I

9-Methyl-∆5(10)-octaline-1,6-dione 49 CYCLOBOND I

(R,S)-(±)-N'-Methylanabasine 69, 72 CYCLOBOND IN-Methylaniline 36 CYCLOBOND IIMethylanthraquinone 39 CYCLOBOND I(R,R)-(S,S)-(±)-N,N'-bis(α- Methylbenzyl)sulfamide 82 CYCLOBOND I Ac

(R,S)-α-Methylbenzylamine 97 CYCLOBOND I SN

1-Methylbutylamine 97 CYCLOBOND I RN[3aR-[2(3′aR*,8aS*),3′aβ,8aβ]-2,2′-methyl-ene-bis[3a,8a-dihydro-8H-indeno[1,2-d]]oxazole

157 CYCLOBOND I 2000 SN

Methylenedioxylated amphetamine – MDA, MDMA, MDEA, MBDB 155 CYCLOBOND I 2000CYCLOBOND I 2000 RSP

Methylidozoxan 88154

CYCLOBOND I SPCYCLOBOND I 2000 DM

1-Methylnaphthalene 52 CYCLOBOND I2-Methylnaphthalene 52 CYCLOBOND IMethylphenidate 85 CYCLOBOND IMethylphenobarbital & Metabolites 75 CYCLOBOND I5-(4-Methylphenyl)-5-phenylhydantoin 49, 154 CYCLOBOND I(D,L)-a-Methyltryptamine 49 CYCLOBOND I(D,L)-O- Methyltyrosine 97 CYCLOBOND I SN

38

Metoprolol 108See page 44119

CYCLOBOND ICYCLOBOND I 2000CYCLOBOND I/AC

Miconazole See page 44 CYCLOBOND I 2000 RSPMonosodium Glutamate, FMOC-Gly 141 CYCLOBOND IIMyosmine 69, 72 CYCLOBOND INadolol 108 CYCLOBOND I1-Naphthol ±1,2,3,4-tetrahydro 88

97CYCLOBOND I SPCYCLOBOND I RN

Naphthol (α,β-) 64, 79 CYCLOBOND I

(D,L)-3-(2-Naphthyl)-alanine 97 CYCLOBOND I SN(D,L)-3-(1-Naphthyl)-alanine 97 CYCLOBOND I SNα-(1-Naphthyl)-ethylamine 88 CYCLOBOND I RSP

N'-(2-Naphthylmethyl)-nornicotine 53 CYCLOBOND IN'-(2-Naphthymethyl)-nornicotine 82 CYCLOBOND I RNNapthalene 47, 64

76CYCLOBOND ICYCLOBOND II

(S)-(-)-Nicotine (homologues) 69, 72 CYCLOBOND I(1R,2S)-anti-Nicotine-N'-oxide 72 CYCLOBOND I(1S,2S)-syn-Nicotine-N'-oxide 72 CYCLOBOND INicotyrine 72 CYCLOBOND INisolidipine 88 CYCLOBOND I SPNitroaniline (m-,o-,p-) 36

64, 79, 103CYCLOBOND IICYCLOBOND I

Nitrophenol (m-,o-,p-) 64 CYCLOBOND IN-Nitrosohydroxyproline 54 CYCLOBOND IIIN-Nitrosoisonipecotic acid 54 CYCLOBOND IIIN-Nitrosonipecotic acid 54 CYCLOBOND IIIN-Nitrosopipecolinic acid 54 CYCLOBOND IIIN-Nitrosoproline 54 CYCLOBOND IIIN-Nitrososarcasine 54 CYCLOBOND IIIN-Nitrosothiazolidine carboxylic acid 54 CYCLOBOND IIINitrotoluene 63 CYCLOBOND INomifensine hydrogen maleate 50 CYCLOBOND I(R,S)-Norcotinine 69, 72 CYCLOBOND I(±)-Norgestrel 85, See

page 44CYCLOBOND II

(R,S)-Nornicotine 64, 72 CYCLOBOND INorphenylephrine See page 44 CYCLOBOND I 2000 AC(R,S)-2-Octanol 97 CYCLOBOND I SNOxazepam 95

119CYCLOBOND I SNCYCLOBOND I RN

Oxazoline 99 CYCLOBOND IOxprenolol 108, LC1003 CYCLOBOND IPemoline 95 CYCLOBOND I SN2,4-Pentadienoate-iron tricarbonyl derivative 99,111 CYCLOBOND I2,4-Pentadienoate, methyl-5-formyl 111 CYCLOBOND I(R,S)-2-Pentanol 97 CYCLOBOND I SNPentaporphyrin 78, 91 CYCLOBOND IPGB1, PGB2, PGB3 43 CYCLOBOND I

Phenanthrene 103 CYCLOBOND I(R,S)-sec-Phenethyl alcohol 97 CYCLOBOND I SN(±)-5-(α-Phenethyl)-semioxamazide 82 CYCLOBOND I Ac

Phenetidine 79 CYCLOBOND IPheniramine 42 CYCLOBOND I

39

Phenobarbital 75 CYCLOBOND IPhenprocoumon 102 CYCLOBOND I(±)-Phensuximide 82

85See page 44

CYCLOBOND I DMPCYCLOBOND ICYCLOBOND I 2000 DMP

1-Phenyl-1-butanol 157 CYCLOBOND I 2000 RSPN-Phenyl-ß-napthylamine 103 CYCLOBOND IN-Phenyl-α-naphthylamine 103 CYCLOBOND I

(±)-γ-Phenyl-γ-butyrolactone 82 CYCLOBOND I DMP

Phenylalanine, FMOC-Gly 142, 143 CYCLOBOND I(D,L)-Phenylalanineamide 45 CYCLOBOND I2-Phenylbutyric acid 157 CYCLOBOND I 2000 RSP(R,S)-2-Phenylbutyric acid 97 CYCLOBOND I SN(±)-2-Phenylbutyrophenone 49 CYCLOBOND I(R,S)-Phenylephrine 42

See page 44CYCLOBOND ICYCLOBOND I 2000 AC

1-Phenylhexane 87 CYCLOBOND I1-Phenyloctane 87, 88 CYCLOBOND I4-Phenyl-2-oxazolidinone 156 CYCLOBOND I 2000 AC2-Phenylphenol 47 CYCLOBOND I4-Phenylphenol 47 CYCLOBOND I3-Phenylphthalide 88

95CYCLOBOND I RSPCYCLOBOND I SN

(D,L)-2-Phenylpropionaldehyde 45 CYCLOBOND I2-Phenylpropionic acid 157, See

page 44CYCLOBOND I 2000 RSP

(R,S)-2-Phenylpropionic acid 97 CYCLOBOND I SN2-Phenylpyrrolidine 69 CYCLOBOND IPhenytoin (PHT) and metabolite (PHPPH) LC1017 CYCLOBOND I 2000 RSPPhosphatidyl ethanolamine (PE) 129,136 CYCLOBOND IPhosphatidylcholine (PC) 129,136 CYCLOBOND IPhosphatidyl inositol (PI) 129,136 CYCLOBOND IPhosphatidic Acid (PA) 129,136 CYCLOBOND IPhosphine Oxides 41 CYCLOBOND IPilocarpic acid 112 CYCLOBOND IPilocarpine 112 CYCLOBOND IPipecolic acid, FMOC 125 CYCLOBOND IPiperoxan 145 CYCLOBOND I 2000 SNPregnalone 138 CYCLOBOND IPrephenate 38 CYCLOBOND IProglumide 119, 145 CYCLOBOND I/AC/RNProline, FMOC See page 44 CYCLOBOND IIProline, metabolites, FMOC 124,125, 143 CYCLOBOND I RNPropionic acid, 2-(-4-chlorophenoxy) 119 CYCLOBOND I RNPropionic acid, 2-phenoxy 119 CYCLOBOND I RN(R,S)-Propranolol 85,108,119,

139See page 45

CYCLOBOND I

CYCLOBOND I 2000Propylbenzene 87 CYCLOBOND IProstaglandin 43 CYCLOBOND IProtoporphyrin 78, 91 CYCLOBOND IPsoralen 51 CYCLOBOND IPyrazole 55 CYCLOBOND IPyrene 33

105107

CYCLOBOND IIICYCLOBOND ICYCLOBOND I/III

40

Quinidine 79 CYCLOBOND I/IIQuinine 79 CYCLOBOND I/II(D,L)-Raffinose 61 CYCLOBOND IRetinal (cis-, trans-) 71 CYCLOBOND IRetinal Palmitate 71 CYCLOBOND IRetinol (cis- trans-) 71 CYCLOBOND IRetinyl Acetate 71 CYCLOBOND I(D,L)-Rhamnose 62 CYCLOBOND I(D,L)-Ribitol (See also Adonitol) 61 CYCLOBOND I(D,L)-Ribose 61 CYCLOBOND IIIRotenolone 40 CYCLOBOND IRotenone 40 CYCLOBOND IRuelene 95

119CYCLOBOND I RNCYCLOBOND I/AC

Scopolamine 44, 98 CYCLOBOND ISelegiline See page 45 CYCLOBOND I 2000 SNSobrerol (cis-, trans-) 73 CYCLOBOND I(D,L)-Sorbitol 61 CYCLOBOND I(D,L)-Sorbose 61 CYCLOBOND ISQ 28 873 88 CYCLOBOND I SPSQ 30 840 85

88CYCLOBOND ICYCLOBOND I SP

SQ 31 236 88 CYCLOBOND I SP/RSPSQ 31 579 88

95CYCLOBOND I SPCYCLOBOND I SN

Stachyose 61 CYCLOBOND IStilbene (cis-, trans-) 103

153CYCLOBOND ICYCLOBOND I 2000 DM

Strigol See page 45 CYCLOBOND I 2000 RSP(D,L)-Sucrose 61 CYCLOBOND ISulfadiazine 67 CYCLOBOND ISulfadimidine 67 CYCLOBOND ISulfamerazine 67 CYCLOBOND ISulfathiazol 67 CYCLOBOND ISuprofen 60

119144

CYCLOBOND ICYCLOBOND I RNCYCLOBOND I 2000 RN

Tagatose 61 CYCLOBOND ITalose 61 CYCLOBOND ITamoxifen (cis-, trans-) 32 CYCLOBOND ITemazepam 95 CYCLOBOND I SNTephrosin 40 CYCLOBOND ITerfenadine 106 CYCLOBOND ITerodiline 95

119CYCLOBOND I SNCYCLOBOND IIICYCLOBOND RSP/AC

Terpenic Alcohols 73 CYCLOBOND ITerphenyl (m-,o-,p-) 103 CYCLOBOND ITestosterone 138 CYCLOBOND I1,2,3,4-Tetrahydro-1-naphthol 156 CYCLOBOND I 2000 RSPTetrahydrozoline 95 CYCLOBOND I SN(D,L)-Tetramisole 95 CYCLOBOND I SNTheanine, FMOC-Gly 152 CYCLOBOND IITheobromine 103 CYCLOBOND IITheophylline 103 CYCLOBOND IIThienopyran 120 CYCLOBOND I

41

(±)-3-Thienylcyclohexylglycolic acid (TCGA) 56 CYCLOBOND I(D,L)-Thrietol 61 CYCLOBOND I3-α-Tigloyloxy-6-ß-acetoxytropane (TAT) 98 CYCLOBOND I AC

Timolol 108,119 CYCLOBOND I ACTocol, 5,7-dimethyl (DMT) 128 CYCLOBOND ITolperisone 95

145CYCLOBOND I SNCYCLOBOND I 2000 SN

Toluene 87 CYCLOBOND IToluidine (m-,o-,p-) 36 CYCLOBOND IIα-Toxicarol 40 CYCLOBOND I

Tramadol See page 45 CYCLOBOND I 2000 DMP(R,S)-N-Trichloroacetyl-1,2,3,4-tetrahydro-1-naphthylamine 82 CYCLOBOND I DMPN'-(2,2,2-Trifluoroethyl)-nornicotine 53 CYCLOBOND ITrihexylphenidyl 119

164CYCLOBOND I/ACCYCLOBOND I 2000

3,3',5-Triiodo-(D,L)-thyronine 88 CYCLOBOND I SP4,5',8-Trimethylpsoralen 51 CYCLOBOND ITriphenylene 103 CYCLOBOND ITropicamide 95 CYCLOBOND I SN(D,L)-Tryptophan 97

145CYCLOBOND I SNCYCLOBOND I 2000 SN

(D,L)-Turanose 61 CYCLOBOND I(D,L)-Tyrosine 45 CYCLOBOND I(D,L)-Tyrosine methy ester 45 CYCLOBOND IUroporphyrin 78, 91 CYCLOBOND IVerapamil 85 CYCLOBOND IIWarfarin 102

154See page 45

CYCLOBOND ICYCLOBOND I 2000 DMCYCLOBOND I 2000

Xylene (m-,o-,p-) 64119

CYCLOBOND ICYCLOBOND I SN

Xylitol 61 CYCLOBOND I(D,L)-Xylose 61

62CYCLOBOND I/IIICYCLOBOND III

Zeaxanthin 71 CYCLOBOND I

42

Astec Chromatogram Library

Amphetamine(N-AQC* Derivative)

Arotinolol Bendroflumethazide Chlorpheniramine

CH 3

NHR

N

NCR=

O

H

Peak 1 – 12.7 min.Peak 2 – 14.5 min.

S

NS

O

H2N S N

OH

C(CH 3)3

H

Peak 1 – 14.0 min.Peak 2 – 15.6 min.

SN

N

H3NO2S

F3C

O O

H

CH 2

H

Peak 1 – 5.9 min.Peak 2 – 7.31 min.

N

N

Cl

H3C

CH 3

Peak 1 – 16.1 min.Peak 2 – 18.1 min.

CYCLOBOND I 2000 DMP45/55: ACN/0.1% TEAA

pH 4.11.0 mL/min.

CYCLOBOND I 200090/10/0.3/0.2:

ACN/MeOH/HOAc/TEA2.0 mL/min.

CYCLOBOND I 2000 SN30/70: CH3CN/1% TEAA,

pH 4.51.2 mL/min.

CYCLOBOND I 200010/90: CH3CN/1% TEAA

pH 4.11.0 mL/min.

Chlorthalidone Ciprofibrate Cresols + Phenol α,α-Di(2-naphthyl-2-pyrrolidine methanol

Cl

O

NHS

HO NH2

O O

Peak 1 – 16.7 min.Peak 2 – 20.3 min.

Cl Cl

COOH

H3C CH 3

O

Peak 1 – 16.4 min.Peak 2 – 17.6 min.

OH

CH 3

OH

CH 3

OHCH 3

OH

Peak 1 – 10.7 min.Peak 2 – 11.7 min.Peak 3 – 14.2 min.Peak 4 – 20.1 min.

OHN CH

Peak 1 – 21.4 min.Peak 2 – 24.2 min.

CYCLOBOND I 2000 RSP5/95: CH3CN/1% TEAA

pH 4.11.0 mL/min.

CYCLOBOND I 200090/10/0.3/0.2:

ACN/MeOH/HOAc/TEA1.0 mL/min.

CYCLOBOND I 200010/90: MeOH/H2O

1.0 mL/min.

CYCLOBOND I 2000 SN98/2/0.03/0.02:

CH3CN/MeOH/HOAc/TEA1.0 mL/min.

43

cis/trans-Dothiepin Enilconazole Fenofibric Acid Flavanone

S

NCH 3

CH 3

NN

O

Cl

Cl

H2C


Cl C

OH

O C COOH

CH 3

CH 3

Peak 1 – 21.3 min.Peak 2 – 24.2 min.

O

O


CYCLOBOND I 2000 AC15/85: CH3CN/1.0% TEAA

pH 4.11.0 mL/min.

CYCLOBOND I 200010/90: ACN/0.1% TEAA

pH 4.10.7 mL/min.

CYCLOBOND I 200080/20: CH3CN/0.1% TEAA

pH 4.01.0 mL/min.

CYCLOBOND I 200080/20: Hex/EtOH

0.6 mL/min.

Methadone Methadone Methamphetamine(N-AQC* Derivative)

α-Methylbenzylamine(N-3,5-DNB derivative)

O

CH 3

CH 3

CH 3

CH 3

N

Peak 1 – 14.1 min.Peak 2 – 15.8 min.

O

CH 3

CH 3

CH 3

CH 3

N


NCH 3

CH 3

R

N

NCR=

O

H

Peak 1 – 36.5 min.Peak 2 – 38.6 min.

CH 3

NHR

NO2

NO2

C

O

R=



pH 4.10.6 mL/min.

CYCLOBOND I 2000 SP15/85: CH3CN/1% TEAA

pH 4.10.6 mL/min.

CYCLOBOND I 2000 DMP35/65: CH3CN/0.1% TEAA

pH 4.11.0 mL/min.

CYCLOBOND I 2000 DMP100% EtOH1.2 mL/min.

44

Metoprolol Miconazole Norgestrel NorphenylephrineOH

NOH

CH 3

CH 3H3CO

Peak 1 – 15.4 min.Peak 2 – 17.0 min.

Cl

Cl

Cl

N

N

O

Cl

Peak 1 – 12.5 min.Peak 2 – 13.7 min.

H3C

O

H

H

H

H

CHOH

Peak 1 – 16.1 min.Peak 2 – 17.5 min.

HOCH CH2 NH2


CYCLOBOND I 200090/10/0.3/0.2:



pH 4.02.0 mL/min.

CYCLOBOND II30/70: CH3CN/H2O

0.8 mL/min.

CYCLOBOND I 2000 AC10/90: CH3OH/0.5%

NaOAc, pH 5.50.4 mL/min.

Phensuximide Phenylephrine 2-Phenylpropionic Acid Proline( FMOC* Derivative)

N

CH 3

O O


HOCH

OH

CH 2NH CH 3

Peak 1 – 10.9 min.Peak 2 – 13.3 min.

CH 3

CH C OH

O

Peak 1 – 11.0 min.Peak 2 – 11.8 min.

R= C O CH 2

O

NR

O

OH

Peak 1 – 14.4 min.Peak 2 – 15.4 min.*R-(9-Fluorenyl-

methoxycarbonyl)

CYCLOBOND I 2000 DMP20/80: Hex/IPA

1.0 mL/min.

CYCLOBOND I 2000 AC10/90: CH3OH/0.5%

NaOAc, pH 5.50.5 mL/min.

CYCLOBOND I 2000 RSP30/70: ACN/20mM NH4OAc

pH 4.00.6 mL/min.

CYCLOBOND II95/5/0.3/0.2:


45

Propranolol Selegiline Strigol Tramadol

OH HO N

CH 3

CH 3

Peak 1 – 20.6 min.Peak 2 – 22.0 min.

CH 3

H3C

CHN


O

H OCH 3

O

O

CH 3

H3C

OH

H

H

O


NH3C CH3

H

OH

H3CO


CYCLOBOND I 200095/5/0.3/0.2:


CYCLOBOND I 2000 SN25/75: CH3CN/1.0% TEAA

pH 4.10.8 mL/min.

CYCLOBOND I 2000 RSP25/75: CH3CN/1.0% TEAA

pH 4.11.0 mL/min.

CYCLOBOND I 2000 DMP20/80: ACN/0.1% TEAA

pH 5.01.2 mL/min.

WarfarinOO

OH O

CH3

Peak 1 – 10.0 min.Peak 2 – 10.8 min.

CYCLOBOND I 200095/5/0.3/0.2

CH3CN/CH3OH/HOAc/TEA0.5 mL/min. *AQC is a trademark of Waters Corporation.

46

CYCLOBOND Bibliography166. High-performance Liquid Chromatographic Enantioseparation of Bicyclic 1,3-Amino Amino Alcohols, Peter, A.,

Kaman, J., Fulop, F., van der Eycken, J., Armstrong, D.W., J. of Chrom. A, 919, 79-86 (2001).

165. Composition and Chirality of Amino Acids in Aerosol/Dust from Laboratory and Residential Enclosures,Armstrong, D.W., Kullman, J.P., Chen, X., Rowe, M., Chirality, 13, 153-158 (2001).

164. Stereoselective Determination of Trihexyphenidyl in Human Serum by LC-ESI-MS, Capka, V., Xu, Y., Chen, Y.H.,J. of Pharm. & Biomed. Analysis, 21, 507-517 (1999).

163. Optimization and Characterization of the Chiral Separation of Citalopram and its Demethylated Metabolites byResponse-Surface Methodology, Carlsson, B., Norlander, B., Chromatographia, 53, March (No.5/6), 266-272(2001).

162. Synthesis and Structure of Biologically Active Ferrocenylalkyl Polyfluoro Benzimidazoles, Snegur, L.V., Boev,V.I., Nekrasov, Y.S., Ilyin, M.M., Davankov, V.A., Starikova, Z.A., Yanovsky, A.I., Kolomiets, A.F., Babin, V.N.,J. of Organometallic Chem., 580, 26-35 (1999).

161. Chiral Separations of Polar Compounds by Hydrophilic Interaction Chromatography with Evaporative LightScattering Detection, Risley, D.S., Strege, M.A., Anal. Chem., 72, 1736-1739 (2000).

160. High-Performance Liquid Chromatographic Separation of Novel Atropic α,α-Disubstituted-β-amino Acids, Eitheron Different β-cyclodextrin Bonded Phases or as their 1-Fluoro-2,4-Dinitrophenyl-5-L-alanine AmideDerivatives,Torok, G., Peter, A., Gaucher, A., Wakselman, M., Mazaleyrat, J-P, Armstrong, D.W., J. ofChromatogr. A, 846, 83-91 (1999).

159. Simultaneous Determination of Amoxycillin and Clavulanic Acid in Pharmaceutical Products by HPLC with β- cyclodextrin Stationary Phase, Tsou, T-L, Wu, J-R, Young, C-W, Wang, T-M, J. Pharm. and Biomed. Analy., 15, 1197-1205 (1997).

158. The effects on Separation of Cephalosporins by HPLC with β-cyclodextrin Bonded Stationary Phase, Tsou, T-L, Wu, J-R, Wang, T-M, J. Liq. Chrom. & Rel. Technol., 19(7), 1081-1095 (1996).

157. Enantiomeric Impurities in Chiral Catalysts, Auxiliaries, Synthons and Resolving Agents. Part 2, Armstrong,D. W., He, L., Yu, T., Lee, J.T., Liu, Y-S, Tetrahedron: Asymmetry, 10, 37-60 (1999).

156. Enantiomeric Impurities in Chiral Catalysts, Auxiliaries and Synthons Used in Enantioselective Synthesis, Armstrong, D.W., Lee, J. T., Chang, L.W., Tetrahedron: Asymmetry, 9, 2043-2064 (1998).

155. Enantiomeric Separation of Four Methylenedioxylated Amphetamines on β-cyclodextrin Chiral Stationary Phases,Sadeghipour, F., Veuthey, J.-L., Chromatographia, 47(5/6), 285-290 (1998).

154. Comparison of the Enantioselectivity of β-cyclodextrin vs. Heptakis-2,3,-O-Dimethyl-β-cyclodextrin LC Stationary Phases, Armstrong, D.W, Chang, L.W.,Chang, S.C., Wang, X., Ibrahim, H., Reid III, G.R., Beesley, T.E., J. Liq. Chrom. & Rel. Technol., 20(20), 3279 -3295(1997).

153. Comparison of the Selectivity and Retention of β-cyclodextrin vs. Heptakis-2,3-O-Dimethyl-β-cyclodextrin LC Stationary Phases for Structural and Geometric Isomers, Armstrong, D.W., Wang, X., Chang, L.W., Ibrahim, H., Reid III, G.R., Beesley, T.E.,J. Liq Chrom. & Rel. Technol., 20(20), 3297-3308 (1997).

152. Varietal Differences in the Total and Enantiomeric Composition of Theanine in Tea, Ekborg-Ott, K.H., Taylor, A., Armstrong, D.W., J. Agric. Food Chem.,45 ,353-363 (1997).

151. Enantioselective High-performance Liquid Chromatography Determination of Methadone Enantiomers and its Metabolite in Human Biological Fluids Using a New Derivatized Cyclodextrin-bonded Phase, Pham-Huy, C., Chikhi-Chorfi, N., Galons, H., Sadeg, N., Laqueille, X., Aymard, N., Massicot, F., Warnet, J., Claude, J., J. of Chrom. B, 700, 155-163 (1997).

47

150. Activities of the Four Optical Isomers of 2´,3´-Dideoxy-3´-Thiacytidine (BCH-189) Against Human Immunodeficiency Virus Type 1 in Human Lymphocytes, Schinazi, R.F., Chu, C.K., Peck, A., McMillan, A., Mathis, R., Cannon, D., Jeong, L., Beach, J.W., Choi, W., Yeola, S., Liotta, D.C., Antimicrobial Agents and Chemotherapy, 672-676 (Mar. 1992).

149. The Separated Enantiomers of 2´-Deoxy-3´-Thiacytidine (BCH 189) Both Inhibit Human Immunodeficiency Virus Replication In Vitro, Coates, J.A.V., Cammack, N., Jenkinson, H.J., Mutton, I.M., Pearson, B.A., Storer, R., Cameron, J.M., Penn, C.R., Antimicrobial Agents and Chemotherapy, 202-205 (Jan. 1992).

148. Enantioresolution of Amphetamine, Methamphetamine, and Deprenyl (Selegiline) by LC, GC and CE, Armstrong, D.W., Rundlett, K.L., Nair, U.B., Current Separations, 15:2, 57-61 (1996).

147. Capillary Electrochromatography: Operating Characteristics and Enantiomeric Separations, Lelièvre, F., Yan, C., Zare, R.N., Gareil, P., J. of Chrom. A, 723, 145-156 (1996).

146. Binding Forces Contributing to Reversed-Phase Liquid Chromatographic Retention on a β-cyclodextrin Bonded Phase, Nah, T.H., Cho, E.H., Jang, M.D., Lee, Y.K., Park, J.H., J. of Chrom. A, 722, 41-46 (1996).

145. Comparison of Liquid and Supercritical Fluid Chromatography Using Naphthylethylcarbamoylated-β-cyclodextrin Chiral Stationary Phases, Williams, K.L., Sander, L.C., Wise, S.A., J. of Chrom. A,746, 91-101 (1996).

144. Detection of non-UV Absorbing Chiral Compounds by High-Performance Liquid Chromatography, Richards, D.S., Davidson, S.M., Holt, R.M., J. of Chrom. A, 746, 9-15 (1996).

143. Evaluation of Concentration and Enantiomeric Purity of Selected Free Amino Acids in Fermented Malt Beverages (Beers), Ekkborg-Ott, K.H., Armstrong, D.W., Chirality, 8, 49-57, (1996).

142. Evaluation of Enantiomeric Purity of Selected Amino Acids in Honey, Pawlowska, M., Armstrong, D.W., Chirality, 6, 270-276 (1994).

141. Evaluation of Free D-Glutamate in Processed Foods, Rundlett, K.L., Armstrong, D.W., Chirality, 6, 277-282 (1994).

140. HPLC Resolution of Hydroxyl Carboxylic Acid Enantiomers Using 2-Quinoxaloyl Chloride as a New Precolumn Derivatizing Agent, Brightwell, M., Pawlowska, M., Zukowski, J., J. of Liq. Chrom., 18(14), 2765-2781 (1995).

139. High-performance Liquid Chromatographic Determination of (S)- and (R)-propranolol in Human Plasma and Urine With a Chiral β-Cyclodextrin Bonded Phase, Pham-Huy, C, Radenen, B, Sahui-Gnassi, A., Claude, J., J. of Chrom. B., 665, 125-132 (1995).

138. Indirect Photodetection of Pregnanolone on a CYCLOBOND Column by High-performance Liquid Chromatography,Agnus, B. Gosselet, N., Sebille, B., J. of Chrom. A., 663, 27-33 (1994).

137. HPLC Determination of o-,m-,p-Methylhippuric Acids and Hippuric Acid in Urine of Xylene and Toluene Exposed Persons, Korn, M., Hennings, R., Heilig, M., 13th Annual Conference on Biochemical Analysis, Annual Meeting of the German Society for Clinical Chemistry.

136. Separations of Major Soybean Phospholipids on β-cyclodextrin-bonded Silica, Abidi, S.L., Mounts, T.L., Rennick, K.A., J. of Liq. Chrom., 17(17), 3705-3725 (1994).

135. Liquid Chromatographic Separation of Radiopharmaceutical Ligand Enantiomers, Green, J., Jones, R., Harrison, R.D., Edwards, D.S., Glajch, J.L., J. of Chrom., 635, 203-209 (1993).

134. Sensitive Enantiomeric Separation of Aliphatic and Aromatic Amines Using Aromatic Anhydrides as Non-chiral Derivatizing Agents, Pawlowska, M., Zukowski, J., Armstrong, D.W., J. of Chrom. A., 666, 485-491 (1994).

133. Direct Enantiomeric Resolution of Monoterpene Hydrocarbons Via Reversed-Phase High-Performance Liquid Chromatography with an α-cyclodetrin Bonded Stationary Phase, Armstrong, D.W., Zukowski, J., J. of Chrom. A, 666, 445-448 (1994).

48

132. Separations of Tocopherols and Methylated Tocols on Cyclodextrin-Bonded Silica, Abidi, S.L., Mounts, T.L.,J. Chromatogr. A, 670, 67-75 (1994).

131. Chiral Recognition of Structurally Related Aminoalkylphosphonic Acid Derivatives on an Acetylated Beta-Cyclodextrin Bonded Phase, Camilleri, P., Reid, C.A., Manallack, D.T., Chromatographia, Vol. 38, No. 11/12, 771-775 (1994).

130. Optimization of the Resolution of the Enantiomers of β-Dimethyl-aminobutyrophenone by HPLC on aβ-Cyclodextrin Column, Barderas, A.V., Duprat, F., J. of Liq. Chrom., 17(8), 1709-1719 (1994).

129. Separation of Porphyrins Using a γ-Cyclodextrin Stationary Phase, Wu, W., Stalcup, A., J. of Liq. Chrom., 17(5), 1111-1124 (1994).

128. Isocratic HPLC Methods to Separate Lipids, Abidi, S.L. and Mounts, T.L., INFORM, Vol. 5, No. 5. 624-627 (1994).

127. Relevance of Enantiomeric Separations in Environmental Science, Armstrong, D.W., Reid III, G.L., Hilton, M.L., Chang, C.-D., Environmental Pollution, 79, 51-58 (1993).

126. Enantiomeric Separation of Fluorescent, 6-amino-quinolyl-N-hydroxysuccinimidyl Carbamate, Tagged Amino Acids, Pawlowska, M., Chen, S., Armstrong, D.W., J. of Chromatogr., 641, 257-265 (1993).

125. D-Amino Acid Levels in Human Physiological Fluids, Armstrong, D.W., Gasper, M., Lee, S.H., Zukowski, J.,Ercal, N., Chirality, 5, 375-378 (1993).

124. Factors Controlling the Level and Determination of D-amino acids in the Urine and Plasma of Laboratory Rodents, Armstrong, D.W., Gasper, M.P., Lee, S.H., Ercal, N., Zukowski, J., Amino Acids, 5, 299-315 (1993).

123. Displacement Chromatograpy on Cyclodextrin Silicas, IV. Separation of the Enantiomers of Ibuprofen, Farkas, G., Irgens, L.H., Quintero, G., Beeson, M.D., Al-Saeed, A., Vigh, G., J. of Chromatogr., 645, 67-74 (1993).

122. High Performance Liquid Chromatographic Determination of the Isomeric Purity of a Series of Dioxolane Nucleoside Analogues, DiMarco, M.P., Evans, C.A., Dixit, D.M., Brown, W.L., Siddiqui, M.A., Tse, H.L.A., Jin, H., Nguyen-Ba, N., Mansour, T.S., J. of Chromatogr., 645, 107-114 (1993).

121. High-Performance Liquid Chromatography of Neutral Oligosaccharides on a β-Cyclodextrin Bonded Phase Column, Simms, P.J., Haines, R.M., Hicks, K.B., J. of Chromatogr., 648, 131-137 (1993).

120. Analytical and Preparative High-Performance Liquid Chromatographic Separation of Thienopyran Enantiomers, Shaw, C.J., Sanfilippo, P.J., McNally, J.J., Park, S.A., Press, J.B., J. of Chromatogr., 631, 173-175 (1993).

119. Evaluation of a New Polar-Organic High-Performance Liquid Chromatographic Mobile Phase for Cyclodextrin- Bonded Chiral Stationary Phases, Chang, S.C., Reid III, G.L., Chen, S., Chang, C.D., Armstrong, D.W., Trends in Anal. Chem. (TRAC), 12(4), 144-153 (1993).

118. High-performance Liquid Chromatographic Enantioseparation of Glycyl di- and tripeptides on Native Cyclodextrin Phases, Mechanistic Considerations, Zukowski, J., Pawlowska, M., Nagatkina, M. Armstrong, D. W., J. of Chromatogr., 629, 169-179 (1993).

117. Liquid Chromatographic Separation of the Enantiomers of Diniconazole Using a β-cyclodextrin-bonded Column, Furuta, R., Nakazawa, H., J. of Chromatogr., 625, 231-235 (1992).

116. Improvement of the Liquid Chromatographic Separation of the Enantiomers of Tetracyclic Eudistomins by the Combination of a ß-cyclodextrin Stationary Phase and Camphorsulphonic Acid as Mobile Phase Additive, Kuijpers, P.H., Gerding T.K., de Jong, G.J., J. of Chromatogr., 625, 223-230 (1992).

115. Liquid Chromatographic Resolution of Enantiomers of Deltahedral Carborane and Metallaborane Derivatives, Plesek, J., Bruner, B., J. of Chromatogr., 626, 197-206 (1992).

114. Optical Resolution of Flavanones by High-performance Liquid Chromatography on Various Chiral Stationary Phases, Krause, M., Galensa, R., J. Chromatogr., 514, 147-159 (1990).

49

113. Facile Resolution of N-tert-Butoxy-Carbonyl Amino Acids: The Importance of Enantiomeric Purity in Peptide Synthesis, Chang, S.C., Wang, L.R., Armstrong, D.W., J. of Liq. Chromatogr., 15(9), 1411-1429 (1992).

112. High-performance Liquid Chromatographic Determination of Pilocarpine Hydrochloride and its Degradation Products Using a ß-cyclodextrin Column, Sternitzke, K.D., Fan, T.Y., Dunn, D.L., J. of Chromatogr., 589, 159-164(1992).

111. Determination of Enantiomers in Human Serum by Direct Injection onto a ß-cyclodextrin HPLC Bonded Phase, Stalcup, A.M., Williams, K.L., J. of Liq. Chrom., 15(1), 29-37 (1992).

110. Armstrong, D.W., Empirical Procedure That Uses Molecular Structure to Predict Enantioselectivity of Chiral Stationary Phases, Berthod, A., Chang, S., Anal. Chem., 64, 395-404 (1992).

109. High-performance Liquid Chromatography of Diastereometric Flavanone Glycosides in Citrus on a ß-cyclodextrin-bonded Stationary Phase (CYCLOBOND I), Krause, M., Galensa, R., J. Chromatogr., 588, 41-45 (1991).

108. A New Approach for the Direct Resolution of Racemic Beta Adrenergic Blocking Agents by HPLC, Armstrong, D.W., Chen, S., Chang C., Chang, S., J. Liq. Chrom., 15(3), 545-556 (1992).

107. Complex Sample Analysis by Column-Switching High Performance Liquid Chromatography, Packham, A.J., LC.GC Intl., Vol. 4, No. 11, 26-29 (1991).

106. Direct Enantiomeric Separation of Terfenadine and its Major Acid Metabolite by High-Performance Liquid Chromatography, and the Lack of Stereoselective Terfenadine Enantiomer Biotransformation in Man, Chan, K.Y., George, R.C., Chen, T., Okerholm, R.A., J.Chromatogr., 571, 291-297 (1991).

105. Column Switching for the High-Performance Liquid Chromatographic Analysis of Polynuclear Aromatic Hydrocarbons in Petroleum Products, Packham, A.J., Fielden, P.R., J. Chromatogr., 552, 575-582 (1991).

104. Multimodal Chiral Stationary Phases for Liquid Chromatography: (R)- and (S)-Naphthylethyl-carbamate-Derivatized β-Cyclodextrin, Armstrong, D.W., Hilton, M., Coffin, L., LC.GC Vol. 9 (9), 646-652 (1991).

103. Microcolumn Liquid Chromatography of Polycyclic Aromatic Hydrocarbons and Some Isomeric Compounds on Cyclodextrin Stationary Phases, Malik, A., Jinno, K., J. of High Res. Chrom. & CC, Vol. 14, 117-122 (Feb. 1991).

102. Fluorometric and Liquid Chromatographic Study of the Binding of Two Coumarins to β-Cyclodextrin, Karnik, N.A., Prankerd, R.J., Perrin, J.H., Chirality, 3, 124-128 (1991).

101. Chiral High-performance Liquid Chromatography of Aromatic Cyclic Dipeptides Using Cyclodextrin Stationary Phases, Florance, J., Konteatis, Z., J. Chromatogr., 543, 299-305 (1991).

100. Liquid Chromatographic Separation of the Enantiomers of Dinitrophenyl Amino Acids Using a β-Cyclodextrin-bonded Stationary Phase, Li, S., Purdy, W.C., J. Chromatogr., 543, 105-112 (1991).

99. High-performance Liquid Chromatographic Separation of Racemic and Diastereomeric Mixtures of 2,4-Pentadienoate Iron Tricarbonyl Derivatives, Xu, M., Tran, C.D., J. Chromatogr., 543, 233-240 (1991).

98. Determination of the Enantiomeric Purity of Scopolamine Isolated From Plant Extract Using Achiral/Chiral CoupledColumn Chromatography, Stalcup, A.M., Faulkner, J. R., Tang, Y., Armstrong, D.W., Levy, L.W., Regalado, E., Biomed. Chromatogr., 5, 3-7 (1991).

97. Effect of the Configuration of the Substituents of Derivatized β-cyclodextrin Bonded Phases on Enantioselectivity in Normal-Phase Liquid Chromatography, Stalcup, A.M., Chang, S.C., Armstrong, D.W., J. Chromatogr., 540, 113-128 (1991).

96. Semi-preparative Separation of Polyhydroxylated Sterols Using a β−cyclodextrin High-Performance Liquid Chromatography Column, West, R.R., Cardellina, J.H., J. Chromatogr., 539, 15-23 (1991).

50

95. (R)- and (S)-Naphthylethylcarbamate Substituted β-cyclodextrin Bonded Stationary Phases for the Reversed-Phase Liquid Chromatographic Separation of Enantiomers, Armstrong, D.W., Chang, C.D., Lee, S.H., J. Chromatogr., 539, 83-90 (1991).

94. Liquid Chromatographic Retention Behavior and Separation of Chlorophenols on a β-Cyclodextrin Bonded Phase Column, Part III. Diaromatic Chlorophenols, Paleologou, M., S. Li, Purdy, W.C., Can. J. Chem., Vol. 68, 1208-1214 (1990).

93. Liquid Chromatographic Retention Behavior and Separation of Chlorophenols on a β-Cyclodextrin Bonded Phase Column, Part II. Mono-aromatic Chlorophenols: Separation, Paleologou, M., S. Li, Purdy, W.C., J. Chrom. Sci., Vol. 28, 319-323 (1990).

92. Liquid Chromatographic Retention Behavior and Separation of Chlorophenols on a β-Cyclodextrin Bonded Phase Column, Part I. Mono-aromatic Chlorophenols: Retention Behavior, Paleologou, M., S. Li, Purdy, W.C., J. Chrom. Sci., Vol. 28, 311-318 (1990).

91. A Study of the Solvent Composition Effects on the Separation of Seven Clinically Important Porphyrins on Cyclodextrin Bonded Phases, Ho, J.W., J. of Liq. Chrom., 13(11), 2193-2205 (1990).

90. Effects of Mobile Phase Composition on the Reversed-Phase Separation of Dipeptides and Tripeptides with Cyclodextrin Bonded-Phase Columns, Chang, C.A., Ji, H., Lin, G., J. Chromatogr., 522, 143-152 (1990).

89. Retention of Benzo(a)pyrene on Cyclodextrin-Bonded Phases, Fielden, P.R., Packham, A.J., J. Chromatogr., 516, 355-364 (1990).

88. (S)-2-Hydroxypropyl-β-cyclodextrin, A New Chiral Stationary Phase for Reversed-Phase Liquid Chromatography, Stalcup, A.M., Chang, S., Armstrong, D.W., Pitha, J. J. Chromatogr., 513, 181-194 (1990).

87. Effect of Alcohol Chain Length, Concentration and Polarity on Separations in High-Performance Liquid Chromatography Using Bonded Cyclodextrin Columns, Atamna, I.Z., Muschik, G.M., Issaq, H.J., J. Chromatogr., 499, 477-488 (1990).

86. Liquid Chromatographic Retention Behavior and Separation of Chlorophenols on a β-Cyclodextrin Bonded Phase Column. Part III. Diaromatic Chlorophenols, Paleologou, M., Li, S., Purdy, W.C., Can. J. Chem., 68, 1208 (1990].

85. Cyclodextrin Chiral Stationary Phases for Liquid Chromatographic Separations of Drug Stereoisomers, Berthod, A., Jin, H.L., Beesley, T.E., Duncan, J.D., Armstrong, D.W., J. of Pharm. & Biomed. Anal., 8(2), 123-130 (1990).

84. Liquid Chromatographic Enantiomeric Resolution of Amino Acids with ß-cyclodextrin Bonded Phases and Derivatization with o-phthalaldehyde, Merino Merino, I., Blanco Gonzalez, E., Sanz-Medel, A., Anal. Chim. Acta, 234, 127-131 (1990).

83. High-Performance Liquid Chromatographic Separation of 3-[(Cyclopentyl-hydroxyphenyl-acetyl)oxy}-1,1-Dimethyl-Pyrrolidinium Bromide Diastereomers, Demian, J., Gripshover, D.F., J. of Liq. Chrom., 13(4), 779-787(1990).

82. Derivatized Cyclodextrins for Normal-Phase Liquid Chromatographic Separation of Enantiomers, Armstrong, D.W., Stalcup, A.M., Hilton, M.L., Duncan, J.D., Faulkner, J.R., Chang, S.C., Anal. Chem. 62, 1610-1615 (1990).

81. Displacement Chromatography on Cyclodextrin-Silicas. III. Enantiomer Separations, Vigh, G., Quintero, G., Farkas, G., J. Chromatogr. 506, 481-493 (1990).

80. Displacement Chromatography on Cyclodextrin-Silicas. II. Separation of cis-trans Isomers in the Reversed Phase Mode on α-Cyclodextrin Silica, Vigh, G., Farkas, G., Quintero, G., J. Chromatogr. 484, 251-257 (1989).

79. Displacement Chromatography on Cyclodextrin-Silicas. I. Separation of Positional and Geometrical Isomers in the Reversed Phase Mode, Vigh, G., Quintero, G., Farkas, G., J. Chromatogr. 484, 237-250 (1989).

78. Separation of Porphyrinson Cyclodextrin-Bonded Phases With a Novel Mobile Phase, Ho, J. W., J. Chromatogr. 508, 375-381 (1990).

51

77. High-Performance Liquid Chromatographic Resolution of Racemic 1,4-benzodiazepin-2-ones by Means of aβ-Cyclodextrin Silica Bonded Chiral Stationary Phase, Bertucci, C., Domenici, E., Uccello--Barretta, Salvadori, P., J. of Chromatogr. 506, 617-625 (1990).

76. Elution Order in Liquid Chromatography on Cyclodextrin Phases. Dependence on the Amount of Organic Modifier in the Eluent, Anderson, J. T., Kaiser, G., Fresenius Z. Anal. Chem., 749-751 (1989).

75. Stereoselective Metabolism and Pharmacokinetics of Racemic Methylphenobarbital in Humans, Lim, W. H., Hooper, W. D., Drug Metabolism and Disposition, Vol. 17(2), 212-217 (1989).

74. Separation of Enantiomers Using a γ-Cyclodextrin Liquid Chromatographic Bonded Phase, Stalcup, A.M.,Jin, H.L., Armstrong, D.W., J. of Liq. Chrom., 13(3), 473-484 (1990).

73. Direct Liquid Chromatographic Separation of Enantiomeric and Diastereomeric Terpenic Alcohols asβ-cyclodextrin Inclusion Complexes, Italia, A., Schiavi, M., Ventura, P., J. Chromatogr., 503, 266-271 (1990).

72. Evaluation of the Effect of Organic Modifier and pH on Retention and Selectivity in Reversed Phase Liquid Chromatographic Separation of Alkaloids on a Cyclodextrin Bonded Phase, Armstrong, D. W., Bertrand, G. L., Ward, K. D., Ward, T. J., Secor, H. V., Seeman, J. I., Anal. Chem., 62, 332-338 (1990).

71. Separation of Carotenes on Cyclodextrin Bonded Phases, Stalcup, A. M., Jin, H. L., Armstrong, D. W., J. Chromatogr., 499, 627-635 (1990).

70. Selective Determination of Benzo(α)pyrene in Petroleum Based Products Using Multi-Column Liquid Chromatography, Fielden, P.R., Packham, A.J., J. Chromatogr. 479, 117 (1989).

69. Separation of Homologous and Isomeric Alkaloids Related to Nicotine on a β-cyclodextrin-bonded Phase, Seeman, J.I., Secor, H.V., Armstrong, D.W., Ward, K.D., Ward, T.J., J. Chromatogr., 483, 169 (1989).

68. High-performance Liquid Chromatographic Separation of Urinary Hippuric and o-, m- and p-methylhippuric Acids with a β−cyclodextrin-bonded Column, Matsui, H., Sekiya, T., J. Chromatogr., 496, 189 (1989).

67. Chemically Bonded Cyclodextrin Stationary Phase for the High-performance Liquid Chromatographic Separation and Determination of Sulphonamides, Ahmed, A.H.N., El-Gizawy, S.M., Analyst, 114, 571 (1989).

66. Rapid High Performance Liquid Chromatographic Separation of Barley Malt α-Amylase on Cyclobond Columns, Henson, C.A., Stone, J.M., J. Chromatogr., 469, 361 (1989).

65. High Performance Liquid Chromatographic Determination of Ibuprofen, its Metabolites and Enantiomers in Biological Fluids, Geisslinger, G., Dietzel, K., Lowe, D., Schuster, O., Lachman, G., Rau, G., Brune, K., J.Chromatogr., 491, 139 (1989).

64. Molecular Modeling of Cyclodextrin-Guest Molecular Interactions, Arnold, E.N., Lillie, T.S. and Beesley, T.E., Arnold, E.N., Lillie, T.S. and Beesley, T.E., J. Liq. Chrom., 12(3), 337 (1989).

63. Determination of 2,6- and 4,6-Dinitrocresols by High Performance Liquid Chromatography on a β-Cyclodextrin Bonded Column, Tripathi, A.M., Mhalas, J.G., Rama Rao, N.V., J. Chromatogr., 466, 442 (1989).

62. Liquid Chromatographic Separation of Anomeric Forms of Saccharides with Cyclodextrin Bonded Phases, Armstrong, D.W., Jin, H.L., Chirality, 1, 27 (1989).

61. Evaluation of the Liquid Chromatographic Separation of Monosaccharides, Disaccharides, Trisaccharides, Tetrasaccharides, Deoxysaccharides and Sugar Alcohols with Stable Cyclodextrin Bonded Phase Columns, Armstrong, D.W., Jin, H.L., J. Chromatogr., 462, 219 (1989).

60. Liquid Chromatographic Separation of Positional Isomers of Suprofen on a Cyclodextrin Bonded Phase, Marziani, F.C., Sisco, W.R., J. Chromatogr., 465, 422 (1989).

52

59. Separation on Cyclodextrins Using Cyclodextrin Bonded Phases, Jin, H.L., Stalcup, A.M., Armstrong, D.W., J. Liq. Chrom., 11(16), 3295 (1988).

58. Effect of Column Dimensions on HPLC Separations Using Constant Volume Columns, Issaq, H.J., Janini, G.M., Schultz, N., Marzo, L., Beesley, T.E., J. Liq. Chrom., 11(16), 3335 (1988).

57. High Performance Liquid Chromatographic Determination of 2',3'-Dideoxycytidine and 3'-Azido-3'-deoxythymidine in Plasma Using A Column Switching Technique, Mathes, L.E., Muschik, G., Demby, L., Polas, P., Mellini, D.W., Issaq, H.J., and Sams, R., J. Chromatogr., 432, 346 (1988).

56. Chiral Resolution of a Series of 3-Thienylcyclohexylglycolic Acids by Liquid or Subcritical Fluid Chromatography, A Mechanistic Study, Macaudiere, P., Caude, M., Rosset, R., Tambute, A., J. Chromatogr., 450, 255 (1988).

55. The Multimodal Cyclodextrin Bonded Stationary Phases for High Performance Liquid Chromatography, Issaq, H.J., J. Liq. Chrom., 11(9&10), 2131 (1988).

54. High Performance Liquid Chromatography Separations of Nitrosamines. III. Conformers of N-Nitrosamino Acids, Issaq, H.J., Williams, D.G., Schultz, N., Saavedra, J.E., J. Chromatogr., 452, 511 (1988).

53. Enantiomeric Resolution and Chiral Recognition of Racemic Nicotine and Nicotine Analogues by β-Cyclodextrin Complexation. Structure-Enantiomeric Resolution Relationships in Host-Guest Interactions, Seeman, J.I., Secor, H.V., Armstrong, D.W., Timmons, K.D., Ward, T.J., Anal. Chem., 60, 2120 (1988).

52. The Influence of Mobile Phase Alcohol Modifiers on HPLC of Polycyclic Aromatics Using Bonded Phase Cyclodextrin Columns, Tarr, M.A., Nelson, G., Patonay, G., and Warner, I.M., Analy. Letters, 21(5) 843 (1988).

51. Improvement in the Fluorimetric Detection of 5- Methoxypsoralen by Using β-Cyclodextrin in the Mobile Phase anda Cross-linked β-Cyclodextrin Column, Cepeda-Saez, A., Prognon, P., Mahuzier, G., Blais, J., Analy. Chim. Acta, 211, 333 (1988).

50. Direct HPLC Resolution of Racemic Nomifensine Hydrogen Maleate Using a Chiral Beta-Cyclodextrin-Bonded Stationary Phase, Aboul-Enein, H.Y., Islam, M.R., and Bakr, S.A., J. Liq. Chrom., 11(7), 1485 (1988).

49. Structural Factors Affecting Chiral Recognition and Separation on β-Cyclodextrin Bonded Phases, Han, S.M., Han, Y.I., Armstrong, D.W., J. Chromatogr., 441, 376 (1988).

48. Enantiomeric Analysis of a New Anti-inflammatory Agent in Rat Plasma Using a Chiral β-Cyclodextrin Stationary Phase, Krstulovic, A.M., Gianviti, J.M., Burke, J.T., Mompon, B., J. Chromatogr., 426, 417 (1988).

47. Mixed Reversed Phase/Beta Cyclodextrin Packings in High Performance Liquid Chromatography: Single Mixed Support Column Versus Two Columns in Series, Issaq, H.J., Mellini, D.W., Beesley, T.E., J. Liq. Chrom., 11(2),333 (1988).

46. Simultaneous Quantitation of a d- and l-Hexobarbital in Rat Blood by High Performance Liquid Chromatography, Chandler, M.H.H., Guttendorf, R.J., Blouin, R.A., Wedlund, P.J., J. Chromatogr., 419, 426 (1987).

45. Liquid Chromatographic Resolution of Enantiomers Containing Single Aromatic Rings with β-Cyclodextrin Bonded Phases, Armstrong, D.W., Han, Y.I., Han, S.M., Analy. Chim. Acta, 208, 275 (1988).

44. Separation of Optical Isomers of Scopolamine, Cocaine, Homatropine, and Atropine, Armstrong, D.W., Han, S.M.,Han, Y.I., Anal. Biochem., 167, 261 (1987).

43. Chromatography of B Prostaglandins on β-Cyclodextrin Silica: Application to Analysis of Major E Prostaglandins in Human Seminal Fluid, Oliw, E.H., J. Chromatogr., 421, 117 (1987).

42. Analysis of Nasal Solutions Containing Phenylephrine Hydrochloride and Pheniramine Maleate by High Performance Liquid Chromatography on a Cyclodextrin Bonded Stationary Phase and Diode Array Spectrophotometry, Pereira-Rosario, R., El-Gizaway, S., Perrin, J.H., Riley, C.M., Drug Dev. and Ind. Pharm., 12(14), 2443 (1986).

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41. Resolution or Racemic Amides and Phosphine Oxides on a β-Cyclodextrin-Bonded Stationary Phase by Subcritical Fluid Chromatography, Macaudiere, P., Caude, M., Rosset, R., and Tambute, A., J. Chromatogr., 405, 135 (1987).

40. Chiral-Phase High Performance Liquid Chromatography of Rotenoid Racemates, Abidi, S.L., J. Chromatogr., 404, 133 (1987).

39. High Performance Liquid Chromatography Using β- Cyclodextrin Bonded Silica Column: Effect of Temperature on Retention, Issaq, H.J., Glennon, M.L., Weiss, D.E., Fox, S.D., Ordered Media and Chemical Separations, Hinze, W.L. and Armstrong, D.W., Editors, ACS Sym. Series #342, Wash., DC, 260-271 (1987).

38. Purification of Chorismate, Prephenate, and Arogenate by HPLC, Connelly, J.A., Siehl, D.L., Methods in Enzymology, 142, 422 (1987).

37. Comparison of Liquid Chromatographic Separations of Geometrical Isomers of Substituted Phenols with β- and γ- Cyclodextrin Bonded Phases, Chang, C.A., Wu, Q., Analy. Chim. Acta, 189, 293 (1986).

36. Facile Liquid Chromatographic Separation of Positional Isomers with a γ-Cyclodextrin Bonded Phase Column, Chang, C.A., Wu, Q. , J. Liq. Chrom., 10(7), 1359 (1987).

35. Bonded Cyclodextrin Stationary Phase Columns for the Separation of Cis/Trans Cyclohexane Derivatives, Tindall,G.W., J. Liq. Chrom., 10, 1077 (1987).

34. Direct Liquid Chromatographic Separation of Racemates with an α-Cyclodextrin Bonded Phase, Armstrong, D.W., Yang, X., Han, S.M., Menges, R.A., Anal. Chem., 59, 2594 (1987).

33. Cyclodextrin Cavity Polarity and Chromatographic Implications, Street, Jr., K.W., J. Liq. Chrom., 10, 655 (1987).

32. Separation of Tamoxifen Geometric Isomers and Metabolites by Bonded Phase β-Cyclodextrin Chromatography, Armstrong, R.D., Ward, T. J., Pattabiraman, N., Benz, C., Armstrong, D.W., J. Chromatogr., 414, 192 (1987).

31. High Performance Liquid Chromatographic Separation of Peptide and Amino Acid Stereoisomers, Florance, J., Galdes, A., Konteatis, Z., Kosarych, Z., Langer, K., Martucci, C., J. Chromatogr., 414, 313 (1987).

30. Optimization of Liquid Chromatographic Separations on Cyclodextrin Bonded Phases, Armstrong, D.W., Li, W., Chromatography, March, 43-48 (1987).

29. Some Structural Requirements for Resolution of Hydantoin Enantiomers with a β-Cyclodextrin Liquid Chromatography Column, Maguire, J.H., J. Chromatogr., 387, 453 (1987).

28. Separation of Cis-Trans Isomers of Prostaglandins with a Cyclodextrin Bonded Column, Snider, B.G.,J. Chromatogr., 351, 548 (1986).

27. High Performance Liquid Chromatographic Determination of the Enantiomeric Composition of Urinary Phenolic Metabolites of Phenytoin, McClanahan, J.S., Maguire, J.H., J. Chromatogr., 381, 438 (1986).

26. Liquid Chromatography of Hydrocarbonaceous Quaternary Amines on Cyclodextrin Bonded-Silica, Abidi, S.L.,J. Chromatogr., 362, 33 (1986).

25. High Performance Liquid Chromatography Separations of Nitrosamines. II. Acyclic Nitrosamines, Issaq, H.J., Glennon, M., Weiss, D.E., Chmurny, G.N., Saavedra, J.E., J. Liq. Chrom., 9(12), 2763 (1986).

24. Normal Phase High Performance Liquid Chromatographic Separations of Positional Isomers of Substituted Benzoic Acids with Amine and β-Cyclodextrin Bonded Phase Columns, Chang, C.A., Wu, Q., Tan, L., J. Chromatogr., 361, 199 (1986).

23. High Performance Liquid Chromatographic Separations of Nitrosamines. I. Cyclic Nitrosamines, Issaq, H.J., McConnel, J.H., Weiss, D.E., Williams, D.G., Saavedra, J.E., J. Liq. Chrom., 9(8),1783 (1986).

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22. The Determination of Aspartame in Diet Soft Drinks by High Performance Liquid Chromatography, Issaq, H.J., Weiss, D., Ridlon, C., Fox, S.D., Muschik, G.M., J. Liq. Chrom., 9(8) 1791 (1986).

21. Applications and Limitations of Commercially Available Chiral Stationary Phases for High Performance Liquid Chromatography, Dappen, R., Arm, H., Myer, V.R., J. Chromatogr.,373, 1 (1986).

20. Coupled β-Cyclodextrin and Reversed-Phase High Performance Liquid Chromatography for Assessing Biphenyl Hydroxylase Activity in Hepatic 9000g Supernatant, Weaver, D.E., van Lier, R.B.L., Analy. Biochem., 154, 590 (1986).

19. Separation of Drug Stereoisomers by the Formation of β-Cyclodextrin Inclusion Complexes, Armstrong, D.W., Ward, T.J., Armstrong, R.D., and Beesley, T.E., Science, 232, 1132 (1986).

18. Reversed Phase High Performance Liquid Chromatographic Separation of Substituted Phenolic Compounds with a β-Cyclodextrin Bonded Phase Column, Chang, C.A., Wu, Q., Armstrong, D.W., J. Chromatogr., 354, 454 (1986).

17. Separation of Selected Dipeptides by High Performance Liquid Chromatography, Issaq, H.J., J. Liq. Chrom., 9(1),229 (1986).

16. Separation of Steroid Epimers and Isomers Using Cyclodextrin HPLC Columns, Kirschbaum, J., Kerr, L., LC Magazine, 4, 30 (1986).

15. Improved Cyclodextrin Chiral Phases: A Comparison and Review, Ward, T.J., Armstrong, D.W., J. of Liq. Chrom., 9(2&3), 407-423 (1986).

14. Liquid Chromatographic Retention Behavior of Organometallic Compounds and Ligands With Amine-, Octadecyl-Silica- and β-Cyclodextrin Bonded-Phase Columns, Chang, C.A., Abdel-Aziz, H., Melchor, N., Wu, Q., Pannell, K.H., J. Chromatogr., 347, 51-60 (1985).

13. Synthesis, Rapid Resolution, and Determination of Absolute Configuration of Racemic 2,2'-Binaphthyldiyl Crown Ethers and Analogues via β-Cyclodextrin Complexation, Armstrong, D.W., Ward, T.J., Czech, A. Czech, B.P., Bartsch, R.A., J. Org. Chem., 50 (26), 5556-5559 (1985).

12. Inclusion Complexing: A New Basis for HPLC Selectivity, Beesley, T.E., Am. Lab., May,78-87 (1985).

11. Chiral Phase Separations - An Update, Fisher, C.M., Chrom. International, 8, 38 (1985).

10. Liquid Chromatographic Separation of Diastereomers and Structural Isomers on Cyclodextrin-Bonded Phases, Armstrong, D.W., DeMond, W., Alak, A., Hinze, W.L., Riehl, T.E., Bui, K.H., Anal. Chem, 57, 234 (1985).

09. Separation of Mycotoxins, Polycyclic Aromatic Hydrocarbons, Quinones, and Heterocyclic Compounds on Cyclodextrin Bonded Phases: An Alternative LC Packing, Armstrong, D.W., Alak, A., DeMond, W., Hinze, W.L., Riehl, T.E., J. Liq. Chrom., 8(2), 261-269 (1985).

08. Separation of Metallocene Enantiomers by Liquid Chromatography: Chiral Recognition via Cyclodextrin Bonded Phases, Armstrong, D.W., DeMond, W., Czech, B.P, Anal. Chem., 57, 481-484 (1985).

07. Liquid Chromatographic Separation of Enantiomers Using a Chiral β-Cyclodextrin-Bonded Stationary Phase and Conventional Aqueous-Organic Mobile Phases, Armstrong, D.W., DeMond, W., Alak, A., Hinze, W.L., Riehl, T.E., Ward, T., Anal. Chem., 57, 237 (1985).

06. Facile Separation of Enantiomers, Geometrical Isomers, and Routine Compounds on StableCyclodextrin LC Bonded Phases, Armstrong, D.W., Alak, A., Bui, K., DeMond, W., Ward, T., Riehl, T.E., Hinze, W.L., J. Inclus. Phenomena, 2, 533 (1984).

05. New HPLC Column Technology: Inclusion Complexing, Fisher, C. M., Chromatography International, Issue 5,10-14 (1984).

04. Chiral Stationary Phases for High Performance Liquid Chromatographic Separation of Enantiomers: A Mini Review, Armstrong, D.W., J. Liq. Chrom., 7(S-2), 353 (1984).

55

03. Cyclodextrin Bonded Phases for the Liquid Chromatographic Separation of Optical, Geometrical, and Structural Isomers, Armstrong, D.W., DeMond, W., J. Chrom. Sci., 22, 411 (1984).

02. HPLC Inclusion Complexing - An Update, THE ASTEC INFORMER, Vol. 4, Issue 2, (1984).

01. New HPLC Column Technology: Inclusion Complexing, THE ASTEC INFORMER, Volume 4, Issue 1 (1984).

For the most up to date information, visit our website atwww.astecusa.com.

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Ordering Information

Where to Order

Advanced Separation Technologies Inc.37 Leslie Court, Post Office Box 297Whippany, NJ 07981 USATel: (973) 428-9080, Fax: (973) 428-0152E-mail: [email protected] Or your local Astec distributor

In the United Kingdom and Ireland Only:Advanced Separation Technologies Ltd.1 Blake Street, Congleton, Cheshire CW12 4DS UKTel: 01260 276276, Fax: 01260 290067E-mail: [email protected]

When Ordering

Please specify shipping address, billing address,catalog number, product description, quantity andprice. A purchase order number is required.

Terms

All prices are f.o.b. Whippany, NJ USA.Payment terms are net 30 days.Prices are subject to change without notice.

Shipping Instructions

All items are shipped via United Parcel Service (UPS)unless otherwise specified.

Returns

Product returns are accepted with prior authorizationonly. Please contact our Customer Service Departmentfor instructions. A 15% restocking charge is applied forproducts returned due to customer error.

Warranty

All Astec products are warranted to freedom of defectsin material and workmanship. We will replace or repairwithout cost any materials which carry such defects.

Availability

All CYCLOBOND phases are available in semi-preparative and preparative scale columns with columndiameters up to 4" I.D. Preparative columns areavailable in 2"x25cm, 2"x 50cm and 4"x25cm packedwith 10µm or 16µm CYCLOBOND media. For prices andavailability please contact our Sales Department.

CYCLOBOND I 2000 (beta) HPLC COLUMNS

MICROBORE CYCLOBOND I 2000 COLUMNS - 5µm

Phase TypeMicrobore Guard

Column2cmx1.0mm

100x2.0mm 150x2.0mm 250x2.0mm

CYCLOBOND I 2000 21010 20018 20019 20020CYCLOBOND I 2000 AC 21011 20118 20119 20120CYCLOBOND I 2000 SP 21012 20218 20219 20220CYCLOBOND I 2000 RSP 21013 20318 20319 20320CYCLOBOND I 2000 SN 21015 20518 20519 20520CYCLOBOND I 2000 RN 21016 20618 20619 20620CYCLOBOND I 2000 DMP 21017 20718 20719 20720CYCLOBOND I 2000 DM 21019 20918 20919 20920

ANALYTICAL CYCLOBOND I 2000 COLUMNS - 5µm

Phase TypeGuard Cartridge*

2cmx4.0mm 50x4.6mm 100x4.6mm 150x4.6mm 250x4.6mm 500x4.6mm

CYCLOBOND I 2000 21100 20021 20022 20023 20024 20026CYCLOBOND I 2000 AC 21101 20121 20122 20123 20124 20126CYCLOBOND I 2000 SP 21102 20221 20222 20223 20224 20226CYCLOBOND I 2000 RSP 21103 20321 20322 20323 20324 20326CYCLOBOND I 2000 SN 21105 20521 20522 20523 20524 20526CYCLOBOND I 2000 RN 21106 20621 20622 20623 20624 20626CYCLOBOND I 2000 DMP 21107 20721 20722 20723 20724 20726CYCLOBOND I 2000 DM 21109 20921 20922 20923 20924 20926Guard Column Holder 21150

57

SEMI-PREPARATIVE CYCLOBOND I 2000 COLUMNS - 5µm

Phase Type 250x10mm 500x10mm 250x22.1mm 500x22.1mm

CYCLOBOND I 2000 20034 20036 20044 20046CYCLOBOND I 2000 AC 20134 20136 20144 20146CYCLOBOND I 2000 SP 20234 20236 20244 20246CYCLOBOND I 2000 RSP 20334 20336 20344 20346CYCLOBOND I 2000 SN 20534 20536 20544 20546CYCLOBOND I 2000 RN 20634 20636 20644 20646CYCLOBOND I 2000 DMP 20734 20736 20744 20746CYCLOBOND I 2000 DM 20934 20936 20944 20946

CYCLOBOND I 2000 HPLC COLUMNS - 10µm "Scout Columns" for Loading Studies for PreparativeSeparations

Phase Type 250x4.6mm

CYCLOBOND I 2000 22024CYCLOBOND I 2000 AC 22124CYCLOBOND I 2000 SP 22224CYCLOBOND I 2000 RSP 22324CYCLOBOND I 2000 SN 22524CYCLOBOND I 2000 RN 22624CYCLOBOND I 2000 DMP 22724CYCLOBOND I 2000 DM 22924

CYCLOBOND II (gamma) and CYCLOBOND III (alpha) HPLC COLUMNS

MICROBORE CYCLOBOND II and III COLUMNS - 5µm

Phase TypeMicrobore Guard

Column2cmx1.0mm

100x2.0mm 150x2.0mm 250x2.0mm

CYCLOBOND II 41001 46018 46019 41021CYCLOBOND II Ac 41002 47018 47019 41024CYCLOBOND III 41005 48018 48019 41031CYCLOBOND III Ac 41006 49018 49019 41034

ANALYTICAL CYCLOBOND II and CYCLOBOND III COLUMNS - 5µm

Phase TypeGuard Cartridge*

2cmx4.0mm 50x4.6mm 100x4.6mm 150x4.6mm 250x4.6mm 500x4.6mm

CYCLOBOND II 42120 46021 40020 46023 41020 40023CYCLOBOND II Ac 42121 47021 47022 47023 41022 41023CYCLOBOND III 42130 48021 40030 48023 41030 40033CYCLOBOND III Ac 42131 49021 49022 49023 41032 41033Guard Column Holder 21150

SEMI-PREPARATIVE CYCLOBOND II and CYCLOBOND III COLUMNS - 5µm

Phase Type 250x10mm 500x10mm 250x22.1mm 500x22.1mm

CYCLOBOND II 40025 40026 40028 41420CYCLOBOND II Ac 40420 40421 40422 41450CYCLOBOND III 40035 40036 40038 41430CYCLOBOND III Ac 40430 40431 40432 41460

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CYCLOBOND II AND III HPLC COLUMNS - 10µm "Scout Columns" for Loading Studies for PreparativeSeparations

Phase Type 250x4.6mm

CYCLOBOND II 44024CYCLOBOND II AC 44124CYCLOBOND III 48121CYCLOBOND III AC 49121

PREPARATIVE COLUMNS AND MEDIA

CYCLOBOND phases are also available in 2" and 4" prepacked columns and bulk 10µm media for preparativeseparations. We also offer contract services for the preparation of purified enantiomers in quantities up to 1 kilogram.Please contact our Sales Department for specific information.

*GUARD COLUMNS

Astec CYCLOBOND guard column system is cartridge type and precision manufactured from 316 stainless steel. Theinlet and outlet of holder connections are made using standard capillary tubing and fittings. Cartridges are packed with5µm materials.

GUARD COLUMN SYSTEM

The Astec guard column system is cartridge type and precision manufactured from 316 stainless steel. The inlet andoutlet of holder connections are made using standard capillary tubing and fittings. Cartridges are packed with 5µmmaterials.

Guard Cartridge Holder

Guard Cartridge Assembly

Advanced Separation Technologies Inc. presents the CYCLOBOND HANDBOOK as an aid to the successful use ofthese products. Exceptions are possible. All statements herein are expressions of opinion which we believe to beaccurate and reliable, but are presented without guarantee or responsibility on our part. While Advanced SeparationTechnologies Inc. has used its best efforts to present useful instructions, no warranty expressed or implied is given withrespect to this material or the products recommended herein.

6th Edition 2002 Advanced Separation Technologies Inc.

All rights reserved.Printed in USA.

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CYCLOBOND™ Handbook - Sigma-Aldrich

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