Effective Organic Compound Purification: Guidelines and Tactics for Flash Chromatography

162 Pages·01.611 MB·English

by  Teledyne Isco, Inc.

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Fourth Edition
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Guidelines & Tactics for
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Teledyne Isco • 4700 Superior Street • Lincoln, NE 68504 • USA
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Publication L-5005 • ©2003, 2005, 2008, 2010 • Printed in the U.S.A.
EffectiveOrganicCompoundPurification
EffectiveOrganicCompoundPurification:
GuidelinesandTacticsforFlashChromatography
FourthEdition
©2003,2005,2008,2010TeledyneIsco,Inc.Allrightsreserved.
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RediSepRf®andRediSepRfGold® i
EffectiveOrganicCompoundPurification
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Contents
Chapter 1
Introduction to Flash Chromatography
ChromatographicPurificationinOrganicChemistry....... 1
Chapter 2
Flash Chromatography Essentials
CompoundSolubility.................................. 4
MobilePhase ........................................ 5
MobilePhaseModifiers ............................... 7
StationaryPhase ..................................... 8
UsingTLCtoPredictSeparation........................ 8
CorrelatingTLCandFlash........................... 8
RetentionFactorandColumnVolumes................ 8
MethodDevelopmentUsingTLC.................... 10
TLCandMobilePhaseTechniques.................. 13
IsocraticElution..................................... 14
GradientElution..................................... 17
SteppedGradient................................. 18
LinearGradient................................... 19
MixedGradients..................................... 21
LoadingCapacityofColumn .......................... 24
ColumnLengthVersusResolutionandPurity............ 26
FlashColumnPackings............................... 26
ParticleShape.................................... 26
ParticleSize...................................... 27
SampleLoadingTechniques .......................... 28
ManualGlassChromatography ..................... 28
AutomatedChromatography ....................... 28
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Chapter 3
From Traditional Glass Columns to
Automated Flash Chromatography
ManualGlassColumnChromatography .................35
BenefitsofAutomation ...............................37
ColumnPacking .....................................38
Manually-packedColumns..........................38
Pre-packedColumns...............................40
TLCPlates........................................42
HighPerformanceFlashChromatography ...............44
ColumnStacking ..................................44
Whysphericalmedia?..............................48
HigherResolutionwithsmallsphericalmedia .........48
Improvedloadcapacity ............................50
Fasterpurifications................................52
Chapter 4
C18 Flash Chromatography
OverviewofReversedPhaseChromatography ...........54
NormalPhaseSilica................................54
ReversedPhaseSilica..............................55
C18MethodDevelopment.............................57
ThinLayerChromatographyPlates ..................57
UsingHPLCSystemstoGenerateFlashMethods.......58
UsingtheFlashInstrumentforMethodDevelopment...59
LoadingCompounds .................................61
ColumnCare ........................................61
SolventModifiers ....................................62
Low-solubilityPolarHeterocycles......................64
PrimaryAmines .....................................65
Carbohydrates ......................................66
Peptides............................................67
CarboxylicAcids.....................................68
IonicCompounds....................................69
RediSepRfGoldHighPerformanceC18Columns .........70
RediSepGoldC18ColumnsatHighpH ..................73
StorageofthecolumnafteruseinhighpH............73
Chapter 5
Advanced Flash Chromatography
AlternativeChromatographicMedia....................75
SpecialtyMedia......................................75
Amine ...........................................76
BasicAlumina ....................................81
iv TeledyneIsco,Inc.
EffectiveOrganicCompoundPurification
Contents
NeutralAlumina.................................. 84
Cyano........................................... 87
Diol............................................. 89
IonExchangeMedia ................................. 93
SCX............................................. 93
SAX............................................. 96
NaturalProducts................................... 100
CytotoxicConstituentsfromButeasuperba.......... 102
AlkaloidsofBanisteriacaapi....................... 103
AdvancedSolventStrategies......................... 105
Chapter 6
Detection Techniques
UVDetection ...................................... 111
DetectionwithUV-Vis............................... 112
All-WavelengthDetection............................ 113
Examplewithacompoundmixture................. 114
Exampleofunknownspectrum .................... 114
Solventspectrumoverlapscompound.............. 116
Sampleoverloadsdetector........................ 117
OtherDetectors.................................... 118
Appendix A
Column Media Selection
MediaSelection.................................... 119
Appendix B
Solvent and UV-Vis Wavelength
Selection Guide
SolventSelection................................... 134
WavelengthSelection............................... 134
Appendix C
Theory & Application of Flash
Chromatography
Elementarytheory.................................. 140
Application........................................ 142
Appendix D
Troubleshooting LC Systems
Basicchecklist..................................... 145
Troubleshooting ................................... 145
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EffectiveOrganicCompoundPurification
List of Figures
1 IllustrationofbasicelementsinatraditionalFlash
columnchromatographyapparatus....................2
2 PhotoofTLCplate...................................5
3 Tableofcommonsolventsinliquidchromatography .....6
4 Illustrationofmobilephasemodifier ...................7
5 TableofRftoCVconversions .........................9
6 Illustrationofsolventstrengthoptimization............10
7 Illustrationofsolventsystemselectivityoptimization....11
8 Illustrationofasolventsystemoptimizedforcompound
selectivity .........................................11
9 TableofsuggestedloadingofRediSepRfsilicagel
columnsbasedonRfdifferencesfromTLCplates........12
10 Illustrationofmobilephasetechniques................13
11 Illustrationofisocratic20%EtOAcinhexane ...........15
12 Illustrationofisocratic30%EtOAcinhexane ...........15
13 Illustrationofisocratic40%EtOAcinhexane ...........16
14 Illustrationofisocratic50%EtOAcinhexane ...........16
15 Illustrationofisocratic70%EtOAcinhexane ...........17
16 Illustrationofasteppedgradientandchromatogram ....19
17 Illustrationofalineargradientandchromatogram ......20
18 Chromatogramsresultingfromvariousgradientslopes ..21
19 Chromatogramsofcatecholandresorcinolseparations..22
20 IllustrationofCombiFlashRfGradientOptimizerand
resultingchromatogram.............................23
21 Chromatogramindicatingcolumnloadingcapacity
nearlimit..........................................24
22 Chromatogramindicatingcolumnloadingcapacity
exceeded..........................................25
23 Illustrationofgradientmobilephase ..................25
24 Photosofparticleshapes............................26
25 Illustrationofsampleinjectiononglasscolumns........29
26 Photoofsyringeinjection............................29
27 Photoofasolidsampleloadcartridge.................30
28 Photoofsolidloadcartridgeconnectedtocolumn ......31
29 Chromatogramsofsyringeinjectionanddriedsolid
loadcartridgetechniques. ...........................32
30 Photoofpre-packedcartridges.......................33
31 Photoofdryloadingsampleontothecolumn...........33
32 TableofRediSepRfsolidloadcartridges...............34
33 PhotoofamanualFlashsysteminuse.................35
34 PhotoofTeledyneIsco’sCombiFlashRfsystem.........37
35 Photoofglasscolumnpreparation....................39
36 Chromatogramsofcompoundsseparatedona
hand-packedcolumn................................40
vi TeledyneIsco,Inc.
EffectiveOrganicCompoundPurification
Contents
37 Photoofpre-packedcolumns........................ 41
38 PhotoofmatchingTLCmedia........................ 42
39 TableofRediSepRfTLCplates....................... 42
40 Tableofsolventmigrationandplatedevelopmenttime
forRediSepC18TLCplates.......................... 43
41 Tableofsolventmigrationandplatedevelopmenttime
forRediSepBasicandNeutralAluminaTLCplates...... 43
42 Chromatogramsshowingimprovedresolution ......... 45
43 Chromatogramscomparingasinglecolumn ........... 46
44 Chromatogramoftwo24gstackedcolumnscompared.. 47
45 Chromatogramofbromotoluenespurification.......... 48
46 Chromatogramofminorcompoundseparation......... 49
47 Diagramofsampleloadcomparison .................. 50
48 Chromatogramsof3-(2-nitrophenylamino)propionitrile
purifications....................................... 51
49 TableofRediSepRfGoldSilicaGel.................... 53
50 Diagramofnormalphasesilica....................... 54
51 Diagramofreversedphasesilica ..................... 55
52 TableofRediSepRfC18ReversedPhasecolumns....... 56
53 Tableofsolventmigrationandplatedevelopmenttime
forRediSepC18TLCplates.......................... 57
54 Illustrationofgallicacidandpyrogallolmethod ........ 58
55 Illustrationofmethyl-andpropyl-parabenmethod...... 60
56 Diagramofinterconversionofdiphenylaceticacid ..... 63
57 Illustrationofesculin/diphenylaceticacidpurifications . 63
58 Chromatogramofquinoxalinemixturepurification ..... 64
59 Chromatogramofprimaryaminemixturepurification... 65
60 Chromatogramofcarbohydratemixturepurification.... 66
61 Chromatogramofpeptidemixturepurification......... 67
62 Chromatogramofcarboxylicacidmixturepurification .. 68
63 Chromatogramofionicmixturepurification ........... 69
64 Chromatogramof10mgcompoundApurification ...... 71
65 Chromatogramof46mgcompoundApurification ...... 71
66 Chromatogramof10mgcompoundApurificationon
aWatersDeltaPrep4000system...................... 72
67 IllustrationofanalyticalHPLC........................ 72
68 TableofReusableRediSepRfGoldC18ReversedPhase
columns,20–40microns............................. 74
69 Diagramoffunctionalizedmedia ..................... 76
70 Diagramofaminestructure.......................... 76
71 Chromatogramofnormalphasecolumnelutionwith
hexane/ethylacetate ............................... 78
72 Chromatogramofaminefunctionalizedcolumnwith
hexane/ethylacetate ............................... 78
73 TableofReusableRediSepRfAmineColumns.......... 79
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EffectiveOrganicCompoundPurification
74 TableofReusableRediSepRfGoldAmineColumns......80
75 Chromatogramofnormalphasesilicacolumn ..........81
76 Chromatogramofbasicaluminacolumn ...............82
77 Tableofsolventmigrationandplatedevelopmenttime
forRediSepBasicAluminaTLCplates .................82
78 TableofRediSepRfAluminaBasicColumns ............83
79 Chromatogramofnormalphasesilicacolumn ..........84
80 Chromatogramofneutralaluminacolumn .............85
81 TableofRediSepRfAluminaNeutralColumns ..........86
82 TableofRediSepRfAluminaAcidicColumns ...........86
83 Diagramofcyanostructure ..........................87
84 TableofReusableRediSepRfGoldCyanoColumns......88
85 Diagramofdiolstructure ............................89
86 Purificationofoleylglycerate ........................90
87 Purificationoftocopherolsfromcornoil...............91
88 Purificationofgreenteaextract ......................91
89 TableofReusableRediSepRfGoldDiolColumns........92
90 DiagramofSCXstructure............................93
91 Chromatogramofnormalphasecolumn ...............94
92 ChromatogramofSCXcolumn........................95
93 TableofReusableRediSepRfSCXColumns.............95
94 DiagramofSAXstructure............................96
95 Chromatogramofnormalphasecolumn ...............97
96 ChromatogramofSAXcolumn........................97
97 TableofReusableRediSepRfSAXColumns.............99
98 Photoofcolumnmount.............................101
99 DiagramofcompoundsextractedfromButeasuperba .. 102
100 Illustrationofharmineandharmalineseparationona
silicagelcolumn...................................104
101 Illustrationofharmineandharmalineseparationona
RediSepaminecolumn .............................104
102 Diagramofsolventselectivity .......................105
103 ChartofUVspectraofethylacetateandacetone. ......107
104 ChartofUVabsorbanceof3-(2-nitrophenylamino)
propionitrile......................................108
105 Chromatogramof3-(2-nitrophenylamino)propionitrile
purificationinhexane/ethylacetate..................109
106 Chromatogramof3-(2-nitrophenylamino)propionitrile
purificationinhexane/acetone ......................109
107 Chartofstigmasterolabsorbance....................110
108 Chromatogramofstigmasterolpurification............110
109 ChartofUVabsorbanceofcatechin..................112
110 Chromatogramofhairdyecompoundspurification.....113
111 Chromatogramshowingdetectionofchlorophyll,
catechinsandcaffeine,andtannins ..................114
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EffectiveOrganicCompoundPurification
Contents
112 Chromatogramshowingdetectionofcatechin......... 115
113 DiagramshowingUVabsorptionofvariouscompounds 115
114 Chromatogramofglucosepentaacetatepurification ... 116
115 Chromatogramshowingpurificationofclosely-eluting,
saturatedpeaks................................... 117
116 ChromatogramshowingELSDdetectionof
2,3-O-isopropylidene-D-ribofuranose................. 118
117 Chartforcolumnmediaselection ................... 120
118 TableofRediSepRfGoldSilicaGelDisposableFlash
Columns,20–40microns ........................... 121
119 TableofRediSepRfSilicaGelDisposableFlash
Columns,40–60microns ........................... 122
120 TableofReusableRediSepRfGoldC18ReversedPhase
columns,20–40microns............................ 123
121 TableofReusableRediSepRfC18ReversedPhase
columns,40–60microns............................ 124
122 TableofReusableRediSepRfGoldAmineColumns,
20–40microns.................................... 125
123 TableofReusableRediSepRfAmineColumns,
40–60microns.................................... 126
124 TableofReusableRediSepRfGoldCyanoColumns,
20–40microns.................................... 127
125 TableofReusableRediSepRfSAXColumns........... 128
126 TableofReusableRediSepRfSCXColumns........... 129
127 TableofReusableRediSepRfGoldDiolColumns,
20–40microns.................................... 130
128 TableofRediSepRfAluminaAcidicColumns.......... 131
129 TableofRediSepRfAluminaNeutralColumns......... 132
130 TableofRediSepRfAluminaBasicColumns........... 133
131 Tableofliquidchromatographysolventsandtheir
characteristics.................................... 135
132 ChartofSolventMiscibility......................... 137
133 Tableofcompoundabsorbancewavelengths ......... 138
134 Tableofcompoundabsorbancewavelengths ......... 139
135 Standardresolutioncharts ......................... 144
136 Tablefortroubleshootingpeakproblems............. 146
137 Tablefortroubleshootingbaselineproblems.......... 148
138 Tablefortroubleshootingrecoveryandretention
problems ........................................ 149
139 Tablefortroubleshootingpressureproblems......... 149
140 Tablefortroubleshootingleaks ..................... 150
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