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TSKgel Size Exclusion Chromatography Columns
Silica-based for protein analysis: Polymethacrylate-based for polar organic-soluble polymers analysis:TSKgel SW mAb TSKgel SW TSKgel Alpha TSKgel SWXL TSKgel SuperAW TSKgel SuperSW Polystyrene-divinylbenzene-based forPolymer-based for desalting: organic-soluble polymers analysis: TSKgel BioAssist DS Columns TSKgel HXL TSKgel HHR Polymethacrylate-based for water-soluble TSKgel SuperH polymers analysis: TSKgel SuperHZ TSKgel SuperMultiporeHZ TSKgel PW TSKgel PWXL TSKgel PWXL-CP TSKgel SuperMultiporePW
30Call customer service: 866-527-3587,
technical service: 800-366-4875
About: TSKgel PW Size Exclusion Columns
TSKgelPWcolumnsarecomposedofspherical,hydrophilicpolymethacrylatebeads.Particlesizesrangefrom12µmforthesmallerporesizecolumnsto17µmforthelargerporesizecolumns.StablefrompH2to12,TSKgelPWcolumnscanbeusedinmobilephasesofwaterorbuffer(upto20%methanol/80%aqueous)andcantoleratetemperaturesupto80˚C.
TheTSKgelPWcolumnlineconsistsofthefollowingcolumns:
• TSKgelG2000PW • TSKgelG2500PW • TSKgelG3000PW • TSKgelG4000PW • TSKgelG5000PW • TSKgelG6000PW • TSKgelGMPW
Themixedbedcolumn,TSKgelGMPW,hasanextendedlinearcalibrationrange,suitableforsampleswithabroadmolarmassdistribution,aswellasforunknownsamples.Theporevolumecanbeaccessedbypolymersranginginmolarmassfrom500to8.0×106Da.Byquicklycategorizingthemolarmassprofileofanunknownsample,thecolumnenablesafastselectionofthebestTSKgelPWcolumnforroutineanalysis.
Attributes and Applications
ProductattributesofalleightTSKgelPWcolumnsareshowninTable12.AllTSKgelPWcolumnshaveabasematerialofhydroxylatedpolymethacrylate,canbeusedinamaximumof20%organic,andareshippedinwater.ThemainapplicationareaforTSKgelPWcolumnsistheanalysisofwater-solublepolymers,suchascelluloses,acrylamides,glycols,dextrans,polyvinylalcohol,andoligosaccharides.TSKgelG2000PW,thelargerparticlesizeequivalentofTSKgelG-Oligo-PW,ismostsuitableforsemi-preparativeandpreparativeisolationofoligosaccharides.RepresentativeapplicationexamplesforthePWcolumnsareillustratedinTable13.ThecalibrationcurveforpolyethyleneglycolandoxidesfortheTSKgelPWcolumnsisshowninFigure32.
Table 12: Product attributes
TSKgel column
Particle size (mean)
Pore size (mean) Calibration range
G2000PW 12 µm 12.5 nmUp to 2,000 Da (polyethylene glycols and oxides)
G2500PW 12 µm and 17 µm <20 nm
Up to 3,000 Da (polyethylene glycols and oxides)
G3000PW 12 µm and 17 µm 20 nm
Up to 5.0 × 104 Da (polyethylene glycols and oxides)
G4000PW 17 µm 50 nmUp to 3.0 × 105 Da (polyethylene glycols and oxides)
G5000PW 17 µm 100 nmUp to 1.0 × 106 Da (polyethylene glycols and oxides)
G6000PW 17 µm >100 nmUp to 8.0 × 106 Da (polyethylene glycols and oxides)
GMPW 17 µm mixed pore sizes
500 - 8.0 × 106 Da (polyethylene glycols and oxides)
Table 13: Representative application examples for TSKgel PW columns
Classification Examples
1.Syntheticpolymers• Nonionic• Cationic• Anionic
•PEG,polyglycerin, polyacrylamide•Polyethyleneimine, polyvinylpyrolidine•Poly(sodiumacrylate), Poly(sodiumstyrene sulfonate)
2.Polysaccharidesandderivatives
•Standarddextran,clinical dextran,pullulan,inulin, heparin,chitosan•Carboxymethylcellulose
3.Verylargebiopolymers• Polynucleotides• Viruses• Proteins
•DNAfragments•TMV,SBMV,TBSV•Lipoprotein(VLDL,LDL), apoferritin,gelatin,sea wormchlorocruorin
4.Smallmolecules• Oligomers• Others
•oligosaccharides(dextran hydrolysate,cyclodoxtrin•hydrolysate),cyclodextrins•oligopeptides•oligonucleotides
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TSKgel Size Exclusion Chromatography Columns
Oligosaccharides
TSKgelPWcolumnsarerecommendedforpolysaccharideanalysisduetotheirabilitytoseparateawidemolarmassdistribution.Aneffectiveseparationoftheanionichydrophilicglucosaminoglycan,hyaluronicacid,isshownin Figure33onaTSKgelG6000PWandTSKgelG4000PWcolumninserieswitha0.2mol/Lsodiumchloridemobilephase.Toobtainshorteranalysistimeandsimilarresolution,werecommendusingTSKgelG3000PWXLandG4000PWXLcolumnsinseries.
Polymers
Sodiumpolyacrylate,ananionicpolymer,iseffectivelyseparatedontwoTSKgelGMPWcolumnsinFigure34.Theadditionof0.01mol/LNaNO3resultsinnormalelutionandpeakshapeovercomingtheionicrepulsionbetweentheanionicsampleandtheresin.
Retention volume (mL)
Column:
Elution:Flow Rate:Detection:
TSKgel PW columns: A. G2000PW, B. G2500PW, C. G3000PW, D. G4000PW, E. G5000PW, F. G6000PW, G. GMPW, all 7.5mm x 60cm distilled water1.0mL/minRI
Log
mol
ar m
ass
10 2015
102
103
104
105
106G
E F
C
D
A B
Figure 32: Polyethylene glycol and oxide calibration curves for TSKgel PW columns
Column: A. G2000PW B. G2500PW C. G3000PW D. G4000PW E. G5000PW F. G6000PW G. GMPW all 7.5 mm ID × 60 cmMobile phase: distilled H2OFlow rate: 1.0 mL/minDetection: RI
30 45Retention time (minutes)
Sample:
Mobile phase:Flow Rate:
mp:
TSKgel G6000PW + G4000PW, two 7.5mm ID x 60cm columns in series
hyaluronic acid
0.2mol/L NaCl0.9mL/min40°C
150
1
2
3
1. hyaluronic acid2. hyaluronic acid3. polyethylene oxide SE30
RI
LSDete
ctor
resp
onse
(V)
Figure 33: Analysis of polysaccharides
Columns: TSKgel G6000PW + G4000PW, two 7.5 mm ID × 60 cm columns in seriesMobile phase: 0.2 mol/L NaClFlow rate: 0.9 mL/minTemperature: 40 °CSample: hyaluronic acid, polyethylene oxide
8040
H2O
Retention time (minutes)
Column:
Sample:
Mobile phase:
Flow Rate:Detection:
TSKgel GMPW, two 17µm, 7.5mm ID x 60cmcolumns in series
0.5mL of 0.05-0.1% of the sodium salt of polyacrylic acid, an anionic polymer
H2O, 0.01mol/L, 0.025mol/L, 0.05mol/L or 0.1mol/L NaNO3 in water
0.5mL/minRI
0.01 mol/L NaNO3
0.025 mol/L NaNO3
0.05 mol/L NaNO3
0.1 mol/L NaNO3
60 100
Dete
ctor
resp
onse
(mV)
Figure 34: Effect of ionic strength on the elution of anionic polymers
Column: TSKgel GMPW, 17 µm, 7.5 mm ID × 60 cm × 2Mobile phase: H2O, 0.01 mol/L, 0.025 mol/L, 0.05 mol/L or 0.1 mol/L NaNO3 in H2OFlow rate: 0.5 mL/minDetection: RISample: 0.5 mL of 0.05-0.1% of the sodium salt of polyacrylic acid, an anionic polymer
32Call customer service: 866-527-3587,
technical service: 800-366-4875
About: TSKgel PWXL Size Exclusion Columns
TSKgel PWXLcolumnsarecomposedofspherical,hydrophilicpolymethacrylatebeads.ThesmallerparticlesizeofTSKgel PWXLcolumnsprovide1.7xhigherresolutionthantheirTSKgelPWcolumnscounterpart,makingTSKgelPWXLcolumnsmoresuitableforanalyticalpurposes.FourspecialtycolumnsareincludedintheTSKgelPWXLcolumnline.
TheTSKgelG-DNA-PWcolumnisdesignedfortheseparationoflargepolynucleotidessuchasDNAandRNAfragmentsof500-5,000basepairs.ThiscolumnisasmallerparticlesizeversionoftheTSKgelG6000PWXLcolumn.TheTSKgelG-Oligo-PWcolumnisdesignedforhighresolutionseparationsofaqueousnonionicandcationicoligomers,andoligosaccharidessuchashydrolyzedcyclodextrins.Becauseofthepresenceofcationicgroupsonthegelmatrix,thiscolumnisnotsuitableforseparatinganionicpolymers.TheTSKgelG-Oligo-PWcolumnhasaPEGandPEOcalibrationcurveidenticaltothatoftheTSKgelG2500PWXLcolumn.Themixed-modecolumn,TSKgelGMPWXL,hasanextendedlinearcalibrationrange,suitableforsampleswithabroadMMdistributionandunknowns.
TheTSKgelSuperOligoPWcolumnisdesignedforthedeterminationofmolarmassofaqueousoligomers,particularlyoligosaccharides,andlowmolarmassaqueouspolymers.Thecombinationofthedecreasedparticlesizeandsemi-microdimensionsoftheTSKgelSuperOligoPWcolumnenableshighspeedseparationwithhighresolutionandloweredsolventconsumption.SincethepackingmaterialintheTSKgelSuperOligoPWcolumnsismorehydrophiliccomparedwithTSKgelG-Oligo-PWcolumns,anevenwiderrangeofwater-solublepolymerscanbeanalyzedwithouttheneedtoaddorganicsolventtotheeluent.
ThefollowingTSKgelPWXLcolumnsareoffered:
• TSKgelG2500PWXL
• TSKgelG3000PWXL
• TSKgelG4000PWXL
• TSKgelG5000PWXL
• TSKgelG6000PWXL
• TSKgelG-DNA-PW • TSKgelGMPWXL
• TSKgelG-Oligo-PW • TSKgelSuperOligoPW
Attributes and Applications
ThemainapplicationareaforTSKgelPWXLcolumnsistheanalysisofwater-solublepolymers,suchascelluloses,acrylamides,glycols,dextrans,polyvinylalcohol,andoligosaccharides.BecauseofthepresenceofcationicgroupsonthebasebeadofTSKgelG2500PWXL,thiscolumnisnotsuitedforseparatinganionicpolymers.ProductattributesofalloftheTSKgelPWXLcolumnsareshowninTable14.AllTSKgel PWXLcolumnshaveabasematerialofhydroxylatedpolymethacrylate,canbeusedinamaximumof20%organicandareshippedinwater.Figures35-39showthecalibrationcurvesforalloftheTSKgelPWXLcolumns.
Table 14: Product attributes
TSKgel column Particle size (mean)
Pore size (mean)
Calibration range
G2500PWXL 7 µm <20 nm
<3,000 Da (polyethylene glycols and oxides)
G3000PWXL 7 µm 20 nm
<4.0 × 104 Da (polyethylene glycols and oxides)
G4000PWXL 10 µm <50 nm
2,000 - 3.0 × 105 Da (polyethylene glycols and oxides)
G5000PWXL 10 µm 100 nm
4,000 - 8.0 × 105 Da (polyethylene glycols and oxides)
G6000PWXL 13 µm >100 nm
4.0 × 104 - 8.0 × 106 Da (polyethylene glycols and oxides)
G-DNA-PW 10 µm >100 nm
4.0 × 104 - 8.0 × 106 Da (polyethylene glycols and oxides)
GMPWXL 13 µm mixed pore sizes
1,000 - 8.0 × 106 Da (polyethylene glycols and oxides)
G-Oligo-PW 7 µm 12.5 nm
Up to 3,000 Da (polyethylene glycols and oxides)
SuperOligoPW 3 µm 12.5 nm 100 - 3,000 Da (PEO,PEG/H2O)
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TSKgel Size Exclusion Chromatography Columns
Retention volume (mL)
Column:
Elution:Flow Rate:Detection:
TSKgel PW columns: A. G2500PWXL, B. G3000PWXL, C. G4000PWXL, D. G5000PWXL, E. G6000PWXL, F. GMPWXL, all 7.8mmID x 30cm distilled water1.0mL/minRI
Log
mol
ar m
ass
102
103
104
105
106EF
C
D
A
B
5 10 15
Figure 35: Polyethylene glycol and oxide calibration curves for TSKgel PWXL columns
Column: A. G2500PWXL B. G3000PWXL C. G4000PWXL D. G5000PWXL E. G6000PWXL F. GMPWXL all 7.8 mm ID × 30 cmMobile phase: distilled H2OFlow rate: 1.0 mL/minDetection: RI
Retention volume (mL)
Sample:
Mobile phase:
Flow Rate:Detection:
TSKgel G-DNA-PW, four 10µm, 7.8mm ID x 30cm columns in series
Eco RI and Bst NI-cleaved pBR322 DNA, void volume determined with λ-DNA
0.3mol/L NaCl in 0.1mol/LTris-HCl, pH 7.5,
0.15mL/minUV@260nm
2824 32 362010
100
400
1,000
2,000
4,000
8,000
Base
Pai
rs
plus 1mmol/L EDTA
Figure 37: Double stranded DNA calibration curves for TSKgel G-DNA-PW column
Column: TSKgel G-DNA-PW, 10 µm, 7.8 mm ID × 30 cm × 4 Mobile phase: 0.3 mol/L NaCl in 0.1 mol/LTris-HCl, pH 7.5, + 1 mmol/L EDTAFlow rate: 0.15 mL/minDetection: UV @ 260 nmSample: Eco RI and Bst NI-cleaved pBR322 DNA, void volume determined with l-DNA
Retention volume (mL)
Sample:
Mobile phase:Flow Rate:Detection:
TSKgel G-Oligo-PW, two 6µm, 7.8mm ID x 30cm columns in series
hydrolyzed β-cyclodextrin
distilled H2O1.0mL/minUV@260nm
181614
Log
mol
ar m
ass
200
400
1,000
2,000
600
800
Figure 38: Oligosaccharide calibration curves for TSKgel G-Oligo-PW column
Column: TSKgel G-Oligo-PW, 7 µm, 7.8 mm ID × 30 cm × 2 Mobile phase: distilled H2OFlow rate: 1.0 mL/minDetection: UV @ 260 nmSample: hydrolyzed b-cyclodextrin
Column: 1. G3000PWXL2. G4000PWXL3. G5000PWXL4. G6000PWXL5. GMPWXL
Sample: a. thyroglobulin (660,000 Da)b. γ-globulin (150,000 Da)c. albumin (67,000 Da)d. ovalbumin (43,000 Da)e. β-lactoglobulin (36,000 Da)f. myoglobin (16,900 Da)g. cytochrome C (12,400 Da)
Elution: 0.2mol/L phosphate buffer (pH 6.8)Flow Rate: 1.0mL/minDetection: UV@280nm
Log
mol
ar m
ass
104
103
105
106
107
51 32 4a
b
cd e
fg
4 6 8 10 12Retention volume (mL)
Figure 36: Protein calibration curves for TSKgel PWXL columns
Column: 1. TSKgel G3000PWXL
2. TSKgel G4000PWXL
3. TSKgel G5000PWXL
4. TSKgel G6000PWXL
5. TSKgel GMPWXL
all 7.8 mm ID × 30 cmMobile phase: 0.2 mol/L phosphate buffer, pH 6.8Flow rate: 1.0 mL/minDetection: UV @ 280 nmSamples: a. thyroglobulin (6.6 × 105 Da) b. γ-globulin (1.5 × 105 Da) c. albumin (6.7 × 104 Da) d. ovalbumin (4.3 × 104 Da) e. b-lactoglobulin (3.6 × 104 Da) f. myoglobin (1.69 × 104 Da) g. cytochrome C (1.24 × 104 Da)
34Call customer service: 866-527-3587,
technical service: 800-366-4875
Oligosaccharides
Figure40demonstratesthehighspeedanalysisofmaltoseoligomersusingaTSKgelSuperOligoPWcolumncomparedtoaTSKgelG-Oligo-PWcolumn.Thefasteranalysistimeisduetothesemi-microdimensions(6.0mmID×15cm)andthesmallparticlesize(3µm)oftheTSKgelSuperOligoPWcolumncomparedtothe7.8mmID×30cmsizeand7µmparticlesizeoftheTSKgelG-Oligo-PWcolumn.
Large DNA fragments
FortheseparationoflargeDNAfragmentsgreaterthan1,000basepairs,afourcolumnsystemistypicallyrequired.BaselineresolutionofDNAfragmentsupto7,000basepairscanbeachieved,providedthereisatwo-folddifferenceinthechainlengthofthefragments.Figure41AshowstheelutionofdoublestrandedDNAfragments,obtainedfrompBR322DNAcleavedbybothEcoRIandBstNI,onfourTSKgelG-DNA-PWcolumnsinseries.Theelutedpeakswerecollectedandsubjectedtopolyacrylamidegelelectrophoresis,whichshowedalmostcompleteseparationofthe1060,1857,and4362basepairfragments.Althoughlowerflowratestypicallyyieldbetterseparationsofmostfragments,theresolutionofthe1857and4362basepairfragmentswasslightlygreateratthehigherflowrate,asshowninFigure41B.
10
102
103
104
105
106
107
1.50 2.50 3.50 4.50 5.50
Retention time (minutes)
Log
mol
ar m
ass
TSKgel SuperOligoPW, 6.0mm ID x 15cmTSKgel SuperMultiporePW-N, 6.0mm ID x 15cmTSKgel SuperMultiporePW-M, 6.0mm ID x 15cmTSKgel SuperMultiporePW-H, 6.0mm ID x 15cm
Mobile phase: H2O Flow rate: 0.60mL/min Detection: RITemperature: 25°CSamples: PEO, PEG and ethylene glycol
Figure 39: Polyethylene glycol, oxide and ethylene glycol calibration curve for TSKgel SuperOligoPW column
Column: TSKgel SuperOligoPW, 6.0 mm ID × 15 cmMobile phase: H2OFlow rate: 0.60 mL/min Detection: RITemperature: 25 °CSamples: PEO, PEG and ethylene glycol
Columns: A: TSKgel SuperOligoPW, 6.0mm ID x 15cm x 4 B: TSKgel G-Oligo-PW, 7.8mm ID x 30cm x 4Mobile phase: H2OFlow rate: A: 0.6mL/min B: 1.0mL/minDetection: RITemperature: 40°CInjection vol.: A: 10µL B: 50µL Samples: 1. maltoheptose 2. maltohexose 3. maltopentose 4. maltotetraose 5. maltotriose 6. maltose 7. glucose
0
50
100
10 14 18 22 26 30 34 38 42
Dete
ctor
resp
onse
(mV)
Retention time (minutes)
176
5432
1
76
5432
A
B
Figure 40: Analysis of maltose oligomers
Columns: A. TSKgel SuperOligoPW, 3 µm, 6.0 mm ID × 15 cm × 4 B. TSKgel G-Oligo-PW, 7 µm, 7.8 mm ID × 30 cm × 4Mobile phase: H2OFlow rate: A: 0.6 mL/min B: 1.0 mL/minDetection: RITemperature: 40 °CInjection vol.: A: 10 µL B: 50 µL Samples: 1. maltoheptose 2. maltohexose 3. maltopentose 4. maltotetraose 5. maltotriose 6. maltose 7. glucose
1
6050 70 80
2
3
45
6
Retention time (minutes)Retention time (minutes)
Sample:
Mobile phase:
Flow Rate:Detection:
TSKgel G-DNA-PW, four 10µm, 7.8mm ID x 30cm columns in series
60µL of Eco RI and Bst NI - cleaved pBR322 DNA, base pairs: 1. 4362, 2. 1857, 3. 1060 & 928, 4. 383, 5. 121, 6. 13
0.3mol/L NaCl in 0.1mol/L Tris-HCl, pH 7.5, plus 1mmol/L EDTA A. 0.15mL/min, B. 0.5mL/minUV@260nm
A. 0.15 mL/min B. 0.5 mL/min
210180 240 270150
1 2
3
4
5
6Dete
ctor
resp
onse
(AU)
Dete
ctor
resp
onse
(AU)
Figure 41A and 41B: Analysis of large DNA fragments
Column: TSKgel G-DNA-PW, 10 µm, 7.8 mm ID × 30 cm × 4 Mobile phase: 0.3 mol/L NaCl in 0.1 mol/L Tris-HCl, pH 7.5, + 1 mmol/L EDTAFlow Rate: A. 0.15 mL/min B. 0.5 mL/minDetection: UV @ 260 nmSamples: 60 µL of Eco RI and Bst NI - cleaved pBR322 DNA, base pairs: 1. 4362 2. 1857 3. 1060 & 928 4. 383 5. 121 6. 13
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TSKgel Size Exclusion Chromatography Columns
Oligomers
TheTSKgelG-Oligo-PWcolumnisdesignedforhighresolutionseparationsofnonionicandcationicoligomers.Figure42demonstratesexcellentresolutionofchito-oligosaccharidesobtainedbyusingthesmaller,6µmparticlesizepackingintheTSKgelG-Oligo-PWcolumn.
Complex Polymers
AnexampleontheinfluenceofporesizeontheseparationofcomplexpolymersisshowninFigure43.WhileonthelargeporeTSKgelG6000PWXLcolumn,gelatinelutesinonenarrowpeak,ontheG4000PWXLcolumnthepeakismuchbroaderandtheshouldernearlyseparatedfromthemainpeak.ThisallowsbetterdeterminationofMw /MnandMz /Mw.
15 20
Samples:
Mobile phase:Flow Rate:Detection:
TSKgel G-Oligo-PW, two 6µm, 7.8mm ID x 30cm
1. chitohexaose 2. chitopentaose3. chitotetraose4. chitotriose 5. chitobiose
distilled H2O1.0mL/minRI
45
3
1
2
Retention time (minutes)
columns in series
Dete
ctor
resp
onse
(mV)
Figure 42: Analysis of large chitooligosaccharides
Column: TSKgel G-Oligo-PW, 7 µm, 7.8 mm ID × 30 cm × 2Mobile phase: distilled H2OFlow rate: 1.0 mL/minDetection: RISamples: 1. chitohexaose 2. chitopentaose 3. chitotetraose 4. chitotriose 5. chitobiose
Retention time (minutes) Retention time (minutes) Retention time (minutes)
A B C
0 10 20 0 10 20 0 10 20
Column: A: TSKgel G6000PWXL, B: TSKgel G5000PWXL, C: TSKgel G4000PWXL; all 7.8mm ID x 30cm LMobile phase: 0.2mol/L phoshpate buffer (pH 6.0)Flow rate: 1.0 mL/minDetection: RISample: Gelatin
Dete
ctor
resp
onse
(mV)
Dete
ctor
resp
onse
(mV)
Dete
ctor
resp
onse
(mV)
Figure 43: Separation of gelatin
Columns: A. TSKgel G6000PWXL B. TSKgel G5000PWXL
C. TSKgel G4000PWXL; all 7.8 mm ID × 30 cm Mobile phase: 0.2 mol/L phoshpate buffer, pH 6.0Flow rate: 1.0 mL/minDetection: RISample: gelatin
36Call customer service: 866-527-3587,
technical service: 800-366-4875
Small Peptides
Figure44demonstratesthattheseparationofsmallpeptidesispossibleonaTSKgelG3000PWXLcolumnunderdenaturingconditions.Usinganaqueouseluentcontaining45%acetonitrileand0.1%trifluoroaceticacid,thepeptideswereretainedonthecolumnusingasizeexclusionmechanism.Anadvantageofthismethodisthattheeluentisvolatile.
Molar Mass
Pullulanstandardsampleswithanarrowmolarmassdistributionarecommerciallyavailable.ThemolarmassofpullulanwasanalyzedbyGFC/LALLSusingaTSKgelGMPWXLcolumn(Figure45).
Retention time (minutes)
Column: TSKgel G3000PWXL, 6µm, 7.8mm ID x 30cm LMobile phase: 0.1% TFA / 45% CH3CNFlow rate: 1.0mL/minSample: peptides; 1= aprotinin, 2= insulin B-chain, 3= a-MSH, 4= bradykinin potentiator C, 5= glutathione
20 4030 50 60 70
1
2
3
4
5
Dete
ctor
resp
onse
(mV)
Figure 44: Analysis of small peptides
Column: TSKgel G3000PWXL, 6 µm, 7.8 mm ID × 30 cm Mobile phase: 0.1% TFA / 45% CH3CNFlow rate: 1.0 mL/minSamples: peptides 1. aprotinin 2. insulin b-chain 3. a-MSH 4. bradykinin potentiator C 5. glutathione
Column: TSKgel GMPWXL, 13µm, 7.8mm ID x 30cm, x 4 Mobile Phase: 0.1 mol/L sodium chloride Flow rate: 1.0mL/minTemperature: 40°CDetection: 1) RI 2) LSInjection vol: 500µLSample: 1) pullulan P400, 2) pullulan P200, 3) pullulan P100, 4) pullulan P50
Reference: Separation Report No. 38 p. 8Title: GFC analysis of water soluble polymers for TSKgel PWXL series
Chromatogram of pullulans by GPC/LALLST00168
25 30 35Retention time (minutes)
LSRI
1
2
3
4
Dete
ctor
resp
onse
(V)
Figure 45: Analysis of pullulan
Column: TSKgel GMPWXL, 13 µm, 7.8 mm ID × 30 cm × 4Mobile phase: 0.1 mol/L sodium chloride Flow rate: 1.0 mL/min Temperature: 40 °CDetection: RI LS Injection vol: 500 µLSamples: 1. pullulan P400 2. pullulan P200 3. pullulan P100 4. pullulan P50
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TSKgel Size Exclusion Chromatography Columns
Nucleic Acids
DesaltingofnucleosidescanbeaccomplishedusingtheTSKgelG2500PWXL asdepictedinFigure46.Clearly,adenosineelutesafterthevoidvolumeintheun-bufferedwatermobilephase.
Sodium Polystrene
SeparationofsodiumpolystyrenesulfonatestandardsbyGFCrequirestheadditionofatleast10%acetonitrileormethanoltoa0.2mol/LNa2SO4mobilephase.Figure47 showschromatogramsforsodiumpolystyrenesulfonatestandardsusingaTSKgelGMPWXLcolumn.Peakshapesforsodiumpolystyrenesulfonatesamplesobtainedbyadding10%acetonitriletoa0.2mol/LNa2SO4 mobile phase remainedconstantuponadditionofmoreacetonitrile.
Column: TSKgel G2500PWXL, 7.8mm ID x 30cm
Sample: 1. 0.5 NaCl, 2. uridine, 3. adenosine
Eluent: distilled waterFlow Rate: 1.0mL/minDetection: UV@260nm
0 10 20Retention time (minutes)
30
1
2
3
Dete
ctor
resp
onse
(AU)
Figure 46: Desalting of nucelosides
Column: TSKgel G2500PWXL, 7 µm, 7.8 mm ID × 30 cmMobile phase: distilled H2OFlow rate: 1.0 mL/minDetection: UV @ 260 nmSamples: 1. 0.5 mol/L NaCl 2. uridine 3. adenosine
Column: TSKgel GMPWXL, 13µm, 7.8mm ID x 30cm, x 4 Mobile Phase: acetonitrile/0.2 mol/L sodium sulfate = 10/90 Flow rate: 1.0mL/minTemperature: 40°CDetection: 1) RI 2) LSInjection vol: 500µLSample: sodium poly(styrene sulfonates)
Reference: Separation Report No. 38 p. 9Title: GFC analysis of water soluble polymers for TSKgel PWXL series
Chromatograms of sodium poly(styrene sulfonates) by GPC/LALLS T00169
20 30Retention time (minutes)
Lot. 1
Lot. 26 Lot. 20
LSRI
Dete
ctor
resp
onse
(V)
Figure 47: Separation of sodium polystyrene sulfonate standards
Column: TSKgel GMPWXL, 13 µm, 7.8 mm ID × 30 cm × 4Mobile phase: ACN/0.2 mol/L sodium sulfate = 10/90 Flow rate: 1.0 mL/min Detection: RI LS Temperature: 40 °CInjection vol: 500 µLSample: sodium poly(styrene sulfonates)
38Call customer service: 866-527-3587,
technical service: 800-366-4875
About: TSKgel PWXL-CP Size Exclusion Columns
TSKgel PWXL-CPcolumnswerespecificallydevelopedfortheanalysisofwater-solublecationicpolymers.Composedofpolymethacrylatebeads,cationicgroupsareintroducedonthesurfaceoftheTSKgelPWXL-CPpackingmaterialtopreventadsorptionofcationicpolymersandallowelutionunderlowsaltconditions.Thesecolumnsshowhightheoreticalplatenumbers,linearcalibrationcurves,andhighdurabilitybecausethebaseresinisthesameasthatusedinthe TSKgel PWXLcolumns.
ThreecolumnsareavailablewithintheTSKgelPWXL-CP series,eachwithadifferentparticlesize,separationrange,andexclusionlimit,allowingpolymerswithinawidemolarmassrangetobeseparatedandcharacterized.
• TSKgelG3000PWXL-CP • TSKgelG5000PWXL-CP • TSKgelG6000PWXL-CP
Attributes and Applications:
Table15showstheproductattributesforeachofthethreeTSKgel PWXL-CPcolumns.Figure48showscalibrationcurvesproducedwithstandardpolyethyleneoxideandpolyethyleneglycolina0.1mol/Laqueoussolutionofsodiumnitrate.
Table 15: Product attributes
TSKgel column G3000PWXL-CP G5000PWXL-CP G6000PWXL-CP
Base material
polymethacrylate polymethacrylate polymethacrylate
Particle size 7 µm 10 µm 13 µm
Pore size 20 nm 100 nm >100 nm
Exclusion limit 1.0 × 105 Da 1.0 × 106 Da 2.0 × 107 Da
Separation range (PEO, PEG)
200 ~ 5.0 × 104 Da 400 ~ 5.0 × 105 Da 1,000 ~ 1.0 × 107 Da
Theoretical plates 16,000 10,000 7,000
Cationic Polymers
VariouscationicpolymerswithdifferentfunctionalgroupsandmolarmasseswereinjectedonthethreeTSKgelPWXL-CPcolumns(TSKgelG6000PWXL-CP,G5000PWXL-CP,andG3000PWXL-CP)connectedinseries.Figure49demonstratesthattheseSECcolumnscanbeutilizedfortheanalysisofawidevarietyofcationicpolymers.
TSKgel G3000PWXL-CP, 7µmTSKgel G5000PWXL-CP, 10µmTSKgel G6000PWXL-CP, 13µm
Mobile phase: 0.1mol/L NaNO3 Flow Rate: 1mL/minDetection: RITemp: 25°CSamples: polyethylene oxides (PEO) standards polyethylene glycols (PEG) standards
10
102
103
104
105
106
107
3 5 7 9 11
Retention time (minutes)
Log
mol
ar m
ass
TSKgel G3000PWXL-CP
TSKgel G5000PWXL-CP
TSKgel G6000PWXL-CP
Figure 48: Polyethylene glycol and oxide calibration curves for TSKgel PWXL-CP columns
Columns: TSKgel G3000PWXL-CP, 7 µm, 7.8 mm ID × 30 cm TSKgel G5000PWXL-CP, 10 µm, 7.8 mm ID × 30 cm TSKgel G6000PWXL-CP, 13 µm, 7.8 mm ID × 30 cmMobile phase: 0.1 mol/L NaNO3 Flow Rate: 1 mL/minDetection: RITemperature: 25 °CSamples: polyethylene oxides (PEO) standards polyethylene glycols (PEG) standards
-20
20
60
100
10 15 20 25 30 35
Dete
ctor
resp
onse
(mV)
Retention time (minutes)
TSKgel G3000PWXL-CP, 7µm, 7.8mm ID x 30cmTSKgel G5000PWXL-CP, 10µm, 7.8mm ID x 30cmTSKgel G6000PWXL-CP, 13µm, 7.8mm ID x 30cm
Mobile phase: 0.1mol/L NaNO3 Flow Rate: 1mL/minDetection: RITemperature: 25°CSample Load: 3g/L, 100µL
PAA (MM:438 kDa)PAA (MM:235 kDa)PEI (MM:266 kDa)P(DADMACI) (MM:204 kDa)PAS (MM:7800 Da)PAS (MM:287 kDa)cationic dextran (MM:11 kDa)chitosan (MM:13.4 kDa)
Figure 49: Analysis of cationic polymers
Columns: TSKgel G3000PWXL-CP, 7 µm, 7.8 mm ID × 30 cm TSKgel G5000PWXL-CP, 10 µm, 7.8 mm ID × 30 cm TSKgel G6000PWXL-CP, 13 µm, 7.8 mm ID × 30 cmMobile phase: 0.1 mol/L NaNO3 Flow Rate: 1 mL/minDetection: RITemperature: 25 °CSample Load: 3 g/L, 100 µL
39For more info visit: www.tosohbioscience.com
TSKgel Size Exclusion Chromatography Columns
PAA
The TSKgel PWXL-CPcolumnseliminateionicadsorptionontotheparticlebyincorporatingacationicfunctionalityontheparticlesurface.ThisisdemonstratedinFigure50 below.PAA[poly(acrylicacid)]wasinjectedontoaTSKgelG5000PWXL-CPcolumn.Eachchromatogram,fromthefirstinjection(red)tothefifthinjection(black),showedsimilarelutionprofileswithoutanyadsorptionofthepolymer.
Small Molar Mass Cationic Polymers
SmallmolarmasscationicpolymerswereanalyzedontwoTSKgelG3000PWXL-CPcolumnsinseries.AsFigure51 shows,thesenarrowmolarmasscationicpolymerselutedinorderoftheirmolarmasses.
-20
20
60
100
140
180
3 5 7 9 11 13Retention time (minutes)
Dete
ctor
resp
onse
(mV)
TSKgel G5000PWXL-CP, 10µm, 7.8mm ID x 30cm
Mobile phase: 0.1mol/L NaNO3Flow rate: 1.0mL/minDetection: RITemperature: 25°CSample: polyallylamine-HCl (PAA)Sample load: 3g/L, 100µL
Figure 50: Analysis of PAA
Column: TSKgel G5000PWXL-CP, 10 µm, 7.8 mm ID × 30 cmMobile phase: 0.1 mol/L NaNO3Flow rate: 1.0 mL/minDetection: RITemperature: 25 °CSample: polyallylamine-HCl (PAA)Sample load: 3 g/L, 100 µL
-20
30
80
130
180
6 8 10 12 14 16 18 20Retention time (minutes)
Dete
ctor
resp
onse
(mV)
PEI (21 kDa)PEI (8,000 Da)PEI (3,000 Da)PAA (16 kDa)
Column: TSKgel G3000PWXL-CP x 2 Mobile phase: 0.1mol/L NaNO3Flow rate: 1.0mL/minDetection: RITemperature: 25°CSamples: Polyethyleneimine (PEI), Polyallylamine-HCl (PAA)
Figure 51: Elution profiles of PAA and PEI polymers
Column: TSKgel G3000PWXL-CP, 7 µm, 7.8 mm ID × 30 cm × 2Mobile phase: 0.1 mol/L NaNO3Flow rate: 1.0 mL/minDetection: RITemperature: 25 °CSamples: polyethyleneimine (PEI) polyallylamine-HCl (PAA)
40Call customer service: 866-527-3587,
technical service: 800-366-4875
About: TSKgel SuperMultiporePW Size Exclusion Columns
Theinnovativemulti-poreparticlesynthesistechnology*,pioneeredbyTosohscientists,isincorporatedintoTSKgelSuperMultiporePWcolumnsforwater-solublepolymeranalysis.Threesemi-microcolumnsvaryinginlinearrangeareavailablewithinthisseries,enablinghighspeedandhighresolutionanalysiswithloweredsolventconsumption.ThebasematerialofeachTSKgelSuperMultiporePWcolumnispolymethacrylate.
AwidemolarmassrangecanbeanalyzedwiththethreedifferentTSKgelSuperMultiporePWcolumns,fromhighmolarmasswater-solublepolymerstooligomers.ThepackingmaterialintheTSKgelSuperMultiporePWcolumnsismorehydrophilicthanthatofTSKgelPWXL series columns,whichfurtherreducesthechanceofadsorptionofhydrophilicpolymers.
• TSKgelSuperMultiporePW-N • TSKgelSuperMultiporePW-M • TSKgelSuperMultiporePW-H
*Usingthisproprietarytechnology,Tosohcanmanufactureparticles,eachcontainingabroadrangeofporesizes.Thisinnovativeapproachessentiallycreatesalinearcalibrationcurvewithineachparticle.Asaresult,columnswithanextendedlinearcalibrationcurvecannowbepreparedwithoutmixingparticlesofdifferentporesizes.
Attributes and Applications:
Table16showstheproductattributesforeachofthethreeTSKgelSuperMultiporePWcolumns.Figure52showspolyethyleneglycol,oxideandethyleneglycolcalibrationcurvesforeachoftheTSKgelSuperMultiporePWcolumns.
Table 16: Product attributes
TSKgel column
SuperMultiporePW-N
SuperMultiporePW-M
SuperMultiporePW-H
Base material
polymethacrylate
Particle size 4 µm* 5 µm* 8 µm*
Pore size 20 nm 100 nm >100 nm
Exclusion limit (PEO, PEG/H2O)
1.0 × 105 - 1.5 × 105 Da
6.0 × 105 - 1.5 × 106 Da -
Separation range 300 ~ 5.0 × 104 Da 500 ~ 1.0 × 106 Da 1,000 ~ 1.0 × 107 Da
Theoretical plates/15cm column
>16,000 >12,000 >7,000
*Particlesizedistributionismonodisperse.
TSKgel SuperOligoPW, 6.0mm ID x 15cmTSKgel SuperMultiporePW-N, 6.0mm ID x 15cmTSKgel SuperMultiporePW-M, 6.0mm ID x 15cmTSKgel SuperMultiporePW-H, 6.0mm ID x 15cm
Mobile phase: H2O Flow rate: 0.60mL/min Detection: RITemperature: 25°CSamples: PEO, PEG and ethylene glycol
10
102
103
104
105
106
107
1.50 2.50 3.50 4.50 5.50
Retention time (minutes)
Log
Mol
ar M
ass
TSKgel SuperMultiporePW-N
TSKgel SuperMultiporePW-M
TSKgel SuperMultiporePW-H
Figure 52: Polyethylene glycol, oxide, and ethylene glycol calibration curves for TSKgel SuperMultiporePW columns
Columns: TSKgel SuperMultiporePW-N, 6.0 mm ID × 15 cm TSKgel SuperMultiporePW-M, 6.0 mm ID × 15 cm TSKgel SuperMultiporePW-H, 6.0 mm ID × 15 cmMobile phase: H2O Flow rate: 0.60 mL/min Detection: RITemperature: 25 °CSamples: polyethylene oxides (PEO) standards polyethylene glycols (PEG) standards ethylene glycol (EG) standards
41For more info visit: www.tosohbioscience.com
TSKgel Size Exclusion Chromatography Columns
Comparison with Conventional GPC Columns
Amixtureofpolyethyleneoxide(PEO)andpolyethyleneglycol(PEG)wasanalyzedonasemi-microTSKgelSuperMultiporePW-Mcolumnandonconventional-sizedTSKgelG3000PWXLandTSKgelG5000PWXLcolumnsinseries.AsshowninFigure53,theanalysisusingtheTSKgelSuperMultiporePW-Mcolumnwascompletedin½thetimeandwithhigherresolutionthantheanalysisperformedusingtheTSKgelG3000PWXLandTSKgelG5000PWXL columns.Thisisduetothesemi-microdimensions(6.0mmID×15cm)andthesmallerparticlesize(4µm)oftheTSKgelSuperMultiporePW-Mcolumncomparedtothe7.8mmID×30cmsizeand7and10µmparticlesizeoftheTSKgelG3000PWXLandTSKgelG5000PWXLcolumnsrespectively.
PVP
Figure54demonstratesthelowerhydrophobicityoftheTSKgelSuperMultiporePWcolumnscomparedtotheconventionalTSKgelPWXLcolumns.HydrophobicinteractioncausespartialadsorptionofPVP-15polymerontheTSKgelG3000PWXLandTSKgelG2500PWXL columns,whiletheabsenceofadsorptionontheTSKgelSuperMultiporePW-Ncolumnsuggeststhattheinternalparticlesurfaceismorehydrophilicthantheconventionalcolumns.
0
40
80
120
1 3 5 7 9 11Retention time (minutes)
Dete
ctor
resp
onse
(mV) 1 (MM = 879 kDa)
1
4 (MM = 194 Da)3 (MM = 3,000 Da)
2 (MM = 39 kDa)
2
3 4
Columns: A: TSKgel SuperMultiporePW-M, 6.0mm ID x 15cm B: TSKgel G5000PWXL + G3000PWXL, each 6.0mm ID x 15cm Mobile phase: H2O Flow rate: 0.6mL/minDetection: RITemperature: 25°CInjection vol.: A: 20µL B: 100µLSamples: mixture of PEO and PEG
Resolution TSKgel PWXL TSKgel SuperMultiporePW-M
Peak 1/Peak 2 3.45 4.25
Peak 2/Peak 3 3.29 3.17
Peak 3/Peak 4 3.30 3.39
A
B
Figure 53: Comparison of analysis
Columns: A: TSKgel SuperMultiporePW-M, 6.0 mm ID × 15 cm B: TSKgel G5000PWXL + G3000PWXL, each 6.0 mm ID × 15 cmMobile phase: H2O Flow rate: 0.6 mL/minDetection: RITemperature: 25 °CInjection vol. : A: 20 µL B: 100 µLSamples: mixture of PEO and PEG
Columns: A: TSKgel SuperMultiporePW-N, 6.0mm ID x 15cm x 2 B: TSKgel G3000PWXL+G2500PWXL, 6.0mm ID x 15cm x 2Mobile phase: 100mmol/L NaNO3 Flow rate: 0.60mL/min Detection: RITemperature: 40°CInjection vol.: 20µLSamples: PVP(K-15)
-5
15
35
55
4 6 8 10 12De
tect
or re
spon
se (m
V)
Retention time (minutes)
A
B
Figure 54: Analysis of a PVP-15 polymer
Columns: A. TSKgel SuperMultiporePW-N, 6.0 mm ID × 15 cm x 2 B. TSKgel G3000PWXL+G2500PWXL, 6.0 mm ID × 15 cm x 2Mobile phase: 100 mmol/L NaNO3 Flow Rate: 0.60 mL/minDetection: RITemperature: 40 °CInjection vol. : 20 µLSamples: PVP(K-15)
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