TSKgel Size Exclusion Chromatography Columns Library... · TSKgel Size Exclusion Chromatography...

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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

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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



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



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.


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


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


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)




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


Sample:

Mobile phase:


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


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)



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


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)


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:


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



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


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



Small Peptides

Figure44demonstratesthattheseparationofsmallpeptidesispossibleonaTSKgelG3000PWXLcolumnunderdenaturingconditions.Usinganaqueouseluentcontaining45%acetonitrileand0.1%trifluoroaceticacid,thepeptideswereretainedonthecolumnusingasizeexclusionmechanism.Anadvantageofthismethodisthattheeluentisvolatile.

Molar Mass

Pullulanstandardsampleswithanarrowmolarmassdistributionarecommerciallyavailable.ThemolarmassofpullulanwasanalyzedbyGFC/LALLSusingaTSKgelGMPWXLcolumn(Figure45).


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



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)



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.


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


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

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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



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.

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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

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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)



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.


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


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



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.

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4 (MM = 194 Da)3 (MM = 3,000 Da)

2 (MM = 39 kDa)

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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)

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V)


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)

TSKgel Size Exclusion Chromatography Columns Library... · TSKgel Size Exclusion Chromatography...

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