columns for hplccolumns for hplc - western · pdf filecolumns for hplccolumns for hplc 95...

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Columns for HPLCColumns for HPLC

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Summary of MN columns for bioanalysis and food analysis

Page Page

GFC

pH 1 – 13

•

NUCLEOGEL GFC 300-8 115

•


pH 1 – 13

•

NUCLEOGEL RP 300-5 110

•


Proteins RPC

pH 2 – 8

•

NUCLEOSIL 300-5 C

4

MPN 107

•

NUCLEOSIL 300-5 C

18

MPN 107

pH 1 – 9

•

NUCLEOSIL 500-5 C

18

PPN 108

IEC

pH 2 – 8.5

•

NUCLEOSIL 4000-7 PEI 101

pH 1 – 13

•

NUCLEOGEL SAX 4000-8 104

•

NUCLEOGEL SCX 4000-8 105

pH 2 – 8.5

•


IEC

pH 1 – 13

•


•

NUCLEOGEL SCX 1000-8 105

•

NUCLEOSIL 100-5 C

18

MPN 107

Peptides

pH 2 – 8

•

NUCLEOSIL 120-3 C

18

MPN 107

•

NUCLEOSIL 300-5 C

4

MPN 107

RPC

•

NUCLEOSIL 300-5 C

18

MPN 107

•

NUCLEOSIL 100-5 C

18

PPN 108

pH 1 – 13

•


Amino acids

also see ”HPLC columns for enantiomer separation“ from page 81

RPC

pH 2 – 8

•

NUCLEOSIL 100-5 C

18

MPN 107

•

NUCLEOSIL 120-3 C

18

MPN 107

Nucleosides

pH 2 – 8

•

NUCLEOSIL 100-5 C

18

MPN 107

•

NUCLEOSIL 300-5 C

18

MPN 107

RPC

pH 1 – 9

•

NUCLEOSIL 100-5 C

18

PPN 108

•

NUCLEOSIL 500-5 C

18

PPN 108

pH 1 – 13

•


•


Nucleotides,oligo-

nucleotides

pH 2 – 8.5

•

NUCLEOGEN 60-7 DEAE 96

•


IEC

pH 1 – 13

•


GFC

pH 1 – 13

•


•


DNA, RNA,restrictionfragments,

plasmids

•


IEC

pH 2 – 8

•


•


pH 1 – 13

•


•

NUCLEOBOND AX *

•


•


Polysaccharides GFC

pH 1 – 13

•


pH 2 – 8

•

NUCLEOSIL Carbohydrate 117

•

NUCLEOSIL 100-5 NH

2

55

Mono- and oli-gosaccharides

•

NUCLEOGEL ION 300 OA 120

•

NUCLEOGEL SUGAR 810 H 118

•

NUCLEOGEL SUGAR Pb 120

•

NUCLEOGEL SUGAR 810 Pb 118pH 1 – 13

•

NUCLEOGEL SUGAR Ca 120

•

NUCLEOGEL SUGAR 810 Ca 118

•

NUCLEOGEL SUGAR Na 120

GFC

= gel filtration chromatography

IEC

= ion exchange chromatography

RPC

= reversed phase chromatography

* Please ask for our catalogue ”Bioanalysis“

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Ion exchange columns for biochemical applications

NUCLEOGEN

®

columns for the separation of oligonucleotides and nucleic acids

The NUCLEOGEN

®

family offers remarkable performancefor biopolymers. It is a series of silica-based DEAE anion ex-changers available with pore sizes of 60, 500 and 4000 Å, re-spectively. The choice of pore sizes and the chemistry of thesurface coating was elaborated for present day problems innucleic acid research.NUCLEOGEN

®

columns guarantee outstanding featureswith respect to

resolution speed reproducibility purity life timerecovery capacity solvent compatibilityregeneration time

The favourable interplay of these qualities is seen best fromthe separation of oligonucleotides, high molecular weightRNA and plasmid DNA.NUCLEOGEN

®

phases show neither swelling with salt or pHgradients nor a break-down of the column packing even withhigh flow rates. Reequilibration to starting conditions takesonly a few minutes. NUCLEOGEN

®

columns are suited forevery day use over a long period of time. It has been report-ed

1

that columns of 1.5 ml bed volume (4 mm ID x 125 mm)have been used with more than 100 l buffer without loss ofresolution; this corresponds to about 1000 repetitive HPLCruns.For numerous separations of deoxyoligonucleotides, plas-mids and DNA restriction fragments see our catalogue “LCApplications” or visit our website:

Length

→

125 mm Guard columns*

NUCLEOGEN

®

60-7 DEAE

DEAE anion exchanger based on silica, particle size 7 µm, pore size 60 Å; eluent in column methanol

EC analytical columns

4 mm ID 736596.40 736400.40

Standard-Prep preparative columns

10 mm ID 736597 736400.40

NUCLEOGEN

®

500-7 DEAE

DEAE anion exchanger based on silica, particle size 7 µm, pore size 500 Å, eluent in column methanol

Valco type analytical columns

6 mm ID 736598 736400.40


10 mm ID 736599 736400.40

NUCLEOGEN

®

4000-7 DEAE

DEAE anion exchanger based on silica, particle size 7 µm, pore size 4000 Å, eluent in column methanol

Valco type analytical columns

6 mm ID 736601 736400.40


10 mm ID 736602 736400.40

Columns are supplied in packs of 1.* ChromCart

®

NUCLEOGEN

®

guard column cartridges are 30 mm long and supplied in packs of 2. They require the CC column holder 30 mm (Cat. No. 721823).

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Capacity of NUCLEOGEN

®

DEAE columns

NUCLEOGEN

®

60-7 DEAE for the separation of oligonucleotides

NUCLEOGEN

®

60-7 DEAE was particularly developed forthe isolation of short nucleic acids. Synthetic oligonucle-otides of defined length and sequence are required for mod-ern genetic engineering and in molecular biology. The HPLCresins applied formerly were very limited with respect to res-olution and recovery. Both disadvantages are overcome withour NUCLEOGEN

® 60-7 DEAE; resolution is extended tochain lengths of 40 bases, and the recovery is > 95% 1, 2. Thepacked columns are ready for use. Elevated temperatures forchromatography are not required. The high capacity of200 A260/ml NUCLEOGEN® DEAE permits the use of smallcolumns.For the isolation of oligonucleotides with chain lengths be-tween 2 and 50 the following buffer systems show a verygood separation efficiency:

For preparative separations LiCl with the last-mentioned buffersystem offers a great advantage, since nucleic acids may bedirectly precipitated from 4 M LiCl with ethanol/acetone with-out co-precipitation of the salt. The volume of the collectedfractions and the LiCl concentration are determined (from thechromatogram) and the concentration is increased to 4 M LiCl

in a vacuum centrifuge (e. g. Speed-Vac). To this concentratedsample five times its volume of ethanol/acetone (1:1) is add-ed, precipitated 5 h at –20 °C and centrifuged 1 h at5000 rpm.

Capacity [A260]Phase column EC 125/4 VA 125/6 SP 125/10

NUCLEOGEN® 60-7 DEAE 300 1875NUCLEOGEN® 500-7 DEAE 730 1940NUCLEOGEN® 4000-7 DEAE 120 350A260: Absorption unit at 260 nm; it corresponds to about 40 µg RNA or 50 µg DNAGood resolution is achieved if not more than 50% of the maximum binding capacity is loaded onto the column

Buffer A (low salt) Buffer B (high salt)

5 M urea 0.02 M K phosphate* pH 5.5 – 6.5

1 M KCl or LiCl, (NH4)2SO4

5 M urea, 0.02 M K phosphate*, pH 5.5 – 6.5

50% formamide, 0.02 M K phosphate* pH 5.5 – 6.5

1 M KCl or LiCl, (NH4)2SO4

50% formamide, 0.02 M K phosphate*, pH 5.5 – 6.5

30% methanol 0.02 M Na acetate pH 5.5 – 6.5

1 M KCl or LiCl, (NH4)2SO430% methanol0.02 M Na acetate, pH 5.5 – 6.5

20% acetonitrile 0.02 M Na acetate pH 5.5 – 6.5

1 M KCl or LiCl, (NH4)2SO420% acetonitrile0.02 M Na acetate, pH 5.5 – 6.5

* stock buffer 1 M K phosphate consists of 0.5 MolKH2PO4 (68.05 g) + 0.5 Mol K2HPO4 (87.09 g) per litreThe pH value is adjusted with H3PO4

Purification of full-length synthetic oligoribonucleotidesK.R. Webster et al. BioTechniques 11 (1991) 658 – 661Column: 125 x 4 mm NUCLEOGEN® 60-7 DEAE,

equilibrated with 20 mM NaAc, 20% acetonitrile (pH 6.5)

Gradient: 0 – 1.5 M KCl in 50 minFlow rate: 2 ml/minDetection: UV, 260 nm

Abs

. 260

nm

0

1

[KC

l] (M

)0

1.5

0 10 30 50 min

12mer

18mer

24mer

30mer

1073

30

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As test mixture, oligo(rA)n, n = 3 – 30 is of advantage be-cause it is easily prepared from poly(rA) with KOH. For a test chromatogram 0.5 A260 oligo(rA)n in 25 – 50 µlbuffer A are injected onto the column and a linear gradientfrom 100% buffer A (0% B) to 100% buffer B (0% A) in50 min is started at a flow rate of 1 ml/min.Under these conditions base line separation can be achievedup to n = 10, and up to n = 25 peaks are discernible. For res-olution of higher chain lengths the gradient time should be in-creased from 50 min to 75 min or 100 min. With increasinglyflatter gradients the resolution for higher chain lengths is im-proved. Better separations can be achieved with convex gra-dients, which allow for the smaller charge differences foundwith higher molecular weights.If the sample contains only long-chain oligonucleotides, it istimesaving to start the separation at higher ionic strength.Thus a separation of oligo(rA)10–25 can be shortened from50 min to 25 min by starting at 25% B (75% A). To preventbreakthrough of the sample, the ionic strength for starting theseparation should be about 50 mM below the ionic strengthfor elution.Before performing preparative separations, an analyticalseparation should be made in order to investigate the com-position of the sample. The gradient required for the prepara-tive separation can then be evaluated from the analyticalchromatogram.For a preparative separation it is important not to exceed themaximum working capacity. The higher the loading of the col-umn, the higher flow rates and longer gradient times are tobe chosen, since every single peak needs a certain volumefor elution if base line separation is required.For the preparative base line separation of 100 A260 oli-go(rA)n the flow rate is increased to 2.5 ml/min using a gradi-ent time of 400 min.

Separation of oligo(rA)nColumn: 125 x 4 mm NUCLEOGEN® 60-7 DEAEBuffer A: 20 mM phosphate, pH 5.5, 5 M ureaBuffer B: buffer A + 1 M KClGradient: 0 – 100% B in 200 minFlow rate: 2 ml/min, 110 bar

ambient temperatureUV detection, 260 nm

A260

0,02

0

0 50 100 min

4

10

20

3037

1151

80

Separation of a deoxyoligonucleotide 18merH. Werntges, Diplomarbeit, University of DüsseldorfSample: d(CGTCGTTTAACAACGTCG)Column: 125 x 4 mm NUCLEOGEN® 60-7 DEAEBuffer A: 40% acetonitrile, 0.02 M NaAc pH 5.5Buffer B: buffer A + 0.7 M KClGradient: 0 – 50% B in 120 min

50 – 100% B in 250 minFlow rate: 3 ml/min, 40 bar

ambient temperatureDetection: 260 nm

A26

0

0 215min

1074

10

For ordering information of NUCLEOGEN® DEAE columns please see page 96.

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NUCLEOGEN® 500-7 DEAE for the separation of tRNA, 5S RNA, viroids and messenger RNA

With the development of NUCLEOGEN® 500-7 DEAE with itspore size of 500 Å it is possible to isolate nucleic acids in theintermediate molecular weight range (25,000 – 1,000,000 Dal-tons).NUCLEOGEN® 500-7 DEAE columns can be used for sin-gle-stranded as well as for double-stranded nucleic acids, forDNA as well as for RNA. Application with tRNA, 5S RNA,messenger RNA, viroids 3, viral DNA and DNA restrictionfragments 1 has been reported. As a consequence of theiroutstanding capacity and very high recovery (> 95%) the col-umns are particularly suitable for large-scale preparations. The isolated nucleic acids are pure with respect to spectro-scopic, hydrodynamic and thermodynamic properties and fullyactive in enzymatic tests.

NUCLEOGEN® 4000-7 DEAE for the separation of plasmids, DNA restriction fragments, ribosomal RNA,messenger RNA and viral RNA

With the introduction of NUCLEOGEN 4000-7 DEAE thescope of HPLC was extended to very high molecular weightnucleic acids (e.g. 1 – 50 megadaltons). This phase is suitedfor the separation of restriction fragments and oligonucle-otides as well as for the separation of different ribonucleic ac-ids. However, the load capacity is significantly lower than forthe narrow-pore materials. A remarkable success of NUCLEOGEN® 4000-7 DEAE isthe isolation of supercoiled plasmid DNA from a crude celllysate in a single HPLC step 1. In a re-chromatography su-percoiled forms can even be separated from the relaxed andlinear forms (see chromatograms on next page). BecauseNUCLEOGEN® 4000-7 DEAE provides very high resolutionfor large as well as for medium-size nucleic acids, cDNA in-serts can be easily separated from the remaining vector. Theplasmids are eluted from the column with more than 95% re-covery and are fully active with respect to digestion with re-striction enzymes, labelling with kinases, PCR, LCR reac-tions, transcription with polymerases and enzymatic ligation.

The following buffer systems show very good separationefficiency for plasmids, viral RNA and restriction fragments.

Preparative separation of a crude RNA extract of viroid (PSTV) infected tomato plants D. Riesner BioEngineering 1 (1988) 42 – 48Column: 125 x 6 mm NUCLEOGEN® 500-7 DEAEBuffer A: 250 mM KCl, 20 mM phosphate buffer pH 6.6,

5 M ureaBuffer B: 1 M KCl, 20 mM phosphate buffer pH 6.6,

5 M ureaGradient: 0 – 50% B in 120 min

50 – 100% B in 250 minFlow rate: 3 ml/min, 40 bar

ambient temperatureDetection: 260 nm

A260

0.04

0

0 50 100 min

tRNA

5S RNA

7S RNA

PSTV

1074

90

Buffer A (low salt) Buffer B (high salt)5 M urea 0.02 M K phosphate* pH 6.5 – 7.0

1.5 M KCl or LiCl, (NH4)2SO4

5 M urea 0.02 M K phosphate*, pH 6.5 – 7.0

50% formamide, 0.02 M K phosphate* pH 6.5 – 7.0

1.5 M KCl or LiCl, (NH4)2SO4

50% formamide 0.02 M K phosphate *, pH 6.5 – 7.0

* for stock buffer K phosphate see page 97

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References1) HPLC of high-molecular weight nucleic acids on the ma-

croporous ion exchanger NUCLEOGEN. M. Colpan, D. Riesner, J. Chromatogr. 296 (1984) 339–353

2) Large-scale purification of viroid RNA using Cs2SO4 gra-dient centrifugation and HPLC M. Colpan, J. Schumacher, W. Brüggemann and D. Ries-ner, Anal. Biochem. 131 (1983) 257 – 265

3) HPLC of DNA restriction fragments. R. Hecker, M. Col-pan, D. Riesner, J. Chromatogr. 326 (1985) 251 – 261

4) HPLC zur Reinigung hochmolekularer RNAs und DNAsR. Dornburg, J. Kruppa, P. Földi, GIT 29 (1985) 505 – 515

5) Application of HPLC technologies in rapid DNA sequenc-ing of kilobase pairs of DNA R. Dornburg, LC.GC 6 (1988) 254 – 259

6) Fractionation of DNA restriction fragments with ion ex-changers for HPLC W. Müller, Eur. J. Biochem. 155 (1986) 203 – 212

7) Isolation of high-molecular nucleic acids for copy numberanalysis using HPLC. S. J. Coppella, C. M. Acheson,P. Dhurjati, J. Chromatogr. 402 (1987) 189 – 199

8) Chromatographic separation of DNA restriction frag-ments (Review). R. Hecker, D. Riesner, J. Chromatogr.418 (1987) 97 – 114

Separation of plasmid pBR 322Sample: 5 µg plasmid pBR 322 containing cleared lysate

from E. coliColumn: 125 x 6 mm NUCLEOGEN® 4000-7 DEAEEluent A: 20 mM K phosphate buffer pH 6.9;

5 M ureaEluent B: eluent A

+ 1.5 M KClGradient: 20% – 100% B in

50 minarrow = ionic strength of 850 mM

Flow rate: 1.0 ml/min, 70 bar, ambient temperature

Detection: UV, 260 nm

A260

0.1

0

0 20 40min

RNA

Plasmid

1074

80

Separation of plasmid DNA[M. Colpan, D. Riesner, private communication]Sample: plasmid pBR 322, supercoiled, relaxed and linearColumn: 125 x 6 mm NUCLEOGEN® 4000-7 DEAEEluent A: 20 mM phosphate buffer pH 6.8; 6 M ureaEluent B: eluent A + 2 M KClGradient: 42% – 100% B in 230 minFlow rate: 1.5 ml/min, 45 bar, ambient temperature

A260

0,04

0

0 15 30 min

supe

rcoi

led

rela

xed

linea

r

1074

80

abcde

fg

h

i

j

k

0 20 40min

Separation of DNA restriction fragmentsSample: 25 µg DNA restriction fragments of plasmid

pSP 64, digested with Hinf I Column: 125 x 6 mm NUCLEOGEN® 4000-7 DEAEGradient: linear, 250 mM – 1250 mM KCl in 200 min, in

30 mM K phosphate buffer pH 6.5, 5.5 M urea

Flow rate: 1 ml/minTemperature: 22 °CDetection: UV, 260 nm

Fragment lengths: a) 19 bp b) 35 bp c) 38 bp d) 41 bp e) 45 bp f) 78 bp g) 168 bp h) 296 bp i) 4 x 344 bp j) 712 bpk) 881 bp

1151

90

For ordering information of NUCLEOGEN® DEAE columns please see page 96.

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NUCLEOSIL® 4000-7 PEI – anion exchanger for the separation of proteins and peptides The purification of biologically active peptides and proteinsbecomes more and more important in research and industry.Ion exchange chromatography is a superior technique in thisregard, because it allows purification of proteins in aqueousbuffers using salt or pH gradients. The low isoelectric point ofmost proteins is the reason why anion exchange chromatog-raphy is preferably applied. The 400 nm pores of the rigid silica matrix of NUCLEOSIL®

4000-7 PEI enable an unrestricted penetration of polypep-tides under very different elution conditions resulting in shortanalysis times. The mechanical stability of the spherical mi-croparticles of this packing allow high flow rates and a long-term column life. The polymeric, covalently bonded poly-ethylene imine network guarantees a good chemical stabilityof the columns towards basic and acidic eluents. The coatingis flexible enough to fit different protein shapes for the forma-tion of specific electrostatic interactions.

NUCLEOSIL® 4000-7 PEI shows a high selectivity for nu-merous proteins, e.g β-lactoglobulins A and B, two proteinsdiffering in just two amino acids, can be separated in only10 minutes.Most of the proteins which can be purified on NUCLEOSIL®

4000-7 PEI are obtained with high yields, preserving their bi-ological activity.

Recovery of enzyme activity · conditionsColumns: 50 x 4 mm NUCLEOSIL® 4000-7 PEIBuffers: A) 20 mM Tris/HCL pH 8.5; B) A + 1.5 M NaClGradient: 0 – 100% B in 5 min, 1 ml/min, 30 barThe eluting peaks were detected and fractionated by theirUV absorbance at 280 nm. The specific enzyme activity wasestimated before and after HPLC by comparison of the UVspectra of the proteins with their volume activity.

Characteristic parameters of the column packing:

Type: weakly basic anion exchanger

Mean particle size: 7 µmMean pore size: 400 nmpH range: 2 – 8.5Restrictions for the use of buffers:

none

Chemical stability with acids or bases:

good

Restrictions for the use of organic solvents:

none

Maximum salt concentration: 8 MIon exchange capacity: 0.15 mmol/gProtein binding capacity: 61 mg BSA/gMaximum working pressure: 250 bar

Length → 50 mm 125 mm 250 mm Guard columns

NUCLEOSIL® 4000-7 PEI polyethyleneimine network, covalently polymer-coated onto NUCLEOSIL® silica, weakly basic anion exchanger, particle size 7 µm, pore size 4000 Å; eluent in column methanol

EC analytical columns 1)

4 mm ID 720401.40 720402.40 720403.40 721091.40

Standard-Prep preparative columns 10 mm ID 720404 720405

ChromCart® guard column cartridges (8 mm) in packs of 2, all other columns in packs of 1.1) As guard columns for EC columns use ChromCart® guard column cartridges with guard column adaptor EC (Cat. No. 721359).

Enzyme Recovery of specific activity after HPLC [%]

Catalase (bovine liver) 93L-Lactic dehydrogenase LDH-1 isoenzyme (porcine heart)

102

Callicrein (porcine pancreas) 98Glucose oxidase (Aspergillus niger)

104

Peroxidase (horseradish) 100

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The flexible anion exchange groups of NUCLEOSIL® 4000-7PEI also show good binding and desorption kinetics for nu-cleotides, as is shown in the figure below.

This silica-based exchanger can be cleaned from sticking im-purities by a 10-min treatment with 0.1 M NaOH or 1 MHNO3.

Recovery of proteinsColumn: 50 x 4 mm NUCLEOSIL® 4000-7 PEIEluent: 10 mM NaH2PO4, 1.5 M NaCl, pH 7.0Flow rate: 1 ml/minSample: 50 µg of each protein

Protein Yield [%]

Myoglobin 100Transferrin 95Ovalbumin 98Bovine serum albumin 100Glucose oxidase 100α-Amylase 100Soybean trypsin inhibitor 100β-Lactoglobulin 97Ferritin 85

Separation of a monoclonal antibody from undialysed ascites fluid46.3 mg/ml of total protein, sample volume 5 µlColumn: 125 x 4 mm NUCLEOSIL® 4000-7 PEIEluent A: 10 mM TRIS/HCl pH 8.2Eluent B: eluent A + 1.5 M NaClGradient: 0 – 13% B in 5 min, then 13 – 70% B in 0.5 minFlow rate: 1 ml/minPressure: 50 barDetection: UV, 280 nm

IgG2b

0 5 min1083

60

Anion exchange chromatography of nucleotidesa) Separation of 9 nucleotidesColumn: 125 x 4 mm NUCLEOSIL® 4000-7 PEIEluent A: 2.5 mM TRIS/phosphate pH 7.2Eluent B: 2.5 mM TRIS/phosphate pH 8.0 + 1.5 M KClGradient: 5 – 95% B in 5 minFlow rate: 1.3 ml/minPressure: 120 barDetection: UV, 260 nmb) Separation of adenine nucleotides in 90 secondsColumn: 50 x 4 mm NUCLEOSIL® 4000-7 PEIEluent A: 20 mM TRIS/acetate pH 8.0Eluent B: eluent A + 1.5 M NaClGradient: 0 – 80% B in 30 sFlow rate: 2 ml/minPressure: 85 barDetection: UV, 260 nmPeaks:1. CMP2. AMP3. GMP4. CDP5. ADP6. GDP7. CTP8. ATP9. GTP

1

2

2

3

4

5

5

6

7

8

8

9

0 5 10 min 0 60 120 s

a) b)

1072

50

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For ordering information of NUCLEOSIL® PEI columns please see page 101.

Operation of NUCLEOSIL® 4000-7 PEI columns

Separation principleSeparation is obtained by reversible adsorption of negativelycharged substances to positively charged groups on the ex-changer material and their subsequent displacement by eitherincreasing ionic strength or pH changes in the mobile phase.Mobile phaseIn order to provide efficient adsorption of a protein to the ani-on exchanger it is usually preferable to select a pH valueabout one unit above the isoelectric point of the protein.The nature of the counterion is an important aspect toachieve high resolution. By using different buffers and/or elu-tion salts (e.g. sodium acetate, ammonium acetate, sodiumchloride, potassium chloride, sodium phosphate, potassiumphosphate) one can easily change the ionic conditions of theseparation process. This procedure can influence the se-quence of elution and the peak shape. Divalent cations (e.g.Mg++, Ca++) usually show a stronger eluting power thanmonovalent ones.When using a salt gradient most of the sample compoundsare eluted from the column at moderate salt concentrations(≤ 1 mol/l). If the compound of interest cannot be eluted achange of pH and/or buffer and salt ions should be consid-ered. For difficult cases non-ionic detergents or organic sol-vents (e.g. 5% acetonitrile) will increase the solubility anddesorption of the polypeptide. Organic modifiers should bepremixed with the aqueous buffer to avoid precipitation ofbuffer. The eluents should be filtered through 0.45 µm filtersin order to prevent the accumulation of particulate matter.Solvents should be degassed to ensure a continuous flowthrough the system.Column installation and operationThe column is supplied with methanol. Use distilled water asinitial solvent to prepare the column for use with aqueous saltbuffers. Equilibration with the starting buffer is finished, whenthe signal of the detector has reached a stable value. Avoidsudden pressure surges since they may destroy the columnpacking. If possible, dissolve the sample in the starting bufferand filter through a 0.45 µm filter.Column cleaning and storageNUCLEOSIL® 4000-7 PEI columns can easily be cleanedfrom sticking impurities by several hours of treatment with2 M sodium acetate pH 8.5, short incubation (10 min, 5 col-umn volumes) with 0.1 M NaOH, 1 M HNO3, 1% CH3COOHor by using organic solvents like acetonitrile, methanol ordimethylsulphoxide. Application of non-ionic detergents likeTriton X 100 or chaotropic reagents like guanidinium hydro-chloride or urea is also possible.Overnight columns should be stored in 0.05% sodium azide /water or methanol/water (80:20, v/v). For a longer storage allsalts have to be washed out from the columns, which arethen equilibrated with methanol.Examples for the purification of different peptides and pro-teins can be found in our catalogue “LC Applications” or onthe internet.

Separation of protein standardsColumn: 125 x 4 mm NUCLEOSIL ®

4000-7 PEIEluent A: 2 mM TRIS/acetate pH 8.0Eluent B: 20 mM TRIS/acetate pH 8.0

+ 1.5 M KClGradient: linear 0 – 40% B in 20 minFlow rate: 1 ml/minPressure: 76 barDetection: UV, 280 nm

Peaks: (Volume 20 µl)1. Catalase2. Myoglobin3. α-Amylase4. Transferrin5. α-Lactalbumin6. Glucose oxidase7. Soybean

trypsin inhibitor

1

2

3

4

5

67

0 10 20 min 1083

10

Tryptic digest of bovine serum albumin

Sample volume 50 µlColumn: 125 x 4 mm NUCLEOSIL® 4000-7 PEIEluent A: 20 mM TRIS/acetate pH 8Eluent B: A) + 1 M KClGradient: 0 – 30% B in 30 min,

then 30 – 80% B in 10 minFlow rate: 0.5 ml/minPressure: 38 barDetection: UV, 280 nm.

0 15 30 45 min 1084

10

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NUCLEOGEL® SAX – strongly basic anion exchanger for biological macromoleculesThis column family features a fully quaternised polyethylene-imine structure coupled to macroporous hydrophilic polymerbeads. Pore sizes of 100 and 400 nm, respectively, allow thepurification of peptides, large proteins and oligonucleotides.This strongly basic anion exchanger possesses a high ca-pacity for proteins even at pH 10; for this reason it can be ap-plied for difficult protein separations in the pH range aroundand above 10.Due to the polymer matrix, pH stability of the exchanger isexcellent, allowing removal of pyrogens by washing with0.1 M NaOH over several hours.

Characteristic parameters of the column packing

Separation of hen’s egg whiteSample: frozen egg white was thawed, filtered and diluted

1:8 with eluent AInjection: 50 µlColumn: 50 x 4.6 mm NUCLEOGEL®

SAX 1000-8Eluent A: 0.01 M Tris HCl, pH 7.5Eluent B: A + 0,5 M NaAc, pH 7.5Gradient: linear, 0 – 100% B in 20 minFlow rate: 1 ml/minDetection: UV, 280 nmPeaks:1. Conalbumin2. Ovalbumin3. not identified 1

2

3

0 20min

1152

00

Type: strongly basic anion exchanger

pH range: 1 – 13restrictions for the use of buffers: none (polar organic

solvents can be used as modifiers)

maximum salt concentration: 8 Mion exchange capacity: > 0.2 mmol/gprotein binding capacity: 40 or 20 mg BSA/g,

respectivelymaximum operating pressure: 200 bar

Length → 50 mm 150 mm Guard columns

NUCLEOGEL® SAX 1000-8 strong anion exchanger -N(CH3)3, polymer-based, gel matrix quaternised PEI; particle size 8 µm, pore size 1000 Å; eluent in column 0.1 M Na2SO4 + 0.2% NaN3

Valco type analytical columns 1)

4.6 mm ID 719469 7196007.7 mm ID 719471 719600

NUCLEOGEL® SAX 1000-10strong anion exchanger as above, particle size 10 µm, pore size 1000 Å

Standard-Prep preparative columns 25 mm ID 719473



4.6 mm ID 719470 7196007.7 mm ID 719472 719600



Columns are supplied in packs of 1.2) Valco type guard column cartridges are 5 x 3 mm, require guard column holder B (Cat. No. 719539) and are supplied in packs of 2.

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NUCLEOGEL® SCX – strongly acidic cation exchanger for biological macromolecules Cation exchangers are an important supplement for the ionchromatographic purification of biological macromolecules.Especially for proteins, peptides and carbohydrates with highisoelectric points this technique is a valuable tool. The ma-croporous structure of NUCLEOGEL® SCX columns guaran-tees unhindered contact of large molecules with the ex-change functionalities. Complex samples (e.g. cell extracts)or solubilised membrane protein mixtures often pose prob-lems because they cannot be recovered quantitatively fromthe column. In this case, the polymer matrix of the NUCLEO-GEL® SCX columns allows use of strongly basic or acidiceluents for an effective column regeneration.

Characteristic parameters of the column packing

Type strongly acidic cation exchanger

pH range 1 – 13restrictions for the use of buffers none (polar organic

solvents can be used as modifiers)

maximum salt concentration 8 Mmaximum working pressure 200 bar

Separation of protein standardsColumn: 50 x 4.6 mm NUCLEOGEL® SCX 1000-8Eluent A: 0.02 M KH2PO4, pH 6.0Eluent B: A + 0.5 M NaCl, pH 6.0Gradient: linear, 0 – 100% B in 20 minFlow rate: 1 ml/minDetection: UV, 280 nm

Peaks:1. Myoglobin2. α-Chymotrypsinogen A3. Cytochrome C4. Lysozyme

1 2

3

4

0 20min 1082

60

Length → 50 mm 150 mm Guard columns

NUCLEOGEL® SCX 1000-8 strong cation exchanger - SO3, hydrophilic gel matrix; particle size 8 µm, pore size 1000 Å; eluent in column 0.1 M Na2SO4 + 0.2% NaN3


4.6 mm ID 719475 7195407.7 mm ID 719477 719540

NUCLEOGEL® SCX 1000-10strong cation exchanger as above, particle size 10 µm, pore size 1000 Å




4.6 mm ID 719476 7195407.7 mm ID 719478 719540


Standard-Prep preparative columns25 mm ID 719480


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Reversed phase columns for biochemical applications

Reversed phase chromatography (RPC) is increasingly usedas an efficient method in peptide and protein analysis. Itgains increasing importance in analytical biochemistry forthe purification of clinically relevant, genetically engineeredpolypeptides. With RPC often small changes in the hydro-phobic surface regions of peptides and proteins can be de-tected, such as e.g. exchange of an amino acid or the pres-ence of deamidation or oxidation products. During work-up ofindustrial scale preparations RPC is increasingly applied forthe final purification. Examples include biosynthetic proteinssuch as human growth hormone, tissue plasminogen activa-tor, human insulin and human malaria vaccine.

We offer three different types of RP columns for biochemicalapplications:

NUCLEOSIL

®

MPN · alkyl chains monomerically bonded to silicaNUCLEOSIL

®

PPN · alkyl chains polymerically bonded to silicaNUCLEOGEL

®

RP · polymer-based phases

Due to their outstanding performance NUCLEOSIL

®

reversedphase materials are very well suited for these applications.We offer a complete RPC column family for analytical as wellas for preparative separations. The silica- and polymer-basedstationary phases differ significantly with respect to their sur-face structure and their chromatographic properties.

Characteristic parameters of the RP phases

For seven proteins tested the mass recovery for all phases is 85 – 100%.

Separation principle

Substances with hydrophobic surface regions are reversiblybonded to the hydrophobic alkyl chains of the RP stationaryphase. By increasing the concentration of the organic com-ponent in the eluent, the hydrate shell of the molecule is de-creased resulting in desorption and chromatographic sepa-ration of substances according to their hydrophobicity. Forbiological macromolecules partition effects have no signifi-cance.

As can be seen from the right figure, the maximum sepa-ration efficiency can be achieved when the injected pro-tein mass does not exceed 1 – 2% of the maximum pro-tein loading capacity.

In practice often sufficient chromatographic resolution is ob-tained for higher injection masses. The sample volume doesnot influence the separation result over a wide range. For thisreason RP chromatography often results in a concentrationof the biomolecule.

Packing pH working range max. working pressure [bar]

dynamic protein binding capacity mg protein per g packing

BSA cytochrome C

Silica-based phases

NUCLEOSIL

®

100-5 C

18

MPN 2 – 8 250 6 110NUCLEOSIL

®

300-5 C

18


®

300-5 C

4


®

100-5 C

18

PPN 1 – 9 250 8 64NUCLEOSIL

®

500-5 C

18

PPN 1 – 9 250 22 40

Polymer-based phases

NUCLEOGEL

®

RP 80-10 C

18

1 – 14 170 – –NUCLEOGEL

®

RP 100-5 1 – 13 180 12 –NUCLEOGEL

®

RP 300-5 1 – 13 180 43 –NUCLEOGEL

®

RP 4000-8 1 – 13 180 11 –

125 x 4 mm NUCLEOSIL® 500-5 C18 PPN, eluent: A) 0.1% TFA in H2O, B) 0.08% TFA in CH3CN

Pea

k vo

lum

e

Protein mass (Cytochrome C)

µl800

600

400

200

µg1 10 100 1 000 10 000

Mass sensitivity

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Columns based on NUCLEOSIL

®

for the separation of proteins/peptides and oligonucleotides

NUCLEOSIL

®

MPN

This type of RP phases is based on NUCLEOSIL

®

silica

monomerically

bonded with alkyl chains. This flexiblebrush-type structure guarantees high selectivities. The pre-dominantly hydrophobic forces are supplemented by a smallportion of hydrophilic interactions. This is a prerequisite forthe successful separation of e.g. peptides with equal molecu-lar weight but different net charges. The 3 µm silica with C

18

modification shows an outstanding selectivity for peptides,while the wide pore C

4

material is especially suited for thepurification of larger, hydrophobic peptides and very differentproteins. If possible the columns should be used in the pHrange between 2 and 8.

Separation of hemoglobin chainsColumn: 250 x 4 mm NUCLEOSIL® 300-5 C4 MPNEluent A: 20% acetonitrile, 80% water, 0.1% TFAEluent B: 60% acetonitrile, 40% water, 0.1% TFAGradient: from 40 to 60% B in 60 minFlow rate: 1 ml/minDetection: UV, 220 nmPeaks:1. Hem2. β-globin3. α-globin4. AγT-globin5. Gγ-globin6. AγI-globin

Abs

orba

nce,

220

nm

1

2

3

4

5

6

0 20 40 min1082

40

Length

→

50 mm 125 mm 250 mm Guard columns


1)

NUCLEOSIL

®

100-5 C

18

MPN

Octadecyl phase, alkyl chains monomerically bonded to silica, brush type structure, particle size 5 µm, pore size 100 Å; eluent in column methanol

2 mm ID 720231.204 mm ID 720230.40 720231.40

NUCLEOSIL

®

120-3 C

18

MPN

Octadecyl phase, alkyl chains monomerically bonded to silica, brush type structure, particle size 3 µm, pore size 120 Å; eluent in column methanol

2 mm ID 720232.204 mm ID 720232.40

NUCLEOSIL

®

300-5 C

4

MPN

Butyl phase, alkyl chains monomerically bonded to silica, brush type structure, particle size 5 µm, pore size 300 Å; eluent in column methanol

2 mm ID 720045.20 721113.304 mm ID 720244.40 720045.40 720245.40 721113.40

ChromCart

®

guard column cartridges (8 mm) in packs of 2, all other cartridges in packs of 1.

1)

As guard columns for EC columns use ChromCart

®

guard column cartridges with guard column adaptor EC (Cat. No. 721359).

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Columns based on NUCLEOSIL

®

for the separation of proteins/peptides and oligonucleotides

NUCLEOSIL

®

PPN

These columns are packed with silica with a

polymeric

alkylmodification on NUCLEOSIL

®

silica. The polymeric layer en-sures that peptides and proteins are exclusively retained byhydrophobic forces. Polypeptides which differ in shape andsize of their hydrophobic surface regions are separated.Thus these columns feature different selectivities comparedto the MPN material, a fact which may improve the solution

of certain separation problems. With NUCLEOSIL

®

100-5C

18

PPN good peak shapes are also found for basic pep-tides. The wide pore NUCLEOSIL

®

PPN phases are available asoctadecyl modification, which is especially suited for largepeptides and medium-size hydrophilic proteins. Memory ef-fects are reduced. The PPN columns can be used with eluentcontaining organic bases up to pH 9.5.

The polymeric coating of NUCLEOSIL

®

100-5 C

18

PPNshows outstanding stability at alkaline pH and the absence ofnonspecific interactions with alkaline or acidic compounds.Therefore, this column is suited for the separation of pep-tides and proteins up to about 40 kD. The column may be pu-rified by treatment with SiO

2

-saturated NaOH. 10 to 20 col-umn volumes of the alkaline eluent should be enough towash away sticking impurities from the column. This proce-dure will have a sterilising effect, too. Retention times, peakshapes and mass recoveries are not affected by passingmore than 300 column volumes of the alkaline eluentthrough the column as is shown on the next page.

Length

→

50 mm 125 mm 250 mm Guard columns

NUCLEOSIL

®

100-5 C

18

PPN

Octadecyl phase, alkyl chains polymerically bonded to silica, particle size 5 µm, pore size 100 Å; eluent in column methanol


1)

2 mm ID 720251.20 720252.20 721594.304 mm ID 720250.40 720251.40 720252.40 721594.40


10 mm ID 720253 720254

NUCLEOSIL

®

500-5 C

18

PPN

Octadecyl phase, alkyl chains polymerically bonded to silica, particle size 5 µm, pore size 500 Å; eluent in column methanol


1)

4 mm ID 720256.40 720257.40 720258.40 721687.40


10 mm ID 720259 720260Corresponding VarioPrep columns can also be supplied on request.

ChromCart

®

guard column cartridges (8 mm) in packs of 2, all other columns in packs of 1.

1)

As guard columns for EC columns use ChromCart

®

guard column cartridges with guard column adaptor EC (Cat. No. 721359).

Amount of NUCLEOSIL

®

packing per column dimension for MPN and PPN phases:

50 x 4 mm 0.24 g125 x 2 mm 0.15 g 125 x 4 mm 0.6 g 125 x 10 mm 3.6 g250 x 2 mm 0.3 g 250 x 4 mm 1.2 g 250 x 10 mm 7.2 g

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Separation of protein standardsleft chromatogram: before treatment, right chromatogram: after treatment with 300 column volumes of the alkaline eluent (= 1 volume 50 mM NaOH + 1 volume n-propanol, saturated with SiO2 by stirring the mixture with 1 g/l unmodified silica gel for 1 day) at a flow rate of 0.4 ml/min Column: 125 x 4 mm NUCLEOSIL® 100-5 C18 PPNEluent A: A) 0.1% TFA in H2O, B) 0.08% TFA in CH3CNGradient 20 – 60% B in 10 minFlow rate: 1.0 ml/minDetection: UV, 280 nmPeaks:1. Ribonuclease2. Cytochrome c3. Lysozyme

4. β-Lactoglobulin5. Ovalbumin

30 min 30 min

1082

20

1

2

3

45

6

7

5 10 15 20 min

1082

80

Separation of pancreatic secretion of pigletsColumn: 125 x 4 mm NUCLEOSIL® 500-5 C18 PPNEluents: A) 0.1% TFA in H2O,

B) 0.08% TFA in CH3CNGradient: linear 30 – 50% B

in 14 min, then 50 – 65% Bin 6 min

Flow rate: 1 ml/minDetection: UV, 215 nmPeaks:1. Trypsin + trypsinogen2. Proelastase3. Lipase + α-chymotrypsin4. Chymotrypsinogen5. α-Amylase6., 7. Procarboxypeptidase

Separation of commercial bacitracinColumn 125 x 4 mm NUCLEOSIL® 100-5 C18 PPNEluent A: 0.1% TFA in H2OEluent B: 0.08% TFA in CH3CNGradient: linear 20 – 40% B in 15 minFlow rate: 1 ml/minDetection: UV, 215 nm

0 10 20min1152

10

Separation of a 20mer oligonucleotide(Sample courtesy of Dr. Essrich, Inst. Prof. Seelig, Karlsruhe, Germany)Column 250x 4 mm NUCLEOSIL® 100-5 C18 PPNEluent A: 0.1 M triethylammonium

acetate pH 7.0 – aceto-nitrile (95:5, v/v)

Eluent B: 0.1 M triethylammonium acetate pH 7.0 – aceto-nitrile (30:70, v/v)

Gradient: linear 15 – 40% B in 20 min

Flow rate: 1 ml/minDetection: UV, 290 nm

1

2

3

0 5 10 15 min1074

20

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Polymer-based RP columns

NUCLEOGEL® RP 80-10 C18 columns

These polymer-based columns have been developed for re-versed phase chromatography in the pH range from 1 to 14.They contain a C18 modified polystyrene-divinylbenzene pol-ymer with outstanding stability, inertness and improved peaksymmetry even for basic substances. Only simple eluent sys-tems are required for the separation. Due to improved sensi-tivity post-column derivatisations are seldom necessary.

NUCLEOGEL® RP columns

This type of reversed phase column is based on a polysty-rene resin cross-linked with DVB. Due to the excellent stabili-ty of the particles compared to other resins operation underreversed phase conditions is possible. The pH range applica-ble with these columns reaches from pH 1 to pH 13.The small pore columns for reversed phase separation ofsmall molecules are especially suited for pharmaceuticalcompounds with basic properties, such as organic heterocy-cles. They can also be used for the separation of nucleosidesand nucleotides up to 5000 dalton and allow gradient as wellas isocratic elution.The wide pore columns are especially recommended forlarge biomolecules. For proteins they show good peakshapes and selectivities. Compared to a silica matrix thehigher background hydrophobicity of the NUCLEOGEL® RPphases can be disadvantageous for the mass recovery ofsome large hydrophobic proteins. The peak capacity for theseparation of complex peptide mixtures is lower than forNUCLEOSIL® MPN or PPN. A working pressure of 180 barshould not be exceeded.

Separation of sympathomimetic aminesColumn: 150 x 4.6 mm NUCLEOGEL® RP 80-10 C18Eluent A: 0.1 M ammonia – acetonitrile (68 : 32)Eluent B: 0.1 M ammonia – acetonitrile (54 : 46)Gradient: linear 0 – 100% B in 5 minFlow rate: 1 ml/minTemperature: ambientDetection: UV, 230 nmPeaks (20 µl injected):1. Phenylpropanolamine2. Pseudoephedrine3. Amphetamine4. Methamphetamine

1

23

4

0 10min1029

60

Separation of cephalosporin antibioticsColumn: 150 x 4.6 mm NUCLEOGEL® RP 80-10 C18Eluent: 0.1% aqueous tetrabutylammonium bromide

solution – acetonitrile (55 : 45)Flow rate: 0.5 ml/minDetection: UV, 254 nmPeaks:1. Cefalexin2. Cefalotin3. Cefazolin4. Cefaloridine

1

2

3

4

0 15 30 min1034

60

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Polymer-based RP columns · ordering information

Length → 50 mm 150 mm 250 mm 300 mm Guard columns

NUCLEOGEL® RP 80-10 C18C18 modified PS/DVB polymer for RP separations in the pH range 1 – 14; pore size 80 Å; eluent in column CH3CN/H2O


4.6 mm ID particle size 10 µm 719500

NUCLEOGEL® RP 100Polystyrene resin cross-linked with divinylbenzene (PS/DVB); pore size 100 Å; eluent in column CH3CN/H2O


4.6 mm ID particle size 5 µm 719454 719455 7195424.6 mm ID particle size 8 µm 719456 719520 7195427.7 mm ID particle size 8 µm 719457 719542

Standard-Prep preparative columns 25 mm ID particle size 10 µm 719458



4.6 mm ID particle size 5 µm 719459 7195424.6 mm ID particle size 8 µm 719460 7195427.7 mm ID particle size 8 µm 719463 719542




4.6 mm ID particle size 8 µm 719461 719510 7195427.7 mm ID particle size 8 µm 719464 719542




4.6 mm ID particle size 8 µm 719462 7195427.7 mm ID particle size 8 µm 719465 719542



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Operation of reversed phase columns

Mobile phase: Besides selection of the column, choice ofthe eluent is very important for the success of a chromato-graphic separation. Adsorption of the biological macromole-cules is usually achieved from aqueous buffer solutions. Ad-ditives often applied are 0.05 – 0.2% trifluoroacetic acid orphosphoric acid in acidic medium and pyridine/formate orammonium acetate in the neutral pH range. These ion pair-ing reagents can either decrease or increase the polarity ofthe peptide. As organic modifiers, mainly acetonitrile, 1-pro-panol or 2-propanol are used. In this series, the elutionstrength of the solvents increases. Often a good selectivitycan be obtained with ternary systems, e.g. water / ace-tonitrile / 1-propanol.Biological macromolecules are almost exclusively obtainedby gradient elution.Column installation and operation: Columns should firstbe washed with 3 – 4 column volumes of water. Then the col-umn can be equilibrated with the corresponding buffer sys-tem. Abrupt flow and pressure surges should be avoided, be-cause they can destroy the column packing. If possible,samples are dissolved in the starting buffer and filteredthrough a 0.45 µm filter. If you have to separate an unknownsample mixture, and if there is no similar separation knownfrom literature, you can choose the chromatographic testconditions of the column as a first attempt.Column cleaning and storage: All columns described inthis chapter can be cleaned with several 10 – 20 min gradi-ents from TFA/water to 80% acetonitrile or 60% 2-propanol.Addition of 0.1 M EDTA can be of advantage in some cases.If possible, the column should be cleaned from unelutedsample components after each day of use. With NUCLEO-GEL® columns, organic impurities can also be removed byflushing with 0.1 M NaOH. The columns should be stored inan acid-free eluent with a high percentage of organic solvent,e.g. 70% methanol.For separations of different peptides, proteins or nucleic ac-ids with these columns please see our catalogue “LC Appli-cations” or visit our website:

Quantitative determination of vancomycinConcentration and decomposition of the glycopeptide antibiotic vancomycin can be quantitatively monitored with NUCLEOGEL® RP columns. The figure shows the chromatogram of a vancomy-cin sample after 15 years at 56 °C.Column: 150 x 4.6 mm NUCLEOGEL® RP 100-8Eluent A: 8% (v/v) acetonitrile

– 0.02 M borate buffer, pH 8.0Eluent B: 16% (v/v) acetonitrile

– 0.2 M borate buffer, pH 8.0Gradient: linear, 0 – 100% B

in 17.5 minFlow rate: 0.5 ml/minDetection: UV, 235 nm

0 30 min

0.05 AU

1037

00

Separation of proteinsColumn: 50 x 4.6 mm NUCLEOGEL® RP 300-8Eluent A: 0.1% TFA in acetonitrile – water (95:5)Eluent B: 0.1% TFA in waterGradient: linear, 20 – 60% A in 22 minFlow rate: 1.5 ml/minDetection: UV, 220 nmPeaks:1. Ribonuclease A2. Insulin3. Cytochrome C4. Lysozyme5. Bovine serum albumin6. Myoglobin7. Hen’s egg white

1

2

3

4

5

6

7

17 min

1082

50

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References:

1) Automated evaluation of tryptic digest from recombinant human growth hormone using UV spectra and numeric peak information H.-J. P. Sievert et al. J. Chrom. 499 (1990) 221 – 234

2) Separation and quantitative determination of high molec-ular-weight subunits of glutenin from different wheat vari-eties and genetic variants of the variety sicco W. Seilmeier, H.-D. Belitz, H. Wieser, Z. Lebensm. Unt-ers. Forsch. 192 (1991)124 – 129

3) Isocratic separation of phenylthiohydantoinamino acidsby RP-HPLC K. Hayakawa, J. Oizumi, J. Chrom. 487 (1989)161 – 166

4) Rapid method based on RP-HPLC for purification of hu-man myelin basic protein and its thrombic and endopro-teinase Lys-C peptidesG. Giegerich, M. Pette, K. Fujita, H. Wekerle, J. T. Epplen,A. Hinkkanen, J. Chrom. 528 (1990) 79 – 90

5) Non-ideal behaviour of silica-based stationary phases inTFA-acetonitrile-based RP-HPLC separations of insulinsand proinsulins S. Linde, B. S. Welinder, J. Chrom. 536 (1991) 43 – 55

6) Effects of eluent composition, ion pair reagent and tem-perature on the separation of histones by HPLC H. Lindner, W. Heiliger, Chromatographia 30 (1990) 518– 522

Rapid reversed phase separation of proteinsColumn: 50 x 4.6 mm NUCLEOGEL® RP 4000-8Eluent A: 0.1% TFA in CH3CN – H2O (5 : 95, v/v)Eluent B: 0.1% TFA in CH3CN – H2O (95 : 5, v/v)Gradient: linear, 18 – 60% B in 60 secondsFlow rate: 4 ml/minDetection: UV, 280 nmPeaks (total protein 0.34 mg)1. Ribonuclease A2. Cytochrome C3. Lysozyme4. Bovine serum albumin5. Myoglobin6. Ovalbumin

1

2

3

4

5

0 60 s

610

8250

Rapid reversed phase separation of peptidesColumn: 50 x 4.6 mm NUCLEOGEL® RP 4000-8Eluent A: 0.1 % TFA in acetonitrile – water (1 : 99)Eluent B: 0.1 % TFA in acetonitrile – water (99 : 1)Gradient: linear, 10 – 60% B in 2 minFlow rate: 4 ml/minDetection: UV, 220 nmPeaks:1. Neurotensin

fragment 1-82. Neurotensin

fragment 8-133. Neurotensin 4. Myoglobin

1 2

3

4

0 60 120s

1079

60

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Gel filtration columns for biochemical separations

NUCLEOGEL® GFC columns for the separation of natural polymers and proteinsGel filtration chromatography (GFC) as a special form ofsize exclusion chromatography requires pressure-stable highperformance columns for the separation of biopolymers ac-cording to size and shape. MACHEREY-NAGEL offers HPLCcolumns for GFC based on polymer resins.NUCLEOGEL® GFC columns can be applied for the analyti-cal and preparative purification of proteins, nucleic acids,polysaccharides and other hydrophilic polymers. These col-umns contain a hydrophilic polymer matrix. Organic solventssuch as methanol or acetonitrile can be used as modifiers.The different pore sizes permit separations up to a molecularweight of 107 daltons (see calibration curves on page 116). The homogenous surface of the hydrophilic polymer parti-cles avoids nonspecific interactions of the macromoleculeswith the column matrix. Salt concentrations up to 0.5 M canbe used without interference of the chromatographic processbe hydrophobic interactions.

The figures on the following pages show characteristic pa-rameters of these stationary phases.

Characteristic parameters or the column packing

working range for polysaccharides/dextran [daltons]NUCLEOGEL® GFC 300 100 – 100 000NUCLEOGEL® GFC 1000 20 000 – 2 000 000NUCLEOGEL® GFC 4000 100 000 – 20 000 000pH working range 2 – 10maximum salt concentration 8 Mpolar organic solvents (modifiers) 0 – 100%maximum temperature 80 °C

Separation of nucleic acidsColumns: 2 times 300 x 7.7 mm NUCLEOGEL® GFC 4000-8Eluent: 0.05 M NaH2PO4, 0.2 M NaCl, pH 6.5, 10% ethanolFlow rate: 0.3 ml/minDetector: UVPeaks:1. Plasmid2. rRNA

1

2

10 30min1075

10

Separation of polysaccharide calibration standards

2 times 300 x 7.7 mm NUCLEOGEL® GFC 4000-8 + 300 x 7.7 mm NUCLEOGEL® GFC 300-8, eluent: 0.02 M NaH2PO4, 0.2 M NaCl, pH 7.0, flow rate 1.0 ml/min, RI detector, ambient temperature1. Pullulan 853 0002. Pullulan 186 0003. Pullulan 23 7004. Pullulan 5 8005. Glucose

1. Dextran T 5002. Dextran T 1503. Dextran T 704. Dextran T 40

1

23

4

5

14 28min

1

2

3

4

18 28min1025

60

1025

70

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Separation principle and operation of NUCLEOGEL® GFC columns

Separation principleDepending on their size and shape substances can pene-trate more or less into the pores of the column packing, re-sulting in different retention times on the column.Mobile phaseChoice of the eluent normally does not cause any problemsin gel filtration chromatography. Frequently phosphate buff-ers (pH 6.5) with NaCl concentrations between 0.1 and0.3 M are applied for the separation of proteins. For certainpolymer mixtures, e.g. chitosan (a carbohydrate), acidic pHvalues and higher salt concentrations can be of advantage.In some difficult cases addition of organic solvents or deter-gents can improve the resolution.

Column installation and operationColumns are supplied equilibrated with water / 0.2% sodiumazide. Thus they can be immediately used with the requiredbuffer. When working with organic modifiers, the eluentshould be checked for possible precipitations prior to use.Drastic pressure and flow changes should be avoided be-cause they can destroy the column packing. Columns can beused in the temperature range between 4 and 80 °C. Thesample volume should not exceed 1 – 2% of the column vol-ume. Optimum separations are obtained with low flow rates.For analytical columns e.g. 0.1 – 0.5 ml/min are recommend-ed. The lifetime of the columns can be considerably in-creased by the use of guard columns.Column cleaning and storageColumns should be flushed with 5 – 10 column volumes wa-ter / 0.2% sodium azide and stored well closed at ambienttemperature.

Length → 300 mm Guard columns

NUCLEOGEL® GFC 300hydrophilic gel matrix; pore size 300 Å; eluent in column H2O + 0.02% NaN3

Valco type analytical columns7.7 mm ID particle size 8 µm 719447 719450


NUCLEOGEL® GFC 1000, as above, but pore size 1000 Å

Valco type analytical columns7.7 mm ID particle size 8 µm 719448


NUCLEOGEL® GFC 4000, as above, but pore size 4000 Å

Valco- type analytical columns7.7 mm ID particle size 8 µm 719449


For NUCLEOGEL® aqua-OH columns for exclusion chromatography of water-soluble compounds please see the chapter ”GPC“ from page 129.

Columns are supplied in packs of 1.Valco type guard columns NUCLEOGEL® GFC are 50 x 7.7 mm and supplied in packs of 1.

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Calibration curves for polysaccharides and proteins on NUCLEOGEL® GFC 300-8Column: 300 x 7.7 mm NUCLEOGEL® GFC 300-8Eluent: 0.02M NaH2PO4, 0.2 M NaCl, pH 7.0Flow rate: 1.0 ml/minDetection: UV 280 nm

Pullulan polysaccharidesProteins

1. Cytochrome C 2. Ribonuclease A3. β-Lactoglobulin4. Ovalbumin5. BSA6. γ-Globulin7. β-Galactosidase

Mol

ecul

ar w

eigh

t

Elution volume [ml]

1 M

100 K

10 K

1 K

4 5 6 7 8 9

12

3

45

67

Purification of a commercial catalase product2 columns 300 x 7.7 mm NUCLEOGEL® GFC 300-8, eluent: 0.02 M NaH2PO4, 0.2 M NaCl pH 7.0, flow rate 0.5 ml/min, UV detector

20 44min

1151

50

Calibration curves for NUCLEOGEL® GFC columns of different pore sizes Columns: 300 x 7.7 mm NUCLEOGEL® GFCEluent: waterFlow rate: 1.0 ml/min

ambient temperatureCalibration substances: pullulan polysaccharides

Mol

ecul

ar w

eigh

t

Elution volume [ml]

10 M

1 M

100 K

10 K

1 K

4 6 8 10

300 Å

1000 Å

4000 Å

Separation of standard proteins300 x 7.7 mm NUCLEOGEL® GFC 300-8, eluent: 20 mM NaH2PO4, 200 mM NaCl pH 7,0, flow rate 0.1 ml/min, UV detec-tor 1. Ferritin2. Albumin (bovine

serum albumin)3. β-Lactoglobulin4. Cytochrome C5. Insulin6. Uridine

1

2

3

4

5

6

60 75 90 min1082

30

columns for hplccolumns for hplc - western · pdf filecolumns for hplccolumns for hplc 95...

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