Basic principles of preparative HPLC - Winlab 2.pdf · MN Columns for HPLCColumns for HPLC Basic...

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Columns for HPLCColumns for HPLC

Basic principles of preparative HPLC

61

The efficiency of HPLC for separation of one or several com-ponents from a complex mixture makes HPLC an importanttechnique for preparative purifications. The difference be-tween analytical and preparative HPLC concerns the aim ofthe separation. In analytical HPLC the aim is to separate allindividual components of a mixture as completely as possi-ble with subsequent identification of the peaks. In general,sample sizes are small. For 4 mm ID, typical sample sizesare 1 – 100 µg analyte per g adsorbent in normal phase col-umns and 10 – 1000 µg analyte per g adsorbent in RP col-umns. For columns with smaller inner diameters correspond-ingly smaller samples are applied. Thus

analytical HPLC

often requires

maximum separation efficiency

of a column.Due to the small inner diameter, expenses for solvents andpackings are low, with the result that in analytical HPLCcosts for separation time (= solvent consumption) and pack-ing material can be almost neglected for method develop-ment. On the contrary, in

preparative HPLC

development ofa separation often involves detailed economical-chemical op-timisation calculations. Due to the column dimensions, costsfor solvents and packings or prepacked columns becomemore and more important with increasing column diameters.The aim of HPLC now is

isolation of the desired productwith defined purity, in maximum amounts and with mini-mum time

. The important parameter is called productionrate or throughput. This chapter discusses the most important parameters,which have to be taken into account in preparative methoddevelopment. Definition of the production rate includes infor-mation about the required purity of the isolated product. Thatis, the user has to define the minimum resolution of the prod-uct peak from neighbouring peaks. The parameters deter-mining the resolution between two peaks are relative reten-tion

α

(ratio of both k’ values), the plate number of thecolumn and the k’ value (formulas see chapter ”Basic termsand definitions“ on page 170). The relative retention is determined by the chromatographicsystem, i.e. mobile and stationary phases. Proper choice ofcolumn and eluent will produce the required selectivity andresolution of the chromatographic system for the sample tobe separated. In practice, one often has to use a non-optimalsystem, e.g. because of sample solubility or because ofincompatibility of a chromatographically desirable eluent withthe following work-up or application steps of the isolatedproduct after chromatography.For a detailed discussion of the term resolution please referto the paper by L.R. Snyder

1)

. For maximising throughput,preparative HPLC columns are often overloaded (for detailsconcerning overload phenomena see below). Under theseconditions the resolution depends on the sample mass andon the injection volume. With increasing feed volume V

0

theresolution remains almost constant, until from a certain valueit decreases linearly with further increase of the feed volume(for details about volume overload see below).

Typical relation between feed volume and resolution

w wmin

R Rmax

V0

wwmin

V0= peak width= minimum peak width

1

= resolution= maximum resolution

= feed volume

RRmax

V0

1

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62

When speaking about the production rate of a preparativeseparation, the term

loadability

of the column should beconsidered, too. According to general understanding, this isthe maximum sample size (with defined sample mass andvolume) under which a column still provides optimum selec-tivity. The chemical literature contains several propositionsfor definition of the loadability

3)

–

6)

. In practice, however,these theoretical approaches are seldom used, because themaximum injection size is determined empirically: one injectsincreasing amounts, until the peaks just touch. However, itshould be noted, that for the same injection size the loadabil-ity for dilute samples is higher than for concentrated ones

7)

.The parameters which are important for the optimisation ofthe

mass loadability

of a column can be described by thefollowing formula.

The significance of the individual parameters can be easilyseen. However, two terms should be noted in detail: columnlength l and particle diameter d

P

. As can be seen from the term d

P2

/l, the mass loadability ofthe column decreases with increasing plate number (l/d

P2

isproportional to the plate number N). Experimentally, this rela-tion can be easily shown with columns of different plate num-bers.

The preceding diagram shows the dependence of the sepa-ration efficiency of two columns from the mass loading. Thetwo columns were run under the same conditions, but withpackings of different particle size (d

P

)

8)

If an increased loadability is required for a given separationefficiency, it is recommended to increase particle size andcolumn length, the increase in column length being thesquare of the increase in particle diameter. This will producea welcome side effect: the relative permeability of the columnwill also increase quadratically, and the pressure drop of thecolumn will decrease quadratically as shown in the tablebelow.

As can be seen from the following formula, the

volume load-ability

linearly depends on the dead volume of the columnused, and otherwise it only depends on the k’ values of thecomponents to be separated and on the separation effi-ciency of the column.

M C1πr2lKdAS C2

dP2

l------=

M C1, C2r l K d AS dP = particle diameter

= adsorbent surface= packing density= partition coefficient= column length= column radius= constants= maximum sample mass

Mass loadability of a column

g (component i)g (adsorbent)

-----------------------------------------

column 1

column 2

mass loading

loadability column 1

loadability column 2

N

Influence of particle size and column length on loadabili-ty and permeability for constant separation efficiency

l [cm] d

p

[µm] rel. loadability rel. permeability

25 10 1.0 1.0

50 14 1.4 2.0

75 17 1.7 3.0

100 20 2.0 4.0

VL V0 α 1–( )k′A2

N--------– 2 k′A k′B+ +( )=

Volume loadability of a column 3)

V0

VL

αNk’A, k’B = capacity factors

= plate number= relative retention (k’B/k’A)= maximum overload volume= dead volume


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63

In addition to the loadability of the column used for a prepar-ative separation another consideration can be important forthe optimisation of the production rate

9)

. The production rateis directly proportional to the column diameter, the linear flowvelocity of the mobile phase, the concentration of the compo-nent to be isolated (unless under mass overload conditions)and the term [1/N–H

0

/l]

1/2

, where H

0

is the plate height of thecolumn under ideal conditions, l is the column length, and Nis the plate number required for separation of the desiredproduct with the purity required. The following limiting casescan be distinguished:

l < H

0

N:

Here a negative value is generated under the squareroot with the result of a physically meaningless produc-tion rate: the plate number of the column is smaller thanrequired.

l = H

0

N:

The column plate number is just sufficient for the separa-tion. The corresponding column length is a critical valuewhere the production rate is zero.

l > H

0

N:

Further increase of the column length results in increas-ing production rates.

The relation between column length and production rate canbe seen in the diagram above, where typical values for pro-duction rates are plotted versus column length. The figureshows different curves for columns with different particlesizes and hence different separation efficiencies. It should benoted, that after a steep rise at relatively short columnlengths the slope of these curves decreases drasticallyapproaching a saturation value. This is reached when 1/N ismuch larger than H

0

/l.

Since in practice optimisation of the production rate of adesired substance often results in overload conditions of thecolumn, we briefly wish to discuss the related phenomena.

Typical cases, which can cause

column overload

condi-tions, are e.g.

samples with low solubility in the mobile phase or injec-tion of too large a sample volume,

highly concentrated samples, samples which are dissolved in a solvent with much bet-ter solvation characteristics than the mobile phase. Heresample concentration at the column inlet can causeproblems.

When considering overload phenomena, one has to distin-guish between concentration overload, volume overload andmass overload conditions.

Concentration overload

The concentration of the analytes in the injected samplesolutions is increased, while the injection volume is kept con-stant. With increasing overload peaks are more and moredistorted, with the peak shape approaching a triangle. Front-ing as well as tailing can occur. Simultaneously, with increas-ing overload the peak maxima are shifted, in most casestowards the dead time. This type of overload is called

con-centration overload

. A concentration overload is only possi-ble, if the solubility of the analytes in the sample solution islarge enough.

Volume overload

If the solubility of the analytes is low, overload can only beobtained if, for a given sample concentration, the injectionvolume is continuously increased. Increasing sample volumeresults in peak broadening, approaching a rectangularshape. However, peaks remain symmetrical and from a cer-tain overload peak heights remain constant. This type ofoverload is called

volume overload.

A characteristic of volume overload is constant retention vol-umes of the peak fronts even under overload conditions

3)

.The rectangular peak shape obtained by volume overload isshown in the following chromatograms.

Pro

duct

ion

rate

[mg/

h]

particle size [µm]

column length [m]

0

10

20

30

0 0,5 1 1,5 2 3 4 5

5 7 10

32,5


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64

Volume overload is demonstrated for the separation of a 3-component mixture of benzene, naphthalene and anthra-cene and a 2-component mixture of naphthalene and anthra-cene. The masses of the solutes injected were kept constantin all cases.

The constant retention volumes of the peak fronts combinedwith the rectangular peak shapes, however, allow a peak as-signment with the aid of frontal analysis even for an other-wise insufficient resolution. When injecting such large vol-umes, certain precautions have to be taken when employinginjection procedures with valve and sample loop. Improperinjection can result in the concentration profile of the sampletaking the form of a Poisson curve which will make frontalanalysis difficult or impossible

3)

. For the decrease of separation efficiency under volume over-load conditions one can make the following estimate:The plate heights will increase by about 20% when the sam-ple volume is about 1.5% of the column void volume, andthey will about double when the sample volume increases toabout 3 – 3.5%

5)

.

3-component mixture 2-component mixture

sample volume 1 µl

sample volume 1 ml sample volume 2 ml



sample volume

sample volume

sample volume 6 ml

sample volume 16 ml

Transition from elution development towardsfrontal analysis caused by volume overload

10 µl 500 µl

2 ml 3 ml

A

BC

A B

C

A B C A B C

A B CB+C

A A+B CA+B+C

B+C


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65

Mass overload

If the injected sample mass (calculated from injection volumeand concentration) exceeds a certain value, the local con-centration of the sample in the column can be so large, thatequilibration is no longer possible: this case is called

massoverload

.

Mass overload

is much more complex than volume overloadand is based on three main effects

3)

:

Dispersion effects.

The sample is distributed along thecolumn by the mobile phase until it contacts sufficientadsorbent to permit equilibrium with the stationaryphase. This phenomenon results in band spreading sim-ilar to volume overload.

Deactivation of the adsorbent.

In addition to disper-sion effects a massive sample charge will occupy a sig-nificant portion of the stationary phase resulting in achanged effective polarity of the mobile phase comparedto the stationary phase. This can cause reduced reten-tion which will also affect components of the sample withlower concentration.

Non-linear adsorption isotherms

, caused by largesample concentrations, result in peak tailing.

Thus, mass overload can be recognised by changed reten-tion volumes of the peak fronts of all components of a mix-ture to be separated. Peak broadening and tailing will mainlyaffect the sample components which are present ”in excess“relative to the column load capacity (see chromatograms onthe left).

The figure shows mass overload for the separation of ben-zene, naphthalene and anthracene. The amount of benzeneinjected is increased from 180 µg via 8.1 mg to 16.9 mg,while the amounts of naphthalene and anthracene are keptconstant.

If the aim of a preparative separation is to obtain as much ofthe pure compound per time unit as possible, overload of thecolumn will in most cases be necessary.

In practice combi-nations of concentration and volume overload are mostoften used.

With diluted samples volume overload will occurmore often, while concentrated sample solutions will show atendency towards concentration and mass overload. Oftenboth effects are present and peaks approach the shape of atrapezoid. Concentration overload is to be preferred,because it allows separation of larger sample amounts.

References:

1) L.R. Snyder, J. Chromatogr. Sci.

10

(1972) 200 and 369

2) A. Wehrli, U. Hermann and J. F. K. Huber, J. Chromatogr. 125

(1976) 59

3) R. P. W. Scott, P. Kucera, J. Chromatogr.

119

(1976) 4674) L. R. Snyder, Anal. Chem.

39

(1967) 6985) W. Beck, I. Halasz, Z. Anal. Chem.

291

(1978) 3406) T. Roumeliotis, K. K. Unger, J. Chromatogr.

185

(1979)445

7) J. J. De Stefano, H. C. Beachell, J. Chromatogr. Sci

10

(1972) 654 8) A. W. J. De Jong, H. Poppe, J. C. Kraak, J. Chromatogr.

209

(1981) 4329) K. P. Hupe, H. H. Lauer, J. Chromatogr.

203

(1981) 4110)V. R. Meyer, Praxis der Hochleistungs-Flüssigchromatog-

raphie, Otto Salle Verlag, Frankfurt, 7. Aufl., 1992

Mass overload for the separation of benzene, naphthalene and anthracene

AB N

A

B

NA

B

N


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Scale-up from analytical to preparative HPLC

66

For separation of larger amounts of a substance there aretwo general approaches:

linear scale-up of the analytical systemoverloading the column

In the case of linear scale-up the column length is kept con-stant and the column cross section is increased proportion-ally to the sample mass. Eluent flow and sample volume areadapted correspondingly. As in analytical HPLC, stationaryphases with small particle sizes are used. The separationefficiency of the prep column is more or less the same com-pared with the analytical column. Peaks remain sharp andsymmetrical.The procedure of linear scale-up is not very economical, sincelarge columns and large amounts of eluents are required, andthe substance yield is relatively low. For example, more than5 kg stationary phase and 50 l mobile phase are necessary toseparate less than 1 g of a substance mixture in one run. Forthis reason linear scale-up alone is only used in exceptionalcases, e.g. if for difficult separations the high separation effi-ciency is needed for the isolation of (in most cases smallamounts of) substances with very high purity.A better way is overloading the column, since for a givenyield less stationary phase and less eluent are required. Thisprocedure allows injection and separation of amounts in thelower milligram range on an analytical HPLC column. Forpreparative separations on the gram or kilogram scale over-loading is combined with a linear scale-up of the chromato-graphic system. However, overloading a column always means a considera-ble loss of separation efficiency. For this reason, overloadingthe chromatographic system must show sufficient resolution.Consequently, for optimising a preparative separation,the chromatographic selectivity has to be optimised

under analytical conditions to reach a resolution as highas possible. The higher the selectivity for a given phase sys-tem, the more a column can be overloaded. For preparativeseparations under overload conditions stationary phaseswith larger mean particle sizes are usually sufficient. Prereq-uisite for a successful transfer from established analyticalmethods to the preparative scale are stationary phases withequal selectivity for both methods.However, heavy overload can also change the chromato-graphic selectivity relative to analytical conditions.

Procedure for scale-upIn general, an analytical chromatogram is used as a startingpoint for a preparative separation. It is necessary to find con-ditions, which separate the sample mixture isocratically withgood resolution, since

gradients are not recommended for preparative separa-tions, because they require large efforts in every re-spect. If necessary, proper sample preparation may berequired.The better the resolution in the analytical chromatogram,the larger the load on the preparative column can be.

After optimisation of the separation on the analytical columnthe maximum sample amount for injection of a concentratedsolution is determined empirically. In the simplest case over-load is increased, until the peaks in the chromatogram of theseparation just do not yet overlap, allowing isolation of thepeaks and, after removal of the mobile phase, obtaining thepure substances with 100% yield. Undiluted solutions are notfavourable. It is not recommended to dissolve the sample in astronger solvent than the mobile phase. Since for preparativework in most cases larger volumes are injected, the solventcan severely interfere with the equilibrium in the column,even making reproducibility of the chromatographic systemimpossible. Solubility of the sample in the mobile phase hasto be good, because otherwise the column may get plugged.

Scale-upfactor

amount of adsorbentsample volume

amount of analyteflow rate

} = f(ID)


MN


Scale-up from analytical to preparative HPLC

67

The scale-up factorAfter optimisation of the analytical separation the preparativecolumn is to be considered. Transfer from an analytical to apreparative separation is easiest, if both are packed with thesame stationary phase (linear scale-up).A key for successful transfer of results gained for the analyti-cal column to the preparative scale is the proper scale-upfactor. If the analytical or the preparative factor is not listed inthe table below, it can be calculated form the following for-mula:

M = sample massL = column lengthd = column diametera analytical columnp preparative column

Now every parameter relevant for the separation has to bemultiplied with the scale-up factor. This includes flow rate,sample volume, sample mass, and the IDs of the capillaries.It is important during scale-up to increase the inner diame-ters of the capillaries proportionally, since otherwise theback-pressure of the system can become to large. For ourprogramme of capillary tubing for HPLC please see thechapter ”Accessories“ on p. 166)

Mp Ma

Lp dp2

×

La da2

×------------------×=

Scale-up factors and parameters for typical MN column dimensions

Column dimensions [mm] 4 x 250 8 x 250 10 x 250 16 x 250 21 x 250 40 x 250 50.8 x 250 80 x 250

Particle sizes [µm]NUCLEOSIL®

NUCLEODUR®5, 7, 10

55, 7, 10,

10, 12, 16, 20, 30, 50

5, 7, 10, 10, 12, 16, 20, 30, 50

5, 7, 10, 10, 12, 16, 20, 30, 50

5, 7, 10, 10, 12, 16, 20, 30, 50

5, 7, 10, 10, 12, 16, 20, 30, 50

5, 7, 10, 10, 12, 16, 20, 30, 50

5, 7, 10, 10, 12, 16, 20, 30, 50

Linear scale-up factor 1 4 6.25 16 27.6 100 161.3 400

Typical sample mass * [mg] 0.02 – 2 0.08 – 8 0.15 – 13 0.3 – 35 0.6 – 60 2 – 210 3 – 350 10 – 850

Amount of packing / column [g ±20%] 2 8 13 35 60 210 350 850

Typical flow rate [ml] 0.5 – 1.5 2 – 6 3 – 9 8 – 24 14 – 40 50 – 150 80 – 250 200 – 600

* for RP material; the maximum amounts given here always depend on the separation problem and on the sample composition.In some cases even half of the amounts given can cause dramatic overload, in other cases the maximum amounts can stillgive acceptable separations.


MN


MN columns for preparative HPLC

68

As with our analytical columns, our preparative columnsfeature capillary connections with UNF inner threads 10-32 (1/16”), thus meeting today’s standard in HPLC technol-ogy. They consist of stainless steel and are packed withNUCLEOSIL® or NUCLEODUR® packings from our rangeof silicas for HPLC. All packed columns are individuallytested and supplied with the corresponding test certificate. We offer three different types of preparative columns: Standard-Prep, VarioPrep® and Ecoprep. Standard-Prep columns are available in lengths of 125 to500 mm and inner diameters of 10 mm, 21 mm and50.8 mm. Standard-Prep guard columns are 50 mm long andavailable with the same inner diameters.Thus for scale-up the user can choose from a range of abouttwo orders of magnitude in column diameters from the ana-lytical 4 mm ID columns up to the 50.8 mm ID prep columns.VarioPrep® columns have been developed in order to allowcompensation of the dead volume, which could result at thecolumn inlet after some time of operation, without need foropening the column. This special column technology is avail-able as columns with one adjustable end fitting at the columninlet and a fixed end fitting at the column end. On request,we can also supply columns with two adjustable end fittings,an option which may e.g. be useful for frequent use of back-flushing techniques.VarioPrep® columns are available with lengths of 125 and250 mm and inner diameters of 10, 21, 40 and 80 mm. Vario-Prep® guard columns are 50 mm long and available withinner diameters of 10, 21 and 40 mm.EcoPrep columns are available with lengths of 30, 70, 125and 250 mm. The column head is an enlarged version of theanalytical EC columns (s. p. 15). The inner diameters areintermediate sizes of 8 and 16 mm. These columns areespecially suited for SMB systems. Shorter columns (individ-ually tested) and guard columns are available on request.On request, our preparative columns can also be cus-tom-packed with other types of NUCLEOSIL® packings as well as with NUCLEODUR® silicas. In addition, they can be made in lengths other than those indicated in the tabulated summary on pages 69 to 72.

Preparative columns

different types of column end fittings

1 2

3

5

1 Standard-Prep column with 10 mm ID2 Standard-Prep column with 21 mm ID3 VarioPrep® column with 21 mm ID4 EcoPrep column with 16 mm ID

Standard-Prep column with 2” ID

(flanged end fitting)

4

EcoPrep columns


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69

Ordering information

Length → 30 mm 70 mm 125 mm 250 mm Guard columns 50 mm

Base deactivated RP phases

NUCLEOSIL® 100-5 C18 HDParticle size 5 ± 1.5 µm, pore size 100 Å; octadecyl phase, endcapped, monomeric coating, 20% C; eluent in column acetonitrile / water

Standard-Prep columns 10 mm ID 71538721 mm ID 715386

VarioPrep® columns 10 mm ID 71584921 mm ID 715697 715851

EcoPrep columns 8 mm ID 715290.80 715291.80 715292.80 715293.80

NUCLEOSIL® 100-7 C18 HDParticle size 7 ± 1.5 µm, pore size 100 Å; octadecyl phase, endcapped, monomeric coating, 20% C; eluent in column acetonitrile / water

EcoPrep columns 16 mm ID 715311.160 715312.160 715313.160 715314.160

NUCLEOSIL® 100-5 PROTECT IParticle size 5 ± 1.5 µm, pore size 100 Å; special RP phase, endcapped, monomeric coating, 11% C; eluent in column acetonitrile / water

VarioPrep® columns10 mm ID 715307

EcoPrep columns 8 mm ID 715304.80

16 mm ID 715304.160On request, all preparative columns are available with any NUCLEOSIL® or NUCLEODUR® packing.

Standard-Prep columns

Preparative columns packed with NUCLEOSIL®

Each column is individually tested and supplied with test chromatogram and test conditions


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70

Standard octadecyl phases

NUCLEODUR® 100-5 C18 ecParticle size 5 ± 1.5 µm, pore size 100 Å; octadecyl phase, endcapped, 17.5% C; eluent in column acetonitrile / water

VarioPrep® columns10 mm ID 76200121 mm ID 762002

NUCLEOSIL® 100-5 C18 Particle size 5 ± 1.5 µm, pore size 100 Å; octadecyl phase, endcapped, 15% C; eluent in column acetonitrile / water



Standard-Prep columns10 mm ID 715002 71520321 mm ID 715020 715205

50.8 mm (2”) ID 715050VarioPrep® columns

10 mm ID 715809 715802 71582721 mm ID 715659 715652 71566640 mm ID 715691

EcoPrep columns 8 mm ID 715330.80 715331.80 715332.80

16 mm ID 715330.160 715331.160 715332.160


EcoPrep columns 16 mm ID 715274.160 715273.160


Standard-Prep columns10 mm ID 715113

NUCLEOSIL® 300-7 C18 Particle size 7 ± 1.5 µm, pore size 300 Å; octadecyl phase, endcapped, 6.5% C; eluent in column acetonitrile / water

Standard-Prep columns10 mm ID 71502521 mm ID 715031




On request, all preparative columns are available with any NUCLEOSIL® or NUCLEODUR® packing.

NEW



MN


71

Standard octyl phases

NUCLEOSIL® 100-7 C8 Particle size 7 ± 1.5 µm, pore size 100 Å; octyl phase, not endcapped, 8.5% C; eluent in column acetonitrile / water




16 mm ID 715630.160

NUCLEOSIL® 300-7 C8 Particle size 7 ± 1.5 µm, pore size 300 Å; octyl phase, not endcapped, ~3% C; eluent in column acetonitrile / water



Standard butyl phases

NUCLEOSIL® 120-7 C4 Particle size 7 ± 1.5 µm, pore size 120 Å; butyl phase, endcapped, eluent in column acetonitrile / water






VarioPrep columns


MN


72

On request, all types of preparative columns can also be packed with other NUCLEOSIL® or NUCLEODUR® phas-es.

On request, VarioPrep columns are also available with two adjustable end fittings, with 80 mm ID or with differ-ent lengths.

For preparative HPLC columns for special separation problems please see the following chapter ”Columns for special applications“

NUCLEOSIL® 300-7 C4 Particle size 7 ± 1.5 µm, pore size 300 Å; butyl phase, endcapped, ~2% C; eluent in column acetonitrile / water


VarioPrep® columns10 mm ID 715804 71597121 mm ID 715654

Standard phenyl phases

NUCLEOSIL® 100-7 C6H5 Particle size 7 ± 1.5 µm, pore size 100 Å; phenyl phase, not endcapped, eluent in column acetonitrile / water



Unmodified silica

NUCLEOSIL® 50-7 Particle size 7 ± 1.5 µm, pore size 50 Å; unmodified, eluent in column n-heptane

Standard-Prep columns10 mm ID 715004 71571121 mm ID 715022

NUCLEOSIL® 100-7 Particle size 7 ± 1.5 µm, pore size 100 Å; unmodified, eluent in column n-heptane

Standard-Prep columns 21 mm ID 715026

VarioPrep® columns 10 mm ID 71580021 mm ID 715650


16 mm ID 715275.160





Each column is individually tested and supplied with test chromatogram and test conditions


MN


Columns for special applications · Summary

73

Certain difficult separation problems can often be solved bet-ter and much more rapidly with chromatographic columnswhich have been specifically developed for the purpose.Environmental analysisThe quantitative determination of inorganic anions requiresion exchange columns which have been optimised for thisseparation. We offer three different types of columns to meetthe different demands of anion analysis.The analysis of polycyclic aromatic hydrocarbons (PAHs)gains increasing importance. For this application too, onlyspecial columns can guarantee the efficiency required forcomplex samples. Enantiomer separationIt is well known, that the two isomers of a natural chiral com-pound can differ substantially in their pharmacological activ-ity depending on their absolute configuration. Often only oneof the antipodes is pharmacologically active, while the othermay be inactive, or even toxic. For synthetic drugs, the sameholds true, and therefore it is necessary to control the opti-cal purity of a substance. This is why we offer a completeline of chiral HPLC columns with different selectivities.Biochemical separationsChromatographic techniques are increasingly applied for theseparation and purification of biologically active mole-cules. These methods take advantage of the chemical, bio-logical and physical properties of the molecules. Often verycomplex mixtures of substances may require a combinationof several chromatographic methods to obtain a pure prod-uct. Depending on the sensitivity of the molecules to be sep-arated the eluents have to be adapted to the natural environ-ment of these molecules. This is especially important for thepurification of proteins or the isolation of long-chain nucleicacids. For these substances ion exchange chromatogra-phy and gel filtration methods are preferred.

Peptides, nucleotides, oligonucleotides, oligo- and monosac-charides and amino acids behave more like conventionalorganic substances. For this reason they can be purifiedunder highly efficient chromatographic conditions withoutconsideration of the natural environment of the molecules.Nevertheless, their biological properties are maintained. Forsome questions in protein analysis, such as the sequenceanalysis of a protein or the determination of the content of anindividual protein in biological fluids, preservation of thethree-dimensional structure of the protein is not necessary.In this case purification can be performed under denaturingconditions with the highest possible selectivity. The same istrue for the purification of nucleic acids.Food analysisA major chapter covers the analysis of mono- and oligosac-charides as well as sugar alcohols and organic acids. Inaddition to special silica phases we offer mixed-mode col-umns based on PS/DVB. These columns allow very efficientseparations due to the combination of different mechanisms(ion exclusion, ion exchange etc.).For the analysis of hop constituents our special columns al-low rapid routine separations without the problems usuallyencountered with this application. We offer a large number of columns for biochemical and foodapplications, which can be used for a wide variety of separa-tion problems. In addition to columns for analytical and semi-preparative separations, we also supply columns andstationary phases for pilot plant and production scale purifi-cations. Thus scale-up from laboratory to production proc-esses is easily possible.The following table presents a summary of our programmeand helps in the selection of the proper column. For adetailed description of the packings used in the different col-umns please see the following pages. The table on page 93 presents a concise selection guide forour columns for biochemical separations. For chromatograms, we refer to our catalogue “LC Applica-tions” which is available on request – or visit our interactivecollection of applications on the internet:

The columns in this chapter have been developed specifi-cally for difficult separation problems. The stationary phasesused in these columns are in most cases not available asbulk packings, but only as packed columns. All columns aretested with special test mixtures for the respective purpose,thus we can guarantee the separation efficiency for thesecolumns. If your separations require a metal-free environment, pleaseask for our custom-packed PEEK columns.

In this chapter you will find HPLC columns for

environmental analysis (from page 75)

enantiomer separations (from page 79)

biochemical separations (from page 93)nucleic acids and nucleic acid constituentsproteins and peptides

food analysis (from page 116)mono-, oligo- and polysaccharideshop constituents

In the following chapter “Products for GPC“ you will find

columns for gel permeation chromatography (from page 124)

polymer standards for GPC (from page 131)

www.mn-net.com


MN


Columns for special applications · Summary

74

Summary of MN HPLC columns for special separations Separation/mechanism recommended column specification of the phase

Environmental analysis

anion exchange chromatogra-phy of inorganic anions

NUCLEOSIL® 10 Anion strongly basic silica-based anion exchanger

NUCLEOSIL® Anion II strongly basic silica-based anion exchanger

NUCLEOGEL® Anion I strongly basic polymer-based anion exchanger

polycyclic aromatic hydrocarbons (PAHs) NUCLEOSIL® 100-5 C18 PAH NUCLEOSIL® 100 polymer-coated with C18 groups

Enantiomer separationligand exchange NUCLEOSIL® CHIRAL-1 covalently bonded amino acid – Cu(II) complexes

charge-transfer-, dipole-di-pole interactions and others

NUCLEOSIL® CHIRAL-2NUCLEOSIL® CHIRAL-3 ”brush type“ phases based on silica

enantioselective binding to chi-ral protein surface structures RESOLVOSIL® BSA-7 protein phase (BSA) based on silica

formation of inclusion com-plexes

NUCLEODEX® α-PM, β-PM, γ-PM and β-OH

permethylated and underivatised cyclodextrins covalently bonded to silica

Biochemical separations

anion exchange chromatogra-phy of biomolecules

NUCLEOGEN® DEAE DEAE anion exchanger based on silica

NUCLEOSIL® 4000-7 PEIpolymeric covalently bonded polyethyleneimine net-work based on NUCLEOSIL® 4000, weakly basic anion exchanger

NUCLEOGEL® SAX strongly basic polymer-based anion exchanger

cation exchange chromatogra-phy of biomolecules NUCLEOGEL® SCX strong cation exchanger based on a macroporous

polymer with sulphonic acid modification

reversed phase chromatogra-phy of biomolecules

NUCLEOSIL® MPN monomerically bonded alkyl chains on silica

NUCLEOSIL® PPN polymerically bonded alkyl chains on silica

NUCLEOGEL® RP polystyrene – divinylbenzene polymer

reversed phase chromatogra-phy of small molecules

NUCLEOGEL® RP 100 small pore macroporous PS-DVB polymer

NUCLEOGEL® RP-C18 C18 modified polystyrene – divinylbenzene polymer

gel filtration of biomoleculesNUCLEOSIL® GFC hydrophilic polyalcohol modification on silica

NUCLEOGEL® GFC macroporous polymer with hydrophilic surface

Food analysismono- and oligosaccharides NUCLEOSIL® Carbohydrate special amino phase based on silica

sugars, alcohols, org. acids

NUCLEOGEL® SUGAR Ca, Na, PbPS-DVB resins with sulphonic acid modification} in different ionic formsNUCLEOGEL® SUGAR 810 H, Ca, Pb

NUCLEOGEL® ION 300 OA

hop constituents NUCLEOSIL® 100-5 C18 Hop silica with C18 modification

Gel permeation chromatography (GPC)water-insoluble substances NUCLEOGEL® GPC gel matrix polystyrene – divinylbenzene

water-soluble polymers NUCLEOGEL® aqua-OH macroporous polymer with hydrophilic surface


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HPLC columns for environmental analysis

75

The determination of environmentally important substancesin very low concentrations and very different matrices suchas surface water, drinking water, soil or waste water requiresapplication of columns which have been tailored for thesespecial separations. Combined with sample preparation andsample enrichment (e.g. with our CHROMABOND® columns

or CHROMAFIX® cartridges for solid phase extraction, whichare described in the chapter ”Solid phase extraction“ frompage 188) such columns provide a high degree of separationefficiency and reproducibility. Our programme comprisesHPLC columns for the determination of inorganic anions, andpolycyclic hydrocarbons (PAH).

Columns for the separation of polycyclic aromatic hydrocarbons (PAHs)

Polycyclic aromatic hydrocarbons (PAHs) are widely distrib-uted in the environment. A number of PAHs (e. g. benzo[a]-pyrene, 3-methylcholanthrene and benzanthracene havebeen proven to be carcinogenic. Thus control of the PAHcontent of food, water and soil is an important task for routineanalysis. For choice and limiting values of the polycyclics werefer to the governmental regulations, which exist in maycountries (e.g. EPA method 610 of the United States Envi-ronmental Protection Agency).

PAH can be determined by different chromatographic tech-niques (TLC, GC, HPLC). Thus the 6 PAHs according to Ger-man drinking water specification (TVO) can e.g. be analysedby TLC (see German Standard DIN 38 409), while a muchlarger number of polycyclic aromatics can be determined byGC or HPLC. For PAH analyses we have developed a spe-cially modified C18 phase, which allows efficient gradientseparation of the 16 PAHs according to EPA.


Length → 50 mm 150 mm 250 mm Guard columns

NUCLEOSIL® 100-5 C18 PAHspecial octadecyl phase on silica for the separation of PAHs, polymeric coating, particle size 5 ± 1.5 µm, pore size 100 Å; eluent in column acetonitrile / water 70:30

ChromCart® columns2 mm ID 721697.20 721684.20 721599.303 mm ID 721697.30 721684.30 721599.304 mm ID 721697.40 721684.40 721599.40

4.6 mm ID 721697.46 721684.46 721599.40

EC columns 1)

2 mm ID 720117.20 721599.303 mm ID 720923.30 720117.30 721599.304 mm ID 720756.40 720923.40 720117.40 721599.40

4.6 mm ID 720117.46 721599.40

PAH standard according to EPA for HPLCPAH standard for HPLC 16 PAHs according to EPA method 610 in acetonitrile (1 ml)

for composition see chromatogram on page 76722393

ChromCart® guard column cartridges (8 mm) in packs of 3, 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).

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76

Detection of the separated PAHs can be achieved by UV(250 to 280 nm), with diode array or with fluorescence detec-tion at different wavelengths for excitation and emission.Acenaphthylene cannot be analysed with fluorescence de-tection. For cost-effective routine PAH analysis we recom-mend application of columns with 2 mm ID, because this willreduce the eluent consumption to 30 – 40% with about dou-bled detection sensitivity compared to the 4 mm ID column.For rapid analysis we recommend a column length of 50 mm.This allows separation of the 16 PAHs according to EPA injust 9 minutes.

References

Determination of PASH in Diesel fuel by HPLC and photodi-ode-array detection; J. Bunot, W. Herbel, H. Steinhart, J.High Res. Chrom. 15 (1992) 682 – 685GIT Spezial Chromat. 2 (1992) 80 – 85

Rapid separation of 16 PAHs according to EPAColumn: EC 50/4 NUCLEOSIL® 100-5 C18 PAH,

50 x 4 mm ID, Cat. No. 720756.40Eluent A: WaterEluent B: AcetonitrileGradient: from 55 to 100% B in 2.5 min; then 3.5 min at

100% B; finally in 0.1 min from 100 to 55% BFlow rate: 1 ml/minPressure: 25 – 30 barTemperature: 25 °CDetection: UV, 260 nmPeaks: (sample volume 10 µl)1. Naphthalene2. Acenaphthylene3. Acenaphthene4. Fluorene5. Phenantrene6. Anthracene7. Fluoranthene8. Pyrene9. Benz[a]anthracene

10. Chrysene11. Benzo[b]fluoranthene12. Benzo[k]fluoranthen13. Benzo[a]pyrene14. Dibenz[ah]anthracene15. Benzo[ghi]perylene16. Indeno[1,2,3-cd]pyrene

1

2

3

4

56

7

8

910

11

12

13

14

15 16

0 10min

1150

30

Separation of the PAH standard according to EPA(Cat. No. 722393)

Column: CC 150/4 NUCLEOSIL® 100-5 C18 PAH,150 x 4 mm ID, Cat. No. 721697.40

Eluent A: Methanol – water (80:20)Eluent B: Acetonitrile – tetrahydrofuran (93:7)Gradient: 0 – 100% B in 10 min, then

5 min at 100% BFlow rate: 1 ml/minPressure: 140 barTemperature: 20 °CDetection: UV, 260 nmPeaks: (10 µg/ml each in acetonitrile)1. Naphthalene2. Acenaphthylene3. Acenaphthene4. Fluorene5. Phenantrene6. Anthracene7. Fluoranthene8. Pyrene9. Benz[a]anthracene

10. Chrysene11. Benzo[b]fluoranthene12. Benzo[k]fluoranthene13. Benzo[a]pyrene14. Dibenz[ah]anthracene15. Benzo[ghi]perylene16. Indeno[1,2,3-cd]pyrene

12

3

4

56

7

8

9

10

1112

13

14

15

16

0 10 20min

1150

40


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77

Columns for the separation of inorganic anions Analytical monitoring of the environment with respect to inor-ganic compounds today is the domain of ion chromatogra-phy. Our columns NUCLEOSIL® 10 Anion and Anion II con-tain special silica-based anion exchangers. They are stablein the pH range 2 – 7.5. For applications which, due to the

sample composition, require pH values outside this range,we offer the polymer-based column NUCLEOGEL® Anion I.A special feature of this column is that it can also be used forthe analysis of fluoride ions.


Length → 120 mm 250 mm Guard columns

NUCLEOSIL® 10 Anionstrongly basic silica-based anion exchanger, particle size 10 ± 1.5 µm, pore size 100 Å, exchange capacity 800 µval/g; eluent in column 25 mM salicylate buffer pH 4

EC columns 1)

4 mm ID 720038.40

NUCLEOSIL® Anion IIstrongly basic silica-based anion exchanger, particle size 10 ± 1.5 µm, pore size 300 Å, exchange capacity 50 µval/g; eluent in column 2 mM potassium hydrogen phthalate buffer pH 5.6

ChromCart® columns4 mm ID 721451.40 721452.40

EC columns 1)

4 mm ID 720094.40 721452.40

NUCLEOGEL® Anion Istrongly basic polymer-based anion exchanger, particle size 10 µm; eluent in column 4 mM salicylate buffer pH 7.8

Valco type columns 2)

4.6 mm ID 719533 719543All columns and ChromCart® guard column cartridges are supplied in packs of 1.1) As guard column for EC columns use ChromCart® guard column cartridges with guard column adaptor EC (Cat. No. 721359). 2) Valco type guard column cartridges are 21 x 4 mm, require guard column holder C (Cat. No. 719538) and are supplied in packs of 2.

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78

NUCLEOSIL® Anion columns based on silica

Our columns EC 250/4 NUCLEOSIL® 10 Anion and EC250/4 NUCLEOSIL® Anion ll and the corresponding Chrom-Cart® columns are packed with spherical silica packingsmodified with quaternary ammonium groups, i.e. stronglybasic anion exchangers; however, they differ considerably intheir exchange capacity and in the pore size of the NUCLE-OSIL® silica.The exchange capacity of the NUCLEOSIL® 10 Anion pack-ing is approximately 800 µval/g, that of the NUCLEOSIL®

Anion II packing is about 50 µval/g. For this reason the col-umns should be operated with buffers of different concentra-tions. The recommended values are 25 mmol/l phthalate forNUCLEOSIL® 10 Anion and 2 mmol/l for NUCLEOSIL®

Anion II, respectively. Another consequence is, that both columns require differentdetection methods and feature different detection limits.NUCLEOSIL® Anion II columns are mainly operated withconductivity detection or negative UV detection and allowdetermination in the ppm range. The column NUCLEOSIL®

10 Anion is mainly used with RI detection resulting in a10fold lower sensitivity. On the other hand – due to the lowercapacity –columns NUCLEOSIL® Anion II require the injec-tion or rather dilute samples, i.e. clearly below 50 ppm percomponent (see upper figure). As eluent we recommend phthalate buffers with weaklyacidic pH, although other buffer systems might be used aswell. In this context we want to mention the corrosive proper-ties of salicylate buffers. Salicylate anions react with ironions, which may originate from the stainless steel of theinstruments used (pumps, capillary tubing etc.) to produce acoloured complex which will interfere with UV detection.

NUCLEOGEL® Anion polymer-based columns It should be noted that it is not possible to determine all inor-ganic anions with the silica-based columns. One exception isfluoride which can only be separated at alkaline pH valuesbecause in an acidic medium it is irreversibly bonded to thesilica.For this purpose we recommend our column NUCLEOGEL®

Anion I, which is very well suited for this separation (seelower figure). It is packed with a macroporous, polymer-based anion exchanger. The base material is stable in the pHrange from 0 to 14. For this reason any aqueous eluent canbe used. Phthalate and salicylate buffers can also beapplied. Using negative UV detection, less than 10 ppm canbe analysed without enrichment of the samples.

For an increased life-time of all above-mentioned columns,we recommend the use of corresponding guard columns.

Separation of an anion standardsColumn: CC 250/4 NUCLEOSIL Anion II

250 x 4 mm ID, Cat. No. 721451.40Eluent: 2 mM potassium hydrogen phthalate, pH 5.7Flow rate: 2 ml/minDetection: UV, 280 nmPeaks:1. H2PO4

–

2. Cl–

3. NO2–

4. NO3–

5. SO42–

1

23

4

5

5 10 15min

1064

40

Separation of inorganic anionsColumn: VA 120/4.6 NUCLEOGEL Anion I,

120 x 4,6 mm ID, Cat. No. 719533Eluent: 4 mM salicylic acid / Tris pH 7.8Flow rate: 1 ml/minDetection: UV, 254 nmPeaks:1. F–

2. Cl–

3. NO2–

4. Br–

5. NO3–

6. PO43–

7. SO42–

1

2 34

5

6 7

0 5 10 15min1150

50


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HPLC columns for enantiomer separation

79

It is well known that the two isomers of a natural chiral com-pound can differ substantially in their pharmacological activ-ity depending on their absolute configuration. Often only oneof the antipodes is pharmacologically active, while the other

may be inactive, or even toxic. For synthetic drugs, the sameholds true, and therefore it is necessary to control the opticalpurity of a substance.

NUCLEODEX® columns– enantiomer separation based on cyclodextrins, covalently bonded to NUCLEOSIL® silica – Our cyclodextrin-based HPLC columns NUCLEODEX® β-OH, α-PM, β-PM and γ-PM are very well suited for the sepa-ration of racemates, constitutional and configurational iso-mers under reversed phase conditions. A large number of

racemates, including pesticides and pharmaceuticals, havebeen separated using these phases which are based on ourwell-known spherical silica NUCLEOSIL®.

Length → 200 mm Guard columns

NUCLEODEX® screening kit (Cat. No. 721920)

consists of one CC 30/4 each with NUCLEODEX® β-OH, α-PM, β-PM and γ-PM and a ChromCart® column holder 30 mm

NUCLEODEX® β-OHβ-cyclodextrin chemically bonded to NUCLEOSIL® silica, particle size 5 ± 1.5 µm, pore size 100 Å, eluent in column CH3OH / 0.1% TEAE pH 4 (55:45)


EC columns 1)

4 mm ID 720124.40 721460.40

NUCLEODEX® α-PMpermethylated α-cyclodextrin chemically bonded to NUCLEOSIL® silica, particle size 5 ± 1.5 µm, pore size 100 Å, eluent in column CH3OH / 50 mM phosphate pH 3 (70:30)


EC columns 1)

4 mm ID 720127.40 721464.40

NUCLEODEX® β-PMpermethylated β-cyclodextrin chemically bonded to NUCLEOSIL® silica, particle size 5 ± 1.5 µm, pore size 100 Å, eluent in column CH3OH / 0.1% TEAE pH 4 (65:35)


EC columns 1)

4 mm ID 720125.40 721462.40

NUCLEODEX® γ-PMpermethylated γ-cyclodextrin chemically bonded to NUCLEOSIL® silica, particle size 5 ± 1.5 µm, pore size 100 Å, eluent in column CH3OH / 0.1% TEAE pH 4 (55:45)


EC columns 1)

4 mm ID 720752.40 721466.40All columns and guard column cartridges (8 mm) 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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80

Separation mechanism

The chiral selectors of the NUCLEODEX® phases are cyclo-dextrins, which are covalently bonded to the silica matrix viaa spacer, resulting in hydrolytically stable adsorbents Themean diameter of the spherical silica particles is 5 µm with apore size of 100 Å.

Cyclodextrins are cyclic oligosaccharides consisting of glu-cose units. They are formed by degradation of starch by bacil-lus macerans or bacillus circulans under action of cyclodex-tringlycosyltransferase. As a first approximation, the cyclicstructure of a cyclodextrin ring can be described as a hollowtruncated cone. β-Cyclodextrin contains seven glucose units,the opening of the cavity is about 7.5 Å. α-Cyclodextrin con-tains only six glucose units resulting in a smaller opening ofthe cavity. γ-Cyclodextrin with its 8 glucose units is the largestcyclodextrin. The inner surface of the cavity is lipophilic, thus anonpolar part of the sample molecule (e.g. phenyl or naphthylsubstituents of suitable size) can penetrate into the cyclodex-trin ring. This forms so-called inclusion complexes, the stabilityof which is responsible for the retention of a compound. Thechiral sugar units of the cyclodextrins allow enantioselectiveinteractions and thus racemate separations.ApplicationThe column NUCLEODEX® β-OH contains β-cyclodextrinwith free hydroxy groups as chiral selector. Thus hydrogenbonds and dipole interactions between functional groups ofthe analyte and the primary hydroxyl groups (small openingof the cyclodextrin) and secondary hydroxyl groups (largeopening of the cyclodextrin) can occur. The phases NUCLEODEX® α-PM, NUCLEODEX® β-PMand NUCLEODEX® γ-PM are based on permethylated cyclo-dextrins. While the chiral selector of the adsorbent NUCLEODEX® β-PM is permethylated β-cyclodextrin, we use the smaller per-methylated α-cyclodextrin for the phase NUCLEODEX® α-PM. Correspondingly, the chiral selector for NUCLEODEX®

γ-PM is permethylated γ-cyclodextrin. For these phases all

hydroxy functionalities are replaced by methoxy groups,resulting in phase with different selectivity characteristicscompared to NUCLEODEX® β-OH. Especially noticeable arethe shorter retention times on NUCLEODEX® β-PM. Per-methylation of the cyclodextrin ring influences the numberand type of possible interactions. The cage is enlarged andthe hydrophobicity of the openings is increased. The ability ofNUCLEODEX® α-PM, β-PM and γ-PM to form hydrogenbonds is considerably lower. As shown in the figure below,the permethylated phases possess only proton acceptorproperties due to the free electron pairs on the oxygen.

Structure of β-cyclodextrin

O

OHHO

O O

O

O

O

O

OO

O

O

O

O

OOH

OH

OH

OH

OH

OH

OH

OH OH

OH

OH

OH

OH

HO

HO

HO

OH

OH

OH

proton acceptor

proton donor

only proton acceptor

OH

N

OH

O

H

OCH3

N

O

CH3

O

H

and

Enantiomer separation of dichlorpropColumn: CC 200/4 NUCLEODEX® α-PM

Cat. No. 721463.40Sample volume: 1 µlEluent: Methanol / 50 mM NaH2PO4

pH 3,0 (65:35, v/v)Flow rate: 0,7 ml/minPressure: 170 barTemperature: 20 °CDetection: UV, 230 nm

0 10 min

Cl

Cl

O CO2HCH

CH3

1110

10

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81

For the example of the barbiturate mephobarbital, alsoknown as prominal, methylation of the cyclodextrin clearly re-sults in a better and more rapid separation of the antipodes.In NUCLEODEX® β-OH, the free hydroxy groups of the oli-gosaccharide result in unnecessary interaction, whichcauses longer retention times without additional chiral dis-crimination. On the one hand, NUCLEODEX® β-PM shows good selec-tivity for some compounds which cannot be separated on β-OH, e.g. the pesticide derivatives mecoprop methyl anddichlorprop methyl. On the other hand, compounds likechlorthalidone require the free hydroxy groups for enantiose-lective interactions. Such compounds can only be separatedon the phase NUCLEODEX® β-OH. NUCLEODEX® α-PM allows separation of the herbicidesmecoprop and dichlorprop as free carboxylic acids. In addi-tion, this phase shows good selectivity for aromatic oxiranessuch as trans-stilbene oxide or styrene oxide.Due to their large molecules, steroids can best be separatedon permethylated γ-cyclodextrin, i.e. on NUCLEODEX® γ-PM.These few examples show that the four phases ideally com-plement each other with respect to selectivity.For numerous separations of the NUCLEODEX® phasesplease ask for our catalogue ”LC Applications“ or on ourwebsite: www.mn-net.com.

Enantiomer separation of mephobarbital (prominal)Column: EC 200/4 NUCLEODEX® β-PM; 200 x 4 mm ID;

Cat. No. 720125.40Eluent: Methanol / 0.1% TEAA pH 4.0 (55 : 45, v/v)Flow rate: 0.7 ml/minPressure: 180 barDetection: UV, 254 nmSample: ProminalInj.volume: 1 µl

0 10min

CH3CH2

O

NH

NCH3

O

O

1058

00

Separation of D,L-estrone on 2 NUCLEODEX® phasesColumn: a) CC 200/4 NUCLEODEX® β-PM,

Cat. No. 721461.40b) CC 200/4 NUCLEODEX® γ-PM, Cat. No. 721465.40

Eluent: Acetonitrile/water (45:55, v/v)Flow rate: 0.8 ml/minDetection: UV, 280 nm

a) b)

0 5 10 15 0 5 10minmin

1061

90/1

0620

0

Enantiomer separation of styrene oxideColumn: EC 200/4 NUCLEODEX® α-PM;

200 x 4 mm ID; Cat. No. 720127.40

Eluent: Methanol / 0.1% TEAA pH 4.0 (60 : 40, v/v)

Flow rate: 0.7 ml/minDetection: UV, 230 nm

O

0 10min

1061

60

For ordering information of NUCLEODEX® columns please see page 79.


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82

As is well known, it is often difficult to predict the chances forseparation of an unknown enantiomeric mixture on a chiralphase. In most cases trial and error is the only way to find theproper column. For this case we offer our

NUCLEODEX® CC screening kit(Cat. No. 721 920)consisting of one ChromCart® column CC 30/4 each with NUCLEODEX® β-OH, α-PM, β-PM, γ-PM and a ChromCart® column holder 30 mmThis kit is especially designed

for method development of enantiomer separationsfor rapid and economical selection of proper chiral phases

Separation of chlorthalidone on NUCLEODEX® β-OHEluent: methanol – 0.1% TEAA (55:45), pH 4Flow rate: 0,7 ml/minDetection: UV, 254 nm

Separation of mecoprop on NUCLEODEX® α-PMEluent: methanol – 50 mmol NaH2PO4 (70:30), pH 3Flow rate: 0,7 ml/minDetection: UV, 230 nm

Separation of mecoprop methyl on NUCLEODEX® β-PMEluent: methanol – 0.1% TEAA (65:35), pH 4Flow rate: 0.7 ml/minDetection: UV, 230 nm

0 5 0 5 10min min

CC 30/4 CC 200/4

CC 30/4 CC 200/4

0 5 0 5 10min min

CC 30/4 CC 200/4

0 5 0 5 10min min

1150

7011

5080

1150

90

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The NUCLEODEX® phases are especially suited for the con-trol of optical purity as is shown below for the detection of 1%D-dansyl-leucine besides 99% of the L-isomer.

It should also be noted that NUCLEODEX® columns can beused for semipreparative separations as is shown below forthe resolution of the isomers of mecoprop methyl.

Enantiomer separation of benzoinColumn: EC 200/4 NUCLEODEX® γ-PM,

200 x 4 mm ID, Cat. No. 720752.40

Eluent: Methanol / 0.1% TEAA pH 4.0 (55:45, v/v)

Flow rate: 0.7 ml/minDetection: UV, 230 nm

0 10 min

OH

O

1151

00

Enantiomer separation of ibuprofenColumn: CC 200/4 NUCLEODEX® β-PM, 200 x 4 mm ID,

Cat. No. 721461.40Eluent: MeOH / 0.1% TEAA,

pH 4.0 (60:40, v/v)Flow rate: 0.7 ml/minPressure: 180 barDetection: UV, 230 nm

20 400min

COOH

1151

30

0 10 0 10

L

D

L

D

min

Enantiomer separation of dansyl-D,L-leucineSample: left: racemate, right: 1% D-

besides 99% L-isomerColumn: EC 200/4 NUCLEODEX®

β-OH, 200 x 4 mm ID, Cat. No. 720124.40

Eluent: MeOH – 1% TEAA, pH 4,0 (65:35, v/v)

Flow rate: 0,7 ml/minDetection: UV, 254 nm

NH

COOH

SO2

NMe2

1151

20

Semipreparative separation of mecoprop methylColumn: CC 200/4 NUCLEODEX® β-PM, 200 x 4 mm ID,

Cat. No. 721461.40Eluent: MeOH / 0.1% TEAA,

pH 4.0 (60:40, v/v)Flow rate: 0.7 ml/minDetection: UV, 254 nm 1 mg

2 µg

0 4 8 12 16 min

OCl

CH3

COOCH3

1151

40



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84

NUCLEODEX® phases can also be used for positional andcis-trans-isomers, as is demonstrated in the following exam-ples.

Separation of cis-trans-isomers of γ-tocotrienolA. M. Drotleff, W. Ternes, Z Lebensm Unters Forsch A 206 (1998) 9 – 13Column: EC 200/4 NUCLEODEX® β-PM, 200 x 4 mm ID

Cat. No. 720125.40Eluent: Acetonitril / water (58:42, v/v)Flow rate: 0.8 ml/minDetection: Fluorescence,

λem = 330 nm, λex = 295 nm

Sample: 3’cis,7’cis-γ-tocotrienol3’cis,7’trans-γ-tocotrienol3’trans,7’cis-γ-tocotrienol3’trans,7’trans-γ-tocotrienolNUCLEODEX® β-PM is suited for the separation of α-, β-, γ- and δ-tocotrienols.The elution sequence of the isomers was only determined for α-Tocotrienol.

mV

50

00 10 20 30 40 50

min

CH3

HO

O

CH3 CH3

CH3

H3C

1122

70

Separation of o-, m- and p-cresolColumn: CC 200/4 NUCLEODEX® β-OH, 200 x 4 mm ID

Cat. No. 721459.40Eluent: Methanol / water

(50:50, v/v)Flow rate: 0.7 ml/minPressure: 170 barDetection: UV, 254 nmPeaks:1. o-Cresol2. m-Cresol3. p-Cresol

1

2

3

0 4 8 12 min1009

50

Separation of positional isomers of nitroanilineColumn: EC 200/4 NUCLEODEX® β-OH,

200 x 4 mm ID, Cat. No. 720124.40Eluent: Methanol / 0.1% triethylammonium acetate pH 4.0,

50 : 50 (v/v)Flow rate: 0.7 ml/minPressure: 180 barDetection: UV, 254 nmPeaks (sample volume 1 µl):1. m-Nitroaniline2. o-Nitroaniline3. p-Nitroaniline

NH2 NH2

NH2

NO2O2NO2N

0 10 20 min

1

2

3

21 3

1014

20


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85

Chromatographic conditions for column operation

HPLC columns NUCLEODEX® β-OH, NUCLEODEX® α-PM,NUCLEODEX® β-PM and NUCLEODEX® γ-PM are normallyoperated under reversed phase conditions. Methanol as wellas acetonitrile in combination with water or buffer solutionsare suited as eluents. The pH value of the eluent should bebetween 3 and 8. The choice of the organic modifier influ-ences the selectivity of the phases. Buffers should be eitherphosphate or triethylammonium acetate (TEAA). As shownbelow for the separation of dansyl-D,L-leucine, the separa-tion efficiency can be improved by increasing the buffer con-centration.The optimum column temperature should be between 0 and50 °C. With increasing temperature the retention time de-creases. Lower temperatures in general increase the selec-tivity (see figure).The influence of the cyclodextrin size on the separation ofenantiomer pairs of very similar compounds is shown belowfor the methyl, ethyl and propyl esters of the herbicide meco-prop. The adsorbent NUCLEODEX® β-PM separates thethree enantiomer pairs of the esters, but not the free carbox-ylic acid. The methyl ester already shows a low selectivity α.On the contrary, NUCLEODEX® α-PM discriminatesbetween the enantiomers of the esters with the same selec-tivity and also separates the free carboxylic acid mecoprop.

Influence of the buffer concentrationColumn EC 200/4 NUCLEODEX® β-OH (Cat. No. 720124.40);Sample dansyl-D,L-leucine; Eluent MeOH/TEAA, pH 4.0, 65:35 (v/v); Flow rate 0.7 ml/min; Detection UV, 254 nm

0.1% TEAA 1% TEAA

0 10 0 10 min1151

60

Influence of the pH valueColumn EC 200/4 NUCLEODEX® β-OH (Cat. No. 720124.40); sample DNP-D,L-methionine; eluent MeOH / 50 mM NaH2PO4 (40:60 v/v); flow rate 0.7 ml/min; detec-tion UV, 254 nm

pH value

6

5

4

3

2

1

capa

city

fact

or

1.6

1.5

1.4

1.3

1.2

1.1

sele

ctiv

ity

6 6.5 7 7.5

k’ (2)

k’ (1)

α

Influence of the temperatureColumn EC 200/4 NUCLEODEX® β-OH (Cat. No. 720124.40); sample dansyl-D,L-threonine; eluent acetonitrile/50 mM NaH2PO4, pH 6.5 (20:80v/v); flow rate 0.8 ml/min; detection UV, 254 nm

capa

city

fact

or

3

2.5

2

1.5

1

0.5

sele

ctiv

ity

1.7

1.6

1.5

1.4

1.3

1.2

Temperature [° C]

15 25 35 45

k’ (2)

k’ (1)

α

Influence of the ester alkyl group on capacity factor and selectivity shown for mecoprop estersColumns EC 200/4 NUCLEODEX® α-PM (Cat. No. 720127.40) and EC 200/4 NUCLEODEX® β-PM, (Cat. No. 720125.40); sample: different mecoprop esters; elu-ent methanol/ 50 mM NaH2PO4, pH 3.0 (70:30 v/v); flow rate 0.7 ml/min; detection UV, 230 nm

5

4

3

2

1

0methyl ethyl n-propyl

1.5

1.4

1.3

1.2capa

city

fact

or

sele

ctiv

ity

α-PM

β-PM

α-PM

β-PM



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86

NUCLEOSIL® CHIRAL columns

NUCLEOSIL® CHIRAL-1 – enantiomer separation based on ligand exchange –

Our HPLC column NUCLEOSIL® CHIRAL-1 has been devel-oped for the separation of optically active compounds andcontrol of optical purity based on ligand exchange chroma-tography. The stationary phase for this column contains asmatrix our well-known, pressure-stable NUCLEOSIL 120,chemically bonded with L-hydroxyproline / Cu2+ complexesas chiral selectors. Separation mechanismLigand exchange chromatography (LEC) for the separationof enantiomers was introduced in liquid chromatography byDavankov et al.1) and extensively studied by Gübitz and co-workers 2). A paper by Davankov describes the enantioselec-tivity of ligand exchange chromatographic systems 3). In LECthe principal interaction mode between solute enantiomersand the chiral selector is the formation of ternary mixed-lig-and complexes with a transition metal cation, in the case ofNUCLEOSIL® CHIRAL-1 Cu(II) 4). The asymmetric centresof the hydroxyproline result in the formation of diastereo-meric complexes. Differences in the stability of the com-plexes cause chromatographic separation.


Separation of amino acid enantiomersColumn: EC 250/4 NUCLEOSIL® CHIRAL-1

250 x 4 mm ID, Cat. No. 720081.40Sample: D,L-alanine D,L-threonineEluent: 0.5 mM CuSO4 0.25 mM CuSO4Flow rate: 1 ml/min 0.8 ml/minPressure: 60 bar 65 barTemperature: 60 °C 60 °CDetection: UV, 250 nm UV, 240 nm

0 5 10min 0 5 10min

1054

10


NUCLEOSIL® CHIRAL-1L-hydroxyproline/Cu2+ complexes chemically bonded to silica, particle size 5 ± 1.5 µm, pore size 120 Å; eluent in column 0.5 mM copper sulphate solution


EC columns 1)


www.mn-net.com


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87


As mobile phases for NUCLEOSIL® CHIRAL-1 we recom-mend CuSO4-containing aqueous eluents. The optical reso-lution can be optimised by changing the mobile phase com-position.

Cu2+ concentrationHigher Cu(II) concentrations shorten retention times ofthe analytes. The Cu(II) concentration should be be-tween 0.2 and 3 mmol/l.

Organic modifiersAddition of organic solvents like acetonitrile or methanoldecreases retention times and improves peak symmetry.TemperatureIncreased temperatures improve column efficiency andresult in shorter retention times. We recommend a tem-perature of 60 °C and a maximum pressure not exceed-ing 140 bar.

Application of NUCLEOSIL® CHIRAL-1A successful separation of enantiomers can be expected ifthe sample molecule has two polar functional groups with thecorrect spacing, which can form chelate complexes with thecopper ions. Therefore, this column is very suitable for sepa-ration of α-amino acids with their amino and carboxylategroups. α-Hydroxycarboxylic acids (e.g. lactic acid), N-alkyl-α-amino acids and similar molecules are also candidates forthis method.Optical isomers separated so far include D,L-alanine, D,L-arginine, D,L-asparagine, D,L-citrulline, D,L-methionine, D,L-phenylalanine, D,L-phenylglycine, D,L-proline, D,L-threonineand D,L-valine.

The enantiomeric resolution of N-methyl-α-amino acids, α-alkyl-α-amino acids and α-amino alcohols on NUCLEOSIL®

CHIRAL-1 columns is reported by H. Brückner et al. 5), 6). H.Skopan et al.7) described the application of NUCLEOSIL®

CHIRAL-1 columns for the control of optical purity of 2-hy-droxycarboxylates from enzymatic reactions. Thomas andSurber described the enantiomer analysis of a HIV antiinfec-tive nucleoside 8).For examples of enantiomeric separations with NUCLE-OSIL® CHIRAL-1 columns please see our catalogue ”LCApplications“.References

1) V.A. Davankov, S.V. Rogozhin, A.V. Semechkin and T.P. Sachkova, J. Chromatogr. 82 (1973) 359V.A. Davankov, Adv. Chromatogr. 18 (1980) 139

2) Separation of the optical isomers of amino acids by lig-and exchange chromatography using chemically bondedchiral phasesG. Gübitz, W. Jellenz and W. Santi, J. Chromatogr. 203(1981) 377 – 384

3) Enantioselectivity of complex formation in ligand-ex-change chromatographic systems with chiral stationaryand/or mobile phasesV.A. Davankov, A.A. Kurganov and T.M. Ponomarova, J.Chromatogr. 452 (1988) 309 – 316

4) Applications and limitations of commercially available chi-ral stationary phases for high performance liquid chroma-tographyR. Däppen, H. Arm and V. R. Meyer, J. Chromatogr. 373(1986) 1 – 20

5) Enantiomeric resolution of N-methyl-α-amino acids andα-alkyl-α-amino acids by ligand exchange chromatogra-phyH. Brückner, Chromatographia 24 (1987) 725

6) Determination of α-alkyl-α-amino acids and α-amino al-cohols by chiral phase capillary gas chromatography andreversed phase high performance liquid chromatographyH. Brückner, I. Bosch, Th. Graser and P. Fürst, J. Chro-matogr. 395 (1987) 569 – 590

7) Ein Biokatalysator zur Herstellung von (R)- und (S)-2-Hy-droxycarbonsäurenH. Skopan, H. Günther and H. Simon, Angew. Chem. 99(1987) 139 – 141

8) Preparative separation and analysis of the enantiomersof [3H] Abbott 69992, an HIV anti-infective nucleoside, byligand exchange HPLCS. B. Thomas and B. W. Surber, J. Chromatogr. 586(1991) 265 – 270

Enantiomer separation of an α-hydroxycarboxylic acidColumn: EC 250/4 NUCLEOSIL® CHIRAL-1

250 x 4 mm ID, Cat. No. 720081.40

Eluent: 0.5 mM copper sulphateFlow rate: 0.8 ml/minTemperature: 80 °CDetection: UV, 240 nm

Sample: (±)-lactic acidSample volume: 1 µl

0 10min1055

60


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88

NUCLEOSIL® CHIRAL-2 and CHIRAL-3 – for organic eluent systems –The chiral columns NUCLEOSIL® CHIRAL-2 and CHIRAL-3have been developed for control of optical purity of com-pounds under normal phase conditions. Main components of

the eluent systems are thus hydrocarbons besides polarorganic modifiers.

NUCLEOSIL CHIRAL-2

Separation mechanismWith the column NUCLEOSIL® CHIRAL-2, as chiral sub-stance D-dinitrobenzoylphenylglycine is covalently bonded tothe NUCLEOSIL® silica matrix via a spacer. NUCLEOSIL®

CHIRAL-2 is a brush type phase. Chiral brush type phasesfor HPLC were first used by Mike et al.1) for the separationof racemic helicenes. Later, the groups of Pirkle and of Oisynthesised a large number of these phases.Although a lot of work has been done in this field, the sepa-ration mechanism is not completely understood in allinstances. However, there is no doubt that charge-transferinteractions, hydrogen bonds, dipole-dipole interactions andsteric effects are involved 2).Chromatographic conditions for column operationThe separation is performed using nonpolar organic mobilephases (n-heptane, isooctane) with polar organic additivessuch as tetrahydrofuran, alcohols, chlorinated hydrocarbonsor similar. Often addition of a small amount of strong acids(e.g. trifluoroacetic acid) to the mobile phase will considera-bly improve separation of the isomers. The solubility of basiccompounds can easily be enhanced by a simple derivatisa-tion step (e.g. with benzoyl chloride or 3,5-dinitrobenzoylchloride).


EC columns

NUCLEOSIL® CHIRAL-2N-(3,5-dinitrobenzoyl)-D-phenylglycine chemically bonded to silica, ”brush type“ phase, particle size 5 ± 1.5 µm;eluent in column n-heptane / isopropanol / trifluoroacetic acid 100:0.5:0.5

4 mm ID 720088.40 721458.40

NUCLEOSIL® CHIRAL-3optical antipode of CHIRAL-2 chemically bonded to silica as above

4 mm ID 720350.40 721458.40As guard columns for EC columns use ChromCart® guard column cartridges with guard column adaptor EC (Cat. No. 721359). ChromCart® guard column cartridges for NUCLEOSIL® CHIRAL-2 and CHIRAL-3 are identical, measure 8 x 4 mm ID and are supplied in packs of 3, all other columns in packs of 1.

Separation of the optical isomers of fenoprop methylColumn: EC 250/4 NUCLEOSIL® CHIRAL-2,

250 x 4 mm ID, Cat. No. 720088.40Sample: Fenoprop methylVolume: 2 µlEluent: n-Heptane / i-propanol / TFA (100:0.5:0.05, v/v)Flow rate: 1.0 ml/minPressure: 80 barDetection: UV, 230 nm

40 8 12 min

Cl

Cl

Cl

O CO2CH3CH

CH3

1060

30

s

^


MN



89

ApplicationNUCLEOSIL® CHIRAL-2 is recommended for the analysis ofstereoisomers such as

separation of enantiomers and diastereomerscontrol of optical purity of plant protectives (pesticides,e.g. propionic acid derived herbicides 3)), pharmaceuti-cals etc.for product control in chiral organic syntheses

NUCLEOSIL CHIRAL-3This HPLC column differs from NUCLEOSIL® CHIRAL-2 onlyin the configuration of the chiral selector. For NUCLEOSIL®

CHIRAL-3 L-dinitrobenzoylphenylglycine is used.The selectivity towards a racemic mixture as well as thechromatographic resolution of the respective enantiomerpairs correspond exactly to the column NUCLEOSIL®

CHIRAL-2, however, the elution sequence of the enantiom-ers is reversed.

For control of the optical purity of a substance, with the twocolumns NUCLEOSIL CHIRAL-2 and NUCLEOSIL CHIRAL-3 the chromatographer now has the option to select condi-tions such that the minor enantiomer, which is present as animpurity, is eluted before the main peak. Thus, overlappingpeaks are avoided. This makes an exact quantification of theimpurity much easier.Conditions for operation are identical for the columns NUCL-EOSIL® CHIRAL-2 and NUCLEOSIL® CHIRAL-3.

References

1) F. Mike , G. Boshart and E. Gil-Av, J.Chromatogr. 122 (1976) 205

2) Chiral stationary phases for high performance liquid chro-matographic separation of enantiomers. A minireview D.W. Armstrong, J.Liquid Chromatography 7 (S-2) (1984)353 – 376

3) Enantiomer resolution and assay of propionic acid-de-rived herbicides in formulations by using chiral liquidchromatography and achiral gas chromatography.M.D. Müller and H.-P. Bosshardt, J. Assoc. Off. Anal.Chem. 71 (1988) 614 – 617

Control of optical purity of mecoprop methyl on NUCLEOSIL® CHIRAL-2 and CHIRAL-3

Column: a) EC 250/4 NUCLEOSIL® CHIRAL-2, Cat. No. 720088.40

b) EC 250/4 NUCLEOSIL® CHIRAL-3,Cat. No. 720350.40

Eluent: n-Heptane / 2-propanol / trifluoroacetic acid (100 : 0.05 : 0.05, v/v)

Flow rate: 1 ml/minTemperature: ambientDetection: UV, 230 nm

Sample: Mecoprop methyl 90% eeSample volume: 1 µl

95% D

95% D

5% L5% L

a) b)

0 10min 0 10min1113

60

s

^

Enantiomer separation of D,L-supidimideColumn: EC 250/4 NUCLEOSIL® CHIRAL-2,

250 x 4 mm ID, Cat. No. 720088.40Eluent: Tetrahydrofuran / n-heptane (10:3, v/v)Flow rate: 1.0 ml/minDetection: UV, 220 nm

0 6 12 18 min24

NC

SN

OO O

O

1056

90


MN



90

RESOLVOSIL® BSA-7

– protein phase for separation of optical isomers based on silica and bovine serum albumin –

The chiral column RESOLVOSIL® BSA-7 has been devel-oped for the separation of optical isomers and demonstratedto be a highly successful tool for determination of the enanti-omeric purity.The chiral stationary phase RESOLVOSIL BSA-7 is basedon bovine serum albumin (BSA) covalently bonded to wide-pore silica.With respect to various chromatographic parameters this col-umn can be characterised as follows:

The main advantages of the RESOLVOSIL® column are extremely high selectivityeasy regulation of retention by small changes in the mo-bile phase composition.

This results in a high flexibility of the chromatographic sys-tem because an optical resolution may be optimised to fitgiven requirements.In combination with high-sensitivity detectors, very smallamounts of a compound need to be injected on the column.Therefore, enantiomeric composition or purity can be deter-mined very accurately in spite of the low capacity.

Separation mechanism

The performance of the RESOLVOSIL® column is based onselective interactions of proteins with low molecular com-pounds.Several proteins can undergo enantioselective interactionswith a large number of pharmacologically active compounds.This effect was first used for chromatographic separations byStewart and Doherty 1), who succeeded in resolving D- andL-tryptophan on bovine serum albumin (BSA) bound to agar-ose. Allenmark et al.2) made the method accessible to HPLC

by binding BSA to HPLC-grade silica. Resolution of enanti-omers on BSA which was irreversibly adsorbed to silica wasstudied by Erlandsson et al. 3).The separation mechanism of protein columns is not known,although there is no doubt that it is based on principles ofbioaffinity. It includes hydrophobic interactions (similar to atrue reversed phase), interactions of polar groups and stericeffects 4). Aubel et al.5) investigated the effect of differentpretreatments on the performance of BSA columns.

Separation factors (α values) for selected representatives of various classes of compounds on RESOLVOSIL® BSA-7

selectivity and resolution: excellentspeed: very good reproducibility: excellent capacity: low to moderate

Length → 150 mm Guard column

EC columns 1)

RESOLVOSIL® BSA-7

protein phase, bovine serum albumin (BSA) chemically bonded to NUCLEOSIL® silica, particle size 7 ± 1.5 µm, pore size 300 Å; eluent in column 0.1 M phosphate buffer pH 7.5, 2% n-propanol


Class Compound α values on BSA-7

Aromatic amino acids D,L-kynurenin 37.5DNP amino acids N-(2,4-dinitrophenyl)-D,L-aspartic acid 7.7Dansyl amino acids dansyl-D,L-glutamic acid 2.3N-Aroyl amino acids N-(p-nitrobenzoyl)-D,L-alanine 17Benzodiazepinones oxazepam 6.8Sulphoxides o-carboxyphenyl-methyl sulphoxide 3.1Coumarin derivatives warfarin 1.5Lactams 4-amino-3-(p-chlorophenyl)butyric acid lactam 28 Aromatic hydroxy ketones benzoin 1.5


MN



91

Applications

Examples for the application of RESOLVOSIL® areamino acid derivativesaromatic amino acidsaromatic sulphoxidebarbituratesbenzodiazepinonesbenzoin and benzoin derivativesβ-blockerscoumarin derivatives

RESOLVOSIL has proven useful for monitoring stereose-lective microbial and enzymatic conversions 6), 8), 9). For examples of separations on the RESOLVOSIL® columnplease see our catalogue ”LC Applications“.


Columns with protein stationary phases are demanding inthat the chiral separation can be easily influenced by thechromatographic conditions such as pH, ionic strength, con-centration of organic modifiers and temperature. Therefore,the optimum combination of these parameters should bedetermined for each separation problem.Mobile phaseThe RESOLVOSIL® column is compatible with mobile phasesystems consisting of aqueous buffers of pH between 5 and8. Phosphate and borate buffers are adequate for this pur-pose. Avoid the use of pH extremes (< 5 or > 8). Retentionand optical resolution can be regulated via pH, bufferstrength (0.01 – 0.2 M) and/or surface tension via smallamounts of 1-propanol (0 – 5%) added as a co-solvent.Retention is always drastically reduced by as little as 1 to 2%1-propanol and >5% is not recommended. pH and ionicstrength will affect retention in a way not generally predicta-ble. To some extent the columns respond like reversed phasecolumns, but please note that the columns will not toleratemobile phase systems containing acetonitrile or metha-nol as used in reversed phase LC since the albumin will bedenatured under such conditions.Upon a change of the mobile phase, please remember toallow sufficient time to elapse for complete re-equilibration.Generally, full reproducibility between runs will e obtainedafter a couple of hours.

The mobile phase systems recommended for RESOLVOSIL®

offer the following advantages:Retention and optical resolution can be readily optimisedby using several independent mobile phase parameters(pH, buffer strength, surface tension).The mobile phase is easily available, inexpensive, non-toxic and biocompatible.All types of high sensitivity detector systems can beused (UV, fluorescence, electrochemical etc.).Injection of aqueous samples can be made directly on tothe column.

The mechanically stable microparticles of the RESOLVOSIL®

column permit chromatography at high flow rates and pres-sures up to about 300 bar (4200 psi).Load capacityFor RESOLVOSIL BSA-7 optimum resolution is achievedwith sample concentrations below 0.2 µmol per injection.Higher concentrations are likely to cause a decrease in reso-lution due to column overload. For biological samples youshould use a reversed phase or RESOLVOSIL guard col-umn or even a column switching technique to improve col-umn life. If a higher column capacity is required we recom-mend using column BSA-7PX.

Ic

Ib

Ia

L L

L

D

D

D

0 10 20 30 40 50 60 70

Enantiomer separation of N-benzoyl-D,L-amino acidsS. Allenmark 7)

Column: EC 150/4 RESOLVOSIL® BSA-7, 150 x 4 mm ID, Cat. No. 720046.40

Eluent: 50 mM phosphate buffer pH 6.5 + 1% 1-propanolFlow rate: 0.70 ml/minDetection: UV, 225 nm

Ia: SerineIb: AlanineIc: Phenylalanine

elution volume [ml]

1054

50


MN



92

Column stabilityChiral recognition of the RESOLVOSIL® column is based onthe interaction between covalently bonded bovine serum albu-min (BSA) and low molecular compounds. Inherently, the pro-tein structure is very susceptible to denaturing. For this reasonRESOLVOSIL® columns should be handled with care. For rec-ommendation concerning the operation of these columnsplease see the chapter “Mobile phase“. When not in use,RESOLVOSIL® columns should be stored in a refrigerator,equilibrated with azide-containing water.

References

1) K.K. Stewart and R.F. Doherty, Proc. Natl. Acad. Sci. USA, 70 (1973) 2850

2) Direct liquid chromatographic separation of enantiomerson immobilized protein stationary phases III. Optical res-olution of a series of N-aroyl D,L-amino acids by high-performance liquid chromatography on bovine serum al-bumin covalently bonded to silica.S. Allenmark, B. Bomgren and H. Borén, J. Chromatogr.264 (1983) 63 – 68

3) Direct analytical and preparative resolution of enantiom-ers using albumin adsorbed to silica as a stationaryphaseP. Erlandsson, L. Hansson and R. Isaksson, J. Chroma-togr. 370 (1986) 475 – 483

4) Direct liquid chromatographic separation of enantiomerson immobilized protein stationary phases IV. Molecularinteraction forces and retention behaviour in chromatog-raphy on bovine serum albumin as a stationary phaseS. Allenmark, B. Bomgren and H. Borén, J. Chromatogr.316 (1984) 617 – 624

5) Effects of pretreatment on the enantioselectivity of silica-bound proteins used as high-performance liquid chroma-tographic stationary phasesM.T. Aubel and L.B. Rogers, J. Chromatogr. 408 (1987)99 – 113

6) Some applications of chiral liquid affinity chromatographyusing bovine serum albumin as a stationary phaseS. Allenmark, B. Bomgren and S. Andersson, Prep. Bio-chem. 14 (1984) 139 – 147

7) Optical resolution of racemic compounds by means ofHPLC on immobilized protein stationary phases - theoryand applicationS. Allenmark, B. Bomgren and H. Borén, in "Affinity chro-matography and biological recognition" (I. Chaiken, M.Wilchek, and I. Parikh. Eds.), Academic Press, New York,1983, S. 259 – 260

8) Enantioselective microbial degradation of racematesstudied by chiral HPLC on silica-bound albumin.S. Allenmark, B. Bomgren, and H. Borén, Enzyme Mi-crob. Technol. 8 (1986) 404 – 408

9) Chiral reversed-phase liquid chromatographic monitoringof asymmetric carbonyl reduction by some yeast organ-ismsS. Allenmark and S. Andersson, Enzyme Microb. Tech-nol. 10 (1988) 177

Separation of the optical isomers of omeprazoleColumn: EC 150/4 RESOLVOSIL® BSA-7, 150 x 4 mm ID,

Cat. No. 720046.40Sample: 135 µM omeprazoleVolume: 20 µlEluent: 0.05 M phosphate buffer pH 7.9

+ 2% propanol-1Flow rate: 1,0 ml/minDetection: UV, 250 nm

N

CH3 O

OCH3

H3COCH3

CH2 SN

N

H

0 2 4 min

1057

60

For ordering information of RESOLVOSIL® columns please see page 90


MN


Summary of MN columns for bioanalysis and food analysis

93

pH 2 – 8 • NUCLEOSIL 125-5 GFC

GFC pH 1 – 13 • NUCLEOGEL GFC 300-8 • NUCLEOGEL GFC 1000-8

pH 1 – 13 • NUCLEOGEL RP 300-5 • NUCLEOGEL RP 4000-8

Proteins RPC pH 2 – 8 • NUCLEOSIL 300-5 C4 MPN • NUCLEOSIL 300-5 C18 MPN

pH 2 – 10 • NUCLEOSIL 500-5 C3 PPN • NUCLEOSIL 500-5 C18 PPN

IEC pH 2 – 8.5 • NUCLEOSIL 4000-7 PEI

pH 1 – 13 • NUCLEOGEL SAX 4000-8 • NUCLEOGEL SCX 4000-8

pH 2 – 8.5 • NUCLEOSIL 4000-7 PEI

IEC pH 1 – 13 • NUCLEOGEL SAX 1000-8 • NUCLEOGEL SCX 1000-8

• NUCLEOSIL 100-5 C18 MPNPeptides

pH 2 – 8 • NUCLEOSIL 120-3 C18 MPN • NUCLEOSIL 300-5 C4 MPN

RPC • NUCLEOSIL 300-5 C18 MPN • NUCLEOSIL 100-5 C18 PPN

pH 1 – 13 • NUCLEOGEL RP 100-5 *

Amino acids also see ”HPLC columns for enantiomer separation“ from page 79

RPC pH 2 – 8 • NUCLEOSIL 100-5 C18 MPN • NUCLEOSIL 120-3 C18 MPN

Nucleosides pH 2 – 8 • NUCLEOSIL 100-5 C18 MPN • NUCLEOSIL 300-5 C18 MPN

RPC pH 2 – 10 • NUCLEOSIL 100-5 C18 PPN • NUCLEOSIL 500-5 C18 PPN

pH 1 – 13 • NUCLEOGEL RP 100-5 * • NUCLEOGEL RP 300-5Nucleotides,

oligo-nucleotides

pH 2 – 8.5 • NUCLEOGEN 60-7 DEAE • NUCLEOSIL 4000-7 PEI

IEC pH 1 – 13 • NUCLEOGEL SAX 1000-8

GFC pH 1 – 13 • NUCLEOGEL GFC 300-8 • NUCLEOGEL GFC 1000-8DNA, RNA,restrictionfragments,

plasmids

• NUCLEOGEL GFC 4000-8

IEC pH 2 – 8 • NUCLEOGEN 4000-7 DEAE • NUCLEOGEN 500-7 DEAE

pH 1 – 13 • NUCLEOGEL SAX 4000-8 • NUCLEOBOND AX*

• NUCLEOGEL GFC 300-8 • NUCLEOGEL GFC 1000-8Polysaccharides GFC pH 1 – 13

• NUCLEOGEL GFC 4000-8

pH 2 – 8 • NUCLEOSIL Carbohydrate • NUCLEOSIL 100-5 NH2**

Mono- and oli-gosaccharides

• NUCLEOGEL ION 300 OA • NUCLEOGEL SUGAR 810 H

• NUCLEOGEL SUGAR Pb • NUCLEOGEL SUGAR 810 PbpH 1 – 13

• NUCLEOGEL SUGAR Ca • NUCLEOGEL SUGAR 810 Ca

• NUCLEOGEL SUGAR Na

Hopconstituents RPC pH 1 – 9 • NUCLEOSIL 100-5 C18 Hop

GFC = gel filtration chromatography IEC = ion exchange chromatography RPC = reversed phase chromatography* Please ask for our catalogue „Bioanalysis“ ** see page 54


MN


Ion exchange columns for biochemical applications

94

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 beenreported 1) that columns of 1.5 ml bed volume (4 mm ID x125 mm) have been used with more than 100 l buffer withoutloss of resolution; this corresponds to about 1000 repetitiveHPLC runs.For numerous separations of deoxyoligonucleotides, plas-mids and DNA restriction fragments see our catalogue “LCApplications” or visit our website:

Length → 125 mm Guard columns1)

NUCLEOGEN® 60-7 DEAE DEAE anion exchanger based on silica, particle size 7 ± 1.5 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 ± 1.5 µm, pore size 500 Å, eluent in column methanol

Valco type analytical columns6 mm ID 736598 736400.40


10 mm ID, IWC 736600 736400.40

NUCLEOGEN® 4000-7 DEAE DEAE anion exchanger based on silica, particle size 7 ± 1.5 µm, pore size 4000 Å, eluent in column methanol

Valco type analytical columns6 mm ID 736601 736400.40


10 mm ID, IWC 736603 736400.40IWC = columns inner wall coated with PTFE

Columns are supplied in packs of 1.1) 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).

www.mn-net.com


MN



95

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).The packed columns are ready for use. Elevated tempera-tures for chromatography are not required. The high capacityof 200 A260/ml NUCLEOGEN® DEAE permits the use ofsmall columns.For the isolation of oligonucleotides with chain lengthsbetween 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 the

chromatogram) and the concentration is increased to 4 M LiClin a vacuum centrifuge (e. g. Speed-Vac). To this concentratedsample five times its volume of ethanol/acetone (1:1) isadded, precipitated 5 h at –20 °C and centrifuged 1 h at5000 rpm.

Capacity [A260]Phase column EC 125/4 VA 125/6 SP 125/10NUCLEOGEN® 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: EC 125/4 NUCLEOGEN® 60-7 DEAE

Cat. No. 736596.40, 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

For ordering information of NUCLEOGEN® DEAE columns please see page 94.


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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 beincreased from 50 min to 75 min or 100 min. With increas-ingly flatter gradients the resolution for higher chain lengthsis improved. Better separations can be achieved with convexgradients, which allow for the smaller charge differencesfound with 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 A260oligo(rA)n the flow rate is increased to 2.5 ml/min using agradient time of 400 min.

Separation of oligo(rA)nColumn: EC 125/4 NUCLEOGEN® 60-7 DEAE

125 x 4 mm ID, Cat. No. 736596.40Buffer A: 20 mM phosphate, pH 5.5, 5 M ureaBuffer B: buffer A + 1 M KClGradient: 0 – 100% B in 200 min

Flow rate 2 ml/min, 110 barambient temperature

UV detection, 260 nmA260

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: EC 125/4 NUCLEOGEN® 60-7 DEAE

125 x 4 mm ID, Cat. No. 736596.40Buffer 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



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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.As some single-stranded nucleic acids can form complexeswith one another, if bivalent metal ions are not excludedusing EDTA (ethylenediamine tetraacetic acid), we supplycolumns with a PTFE inner surface coating to prevent corro-sion by the EDTA.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 ribonucleicacids. However, the load capacity is significantly lower thanfor the 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-chromatographysupercoiled forms can even be separated from the relaxedand linear forms (see chromatograms on next page).Because NUCLEOGEN® 4000-7 DEAE provides very highresolution for large as well as for medium-size nucleic acids,cDNA inserts can be easily separated from the remainingvector. The plasmids are eluted from the column with morethan 95% recovery and are fully active with respect to diges-

tion with restriction enzymes, labelling with kinases, PCR,LCR reactions, transcription with polymerases and enzy-matic ligation.The following buffer systems show very good separationefficiency for plasmids, viral RNA and restriction fragments.

Preparative separation of a crude RNA extract of vi-roid (PSTV) infected tomato plants D. Riesner BioEngineering 1 (1988) 42 – 48Column: VA 125/6 NUCLEOGEN® 500-7 DEAE

125 x 6 mm ID, Cat. No. 736598Buffer 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 95


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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: VA 125/6 NUCLEOGEN® 4000-7 DEAE

125 x 6 mm ID, Cat. No. 736601Eluent 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: VA 125/6 NUCLEOGEN® 4000-7 DEAE

125 x 6 mm ID, Cat. No. 736601Eluent 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: VA 125/6 NUCLEOGEN 4000-7 DEAE,

125 x 6 mm ID, Cat. No. 736601Gradient: 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


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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 sphericalmicroparticles of this packing allow high flow rates and along-term column life. The polymeric, covalently bondedpolyethylene imine network guarantees a good chemical sta-bility of the columns towards basic and acidic eluents. Thecoating is flexible enough to fit different protein shapes forthe formation 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: EC 50/4 NUCLEOSIL® 4000-7 PEI,

50 x 4 mm ID, Cat. No. 720401Buffers: 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 to strongly 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


NUCLEOSIL® 4000-7 PEI polyethyleneimine network, covalently polymer-coated onto NUCLEOSIL® silica, weakly basic anion exchanger, particle size 7 ± 1.5 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

Standard columns are manufactured from stainless steel. Metal-free columns (PEEK) can be custom-packed with 4.6 or 7.5 mm ID and lengths of 50, 150 and 250 mm.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

www.mn-net.com


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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 stickingimpurities by a 10-min treatment with 0.1 M NaOH or 1 MHNO3.

Recovery of proteinsColumn: EC 50/4 NUCLEOSIL 4000-7 PEI,

50 x 4 mm ID, Cat. No. 720401.40Eluent: 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 undialysedascites fluid46.3 mg/ml of total protein, sample volume 5 µlColumn: EC 125/4 NUCLEOSIL 4000-7 PEI,

125 x 4 mm ID, Cat. No. 720402.40Eluent 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: EC 125/4 NUCLEOSIL 4000-7 PEI,

125 x 4 mm ID, Cat. No. 720402.40Eluent 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: EC 50/4 NUCLEOSIL 4000-7 PEI,

50 x 4 mm ID, Cat. No. 720401.40Eluent 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 99.

Operation of NUCLEOSIL 4000-7 PEI columns

Separation principleSeparation is obtained by reversible adsorption of negativelycharged substances to positively charged groups on theexchanger material and their subsequent displacement byeither increasing ionic strength or pH changes in the mobilephase.Mobile phaseIn order to provide efficient adsorption of a protein to theanion 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 thesequence 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: EC 125/4 NUCLEOSIL 4000-7 PEI, 125 x 4 mm ID, Cat. No. 720402.40Eluent 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: EC 125/4 NUCLEOSIL 4000-7 PEI,

125 x 4 mm ID, Cat. No. 720402.40Eluent 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 highcapacity for proteins even at pH 10; for this reason it can beapplied for difficult protein separations in the pH rangearound and 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: VA 50/4.6 NUCLEOGEL

SAX 1000-8, 50 x 4.6 mm ID, Cat. No. 719469

Eluent 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


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. Themacroporous structure of NUCLEOGEL® SCX columnsguarantees unhindered contact of large molecules with theexchange functionalities. Complex samples (e.g. cellextracts) or solubilised membrane protein mixtures oftenpose problems because they cannot be recovered quantita-tively from the column. In this case, the polymer matrix of theNUCLEOGEL® SCX columns allows use of strongly basic oracidic eluents 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: VA 50/4.6 NUCLEOGEL SCX 1000-8,

50 x 4.6 mm ID, Cat. No. 719475Eluent 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


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

104

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 bedetected, such as e.g. exchange of an amino acid or thepresence of deamidation or oxidation products. During work-up of industrial scale preparations RPC is increasinglyapplied for the final purification. Examples include biosyn-thetic proteins such as human growth hormone, tissue plas-minogen activator, human insulin and human malaria vac-cine.

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 isdecreased resulting in desorption and chromatographic sep-aration of substances according to their hydrophobicity. Forbiological macromolecules partition effects have no signifi-cance.

As can be seen from the left figure below, the maximumseparation efficiency can be achieved when the injectedprotein mass does not exceed 1 – 2% of the maximumprotein loading capacity. In practice often sufficient chroma-tographic resolution is obtained for higher injection masses.The sample volume does not influence the separation resultover a wide range. For this reason RP chromatography oftenresults in a concentration of the biomolecule (see right figure).

Packing pH working range max. working pressure [bar]

dynamic protein binding capacity mg protein per g packing

BSA cytochrome CSilica-based phasesNUCLEOSIL® 100-5 C18 MPN 2 – 8 250 6 110NUCLEOSIL® 300-5 C18 MPN 2 – 8 250 16 55NUCLEOSIL® 300-5 C4 MPN 2 – 8 250 14 27NUCLEOSIL® 100-5 C18 PPN 2 – 10 250 8 64NUCLEOSIL® 500-5 C18 PPN 2 – 10 250 22 40NUCLEOSIL® 500-5 C3 PPN 2 – 10 250 23 29Polymer-based phasesNUCLEOGEL® RP 80-10 C18 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 –

EC 125/4 NUCLEOSIL 500-5 C18 PPN, Cat. No. 720257.40, 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 Chromatographic resolution as a function of sample volume

EC 125/4 NUCLEOSIL® 500-5 C3 PPN, Cat. No. 720263.40, eluent A) 0.1% TFA in H2O, B) 0.08% TFA in CH3CN, gradient 20% – 60% B in 10 min, proteins cytochrome C, bovine serum albumin

chro

mat

ogra

phic

res

olut

ion

sample 1 10 100 1 000 µl

Rs5

4

3

2

1

volume


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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 silicamonomerically 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 C18modification shows an outstanding selectivity for peptides,while the wide pore C4 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: EC 250/4 NUCLEOSIL® 300-5 C4 MPN

250 x 4 mm ID, Cat. No. 720245.40Eluent 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



NUCLEOSIL® 100-5 C18 MPN Octadecyl phase, alkyl chains monomerically bonded to silica, brush type structure, particle size 5 ± 1.5 µm, pore size 100 Å; eluent in column methanol

2 mm ID 720231.204 mm ID 720230.40 720231.40

NUCLEOSIL® 120-3 C18 MPNOctadecyl phase, alkyl chains monomerically bonded to silica, brush type structure, particle size ~ 3.5 µm, pore size 120 Å; eluent in column methanol

2 mm ID 720232.204 mm ID 720232.40

NUCLEOSIL® 300-5 C4 MPNButyl phase, alkyl chains monomerically bonded to silica, brush type structure, particle size 5 ± 1.5 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

The above columns are manufactured from stainless steel. Metal-free columns (PEEK) with 4.6 or 7.5 mm ID and lengths of 50, 150 and 250 mm can be custom-packed on request.

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 layerensures that peptides and proteins are exclusively retainedby hydrophobic forces. Polypeptides which differ in shapeand size of their hydrophobic surface regions are separated.Thus these columns feature different selectivities comparedto the MPN material, a fact which may improve the solutionof certain separation problems. With NUCLEOSIL 100-5C18 PPN good peak shapes are also found for basic pep-tides.

The wide pore NUCLEOSIL PPN phases are available intwo different alkyl chain lengths (octadecyl and propyl),which differ in hydrophobicity and rigidity of the alkyl network.While the C18 material is especially suited for large peptidesand medium-size hydrophilic proteins, the C3 phase is rec-ommended for larger and hydrophobic proteins. Memoryeffects are reduced. The PPN columns can be used with elu-ent containing organic bases up to pH 9.5.


NUCLEOSIL® 100-5 C18 PPN Octadecyl phase, alkyl chains polymerically bonded to silica, particle size 5 ± 1.5 µm, pore size 100 Å; eluent in column methanol


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


NUCLEOSIL® 500-5 C3 PPN Propyl phase, alkyl chains polymerically bonded to silica, particle size 5 ± 1.5 µm, pore size 500 Å; eluent in column methanol


2 mm ID 720263.20 721118.304 mm ID 720262.40 720263.40 721118.40


NUCLEOSIL® 500-5 C18 PPN Octadecyl phase, alkyl chains polymerically bonded to silica, particle size 5 ± 1.5 µm, pore size 500 Å; eluent in column methanol


4 mm ID 720256.40 720257.40 720258.40 721687.40


The above columns are manufactured from stainless steel. Metal-free columns (PEEK) with 4.6 or 7.5 mm ID and lengths of 50, 150 and 250 mm can be custom-packed on request. Corresponding 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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The polymeric coating of NUCLEOSIL 100-5 C18 PPN showsoutstanding stability at alkaline pH and the absence of nonspe-cific interactions with alkaline or acidic compounds. Therefore,this column is suited for the separation of peptides and proteinsup to about 40 kD. The column may be purified by treatmentwith SiO2-saturated NaOH. 10 to 20 column volumes of the al-kaline eluent should be enough to wash away sticking impuritiesfrom the column. This procedure will have a sterilising effect,too. Retention times, peak shapes and mass recoveries are notaffected by passing more than 300 column volumes of the alka-line eluent through the column as is shown on the right.

1

2

3

45

6

7

5 10 15 20 min

Separation of pancreatic secretion of pigletsColumn: EC 125/4 NUCLEOSIL 500-5 C18 PPN

125 x 4 mm ID, Cat. No. 720257.40Eluents: 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

1082

80

Separation of commercial bacitracinColumn EC 125/4 NUCLEOSIL 100-5 C18 PPN125 x 4 mm ID, Cat. No. 720251.40Eluent 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 20min

1152

10

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: EC 125/4 NUCLEOSIL 100-5 C18 PPN,

125 x 4 mm ID, Cat. No. 720251.40Eluent 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

Separation of a 20mer oligonucleotide(Sample courtesy of Dr. Essrich, Inst. Prof. Seelig, Karlsruhe, Germany)Column EC 250/4 NUCLEOSIL 100-5 C18 PPN, 250 x 4 mm ID, Cat. No. 720252.40Eluent 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 forreversed phase chromatography in the pH range from 1 to14. They contain a C18 modified polystyrene-divinylbenzenepolymer with outstanding stability, inertness and improvedpeak symmetry even for basic substances. Only simple elu-ent systems are required for the separation. Due to improvedsensitivity post-column derivatisations are seldom neces-sary.

NUCLEOGEL® RP columns

This type of reversed phase column is based on a polysty-rene resin cross-linked with DVB. Due to the excellent stabil-ity 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: VA 150/4.6 NUCLEOGEL® RP 80-10 C18,

150 x 4,6 mm, Cat. No. 719500Eluent 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: VA 150/4.6 NUCLEOGEL® RP 80-10 C18,

150 x 4,6 mm, Cat. No. 719500Eluent: 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 125 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

Valco- type analytical columns 1)

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 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 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.Additives 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,because 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 nucleicacids with these columns please see our catalogue “LCApplications” 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: VA 150/4.6 NUCLEOGEL RP 100-8,

150 x 4,6 mm ID, Cat. No. 719456Eluent 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: VA 50/4.6 NUCLEOGEL RP 300-8,

50 x 4,6 mm ID, Cat. No. 719460Eluent 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: VA 50/4.6 NUCLEOGEL® RP 4000-8,

50 x 4,6 mm, Cat. No. 719462Eluent 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: VA 50/4.6 NUCLEOGEL® RP 4000-8,

50 x 4,6 mm, Cat. No. 719462Eluent 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

112

Gel 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 HPLC columns for GFC basedon silica or polymer resins.

NUCLEOSIL® GFC silica-based columnsNUCLEOSIL® GFC is a silica-based column with a hy-drophilic polyalcohol modification.

Characteristic parameters of the column packingWorking range for globular proteins 5 000 – 150 000pH working range 2 – 8minimum salt concentration 100 mMpolar organic solvents (modifiers) 0 – 100%maximum pressure 100 bartypical flow rate 0.5 – 1 ml/mintypical temperature 10 – 30 °C


NUCLEOSIL® 125-5 GFChydrophilic polyalcohol modification on silica; particle size 5 µm, pore size 125 Å; eluent in column 0.1 M NaH2PO4 pH 6.7 / 0.1 M Na2SO4 (1:1, v/v) + 0.05% NaN3

Valco type analytical columns7.7 mm ID 719590 721655.40

Columns are supplied in packs of 1. ChromCart® guard column cartridges CC 30/4 NUCLEOSIL® 125-5 GFC are 30 mm long and supplied in packs of 2. They require the CC column holder 30 mm (Cat. No. 721823).

Separation of standard proteinsColumn: VA 300/7.7 NUCLEOSIL®

125-5 GFC, 300 x 7.7 mm ID, Cat. No. 719590Eluent: 0.05 M NaH2PO4,

0.3 M NaCl, pH 7.0Flow rate: 1 ml/minDetection: UV, 220 nm

Peaks:1. Thyroglobulin2. γ-Globulin3. Ovalbumin4. RNase A5. p-Amino-

benzoic acid

Separation of standard proteinsColumn: VA 300/7.7 NUCLEOSIL®

125-5 GFC, 300 x 7.7 mm ID, Cat. No. 719590

Eluent: 0.02 M NaH2PO4, 0.2 M NaCl, pH 7.0

Flow rate: 1 ml/minDetection: UV, 220 nmPeaks:1. Ferritin2. BSA3. β-Lactoglobulin4. Cytochrome C5. Uridine

Separation of hemoglobin and myoglobin

Column: VA 300/7.7 NUCLEOSIL® 125-5 GFC, 300 x 7.7 mm ID, Cat. No. 719590

Eluent: 0.1 M NaH2PO4, 0.3 M NaCl, pH 7.0

Flow rate: 1 ml/minDetection: UV, 280 nmPeaks:1. Hemoglobin2. Myoglobin

1

2

3

4

5

0 10 min

1

2

3

4 5

0 10min

1

2

0 8min1152

20

1152

30

1152

40


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113

NUCLEOGEL® GFC columns for the separation of natural polymers and proteinsNUCLEOGEL® 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 and can easily beused in the pH range from 1 to 13. Thus they allow theremoval of pyrogens and sterilisation of the columns with0.1 M NaOH. Organic solvents such as methanol or ace-tonitrile can be used as modifiers. The different pore sizespermit separations up to a molecular weight of 107 daltons(see calibration curves on page 115). 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 characteristicparameters 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 1 – 13maximum salt concentration 8 Mpolar organic solvents (modifiers) 0 – 100%maximum temperature 80 °C

Separation of nucleic acidsColumns: 2 x VA 300/7.7 NUCLEOGEL GFC 4000-8,

300 x 7.7 mm ID, Cat. No. 719449Eluent: 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


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 130.

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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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,resulting 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 avoidedbecause they can destroy the column packing. Columns canbe used 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 recom-mended. The lifetime of the columns can be considerablyincreased by the use of guard columns.Column cleaning and storageFor cleaning, columns can be flushed with 0.1 M HCl, 0.1 MNaOH or with organic solvents, which allow an efficientremoval of sample residues from the column. Columnsshould be flushed with 5 – 10 column volumes water / 0.2%sodium azide and stored well closed at ambient temperature.

Wash cycles

New column

Myoglobin elution volume [ml]

plate number 12500 m–1

0.1 M HCl0.1 M NaOH10% methanol0.5 M HCl20% acetic acid50% methanol100% methanol plate number 13000 m

–1

0 1 2 3 4 5 6 7 8

New column plate number 10000 m

–1


–1

0 1 2 3 4 5 6 7 8

New column plate number 11800 m

–1


–1

0 1 2 3 4 5 6 7 8

Ovalbumin elution volume [ml]

Chymotrypsinogen A elution volume [ml]

Stability of NUCLEOGEL

GFC columns

Purification of a commercial catalase product2 columns VA 300/7.7 NUCLEOGEL GFC 300-8 (Cat. No. 719447), eluent: 0.02 M NaH2PO4, 0.2 M NaCl pH 7.0, flow rate 0.5 ml/min, UV detector

20 44min

1151

50

Separation of standard proteinsVA 300/7.7 NUCLEOGEL GFC 300-8 (Cat. No. 719447), eluent: 20 mM NaH2PO4, 200 mM NaCl pH 7,0, flow rate 0.1 ml/min, UV detector 1. Ferritin2. Albumin (bovine

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

1

2

3

4

5

6

60 75 90 min1082

30


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Calibration curves for polysaccharides and proteins on NUCLEOGEL® GFC 300-8Column: VA 300/7.7 NUCLEOGEL GFC 300-8,

300 x 7.7 mm ID, Cat. No. 719447Eluent: 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

Separation of polysaccharide calibration standards 2 x VA 300/7.7 NUCLEOGEL

GFC 4000-8 (Cat. No. 719449) + VA 300/7.7 NUCLEOGEL

GFC 300-8 (Cat. No. 719447), eluent: 0.02 M NaH

2

PO

4

, 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 28min

1025

60

1025

70

Calibration curves for NUCLEOGEL® GFC columns of different pore sizes Columns: VA 300/7.7 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


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HPLC columns for food analysis

116

Columns for the separation of carbohydrates

Frequently in practice oligo- and monosaccharides have tobe analysed together with organic acids and alcohols. Theanalysis of carbohydrate-containing solutions is an importanttask in biochemistry, medicine, pharmaceutical and otherindustries, especially food and beverage industries. One diffi-culty arises from the fact that often molecules have to beseparated based on stereochemical differences or different

monomer linkages, only. Increasingly, HPLC methods areused for this purpose. This chapter describes columns whichhave been specially developed for the analysis of carbohy-drates, organic acids and alcohols in food, alcoholic bever-ages, fermentation broths and other complex matrices.MACHEREY-NAGEL offers three different column families forthis field:

NUCLEOSIL

®

Carbohydrate

NUCLEOSIL

®

Carbohydrate is a special silica phase withamino modification, which is recommended for the RP sepa-ration of different mono- and disaccharides. The silica matrixenables high working pressures (> 200 bar) and thus shortanalysis times.

Column type matrix modification recommended application

NUCLEOSIL

®

Carbohydrate

silica amino mono- + disaccharides

NUCLEOGEL

®

SUGAR 810 H

PS/DVB SO

3

H sugars, sugar alcohols, organic acids

NUCLEOGEL

®

SUGAR 810 Ca

PS/DVB SO

3

Ca mono-, di- and oligosaccharides

NUCLEOGEL

®

SUGAR 810 Pb

PS/DVB SO

3

Pb mono-, di- and oligosaccharides

NUCLEOGEL

®

ION 300 OA

PS/DVB SO

3

H sugars, alcohols, organic acids

NUCLEOGEL

®

SUGAR Ca

PS/DVB SO

3

Ca mono- and oligosaccharides, sugar alcohols

NUCLEOGEL

®

SUGAR Na

PS/DVB SO

3

Na oligosaccharides from starch hydrolysates and food

NUCLEOGEL

®

SUGAR Pb

PS/DVB SO

3

Pb mono- and disaccharides from food and biological samples

Separation of sugarsColumn: EC 250/4 NUCLEOSIL® CarbohydrateCat. No. 720905.40

Sample volume 10 µlEluent: acetonitrile – water

(79:21, v/v)Flow rate: 2 ml/minTemperature: 25 °CDetector: RI

Peaks:1. Fructose2. Glucose3. Saccharose4. Maltose5. Lactose

1

2 3

45

0 5 10 min 1024

80

Length

→

250 mm Guard columns

NUCLEOSIL

®

Carbohydrate

special amino phase based on NUCLEOSIL

®

silica, eluent in column CH

3

CN:H

2

O (79:21)

ChromCart

®

columns

4 mm ID 721467.40 721595.40

EC columns

1)

4 mm ID 720905.40 721595.40

All

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).


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For the separation of oligosaccharides with longer chains(10 < n < 40) our phase NUCLEOSIL

®

300-5 C

18

can besuccessfully applied (see figure below). In this case a veryflat gradient allows good resolution of the carbohydrates.

For ordering information of this phase please see page 40.

HPLC separation of an amidase digest of 150 µg [

3

H]GlcNAc-murein from E. coli EH3247

H. Harz, K. Burgdorf, J.-V. Höltje, Anal. Biochem.

190

(1990) 120 – 128Column EC 250/4.6 NUCLEOSIL

®

300-5 C

18

, 250 x 4.6 mm ID, Cat. No. 720065.46; eluent: convex gradient from 100 mM sodium phosphate, pH 2.0 with 5% acetonitrile to 100 mM sodium phosphate, pH 2.0 with 11% acetonitrile, followed by 100% methanol; flow rate 0.5 ml/min; temperature 50 °C; detection: upper chromatogram UV at 202 nm, lower chromatogram radioactivity of 31-µl portions of fractions (125 µl). The numbers above the peaks indicate the length in disaccharide units.

Abs

orpt

ion

at 2

02 n

mR

adio

activ

ity [c

pm]

0,025

0,01

3200

2600

2000

1400

1000

750

500

250

0

1 25 50 75 100 125 150 175 200 225 250 275 300 325fraction number

350 375 400 425 450

23000

1 23

45

6 7 8 9 1011 12

1027

30


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118


NUCLEOGEL

®

SUGAR 810 columns

The adsorbents of the NUCLEOGEL

®

SUGAR 810 familyare based on a sulphonated polystyrene / divinylbenzeneresin.

Due to a selectivity pattern which differs from theNUCLEOGEL

®

SUGAR columns, the range of applica-tion is considerably enlarged.

The interplay of differentseparation mechanisms such as ion exclusion, ionexchange, size exclusion, ligand exchange as well as NPand RP chromatography allows the efficient separation oforganic acids, carbohydrates and other polar small mole-cules.

Advantages of NUCLEOGEL

®

SUGAR 810 columns areThey allow up to 30% acetonitrile in the eluent.They feature pressure stability, long column life and ex-cellent pH stability.Guard columns in the convenient ChromCart

®

systemdon’t require tools for installation

Length

→

300 mm Guard columns

Valco type columns

NUCLEOGEL

®

SUGAR 810 H

polystyrene / divinylbenzene resin, –SO

3

H modification, eluent in column 0.01 N H

2

SO47.8 mm ID 719574 719575

NUCLEOGEL® SUGAR 810 Capolystyrene / divinylbenzene resin, –SO3Ca modification, eluent in column H2O

7.8 mm ID 719570 719571

NUCLEOGEL® SUGAR 810 Pbpolystyrene / divinylbenzene resin, –SO3Pb modification, eluent in column H2O

7,8 mm ID 719572 719573Columns are supplied in packs of 1.ChromCart® guard column cartridges NUCLEOGEL® SUGAR 810 are 30 x 4 mm and supplied in packs of 2. They are installed using CC col-umn holder 30 mm (Cat. No. 721823).

Separation of tobacco constituentsColumns: VA 300/7.8 NUCLEOGEL® Sugar 810 Ca

300 x 7.8 mm ID, Cat. No. 719570Volume: 5 µlEluent: WaterFlow rate: 0.6 ml/minDetector: RITemperature: 85 °CPeaks:1. Sucrose2. Glucose3. Fructose4. Glycerol5. 1,2-Propanediol6. Sorbitol

1

2

34

5

6

0 10 20min11

4140

Separation of organic acidsColumns: VA 300/7.8 NUCLEOGEL® Sugar 810 H

300 x 7.8 mm ID, Cat. No. 719574Volume: 5 µlEluent: 5 mM H2SO4Flow rate: 0.6 ml/minDetector: RITemperature: 35 °CPeaks:1. Oxalic acid2. Citric acid3. Malic acid4. Succinic acid5. Formic acid6. Acetic acid 1

2

3

4

5 6

0 10 15 min51135

70


MN



119


NUCLEOGEL® ION 300 OA and NUCLEOGEL® SUGAR columns

These NUCLEOGEL® columns are packed with sulphonatedspherical polystyrene-divinylbenzene particles in differentionic forms with a mean particle size of 10 µm and a poresize of 10 nm. For separation of the carbohydrate moleculesdifferent mechanisms such as steric exclusion, ligandexchange and partition effects play together. Ligandexchange, however, is the predominant force, since thehydrated metal ions form strong interactions with the hydroxygroups of the sample molecules. The intensity of these inter-actions decreases in the sequence Pb, Ca, Na.These columns are operated with deionised water or – forNUCLEOGEL® ION 300 OA – with diluted sulphuric acid. Upto 20% or polar organic modifiers can be added to the elu-ent. If salts are added to sample or eluents, this will alter themetal coating and thus deteriorate column performance. Formaximum separation efficiency the columns should be oper-ated at temperatures above 60 °C. The maximum workingtemperature is 95 °C.For a long column life it is important to use prepurified sam-ples. The literature cites different methods, e. g. using solidphase extraction cartridges (see the chapter ”Sample prepa-ration“ for our CHROMABOND® programme).The maximum working pressure should not exceed 100 bar.

Separation of carbohydrates and organic acidsColumn: VA 300/7.8 NUCLEOGEL ION 300 OA

Cat. No. 719501Eluent: 0.01 N H2SO4Flow rate: 0.6 ml/minTemperature: 30 °CDetection: RIPeaks:1. Sucrose2. Glucose3. Citric acid4. Fructose5. Tartaric acid 6. Malic acid7. Glycerol8. Acetic acid9. Lactic acid10. Methanol11. Ethanol

1 2

3

4

5 67

8

9

10

11

0 10 20 min1024

60


Valco type columns

NUCLEOGEL® ION 300 OApolystyrene / divinylbenzene resin, –SO3H modification, particle size ~ 10 µm, pore size 100 Å; eluent in column 0.01 N H2SO4

7.8 mm ID 719501 719537

NUCLEOGEL® SUGAR Ca polystyrene / divinylbenzene resin, –SO3Ca modification, particle size ~ 10 µm, pore size 100 Å; eluent in column H2O + 0.02% azide

6.5 mm ID 719531 719535

NUCLEOGEL® SUGAR Pbpolystyrene / divinylbenzene resin, –SO3Pb modification, particle size ~ 10 µm, pore size 100 Å; eluent in column H2O + 0.02% azide

7.8 mm ID 719530 719534

NUCLEOGEL® SUGAR Napolystyrene / divinylbenzene resin, –SO3Na modification, particle size ~ 10 µm, pore size 100 Å; eluent in column H2O + 0.02% azide

7.8 mm ID 719532 719536Columns are supplied in packs of 1.Valco type guard column cartridges are 21 x 4 mm and supplied in packs of 2. They are installed using guard column holder C (Cat. No. 719538).


MN



120

Separation of carbohydratesColumn: VA 300/7.8 NUCLEOGEL® SUGAR Pb

Cat. No. 719530Eluent: deionised waterFlow rate: 0.4 ml/minTemperature: 80 °CDetection: RIPeaks:1. Sucrose2. Maltose3. Glucose4. Xylose5. Galactose6. Arabinose7. Mannose

1 2

34

56

7

0 5 10 15 20 25 30min

1024

30

Analysis of a starch hydrolysateColumn: VA 300/7.8 NUCLEOGEL® SUGAR Na

Cat. No. 719532Eluent: deionised waterFlow rate: 0.5 ml/minTemperature: 70 °CDetection: RISample: 20 µl starch hydrolysate

Fruc

tose

Glu

cose

DP

2

DP

3D

P4

DP

5D

P6

DP

7D

P8

DP

9D

P10

0 5 10 15 20 25 min

1151

70

Separation of organic acids and sugars in wineColumn: VA 300/7.8 NUCLEOGEL® ION 300 OA

Cat. No. 719501Eluent: 0.005 N H2SO4Flow rate: 0.3 ml/minTemperature: 60 °CDetection: RIPeaks:1. Citric acid2. Tartaric acid3. Glucose4. Malic acid5. Fructose6. Acetic acid7. Glycerol8. Lactic acid9. Methanol

10. Ethanol

1

2

3

4

5

6

7

8 9

10

0 10 20 30 40 50min

1023

70

Separation of tartaric acid, sugars and sugar alcoholsColumn: VA 300/7.8 NUCLEOGEL® SUGAR Ca

Cat. No. 719531Eluent: WaterFlow rate: 0.5 ml/minTemperature: 70 °CDetection: RISample (10 µl of a 10 mg/ml solution)1. Tartaric acid2. Saccharose3. Glucose4. Fructose5. Sorbitol

1

2

34

5

0 4 8 12 16 min

1151

10


MN



121

RP column for the separation of hop constituentsDetermination of hop constituents

Reversed phase HPLC with C18 modified silica phases is themethod of choice for the analysis of hop constituents. Espe-cially for hop analysis we have developed our phase NUCLE-OSIL® 100-5 C18 Hop. The „European Brewery ConventionMethods of Analysis“ explicitly recommend this phase for theanalysis of iso-alpha-, alpha- and beta-acids as well as isom-erised hop extracts [J. Inst. Brew. 100 (4) 239 – 311 (1994)].

As can be seen in the two chromatograms below, separationof the hop constituents is most frequently performed underisocratic conditions. For this reason proper sample prepara-tion is a very important step for this analysis. Each column is

supplied with detailed instructions for use. thus operation ofthe columns under very different conditions is possible with-out any problems. For more chromatograms using these col-umns please see our catalogue "LC Applications".

Hops have been used for more than a thousand years,especially for beer brewing. The contents of the cone-shaped seed case are rich in flavour and colour sub-stances. As main constituents of hop oil and lupulin theisoprenoid hop bitter acids lupulon (β-lupulinic acid, β-hopbitter acid) and humulon (α-hop bitter acids, α-lupulinicacid) and their isomerisation products have to be routinelymonitored during hop processing.


NUCLEOSIL® 100-5 C18 Hopspecial octadecyl phase based on silica for the separation of hop constituents, particle size 5 ± 1.5 µm, pore size 100 Å; eluent in columns methanol

EC columns 3 mm ID 720217.30 721489.304 mm ID 720044.40 721489.40

ChromCart® guard column cartridges (8 mm) in packs of 2, all other columns in packs of 1 As guard columns for EC columns use ChromCart® guard column cartridges with guard column adaptor EC (Cat. No. 721359).

Determination of hop constituentsColumn: EC 125/3 NUCLEOSIL 5 C18 Hop,

125 x 3 mm ID, Cat. No. 720217.30Eluent: MeOH – H2O – H3PO4

(75:20:0,25)Injectionvolume: 5 µlFlow rate: 0.4 ml/minPressure: 90 barDetection: UV, 314 nmPeaks: 1. Co-humulon2. N+Ad-humulon3. Co-lupulon4. N+Ad-lupulon

12

34

0 10 20 min

Separation of α- and β-hop bitter acidsColumn: EC 250/4 NUCLEOSIL 5 C18 Hop,

250 x 4 mm ID, Cat. No. 720044.40Eluent: MeOH – H2O – H3PO4

(85:17:0,25)Flow rate: 0.8 ml/minPressure: 140 barDetection: UV, 314 nmPeaks: 1. Co-humulon2. N+Ad-humulon3. Co-lupulon4. N+Ad-lupulon

1

2

34

0 10 20 30min

1149

20

1000

50


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Basic principles of preparative HPLC - Winlab 2.pdf · MN Columns for HPLCColumns for HPLC Basic...

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