columns for hplccolumns for hplc - western · pdf file mn columns for hplccolumns for hplc 20...

MNwww.mn-net.com

Columns for HPLCColumns for HPLC

20

6.7.

05

In this chapter we describe analytical HPLC columns withour NUCLEOSIL

®

silica phases. Silica as well as polymer-based phases for special separation problems are describedin the chapter “Columns for special applications” from p. 73.

For our silica bulk packing materials, spherical NUCLEOSIL

®

and NUCLEODUR

®

materials and the irregular POLY-GOSIL

®

and POLYGOPREP silicas, please see the chapter“Packings for HPLC” from p. 143.

NUCLEOSIL

®

silica · the standard in HPLC

NUCLEOSIL

®

is a family of totally porous spherical silicas.They feature a very pure and uniform SiO

2

structure andhave gained wide acceptance as outstanding chromato-graphic packing for very different fields of modern chroma-tography. Due to its particle sizes NUCLEOSIL

®

finds application in an-alytical as well as in preparative columns. It allows

high bed stability

due to spherical particles

high efficiency

due to narrow particle size distribution

high separation performance

due to optimized bindingtechniques

high chemical and mechanical stabilityhigh load capacity and recovery rateshigh reproducibility

from lot to lot

As can be seen in the figure, NUCLEOSIL

®

is a sphericallyshaped totally porous silica. We manufacture this packingmaterial with different pore diameters (50, 100, 120, 300,500, 1000 and 4000 Å) and particle sizes from 3 µm (onlyNUCLEOSIL

®

50, 100 and 120) to 10 µm with very narrowfractionation. The different groups of NUCLEOSIL

®

packing materials andcolumns packed with these phases are described in detail inthe following chapters. All narrow-pore NUCLEOSIL

®

packings are stable up to 600bar (8 500 psi), for NUCLEOSIL

®

120 even pressures of upto 800 bar (11 500 psi) can be applied. The wide-pore NU-CLEOSIL

®

silicas are stable up to 300 or 400 bar (4 200 or5 600 psi). Thus all NUCLEOSIL

®

packings can be used inevery field of HPLC without limitations.

Modified packings

NUCLEOSIL

®

packings are available as unmodified silica orwith numerous chemically bonded phases. Silica packingswith chemically bonded phases can be classified in severalgroups, namely those intended mainly for reversed phasechromatography (the base deactivated RP phases C

18

AB,C

18

HD, C

8

HD, NAUTILUS and PROTECT I and the stand-ard RP phases C

18

, C

8

ec, C

8

, C

4

, C

2

and Phenyl) as well aspackings with chemically bonded polar groups with selectiveseparation performance (such as CN, NO

2

, NH

2

, N(CH

3

)

2

,OH types). Furthermore, for NUCLEOSIL

®

100 we produceion exchangers (NUCLEOSIL

®

SA and SB), which are stablefrom pH 2 to 8,5 and do not swell. Compared to resin-basedion exchangers they offer the advantage of constant permea-bility, even when the ionic strength and/or pH of the eluentare changed.

SEM photograph of NUCLEOSIL®

(magnification see scale, 1 cm ≈ 40 µm)

NUCLEOSIL

®

silica · summary of available modifications

Type of modification functional grouporganic Si-C group

Pore diameter [Å] Parti-cles50 100 120 300 500 1000 4000

base deactivated RP phases

C

18

HD

see p. 28

Octadecyl, base deactivatedmonomer modification

-(CH

2

)

17

-CH

3

✔

3 µm

✔

5 µm

✔

7 µm

C

8

HD

see p. 28

Octyl, base deactivatedmonomer modification

-(CH

2

)

7

-CH

3

✔

3 µm

✔

5 µm

C

18

NAUTILUS

see p. 30

Octadecyl, base deactivatedembedded polar groupfor up to 100% aqueous eluents

✔

3 µm

✔

5 µm

PROTECT I

see p. 32

special RP phasebase deactivated, monomer modification

✔

3 µm

✔

5 µm

✔

= as packed column in our standard programme;

✗

= available as bulk material

Analytical columns packed with NUCLEOSIL

®

MN www.mn-net.com


21

6.7.

05

C

18

AB

see p. 34

Octadecyl, base deactivatedpolymer modification

-(CH

2

)

17

-CH

3

✔

✗

5 µm

Standard RP phases

C

18

see p. 39

Octadecyl, endcapped

-(CH

2

)

17

-CH

3

✔✗ ✔✗

3 µm

✔✗ ✔✗ ✔✗ ✔✗

5 µm

✔✗ ✔✗ ✗ ✔✗ ✔✗ ✔✗

7 µm

✔✗ ✔✗ ✗

10 µm

C

8

ec

see p. 43Octyl, endcapped

-(CH

2

)

7

-CH

3

✔✗ ✔✗

5 µm

C

8

see p. 43

Octyl, not endcapped

-(CH

2

)

7

-CH

3

✔✗

3 µm

✔✗ ✔✗ ✔✗

5 µm

✔✗ ✗ ✗ ✗

7 µm

✔✗ ✗ ✗

10 µm

C

6

H

5

ec

see p. 46

Phenyl, endcapped-(CH

2

)

3

✔✗

5 µm

C

6

H

5

see p. 46

Phenyl-(CH

2

)

3

✔✗

5 µm

✔✗ ✗ ✗ ✗ ✗ 7 µm

C4see p. 47

Butyl

-(CH2)3-CH3

✔✗ ✔✗ 5 µm✔✗ ✗ ✗ ✗ 7 µm✗ 10 µm

C2see p. 48

Dimethyl-(CH3)2

✔✗ 7 µm

Polar NUCLEOSIL® phases and NUCLEOSIL® ion exchangers

CNsee p. 51

Cyano (Nitrile)

-(CH2)3-CN

✔✗ 5 µm✔✗ ✗ ✗ 7 µm

✔✗ 10 µm

NO2see p. 52

Nitro-(CH2)3 NO2

✔✗ 5 µm✗ 10 µm

OHsee p. 53

Diol ✔ 5 µm✔✗ ✗ ✗ ✗ ✗ 7 µm

NH2see p. 55

Amino

-(CH2)3-NH2

✔✗ 3 µm✔✗ 5 µm

✔✗ ✗ 7 µm✗ 10 µm

N(CH3)2see p. 56

Dimethylamino-(CH2)3-N(CH3)2

✔✗ 5 µm✗ 10 µm

SAsee p. 57

Sulphonic acid-(CH2)3 SO3Na

✔✗ 5 µm✔✗ 10 µm

SBsee p. 58

quaternary ammonium groups

-(CH2)3 CH2-N+(CH3)3Cl–✔✗ 5 µm✔✗ 10 µm

NUCLEOSIL® silica · summary of available modificationsType of modification functional group

organic Si-C groupPore diameter [Å] Parti-

cles50 100 120 300 500 1000 4000

✔ = as packed column in our standard programme; ✗ = available as bulk material

-(CH2)3-O-CH2-CH-CH2

OH OH

Analytical columns packed with NUCLEOSIL®

MNwww.mn-net.com


22

6.7.

05

Base deactivated phases for RP chromatographyIt is a known fact that the surface of silica contains activegroups that can cause unwanted interactions with the ana-lytes, even after derivatization and endcapping. For NUCLEOSIL®, MACHEREY-NAGEL use four differentapproaches to reduce silanol interactions. This results in fourtypes of base deactivated RP phases, which cover a verywide range of selectivities solving almost all reversed phaseseparation problems.

NUCLEOSIL® 100-5 C18 ABthe classical Acid and Base deactivated octadecyl-phasewith polymeric coatingThis phase exhibits a high steric selectivity.

NUCLEOSIL® 100-5 C8 HD · NUCLEOSIL® 100-3 C18 HD · NUCLEOSIL® 100-5 C18 HDa family of base deactivated phases with a High Densitymonomeric coatingavailable as octyl phase or octadecyl phase with 3 or 5 µmparticlesThe dense coverage with alkyl groups results in phases withvery low bleeding, which are ideal for trace analyses and es-pecially recommended for LC/MS.

NUCLEOSIL® 100-3 C18 NAUTILUS, NUCLEOSIL® 100-5 C18 NAUTILUSbase deaktivated octadecyl phases for up to 100% aque-ous eluents

An embedded polar functional group allows chromatographyunder purely aqueous elution conditions.

NUCLEOSIL® 100-3 PROTECT I, NUCLEOSIL® 100-5 PROTECT Ia base deactivated silica RP phase with special mono-meric coatingA polar protective group in the carbon skeleton strongly inter-acts with the silanol groups, effectively shielding them fromthe analytes.While featuring an outstanding base deactivation, the phaseNUCLEOSIL® PROTECT I shows higher hydrophilicity thanthe above phases.

Si OSi(CH3)3

Si OH conventional NUCLEOSIL® 100-5 C18

Si OSi(CH3)3

Pol

Pol

Si OH

Si OSi(CH3)3

Si OH

pro

pro


MN www.mn-net.com


23

6.7.

05

Base deactivated phases for RP chromatography

Why is a good base deactivation of the silica so important?

Due to interactions with residual silanol groups the RP chro-matography of basic components on standard silica materi-als is subject to severe problems concerning peak symmetryand detection sensitivity. A ”normal” surface modification re-action leaves many unreacted silanol groups on the surface

of the silica, which can severely disturb the chromatographyof ionizable compounds, especially if one works without buff-ers or PIC reagents. Even simple endcapping does not com-pletely solve this problem.

Our adsorbents are manufactured according to very stringent quality criteria.

All base deactivated NUCLEOSIL® phases feature:

versatile selectivity

optimised endcapping

high purity silica

outstanding batch-to-batch reproducibility

excellent peak shape for basic compounds

NUCLEOSIL®

C18 HDNUCLEOSIL®

C8 HD

NUCLEOSIL®

PROTECT I

polymer-coatedoctadecyl phase

monomer-coatedoctadecyl phase

monomer-coatedoctyl phase

monomer-coatedoctyl phase with embedded

polar group

NUCLEOSIL®

C18 NAUTILUS

monomer-coated octadecyl phase with embedded polar group

for every separation the optimised RP phasefor every separation the optimised RP phase

incr

easing carbon content

relativelymore

hydrophobic

relativelymore

hydrophilic

basedeactivatedsilicas for

RPLCNUCLEOSIL®

C18 AB


MNwww.mn-net.com


24

6.7.

05

RP method development kitsFor selection of the RP phase best suited for your separationwe offer two RP method development kits with 30 mmChromCart® columns: each kit consists of a CC column holder 30 mm and one col-umn each 30 x 2 or 30 x 4 mm, respectively, packed with:NUCLEOSIL® PROTECT I, NUCLEOSIL® 100-5 C18 HD,NUCLEOSIL® 100-5 C8 HD, NUCLEOSIL® 100-5 C18 ABand NUCLEOSIL® 100-5 C18

Comparison of selectivities for base deactivated RP phases

Fast LC of anilinesColumn: 30 x 4 mm NUCLEOSIL® 100-5 PROTECT IEluent: methanol – water

(45:55, v/v)Flow rate: 2 ml/minDetection: UV, 254 nmPeaks:1. Aniline2. N,N-Dimethylaniline3. N,N-Diethylaniline

1

2

3

0 1 2min1149

80

Ordering information

Designation Cat. No.

Method development kit ChromCart® 2 mm 721125.20

Method development kit ChromCart® 4 mm 721125.40

Method development kit ChromCart® 4.6 mm 721125.46

0 10 20 min

1

2

3

4

5

6

0 10 20 30min

12

3 4

5

67

0 20 min

1

2,3

4

5

7

6

10 0 10 min

1

2,3

4

5

7

6

Separation of a test mixture on four base deactivated NUCLEOSIL® phasesThese chromatograms show the differences in retention times and selectivities of the four phases. Columns: A) 250 x 4 mm NUCLEOSIL® 100-5 C18 HD; B) 250 x 4 mm NUCLEOSIL® 100-5 C18 AB;

C) 250 x 4 mm NUCLEOSIL® 100-5 C8 HD; D) 250 x 4 mm NUCLEOSIL® 100-5 PROTECT IEluent: methanol – water (60:40); flow rate 0.8 ml/min; temperature: 35 °CDetection: UV, 230 nm

Peaks:1. p-Ethylaniline2. Diethyl phthalate3. N,N-Dimethylaniline4. Ethyl benzoate

5. Toluene6. Naphthalene7. Ethylbenzene

C18 HD C18 AB C8 HD PROTECT


www.mn-net.com

MN www.mn-net.com


25

6.7.

05

Characterization of different NUCLEOSIL® RP phasesThe separation of a substance mixture depends on many factors. Based on suggestions of Tanaka1) and Johnson2) we compareour RP adsorbents with respect to the following parameters:

The capacity factor of pentylbenzene (axis A) and the selec-tivity between pentyl- and butylbenzene (axis B) are a meas-ure for the hydrophobic interactions of a stationary phase.Naturally, octadecyl modified silica phases show very largevalues for these parameters. The capacity factor ofNUCLEOSIL® 100-5 C18 HD proves the high density of thealkyl groups of this phase compared to NUCLEOSIL® 100-5C18 AB and the conventional NUCLEOSIL® 100-5 C18phase.Shortening of the alkyl chain from C18 to C8 results in a lowercarbon content and a reduction of the hydrophobic interac-tions.

The steric selectivity (axis C), i.e. the ability of an adsorbentto differentiate between molecules of equal hydrophobicity,but different shape (planar, non-planar) is especially large forthe phase NUCLEOSIL® 100-5 C18 AB.Axes D – F describe the quality of the base deactivation un-der different conditions. 1. N. Tanaka et al., J. Chromatogr. Sci. 27, 721 (1989)2. C. M. Johnson et al., Chromatographia 44, 151 (1997)

A capacity k’ (pentylbenzene) number of alkyl chains, C contentB hydrophobicity α (pentylbenzene, butylbenzene) CH2 group selectivityC steric selectivity α (triphenylene, o-terphenyl) steric differences of the analytesD silanol capacity α (caffeine, phenol) residual silanol groupsE ion exchange capacity at pH 7.6 α (benzylamine, phenol) residual silanol groupsF ion exchange capacity at pH 2.8 α (benzylamine, phenol) residual silanol groups

Characteristics of some of our base deactivated NUCLEOSIL® phases

Phase Tanaka plot Hydrophobicity Base deactivation

Steric selectivity

NUCLEOSIL® 100-5 C8 HD

for comparison:NUCLEOSIL®

100-5 C8

please note: this phase is not endcapped

medium very good low

NUCLEOSIL® 100-5 C18 HD

for comparison:NUCLEOSIL®

100-5 C18

this phase is endcapped

very high very good medium

NUCLEOSIL® 100-5 C18 AB

high good high

A

D

B

C

F

E

6.5

3.251.5

1.375

2.2

0.2

1.50.7

0

0

1.5

0.1

A

D

B

C

F

E

6.5

3.251.5

1.375

2.2

0.2

1.50.7

0

0

1.5

0.1

A

D

B

C

F

E

6.5

3.251.5

1.375

2.2

0.2

1.5

0.70

0

1.5

0.1

A

D

B

C

F

E

6.5

3.251.5

1.375

2.2

0.2

1.50.7

0

0

1.5

0.1A

D

B

C

F

E

6.5

3.251.5

1.375

2.2

0.2

1.50.7

0

0

1.5

0.1


MNwww.mn-net.com


26

6.7.

05

Base deactivated phases · particle size and separation efficiencyDue to the basic principles of HPLC 1) short diffusion pathsin the pores of the stationary phase are required for achiev-ing high column efficiency.According to the van Deemter equation

A: band broadening caused by eddy diffusion and streamsplitting of the mobile phase

B: band broadening caused by longitudinal diffusionC: band broadening caused by equilibrium distribution

between stationary and mobile phase ν: velocity of the mobile phase

the C-term describes the influence of band broadening,which is caused by the equilibrium distribution of the mole-cules between stationary and mobile phase. In the pores ofsmaller particles the diffusion paths are shorter.As a result the C-term is reduced, which means a favorableeffect for gaining low HETP values (height equivalent to onetheoretical plate). Provided that the velocity of the mobilephase is in its optimum range, smaller particles exhibit a con-siderably increased column efficiency.

It is a rule of thumb, that a well-packed 3 µm HPLCcolumn has about twice the separation efficiencyof a 5 µm column.

The choice of smaller particle size packings allows the use ofshorter columns for rapid separations without loss of resolu-tion. This is shown in the separation of vanillin compoundson the right. Analysis time can be reduced to a fifth by the re-placement of a conventional 250 mm/5 µm NUCLEOSIL®

NAUTILUS column with a 70 mm high-speed column packedwith the 3 µm counterparts. The drastic shortening of theanalysis time is not only due to reducing column length, butalso to increasing the flow rate by 50%. Problems with bandbroadening do not occur, because of the increased flow ratefor smaller-sized packings. An additional benefit in the use ofrapid resolution columns is the remarkable reduction of sol-vent consumption.The following 3 µm NUCLEOSIL® base deactivated silicasare available:

NUCLEOSIL® 100-3 C18 NAUTILUSNUCLEOSIL® 100-3 PROTECT INUCLEOSIL® 100-3 C8 HDNUCLEOSIL® 100-3 C18 HD

Besides the range of our well established standard materialsNUCLEOSIL® 100-3 C18, NUCLEOSIL® 120-3 C18 and NU-CLEOSIL® 120-3 C8 are still available.All 3 µm packings are available in a variety of column config-urations and can be used for standard and high-throughputapplications.

The currently available high performance NUCLEOSIL®

3 µm RP packings are characterised by the following fea-tures:

high purity NUCLEOSIL® silicaderivatised with unique monofunctional silanesoptimised endcappingoutstanding peak symmetry for basic compoundsexcellent reproducibility from batch to batch and columnto column

1) V. R. Meyer, Practical High Performance Liquid Chroma-tography (John Wiley & Sons, New York, 3rd. ed. 1999)

h A ν0.33

⋅Bν---- C ν⋅+ +=

Separation of isovanillin, vanillin and ethoxyvanillinColumn: a) 70 x 4 mm NUCLEOSIL® 100-3 C18 NAUTILUS

b) 250 x 4 mm NUCLEOSIL® 100-5 C18 NAUTILUSEluent: ACN – water – H3PO4 (20:80:0.1, v/v/v)

ambient temperatureFlow rate: a) 1.5 ml/min, b) 1 ml/minDetection: UV, 280 nm

Peaks:1) Isovanillin2) Vanillin3) Ethoxyvanillin

2 4 6

0 5 10 15 20 25min

a)

b)11

5770


MN www.mn-net.com


27

6.7.

05

Base deactivated phases for RP chromatography: NUCLEOSIL® HD

NUCLEOSIL® HD is a family of RP phases with optimisedbase deactivation, which is available with octyl or octadecylmodification and 3 or 5 µm particles. NUCLEOSIL® 100-5 C8 HD is an octyl modified RP phasewith 5 µm particles. As can be seen in the figure below, thebasic compound lidocaine is eluted without tailing. Com-pared to NUCLEOSIL® 100-5 C18 HD introduction of theshorter carbon chain results in a reduction of hydrophobic in-teractions.

The chromatograms show the separation of compounds withdifferent functional groups on both phases. The less polar ar-omatics toluene and naphthalene are eluted earlier from theC8 HD phase resulting in a reversed elution sequence of tol-uene and lidocaine.

Comparison of selectivities between NUCLEOSIL® HD octadecyl and octyl phases

Highest quality! · Batch-to-batch reproducibility of NUCLEOSIL® HD

0 5 10 15 min

1

2

3

5

4

6

Separation of a test mixture on NUCLEOSIL® C8 HDColumn: 250 x 4 mm NUCLEOSIL® 100-5 C8 HDEluent: MeOH – 25 mM NaH2PO4 pH 7.0 (65:35)Flow rate: 0.8 ml/minTemperature: 25 °CDetection: UV, 254 nmPeaks:1. Phenol2. 5’-(p-Methylphenyl)-

5-phenylhydantoin3. Diethyl phthalate4. Lidocaine5. Toluene6. Naphthalene

1100

00

0 10 20 min

1

2

3

4

5

6

Separation of a test mixture on NUCLEOSIL® C18 HDColumn: 250 x 4 mm NUCLEOSIL® 100-5 C18 HDEluent: MeOH – 25 mM NaH2PO4 pH 7.0 (65:35)Flow rate: 0.8 ml/minTemperature: 25 °CDetection: UV, 254 nmPeaks:1. Phenol2. 5’-(p-Methylphenyl)-

5-phenylhydantoin3. Diethyl phthalate4. Lidocaine5. Toluene6. Naphthalene

1106

50

1

2

3

4

5

6

7

0

2

4

6

8

FEDCBA

Capacity factor Selectivity

NUCLEOSIL® 100-5 C18 HD, acetonitrile – water 50:50(v/v), 1 ml/min, 25 °C, UV 254 nm

k’ (toluene)

k’ (ethyl benzoate)

k’ (MPPH)α (MPPH/toluene)

α (ethyl benzoate/toluene)

Lot

Separation of a test mixture on three different batches of NUCLEOSIL® 100-5 C8 HDColumn 250 x 4 mm NUCLEOSIL® 100-5 C8 HDEluent methanol – water (55:45, v/v); flow rate 1 ml/mintemperature 40 °C; detection: UV, 254 nm

Peaks:1. Thiourea2. Aniline3. Phenol4. o,m,p-Toluidine5. N,N-Dimethylaniline6. Methyl benzoate7. Toluene8. Ethylbenzene

1

2

3

4

5

6 78

0 10 20 min 1108

90


www.mn-net.com

MNwww.mn-net.com


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

28

6.7.

05

NUCLEOSIL® 100-3 C8 HD Octyl 3 µm

spherical 3 µm silica particles with monomeric octylmodification

NUCLEOSIL® 100-5 C8 HD Octyl 5 µm

spherical 5 µm silica particles with monomeric octylmodification

NUCLEOSIL® 100-3 C18 HD Octadecyl 3 µm

spherical 3 µm silica particles with monomeric octadecylcoating

NUCLEOSIL® 100-5 C18 HD Octadecyl 5 µm

spherical 5 µm silica particles with monomeric octadecylcoating

These packings are manufactured from NUCLEOSIL® 100with 3 or 5 µm particles, respectively. Thus, surface shielding,capacity factors and selectivities are the same for both materi-als. However, the number of theoretical plates is higher whenusing the 3 µm starting silica. The 3 µm particle allows appli-cation of shorter columns (= reduction of time required for theanalysis) and improves the detection sensitivity due to smallerpeak widths.The following chromatogram exhibits the higher efficiency ofthe 3 µm NUCLEOSIL® C8 HD column compared to the 5µmequivalent for identical column lengths (curves a and b). Theimproved sensitivity of the 3 µm packing becomes evident bythe increased peak heights in the chromatogram. However, ifthe column length is reduced from 250 mm to 150 mm, base-line separation, under the given chromatographic conditions,cannot be achieved (c). In this case the use of the 250 mmNUCLEOSIL® 100-3 C8 HD column is strongly recommend-ed to obtain optimal results.

Separation of paracetamol, caffeine and acetylsalicylic acidSample preparation: dissolve 1 tablet in 20 ml ACN/water (50:50), dilute 1:10.Columns: a) 250 x 4 mm NUCLEOSIL® 100-5 C8 HD

b) 250 x 4 mm NUCLEOSIL® 100-3 C8 HDc) 150 x 4 mm NUCLEOSIL® 100-3 C8 HD

Eluent: ACN – water – H3PO4 (20:80:0.1, v/v/v)Temperature: 30 °CFlow rate: 1 ml/minDetection: UV, 254 nm Peaks:

1. Paracetamol2. Caffeine3. Acetylsalicylic

acid

1

2

3

2 4 6 8 10 12 2 4 6 8 10 12 2 4 6 8 min

1169

60Ordering information · Analytical columns with NUCLEOSIL® HD

Length → 30 mm 1) 70 mm 100 mm 125 mm 150 mm 250 mm Guard columns

NUCLEOSIL® 100-3 C8 HDParticle size 3 µm, pore size 100 Å; octyl phase, endcapped, monomer coating, 13% C; eluent in column acetonitrile / water

ChromCart® columns2 mm ID 721656.20 721657.20 721658.20 721669.20 721612.303 mm ID 721656.30 721657.30 721658.30 721669.30 721612.304 mm ID 721656.40 721657.40 721658.40 721669.40 721612.40

4.6 mm ID 721656.46 721657.46 721658.46 721704.46 721669.46 721612.40

Microbore columns 2)

1 mm ID 717115.10 717116.10 717117.10 717118.10

EC columns 3)

2 mm ID 720526.20 720528.20 721612.303 mm ID 720526.30 720528.30 721612.304 mm ID 720526.40 720528.40 721612.40

4.6 mm ID 720526.46 720527.46 720528.46 721612.40

Base deactivated NUCLEOSIL® phases for RP chromatography

MN www.mn-net.com


Custom-packed columns with other lengths are available on request

29

6.7.

05

NUCLEOSIL® 100-5 C8 HDParticle size 5 µm, pore size 100 Å; octyl phase, endcapped, monomeric coating, 13% C; eluent in column acetonitrile / water


4.6 mm ID 721071.46 721499.46 721497.46 721501.46 721498.46 721500.40


1 mm ID 717043.10 717046.10 717049.10 717057.10

EC columns 3)

2 mm ID 720195.20 720196.20 721500.303 mm ID 720195.30 720196.30 721500.304 mm ID 720195.40 720196.40 721500.40

4.6 mm ID 720195.46 720194.46 720196.46 721500.40

NUCLEOSIL® 100-3 C18 HDParticle size 3 µm, pore size 100 Å; octadecyl phase, endcapped, monomeric coating, 20% C; eluent in column acetonitrile / water


4.6 mm ID 721196.46 721493.46 721491.46 721495.46 721492.46 721494.40


1 mm ID 717037.10 717038.10 717039.10 717040.10

EC columns 3)

2 mm ID 720191.20 720192.20 721494.303 mm ID 720191.30 720192.30 721494.304 mm ID 720191.40 720192.40 721494.40

4.6 mm ID 720191.46 720193.46 720192.46 721494.40

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


4.6 mm ID 721072.46 721851.46 721852.46 721854.46 721850.46 721853.40


1 mm ID 717033.10 717024.10 717015.10 717001.10

EC columns 3)

2 mm ID 720296.20 720280.20 721853.303 mm ID 720296.30 720280.30 721853.304 mm ID 720296.40 720280.40 721853.40

4.6 mm ID 720296.46 720294.46 720280.46 721853.4030 mm ChromCart® columns and CC guard column cartridges (8 mm) in packs of 3, all other columns in packs of 1.1) 30 mm ChromCart® columns require the CC column holder 30 mm (Cat. No. 721823). 2) On request, microbore columns are also available in lengths of 40, 60, 200 and 300 mm and with 0.15, 0.3, 0.4, 0.5, 0.75 and 1.5 mm ID.

Guard columns for microbore columns on request.3) As guard columns for EC columns use ChromCart® guard column cartridges with guard column adaptor EC (Cat. No. 721359).

Ordering information · Analytical columns with NUCLEOSIL® HDLength → 30 mm 1) 70 mm 100 mm 125 mm 150 mm 250 mm Guard columns


MNwww.mn-net.com



30

6.7.

05

OH HOH

H

O H

H

Pol SO S

ii

NUCLEOSIL® 100 C18 Nautilus

the formula for RP chromatography in aqueous media

With highly aqueous mobile phases (> 95 %) conventionalreversed phase columns very often exhibit a plainly visibledeterioration of column performance after a certain period oftime. The non-polar alkyl chains lose their "brush-type struc-ture" for maintaining the hydrophobic interactions betweenstationary phase and analyte. The result of this phase col-lapse is a drastic decrease of retention time and poor resolu-tion. This phenomenon may occur very fast or after a while.

NUCLEOSIL® 100-5 C18 Nautilus is a reversed phase C18column based on highly pure silica with thorough endcap-ping, which is totally stable if the mobile phase is 100%aqueous without any organic modifiers. This property isachieved by a covalently bonded C18 silane (synthesised inMACHEREY-NAGEL’s R&D laboratories) with an embeddedfunctional group, which is the driving force for the excellentwater stability of the RP surface. Compared to standard C18phases where hydrophobic interactions between analyte andstationary phase predominate, NUCLEOSIL® 100-5 C18Nautilus also shows polar interaction via hydrogen bondingactivities and dipole-dipole forces. This can influence reten-tion time (tR) and improve selectivity (α) particularly for theseparation of polar compounds.

Even after days or weeks of operation in purely aqueous elu-ents the C18 chains of NUCLEOSIL® 100-5 C18 Nautilus nei-ther are folded nor show any collapsing. A significant reduc-tion of retention time cannot be observed as is shown in thefollowing diagrams.

In spite of the polar character of the embedded functionalgroup NUCLEOSIL® C18 Nautilus exhibits sufficient hydro-phobic properties and is very well suited for analysing basiccompounds just like NUCLEOSIL® HD or NUCLEOSIL®

Protect I. It may be worthwhile to try this chemistry for diffi-cult separations.

H2O

H2O

H2O

H2O

H2O

H2OH2O

H2O

A

A

AA

H2O

H2O

H2O

H2O

H2O

H2O

H2O

H2O

A

A

A

A

A

Sta

rt

Sta

rt2 4 6 8 2 4 6

Phase collapse and decrease in retention times for a con-ventional RP phase with 100 % aqueous eluents

A = analyte

0 2 4 6 8

0

20

40

60

80

100

Phase collapse in aqueous media

% o

f ini

tial r

eten

tion

time

conventional RP phase

NUCLEOSIL® 100-5 C18 Nautilus

change of retention time with 100% aqueous eluents

day

Separation of nucleobases in a purely aqueous eluentColumn: 125 x 4 mm NUCLEOSIL®

100-5 C18 NautilusEluent: 20 mM ammonium formate,

pH 4.1Flow rate: 1 ml/minTemperature: 20 °CDetection: UV, 254 nm

Peaks:1. Cytosine2. Uracil3. Hypoxanthine4. Xanthine5. Adenine

1 2

3

4

5

4 8 min

1156

40


MN www.mn-net.com



31

6.7.

05

In the selectivity test on the right the strong base p-ethyl-aniline is eluted as a sharp signal with remarkably good peakshape. Another example for the excellent base deactivationof NUCLEOSIL® Nautilus is the tailing-free elution of pyrid-ine.Chromatographic conditions:Column 250 x 4 mm NUCLEOSIL® 100-5 C18 Nautilus; elu-ent methanol – water (left 55:45, right 30:70, v/v); flow rateleft 0.8 ml/min, right 1 ml/min; temperature 40 °C; detectionUV, 254 nmPeaks (left): 1. uracil, 2. aniline, 3. phenol, 4. p-ethylaniline, 5. N,N-dimethylaniline, 6. toluene, 7. ethylbenzeneReferences D. Rieger, H. Riering, Int. Laboratory, Vol. 30, p. 41

Selectivity test

OHN

Pyridine/phenol

4 8 1220 24 28 min

1 2

3

4

5

6

7

48 12 16 16

Ordering information · Analytical columns with NUCLEOSIL® 100-5 C18 NautilusLength → 30 mm 1) 70 mm 100 mm 125 mm 150 mm 250 mm Guard columns

NUCLEOSIL® 100-3 C18 NautilusParticle size 3 µm, pore size 100 Å; octadecyl phase, endcapped, embedded polar group, 16% C; eluent in column acetonitrile / water


4.6 mm ID 721649.46 721650.46 721651.46 721703.46 721652.46 721611.40


1 mm ID 717110.10 717111.10 717112.10 717113.10

EC columns 3)

2 mm ID 720472.20 720470.20 721611.303 mm ID 720472.30 720470.30 721611.304 mm ID 720472.40 720470.40 721611.40

4.6 mm ID 720472.46 720471.46 720470.46 721611.40

NUCLEOSIL® 100-5 C18 Nautilus as above, however, particle size 5 µm


4.6 mm ID 721133.46 721134.46 721131.46 721132.46 721130.46 721140.40


1 mm ID 717066.10 717065.10 717067.10 717068.10

EC columns 3)

2 mm ID 720430.20 720431.20 721140.303 mm ID 720430.30 720431.30 721140.304 mm ID 720430.40 720431.40 721140.40

4.6 mm ID 720430.46 720432.46 720431.46 721140.4030 mm ChromCart® columns and CC guard column cartridges (8 mm) in packs of 3, all other columns in packs of 1.1) 30 mm ChromCart® columns require the CC column holder 30 mm (Cat. No. 721823). 2) On request, Microbore columns are also available in lengths of 40, 60, 200 and 300 mm and with 0.15, 0.3, 0.4, 0.5, 0.75 and 1.5 mm ID.

Guard columns for Microbore columns on request.3) As guard columns for EC columns use ChromCart® guard column cartridges with guard column adaptor EC (Cat. No. 721359).


MNwww.mn-net.com



32

6.7.

05

NUCLEOSIL® 100-3 Protect I Special RP phase 3 µm

spherical 3 µm silica particles with monomeric specialRP modification

NUCLEOSIL® 100-5 Protect ISpecial RP phase 5 µm

spherical 5 µm silica particles with monomeric specialRP modification

Although hydrophobic interactions of NUCLEOSIL® 100-5Protect I are comparatively low and closer to the standardoctyl phase NUCLEOSIL® 100-5 C8, the special surfacemodification combined with a rigorous endcapping of the un-reacted silanol groups reduces interaction successfully andensures excellent peak shapes with basic compounds.In comparison with the well-established NUCLEOSIL® 100-5C18 AB, NUCLEOSIL® 100-5 C18 HD and NUCLEOSIL®

100-5 C8 HD a key characteristic of the NUCLEOSIL® 100-5protect I is its higher polarity. Nevertheless, critical com-pounds like amines show outstanding peak symmetries. Thisstationary phase is well suited for the separation of low mo-lecular-weight compounds (up to 5000 dalton), e. g. drugs orpesticides.For example, the tricyclic amines protriptyline and amitriptyl-ine show especially strong interactions with residual silanolgroups on a conventional C18 column, while ourNUCLEOSIL® 100-5 Protect I features✔ early elution and✔ excellent peak symmetryfor this critical pair of compounds (see chromatogram).The batch test underlines the outstanding base deactivationand unique selectivity of NUCLEOSIL® 100-3 Protect I forthe successful separation of critical bases such as amitriptyl-ine and protriptyline, which are eluted with excellent peakshapes as for the aromatic hydrocarbons.

4 8 12 min

1

23

4

5

6

Separation of a batch test mixtureColumn: 250 x 4 mm NUCLEOSIL® 100-3 Protect I,

Cat. No. 720542.40Eluent: acetonitrile – 25 mM KH2PO4, pH 7.5 (50:50)Flow rate: 1 ml/minDetection: UV, 254 nmTemperature: 25 °CPeaks:1. Uracil2. Protriptyline3. Amitriptyline4. Ethyl benzoate5. Toluene6. Ethylbenzene

1169

70

www.mn-net.com


MN www.mn-net.com



33

6.7.

05

Ordering information · Analytical columns with NUCLEOSIL® 100-5 Protect ILength → 30 mm 1) 70 mm 100 mm 125 mm 150 mm 250 mm Guard columns

NUCLEOSIL® 100-3 Protect IParticle size 3 µm, pore size 100 Å; special RP phase, endcapped, monomer coating, 11% C; eluent in column acetonitrile / water


4.6 mm ID 721670.46 721671.46 721672.46 721705.46 721673.46 721613.40


1 mm ID 717120.10 717121.10 717122.10 717123.10

EC columns 3)

2 mm ID 720540.20 720542.20 721613.303 mm ID 720540.30 720542.30 721613.304 mm ID 720540.40 720542.40 721613.40

4.6 mm ID 720540.46 720541.46 720542.46 721613.40

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


4.6 mm ID 721157.46 721152.46 721151.46 721153.46 721150.46 721154.40


1 mm ID 717034.10 717025.10 717016.10 717007.10

EC columns 3)

2 mm ID 720175.20 720170.20 721154.303 mm ID 720175.30 720170.30 721154.304 mm ID 720175.40 720170.40 721154.40

4.6 mm ID 720175.46 720174.46 720170.46 721154.4030 mm ChromCart® columns and CC guard column cartridges (8 mm) in packs of 3, all other columns in packs of 1.1) 30 mm ChromCart® columns require the CC column holder 30 mm (Cat. No. 721823). 2) On request, Microbore columns are also available in lengths of 40, 60, 200 and 300 mm and with 0.15, 0.3, 0.4, 0.5, 0.75 and 1.5 mm ID.



MNwww.mn-net.com



34

6.7.

05

NUCLEOSIL® 100-5 C18 ABOctadecyl 5 µm

spherical 5 µm silica with polymeric octadecyl coatingTailing, changes of elution sequence and decreased sensitiv-ity are common problems encountered in the separation ofacid as well as basic substances on conventional RP phas-es. The polymer-coated octadecyl packing materialNUCLEOSIL® 100-5 C18 AB features an outstanding shield-ing of the silica matrix and is inert towards acidic and basicsubstances, which have a high affinity for silica. The carboncontent of 25% is considerably higher than for monomericallycoated standard octadecyl phases. NUCLEOSIL® 100-5 C18 AB is a nonpolar, hydrophobic RPsilica for HPLC with an increased stability to hydrolysis in al-kaline media compared to standard RP phases.Moreover, the distinct steric selectivity of this phase can beemphasized.

Steric selectivity of NUCLEOSIL® C18 ABColumns: a) 250 x 4 mm NUCLEOSIL® 100-5 C18, b) 250 x 4 mm NUCLEOSIL® 100-5 C18 AB, 250 x 4 mm ID; eluent MeOH – H2O (80:20, v/v), flow rate 1 ml/min, temperature 40 °C, detection UV, 254 nmPeaks:1. Phenol2. Diethyl phthalate3. Naphthalene4. Dibutyl phthalate5. Butyl benzene

6. o-Terphenyl7. Anthracene8. Pentylbenzene9. Triphenylene

1176

70

0

1

2

34 5

6

7

8

9

1

2

3

45

6

7

8

9

a)

b)

5 10 15 20 min

Ordering information · Analytical columns with NUCLEOSIL® 100-5 C18 AB

packing: particle size 5 µm, pore size 100 Å; octadecyl phase, endcapped, polymeric coating, 25% C; eluent in column acetonitrile / water

Length → 30 mm 1) 70 mm 100 mm 125 mm 150 mm 250 mm Guard columns


4.6 mm ID 721073.46 721719.46 721623.46 721643.46 721663.46 721603.40

Microbore columns 2) 1 mm ID 717032.10 717023.10 717014.10 717006.10

EC columns 3)

2 mm ID 720935.20 720936.20 721603.303 mm ID 720935.30 720936.30 721603.304 mm ID 720935.40 720936.40 721603.40

4.6 mm ID 720935.46 720305.46 720936.46 721603.4030 mm ChromCart® columns and CC guard column cartridges (8 mm) in packs of 3, all other columns in packs of 1.1) 30 mm ChromCart® columns require the CC column holder 30 mm (Cat. No. 721823). 2) On request, Microbore columns are also available in lengths of 40, 60, 200 and 300 mm and with 0.3, 0.4, 0.5, 0.75 and 1.5 mm ID.



MN www.mn-net.com


35

6.7.

05

Applications

0 10 20 min

1

2

3

4

5

6

7 8

9 10

11

Separation of phenolsColumn: 250 x 4 mm NUCLEOSIL® 100-5 Protect I Eluent: acetonitrile – 25 mM KH2PO4 pH 7 (50:50, v/v)Flow rate: 1 ml/minTemperature: 20 °CDetection: UV, 235 nm

Peaks:1. p-Benzoquinone2. Phenol3. o-Nitrophenol4. p-Nitrophenol5. 2,3-Dinitrophenol6. m-Nitrophenol7. 2,5-Dinitrophenol8. 3,4-Dinitrophenol9. 2,6-Dinitrophenol

10. 2,4-Dinitrophenol11. 2,4,6-Trinitrophenol

1115

40

10 20 30min

1

2

3

4

5

6

Analysis of customary heroin, stretched with paracetamol/caffeine

Column 250 x 2 mm NUCLEOSIL® 100-5 C18 AB, with guard column 8 x 3 mm NUCLEOSIL® 100-5 C18 ABInjection volume 30 µlEluents: A) 2 mmol NaH2PO4, 1 g/l 1-hexanesulphonic acid adjusted to pH 2.5 with H3PO4, B) acetonitrileGradient: 8 – 33% B in 30 min, then 5 min 90% BFlow rate: 0.32 ml/min, temperature: 40 °CDetection: DAD, 210 nm

Peaks:1. Paracetamol2. Caffeine3. 6-Monoacetyl-

morphine4. Heroin5. Papaverine6. Noscapinecourtesy of Mr. Kothe, Laboratory Dr. Krone, Prof. Hagedorn, Medizinal-Untersu-chungsstelle Herford

1101

80

AB

CD

E

0 5 10 15 20

Separation of sunscreen agentsV. Vanquerp et al. J. Chromatogr. 832 (1999) 273 – 277Column: 250 x 4 mm NUCLEOSIL®

100-5 C8 HDEluents: A) 1% acetic acid

in water, B) methanol

Gradient: 80 – 100% B in 10 min

Flow rate: 1 ml/minDetection: UV, 300 nmPeaks (Injection volume 10 µl):A) Benzophenone-3B) Methylbenzylidene camphorC) Octyl methyl PABAD) Butyl methoxydibenzoyl-

methaneE) PEG-25 PABA

1147

40

0 10 20 30min

0

10

20

30

mAU2

1

Analysis of customary ecstasyColumn: 250 x 4 mm NUCLEOSIL® 100-5 C18 HDEluents: A) 2 mmol NaH2PO4 adjusted to pH 2.5

with H3PO4 B) acetonitrile

Gradient: 8– 33% B in 30 minFlow rate: 0.8 ml/minDetection: UV, 235 nmPeak:1. Caffeine ?2. S(+)-3,4-Methylenedioxymethamphetamine

hydrochloride (Ecstasy)courtesy of Mr. Kothe, Laboratory Dr. Krone, Prof. Hagedorn, Medizinal-Untersuchungsstelle Herford

1101

30


MNwww.mn-net.com


36

6.7.

05

Applications

0 5 10 15 min

1

2

3

4

5

6

Separation of tricyclic antidepressantsColumn: 250 x 4 mm NUCLEOSIL® 100-5 Protect IEluent: acetonitrile – 25 mM KH2PO4, pH 7.0 (35 : 65, v/v)Flow rate: 1 ml/minTemperature: 25 °CDetection: UV, 254 nm

Peaks:1. Protriptyline

2. Nortriptyline

3. Doxepin

4. Imipramine

5. Amitriptyline

6. Trimipramine

NHCH3

NHCH3

N(CH3)2

O

N(CH3)2

N

N(CH3)2

N(CH3)2

N

1100

30

0 10 20 30

1

2

3

4

5

min

Separation of tricyclic antidepressantsColumn: 250 x 4 mm NUCLEOSIL® 100-5 C8 HDEluent: MeOH – 25 mM NaH2PO4 pH 7.0 (70:30)Flow rate 1 ml/minTemperature 30 °CDetection UV, 254 nmPeaks:1. Protriptyline2. Nortriptyline3. Doxepin4. Amitriptyline5. Trimipramine

1107

50

0 10 20min

1

2 34

5

6

Separation of tricyclic antidepressantsColumn: 250 x 4 NUCLEOSIL® 100-5 C18 HDInjection volume 5 µlEluent acetonitrile – 0.1% TEAPO4 pH 6.9 (45:55)Flow rate 1 ml/minTemperature 25 °CDetection UV, 254 nmPeaks:1. Protriptyline2. Nortriptyline3. Doxepin4. Imipramine5. Amitriptyline6. Trimipramine

1107

60


MN www.mn-net.com


37

6.7.

05


®

Standard phases for RP chromatography

Reversed phase silica phases are by far the most widelyused packings. Our base deactivated high performance RPmaterials are described in the preceding chapter. In thischapter you will find columns packed with standard silicaphases for RPLC. For ordering information of the corre-sponding bulk packings please see the chapter starting onpage 143. Octadecyl, Octyl, Butyl, Dimethyl and Phenylphases differ in retention time for a given solute. Under iden-tical conditions (mobile phase, flow rate, temperature etc.),e. g. the retention times on NUCLEOSIL

®

C

18

are, for quite anumber of compounds, by a factor of up to 2 larger than onNUCLEOSIL

®

C

8

. The mechanism of separation with all NUCLEOSIL

®

andNUCLEODUR

®

RP phases is the attraction between hydro-phobic groups of the test molecule and the bonded alkyl

groups of the stationary phase by van der Waals forces. theless polar the sample molecules, the more they are attracted.Differences in the chemical structure of the sample mole-cules result in a stronger or weaker adsorption to the station-ary phase and hence in a separation.The more polar the sample, the shorter are the retentiontimes and the weaker are the van der Waals forces. The fol-lowing is roughly the order of increasing polarity (reverse or-der of elution): aliphatic hydrocarbons, induced dipoles(CCI

4

), permanent dipoles (CHCI

3

), weak Lewis bases(ethers, aldehydes, ketones), strong Lewis bases (amines),weak Lewis acids (alcohols, phenols) and strong Lewis acids(carboxylic acids). Also of considerable importance for reten-tion times is the chain length of the molecules which are tobe separated.

The mobile phase in RP chromatography

The mobile phase for reversed phase chromatography is amixture of water and an organic solvent which is misciblewith water. The time of elution of the substances to be sepa-rated depends to a large degree on the water content of theorganic solvent and is therefore easily adjusted to a suitable

value. Higher concentrations of organic solvent will causeshorter retention times. RP separations can be optimised byutilising the specific selectivity of organic solvents. The viscosity of the eluent should not be neglected. It is oftennot realised in practice, that eluents have very different vis-cosities (

η

) and that these differences in viscosity (with thesame flow rate) cause changes in back pressure. The backpressure changes according to the following equation:

The viscosity of a solvent mixture is often higher than that ofthe single components, i.e. a higher pressure is needed toachieve the same flow rate.The graph on the left illustrates how strongly the viscosity ofthe eluent is influenced by its composition. For example theviscosity (and consequently the back pressure of the col-umn) increases more than double when the eluent ischanged from acetonitrile – water (70:30) to methanol – wa-ter (70:30).When water is used as a solvent or as a component in gradi-ent mixtures, it is essential that it is completely degassed.Apparent instability can be caused by air bubbles (from notproperly degassed solvents) which accumulate at the detec-tor. The best way to remove such air is to first pump de-gassed solvent through the column and then follow up withdegassed water. One can degas solvents with a vacuumpump, by passing helium through or by ultrasonic treatment.

water

n-propanol

ethanol

methanol

acetonitrile

0 20 40 60 80 100% [w/w]organic

η

[cP]at

20 °C

3

2

1

3

2

1

visc

osity

Viscosity of solvent mixturesas a function of composition

p1η1

η2------ p2⋅=

MNwww.mn-net.com


38

6.7.

05


®

Test mixture for reversed phase columns

For reversed phase HPLC columns we supply a test mixturein acetonitrile to determine the quality of the packing. The fig-ure on the right shows a typical test chromatogram of a col-umn with the stationary phase NUCLEOSIL

®

100-5 C

18

.

Ordering information

Designation Pack of

Cat. No.

Test mixture for reversed phase columns in acetonitrile

1 ml

722394

Test mixture for reversed phase HPLC columns (Cat. No. 722394)

Column: 250 x 4 mm NUCLEOSIL® 100-5 C18

Sample volume: 20 µlEluent: acetonitrile – water (70:30)Flow rate: 1 ml/minPressure: 105 barTemperature: 22 °CDetection: UV, 254 nmPeaks: (concentration in µg/ml eluent)1. Phenol (400)2. Naphthalene (180)3. Anthracene (12)

1

2 3

4 8 min

Individual test certificate enclosed witheach NUCLEODUR

®

and NUCLEOSIL

®

column, respectively.

MN www.mn-net.com


Custom-packed columns with other phases and other lengths are available on request

39

6.7.

05


®

Octadecyl phases

-(CH

2

)

17

– CH

3

C

18

phases are suited for reversed phase chromatographyand ion-pairing chromatography for separation of nonpolar tomoderately polar compounds such as

fatty acidsglyceridespolycyclic aromaticsesters (phthalates)fat-soluble vitaminssteroidsprostaglandinsPTH amino acids etc.

Analysis of nitroaromatic compounds (explosives)

Column 250 x 3 mm NUCLEOSIL® 120-3 C18; eluents: A) meth-anol, B) water, gradient 35 – 55% A in 12 min, then 40 min iso-cratic, finally 100% A, flow rate 0.35 ml/min; temperature 30 °C, detection DAD, 230 nm; injection volume 40 µlPeaks:1) 2,6-Diamino-4-nitrotoluene2) 2,4-Diamino-4-nitrotoluene3) Hexogene (Hexahydro-

1,3,5-trinitro-1,3,5-triazine)4) 1,3,5-Trinitrobenzene5) 2-Methyl-3-nitroaniline6) 2-Methyl-5-nitroaniline7) 4-Methyl-3-nitroaniline

8) 1,3-Dinitrobenzene9) 2,4,6-Trinitrotoluene10)4-Amino-2,6-dinitrotoluene11)2-Amino-4,6-dinitrotoluene12)2,6-Dinitrotoluene13)2,4-Dinitrotoluene14)2-Nitrotoluene15)4-Nitrotoluene16)3-Nitrotoluene

courtesy of Dr. Steinbach, Inst. f. Org. Chemistry, Univ. Marburg

200

100

0

mAU

20 40 min

1

2

3

4

6

78

9

1011

1213

14 15 16

5

1148

10

Separation of triazinesColumn: 250 x 4 mm NUCLEOSIL® 50-5 C18 ecEluent: methanol – acetonitrile – water (40:20:40, v/v/v)Flow rate: 1 ml/min, 30 °C, 220 barDetection: UV, 220 nmPeaks (injection volume 5 µl, 10 µg/ml each):1. Simazine2. Atrazine3. Prometon4. Ametryne5. Propazine6. Prometryne7. Terbutryne

1

2

3 4 5

6 7

10 20 min0

1115

70

Ordering information · Analytical columns with NUCLEOSIL

®

C

18

Length

→

30 mm

1)

70 mm 100 mm 125 mm 150 mm 250 mm Guard columns

NUCLEOSIL

®

50-5 C

18

ec

Particle size 5 µm, pore size 50 Å; octadecyl phase, endcapped, 14.5% C; eluent in column acetonitrile / water

ChromCart

®

columns

2 mm ID 721825.20 721826.20 721828.20 721829.303 mm ID 721825.30 721826.30 721828.30 721829.304 mm ID 721825.40 721826.40 721828.40 721829.40

4.6 mm ID 721825.46 721826.46 721827.46 721828.46 721829.40

EC columns

3)

4 mm ID 720098.40 721829.404.6 mm ID 720098.46 721829.40

30 mm ChromCart

®

columns and CC guard column cartridges (8 mm) in packs of 3, all other columns in packs of 1.

1)

30 mm ChromCart

®

columns require the CC column holder 30 mm (Cat. No. 721823).

2)

On request, Microbore columns are also available in lengths of 40, 60, 200 and 300 mm and with 0.15, 0.3, 0.4, 0.5, 0.75 and 1.5 mm ID.Guard columns for Microbore columns on request.

3)

As guard columns for EC columns use ChromCart

®

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

MNwww.mn-net.com



40

6.7.

05


®

NUCLEOSIL

®

100-3 C

18

Particle size 3 µm, pore size 100 Å; octadecyl phase, endcapped, 15% C; eluent in column acetonitrile / water

ChromCart

®

columns

2 mm ID 721883.20 721865.20 721866.303 mm ID 721883.30 721865.30 721866.304 mm ID 721883.40 721865.40 721866.40

4.6 mm ID 721883.46 721806.46 721865.46 721866.40

Microbore columns

2)

1 mm ID 717029.10 717020.10 717011.10 717003.10

EC columns

3)

2 mm ID 720150.20 720133.20 721866.303 mm ID 720150.30 720133.30 721866.304 mm ID 720150.40 720133.40 721866.40

4.6 mm ID 720150.46 720949.46 720133.46 721866.40

NUCLEOSIL

®

100-5 C

18


ChromCart

®

columns

2 mm ID 721074.20 721781.20 721622.20 721662.20 721602.303 mm ID 721074.30 721781.30 721622.30 721662.30 721602.304 mm ID 721074.40 721781.40 721622.40 721662.40 721602.40

4.6 mm ID 721074.46 721781.46 721622.46 721642.46 721662.46 721602.40

Microbore columns

2)

1 mm ID 717028.10 717019.10 717010.10 717002.10

EC columns

3)

2 mm ID 720002.20 720014.20 721602.303 mm ID 720002.30 720014.30 721602.304 mm ID 720002.40 720014.40 721602.40

4.6 mm ID 720002.46 720120.46 720014.46 721602.40

NUCLEOSIL

®

100-7 C

18


ChromCart

®

columns

3 mm ID 721878.30 721609.304 mm ID 721878.40 721609.40

4.6 mm ID 721609.46

EC columns

3)

4 mm ID 720018.404.6 mm ID 720018.46


®

C

18

Length

→

30 mm

1)

70 mm 100 mm 125 mm 150 mm 250 mm Guard columns

30 mm ChromCart

®

columns and CC guard column cartridges (8 mm) in packs of 3, all other columns in packs of 1.

1)

30 mm ChromCart

®

columns require the CC column holder 30 mm (Cat. No. 721823).

2)

On request, Microbore columns are also available in lengths of 40, 60, 200 and 300 mm and with 0.15, 0.3, 0.4, 0.5, 0.75 and 1.5 mm ID.Guard columns for Microbore columns on request.

3)


®


MN www.mn-net.com



41

6.7.

05


NUCLEOSIL® 100-10 C18 Particle size 10 µm, pore size 100 Å; octadecyl phase, endcapped, 15% C; eluent in column acetonitrile / water

ChromCart® columns3 mm ID 721681.30 721689.304 mm ID 721681.40 721689.40

4.6 mm ID 721689.46

EC columns 3)

4 mm ID 720023.404.6 mm ID 720023.46



4.6 mm ID 721075.46 721721.46 721626.46 721646.46 721666.46 721606.40


1 mm ID 717031.10 717022.10 717013.10 717005.10

EC columns 3)

2 mm ID 720040.20 720055.20 721606.303 mm ID 720040.30 720055.30 721606.304 mm ID 720040.40 720055.40 721606.40

4.6 mm ID 720040.46 720740.46 720055.46 721606.40



4.6 mm ID 721070.46 721723.46 721629.46 721659.46 721712.46 721783.40


1 mm ID 717030.10 717021.10 717012.10 717004.10

EC columns 3)

2 mm ID 720051.20 720041.20 721783.303 mm ID 720051.30 720041.30 721783.304 mm ID 720051.40 720041.40 721783.40

4.6 mm ID 720051.46 720730.46 720041.46 721783.40

Ordering information · Analytical columns with NUCLEOSIL® C18Length → 30 mm 1) 70 mm 100 mm 125 mm 150 mm 250 mm Guard columns

30 mm ChromCart® columns and CC guard column cartridges (8 mm) in packs of 3, all other columns in packs of 1.1) 30 mm ChromCart® columns require the CC column holder 30 mm (Cat. No. 721823). 2) On request, Microbore columns are also available in lengths of 40, 60, 200 and 300 mm and with 0.15, 0.3, 0.4, 0.5, 0.75 and 1.5 mm ID.


MNwww.mn-net.com



42

6.7.

05



EC columns 3)

4 mm ID 720042.404.6 mm ID 720042.46


EC columns 3)

4 mm ID 720043.404.6 mm ID 720043.46

NUCLEOSIL® 300-5 C18 Particle size 5 µm, pore size 300 Å; octadecyl phase, endcapped, 6.5% C; eluent in column acetonitrile / water

ChromCart® columns2 mm ID 721790.20 721628.20 721668.20 721608.303 mm ID 721628.30 721668.30 721608.304 mm ID 721628.40 721668.40 721608.40

4.6 mm ID 721628.46 721648.46 721668.46 721608.40


1 mm ID 717045.10 717048.10 717056.10 717059.10

EC columns 3)

2 mm ID 720713.20 721608.303 mm ID 720713.30 721608.304 mm ID 720065.40 721608.40

4.6 mm ID 720065.46 721608.40


EC columns 3)

4 mm ID 720074.404.6 mm ID 720074.46

NUCLEOSIL® 1000-7 C18 Particle size 7 µm, pore size 1000 Å; octadecyl phase, endcapped, ~1% C; eluent in column acetonitrile / water

EC columns 3)

4 mm ID 720077.404.6 mm ID 720077.46

NUCLEOSIL® 4000-7 C18 Particle size 7 µm, pore size 4000 Å; octadecyl phase, endcapped, <1% C; eluent in column acetonitrile / water

EC columns 3)

4 mm ID 720085.404.6 mm ID 720085.46




MN www.mn-net.com



43

6.7.

05


Wide pore silica packings

Silica as HPLC packing material has several advantages:pore size and surface are definedit is mechanically stablenumerous methods for surface modification areavailable

Many biologically interesting molecules can not be separatedusing conventional narrow pore silicas with pore sizes of about100 Å. This is why we offer a complete line of wide pore pack-ings with pore sizes of 300, 500, 1000 and 4000 Å. These ma-terials can also be used for size exclusion chromatography(SEC).

Octyl phases -(CH2)7 – CH3C8 phases are suited for reversed phase chromatographyand ion-pairing chromatography for separation of moderatelyto highly polar (water-soluble) compounds such as

steroidsnucleosidescyclodextrinspharmacological plant constituents


NUCLEOSIL® 50-5 C8 ecParticle size 5 µm, pore size 50 Å; octyl phase, endcapped, 9% C; eluent in column acetonitrile / water

ChromCart® columns2 mm ID 721830.20 721831.20 721833.20 721834.303 mm ID 721830.30 721831.30 721833.30 721834.304 mm ID 721830.40 721831.40 721833.40 721834.40

4.6 mm ID 721830.46 721831.46 721832.46 721833.46 721834.4030 mm ChromCart® columns and CC guard column cartridges (8 mm) in packs of 3, all other columns in packs of 1.1) 30 mm ChromCart® columns require the CC column holder 30 mm (Cat. No. 721823). 2) On request, Microbore columns are also available in lengths of 40, 60, 200 and 300 mm and with 0.15, 0.3, 0.4, 0.5, 0.75 and 1.5 mm ID.


1. Aloe emodin-8-glucoside2. Rhein-8-glucoside3: Sennoside D14: Sennoside D5: Sennoside B6: Sennoside A17: Sennoside C8: Sennoside A

9: Sennidin-B-monoglucoside10: Sennidin-A-monoglucoside11: Aloe emodin12: Rhein13: Sennidin A14: Emodin15: Sennidin B

Anthranoids in senna based drugsL. Kabelitz, K. Reif, Dtsch. Apotheker Ztg. 134 (1994) 37 – 40Column: 250 x 4 mm NUCLEOSIL® 100-5 C8Eluents: A) 0.02 M KH2PO4, adjusted to pH 2 with H3PO4 (85%), B) acetonitrileGradient: flow rate 1 ml/min: 10 min 14% B, in 5 min to 20% B, 10 min at 20% B, flow rate 0.8 ml/min: in 1 min to 21% B, in 29 min to 80% B,

flow rate 1 ml/min: in 2 min to 14% B, then 13 min at 14% BDetection: Photodiode array: dianthrones at 270 nm, anthraquinones at 435 nmPeaks:

1

2

34

5

6

7

8

910

11

12

13

14

15

0 10 20 30 40 min

0

0.1

0.2

0.3

0.4

0.5

0.6

1026

30

MNwww.mn-net.com



44

6.7.

05


EC columns 3)

4 mm ID 720092.40 721834.404.6 mm ID 720092.46 721834.40

NUCLEOSIL® 100-5 C8 ecParticle size 5 µm, pore size 100 Å; octyl phase, endcapped, 9% C; eluent in column acetonitrile / water


4.6 mm ID 721804.46 721795.46 721797.46 721796.46 721805.40


1 mm ID 717035.10 717026.10 717017.10 717008.10

NUCLEOSIL® 100-5 C8 Particle size 5 µm, pore size 100 Å; octyl phase, not endcapped, 8.5% C; eluent in column acetonitrile / water


4.6 mm ID 721194.46 721780.46 721621.46 721641.46 721661.46 721601.40


1 mm ID 717036.10 717027.10 717018.10 717009.10

EC columns 3)

3 mm ID 720001.30 720013.30 721601.304 mm ID 720001.40 720013.40 721601.40

4.6 mm ID 720001.46 720990.46 720013.46 721601.40


EC columns 3)

4 mm ID 720017.404.6 mm ID 720017.46


EC columns 3)

4 mm ID 720022.404.6 mm ID 720022.46




MN www.mn-net.com



45

6.7.

05


www.mn-net.com



4.6 mm ID 721720.46 721786.46 721722.46 721782.46 721785.40

EC columns 3)

2 mm ID 720071.20 720703.20 721785.303 mm ID 720071.30 720703.30 721785.304 mm ID 720071.40 720703.40 721785.40

4.6 mm ID 720071.46 720214.46 720703.46 721785.40



4.6 mm ID 721868.46 721892.46 721521.46 721801.46 721787.40

EC columns 3)

2 mm ID 720050.20 720052.20 721787.303 mm ID 720050.30 720052.30 721787.304 mm ID 720050.40 720052.40 721787.40

4.6 mm ID 720050.46 720735.46 720052.46 721787.40

NUCLEOSIL® 300-5 C8 Particle size 5 µm, pore size 300 Å; octyl phase, not endcapped, ~3% C; eluent in column acetonitrile / water

ChromCart® columns3 mm ID 721103.30 721098.30 721101.304 mm ID 721103.40 721098.40 721101.40

EC columns 3)

4 mm ID 720062.40 721101.404.6 mm ID 720062.46 721101.40




MNwww.mn-net.com



46

6.7.

05


Phenyl phases -(CH2)3

C6H5 phases are suited for reversed phase chromatographyand ion pairing chromatography for separation of moderatelypolar compounds. The retention characteristics are similar to

the C8 phase, but with different selectivity for polycyclic aro-matic hydrocarbons, polar aromatics, fatty acids etc.

Ordering information · Analytical columns with NUCLEOSIL® C6H5Length → 125 mm 150 mm 250 mm Guard columns

NUCLEOSIL® 100-5 C6H5 ecParticle size 5 µm, pore size 100 Å; phenyl phase, endcapped, 8 % C; eluent in column acetonitrile / water


NUCLEOSIL® 100-5 C6H5 Particle size 5 µm, pore size 100 Å; phenyl phase, not endcapped, 8% C; eluent in column acetonitrile / water


4.6 mm ID 721860.46 721887.46 721861.46 721862.40

EC columns 1)

2 mm ID 720695.20 721862.303 mm ID 720695.30 720956.30 721862.304 mm ID 720956.40 721862.40

4.6 mm ID 720956.46 721862.40ChromCart® 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).

Separation of opium alkaloids and meconic acidN.R. Ayyangar, S. R. Bhide, J. Chromatogr. 436 (1988) 455-465Column: 250 x 4 mm NUCLEOSIL® 100-7 C6H5Eluent A: methanol – water (5:95) with 1% triethyl-

ammonium phosphate stock buffer, pH 3.2Eluent B: methanol – water (80:30) with 1% triethyl-

ammonium phosphate stock buffer, pH 3.95Gradient: linear from 100% A to 100% B in 20 min,

then 12 min eluent BFlow rate: 1,5 ml/minDetection: UV, 280 nmPeaks:1. Morphine2. Meconic acid3. Codeine4. Thebaine5. Laudanosine6. Cryptopine7. Noscapine8. Narceine9. Papaverine

% v

CH

3OH

100

90

80

70

60

50

40

30

20

10

0

0 2 4 6 8 10 12 14 16 18 20 22 24 26min

1

2

3

4

5 6

7

8

9

1012

00

MN www.mn-net.com



47

6.7.

05


Butyl phases -(CH2)3 – CH3C4 phases are suited for reversed phase chromatographyand ion-pairing chromatography for separation of macromol-ecules and hydrophobic substances. Retention times on

butyl phases are shorter than on C8 and C18 phases. Forbutyl phases for biochemical separation problems please re-fer to page 107.

NUCLEOSIL® 100-7 C6H5 Particle size 7 µm, pore size 100 Å; phenyl phase, not endcapped, 8% C; eluent in column acetonitrile / water

EC columns 1)

4 mm ID 720019.404.6 mm ID 720019.46

Ordering information · Analytical columns with NUCLEOSIL® C6H5Length → 125 mm 150 mm 250 mm Guard columns

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).

Ordering information · Analytical columns with NUCLEOSIL® C4Length → 70 mm 100 mm 125 mm 150 mm 250 mm Guard columns

NUCLEOSIL® 120-5 C4 Particle size 5 µm, pore size 120 Å; butyl phase, endcapped, ~2 % C; eluent in column acetonitrile / water


EC columns 2)

4 mm ID 720096.40 721889.404.6 mm ID 720096.46 721889.40

ChromCart® guard column cartridges (8 mm) in packs of 3, all other columns in packs of 1.1) On request, Microbore columns are also available in lengths of 40, 60, 200 and 300 mm and with 0.15, 0.3, 0.4, 0.5, 0.75 and 1.5 mm ID.


Hemoglobin analysis of the adult Sumatran tiger

M. Jahan et al. Biol. Chem. Hoppe-Seyler 370 (1989) 27 – 33Column: 250 x 4.6 mm NUCLEOSIL® 300-5 C4Eluents: A) 0.1% trifluoroacetic acid, B) acetonitrileGradient: 0 – 35% B in 2 min, then 35 – 60% B in 60 minFlow rate: 1 ml/minDetection: UV, 230 nm

A23

0

Hemeα

βII

βI

0 10 20 30 40 50 60 min

1135

00

MNwww.mn-net.com



48

6.7.

05


Dimethyl phases -(CH3)2Dimethyl phases are suited for reversed phase chromatogra-phy and ion-pairing chromatography. Retention times on thedimethyl phase are much shorter than for the other RP phas-es.

NUCLEOSIL® 300-5 C4 Particle size 5 µm, pore size 300 Å; butyl phase, endcapped, ~2% C; eluent in column acetonitrile / water


4.6 mm ID 721627.46 721647.46 721667.46 721607.40


1 mm ID 717044.10 717047.10 717055.10 717058.10

EC columns 2)

4 mm ID 720059.40 721607.404.6 mm ID 720059.46 721607.40

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

EC columns 2)

4 mm ID 720060.404.6 mm ID 720060.46

Ordering information · Analytical columns with NUCLEOSIL® C4Length → 70 mm 100 mm 125 mm 150 mm 250 mm Guard columns

ChromCart® guard column cartridges (8 mm) in packs of 3, all other columns in packs of 1.1) On request, Microbore columns are also available in lengths of 40, 60, 200 and 300 mm and with 0.15, 0.3, 0.4, 0.5, 0.75 and 1.5 mm ID.


Ordering information · Analytical columns with NUCLEOSIL® C2Length → 125 mm 250 mm Guard columns

NUCLEOSIL® 100-7 C2 Particle size 7 µm, pore size 100 Å; dimethyl phase, not endcapped, 3.5 % C; eluent in column acetonitrile / water

ChromCart® columns 4 mm ID 721873.40 721874.40 721069.40

EC columns 1)

4 mm ID 720089.40 721069.404.6 mm ID 720089.46 721069.40


MN www.mn-net.com


49

6.7.

05


®

Unmodified NUCLEOSIL

®

, polar NUCLEOSIL

®

phases and ion exchangers

Packings with chemically bonded polar phases

These materials are characterised by fast mass transfer, be-cause the bonded organic layer of these stationary phases ismonomeric, thus minimising diffusion paths.Due to the availability of different functional groups it is possi-ble to vary the chemical characteristics of the surface andconsequently the adsorption characteristics of the stationaryphase. Silica packings with bonded polar phases thus com-bine the advantages of partition chromatography (variation ofadsorption properties) and adsorption chromatography (rap-id mass transfer in the stationary phase). Further advantag-es of chemically bonded polar phases are:

They do not “bleed”; equilibration with a liquid stationaryphase is not necessary as is the case in classical parti-tion chromatography.

The influence of impurities in the eluent (e. g. water) onthe separation qualities of bonded polar phases is muchsmaller than is the case with unmodified silica gels andaluminium oxides for adsorption chromatography.Swelling and consequently variation in the permeabilityof the packed columns never occurs with chemicallybonded polar silica phases. Even the use of water orother aqueous eluents does not present any problems.

Although the chemically bonded phases on the surface orthe silica gel form a monomeric organic layer, they have alarge surface concentration of functional groups, which isabout 3 to 4 functional groups per 100 Å

2

, i. e. about5 µeq/m

2

.

Influence of functional groups on the separation characteristics of a stationary phase

The figure on the left shows the influence of the modificationof silica gel on the k’ values (capacity factors) of aromatic hy-drocarbons, with

n

-heptane as eluent on unmodified NUCL-EOSIL

®

as well as three chemically bonded polar phases,namely NUCLEOSIL

®

CN, NH

2

and NO

2

. On the y-axis thek’ values of 9 aromatic hydrocarbons have been plotted. Theinfluence of the nitroaromatic groups on the separation is es-pecially evident. A strong interaction between the electronicsystems results in the formation of charge transfer complex-es with considerably larger k’ values than for unmodifiedNUCLEOSIL

®

.

k’

10,0

9,0

8,0

7,0

6,0

5,0

4,0

3,0

2,0

1,0

0

Benze

ne

Napht

halen

e

Biphen

yl

Anthr

acen

e

Phena

nthr

ene

Pyren

e

Chrys

ene

Benzo

[a]p

yren

e

Benzo

[e]p

yren

e

NO2 phase

NH2 phase

NUCLEOSILunmodified

CN phase

Effect of modification on the separationof aromatic hydrocarbons

MNwww.mn-net.com


50

6.7.

05


®

NUCLEOSIL

®

ion exchangers

These phases have been especially developed for HPLC.Contrary to adsorptions or reversed phase chromatographywith ion exchange chromatography there are three variableparameters for the mobile phase, which can influence theseparation

the

type of buffer

,the

ionic strength

and the

pH value

.By changing the ionic strength of the eluent, the competitionbetween mobile phase and sample molecules for the func-tional ionic groups on the stationary phase is influenced.When the retention time of the sample molecules is too long,an increase in the ionic strength of the mobile phase will re-duce retention times and vice versa.A change in the pH value of the mobile phase will alter thedissociation of analyte and buffer salt. These two effects in-fluence the equilibrium between the sample molecules andthe functional groups of the stationary phase. Thus the reten-tion of weak acids on an anion exchanger will increase whenthe pH value is raised, because a larger part of the acid ispresent in ionic form at higher pH values. If, on the otherhand, e.g. a phthalate buffer is used, increase of the pH val-ue can increase the concentration of phthalate ions and thusthe ionic strength. This results in a decreasing retention time.Proper selection of buffer types and pH values can thus re-sult in enormous selectivity differences, which can be utilisedfor optimisation of a separation.Even for equal ionic strength of the mobile phase, differentions show different degrees of affinity to the ion exchangegroups. The following orders of increasing affinity can be giv-en for anion exchangersF

–

< OH

–

< Cl

–

< CN

–

< Br

–

< NO

3–

< HSO

4–

< I

–

and for cation exchangersH

+

< Na

+

< NH

4+

< K

+

< Cs

+

< Ag

+

Divalent ions are more strongly attracted to the ion exchangegroups than monovalent, and trivalent more than divalentions. Generally speaking, the greater the charge and effec-tive radius of ions, the stronger is the attraction to the ion ex-change groups.

+ ++

++

+

+

++

++

+

++

++

+++

+ ++

+

+++

+++

++

www.mn-net.com

MN www.mn-net.com



51

6.7.

05


®

Cyano phases (nitrile)

-(CH

2

)

3

– CN

CN phases are suited for reversed phase chromatographyand for normal phase chromatography:

Normal phase chromatography:

with low-polarity solvents for many compounds, which can al-so be separated on unmodified silica, however, due to therapid equilibration much more suitable for gradient separa-tions

Reversed phase chromatography:

with different selectivity than C

18

, C

8

or phenyl modified pack-ingsPlease note: the eluent in the columns (except withNUCLEOSIL

®

100-5 CN-RP) is

n

-heptane. If you want to usean eluent which is not miscible with

n

-heptane (e. g. water), itis necessary to rinse the column with THF first.

Trace analysis of tetracyclinesC. Fu, H. Xu, Proc. 6th Natl. Symp. Chromatogr. (1987) 412 – 413Column 250 x 4.6 mm NUCLEOSIL® 100-5 CNEluent acetonitrile – 0.015 M oxalic acid pH 2.35 (20:80); flow rate 1 ml/minDetection UV, 363 nmPeaks:1. Terramycin2. E-Tetracycline3. Tetracycline4. E-Aureomycin5. Aureomycin6. Deoxyterramycin

12 3

4 5 6

1037

60

0 4 8 12 min


®

CN

Length

→

70 mm 125 mm 150 mm 250 mm Guard columns

NUCLEOSIL

®

100-5 CN

Particle size 5 µm, pore size 100 Å; cyano phase (nitrile), eluent in column

n

-heptane

ChromCart

®

columns

2 mm ID 721788.20 721624.20 721664.20 721604.303 mm ID 721624.30 721664.30 721604.304 mm ID 721624.40 721664.40 721604.40

4.6 mm ID 721624.46 721644.46 721664.46 721604.40

EC columns

1)

4 mm ID 720090.40 721604.404.6 mm ID 720090.46 721604.40

NUCLEOSIL

®

100-5 CN-RP

Particle size 5 µm, pore size 100 Å; cyano phase (nitrile), eluent in column acetonitrile / water

EC columns

1)

4 mm ID 720205.40 721917.404.6 mm ID 720205.46 721917.40

NUCLEOSIL

®

100-10 CN


n

-heptane

EC columns

1)

4 mm ID 720024.404.6 mm ID 720024.46

NUCLEOSIL

®

120-7 CN


n

-heptane

EC columns

1)

4 mm ID 720057.404.6 mm ID 720057.46

ChromCart

®

guard column cartridges (8 mm) in packs of 3, all other columns in packs of 1.

1)


®


MNwww.mn-net.com



52

6.7.

05


®

Cyano phases · Application Nitro phases · Application

LC/MS of the oxidation products of cholesterolM. Careri et al. J. Chromatogr. 794 (1998) 253 – 262Column: 250 x 2 mm NUCLEOSIL® 100-5 CNEluent: heptane – propan-2-ol (94:6)Flow rate: 0.3 ml/minDetection: MS; (a) EI mode, (b) PCI mode, (c) NCI modeElution sequence: 1 cholesterol, 2 5,6α-epoxycholesterol, 3 25-hydroxycholesterol, 4 7-ketocholesterol, 5 7β-hydroxycholesterol, 6 Cholestan-3β-5α-6β-triol

100% = 78315100

50

00 2 4 6 8 10 12 14 16

100% = 12136100

50

00 2 4 6 8 10 12 14 16

100% = 19350100

50

00 2 4 6 8 10 12 14 16 min

(a)

(b)

(c)

1120

30

min

min

Separation of gibberellins by normal phase HPLCP.C. Odén et al. J. Chromatogr. 464 (1989) 195 – 200Column: 125 x 4.6 mm NUCLEOSIL® 100-5 NO2Eluent A: n-heptane half saturated with 1 M formic acidEluent B: ethyl acetate with 1% water and 0.5% formic acidGradient: 100% A – 100% B in 60 minFlow rate: 2 ml/minDetection: radioactivity

Rad

ioac

tivity ka

uren

eka

uren

al

kaur

enol

kaur

enoi

c ac

id

GA

12-a

ldG

A9

GA

15G

A12 G

A4

GA

20

GA

44G

A53

GA

1

GA

19 GA

29

GA

8

10 20 30 40 50 60 70 80 90 100 110 120

Elution volume (ml)

GA

3

1062

80

Nitro phases -(CH2)3 NO2NO2 phases are suited for the separation of compounds withdouble bonds or for aromatic compounds.Please note: the eluent in the columns is n-heptane. If youwant to use an eluent which is not miscible with n-heptane(e. g. water), it is necessary to rinse the column with THFfirst.

Ordering information · Analytical columns with NUCLEOSIL® NO2Length → 125 mm 250 mm Guard columns

NUCLEOSIL® 100-5 NO2Particle size 5 µm, pore size 100 Å; nItro phase, eluent in column n-heptane


EC columns 1)

4 mm ID 720993.40 721863.404.6 mm ID 720993.46 721863.40


MN www.mn-net.com



53

6.7.

05


Diol phases

OH phases are suited for reversed phase chromatographyand normal phase chromatography; they are less polar thanunmodified silica and very easily wettable with water.Please note: the eluent in the columns is n-heptane. If youwant to use an eluent which is not miscible with n-heptane(e. g. water), it is necessary to rinse the column with THFfirst.

-(CH2)3 – O – CH2 – CH – CH2

– –

OH OHSFC separation of steroidsF. Vérillon et al. LC · GC int. 7 (1994) 710 – 716Column: 250 x 2 mm NUCLEOSIL® 100-7 OH (Diol)Eluent: CO2 with 1 – 12% methanolFlow rate: 2.5 ml/minOutlet pressure: 14 – 35 MpaTemperature: 85 °CDetection: UV, 210 nmPeaks:A) CholesterolB) ProgesteroneC) AndrosteroneD) Testosterone

E) EstroneF) EstradiolG) CortisoneH) HydrocortisoneI) PrednisoneJ) PrednisoloneK) Estriol

The hollow in the upper trace is caused by instantaneous detection of the pressure gradient and delayed detection of the methanol gradient. The lower trace shows a software-corrected chromatogram.

A

B

C

D

E

F

G

HI J

K

0 2 4 6min

1098

40

Ordering information · Analytical columns with NUCLEOSIL® OH (Diol)Length → 125 mm 250 mm Guard columns

NUCLEOSIL® 100-5 OH (Diol)Particle size 5 µm, pore size 100 Å; diol phase, eluent in column n-heptane


EC columns 1)

4 mm ID 720143.40 721478.404.6 mm ID 720143.46 721478.40

NUCLEOSIL® 100-7 OH (Diol)Particle size 7 µm, pore size 100 Å; diol phase, eluent in column n-heptane

EC columns 1)

4 mm ID 720070.404.6 mm ID 720070.46

ChromCart® guard columns 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).

MNwww.mn-net.com



54

6.7.

05


Unmodified silica SiOHDue to its narrow pore structure, NUCLEOSIL® 50 is recom-mended for adsorption chromatography, while NUCLEOSIL®

100 can be used for adsorption as well as for partition chro-matography.

Please note: the eluent in the columns is n-heptane. If youwant to use an eluent which is not miscible with n-heptane(e. g. water), it is necessary to rinse the columns with THFfirst.

Ordering information · Analytical columns with unmodified NUCLEOSIL® Length → 70 mm 125 mm 150 mm 250 mm Guard columns

NUCLEOSIL® 50-5 Particle size 5 µm, pore size 50 Å; unmodified, eluent in column n-heptane


4.6 mm ID 721620.46 721640.46 721660.46 721600.40

EC columns 1)

4 mm ID 720093.40 721600.404.6 mm ID 720093.46 721600.40


EC columns 1)

4 mm ID 720015.404.6 mm ID 720015.46


ChromCart® columns2 mm ID 721871.20 721870.20 721872.303 mm ID 721871.30 721870.30 721872.304 mm ID 721871.40 721870.40 721872.40

4.6 mm ID 721871.46 721516.46 721870.46 721872.40

EC columns 1)

3 mm ID 720099.30 721872.304 mm ID 720099.40 721872.40

4.6 mm ID 720099.46 721872.40


EC columns 1)

4 mm ID 720016.404.6 mm ID 720016.46


EC columns 1)

4 mm ID 720021.404.6 mm ID 720021.46


MN www.mn-net.com



55

6.7.

05


Amino phases -(CH2)3 – NH2NH2 phases feature versatile applicability:– in normal phase chromatography with hexane, CH2CI2 or

isopropanol as mobile phase for polar compounds suchas substituted anilines, esters, chlorinated pesticides etc.

– in aqueous-organic eluent systems for reversed phasechromatography of polar compounds like carbohydrates

– as weak anion exchanger for anions and organic acidsusing common buffers (e. g. acetate or phosphate) inconjunction with organic modifiers (e. g. acetonitrile)

Please note: the eluent in the columns is n-heptane. If youwant to use an eluent which is not miscible with n-heptane(e. g. water), it is necessary to rinse the column with THFfirst.

NUCLEOSIL® 100-5 NH2 RP– the amino phase for reversed phase applications; eluent

in column: acetonitrile / water– rinsing with THF is not required

Ordering information · Analytical columns with NUCLEOSIL® NH2Length → 70 mm 125 mm 150 mm 250 mm Guard columns

NUCLEOSIL® 100-3 NH2Particle size 3 µm, pore size 100 Å; amino phase, eluent in column n-heptane


4.6 mm ID 721121.46 721120.46 721122.40

EC columns 1)

4 mm ID 720275.40 721122.404.6 mm ID 720275.46 721122.40



4.6 mm ID 721625.46 721645.46 721665.46 721605.40

EC columns 1)

4 mm ID 720095.40 721605.404.6 mm ID 720095.46 721605.40

NUCLEOSIL® 100-5 NH2 RPParticle size 5 µm, pore size 100 Å; amino phase, eluent in column acetonitrile / water (80:20)

ChromCart® columns3 mm ID 721625.30RP 721665.30RP 721605.30RP4 mm ID 721665.40RP 721605.40RP

4.6 mm ID 721645.46 RP 721605.40RP

EC columns1)

3 mm ID 720121.30RP 720095.30RP 721605.30RP


EC columns 1)

4 mm ID 720058.404.6 mm ID 720058.46


MNwww.mn-net.com



56

6.7.

05


Dimethylamino phases -(CH2)3 – N(CH3)2The DMA phase is a weakly basic anion exchanger for theseparation of many anions; it can also be used in a similarway as the NH2 phase.Please note: the eluent in the columns is n-heptane. If youwant to use an eluent which is not miscible with n-heptane(e. g. water), it is necessary to rinse the column with THFfirst.

Separation of sennosides from rhubarbY. Ohshima, K. Takahashi, J. Chromatogr. 258 (1983) 292 – 296Column: NUCLEOSIL® 100-5 N(CH3)2, 250 x 8 mm IDEluent: THF – water – acetic acid (8:2:1)Flow rate: 3,5 ml/minDetection: UV, 280 nmSamples: methanolic extracts ofa) Rheum palmatum var. tanguticumb) Rheum coreanumc) Rheum palmatumd) Rheum coreanum x Rheum palmatum (Shinshu-rhubarb)

(a)(b)(c)(d)

010min 010min 010min 010min

1025

90

Ordering information · Analytical columns with NUCLEOSIL® N(CH3)2Length → 250 mm Guard columns

NUCLEOSIL® 100-5 N(CH3)2Particle size 5 ± 1.5 µm, pore size 100 Å; dimethylamino phase, eluent in column n-heptane

EC columns 4 mm ID 720994.40 721610.40

4.6 mm ID 720994.46 721610.40ChromCart® guard column cartridges (8 mm) in packs of 3, 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).

MN www.mn-net.com



57

6.7.

05


Cation exchangers, strongly acidic -(CH2)3 SO3Na

The cation exchanger NUCLEOSIL® SA is used for ion ex-change chromatography.

It is a strongly acidic cation exchanger with sulphonic acidmodification and a capacity of about 1 meq/g.

Ordering information · Analytical columns with NUCLEOSIL® SA

Length → 125 mm 150 mm 250 mm Guard columns

NUCLEOSIL® 100-5 SAParticle size 5 µm, pore size 100 Å; cation exchanger, strongly acidic, eluent in column 0.15 M (NH4)2HPO4, pH 5


4.6 mm ID 721486.46 721525.46 721342.46 721487.40

EC columns 1)

4 mm ID 720097.40 721487.404.6 mm ID 720097.46 721487.40

NUCLEOSIL® 100-10 SA Particle size 10 µm, pore size 100 Å; cation exchanger, strongly acidic, eluent in column 0.15 M (NH4)2HPO4, pH 5


EC columns 1)

4 mm ID 720028.40 721706.404.6 mm ID 720028.46 721706.40


Separation of rare-earth elementsNuryono et al. Chromatographia 48 (1998) 407 – 414Column: 150 x 4.6 mm NUCLEOSIL® 100-5 SAEluent A: 20 mM α-hydroxyisobutyric acid (HIBA), pH 4.6Eluent B: 12 mM oxalic acid, pH 4.6Gradient: 8 min 5% B, 5 – 50% B in 10 min, 50 – 75% B in 2 min, 5 min 75%

BFlow rate: 1 ml/minTemperature: 25 °CDetection: post-column derivatization with Arsenazo I, measurement in visible

light at 600 nmSample: rare-earth elements, 10 ppm each, 20 µl injected

mAU600 nm

80

60

40

20

0

0 5 10 15 20 min

Lu

Yb

Tm

ErHo

Dy

Y

Tb

Gd

EuSm Nd Pr

Ce

La

1140

30

MNwww.mn-net.com



58

6.7.

05


Anion exchangers, strongly basic -(CH2)3 CH2 – N+(CH3)3Cl–

The anion exchanger NUCLEOSIL® SB is used for ion ex-change chromatography. It is a strongly basic anion exchanger with a quaternary am-monium modification and a capacity of about 1 meq/g.

HPLC of inorganic and organic anionsD. Yan, G. Schwedt, J. Chromatogr. 516 (1990) 383 – 393Column 250 x 4 mm NUCLEOSIL® 100-10 SBEluent 1 mM diethylenetriamine-pentaacetic acid (DTPA)Flow rate 2 ml/minDetection conductivity

Ace

tate

For

mat

eC

l–

NO

2–

Br– N

O3–

SO

42–

Tart

rate

Oxa

late

0.2

µS

0 8 16 min

1065

40

Ordering information · Analytical columns with NUCLEOSIL® SB Length → 125 mm 150 mm 250 mm Guard columns

NUCLEOSIL® 100-5 SBParticle size 5 µm, pore size 100 Å; anion exchanger, strongly basic, eluent in column 0.15 M (NH4)2HPO4, pH 5


4.6 mm ID 721688.46 721523.46 721884.46 721885.40

EC columns 1)

4 mm ID 720996.40 721885.404.6 mm ID 720996.46 721885.40

NUCLEOSIL® 100-10 SBParticle size 10 µm, pore size 100 Å; anion exchanger, strongly basic, eluent in column 0.15 M (NH4)2HPO4, pH 5


EC columns 1)

4 mm ID 720029.40 721886.404.6 mm ID 720029.46 721886.40


www.mn-net.com

columns for hplccolumns for hplc - western · pdf file mn columns for hplccolumns for hplc 20...

Documents