Columns for HPLCColumns for HPLC - Western … · MN Columns for HPLCColumns for HPLC 8 6.7.05 NUCLEODUR ® C 18 Gravity and C 8 Gravity · high-performance silicas for …

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Columns for HPLCColumns for HPLC

6

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NUCLEODUR

®

reversed phase packings

In reversed phase liquid chromatography the efficiency of thepacking is strongly affected by the quality of the base silica it-self. Shortcomings in the surface geometry of the particles ormetal contaminants are the main reasons for inadequatecoverage with the covalently bonded alkylsilanes in the sub-sequent derivatization steps. It is well known, that poor sur-face coverage and, in consequence, high activity of residualfree silanols often results in peak tailing or adsorption, partic-ularly with basic compounds.NUCLEODUR

®

silicas are synthesized in a unique and care-fully controlled manufacturing process which provides silicaparticles, which are totally spherical. The important advan-tages of NUCLEODUR

®

are:

ideal particle shape and surface symmetry

high purity · minimized metal residues

high pressure stability

For a description of the properties of the NUCLEODUR

®

base silica please see page 141.

NUCLEODUR

®

Sphinx RP · the bifunctional RP phase

distinct selectivity based on bifunctional surface coverage

widens the scope in method development

high density of covalently bonded silanes

guarantees sharp peaks without tailing

pH stabilility

can be used from pH 1 – 10

low bleeding characteristics

in LC/MS applications

high reproducibility

· tight QC procedures provide consistant quality

NUCLEODUR

®

Sphinx RP, 5 µm is characterized by excep-tional selectivity features generated by a well-balanced ratioof covalently bonded octadecyl and phenyl groups. The com-bination of classical hydrophobic with

π

-

π

interactions (aro-matic ring system) expands the scope of selectivity in com-parison with conventional reversed phase packings.NUCLEODUR

®

Sphinx RP is particularly suited for the sepa-ration of molecules containing aromatic and multiple bonds.For the separation of polar compounds NUCLEODUR

®

Sphinx RP can be especially recommended and can alsooutperform many customary C18 phases. In addition, ex-haustive endcapping steps minimize unwanted surface sila-nol activity and guarantee excellent peak shapes even forstrong basic analytes. Different from standard phenyl phases, NUCLEODUR

®

Sphinx RP is far more stable towards hydrolysis and is alsosuggested for LC/MS applications. Due to the additional intermolecular interactions NUCLEO-DUR

®

Sphinx RP is an interesting replenishment to the highdensity bonded phases NUCLEODUR

®

C

8

/C

18

Gravity andthe polar endcapped NUCLEODUR

®

C

18

Pyramid.

Analytical columns packed with NUCLEODUR

®

SiSi Si Si SiSi

Si Si Si

O O O OH O

SiSi Si Si SiSi

Si Si SiSi(CH 3

) 3

Si(CH 3

) 3

O O

Si(CH 3

) 3

OO OH O

π – π

NEWNEW

hydrophobic

interaction

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0,0 2,5 5,0 7,5 10,0min

1 2 3 4 5 6

NUCLEODUR® C8 Gravity, 5 µm


NUCLEODUR® Sphinx RP, 5 µm

Separation of flavonoids on 3 different NUCLEODUR® phasesColumns: 150 x 4.6 mmEluent: water – methanol (40:60, v/v)Flow rate: 1 ml/minTemperature: 30 °CInj. volume: 3 µlDetection: 270 nm

Peaks:1. Catechin

2. Rutin R1 = R3 = OH, R2 = O-rutinose3. Fisetin R1 = R2 = OH, R3 = H4. Quercetin R1 = R2 = R3 = OH5. Kaempferol R1 = H, R2 = R3 = OH6. Isorhamnetin R1 = OCH3, R2 = R3 = OH

O

OH

OH

OH

OH

HO

O

R1

OH

R2

R3

HO

O

Ordering information · Analytical columns with NUCLEODUR

®

Sphinx RP

Length

→

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

NUCLEODUR

®

Sphinx RP, 5 µm

Particle size 5 µm, pore size 110 Å; special bifunctional RP phase, 15% C;

eluent in column acetonitrile / water

ChromCart

®

columns

2 mm ID 761551.20 761552.20 761554.20 761550.303 mm ID 761551.30 761552.30 761554.30 761550.304 mm ID 761551.40 761552.40 761554.40 761550.40

4,6 mm ID 761551.46 761552.46 761553.46 761554.46 761550.40

Microbore columns

1)

1 mm ID 717680.10 717681.10 717682.10 717683.10 717684.10

EC columns

2)

2 mm ID 760800.20 760801.20 760802.20 760803.20 761550.303 mm ID 760800.30 760801.30 760802.30 760803.30 761550.304 mm ID 760800.40 760801.40 760802.40 760803.40 761550.40

4,6 mm ID 760800.46 760801.46 760802.46 760803.46 761550.40

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.Guard columns for Microbore columns on request.

2)

As guard columns for EC columns use ChromCart

®

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


®

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NUCLEODUR

®

C

18

Gravity and C

8

Gravity · high-performance silicas for RP-HPLC

for method development

for HPLC at pH extremes

for overall sophisticated analytical separations

Base deactivation

NUCLEODUR

®

C

18

Gravity and C

8

Gravity

are based onthe new ultra-pure NUCLEODUR

®

silica. A unique derivati-zation process generates a homogeneous surface with ahigh density of bonded silanes (carbon content ~ 18% for theC

18

phase, ~ 11% for the C

8

phase). The following thoroughend-capping suppresses any unwanted polar interactionsbetween the silica surface and the sample, which makesGravity phases particularly suitable for the separation of ba-sic and other ionizable analytes. The figure on the rightshows a comparison study, where the strongly basic am-itriptyline is eluted on various highly base deactivated C18phases under isocratic conditions.

Enhanced pH stability

One major disadvantage of using silica stationary phases isthe limited stability at strong acidic or basic pH-ranges.Cleavage of the siloxane bonding by hydrolysis, or dissolu-tion of the silica will rapidly lead to a considerable loss in col-umn performance. Therefore conventional RP phases areusually not recommended to be run with mobile phases atpH > 8 or pH < 2 for extended periods of time.

The special surface bonding technology and the lowcontent of trace elements of NUCLEODUR

®

Gravity al-low for use at an expanded pH range from pH 1 to 11.

Surface silanols at different pH values

When is enhanced pH stability beneficial?

The option to work at an expanded pH range is often re-quired in method development. Many nitrogen containingcompounds like basic drugs are protonated at acidic or neu-tral pH and exhibit poor retention on a standard C18 phase.The retention behavior can be improved by working at ahigher pH, where the analyte is no longer protonated, but for-mally neutrally charged, as is the rule at pH 9 – 10. For acidicanalytes it is exactly in inverse proportion, maximum reten-tion can be attained at low pH. The figure above shows theextent of protonation of surface silanols and of two exempla-ry analytes at acidic and alkaline pH. The following graph ex-plains the general correlation between retention and pH.

Comparison of different base deactivated phasesColumns: 250 x 4 mmEluent: methanol/20 mM KH2PO4, pH 7.0 (75 : 25, v/v)Flow rate: 1.0 ml/min; temperature: 30 °C

Peaks:1. Dibutyl phthalat2. Acenaphthene3. Amitriptyline


Phase I (C18, 5 µm)

Phase L (C18, 5 µm)1 and 2 overlap

Phase P (C18, 5 µm)

Phase S (C18, 5 µm)

1

2

3

10 15 20 25 min


®

Si

Si

Si

Si

Si

Si

Si

Si

Si

OHOH

OOHOHOH

O

OH

O

H+

H+

H+

H+

HOOC

+NH2Me

Si

Si

Si

Si

Si

Si

Si

Si

Si

O–

O–

OO–

O–

O–

O

O–

O

OH–

OH–

OH–

OH–

–OOC

MeHN

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The figure below shows the excellent column stability ofNUCLEODUR

®

C

18

Gravity in acidic conditions. Retentiontimes of all 3 compounds in the column performance test re-main consistent and virtually unchanged, even after the col-umn is run with 5000 ml eluent. Due to the extremely stablesurface modification, no cleavage of Si-O-Si bonding occurs,column deterioration is therefore successfully prevented.

Another example for the effect of pH on selectivity is the sep-aration of the acid ketoprofen, the base lidocaine and ofbenzamide. Under acidic conditions the protonated lidocaineis eluted very fast due to lack of sufficiently strong hydropho-bic interactions between analyte and C

18

chains, whereasthe formally neutral ketoprofen is eluted after about 3 min.Contrary, at pH 10 a reversal of elution order is observed,with a notably longer retention time for the basic lidocaine.

k’

pH0 4 8 12

HA

B

BH+A–

Correlation between retention and pH for basic and acidic compounds

Stability of NUCLEODUR® C18 Gravity at pH 1.5Column: 125 x 4 mm NUCLEODUR® C18 Gravity, 5 µmeluent: acetonitrile / 1% TFA in water (50:50, v/v), pH 1.5 flow rate: 1.0 ml / min, temperature: 30°C, detection: UV, 230 nm, inj.volume: 5 µl, peaks: 1. pyridine, 2. toluene, 3. ethylbenzene

1 2

3

0 5 10 min

after 5000 ml eluent

1st injection

NH N(C2H5)2

O

CH3

H3C

O

O

OHH3C

O NH2

Influence of the pH value on selectivityColumn: 125 x 4 mm NUCLEODUR® C18 Gravity, 5 µm

eluents: A) acetonitrile/10 mM NH4HCO2, pH 3.0 (50:50, v/v)B) acetonitrile/10 mM NH4HCO3, pH 10.0 (50:50, v/v)

flow rate: 1 ml/mintemperature: 30 °Cdetection: UV, 240 nm inj. volume: 2 µl

Peaks:1. Lidocaine

2. Benzamide

3. Ketoprofen

A)

B)

12

3

1

2

3

0 2 4 min

0 2 4 min

0 5 10 min

1 24

6

3

5

SteroidsColumn: 125 x 4 mm NUCLEODUR® C8 Gravity, 5 µmEluent: acetonitrile – water (60:40, v/v)Flow rate: 1.0 ml/minTemperature: 25 °CDetection: UV, 240 nmPeaks:1. Cortisone2. Hydrocortisone3. Hydrocortisone 21-acetate4. 6α-Methyl-11β-hydroxyprogesterone5. 6α-Methyl-17α-hydroxyprogesterone6. 6α-Methyl-17α-hydroxyprogesterone acetate

1185

40


®

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The figure demonstrates the stability of NUCLEODUR® C18Gravity under alkaline conditions in comparison with 4 com-mercially available modern RP18 phases. Again, the ultra-pure Gravity with its unique high density surface bonding

technology withstands strongly alkaline mobile phase condi-tions. Even after 300 injections no loss of column efficiency,identified e.g. by peak broadening or decrease in retentiontimes, is observed.

As mentioned above, enhanced pH stability of the stationaryphase can be helpful for improving selectivity in method de-velopment. The two chromatograms on the right show theseparation of 4 basic drugs under acid and basic conditions.At pH 2.5 the protonated analytes exhibit poor retention (ear-ly elution) and in addition an inadequate resolution for pa-paverine and noscapine, whilst the formally non ionized mol-ecules can be baseline separated due to the better retentionpattern at alkaline pH.

The pH stability of sillica under alkaline conditions is mainly akinetic effect and based on the velocity of the dissolution ofthe silica support. It is worth mentioning, that this phenome-non also depends on type and concentration of buffers, aswell as on temperature. It is well known, that the use of phos-phate buffers, particularly at elevated temperatures, can re-duce column lifetime even at moderate pH. If possible, phos-phate buffers should be replaced by less harmfulalternatives.For a detailed discussion of the different retention character-istics of NUCLEODUR® octyl phases compared to the octa-decyl phases see page 14.

Stability of NUCLEODUR® C18 Gravity under alkaline conditions compared

with different C18 phasesfirst injectionafter 300 injections and 10 l eluent

Columns: 50 x 4.6 mmEluent: methanol / water / ammonia

(20:80:0.5, v/v/v), pH 11Flow rate: 1.3 ml /minTemperature: 30 °CDetection: UV, 254 nmInj. volume: 2.0 µlPeaks:1. Theophylline2. Caffeine

Phase X

Phase K

Phase L

Phase I

NUCLEODUR®

C18 Gravity

0 1 2 min

0 2 4 min

0 1 2 3 min

0 1 2 3 min

0 1 2 min

Separation of basic alkaloidsColumn: 125 x 4 mm NUCLEODUR® C18 Gravity, 5 µm

Eluent A: acetonitrileEluent B: 20 mM (NH4)2HPO4,

pH 2.5 / 10.0Gradient: 10% A (1 min)

→ 75% A in 10 minFlow rate: 1.0 ml/minTemperature: 25 °CDetection: UV, 254 nmInj. volume: 2 µl

Peaks:1. Lidocaine2. Papaverine3. Noscapine4. Diphen-

hydramine

pH 2.5

0 5

pH 10.0

0 5 10 min

10 min

1

2

34

1

2

3

4

Analytical columns packed with NUCLEODUR®

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Ordering information · Analytical columns with NUCLEODUR® Gravity

Length → 30 mm 1) 50 mm 70 mm 100 mm 125 mm 150 mm 250 mmGuard

columns

NUCLEODUR® C18 Gravity, 3 µmParticle size 3 µm, pore size 110 Å; octadecyl phase, endcapped, 18% C; eluent in column acetonitrile / water

ChromCart® columns2 mm ID 761450.20 761451.20 761452.20 761453.20 761124.303 mm ID 761450.30 761451.30 761452.30 761453.30 761124.304 mm ID 761450.40 761451.40 761452.40 761453.40 761124.40

4.6 mm ID 761450.46 761451.46 761452.46 761454.46 761453.46 761124.40

Microbore columns1 mm ID 717714.10 717715.10 717716.10 717717.10

EC columns 3)

2 mm ID 760080.20 760081.20 760082.20 761124.303 mm ID 760080.30 760081.30 760082.30 761124.304 mm ID 760080.40 760081.40 760082.40 761124.40

4.6 mm ID 760080.46 760081.46 760083.46 760082.46 761124.40

NUCLEODUR® C18 Gravity, 5 µmParticle size 5 µm, pore size 110 Å; octadecyl phase, endcapped, 18 % C; eluent in column acetonitrile / water


4.6 mm ID 761502.46 761503.46 761500.46 761504.46 761501.46 761125.40


EC columns 3)

2 mm ID 760102.20 760100.20 760101.20 761125.303 mm ID 760102.30 760100.30 760101.30 761125.304 mm ID 760102.40 760100.40 760101.40 761125.40

4.6 mm ID 760102.46 760100.46 760103.46 760101.46 761125.40

NUCLEODUR® C8 Gravity, 5 µmParticle size 5 µm, pore size 110 Å; octyl phase, endcapped, 11 % C; eluent in column acetonitrile / water

ChromCart® columns2 mm ID 761755.20 761751.20 761753.20 761754.303 mm ID 761755.30 761751.30 761753.30 761754.304 mm ID 761755.40 761751.40 761753.40 761754.40

4.6 mm ID 761755.46 761751.46 761752.46 761753.46 761754.40

EC columns 3) 2 mm ID 760750.20 760751.20 760753.20 761754.303 mm ID 760750.30 760751.30 760753.30 761754.304 mm ID 760750.40 760751.40 760753.40 761754.40

4.6 mm ID 760750.46 760751.46 760752.46 760753.46 761754.4030 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 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).


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NUCLEODUR® C18 Pyramid · RP-HPLC with highly aqueous mobile phases stable in 100% aqueous mobile phase systemsinteresting polar selectivity featuresexcellent base deactivation

The efforts to neutralize unwanted activity of unreacted sur-face silanols often results in well base-deactivated phaseswith high carbon load, but a limited scope of selectivity be-yond non-polar interactions. In particular polar compoundslike carboxylic acids, drug metabolites, etc. show only weakretention on densely bonded reversed phase columns due todistinct hydrophobic properties but low polar selectivity. Verypolar analytes require highly aqueous mobile phases for sol-ubility and retention. Conventional reversed phase columnsoften display stability problems in eluent systems with highpercentage of water (> 95%) as evidenced by a sudden de-crease of retention time and overall poor reproducibility. Thisphenomenon is described as phase collapse.Different approaches can be used to increase column stabili-ty with highly aqueous mobile phase systems. The mostpromising concepts are incorporating a polar group in the hy-drophobic alkyl chain, or using hydrophilic endcapping pro-cedures to improve the wettability of the reversed phasemodification. NUCLEOSIL® NAUTILUS may be taken as anexample for the embedded polar group strategy, in which aC18 silane with a polar function is successfully linked to thesilica surface 1) (also see page 30).Stability features of NUCLEODUR® C18 PyramidNUCLEODUR® C18 Pyramid is a silica phase with hydro-philic endcapping, designed especially for use in eluent sys-tems of up to 100% water. The figure below shows the reten-tion behaviour of tartaric, acetic and maleic acid under purelyaqueous conditions on NUCLEODUR® C18 Pyramid in com-parison with a conventionally bonded RP phase. It can beshown that the retention times for NUCLEODUR® C18 Pyra-mid remain nearly unchanged between initial injection and

restart after the flow has been stopped for 12 hours, whilstthe performance of the conventional RP column collapsedtotally after the same period.Retention characteristics of NUCLEODUR® C18 PyramidBased on the ultrapure NUCLEODUR® silica 2) (also seepage 141) the polar surface derivatisation exhibits retentioncharacteristics, which differentiate the ”Pyramid“ from con-ventional C18 stationary phases. The following chromato-gram shows the improved retention behaviour of very polarcompounds such as short chain organic acids, which are in-sufficiently retained on RP columns with predominantly hy-drophobic surface properties.

The figure below shows the separation of various organic ac-ids at acidic pH under 100% aqueous eluent conditions.

In addition to the exceptional polar selectivity NUCLEODUR®

C18 Pyramid also provides adequate hydrophobic retention,which can be illustrated in the selectivity test according toEngelhardt 3) (see below). The capacity factors of the non-polar, alkyl-substituted benzenes toluene and ethylbenzenedo not go too far in comparison with standard C18 phases.Base deactivation of NUCLEODUR® C18 PyramidThe perceptible increase in polarity has no impact on the re-tention behaviour of ionizable analytes. Even with the strong-ly basic compounds of the tricyclic antidepressant test mix-ture, no unwanted interactions or a so-called lack in basedeactivation are observed.

NUCLEODUR® C18 Pyramid conventional RP column

initialinjection

restartafter 12 h

0 5 min

0 5 min

0 5 min

0 5 min

both columns 125 x 4 mm ID, injection volume 1 µl50 mM KH2PO4 pH 2.5, 0.7 ml/min; 25 °C; UV, 210 nmPeaks: 1. tartaric acid, 2. acetic acid, 3 maleic acid

12

3

12

3

12

3

pumpstopped!

t0

Separation of very polar compoundsColumn: NUCLEODUR® C18 Pyramid, 5 µm, 125 x 4 mm IDEluent: 0.2% H3PO4Flow rate: 1 ml/minTemperature: 22 °CDetection: UV, 202 nmPeaks: (injection volume 2 µl)1. Formic acid2. Acetic acid

1

20

2

4 min

t0

Separation of organic acidsColumn: NUCLEODUR® C18 Pyramid, 5 µm, 250 x 4 mm ID

Eluent: 0.2% H3PO4Flow rate: 0.7 ml/minTemperature: 25 °CDetection: UV, 210 nmPeaks: (injection volume 2 µl)1. Tartaric acid2. Malic acid3. Lactic acid4. Succinic acid

1

23

4

0 5 10 min


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References1) D. Rieger and H. Riering, Int. Laboratory Aug. 2000, Vol.

30 (4A), 12

2) D. Rieger, J. Pfeiffer, LaborPraxis 26(2) (2002) 20 – 213) H. Engelhardt, M. Arangio, T. Lobert, LCGC Int. 10 (12),

803 – 812 (1997)

Selectivity testColumn: NUCLEODUR® C18 Pyramid, 5 µm, 250 x 4 mm IDEluent: MeOH – H2O (55:45, v/v)

Flow rate: 1 ml/minTemperature: 40 °CDetection: UV, 254 nmInj. volume: 3 µl

Peaks:1. Uracil2. Aniline3. Phenol4. Dimethylaniline5. p-Ethylaniline6. Toluene7. Ethylbenzene

1 2

3

4

5

6

7

0 10 20 30 min

12

3

4

5

0 5 10 min

Tricyclic antidepressantsColumn: NUCLEODUR® C18 Pyramid, 5 µm, 125 x 4 mm IDEluent: MeOH – 20 mM NH4H2PO4 pH 6.95 (70:30, v/v)Flow rate: 1 ml/minTemperature: 40 °CDetection: UV, 254 nmPeaks: (injection volume 5 µl)1. Protriptyline2. Nortriptyline3. Doxepin4. Imipramine5. Amitriptyline

Ordering information · Analytical columns with NUCLEODUR® C18 PyramidLength → 50 mm 70 mm 100 mm 125 mm 150 mm 250 mm Guard columns

NUCLEODUR® C18 Pyramid, 3 µmParticle size 3 µm, pore size 110 Å; octadecyl phase with hydrophic endcapping, 14% C; eluent in column acetonitrile / water


4.6 mm ID 761853.46 761850.46 761851.46 761852.46 761854.40

Microbore columns 1)

1 mm ID 717740.10 717741.10 717742.10 717743.10 717744.10

EC columns 2)

2 mm ID 760263.20 760260.20 760261.20 760262.20 761854.303 mm ID 760263.30 760260.30 760261.30 760262.30 761854.304 mm ID 760263.40 760260.40 760261.40 760262.40 761854.40

4.6 mm ID 760263.46 760260.46 760261.46 760262.46 761854.40

NUCLEODUR® C18 Pyramid, 5 µm as above, however, particle size 5 µm


4.6 mm ID 761801.46 761802.46 761803.46 761800.40

Microbore columns 1)

1 mm ID 717722.10 717723.10 717724.10 717725.10

EC columns 2)

2 mm ID 760200.20 760201.20 760202.20 761800.303 mm ID 760200.30 760201.30 760202.30 761800.304 mm ID 760200.40 760201.40 760202.40 761800.40

4.6 mm ID 760200.46 760201.46 760203.46 760202.46 761800.40ChromCart® 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.



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NUCLEODUR® C18 ec and C8 ecfor daily routine analyses and up-scaling for preparative HPLC

The efficiency of a separation is controlled by particle sizeand selectivity of the stationary phase. The exceptional sur-face coverage of monomeric bonded alkylsilanes, combinedwith an exhaustive endcapping, results in a surface with low-est silanol activity. This allows the tailing-free elution of polarcompounds such as basic drugs. NUCLEODUR® C18 ec is available in 9 different particle siz-es (3, 5, 7, 10, 12, 16, 20, 30 and 50 µm) which cover thewhole range from high speed analytical HPLC up to mediumand low pressure prep LC. NUCLEODUR® C18 ec is also an ideal tool for scale-up pur-poses.

NUCLEODUR® Gravity and NUCLEODUR® ec also available as octyl phases!In addition to the program of NUCLEODUR® C18 phasesMACHEREY-NAGEL introduces the corresponding octylmodified NUCLEODUR® C8 Gravity and C8 ec columns toexpand the reversed phase tool box effectively. The C8 phas-es exhibit the same excellent chemical and mechanical sta-bility features as the C18 counterparts. Due to the shorterchain and less hydrophobic properties of the stationaryphase the retention of non-polar compounds is decreasedand in consequence a reduction in time of analysis can beachieved. Moreover a stronger polar selectivity, particularlywith the separation of ionizable analytes is frequently ob-served (as distinct from the C18 phases). NUCLEODUR® C8 ec and NUCLEODUR® C8 Gravity (for or-dering information see page 11) are most suitable for the de-velopment of new methods but also for robust routine analy-sis .

C18 or C8, the best of both worldsChromatographers now might wonder about the differencesbetween C8 and C18 phases and the preferred range of ap-plication. Indeed there are no general guidelines which couldmake the choice easier but it will always be beneficial to addboth phases to the existing pool of reversed phase columnsin the laboratory. The following comparative study reveals the

different selectivity patterns of NUCLEODUR® C8 ec andNUCLEODUR® C18 ec. The separation of various nonsteroi-dal anti-inflammatory drugs illustrates the differences in po-larity between C8 and C18 and the resulting impact on effi-ciency. NUCLEODUR® C8 ec exhibits enhanced selectivityand excellent resolution for the polar compounds piroxicamand suprofen which co-elute on the C18 column. Howeverdue to the longer alkyl chain NUCLEODUR® C18 ec shows adistinct hydrophobic selectivity that leads to baseline separa-tion of the more non-polar analytes carprofen and fenoprofenwith superior peak shapes.

Anti-inflammatory drugsColumn: 250 x 4 mm NUCLEODUR® 100-5 C8 ec / C18 ecEluent: acetonitrile – water, 1% acetic acid (48 : 52, v/v) Flow rate: 1 ml/minTemperature: 25 °CDetection: UV, 230 nmPeaks: (injection volume 10 µl)1. Piroxicam2. Suprofen3. Ketoprofen4. Carprofen5. Fenoprofen6. Diclofenac

12

3

4+5

6

1+2 3 4 56

0 10 15 20min

5

0 10 15 20min5

C8

C18

Ordering information · Analytical columns with NUCLEODUR® C8 and C18

Length → 30 mm 1) 50 mm 70 mm 100 mm 125 mm 150 mm 250 mm Guard columns

NUCLEODUR® 100-3 C18 ec Particle size 3 µm, pore size 110 Å; octadecyl phase, endcapped, 17.5% C; eluent in column acetonitrile / water


4.6 mm ID 761001.46 761002.46 761003.46 761006.46 761004.46 761005.40



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EC columns 3)

2 mm ID 760050.20 760051.20 760052.20 761005.303 mm ID 760050.30 760051.30 760052.30 761005.304 mm ID 760050.40 760051.40 760052.40 761005.40

4.6 mm ID 760050.46 760051.46 760053.46 760052.46 761005.40

NUCLEODUR® 100-5 C18 ec as above, however, particle size 5 µm


4.6 mm ID 761200.46 761150.46 761350.46 761380.46 761400.46 761100.40


EC columns 3)

2 mm ID 760004.20 760001.20 760002.20 761100.303 mm ID 760004.30 760001.30 760002.30 761100.304 mm ID 760004.40 760001.40 760002.40 761100.40

4.6 mm ID 760004.46 760001.46 760008.46 760002.46 761100.40

NUCLEODUR® 100-3 C8 ecParticle size 3 µm, pore size 110 Å; octyl phase, endcapped, 10.5 % C; eluent in column acetonitrile / water


4,6 mm ID 761018.46 761019.46 761015.46 761016.46 761017.46 761012.40

EC columns 3)

2 mm ID 760063.20 760060.20 760062.20 761012.303 mm ID 760063.30 760060.30 760062.30 761012.304 mm ID 760063.40 760060.40 760062.40 761012.40

4,6 mm ID 760063.46 760060.46 760061.46 760062.46 761012.40

NUCLEODUR® 100-5 C8 ec as above, however, particle size 5 µm


4.6 mm ID 761705.46 761701.46 761702.46 761703.46 761704.40

EC columns 3)

2 mm ID 760700.20 760701.20 760703.20 761704.303 mm ID 760700.30 760701.30 760703.30 761704.304 mm ID 760700.40 760701.40 760703.40 761704.40

4.6 mm ID 760700.46 760701.46 760702.46 760703.46 761704.4030 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 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.


Ordering information · Analytical columns with NUCLEODUR® C8 and C18

Length → 30 mm 1) 50 mm 70 mm 100 mm 125 mm 150 mm 250 mm Guard columns


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NUCLEODUR® CN · cyano modified high-purity silica

Widen your scope in selectivity! multi-mode column (RP and NP)different retention characteristics in comparison to C8 / C18 stable against hydrolysis at low pHhigh reproducibility from lot to lot

In reversed phase HPLC it is fairly common to start with C18or C8 columns, if new methods have to be developed. How-ever, superior polarity and selectivity properties often re-quired for more sophisticated separations, are not alwayssufficiently provided by classical RP phases, which are usu-ally characterised by a hydrophobic layer of monomeric orpolymeric bonded alkylsilanes.

The polarity of the NUCLEODUR® 100 CN-RP phases canbe classified as intermediate based on multiple retentionmechanisms such as dipole-dipole, π-π, and also hydropho-bic interactions 1). Therefore, this phase shows a distinct se-lectivity for polar organic compounds as well as for mole-cules containing π-electron systems (e.g. analytes withdouble bonds, tricyclic antidepressants) 2). Short-chainbonded phases are sometimes suspected of revealing short-comings in stability towards hydrolysis at low pH 3). The chro-matogram on the right shows that even after 100 sample in-jections and four weeks storage at pH 1 (curve 2), neither aconsiderable shift in retention, nor a visible change in peaksymmetry could be noticed (curve 1 = new column).

One approach to improve the resolution of compounds poor-ly separated on nonpolar stationary phases, is to changebonded-phase functionality. The fully endcapped and highlyreproducible NUCLEODUR® 100 CN-RP phases have cyan-opropyl groups on the surface able to generate a clearly rec-ognizable different retention behaviour compared to purelyalkyl-functionalized surface modifications.

Due to the exceptional polarity features the cyano phase canalso be run in the normal phase mode. NUCLEODUR® CNcolumns for normal phase applications are shipped in n-hep-tane. The drastic change in selectivity and order of elution fora mixture of various steroids in normal and reversed phasemode is displayed in the chromatogram below. Moreover thehigh coverage combined with a thorough endcapping makesNUCLEODUR® 100 CN-RP suitable for the separation ofionizable compounds such as basic drugs.

References

1) C. S. Young and R. J. Weigand, LCGC 20 (5), 464 – 473(2002)

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

3) J. J. Kirkland, LCGC 14 (6), 486 – 500 (1996).

Reproducibility of NUCLEODUR® CN-RPColumn: NUCLEODUR® 100-5 CN-RP, 250 x 4 mm IDEluent: acetonitrile – water (60:40, v/v)Flow rate: 1 ml/minTemperature: 20 °CDetection: UV, 254 nmInj. volume: 15 µlPeaks:1. Benzamide2. Dimethyl phthalate3. Phenetol4. o-Xylene5. Biphenyl 1

2

3

4

5

0

4

8min

lot A

lot B

lot C

lot D

Comparison of NUCLEODUR® C18 ec and CN-RP for a separation of coldmedicine ingredients

Columns: A) NUCLEODUR® 100-5 CN-RP, 250 x 4 mm IDB) NUCLEODUR® 100-5 C18 ec, 250 x 4 mm ID

Eluent: acetonitrile – 100 mM sodium citrate pH 2.5 (15:85, v/v)

Flow rate: 1 ml/min Temperature: 25 °CDetection: UV, 270 nmInj. volume: 10 µlPeaks:1. Maleic acid2. Norephedrine3. Ephedrine4. Acetaminophen5. Chlorpheniramine6. Brompheniramine

1

23

4

5

6

1

23

4

5

6

0 4 8 12 min


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Ordering information · Analytical columns with NUCLEODUR® CNLength → 30 mm 50 mm 125 mm 150 mm 250 mm Guard columns

NUCLEODUR® 100-3 CN-RP Particle size 3 µm, pore size 110 Å; cyano phase (nitrile), 7% C; eluent in column acetonitrile / water

ChromCart® columns3 mm ID 761430.304 mm ID 761430.40

4.6 mm ID 761431.46 761430.40

EC columns 1)

2 mm ID 760159.20 761430.303 mm ID 760157.30 761430.304 mm ID 760156.40 761430.40

4.6 mm ID 760156.46 761430.40

NUCLEODUR® 100-5 CN-RP as above, however, particle size 5 µm

ChromCart® columns4 mm ID 761424.40 761423.40 761420.40

4.6 mm ID 761424.46 761423.46 761420.40

EC columns 1)

4 mm ID 760153.40 760152.40 761420.404.6 mm ID 760153.46 760154.46 760152.46 761420.40

NUCLEODUR® 100-5 CNParticle size 5 µm, pore size 110 Å; cyano phase (nitrile), 7% C; eluent in column n-heptane

ChromCart® columns4 mm ID 761422.40 761421.40 761419.40

4.6 mm ID 761422.46 761421.46 761419.40

EC columns 1)

4 mm ID 760151.40 760150.40 761419.404.6 mm ID 760151.46 760150.46 761419.40

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).

Separation of steroids in normal phase and reversed phase modeReversed phase modeColumn: NUCLEODUR® 100-5 CN-RP, 250 x 4 mm IDEluent: acetonitrile – water (25:75, v/v)Flow rate: 1 ml/minTemperature: 25 °CDetection: UV, 254 nm

Normal phase modeColumn: NUCLEODUR® 100-5 CN, 250 x 4 mm IDEluent: n-heptane – 2-propanol (90:10, v/v)other conditions as for reversed phase mode

Peaks (injection volume 10 µl):1. Methyltestosterone2. Testosterone3. Norgestrel4. Medrysone5. Cortisone6. Hydrocortisone7. Prednisolone

1

2

3

4 56

7

0 10 20 30 min

1

2

34

5

6+7

0 10 20 min


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Selectivity testColumn: 250 x 4 mm NUCLEODUR® C18 Gravity, 5 µmEluent: methanol / 20 mM KH2PO4, pH 7.0 (75:25, v/v)Flow rate: 1.0 ml/minTemperature: 30 °C

Detection: UV, 254 nmInj. volume: 5 µl

Peaks:1. Uracil2. 2,7-Dihydroxynaphthalene3. 2,3-Dihydroxynaphthalene4. Lidocaine5. Toluene

6. Naphthalene7. Ethylbenzene8. Dibutyl phthalate9. Acenaphthene

10. Amitriptyline

1

23

4 5

6

7 8

9

10

0 5 10 15 20min

Separation of antidepressants Column: 125 x 4 mm NUCLEODUR® C18 Gravity, 5 µmEluent A: methanol / acetonitrile (50:50, v/v)Eluent B: 20 mM KH2PO4, pH 7.0Gradient: 50% A → 65% A in 6 min, then 20 min 65% AFlow rate: 1.2 ml/minTemperature: 30 °CDetection: UV, 230 nmInj. volume: 5 µl

Peaks:1. Nordoxepin2. Protriptyline3. Maprotiline4. Nortriptyline5. Norclomipramine6. Doxepin7. Imipramine8. Amitriptyline9. Clomipramine

10. Trimipramine

1

2

3

4

5

6

7

8

910

0 5 10 15 min

1177

90

Separation of anti-inflammatory drugs Column: 250 x 4 mm NUCLEODUR® C18 Gravity, 5 µmEluent: acetonitrile / 20 mM KH2PO4, pH 2.5 (45:55, v/v)Flow rate: 1.0 ml/minTemperature: 22 °CDetection: UV, 230 nmInj. volume: 5 µl

Peaks:1. Acetylsalicylic acid2. Sulindac3. Piroxicam4. Suprofen5. Tolmetin6. Naproxen7. Diflunisal8. Flurbiprofen9. Indomethazin

10. Ibuprofen

1

2

3 4 5

6

7

8 9

10

0 5 10 min

1178

40

Separation of a coldmedicineColumn: 125 x 4 mm NUCLEODUR® C18 Gravity, 5 µmEluent A: 50 mM KH2PO4 + 5 mM Na pentansulfate, pH 2.5Eluent B: methanolGradient: 65% A → 45% A in 5 minFlow rate: 1.0 ml/minTemperature: 40 °CDetection: UV, 214 nmInj. volume: 5 µl

Peaks:1. Maleic acid2. Acetaminophen3. Pseudoephedrine4. Benzoic acid5. Chlorpheniramine6. Dextromethorphan

1

2

3

4

5 6

0 5 min 1178

10

Analytical columns packed with NUCLEODUR® · Applications

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1

3

2

4

5

6

0 4 8 12 min

Separation of antibacterialsColumn: 250 x 4 mm NUCLEODUR® 100-5 C18 ecEluent: acetonitrile – water (40:60, v/v) 0.05% TFAFlow rate: 1,0 ml/minTemperature: 25 °CDetection: UV, 254 nmInj. volume: 5 µlPeaks:1. Ofloxacin2. Ciprofloxacin3. Cinoxacin4. Penicillin G5. Penicillin V6. Cloxacillin

1178

70

0 5 10 15 20 25min

1

2

3 4

5

6

7

8

9

10

SteroidsColumn: 125 x 4 mm NUCLEODUR® 100-5 C8 ec

Cat. No. 760701.40Eluents: A) water, B) methanolGradient: 1 min at 20% B, 20 – 35% B in 10 min, 3 min at

35% B, 35 – 60% B in 6 min, 5 min at 60% BFlow rate: 1 ml/minTemperature: 30 °CDetection: UV, 230 nm Peaks (each env. 10 – 50 µg/ml, inj. volume 10 µl):

1. Estriol 2. Prednisolone3. Cortisone4. Testosterone5. 6α-Methyl-11β-hydroxy-

progesterone6. 6α-Methyl-17α-hydroxy-

progesterone7. 6α-Methyl-17α-hydroxy-

progesterone acetate8. Estradiol9. Estrone

10. Progesterone

1185

50

0 10 20 30 40 50 60min

12

3

4

5

67 8

9 11

12

13

1415

16

17 18

10

Separation of pesticides Column: 250 x 4 mm NUCLEODUR® 100-3 C8 ecEluents: A) water (1% acetic acid), B) acetonitrileGradient: 10 – 25% B in 10 min, 25 – 30% B in 10 min,

5 min at 30% B, 30 – 40 % B in 20 min40 – 50% B in 10 min, 10 min at 50% B

Flow rate 1 ml/min, temperature 35 °C, detection UV, 230 nmPeaks:

1. Desisopropylatrazine2. Metamitron3. Desethylatrazine4. Bromoxynil5. Simazine6. Cyanazine7. Metabenzthiazuron8. Atrazine9. Monolinuron

10. Isoproturon11. Diuron12. Metobromuron13. Metazachlor14. Sebutylazine15. Terbuthylazine16. Linuron17. Chloroxuron18. Metolachlor

1186

10

0 5 10 15 min

1

2

3

4

5

6

78

9

10

Analgesic and antiinflammatory drugsColumn: 250 x 4 mm NUCLEODUR® 100-5 C8 ec

Cat. No. 760703.40Eluent: acetonitrile – 1% acetic acid (48:52, v/v)Flow rate: 1 ml/minTemperature: 25 °CDetection: UV, 230 nmInj. volume: 10 µlPeaks:

1. Acetylsalicylic acid (1,6 µg/ml)2. Tolmetin (26 µg/ml)3. Piroxicam (26 µg/ml)4. Suprofen (26 µg/ml)5. Naproxen (0,64 µg/ml)6. Diflunisal (1,6 µg/ml)7. Fenoprofen

(26 µg/ml)8. Flurbiprofen

(26 µg/ml)9. Indomethazin

(52 µg/ml)10. Ibuprofen

(52 µg/ml)

1185

90

Analytical columns packed with NUCLEODUR® · Applications

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