stationary phase specifications introduction 北京慧德易科技

2

This Guide provides the following comparison data on commonly used C18 phases:

Stationary Phase Specifications Specifications provided by column manufacturers

Phases Compared According to RelativeHydrophobicity

Retention data for hydrophobic and neutral compounds

Phases Compared According to RelativePolarity

Categorization of Phases According toHydrophobicity and Polarity

Comparison of Column Efficiency for a Neutral Compound

Comparison of Column Efficiency forBasic Compounds

Also measures peak tailing

Phases Grouped According to Silanol Activity

Phases Compared According to Metal Activity

Stationary Phase SpecificationsStationary phase specifications providebasic information that can be helpful indeciding which phases to select for evaluation. For example, phases with high surface area and high carbon loadwill generally retain hydrophobic com-pounds longer than phases with low surface area and low carbon load. If youare analyzing macromolecules, such aspeptides and proteins, a wider pore (200 — 300 Å) phase usually provides better performance than a phase withsmall pores. New high purity silicas usually provide better peak shape forbasic compounds than older, more acidicsilicas (see Figure 2). Stationary phasespecifications, however, will not give youenough information to accurately predictretention or band spacing (selectivity).This is especially true when separatingpolar compounds.

IntroductionThere are so many different C18 columnsto choose from, that finding the right column for a particular separation can be very time consuming and expensive. Two apparently similar C18 phases cangive very different results. For example,Figure 1 compares the separation of the same sample mixture on a HypersilHyPURITY C18 and a Symmetry C18 column under identical mobile phase conditions. Even though both columnsare packed with “new generation” C18stationary phases, the band spacing(selectivity) between peaks is very different on the two columns. Without

more information, it is impossible to predict how the performance of differentstationary phases will compare.

This Comparison Guide to C18 Reversed

Phase HPLC Columns provides basic comparison information on commonlyused C18 columns to help you more easily identify similarities and differencesbefore investing time and money in chromatographic testing. Hopefully, thisinformation will help you find the rightcolumn for your application quicker.

Only silica based C18 bonded phases areevaluated in this Guide. Other bondedphases, such as C8, CN, Phenyl and polarembedded phases, are excluded.

This Guide does not identify an overall“best” column. The column that worksbest for one application will not necessar-ily be the column that will work best forother applications. And, there certainly isnot a single column that will work bestfor all applications. However, this Guidecan help you identify columns that arelikely to perform well so that at least youcan narrow the number of columns forchromatographic testing. You may findthat this Guide helps you identify severalcolumns that provide good separationsand performance. It is always desirable tohave more than one column identified foran application, especially if you are run-ning routine assays.

Increasingly, chromatographers are seek-ing to identify alternate brands of HPLCcolumns suitable for their assays. Havingan alternate column choice for a methodreduces the risk of “down time” due tocolumn problems such as a change inselectivity from one manufactured lot toanother or slow supplier delivery. Findingan alternate or back-up column that willprovide acceptable selectivity and performance when substituted into amethod can be as expensive and time consuming as finding the right column fordeveloping an initial separation. It is ourhope that this Guide will make that jobeasier by identifying columns with similarchromatographic characteristics.

0 1 2 3 4 5 6 7 8 9 10 11Time (min)

12

3 45

6

7

0 1 2 3 4 5 6 7 8 9 10 11Time (min)

1

2

3 4

5

6

7

Figure 1

Apparently Similar C18 Phases Can GiveVery Different Chromatographic Results

Mobile Phase: 60% CH3CN40% 50mM KH2PO4, pH 3.2

Sample: 1. Uracil 5. N,N-Dimethylaniline2. Pyridine 6. 4-Butylbenzoic acid3. Phenol 7. Toluene4. Dimethyl phthalate

Both Symmetry C18 and Hypersil HyPURITY C18 are new generation phases. You would expect them to provide similar performance, and in some casesthey do. However, in the example given here you cansee significant differences in peak retention times,selectivity, and even peak shape.

Column: Symmetry C18

Column: Hypersil HyPURITY C18

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Particle Pore Surface Carbon High PurityStationary Phase Size (µm) Size (Å) Area (m2/g) Load (%) Endcapped Silica

Ace C18 5 100 300 15.5 yes yesCapcell Pak AG C18 5 120 300 15 yes noCapcell Pak SG C18 5 120 300 14 yes noCapcell Pak UG C18 5 120 300 15 yes yesCosmosil C18-AR 5 120 300 17 yes yesDevelosil ODS-HG 5 140 300 18 yes yesDevelosil ODS-MG 5 100 450 15 yes yesDevelosil ODS-UG 5 140 300 18 yes yesExsil ODS 5 100 200 11 yes noExsil ODS1 5 100 200 11 yes noExsil ODSB 5 100 200 12 yes noGenesis C18 4 120 300 ? yes yesHichrom C18 5 150 250 15 yes yesHichrom RPB 5 110 340 14 yes yesHypersil BDS C18 5 130 170 11 yes noHypersil HyPURITY C18 5 180 200 13 yes yesHypersil ODS 5 120 170 10 yes noInertsil ODS 5 100 350 14 yes noInertsil ODS3 5 100 450 15 yes yesInertsil ODS2 5 150 320 18.5 yes yesKromasil C18 5 100 340 19 yes yesLiChrosorb RP-18 10 100 300 17 no noLiChrospher RP-18 5 100 350 21.6 no noLuna 5 C18(2) 5 100 400 17.5 yes yesNovapak C18 4 60 120 7.3 yes noNucleosil C18 5 100 350 15 yes noNucleosil C18 HD 5 100 ? 20 yes yesNucleosil C18AB 5 100 350 24 yes noPartisil ODS 10 85 350 5 no noPartisil ODS2 10 85 350 15 yes noPartisil ODS3 10 85 350 10.5 yes noProdigy ODS2 5 150 310 18.4 yes yesProdigy ODS3 5 100 450 15.5 yes yesPurospher RP18-e 5 80 500 ? yes yesResolve C18 5 90 200 10 no noSymmetry C18 5 100 335 19 yes yesSynchropak CR101 5 100 ? ? no noTSK ODS-120T 5 120 ? 22 yes noTSK ODS-80TM 5 80 ? 15 yes noµBondapak C18 10 125 330 10 yes noUltrasphere ODS 5 80 ? 12 yes noVydac 218MS 5 300 70 ? yes noVydac 218TP 5 300 70 8 yes noVydac Selectapore 300M 5 300 70 ? yes yesVydac Selectapore 300P 5 300 70 ? yes yesVydac Selectapore 90M 5 90 250 ? yes yesWaters Spherisorb ODS1 5 80 220 6.2 no noWaters Spherisorb ODS2 5 80 220 11.5 yes noWaters Spherisorb ODSB 5 80 220 11.5 yes noXTerra MS C18 5 125 ? 15.5 yes —YMC J’Sphere ODS H80 4 80 510 22 yes noYMC J’Sphere ODS M80 4 80 510 14 yes noYMC ODS A 5 120 300 17 yes noYMC ODS AM 5 120 300 17 yes noYMC Pro C18 5 120 335 16 yes yesZORBAX Extend C18 5 80 180 12.5 yes yesZORBAX ODS 5 70 330 20 yes noZORBAX Rx-C18 5 80 180 12 no yesZORBAX SB-C18 5 80 180 10 no yesZORBAX XDB-C18 5 80 180 10 yes yes

0 1 2 3 4 5Time (min)

1

2

3

0 1 2 3 4 5 6 7Time (min)

1

2

3

Figure 2

High Purity Silicas Provide Better PeakShape for Basic Compounds

Figure 3

Specifications of C18 Stationary Phases

Mobile Phase: 60% CH3OH, 40% H2OSample: 1. Uracil

2. Pyridine3. Phenol

3

Interaction between cationic compounds and acidic silanolsites on the surface of silica stationary phase supports cancontribute to retention and peak tailing. Phases made withhigh purity silica (less acidic silica) generally can be expectedto provide better peak shape for basic compounds.

Specifications were obtained from manufacturer’s literature.

Acidic Silica (Waters Spherisorb ODS2)

High Purity Silica (Ace C18)


Inertsil ODS3

LiChrospher RP-18

YMC J’Sphere ODS H80

Nucleosil C18

Kromasil C18

Develosil ODS-MG

Partisil ODS2

LiChrosorb RP-18

Symmetry C18

Inertsil ODS

Develosil ODS-UG

Purospher RP18-e

YMC Pro C18

Prodigy ODS3

Zorbax ODS

Nucleosil C18HD

YMC ODS AM

YMC ODS A

Inertsil ODS2

Cosmosil C18-AR

Prodigy ODS2

TSK ODS-80TM

Ace C18

Luna C18(2)

Genesis C18

Nucleosil C18AB

Waters Spherisorb ODS2

Hichrom C18

Zorbax Extend C18

Develosil ODS-HG

Resolve C18

Zorbax RX C18

Zorbax XDB C18

Waters Spherisorb ODSB

Hypersil BDS C18

Hypersil HyPurity C18

Ultrasphere ODS

TSK ODS-120T

Partisil ODS3

Hichrom RPB


u Bondapak C18

Exsil ODS

Capcell Pak AG C18

YMC J’Sphere ODS M80

Capcell Pak SG C18

Capcell Pak UG C18

Zorbax SB C18

Exsil ODSB

XTerra MS C18

Hypersil ODS

Novapak C18

Synchropak CR101

Exsil ODS1

Vydac SelectaPore 90M

Vydac SelectaPore 300P

Vydac 218TP

Partisil ODS

Vydac 218MS

Vydac SelectaPore 300M

Figure 4

C18 Phase Compared According to Relative Hydrophobicity

Mobile Phase: 90% CH3OH, 10% H2OElution Order: 1. Dimethyl phthalate

2. Toluene3. Biphenyl4. Phenanthrene

Column Temperature: 24°C

Phases Compared According to Relative HydrophobicityHydrophobicity is measured as the retention of a hydrophobic solute, phenanthrene.Figure 4 gives a comparison of hydrophobicity with the C18 phases listed according to hydrophobicity. Notice, however, that the retention for dimethyl phthalate, the least hydrophobic solute in the mixture, cannot always be predicted from thehydrophobicity ranking. Some low hydrophobicity phases actually have greater retention for dimethyl phthalate than some high hydrophobicity phases. We find thatthis is not unusual when separating polar compounds. Phases that are significantlymore retentive for hydrophobic analytes may show only slightly more retention forpolar compounds than low hydrophobicity phases, and sometimes they show less.

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Synchropak CR101

0.0 0.5 1.0 1.5 2.0 2.5

Vydac Selectapore 90M


Hypersil HyPURITY C18

Partisil ODS3

Capcell Pak SG C18

TSK ODS-120T

Hypersil BDS

Hypersil ODS

Capcell Pak UG C18

Nucleosil C18AB

Exsil ODS

Develosil ODS-HGLiChrosorb RP-18

Hichrom RPB


TSK ODS-80TM

ZORBAX SB-C18

Resolve C18

Ultrasphere ODS

Cosmosil C18-AR

Capcell Pak AG C18

XTerra MS C18

YMC ODS AM

YMC Pro C18

YMC ODS A

Ace C18

Partisil ODS2

Inertsil ODS

ZORBAX Rx C18

Develosil ODS-UG

Prodigy ODS2

ZORBAX XDB-C18

Nucleosil C18 HD

Nucleosil C18

Inertsil ODS2

Genesis C18

ZORBAX Extend C18

Luna C18(2)

LiChrospher RP-18

ZORBAX ODS

Prodigy ODS3

Symmetry C18

Develosil ODS-MG

YMC J'Sphere ODS H80

Kromasil C18

Purospher RP-18e

Inertsil ODS3

k (Toluene)

Partisil ODS


Vydac Selectapore 300P

Vydac 218TP

Vydac 218MS

Exsil ODSB

µBondapak C18

Exsil ODS1

Novapak C18

Hichrom C18


YMC J'Sphere ODS M80

Mobile Phase: 80% CH3OH20% 25mM KH2PO4, pH 6.0

Sample: TolueneTemperature: 24°C

Figure 5

C18 Phases Ranked According toRetention for Toluene

Alternative Test forHydrophobicityToluene can also be used as a probe to measure hydrophobicity. Notice that the ranking of C18 phases accord-ing to retention for toluene (Figure 5)is slightly different from the rankingaccording to retention for phenanthrene(Figure 4).



Exsil ODS1


Hypersil ODS

Resolve C18

Zorbax ODS

Partisil ODS

Hypersil BDS C18

Vydac 218TP


LiChrospher RP-18

Cosmosil C18-AR

Vydac 218MS

µBondapak C18

TSK ODS-80TM

Novapak C18

LiChrosorb RP-18

Zorbax SB-C18

Zorbax Rx-C18


TSK ODS-120T

Purospher RP18-e


Ultrasphere ODS

Nucleosil C18

Partisil ODS3

Capcell Pak AG C18

Capcell Pak SG C18

Nucleosil C18AB

YMC ODS A

YMC ODS AM

Synchropak CR101

Symmetry C18

Partisil ODS2

Inertsil ODS2

XTerra MS C18

Inertsil ODS

Genesis C18

Zorbax Extend C18

Prodigy ODS3

Nucleosil C18 HD

Kromasil C18

Inertsil ODS3

Hichrom C18

Prodigy ODS2

Hichrom RPB

Develosil ODS-HG

Ace C18

Develosil ODS-MG

Zorbax XDB-C18

Exsil ODSB

Luna C18(2)

Develosil ODS-UG

YMC Pro C18

0 1 2 3 4 5

k (Pyridine) / k (Phenol)

HighPolarity

LowPolarity

Capcell Pak UG C18

Hypersil HyPurity C18





Exsil ODS

Phases Ranked According toRelative PolarityThere have been several chromatographictests suggested for measuring polarity of stationary phases. Although there isno one test that we think provides adefinitive measurement, we have chosento use the ratio of k values for pyridineand phenol as our measure of relativepolarity for these C18 phases.

Figure 6 ranks stationary phases according to relative polarity using thetest conditions given. In this ranking,there is not necessarily a significant difference between consecutive listings.If a different mobile phase condition wasused for the test, e g., a lower mobilephase pH, or if different probes wereused, the ranking may be somewhat different. However, phases at the highpolarity end of the ranking and phases at the low polarity end of the ranking arelikely to test that way under most polaritytests conditions. Therefore, this rankingcan be used to identify relative differ-ences and similarities in polarity that canaffect selectivity for polar compounds

Since silanol activity is a major contribu-tor to phase polarity, the test conditionsused here to measure polarity have alsobeen used by some chromatographers as an indication of silanol activity. This seems consistent with the fact thatmost phases at the high polarity end ofthe ranking use more acidic silicas as stationary phase supports where phasesat the low polarity end of the ranking useless acidic (high purity) silicas. However,there are other factors that contribute to the retention of pyridine and phenol that prevent us from using their relativeretention as a reliable measure of silanolactivity. For example, Inertsil ODS hasmoderate polarity but shows significantsilanol activity in other tests. Also, wesee that Prodigy ODS2 tests with similarpolarity as the Ace C18, but in tests forsilanol activity, the Ace C18 shows signifi-cantly less silanol activity (see Figure 14).

Figure 6

C18 Phases RankedAccording to Polarity

Mobile Phase: 80% CH3OH20% H2O

Polarity = k Pyridine / k PhenolTemperature: 24°C



High Polarity/ High Polarity/ High Polarity/Low Hydrophobicity Moderate Hydrophobicity High Hydrophobicity

Exsil ODS1 Cosmosil C18-AR LiChrosorb RP-18Hypersil ODS Exsil ODS LiChrospher RP-18Novapak C18 Hypersil BDS C18Partisil ODS Resolve C18Vydac 218MS TSK ODS-80TM Vydac 218TP µBondapak C18 Vydac Selectapore 300P Waters Spherisorb ODS1 Waters Spherisorb ODS2 Zorbax ODS

Moderate Polarity/ Moderate Polarity/ Moderate Polarity/Low Hydrophobicity Moderate Hydrophobicity High Hydrophobicity

Synchropak CR101 Capcell Pak AG C18 Nucleosil C18Vydac Selectapore 300M Capcell Pak SG C18 Partisil ODS2Vydac Selectapore 90M Genesis C18 Symmetry C18Xterra MS C18 Inertsil ODS YMC J’Sphere ODS H80

Inertsil ODS2Nucleosil C18 ABPartisil ODS3Prodigy ODS3Purospher RP18-eTSK ODS-120TUltrasphere ODSWaters Spherisorb ODSBYMC J’Sphere ODS M80YMC ODS AYMC ODS AMZorbax Extend C18Zorbax Rx-C18Zorbax SB-C18

Low Polarity/ Low Polarity/ Low Polarity/Low Hydrophobicity Moderate Hydrophobicity High Hydrophobicity

Exsil ODSB Ace C18 Develosil ODS-MGCapcell Pak UG C18 Inertsil ODS3Develosil ODS-HG Kromasil C18Develosil ODS-UGHichrom C18Hichrom RPBHypersil HyPURITY C18Luna C18(2)Nucleosil C18 HDProdigy ODS2YMC Pro C18Zorbax XDB-C18

Figure 7

C18 Phases Grouped According to Hydrophobicity and PolarityPhases are listed in alphabetical order by category.

Categorization of phasesaccording to hydrophobicityand polarity.The hydrophobicity and polarity data can be used to group phaseswith similar characteristics into categories. The following criteriawas used for the categories:

Hydrophobicityk for phenanthrene

High > 7.50

Moderate 5.50 to 7.49

Low < 5.50

Polarityk pyridinek phenol

High > 1.00

Moderate 0.50 to 0.99

Low < 0.50



Mobile Phase: 80% CH3OH, 20% 25mM KH2PO4, pH 6.0

Sample:1. Norephedrine 4. Imipramine2. Nortriptyline 5. Amitriptyline3. Toluene

Figure 8 provides an example ofhow columns from different polarity/hydrophobicity categorieswill compare. In this separationof antidepressants, Symmetry C18(high hydrophobicity) is slightlymore retentive than Hypersil BDSC18 (moderate hydrophobicity),and the band spacing of Ace C18(low polarity) is more similar toSymmetry C18 (moderate polarity)than it is to Hypersil BDS C18(high polarity).

Figure 8

Chromatographic Comparisonof Stationary Phases fromDifferent Categories

Hypersil BDS C18High Polarity/Moderate Hydrophobicity

Symmetry C18Moderate Polarity/High Hydrophobicity

Ace C18Low Polarity/Moderate Hydrophobicity



0 20,000 40,000 60,000 80,000 100,000 120,000

N/Meter (Toluene)

Capcell Pak UG C18


Novapak C18

Inertsil ODS3

TSK ODS-80TM

Prodigy ODS3

Vydac 218TP


Capcell Pak SG C18

Develosil ODS-MG

Genesis C18Hypersil HyPURITY C18

Inertsil ODS

LiChrosorb RP-18

Hypersil BDS C18

Vydac 218MS

Ultrasphere ODS

Cosmosil C18-AR


LiChrospher RP-18Zorbax Extend C18


Exsil ODSB

YMC ODS AM

Nucleosil C18 HD

Develosil ODS-HG

Zorbax ODS

Nucleosil C18AB

Luna C18(2)

Zorbax Rx-C18



Develosil ODS-UG

Symmetry C18

Exsil ODS

Hypersil ODS

Zorbax XDB-C18

Hichrom RPB

Kromasil C18

YMC ODS A


Hichrom C18

Nucleosil C18

Synchropak CR101

Zorbax SB-C18

YMC Pro C18

Ace C18

Exsil ODS1

Inertsil ODS2

µBondapak C18

Partisil ODS2

Purospher RP18-e

Resolve C18

XTerra MS C18

Partisil ODS

Prodigy ODS2

Capcell Pak AG C18

Partisil ODS3


TSK ODS-120T

Comparison of Column Efficiencyfor a Neutral CompoundColumn efficiency is reported as platesper meter (N/Meter). Using a neutralcompound (toluene) for the measurementgreatly reduces the effects of secondaryretention on the measurement of N andallows us to obtain data that is a betterindication of the following factors:

• Particle sizeSmaller average packing particle size =Larger N

• Particle size distributionBroader particle size distribution =Smaller N

• Packing efficiencyBetter packing procedures = Larger N

Figure 9

Comparison of Column Efficiencyfor a Neutral CompoundColumn efficiency reported as Platesper meter (N/Meter)

Mobile Phase: 80% CH3OH20% 25mM KH2PO4, pH 6.0

Sample: TolueneTemperature: 24°C



Nucleosil C18AB

Partisil ODS2

Vydac 218TP

Vydac 218MS


LiChrosorb RP-18


Cosmosil C18-AR

TSK ODS-80TM

Novapak C18

Prodigy ODS2

Partisil ODS

Zorbax SB-C18

Nucleosil C18



Inertsil ODS2

Purospher RP18-e


µBondapak C18

Prodigy ODS3

Partisil ODS3

Zorbax Rx-C18

Genesis C18

Kromasil C18

TSK ODS-120T

XTerra MS C18

Capcell Pak SG C18


YMC ODS AM

Nucleosil C18 HD

Luna C18(2)Zorbax Extend C18

YMC ODS A

Inertsil ODS


Inertsil ODS3

Exsil ODSB

Capcell Pak UG C18

Hichrom C18

Develosil ODS-HG

Zorbax XDB-C18

Hichrom RPB

Develosil ODS-MG

Synchropak CR101

YMC Pro C18

Develosil ODS-UG

Ace C18

0 10,000 20,000 30,000 40,000 50,000 60,000 70,000 80,000

N/Meter (Pyridine)

Resolve C18

LiChrospher RP-18

Hypersil ODS

Zorbax ODS


Exsil ODS

Exsil ODS1

Hypersil BDS C18


Ultrasphere ODS

Symmetry C18

Capcell Pak AG C18

Comparison of ColumnEfficiency for a Basic CompoundMeasuring column efficiency using aneutral compound is not very useful inpredicting column performance whenseparating ionic compounds. Interactionbetween polar solutes and silanol siteson the stationary phase can cause tailingpeaks and poor column efficiency. Togain a better understanding of columnperformance with basic compoundscolumns were tested using pyridine andamitriptyline as probes. Althoughcolumns are ranked somewhat different-ly on the two tests, phases at the higherend of the ranking scale can be expectedto give better peak shape and higher resolution for basic compounds thanphases at the lower end of the scale.Not surprisingly, stationary phases thatuse high purity silicas exhibit betterpeak shape and higher column efficiencythan stationary phases that use moreacidic silicas as their stationary phase supports.

Figure 10

Comparison of Column Efficiencyfor a Basic Compound: Pyridine


Sample: PyridineTemperature: 24°C

Ace

Hypersil HyPURITY

Symmetry

Figure 11

Comparison of Peak Shape


Sample: PyridineTemperature: 24°C

Ace C18 gave the best peak shape and highest columnefficiency for pyridine.



Capcell Pak UG C18

Hichrom C18

Develosil ODS-HG

Zorbax XDB-C18

Hichrom RPB

Develosil ODS-MG

Synchropak CR101

YMC Pro C18

Develosil ODS-UG

Ace C18

0 10,000 20,000 30,000 40,000 50,000 60,000 70,000

N/Meter (Pyridine)

Nucleosil C18 HD


Hichrom RPB

Prodigy ODS3

Luna C18(2)Develosil ODS-UG

YMC Pro C18

YMC J'Sphere ODS M80Hichrom C18

Ace C18

0 10,000 20,000 30,000 40,000 50,000 60,000 70,000

N/Meter (Amitriptyline)

Zorbax ODS

Resolve C18

Partisil ODS2

Hypersil ODS

Exsil ODSCosmosil C18-AR

Capcell Pak AG C18

Ultrasphere ODS

Zorbax Rx-C18Nucleosil C18

µBondapak C18

LiChrosorb RP-18Partisil ODS3

Partisil ODS


Capcell Pak SG C18

LiChrospher RP-18

Vydac 218TP

TSK ODS-120T


TSK ODS-80TM

Novapak C18

Exsil ODSBInertsil ODS

Inertsil ODS2

Zorbax SB-C18

Exsil ODS1

Vydac 218MS


Zorbax Extend C18

Synchropak CR101

YMC ODS AM

Zorbax XDB-C18

YMC ODS A

Nucleosil C18AB

Kromasil C18Purospher RP18-e

Genesis C18

Develosil ODS-HG

Symmetry C18Hypersil BDS C18

XTerra MS C18


Capcell Pak UG C18

Prodigy ODS2

Develosil ODS-MG

Inertsil ODS3

Nucleosil C18 HD

Hichrom RPB

Prodigy ODS3

Luna C18(2)

Develosil ODS-UG

YMC Pro C18


Hichrom C18

Ace C18

0 10,000 20,000 30,000 40,000 50,000 60,000 70,000

N/Meter (Amitriptyline)





Figure 12

Comparison of Column Efficiency for aBasic Compound: Amitriptyline

Plate count is measured at 10% of peakheight to include peak tailing in the calculation. Both tests use mobile phases at neutral pH to encourage interaction between the basic probesand silanols on the stationary phase.

Figure 13

Top 10 Columns Ranked According toPeak Shape and Efficiency

Mobile Phase: 80% CH3OH20% 0.025M KH2PO4, pH 6.0

Sample: AmitriptylineTemperature: 24°C

Sample: AmitriptylineMobile Phase: 80% CH3OH

20% 0.025M Phosphate, pH 6.0

Sample: PyridineMobile Phase: 60% CH3OH

40% H2O

Note: All columns are packedwith 5 micron size material.

Column ranking does differ in the two tests of columnefficiency for a basic compound (Figures 10 and 12).However, of the 15 columns that ranked in the top 10on at least one of the tests, 5 ranked in the top 10 onboth tests.



Phases Grouped According toSilanol ActivityAmitriptyline and pyridine are both goodtest probes to use for measuring silanolactivity of stationary phases. Even asmall amount of silanol exposure by thestationary phase can cause measurablepeak broadening on one or both of thesecompounds. Chromatographic testsusing these two probes are the primarymeasurements used to group these C18phases according to silanol activity. Ingeneral, phases identified as having “verylow” silanol activity will give the highestcolumn efficiency in the pyridine andamitriptyline tests (Figures 10 and 12).

Phases Compared According toMetal ActivityThe presence of metals on the surface ofstationary phases can have a significanteffect on chromatographic performance.Even trace levels of metal impurities

can contribute to peak tailing of somecompounds. In addition, subtle lot-to-lot variations in the amount of trace metalsare another cause of poor column reproducibility.

The National Institute of Standards &Technology (NIST) uses peak asymmetryof quinizarin, a strong metal chelatingagent, in their column performance testmixture SRM 870 to measure metal activi-ty of stationary phases. Their internetsite (http://ois.nist.gov/srmcatalog/certifi-cates/870.pdf) has data for 41 C18 station-ary phases evaluated with test mixtureSRM 870. Figure 15 was produced usingthe quinizarin asymmetry data obtainedfrom the NIST internet site. In thisFigure, the 20 columns with the lowestmetal activity are ranked according topeak asymmetry for quinizarin. Thecolumns with lower asymmetry valueshave lower metal activity.

Material Silanol Activity

Ace C18Develosil ODS-MGHichrom C18Hypersil HyPURITY C18Inertsil ODS3Luna C18(2)Nucleosil C18 HDXTerra MS C18YMC Pro C18

Capcell Pak UG C18Develosil ODS-HGDevelosil ODS-UGGenesis C1Hichrom RPBInertsil ODS2Kromasil C18Prodigy ODS2Prodigy ODS3Purospher RP18-eSymmetry C18YMC ODS AYMC ODS AMZorbax Extend C18Zorbax XDB-C18

Capcell Pak C18 SGCosmosil C18-ARExsil ODSBHypersil BDS C18Inertsil ODSNova-Pak C18Nucleosil C18ABPartisil ODS3Synchropak CR101TSK ODS-120TTSK ODS-80TMµBondapak C18Vydac 218MSVydac 218TPVydac Selectapore 300MVydac Selectapore 300PVydac Selectapore 90MWaters Spherisorb ODSBYMC J’Sphere ODS H80YMC J’Sphere ODS M80Zorbax Rx-C18Zorbax SB-C18

Capcell Pak C18 AGExsil ODSExsil ODS1Hypersil ODSLiChrosorb RP-18LiChrospher RP-18Nucleosil C18Partisil ODSPartisil ODS2Resolve C18Ultrasphere ODSWaters Spherisorb ODS1Waters Spherisorb ODS2Zorbax ODS

Very Low

Low

Moderate

High

Figure 14

Grouping of C18 Columns According toSilanol Activity

Partisil ODS2

µBondapak C18

Novapak C18

Nucleosil C18

OmniSpher C188


Luna C18(2)

Zorbax StableBond SB-C18

Discovery C18


Symmetry C18

Inertsil ODS2

Hypersil BDS C18

Inertsil ODS3

ProtoSIL C18SH

Cosmosil C18 AR-II

XTerra MS C18

Hypersil Elite C18

Zorbax Eclipse XDB-C18

Ace C18

1.0 1.5 2.0 2.5 3.0 3.5

Peak Asymmetry

HigherMetalActivity

LowerMetalActivity

Figure 15

Comparison of Peak Asymmetry for Quinizarin: A Measure of Metal Activity

MAC-MOD Analytical127 Commons CourtP.O. Box 2600Chadds Ford, PA 19317

Phone: 1-800-441-7508Fax: 1-610-358-5993E-mail: [email protected] Site: www.mac-mod.com

Mobile Phase: 80% CH3OH20% 5mM Potassium

Phosphate, pH 7.0Sample: QuinizarinFlow Rate: 2 mL/minTemperature: 23°C

analytical inc.


stationary phase specifications introduction 北京慧德易科技

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