Classification test for reversed-phase HPLC columns
Many scientists have sought to characterise and classify liquid chromatography stationary phases in order to provide analysts with the main principles of their use.
A number of our HPLC stationary phases were developed through the study of these tests and the collaboration established with LETIAM (IUT Orsay), the laboratory of the analytical chemistry group of the University of Paris-Sud 11.
1. Lesellier & Tchapla test - subcritical chromatographic test of carotenoid isomers
This test compares the main properties of bonded silica particles, primarily C18 silica particles. It makes it possible to discriminate more than a dozen stationary phase groups with very different properties. Approx. 200 columns have been evaluated to date.
List of the HPLC columns evaluated
| Ace | ACE 5 C18 | Higgins | Targa C18 | Shiseido | Capcell Pak C18 | ||||
| Ace | ACE C18 AQ | Interchim | Strategy Pro | SMT | SMT C18 | ||||
| Ace | ACE C18 HL | Interchim | Uptisphere BioP I | Supelco | Acclaim Polar Advantage II | ||||
| Agilent Technologies | Zorbax 300 SB C18 | Interchim | Uptisphere BioP II | Supelco | Ascentis C18 | ||||
| Agilent Technologies | Zorbax Bonus-RP | Interchim | Uptisphere HDO | Supelco | Ascentis Express C18 - 2,7µm | ||||
| Agilent Technologies | Zorbax Eclipse Plus | Interchim | Uptisphere HSC | Supelco | Ascentis RP Amide | ||||
| Agilent Technologies | Zorbax Eclipse Plus C18 | Interchim | Uptisphere NEC | Supelco | Discovery C18 | ||||
| Agilent Technologies | Zorbax Eclipse Plus PAH | Interchim | Uptisphere ODB | Supelco | Discovery HS C18 | ||||
| Agilent Technologies | Zorbax Eclipse Plus Phenyl hexyl | Interchim | Uptisphere Phenyl | Supelco | Discovery RP amide C16 | ||||
| Agilent Technologies | Zorbax Eclipse XDB C18 | Interchim | Uptisphere PLP | Supelco | Supelcosil ABZ | ||||
| Agilent Technologies | Zorbax Extend C18 | Interchim | Uptisphere Strategy C18-2 | Supelco | Supelcosil ABZ + |
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| Agilent Technologies | Zorbax ODS | Interchim | Uptisphere Strategy C18-2 - 2,2µm | Supelco | Supelcosil LC 18-DB | ||||
| Agilent Technologies | Zorbax RX C18 | Interchim | Uptisphere Strategy C18-3 | Supelco | Supelcosil LC-18 | ||||
| Agilent Technologies | Zorbax SB C18 | Interchim | Uptisphere Strategy RP | Supelco | Supelcosil LC-18S | ||||
| Agilent-Varian | Pursuit Diphenyl | Interchim | Uptisphere TF | Supelco | Supelcosil LC-18T | ||||
| Agilent-Varian | Pursuit XRs C18 | Interchim | Uptisphere WOD | Supelco | Suplex PKB | ||||
| AMT | Halo C18 - 2,7µm | Interchim | Uptisphere WRP | Tessek | Separon C18 | ||||
| Baker | Baker C18-NP | Macherey Nagel | Nautilus C18 | Tessek | Separon C18 ec |
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| Baker | Baker C18-WP | Macherey Nagel | Nucleodur 100 C18ec | Thermo separation | Aquasil C18 | ||||
| Beckmann | Ultrasphere ODS | Macherey Nagel | Nucleodur Gravity C18 | Thermo separation | Betabasic C18 | ||||
| Beckmann | Ultrasphere XL ODS | Macherey Nagel | Nucleodur Isis | Thermo separation | Hypersil 100 C18 | ||||
| Biotage | Unisphere C18 | Macherey Nagel | Nucleodur Pyramid | Thermo separation | Hypersil BDS C18 | ||||
| Cluzeau | Satisfaction RP 18-AB | Macherey Nagel | Nucleodur Sphinx | Thermo separation | Hypersil Elite C18 | ||||
| Cluzeau | Stability BS C23e | Macherey Nagel | Nucleosil 100 C18 | Thermo separation | Hypersil Gold | ||||
| Cluzeau | Stability BS C23ne | Macherey Nagel | Nucleosil 100 C18-HD | Thermo separation | Hypersil Green-PAH | ||||
| Cluzeau | Stability ODS2 | Macherey Nagel | Nucleosil 100 C18-PAH | Thermo separation | Hypersil ODS | ||||
| Cogent | C18 bidentate | Macherey Nagel | Nucleosil 300 C18 | Thermo separation | Hypersil PAH | ||||
| Colochrom | Colosphere C18 | Macherey Nagel | Nucleosil 50 C18 | Thermo separation | HyPurity Aquastar | ||||
| Colochrom | Excelsphere 120 C18-H | Macherey Nagel | Nucleosil 50 C18ec | Thermo separation | HyPurity C18 | ||||
| Colochrom | Exelsphere 120 ODS2 | Macherey Nagel | Nucleosil C18-AB | Thermo separation | HyPurity C8 Advance | ||||
| Colochrom | Normasphere ODS2 | Macherey Nagel | Nucléosil Nautilus C18 | Tosoh Biosciences | TSK OD 80-TM | ||||
| Dionex | Acclaim C18 | Macherey Nagel | Nucleosil Protect C8 | Tosoh Biosciences | TSK ODS-120A | ||||
| Dionex | Acclaim Polar Advantage | Merck | Chromolith C18 | Tosoh Biosciences | TSK ODS-120T | ||||
| Eichrom | Synchropak C18 | Merck | Lichrosorb RP18 | Tosoh Biosciences | TSK ODS-80TS | ||||
| Eka Nobel | Kromasil C18 | Merck | Lichrospher 100 RP18 | Varian | Omnisphere C18 | ||||
| ES Industries | Chromegabond C22 | Merck | Lichrospher 100 RP18e | Varian | Polaris A C18 | ||||
| ES Industries | Gamma-bond C18 | Merck | Lichrospher PAH | Varian | Polaris amide C18 | ||||
| GL sciences | Inertsil ODS-2 | Merck | Purospher 100 RP18 | Varian | Polaris B C18 | ||||
| GL sciences | Inertsil ODS-3 | Merck | Purospher 100 RP18e | Varian | Polaris ether C18 | ||||
| Grace - Alltech | Adsorbosil C18 | Merck | Purospher star RP18e | Varian | Pursuit C18 | ||||
| Grace - Alltech | Adsorbosphere HS C18 | Merck | Superspher 100 RP18 | Varian | ResElut C18 | ||||
| Grace - Alltech | Adsorbosphere XL C18 | Merck | Superspher 100 RP18e | Waters | Atlantis dcC18 | ||||
| Grace - Alltech | Alltima C18 | Nacalai Tesque | Cosmosil C18-AR II | Waters | Delta-Pak C18 | ||||
| Grace - Alltech | Alltima HP C18 | Nacalai Tesque | Cosmosil C18-MS II | Waters | microBondapak C18 | ||||
| Grace - Alltech | Alltima HP C18 amide | Nacalai Tesque | Cosmosil C18-PAQ | Waters | Nova-Pak C18 | ||||
| Grace - Alltech | Alltima HP C18 HL | Nacalai Tesque | Cosmosil Cholester | Waters | Resolve C18 | ||||
| Grace - Alltech | Alphabond C18 | Nacalai Tesque | Cosmosil Pi naphtyl | Waters | Spherisorb ODB | ||||
| Grace - Alltech | Brava BDS C18 | Nomura | Develosil C18 | Waters | Spherisorb ODBS1 | ||||
| Grace - Alltech | Econosil C18 | Perkin Elmer | PE CR C18 | Waters | Spherisorb ODBS2 | ||||
| Grace - Alltech | Econosphere C18 | Perkin Elmer | Spheri-5 ODS | Waters | Symmetry C18 | ||||
| Grace - Alltech | Platinium EPS C18 | Phenomenex | Gemini C18 | Waters | Symmetry Shield RP18 | ||||
| Grace - Alltech | Platinum C18 | Phenomenex | Gemini NX | Waters | X Bridge | ||||
| Grace - Alltech | Prevail amide C18 | Phenomenex | Gemini Phenyl hexyl | Waters | XTerra MS C18 | ||||
| Grace - Alltech | Prevail C18 | Phenomenex | Luna C18 (1) | Waters | Xterra RP 18 | ||||
| Grace - Alltech | Prosphere 300 C18 | Phenomenex | Luna C18-2 | Whatman | Partisil ODS1 | ||||
| Grace - Jones | Apex C18 | Phenomenex | Luna Phenyl hexyl | Whatman | Partisil ODS2 | ||||
| Grace - Jones | Genesis C18 | Phenomenex | Prodigy ODS 2 | Whatman | Partisil ODS3 | ||||
| Grace - Vydac | Vydac 201HS | Phenomenex | Prodigy ODS 3 |
YMC | Hydrosphere C18 | ||||
| Grace - Vydac | Vydac 201TP | Phenomenex | Synergy Fusion RP | YMC | J´Sphere 80H | ||||
| Grace - Vydac | Vydac 202TP | Phenomenex | Synergy Hydro RP | YMC | J´Sphere 80L | ||||
| Grace - Vydac | Vydac 218MR | Restek | Allure C18 | YMC | J´Sphere 80M | ||||
| Grace - Vydac | Vydac 218TP | Restek | Ultra C18 | YMC | ODS A 120 A | ||||
| Grace - Vydac | Vydac 238TP | SFCC | Bondasorb C18 | YMC | ProC18 RS | ||||
| Higgins | Clipeus C18 | SGE | Exsil ODS | YMC | YMC Pack ODS-AQ | ||||
| Higgins | HAIsil C18 | SGE | SGE-250 GL4 P-C18 | YMC | YMC Pack ProC18 | ||||
| Higgins | HAIsil C18-HL | SGE | Wakosil C18-RS |
Results diagram
In the range of values between 20 and 10 along the Y-axis, the accessibility of the stationary phases to the polar sites is very low. This accessibility is average between 10 and 5, and rises to very high in the range of less than 5.
References
- E. Lesellier, A. Tchapla, J. Chromatogr. A 1100 (2005) 45
- E. Lesellier, C. West, A. Tchapla, J. Chromatogr. A 1111 (2006) 62
- E. Lesellier, C. West, J. Chromatogr. A 1149 (2007) 345
- C. West, L. Fougère, E. Lesellier, J. Chromatogr. A 1189 (2008) 227
- E. Lesellier, J. Sep. Sci. 33 (2010) 3097
A selection of 45 HPLC columns representative of those available on the market were compared using the following tests.
| Column No. | Manufacturer | Column name | Code |
| 6 | Agilent Technologies | Zorbax RX C18 | ZRx |
| 11 | Agilent Technologies | Zorbax Eclipse XDB | ZXDB |
| 35 | Agilent Technologies | Extend C18 | ZExt |
| 63 | Agilent Technologies | Zorbax Bonus RP | ZBon |
| 31 | Dionex | Acclaim Mixed-Mode Hilic1 | DHil |
| 32 | Dionex | Acclaim Polar Advantage | DAPA |
| 10 | EKA Nobel | Kromasil C18 | K18 |
| 12 | GL Sciences | Inertsil ODS 3 | GLIn |
| 14 | Interchim | Uptisphere HSC | UHSC |
| 15 | Interchim | Uptisphere TF | UTF |
| 16 | Interchim | Uptisphere NEC | UNEC |
| 17 | Interchim | Uptisphere HDO | UHDO |
| 18 | Interchim | Uptisphere ODB | UODB |
| 19 | Interchim | Uptisphere Strategy C18(2) | US2 |
| 20 | Interchim | Uptisphere Strategy RP | URP |
| 21 | Interchim | Uptisphere Strategy (C18(3)) | US3 |
| 22 | Interchim | Uptisphere PLP | UPLP |
| 23 | Interchim | Uptisphere PRO | U38 |
| 93 | Interchim | Uptisphere MM1 | UMM1 |
| 40 | Macherey Nagel | Nucleodur C18 Gravity | MNGr |
| 41 | Macherey Nagel | Nucleodur C18 Isis | MNIs |
| 42 | Macherey Nagel | Nucleodur C18 Pyramid | MNPy |
| 64 | Macherey Nagel | Nucleodur C18 Sphinx | MNSp |
| 4 | Phenomenex | Luna C18(2) | PL |
| 9 | Phenomenex | Synergi MaxRP 4µm | Pmax |
| 37 | Phenomenex | Gemini | PGem |
| 39 | Phenomenex | Luna Phenyl Hexyl | PLPH |
| 58 | Phenomenex | Synergy Fusion RP | PFu |
| 59 | Phenomenex | Synergy Hydro RP | PHy |
| 34 | SAF | Discovery RP Amid C16 | SA16 |
| 55 | SAF | Supelco ABZ+ | SBZ+ |
| 28 | Shiseido | Capcell PAK C18 UG | ShUG |
| 45 | Sielc | Primesep 100 | SIP1 |
| 70 | Thermo Scientific | Hypersil Gold | HG |
| 94 | Thermo Scientific | Hypersil Gold AQ | HGAQ |
| 76 | Tosoh Bioscience | TSK ODS 120T | T1T |
| 77 | Tosoh Bioscience | TSK ODS 120A | T1A |
| 7 | Waters | Symmetry C18 | Wsy |
| 13 | Waters | XTERRA MS | WXMS |
| 71 | Waters | Xbridge Shield | WXBS |
| 72 | Waters | Xbridge C18 | WXBO |
| 80 | Waters | XTERRA C18 | WXTO |
| 92 | Waters | XTERRA Phenyl | WXPh |
| 3 | YMC | YMC PRO C18 | YP18 |
| 5 | YMC | YMC Pack ODS-AQ | YAQ |
2. Engelhardt Test
Butylparaben/Dipropylphthalate selectivity (α) is an indicator of polar selectivity of stationary phases.
3. Sander and Wise Test
Benzo-α-pyrene/Tetrabenzonaphthalene selectivity (α) is an indicator of steric resistance and shape recognition.
4. Tanaka Test - Methylene Selectivity
Amylbenzene/Butylbenzene selectivity (α) is an indicator of stationary phase hydrophobicity.
5. Tanaka Test - Steric selectivity
Triphenylene/o-Terphenyl selectivity (α) is an indicator of steric resistance and shape recognition.
6. Tanaka Test - Neutral
Caffeine/Phenol selectivity (α) is an indicator of polar selectivity – analysis conducted in a neutral medium.
7. Tanaka Test - Acidic
Benzylamine/Phenol selectivity (α) is an indicator of polar selectivity – analysis conducted in an acidic medium.
8. Tanaka Test - Basic
Benzylamine/Phenol selectivity (α) is an indicator of polar selectivity – analysis conducted in a basic medium.
9. LCAP Geneva Test
Since 1993, the Pharmaceutical Chemical Analysis Laboratory (LCAP) at the University of Geneva has been developing and providing chromatographic tests for classifying columns for basic compound separation.
Developed using a combination of seven basic compounds with different yet complementary properties, these tests make it possible to quickly determine stationary phase behaviour under real conditions (hydro-organic mixtures buffered to pH 3 and 7).
Chemometric tools such as principal component analysis (PCA) and ascending hierarchical classification (HAC) were used in order to provide a better picture of the performance of the columns and be able to rank them in order of similarity.
The results obtained are used to rank the columns by their hydrophobic retention capacity and their residual interaction with basic compounds.
For more information, please visit the LCAP´s website.