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 +
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
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.


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
71 Waters Xbridge Shield WXBS
72 Waters Xbridge C18 WXBO
80 Waters XTERRA C18 WXTO
92 Waters XTERRA Phenyl WXPh

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.