The Engelhardt Test
This 8-compound test, developed by Engelhardt et al.*, has become one of the standard procedures for evaluating the hydrophobicity and silanophilic activity of reversed-phase column packings. Toluene and ethylbenzene demonstrate the hydrophobic properties, phenol and ethylbenzoate the neutral polar interactions. The important behaviour towards basic compounds is evaluated by the injection of 5 different anilines. Aniline should be eluted before phenol with excellent peak shape and the geometric isomers of ethyl aniline should co-elute if there is a good suppression of the silanolic activity of the stationary phase.
* Engelhardt et al., Chromatographia 29, 59, 1990
| Purospher® RP-18 | |||
|---|---|---|---|
| Due to the amino endcapping of Purospher® RP-18 with polar interactions to the analytes, co-elution of the geometric isomers of (p-, m-, o-) ethyl aniline (4) could not be observed. Both the anilines (2, 5) elute as symmetrical peaks, thus indicating excellent suitability of Purospher® RP-18 for the separation of strong basic analytes. |
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| Column | LiChroCART 125-4 Purospher® RP-18, 5 µm |
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| Mobile Phase | Methanol/Water 55/45 (v/v) | ||
| Flow rate | 1.0 mL/min | ||
| Temperature | ambient | ||
| Detection | UV 254 nm | ||
| Sample | 1. Thiourea 2. Aniline 3. Phenol 4. p-, m-, o-ethyllaniline 5. N,N-Dimethylaniline 6. Ethylbenzoate 7. Toluene 8. Ethylbenzene |
to basic acidic basic basic neutral neutral neutral |
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| Purospher® RP-18 endcapped | |||
|---|---|---|---|
| As there are no polar interactions on the surface, Purospher® RP-18 endcapped shows perfect co-elution of p-, m- and o-ethyl anilines (4). The anilines (2, 5) both elute with a symmetrical peak shape which demonstrates the excellent suitability for the separation of strong basic analytes. The high retention time of ethyl benzene (8) indicates the hydrophobicity of the stationary phase. |  |
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| Column | LiChroCART 125-4 Purospher® RP-18 endcapped, 5 µm |
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| Mobile Phase | Methanol/Water 55/45 (v/v) | ||
| Flow rate | 1.0 mL/min | ||
| Temperature | ambient | ||
| Detection | UV 254 nm | ||
| Sample | 1. Thiourea 2. Aniline 3. Phenol 4. p-, m-, o-ethyllaniline 5. N,N-Dimethylaniline 6. Ethylbenzoate 7. Toluene 8. Ethylbenzene |
to basic acidic basic basic neutral neutral neutral |
|
| Purospher® STAR RP-18 endcapped | |||
|---|---|---|---|
| Purospher® STAR RP-18 endcapped shows perfect co-elution of p-, m- and o-ethylanilines (4), indicating no polar interactions due to silanol activity on the surface. Both the anilines (2, 5) elute symmetrically showing the excellent suitability for separation of strong basic compounds. Purospher® STAR RP-18 endcapped exhibits a similar selectivity as Purospher® RP-18 endcapped, but has an extended pH range. |  |
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| Column | Purospher® STAR RP-18 endcapped, 5 µm | ||
| Mobile Phase | Methanol/Water 55/45 (v/v) | ||
| Flow rate | 1.0 mL/min | ||
| Temperature | RT | ||
| Detection | UV 254 nm | ||
| Sample | 1. Thiourea 2. Aniline 3. Phenol 4. p-, m-, o-ethyllaniline 5. N,N-Dimethylaniline 6. Ethylbenzoate 7. Toluene 8. Ethylbenzene |
to basic acidic basic basic neutral neutral neutral |
|
| Purospher® RP-18 HC | |||
|---|---|---|---|
| As there is no endcapping of the bonded phase, Purospher® RP-18 HC shows very clear polar interactions with the residual silanols on the surface. Aniline and phenol (2,3) elute as one peak whereas basic compounds show increased retention but loss in performance. Therefore, this column is well suitable for the separation of polar, non-basic analytes, such as e.g. explosives. |  |
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| Column | LiChroCART 125-4 Purospher® RP-18 HC, 5 µm |
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| Mobile Phase | Methanol/Water 55/45 (v/v) | ||
| Flow rate | 1.0 mL/min | ||
| Temperature | RT | ||
| Detection | UV 254 nm | ||
| Sample | 1. Thiourea 2. Aniline 3. Phenol 4. p-, m-, o-ethyllaniline 5. N,N-Dimethylaniline 6. Ethylbenzoate 7. Toluene 8. Ethylbenzene |
to basic acidic basic basic neutral neutral neutral |
|
| Purospher® STAR RP-8 endcapped | |||
|---|---|---|---|
| As there are polar interactions due to the thin layer of hydrophobic coverage with short C8-chains, Purospher® STAR RP-8 endcapped shows no co-elution of p-, m- and o-ethyl anilines (4).The anilines (2, 5) elute as symmetrical peaks demonstrating the very good suitability for separation of strong basic compounds. Both ethylbenzoate (6) and toluene (7) elute in one peak - this is typical for C-8 phases. |
 |
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| Column | LiChroCART 125-4 Purospher® STAR RP-8 endcapped, 5 µm |
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| Mobile Phase | Methanol/Water 55/45 (v/v) | ||
| Flow rate | 1.0 mL/min | ||
| Temperature | RT | ||
| Detection | UV 254 nm | ||
| Sample | 1. Thiourea 2. Aniline 3. Phenol 4. p-, m-, o-ethyllaniline 5. N,N-Dimethylaniline 6. Ethylbenzoate 7. Toluene 8. Ethylbenzene |
to basic acidic basic basic neutral neutral neutral |
|
| Chromatographic properties of Purospher® stationary phases | ||||||
|---|---|---|---|---|---|---|
| Peak symmetry of complexing agents | Polar interactions | Steric selectivity | CH2-group selectivity | Silanol group activity | Hydrophobicity | |
| Purospher® RP-18 | +++ | ++ | +++ | +++ | ++ | + |
| Purospher® RP-18 endcapped | +++ | - | +++ | +++ | - | +++ |
| Purospher® STAR RP-18 endcapped | +++ | - | +++ | +++ | - | +++ |
| Purospher® STAR RP-8 endcapped | +++ | + | + | ++ | ++ | + |
| Purospher® RP-18 HC |
+++ | ++ | +++ | +++ | + | ++ |
