Tanaka Test
Chromatographic characterization of column properties
Controlling the physical and chemical properties of stationary phases is important, but only a comprehensive chromatographic characterization ensures a consistently high level of reproducibility. To characterize selectivity, different approaches have been applied by leading HPLC scientists.
The Tanaka* test is established worldwide as industrial standard test which assesses selectivity and performance differences between HPLC columns. These column parameters should be known for effectively choosing the appropriate HPLC column for a particulate separation and allow comparing columns easily.
A set of seven selected substances is used to describe capacity, hydrophobicity, steric selectivity, and silanophilic properties. To facilitate the illustration and to recognize the quality of a sorbent at one glance, the values of these parameters are outlined on the six axes of a hexagon. The more symmetrical the hexagon appears and the larger its area, the more balanced the stationary phase is in the sum of its chromatographic properties.
(* Prof. Tanaka, Kyoto Institute of Technology, J. of Chrom. Sci. 27, 725, 1989)
Parameters according to Tanaka for the characterization of Purospher® HPLC sorbents
| Parameters | Property of stationary phase | Factors in preparation of the stationary phase |
|
|---|---|---|---|
| k (Pentylbenzene) 80% Methanol |
Retention capacity (A) |
Amount of alkyl chains | Silica surface Silica coverage |
| k (Pentylbenzene) / k (Butylbenzene) 80% Methanol |
Hydrophobicity (B) | Hydrophobic capacity (CH2-group selectivity) |
Surface coverage |
| k (Triphenylene) / k (o-Terphenyl) 80% Methanol |
Steric selectivity (C) | Steric (shape) selectivity | Silane functionality Surface coverage |
| k (Caffeine) / k (Phenol) 30% Methanol |
Silanol capacity (D) | Silanol capacity (content and type of silanols) | Residual silanols Endcapping Surface coverage |
| k (Benzylamine) / k (Phenol) 30% Methanol / 70% Phosphate buffer pH 7.6 |
Ion exchange capacity (E) |
Ion exchange capacity at pH 7 |
Residual silanols Active sites at pH 7 |
| k (Benzylamine) / k (Phenol) 30% Methanol / 70% Phosphate buffer pH 2.7 |
Ion exchange capacity (F) |
Ion exchange capacity at pH 3 |
Active sites at pH 3 Treatment of basic silica (metal impurities) |
| Tanaka 1 – Retention capacity, hydrophobicity, steric selectivity | ||
|---|---|---|
| The Tanaka 1 Test describes the retention capacity, the hydrophobicity, and the steric selectivity of RP-phases. |  |
|
| Column | LiChroCART® 150-4,6 Purospher® STAR RP-18 endcapped, 5μm |
|
| Mobile Phase | Methanol / Water 80:20 | |
| Flow rate | 1.0 ml/min | |
| Detection | UV 254 nm | |
| Temperature | 30° C | |
| Inj. Volume | 10 μl | |
| Sample | 1. Uracil 2. Butylbenzene 3. o-Terphenyl 4. Pentylbenzene 5. Triphenylene |
|
| Tanaka 2 – Silanol capacity | ||
|---|---|---|
| The Tanaka 2 Test (Chromatogram A) illustrates the silanophilic properties of stationary phases. Chromatogram B is not part of Tanaka 2 Test. The Phenol/Pyridine test measures silanol activities. |  |
|
| Column | LiChroCART® 125-4 Purospher® STAR RP-18 endcapped, 5 μm |
|
| Mobile Phase | A: Methanol / Water 30:70 (v/v) B: Acetonitrile / Water 30:70 (v/v) |
|
| Flow rate | 1.0 ml/min | |
| Detection | UV 254 nm | |
| Sample | A: Uracil (1), Caffeine (2), Phenol (3) B: Pyridine (1), Phenol (2) |
|
| Tanaka 3+4 – Ion exchange capacities | ||
|---|---|---|
| The result obtained from Tanaka 3 (at low pH) shows if the deactivation of the residual silanol groups is complete. Tanaka 4 test at high pH indicates the complete absence of metal in the base sorbent. |
 |
|
| Column | LiChroCART® 125-4 Purospher® STAR RP-18 endcapped, 5 μm |
|
| Mobile Phase | Methanol / 0.02 mol/L Phosphoric acid 30:70 (v/v) |
|
| Flow rate | 0.6 ml/min | |
| Detection | UV 254 nm | |
| Sample | 1. Uracil 2. Benzylamine 3. Phenol |
|
| Results from the Tanaka Test for Purospher® STAR RP-18 endcapped | |||
|---|---|---|---|
| In comparison to competitive “high purity” RP-18 endcapped columns, Purospher® STAR RP-18 endcapped shows the best over-all selectivity. It is the chromatographer’s first choice for a successful separation. |
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 |
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| A: | K (Pentylbenzene) | 9.59 | |
| B: | α (Pentyl-/ Butylbenzene) | 1.51 | |
| C: | α (Triphenylene/ o-Terphenyl) | 1.63 | |
| D: | α (Caffeine/ Phenol) | 0.44 | |
| E: | α (Benzylamine/ Phenol; pH 7.6) | 0.23 | |
| F: | α (Benzylamine/ Phenol; pH 2.7) | 0.02 | |
