Tanaka Test for the Characterization of C18 Stationary Phase in HPLC Columns

Section Overview:

• Introduction
• Tanaka Tests for Purospher^® STAR RP-18 endcapped
     - Tanaka Test 1: Retention Capacity, Hydrophobicity, Steric Selectivity
     - Tanaka Test 2: Silanol Capacity
     - Tanaka Test 3 and 4: Ion Exchange Capacities
• Results
• Conclusion

Introduction

The properties of stationary phases, both physical and chemical, play a crucial role in the performance of HPLC. However, achieving reproducibility requires comprehensive chromatographic characterization. Consequently, prominent HPLC scientists have employed a range of approaches to assess selectivity. The Tanaka Test is widely recognized as the industry standard for evaluating column selectivity and performance.

The test facilitates the comparison of HPLC columns and assists in choosing the most suitable column for specific separations by assessing essential parameters. Seven substances are utilized to assess capacity, hydrophobicity, steric selectivity, and silanophilic characteristics. The parameters are displayed on a hexagonal grid, offering a visual depiction of the sorbent’s characteristics. A hexagon that exhibits greater symmetry and a larger area indicates a more balanced stationary phase in terms of its chromatographic properties.

Table 1.Tanaka test parameters for the characterization of revered-phase HPLC columns

Parameter/Property			Property of stationary phase	Factors in preparation of the stationary phase
A	k (Pentylbenzene) 80% Methanol	Retention capacity	Amount of alkyl chains	Silica surface Silica coverage
B	k (Pentylbenzene) / k (Butylbenzene) 80% Methanol	Hydrophobicity	Hydrophobic capacity (CH2-group selectivity)	Surface coverage
C	k (Triphenylene) / k (o-Terphenyl) 80% Methanol	Steric selectivity	Steric (shape) selectivity	Silane functionality Surface coverage
D	k (Caffeine) / k (Phenol) 30% Methanol	Silanol capacity	Silanol capacity (content and type of silanols)	Residual silanols Endcapping Surface coverage
E	k (Benzylamine) / k (Phenol) 30% Methanol / 70% Phosphate buffer pH 7.6	Ion exchange capacity	Ion exchange capacity at pH 7	Residual silanols Active sites at pH 7
F	k (Benzylamine) / k (Phenol) 30% Methanol / 70% Phosphate buffer pH 2.7	Ion exchange capacity	Ion exchange capacity at pH 3	Active sites at pH 3 Treatment of basic silica (metal impurities

Tanaka Tests for Purospher^® STAR RP-18 endcapped

Tanaka Test 1: Retention Capacity, Hydrophobicity, Steric Selectivity

The Tanaka 1 Test describes the retention capacity, the hydrophobicity, and the steric selectivity of RP-phases.

Table 2.HPLC conditions for the Tanaka test 1

HPLC Parameters
Column	Purospher® STAR RP-18 endcapped (5μm) LiChroCART® 150-4,6 HPLC column
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 Test 2: Silanol Capacity

The Tanaka 2 Test illustrates the silanophilic properties of stationary phases. The Phenol/Pyridine test measures silanol activities.

Table 3.HPLC conditions for the Tanaka test 2

HPLC Parameters
Column	Purospher® STAR RP-18 endcapped (5μm) LiChroCART® 125-4 HPLC column
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 Test 3 and 4: Ion Exchange Capacities

The results obtained from the Tanaka 3 Test (at low pH) demonstrate whether the deactivation of the residual silanol groups is complete. The Tanaka 4 Test (at high pH) indicates the complete absence of metal in the base sorbent.

Table 4.HPLC conditions for the Tanaka test 3 and 4

HPLC Parameters
Column	Purospher® STAR RP-18 endcapped (5μm) LiChroCART® 125-4 HPLC column
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

In order to facilitate a clear comparison of the chromatographic properties of various phases, a hexagonal net diagram was implemented to visually depict the measured Tanaka parameters. To standardize the display, the measured values are multiplied by certain factors for this diagram. Table 5 contains the measured, un-modified values and the Figure 1 shows the Tanaka plots for the tested stationary phases, demonstrating their relative performance across the selected parameters.

The Purospher^® STAR RP-18 endcapped column demonstrated superior selectivity across all critical parameters in a comparative analysis with other "high-purity" RP-18 endcapped columns, as illustrated in Figure 1. The Tanaka Test results showed that this column offers a balanced performance in terms of hydrophobicity, retention, steric selectivity, and silanol activity, making it a column of choice for a wide range of HPLC separations.

Figure 1. Six axes radar Tanaka plots for Purospher^® STAR RP-18 endcapped column and seven competitor columns.

Table 5.Measured Tanaka values without multipliers for Purospher^® STAR RP-18 endcapped column

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

Conclusion

The Tanaka Test is an indispensable tool for chromatographic characterization, as it enables a comprehensive assessment of HPLC columns. The Purospher^® STAR RP-18 endcapped column is a robust option for high-performance, reliable separations, with its balanced chromatographic properties confirmed through rigorous testing. By understanding the significance of each parameter assessed in the Tanaka Test, it is possible to make well-informed decisions regarding column selection for particular analytical requirements.

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