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APPLICATION NOTE

AN032208-52
 

STYROS™ Amino HILIC Simulated Monolith™  Polymeric: Separation of Benzoic Acid and Derivatives

 

HILIC or Hydrophilic Interaction Chromatography is a variation of normal phase chromatography with the major difference of providing the possibility of using solvents that are not miscible with water.

It also provides complementary selectivity compared to reversed phase chromatography.

The following chromatogram shows the separation of benzoic acid and its derivatives on a STYROS™ Amino-HILIC Simulated Monolith™  column at 60˚ C.

 

Chromatogram 1

Separation of Benzoic Acid and its derivatives on STYROS™ Amino HILIC

 

HPLC System.

Agilent 1100 with thermostatted column compartment.

Columns

STYROS™ Amino HILIC 4.6 X 100 mm (V=1.66 ml)

Mobile phase.

A: ACN
B: 200 mM HCOONH4, pH=9.6

Flow rate

1 ml/min (360 cm/hr of linear flow rate )

Gradient

Isocratic: 75 % A, 25 % B

Temperature

60°C

Detection

230 nm

Injection volume

5 ul

Sample:

1- Acetylsalicylic acid, 2- Benzoic acid, 3- Salicylic acid,
4- 4-Hydroxybenzoic acid, 5- 2,4-Dihydroxybenzoic acid,
6- 3,5-Dihydroxybenzoic acid (170 ug/ml each) in ACN:H2O 50:50.

Ammonium formate is used in the present case at a concentration of 200 mM. The actual concentration is 50 mM when used with 75 % of ACN.

Phosphates should be avoided due to their low solubility in organic solvents. Ion pairing agents such as TFA should be avoided as well.

No tailing is observed at the present concentration even with the most retained eluent at 8.45 min.

The column bed remains stable at 60˚ C and the pressure drops as a result of lower viscosity.

 

Chromatogram 2

Loadability Study on STYROS™ Amino HILIC
 

Table 1. Operating parameters.

HPLC System.

Agilent 1100 with thermostatted column compartment.

Columns

STYROS™ Amino HILIC 4.6 X 100 mm (V=1.66 ml)

Mobile phase.

A: ACN
B: 200 mM HCOONH4, pH=9.6

Flow rate

1 ml/min (360 cm/hr of linear flow rate )

Gradient

Isocratic: 75 % A, 25 % B

Temperature

60°C

Detection

230 nm

Injection volume

5 to 100 ul

Sample:

1- Acetylsalicylic acid, 2- Benzoic acid, 3- Salicylic acid,
4- 4-Hydroxybenzoic acid, 5- 2,4-Dihydroxybenzoic acid,
6- 3,5-Dihydroxybenzoic acid (170 ug/ml each) in ACN:H2O 50:50.

All the components of the mixture remain baseline separated. There is no indication of column saturation seen at the void volume.

 

STYROS™ Amino-HILIC Simulated Monolith™  is stable in the full pH range and high temperature.

 

Unlike Monolith, STYROS™ Simulated Monolith™ ™ columns are available in many sizes for additional resolving capabilities.

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