N-(4-Aminobenzoyl)-L-Glutamic Acid CAS 4271-30-1 Purity ≥99.0% (HPLC) Folic Acid Intermediate Factory

Purity and Related substance by HPLC
Equipment:
Balance
HPLC
Reagents
Water HPLC grade
Disodium hydrogen orthophosphate
Potassium dihydrogen phosphate
ortho phosphoric acid
Methanol HPLC grade
Procedure
Preparation of buffer:
Transfer accurately weighed about 1.277gm Disodium hydrogen orthophosphate & 0.136gm Potassium dihydrogen phosphate to a500ml graduated cylinder and dissolve in 300ml of water. Dilute with water to 500ml Adjust pH 6.5 with diluted ortho phosphoric acid.
Preparation of Mobile phase:
Prepare a filtered and degassed mixture of phosphate buffer and Methanol (2:8)
Freshly Standard Preparation:
Weigh accurately about 25.0mg of the In-house working reference standard and transfer to a 25ml volumetric flask. Add 15.0ml of mobile phase to dissolve the substance and dilute with mobile phase to volume and mix. Transfer 1.0ml of above solution in to 10ml of volumetric flask and make up the volume with mobile phase and mix well.
Freshly Test Preparation:
Weigh accurately about 25.0mg of the test substance and transfer to a 25ml volumetric flask. Add 15.0ml of mobile phase to dissolve the substance and dilute with mobile phase to volume and mix. Transfer 1.0ml of above solution in to 10ml of volumetric flask and make up the volume with mobile phase and mix well.
Chromatographic System:
Column: C-18 Inertsil (ODS), 4.6mm X 25cm 5µ
Wavelength: 220nm
Flow Rate: 1.0ml/minute.
Injection Volume: 20µl each blank, standard and test preparation
Run Time: 20 minutes.
Procedures:
Inject equal volume of blank, standard and test preparation, Record the Chromatograph and measure the peak response for major peak and Unknown Peaks of test preparation.
1) Blank: Mobile Phase
2) Standard preparation : For Retention time of the in-house working reference standard
3) Test Preparation: For purity and related substance.
% Purity and impurities on chart paper basis