SI No.

CONTENTS

1 Scope

2 Principle

3 Apparatus and Materials

4 Chemicals and Solvents

5 Sample Preparation

6 HPLC Conditions

7 Reference
1. Scope: The method describes a procedure for the quantitative determination of butyric acid content in sample
containing butyric acid as active component.

2. Principle: The sample is separated on HPLC and detected using an UV detector.

3. Apparatus

1. Standard Flasks.
2. Filtration apparatus for HPLC injection

4. Chemicals and Solvents

1. HPLC grade water, Acetonitrile

2. Butyric acid Standard
3. Sodium phosphate monobasic

5. Sample Preparation
Weigh accurately 100 mg of sample and make up the volume to 50 ml using water the filtered for injection.

5.1 Standard preparation

A 1000-ppm working standard solution was prepared by adding 50 µL of acid standard to a 50-mL volumetric cylinder
and filling to volume with diluent. Six calibration levels were prepared via serial dilution of the working standard
solution. Each standard level was injected in duplicate.

6. HPLC condition:

Column Aqueous C18 5-µm, 250 x 4.6-mm

Detector UV detection at 210 nm
Diluents Water
Mobile Phase Mobile Phase A: 10-mM KH2PO4, pH 2.4 with phosphoric acid
Mobile Phase B: Acetonitrile (ACN)
Time in Flow A% B %
min rate
1 Initial 1.5 80 20
2 10.0 1.5 40 60
3 12.5 1.5 40 60
4 12.6 1.5 80 20
Flow Rate and column temperature 1.5 mL/min, 30 ºC
Run Time 12.5 minutes.

7. Reference:
The Analysis of a Broad Range of Organic Acids by HPLC with UV Detection, Author: Wilhad M. Reuter
PerkinElmer, Inc. Shelton, CT.

https://www.perkinelmer.com/lab-solutions/resources/docs/APP_The-Analysis-of-a-Broad-Range-of-Organic-
Acids-by-HPLC-with-UV-Detection-012379_01.pdf.