CHEMICAL SYNTHESIS OF HYDROXYAPATITE FOR

APPLICATION IN CHROMATOGRAPHY

BY

Jagjit Singh Kahlon Sec- F77E1 Roll No. 49

Faculty Adviser:Anjuvan Singh

15097Biotechnology

LSSPDepartment of Biotechnology

Lovely Institute of Engineering and Computer Applications (LIECA)LPU, Phagwara, India

CONTENTS

INTRODUCTIONOBJECTIVEREVIEW LITERATURE RESEARCH METHODOLOGYRESULT

INTRODUCTION

Hydroxyapatite is a Calcium phosphate ceramic that is used as a

biomaterial.

Hydroxyapatite [Ca10(PO4)6(OH)2] is a newly introduced bio-ceramic

with composite similar to bone and teeth.

Inorganic phase of our bones is apatite.

It is chemically similar to mineral compound of bones

and tissues in mammals.

What type of material is Hydroxyapatite?

Exhibiting an excellent biocompatibility

Bone and teeth 50 wt.% mineral

(mainly Hydroxyapatite) 50 wt.% organic molecules + water

(collagen, mucopolysaccharides) and water

OBJECTIVE

To synthesized Hydroxyapatite powder by Wet chemical

precipitation method

Study of chromatographic behaviour of hydroxyapatite.

Literature Review

Protein chromatography on hydroxyapatite columns.Cummings LJ, Snyder MA, Brisack K.Bio-Rad Laboratories, Inc., Hercules, California, USA.

AbstractThe introduction of spherical forms of hydroxyapatite has enabled protein scientists to separate and purify proteins multiple times with the same packed column. Biopharmaceutical companies have driven single column applications of complex samples to simpler samples obtained from upstream column purification steps on affinity, ion exchange or hydrophobic interaction columns. Multiple column purification permits higher protein loads to spherical forms of hydroxyapatite and improved reduction in host cell protein, aggregates, endotoxin, and DNA from recombinant proteins. The chemical interactions of hydroxyapatite surface with common ions, metals, and phosphate species affect column lifetimes. Column packing methods for the robust spherical particles as well as the microcrystalline hydroxyapatite particles are reviewed. Applications covering extracted proteins and recombinant protein purification, especially monoclonal antibodies, with multiple chromatography media in concert with hydroxyapatite are reviewed.

Literature Review

Purification of lactoferrin using hydroxyapatite.Ng PK, Yoshitake T.Process Applications R&D, Process Chromatography Division, Bio-Rad Laboratories, Hercules, CA 94547, USA. paul ng@bio-rad.com

AbstractLactoferrin is an important nutriceutical with various physiological functions. It is present in whey at very low concentrations. This work describes a mixed-mode (hydroxyapatite) chromatography method for one-column fractionation of lactoferrin from whey. Lactoperoxidase, a protein with similar molecular weight and isoelectric point, was initially desorbed from the matrix under isocratic conditions. Lactoferrin was obtained in homogeneity without lactoperoxidase activity and free from other major whey proteins such as alpha lactoalbumin and beta lactoglobulin.

Literature Review

It tells how the slope of gradient and column of the length can affect the separation process.

It discusses about the similarity between HAP chromatography , ion-exchange chromatography and affinity chromatography.

Reaction of HAP Synthesis

• 10Ca(NO3)2.4H2O + 6(NH4)2HPO4 + 8NH4OH

=> Ca10(PO4)6(OH)2 + 20NH4NO3 + 46H2O

Research Methodology

In the Titration process

In Burette Ammonium Solution

300 ml Calcium Nitrate 12

gms Water 260 ml

Then after placing in at 4oC & powdered form of HAP.

In Beaker Ammonium Solution

300 ml Water 400 ml (NH4)2HPO4 : 120 gms

incubation overnight we got a

Finally we get HAP in powdered form To get smoother powder Pestle motor

and fine sieve were used.

FTIR Spectroscopy

Fourier transform infrared spectroscopy (FTIR) is a technique which is used to obtain an infrared spectrum of absorption of a solid, liquid or gas.

FTIR of powder is done to analyse the compatibility of our product which comfirmed it valuable.

Result Reference

Pallet Formation

Now this powder was converted into pellets (each of 0.8 gms) with help of Hydraulic Press under 1.5 tons pressure for 1 minute.

Calcination

It is a thermal treatment process applied in order to bring about a thermal decomposition, phase transition, or removal of a volatile fraction.

The calcination process normally takes place at temperatures below the melting point of the product materials.

Chromatographic Coloumn

HAP pellets were added to the syringe making a chromatographic column

Known amount of BSA was loaded to the column for separation

Chromatographic Action

"mixed-mode" ion exchange or “pseudo affinity” chromatography

It involves nonspecific interactions between positively charged calcium ions and negatively charged phosphate ions with proteins

Useful for chromatographic separation of biopolymers like proteins, nucleic acids etc

Results

Synthesis of HAP through wet method

Confirmation of HAP through FTIR

Formation of pellets using Hydraulic press

Column preparation for separation of proteins using HAP as matrix

Proteins separated according to their affinity towards matrix(HAP)

THANK YOU