The document summarizes the chemical synthesis of hydroxyapatite (HAP) for use in chromatography. HAP was synthesized through a wet chemical precipitation method involving calcium nitrate, ammonium phosphate, and ammonium hydroxide solutions. The resulting HAP powder was characterized using FTIR spectroscopy and formed into pellets for use as a chromatographic matrix. Bovine serum albumin was separated on a column packed with the HAP pellets, demonstrating the potential of synthesized HAP for protein chromatography applications.

1. CHEMICAL SYNTHESIS OF HYDROXYAPATITE FOR APPLICATION IN CHROMATOGRAPHY BY Jagjit Singh Kahlon Sec- F77E1 Roll No. 49 Faculty Adviser: Anjuvan Singh 15097 Biotechnology LSSP Department of Biotechnology Lovely Institute of Engineering and Computer Applications (LIECA) LPU, Phagwara, India

2. CONTENTS INTRODUCTION OBJECTIVE REVIEW LITERATURE RESEARCH METHODOLOGY RESULT

3. INTRODUCTION Hydroxyapatite is a Calcium phosphate ceramic that is used as a biomaterial. Hydroxyapatite [Ca 10 (PO 4 ) 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.

4. 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

5. OBJECTIVE To synthesized Hydroxyapatite powder by Wet chemical precipitation method Study of chromatographic behaviour of hydroxyapatite.

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

7. 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 Abstract Lactoferrin 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.

8. 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.

9. Reaction of HAP Synthesis 10Ca(NO 3 ) 2 .4H 2 O + 6(NH 4 )2HPO 4 + 8NH 4 OH => Ca10(PO 4 )6(OH) 2 + 20NH 4 NO 3 + 46H 2 O

10. Research Methodology

11. In the Titration process In Burette Ammonium Solution 300 ml Calcium Nitrate 12 gms Water 260 ml Then after placing in at 4 o C & powdered form of HAP. In Beaker Ammonium Solution 300 ml Water 400 ml (NH 4 ) 2 HPO 4 : 120 gms incubation overnight we got a

12. Finally we get HAP in powdered form To get smoother powder Pestle motor and fine sieve were used.

13. 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

14. 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.

15. 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.

16. Chromatographic Coloumn HAP pellets were added to the syringe making a chromatographic column Known amount of BSA was loaded to the column for separation

17. 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

18. 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)

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