is applicable to all personnel in the Analytical Method Development (AMD) department involved in developing, optimizing, or validating chromatographic methods for APIs, intermediates, excipients, and finished products.

3. Responsibilities

• Analytical Scientist: Designs experiments and selects mobile phase candidates based on analyte and matrix characteristics.
• Team Leader: Reviews mobile phase suitability and approves finalized compositions.
• QA Department: Ensures mobile phase selection rationale is documented and traceable.
• Head – AMD: Authorizes the final method incorporating the selected mobile phase.

4. Accountability

The Head of AMD is accountable for ensuring scientifically sound, reproducible, and well-documented selection of mobile phases during chromatographic method development.

5. Procedure

5.1 Assess Physicochemical Properties of Analyte

1. Determine the following parameters:
   • Molecular structure and functional groups
   • pKa, logP, solubility in various solvents
   • UV absorption range or detection feasibility
2. Record in Annexure-1: Analyte Evaluation Sheet.

5.2 Mobile Phase Selection Strategy

1. Choose between Reversed-phase, Normal-phase, or Ion-exchange chromatography based on analyte polarity:
   • RP-HPLC: Preferred for non-polar to moderately polar compounds
   • NP-HPLC: Suitable for polar analytes (e.g., sugars, steroids)
   • Ion-pairing: Used for ionizable compounds with poor retention
2. Base initial selection on literature, prior data, or pharmacopoeial methods.

5.3 Common Mobile Phase Components

1. Aqueous Phase: Water, phosphate buffers, acetate buffers (pH 2–7.5)
2. Organic Modifiers: Acetonitrile, methanol, ethanol (HPLC grade)
3. pH Adjusters: Orthophosphoric acid, triethylamine, ammonia solution
4. Ion Pairing Reagents (if applicable): Sodium dodecyl sulfate, tetrabutylammonium hydroxide

5.4 Buffer Preparation and Selection

1. Select a buffer that:
   • Maintains desired pH near analyte pKa (±1 unit)
   • Has low UV absorbance if UV detection is used
   • Is chemically compatible with column packing
2. Prepare using analytical grade reagents and HPLC-grade water.
3. Filter and degas mobile phase through 0.45 µm filters prior to use.

5.5 Gradient vs. Isocratic Mode

1. Choose gradient mode when:
   • Analyte mixture has wide polarity range
   • Late eluting peaks need separation
2. Choose isocratic mode for simple APIs or when analyte retention is stable.
3. Optimize gradient program by trial runs using different slope conditions.

5.6 Evaluation of System Suitability

1. Assess retention time (RT), resolution (Rs), theoretical plates (N), tailing factor (T), and reproducibility.
2. Ensure system suitability meets acceptance criteria before finalizing mobile phase.
3. Record observations in Annexure-2: Mobile Phase Optimization Log.

5.7 Documentation and Approval

1. Document selected mobile phase composition, preparation instructions, stability, and storage conditions.
2. Attach chromatograms from each phase of optimization.
3. Obtain QA and Head – AMD approval before proceeding to validation.

6. Abbreviations

• AMD: Analytical Method Development
• RP-HPLC: Reversed-Phase High Performance Liquid Chromatography
• UV: Ultraviolet
• pKa: Acid Dissociation Constant
• LOD: Limit of Detection
• Rs: Resolution
• RT: Retention Time

7. Documents

1. Analyte Evaluation Sheet – Annexure-1
2. Mobile Phase Optimization Log – Annexure-2

8. References

• ICH Q2(R1) – Validation of Analytical Procedures
• USP General Chapter <621> – Chromatography
• Journal of Chromatography A and B
• Pharmacopoeial Monographs (USP, IP, BP)

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Analyte Evaluation Sheet

Analyte	pKa	Solubility	UV Max (nm)	Polarity
Loratadine	5.1	Soluble in ACN	248	Moderately Lipophilic

Annexure-2: Mobile Phase Optimization Log

Date	Mobile Phase	pH	Flow Rate	Resolution	Retention Time	Remarks
12/05/2025	0.1% OPA:ACN (60:40)	3.0	1.0 mL/min	2.5	6.2 min	Acceptable profile

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
04/05/2025	2.0	Added buffer guidelines and annexure structure	Internal standardization