The document outlines the process of HPLC method development, focusing on the selection of stationary and mobile phases based on molecular characteristics of the sample, including charge, hydrophobicity, and solubility. It also details data analysis techniques for qualitative and quantitative assessment, utilizing retention time and spectral profiles for peak identification. Troubleshooting common issues such as unusual peak shapes and fluctuating retention times is also discussed.

1. Prepared by
Sumon Dey

2. Molecular characteristics of the sample.
Selection of Stationary phase & Mobile Phase
Troubleshooting & Optimization
Data Analysis: Qualitative & Quantitative.
Steps for HPLC method development

3. What are the molecular characteristics
of the analyte or sample?
CHASM
1. Charge
Positive/negative
2. Hydrophobicity
3. Affinity
“lock and key” sites
4. Solubility & stability
pH, ionic strength, organic solvents
5. Molecular weight

4. Bulk (SiO2)x Bulk (SiO2)xSurface Surface
Selection of Stationary Phase
Normal Phase Reverse Phase
SurfaceBulk (SiO2)x
Ion Exchange

5. Normal Phase Reverse Phase Ion
Exchange
Stationary Phase Polar Non polar Silica having
functional
group
Mobile Phase Non polar
(hexane,heptan)
& slightly polar
(isopropanol,
ethylacetate)
Polar (water,
methanol,
acetonitrile,
tetrahydrofuran)
Depending
on analyte,
may salt
gradient or
pH gradient
Sample
Movement
Non polar
fastest
Polar fastest More
cation/anion
Separation based
on
Different
polarities
(functionality)
Different
hydrocarbon
content
Ion
exchange.
Selection of Mobile Phase

6. Characteristics of Solvent

7. Solvent used in Reverse Phase HPLC

8. Retention strength in Reverse Phase HPLC

9. Reverse Phase Normal Phase
1. Neutral & Nonpolar.
compound (MW<2000 Da).
2. Homo-logs & Benzologs.
3. Weak Acid & Base.
4. Protein & Peptide.
1. Too hydrophilic/
hydrophobic.
2. Not soluble in water.
3. Isomers .
Choice of columns

10. Data Analysis
Qualitative Quantitative
By Retention time
By Spectral profile
By Peak Area
measurement

11. Use of reference standards for peak identification

12. Use of spectral detectors

13. Area Estimation

14. Amount of analyte
Peak Area
Response Factor
Amount (Std.)
Response Factor
Peak Area (Std.)
Calculation
Std. Amount = 2,000 µg/ml
Std. Peak Area = 100,000
Analyte Peak Area = 57,000
Amount (analyte) = ?
Response Factor = 100,000/2,000 = 50
Amount (analyte) = 57,000/50 = 1,140 µg/ml

15. Troubleshooting
 Unusual Peak Shapes:
1. Broad Peak
2. Ghost Peak
3. Negative Peak
4. Peak Doubling
5. Peak Fronting & tailing
 Fluctuating Retention time:

16. Broad Peak

17. Ghost Peak

18. Negative peak

19. Peak doubling

20. Peak fronting & tailing

21. Fluctuating retention time