HPLC in Pharmaceutical Development: Principles, Benefits, Case Studies & Expert FAQs

HPLC in Pharmaceutical Development: HPLC (High-Performance Liquid Chromatography) is a powerful analytical technique used to separate, identify, and quantify components within a liquid sample. The term “chromatography” refers to the general process of separation, while a “chromatograph” is the instrument used, and a “chromatogram” is the visual output or result of the analysis.

HPLC systems rely on several advanced technologies, including specialised columns designed for molecular separation and high-precision pumps that deliver solvents at a consistent and stable flow rate. As these technologies evolved, the term “High-Performance Liquid Chromatography” became commonly abbreviated to “LC” in laboratory settings. More recently, Ultra High-Performance Liquid Chromatography (UHPLC) – which allows for faster and more efficient analysis – has become increasingly widespread.

HPLC is limited to compounds that are soluble in liquids, as it requires the sample to be dissolved in a solvent. The technique separates individual components based on their interactions with the mobile and stationary phases, enabling both qualitative (what is present) and quantitative (how much is present) analysis. This makes HPLC an essential tool in pharmaceutical development.

HPLC Principle

High-Performance Liquid Chromatography (HPLC) is an advanced analytical separation technique that utilises a solid stationary phase and a liquid mobile phase to separate components within a sample or sample mixture. The separation is primarily governed by the polarity of the stationary phase, mobile phase, and the analyte. Depending on the nature of the interaction between the analyte and the stationary phase, separation can occur through one or more of the following mechanisms: partition, adsorption, or ion exchange. HPLC is widely used in pharmaceutical development for its high resolution, sensitivity, and reproducibility in analysing complex mixtures.

Related: Relative Response factor (RRF) In Pharmaceutical Analysis

Types of HPLC (High-Performance Liquid Chromatography)

The following HPLC mode of separation is highly used in the Pharmaceutical industry:

1. Reverse-phase HPLC (RP-HPLC)
2. Normal phase HPLC (NP-HPLC)
3. Ion exchange HPLC and
4. Size exclusion HPLC

Note: Reverse-phase HPLC and normal-phase HPLC are widely used in the pharmaceutical industry.

Reverse-phase HPLC (RP-HPLC)

In reverse-phase high-performance Liquid Chromatography (RP-HPLC), the stationary phase is nonpolar (hydrophobic), and the mobile phase is polar. The mobile phase typically consists of a mixture of water and an organic solvent such as methanol or acetonitrile.

Common nonpolar stationary phases include C18 (octadecylsilane), C8 (octylsilane), and C4 (butylsilane) bonded phases, which retain nonpolar compounds through hydrophobic interactions. For effective separation in reverse phase chromatography, the sample must be soluble in the mobile phase, which is usually water or a water-organic solvent mixture.

Normal phase HPLC (NP-HPLC)

Normal Phase High-Performance Liquid Chromatography (NP-HPLC) is a chromatographic technique in which the stationary phase is polar and the mobile phase is non-polar. The mobile phase typically consists of organic solvents such as hexane, chloroform, or mixtures of non-polar solvents.

Common polar stationary phases used in normal phase chromatography include silica (SiO₂), diol, and cyano (CN) bonded phases. Separation is based on the differences in polarity between analytes, with more polar compounds interacting more strongly with the stationary phase and thus eluting later.

For effective separation in NP-HPLC, the sample must be soluble in the mobile phase, which means it must dissolve in non-polar or moderately polar organic solvents. This technique is particularly useful for separating polar compounds that are not well retained in reverse-phase systems.

HPLC Machine: Components

The following are the various components of HPLC machine:

1. Mobile Phase reservoir: It contains the mobile phase
2. Mobile phase: The Mobile phase is a mixture of different solvents in different proportions. In some cases, the single solvent is also used as a mobile phase. It carries the analytes from the injector port to the column to the detector, and it is also responsible for the separation.
3. Degasser: It removes dissolved gases from the mobile phase
4. Pump: It sucks mobile phase from the Mobile Phase reservoir and pumps it towards the column at high pressure.
5. Flow controller: It manages the flow rate of the mobile phase as per the method
6. Guard column: It is packed with the stationary phase similar to that of an HPLC column, but the size of the stationary phase is larger.
7. Sample injection port: The Injector inject the sample into the injector port.
8. Injector: The function of the injector is to inject the analyte mixture in the injector port. In most of the systems precise syringe is used as an injector.
9. HPLC Column: It is one of the most vital parts of the HPLC instrument. In column separation of analytes take place. It is packed with the stationary phase. Typical columns are C18, C8, Silica and Cyno.
10. Column temperature controller: It controls the temperature of the HPLC column to avoid variation in retention time.
11. Waste collector: It collects the waste mobile phase
12. Data processor: It converts the analyte signal (sent by the detector) into a peak.
13. Detector: It detects the analytes and converts them into signals. Several detectors are available for the analysis, but UV and PDA detectors are widely used in the Pharmaceutical industry. Detectors like mass, RI and ECD are also used in some of the pharmaceutical analysis.
14. HPLC Chromatogram: A chromatogram is a graph that plots detector response (usually absorbance in milli-absorbance units, mAU) vs. time (in minutes). Each peak represents a compound eluted from the column.

Step-by-Step HPLC Operation

• Precautions: Must ensure
  • You’ve read the system SOP (Standard Operating Procedure) and MSDS.
  • The instrument is maintained and calibrated.
  • You’re using the correct column and method for your analysis.

The following are step-by-step guide for basic HPLC operation, assuming you have a standard system with a quaternary pump, autosampler, UV detector, and data software (like Empower, ChemStation, or OpenLab):

1. Prepare Mobile Phase
2. Prime the System
3. Install the Column
4. Prepare the Sample
5. Set Up the Method
6. Run the System
7. Data Analysis and documentation
8. Shutdown Procedure
9. Regular Maintenance

1. Prepare Mobile Phase

• Use HPLC-grade solvents (e.g., water, acetonitrile, methanol).
• Degas solvents (using helium sparging, sonication, or a built-in degasser).
• Filter through a 0.22 or 0.45 µm membrane filter.
• Label and place bottles on the solvent tray (A, B, C, D lines as needed).

2. Prime the System

• Turn on the pump and prime each solvent line to remove air bubbles.
• Use the purge valve or priming function in software to flush the lines.
• Set flow rate to ~2–5 mL/min for priming, depending on system specs.

3. Install the Column

• Check that the column is appropriate (e.g., C18 for reversed-phase).
• Connect it in the correct flow direction (usually marked with an arrow).
• Equilibrate with mobile phase for 10–30 minutes until baseline stabilizes.

4. Prepare the Sample

• Filter your sample through a 0.22 or 0.45 µm syringe filter.
• Dilute to required concentration.
• Transfer to HPLC vials with caps and septa.

5 .Set Up the Method

Using your HPLC software:

• Mobile phase composition (gradient or isocratic).
• Flow rate (e.g., 1 mL/min).
• Column temperature (if using a column oven).
• Injection volume (e.g., 10–50 µL).
• Detector wavelength (e.g., 254 nm for UV).
• Run time (e.g., 10–30 min per sample).

6. Run the System

• Start the sequence or manual injection.
• Monitor pressure, baseline, and detector signal.
• Save and label chromatograms/data files.

7. Data Analysis

• Identify peaks using retention time.
• Integrate peak areas or heights.
• Quantify using calibration curves if needed.
• Shutdown Procedure
• Flush the column with a compatible solvent (usually water → organic).
• Store the column in suitable solvent (e.g., 100% methanol or acetonitrile).
• Turn off pump flow.
• Shut down software and power down system.

8. Shutdown Procedure

• Flush the column with a compatible solvent (usually water → organic).
• Store the column in suitable solvent (e.g., 100% methanol or acetonitrile).
• Turn off pump flow.
• Shut down software and power down system.

9. Regular Maintenance

• Regularly clean the injector, pump seals, and detector cell.
• Replace inline filters and frits when needed.
• Keep logs of maintenance and troubleshooting.

HPLC Method/Chromatographic Condition

An HPLC method contains the following details:

• Chemicals and reagents: This section contains all the chemicals and reagents and their grade, and part numbers used in the mobile phase preparation
• Mobile phase preparation procedure: This section contains a details procedure for mobile phase preparation
• Instrument details: This section contains instrument and detector details required for analysis
• HPLC column details: This section contains the column name, its part number, make and dimension or equivalent column details. For example: C18 column,(250×4.6)mm, micron, make: xxxx (as applicable)
• HPLC column temperature: It contains the temperature required by the method
• Mobile phase mode: This section contains isocratic mode or gradient mode details
• Wavelength: It contains the avelength at which the analysis will be performed
• Flow rate of mobile phase: It contains the flow rate of the mobile phase required by the method
• Injection volume: It contains injection details like 10µl, 20µl, 50µl or 100µl (as required by the method)
• Run time: It contains the analysis run time (in minutes) required by the method
• Column equilibration time: It contains the column temperature at which the analysis will be performed
• Diluent: It contains the solvent details in which the sample will be prepared
• Sample, standard, sensitivity solution and system suitability preparation procedure: It contains solution preparation details with weight and glassware (e.g volumetric flask details)
• Retention time and Relative retention time table: It contains Retention time(RT) and Relative retention time (RRT), structures of different analyte components
• System suitability acceptance criteria: It contains the System suitability test (SST) acceptance criteria required by the method. For example Resolution should not be less than 2, the theoretical plate should not be less than 10000, etc.
• Procedure: It contains blank, standard, sample and SST injection order and calculation procedure
• Typical chromatogram: This section contains Blank, SST, sensitivity, standard and sample typical chromatogram

How to Read an HPLC Chromatogram?

It involves the following steps:

1. Understand the Axes
   • X-axis: Retention time (tR) — the time a compound takes to pass through the column.
   • Y-axis: Detector response — usually UV absorbance, indicating compound concentration.
2. Identify the Peaks
   • Each peak corresponds to a different analyte (compound).
   • Earlier peaks usually represent more polar (or smaller) molecules (in reverse-phase HPLC).
   • Later peaks suggest more hydrophobic or larger molecules.
3. Retention Time (T)
   • This is the time at which the peak appears.
   • Use known standards to match retention times and identify compounds.
   • For qualitative analysis, retention time consistency is critical.
4. Peak Area vs. Peak Height
   • Peak Area is used for quantification because it better reflects the total amount.
   • Peak Height is less reliable, especially for broad or tailing peaks.
5. Peak Shape: Peak may be
   • Symmetrical
   • Tailing
   • Fronting
   • Broad
   • Split
6. Baseline
   • A stable, flat baseline indicates good system performance.
   • Noisy or drifting baseline may suggest detector, solvent, or temperature issues.
7. System Suitability Test (SST): Evaluate SST suing the follwing parametrs:
   • Retention time repeatability
   • Theoretical plates (efficiency)
   • Tailing factor
   • Resolution
   • %RSD of peak area for standards

Applications

HPLC is used for qualitative tests such as identification, purity, reaction monitoring and quantitative tests such as assay, and content tests in the:

• Pharmaceuticals development and routine analysis
• Polymer industries
• Environmental analysis
• Analysis of pollutant
• Isolation of impurities and characterisation
• Water treatment plant (to check purity of treated water)
• Food and beverage industries
• Forensic analysis

Advantages

The following are the advantages of HPLC

• Specific and selective: In a single run, several components can be separated and analysed with better resolution
• Efficient: HPLC is a highly efficient technique
• Accurate and precise: The Result obtained by HPLC is highly accurate and precise
• Sensitive: HPLC is a highly sensitive instrument, and impurities at lower levels can be easily detected, and which is why it is widely used in pharmaceutical development
• Robust and reproducible: HPLC is a robust technique, and the results obtained by it is reproducible in any lab
• Acceptability: HPLC is acceptable to all regulators and all pharmacopoeias
• Speed

Disadvantages

• Cost: It is a costly instrument and difficult to afford by a small organisation
• Complexity: To handle the HPLC requires skills as well as chemistry knowledge
• Analysis time: Analysis time is longer than traditional analyses, like chemical analysis and spectroscopy analysis

Basic requirements for handling the HPLC

HPLC is a complex instrument, and its instrumentation needs both knowledge and dedicated skills. Chemistry or pharmacy graduate or post postgraduate, or doctoral qualifications are preferable for it.

Case studies:

Elution pattern of Benzene, Naphthalene and in Reverse phase chromatographic mode

• Column: C18
• Mobile phase: Methanol: H₂O (90:10);
• Flow rate: 1 ml/ min
• Column temp: 30℃,
• UV detection:254 nm

Elution order: First Benzene then Naphthalene and then Anthracene. As the polarity of the molecule decreases retention time increases.

Elution pattern of Benzene, Benzaldehyde, and Benzyl alcohol in Normal phase chromatographic mode

• Column: Si,150×4.6mm, 5µm
• Mobile phase: 96% hexane % IPA
• Flow: 1.0 ml/min
• Temp.: ambient
• Detection: UV @ 254

Elution order: Benzene, then Benzaldehyde and then Benzyl alcohol. As the polarity of the molecule increases retention time increases.

Conclusion

Just as a mirror reflects the true beauty of the human face, an HPLC chromatogram reflects the purity, composition, and overall quality of pharmaceuticals. The insights provided by HPLC not only help in identifying and quantifying compounds but also play a vital role in ensuring the safety, efficacy, and consistency of pharmaceutical products. By understanding and interpreting HPLC results effectively, professionals can enhance their pharmaceutical development processes and guarantee the highest standards of quality control.

We hope this article has deepened your understanding of HPLC and its critical role in pharmaceutical development. For any queries or suggestions, feel free to leave a comment below or reach out to us using the contact form. Let’s continue to innovate and improve pharmaceutical quality together!

You may also want to check out other articles on my blog, such as:

1. How to select an equivalent HPLC column
2. How to select HPLC Detector
3. How to prepare and Optimise mobile phase
4. Peak integration in HPLC
5. How to remove Ghost Peak in HPLC
6. HPLC Column: Types, Working Principles, Expert Tips, and …
7. Void Volume, Dead Volume and Dwell Volume In HPLC: Learn Easily In 11 Minutes

Expert FAQs

Precautions

• Keep the instrument neat and clean
• Calibrate the instrument as per the schedule
• Use a log book to manage HPLC work and HPLC maintenance work

Abbreviations

• HPLC: High performance liquid chromatography
• UV: Ultra-violet
• PDA: Photodiode array
• MS: Mass spectroscopic detector
• RI: Refractive index
• µl: Microliter

Further Reading

• USP<621>
• Pharma knowledge forum YouTube channel