headerbuttonimg 9910157319
October 13, 2023 Dr Pramod Kr Pandey
1 Comment
Analytical Research

How to Understand the Principles of HPLC?

Introduction and outcome

HPLC or High Performance Liquid Chromatography is required to control the quality of the pharmaceuticals. Be it a raw material analysis or reaction monitoring or release of the pharmaceuticals HPLC is required at every stage. Hence, we can say that HPLC is the backbone of the pharmaceuticals development. That is why I decided to share skill-based knowledge on this topic. In this post, I will discuss principles, components, types, advantages, disadvantages, applications, case studies and frequently asked questions of HPLC.

Table of content

HPLC Principle

HPLC or high performance liquid chromatography is separation technique based on solid stationary phase and liquid mobile phase and separation is achieved by partition, adsorption or ion exchange process. Separation is governed by polarity of stationary phase, mobile phase and the analyte and may be achieved by the following mechanism:

  • Partition
  • Adsorption and Ion
  • Exchanges process

Components of HPLC

HPLC
HPLC: Pharma Knowledge Forum

The following are the various components of HPLC:

  1. Mobile Phase reservoir: It contains mobile phase
  2. Mobile phase: Mobile phase is mixture of different solvents in different proportions. In some cases, single solvent is also used as a mobile phase. It carries the analytes from injector port to 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: Injector inject the sample in injector port.
  8. Injector: The function of injector is to inject the analyte mixture in the injector port. In most of the system precise syringe is used as a injector.
  9. Column: It is one of the most vital part of the HPLC instrument. In column separation of analytes take place. It is packed with stationary phase. Typical columns are C18, C8, Silca and Cyno.
  10. Column temperature controller: It control the temperature of the HPLC column to avoid variation in the retention time.
  11. Waste collector: It collects the waste mobile phase
  12. Data processor: It converts the analyte signal (sent by detector) into a peak.
  13. Detector: It detects the analytes and converts them into the signals. Several detectors are available for the analysis but UV and PDA are widely used in the Pharmaceutical industries. Detector likes mass, RI and ECD are also used in some of the the pharmaceuticals analysis.

Types of HPLC

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

  • Reverse phase chromatography or RPC
  • Normal phase chromatography or NPC
  • Ion exchange and
  • Size exclusion

RPC and NPC separation mode are widely used in the pharmaceutical industries.

Reverse phase chromatography or RPC

In RPC mobile phase is polar and stationary phase is non polar. Mixture of aqueous and organic solvents are used in the mobile phase. Non polar stationary phase like C18, C8, C4 are used. For separation in the reverse phase sample must be soluble in the mobile phase or water or mixture water and organic solvents.

Normal phase chromatography or NPC

In NPC mobile phase is non-polar and stationary phase is polar. Mixture of organic solvents or are used in the mobile phase. Polar stationary phase like Silica, Diol,cyno are used. For separation in the NPC sample must be soluble in the mobile phase or organic solvents.

Components of HPLC method:

An HPLC method contains the following components:

  • Chemical and reagents: This section contains all the chemicals and reagents and their grade and parts number used in the mobile phase preparation
  • Mobile phase preparation procedure: This section contains details procedure of mobile phase preparation
  • Instrument details: This section contains instrument and detectors details required for analysis
  • HPLC column details: This section contains column name, its part number, make and dimension or equivalents column details. For example: C18 column,(250×4.6)mm, micron, make: xxxx (as applicable)
  • HPLC column temperature: It contains temperature required by the method
  • Mobile phase mode: This section contains isocratic mode or gradient mode details
  • Wavelength : It contains wavelength at which analysis will be performed
  • Flow rate of mobile phase: It contains 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 analysis run time (in minutes) required by the method
  • Column equilibration time: It contains column temperature at which analysis will be performed
  • Diluent: It contains solvent details in which 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 System suitability test (SST) acceptance criteria required by the method. For example Resolution should not be less than 2, 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

Applications of HPLC

It is require for qualitative tests such as identification , purity , reaction monitoring and quantitative test such as assay, content test 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

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 is highly efficient technique
  • Accurate and precise: Result obtained by HPLC is highly accurate and precise
  • Sensitive: HPLC is highly sensitive instrument and impurities at lower level can be easily detected and that is why it is widely used in the pharmaceuticals development
  • Robust and reproducible: HPLC is robust technique and result obtained by it is reproducible in any lab
  • Acceptability: HPLC is acceptable by all regulators and all pharmacopoeias

Disadvantages of HPLC

  • Cost: It is costly instrument and difficulty to affords by small organisation
  • Complexity: To handle the HPLC needs skill as well as chemistry knowledge
  • Analysis time: Analysis time is longer than traditional analysis 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 graduate or doctorate are preferable qualifications for it.

HPLC chromatographic terms

The following chromatographic terms are widely used in the HPLC system suitability test:

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 tem: 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, Benzyl alcohol in Normal phase chromatographic mode

  • Column: Si,150×4.6mm, 5µm
  • Mobile phase: 96% hexane:4% 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

As a mirror tells about the beauty of the human face in the same way HPLC result tells about the quality of the pharmaceuticals. I hope this post has increased your knowledge to the next level and now you can use it more effectively in pharmaceutical development. For any query or suggestion related to this article write in the comment section or contact me using contact form.

Precautions

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

FAQS

Read this article

Abbreviations
  • HPLC: High performance liquid chromatography
  • UV: Ultra-violet
  • PDA: Photodiode array
  • MS: Mass spectroscopic detector
  • RI: Refractive index
  • µl: Microliter
References
  • USP
  • IP
  • Pharma knowledge forum Youtube channel

Analytical Research
Analytical Research

One Response to “How to Understand the Principles of HPLC?”

Leave a Reply

Your email address will not be published. Required fields are marked *

You cannot copy content of this page

×