What is High-performance liquid chromatography (HPLC)?
HPLC (High-Performance Liquid Chromatography) is an analytical technique that separates, identifies and quantifies the components of a liquid mixture by pumping it at high pressure through a column packed with fine particles. It is the most widely used analytical technique in pharmaceutical quality control.
Before understanding the principle of HPLC, first, we need to know about chromatography. Chromatography is an analytical process of separating components in a mixture. To initiate the process, a mixture of unknown components is dissolved in a substance known as mobile phase, which carries it through a solid second substance called the stationary phase. This mixture of unknown components travels through the stationary phase at variable speed, causing them to separate from one another. These different travel times are popularly known as the retention time (T + 15mins as shown in figure). The speed of the mixture is based on the polarity of the components in the mobile and stationary phases. In reverse phase HPLC, the most common mode, more polar components move faster and elute first. In normal phase HPLC, the order is reversed. The stationary phase constrains many of the components in a mixture, slowing them down to move slower than the mobile phase.
Out of all chromatography techniques, liquid chromatography (LC) is widely used across different industries. It is a separation technique in which the mobile phase is a liquid, where sample ions or molecules are dissolved. It is carried out either in a column or a plane. HPLC is an advanced and modified LC technique performed under a significantly higher operational pressure than LC. The conventional LC technique relies on the force of gravity to pass the mobile phase through the column resulting in a slow flow rate. On the other hand, HPLC solvent is forced through the column under high pressures up to 400 atmospheres, which increases the mobile phase flow rate, fastens the separation process, and thus increases efficiency. Ultra-high-performance liquid chromatography (UHPLC) systems work at even higher pressures, often above 1,000 bar
Principle of High-Performance Liquid Chromatography (HPLC):
- The principle of separation on HPLC is based on the distribution of analyte (sample with a different unknown amount of compounds) between the mobile phase and stationary phase (column).
- Depending on the chemical structure of the analyte, the molecules are retarded in the column stationary phase due to specific intermolecular interactions between the analyte and the packing material on the column. This defines the analyte’s retention time on the column, and therefore different substances elute at different time intervals, thereby achieving the separation of different compounds in an analyte.
- The mobile phase is pressurized into the column using solvent delivery pumps with the stationary phase.
- An analyte sample with unknown compounds is injected into the mobile phase before entering the column.
- Subsequently, individual compounds in the analyte migrate through the column at different rates achieving separation.
- Once the analytes exit the column, the detector unit recognizes the compounds in the analyte and exhibits them in electrical signals. Such signals are fed to the computer data station in the HPLC to generate the chromatogram.
- Chromatograms are used to identify and quantitate the concentration of analyte constituents.
Instrumentation in the High-Performance Liquid Chromatography (HPLC)
Solvent delivery pump:
- Also known as a solvent delivery system, it is used to deliver the mobile phase at a constant, pulse-free flow rate.
- The purpose of the pump is to force the mobile phase through the column while maintaining a specific flow rate.
- There are three HPLC pumps categories: Syringe, Reciprocating, and Pneumatic pumps. Out of these, reciprocating pumps are most commonly used due to their delivery of the mobile phase at a constant, pulse-free flow rate, small footprint, constant and reproducible flow rate,
- Before selecting a pump, certain things like instruments compatibility with different solvents, price, ease of use, cleanability, maintenance costs, etc., shall be considered.
Injector:
- An HPLC injector allows the introduction of samples onto the column. These injectors inject the sample without disturbing the flow rate and pressure of the HPLC system.
- The role of the injector has a lot of significance because direct injection of the sample is not suggested as the working pressure of the HPLC is adequately high that we cannot inject the sample into the mobile phase.
- Care must be taken while injecting the sample. Points that must be kept in mind like introducing a sample without air bubbles, a sample introduced with constant pressure and flow rate, injection volume of the sample is in microliters, and the sample must be free from any particulate matter.
- There are many conventional types of injectors like Rheodyne injector, Septum injector, and Stopflow injector; however, an advanced injector known as Auto-sampler injector is used to deliver an aliquot of sample to the HPLC column. It is high-tech automation equipment with high precision, variable volume, and long-term reliability.
HPLC Column:
- The hplc column is an integral part of the HPLC system that performs the critical task of separating molecular compounds during analysis.
- A typical analytical HPLC column is 150–250 mm long with an internal diameter of 4.6 mm, packed with 3–5 µm particles. UHPLC columns use particles smaller than 2 µm. Read more about HPLC columns and their types.
- Several types of columns are used in the pharmaceutical industry; however, the most commonly used ones are C18 and C8 columns.
- Common packing materials in columns include silica or hydroxyapatite media and polymeric resins such as polystyrene divinylbenzene.
- Columns are made of stainless-steel housing or glass casing; however, housing made SS is considered the most stable and is tolerant towards a wide variety of solvents.
Detector:
- Detectors are used to sense the presence of separated compounds as they leave the column. The separates are monitored and expressed electronically by the detectors (Refer below table for types of detectors).
- Detectors used shall be sensitive, have good stability, reproducibility, inexpensive.
- Detectors measure the difference in some physical properties of the solute in the mobile phase compared to the mobile phase alone.
Common types of HPLC detectors
| Detector | What it measures | Typical use |
|---|---|---|
| UV-Visible (UV-Vis) | Absorbance of light at a set wavelength | Most widely used; assay and impurities of drugs with a chromophore |
| Photodiode array (PDA / DAD) | Absorbance across a full UV-Vis spectrum at once | Peak purity checks and identification by spectrum |
| Fluorescence | Light emitted by fluorescent compounds | Very sensitive analysis of naturally fluorescent or derivatised compounds |
| Refractive index (RI) | Change in refractive index of the eluent | Sugars and other compounds with no UV absorbance; isocratic methods only |
| Evaporative light scattering (ELSD) / Charged aerosol (CAD) | Particles left after the mobile phase is evaporated | Non-volatile compounds without a chromophore, such as lipids and some excipients |
| Mass spectrometer (LC-MS) | Mass-to-charge ratio of ionised molecules | Identifying unknown impurities and degradation products; bioanalysis |
Computer Data Station (Recorder)
- Electronic data signals expressed by the detectors are interpreted and processed into a meaningful inference in the form of chromatograms.
- They record the baseline and all the peaks obtained with respect to time.
- Present date data stations perform multiple complex tasks like data acquisition, interpretation, data processing, data storage, and distribution across the connected systems within an organization.
Degasser:
- The mixing of liquids involves the entrapment of gases like oxygen, which contributes to noise and causes an unstable baseline.
- Degasser is a high-efficiency in-line system designed to remove dissolved gases from a solvent.
- The degasser is easy to use, provides reliable continuous operation, and eliminates the need for helium sparging to remove gases.
- Polymeric Teflon tubing inside the degasser has micropores, which contribute to outward gaseous exchange to the environment while preventing any liquid from passing through the pores.
Column Heater:
- This accessory is used to precisely control the temperature of the analysis to improve the sensitivity, analysis time, and peak separation and ensure the accuracy of sample results.
- Column Heater provides consistent, reproducible temperature control for HPLC columns.
- The heater will produce temperatures between ambient +5° C to 150° C with an accuracy of ±2°C while operating in environments with temperatures between -5° C to 50° C.
Types of High-Performance Liquid Chromatography (HPLC)
Normal Phase chromatography:
- This chromatography type uses columns packed with a polar stationary phase and a nonpolar or moderately polar mobile phase to separate polar compounds.
- The polar stationary phase includes silica, alumina, or polar-bonded phase, whereas the nonpolar mobile phase includes hexane, heptane, etc.
- This method separates analytes based on polarity. Less polar solutes move the fastest and therefore exit the column and are detected first, followed by solutes of increasing polarity, which move more slowly.
- This technique is used for water-sensitive compounds, geometric isomers, class separation, and chiral compounds.
Reverse Phase Chromatography:
- This chromatography type uses columns packed with a nonpolar stationary phase and a moderately polar, aqueous mobile phase to separate polar, nonpolar, ionic, and ionizable compounds.
- It works on the principle of hydrophobic interactions.
- In this chromatography, the surface of the column stationary phase is covalently bound with alkyl or aromatic ligands to provide a hydrophobic surface. Hydrophobic solutes present in the mobile phase tend to get bound to the stationary phase via hydrophobic interactions, forming the basics of separation.
Ion Exchange Chromatography:
- Ion exchange chromatography separates ions and charged molecules, from small inorganic ions to proteins, based on their attraction to oppositely charged groups on the stationary phase.
Size Exclusion Chromatography:
- Size exclusion chromatography (SEC), also called gel permeation or gel filtration chromatography, separates molecules by size.
- The column is packed with porous particles: large molecules cannot enter the pores and elute first, while smaller molecules enter the pores and elute later.
- It is used for proteins, polymers and aggregates
Normal phase vs reverse phase HPLC
| Feature | Normal phase | Reverse phase |
|---|---|---|
| Stationary phase | Polar (silica, alumina) | Non-polar (C18, C8 bonded silica) |
| Mobile phase | Non-polar (hexane, heptane) | Polar (water or buffer with methanol or acetonitrile) |
| Elutes first | Least polar compounds | Most polar compounds |
| Use in pharma | Isomers, water-sensitive compounds | Most assay, impurity and dissolution methods |
Most pharmaceutical HPLC methods use reverse phase with a C18 column. Learn more in our guides to the C18 column and reverse phase chromatography.
System Suitability in HPLC
Before any samples are analysed, the HPLC system must pass system suitability: a set of checks that confirm the instrument, column and method are performing as required. The usual parameters are:
- Resolution (Rs): how well two neighbouring peaks are separated. Many methods require a resolution of not less than 2.0 between critical peaks.
- Tailing factor (T): how symmetrical a peak is. A limit of not more than 2.0 is common.
- Theoretical plates (N): a measure of column efficiency. Higher is better, and each method sets its own minimum.
- %RSD of replicate injections: the precision of repeated injections of the standard, commonly not more than 2.0% for assay methods.
If system suitability fails, no sample results from that run can be reported. The cause is investigated and fixed before the analysis is repeated.
The exact limits always come from the method or pharmacopoeia monograph. The values above are typical examples.
Applications of the High-Performance Liquid Chromatography (HPLC)
- Detection of impurities in the pharmaceutical industry
- Water purification
- Qualitative and quantitative analysis
- To identify the purity of compounds
- Determination of mixture of drugs or multicomponent analysis
- Biochemical separations
- Find pollutants in Environmental samples
- Analysis of synthetic polymers
- Purification of compounds
- Analysis of products in the Pharmaceutical, biotech, and food industries
- Pre-concentration of trace elements
- Ion-exchange chromatography of proteins
- High-pH anion-exchange chromatography of carbohydrates and oligosaccharides
- Ligand-exchange chromatography
Advantages of High-Performance Liquid Chromatography (HPLC)
- Fast, precise and reproducible analytical instrument to separate targeted compounds.
- Accurate and reliable
- Powerful and adaptable
Limitations of High-Performance Liquid Chromatography (HPLC)
- High maintenance costs
- Very complex to operate
- Needs competent and well-trained personnel to operate
- Volatile substances are not suitable for separation via HPLC
- Compounds without a UV chromophore need other detectors, such as RI, ELSD or mass spectrometry.
Frequently Asked Questions
What is the full form of HPLC?
HPLC stands for High-Performance Liquid Chromatography. It was earlier also called high-pressure liquid chromatography, because the mobile phase is pumped through the column at high pressure.
What is the principle of HPLC?
HPLC separates the components of a mixture based on how strongly each one interacts with the stationary phase in the column compared with the mobile phase. Components that interact more strongly with the stationary phase move more slowly, so each component leaves the column at a different retention time and is detected as a separate peak.
What are the main parts of an HPLC system?
An HPLC system has a solvent reservoir, degasser, pump, injector or autosampler, column (often in a column oven), detector and a computer data system that records the chromatogram.
What is the difference between normal phase and reverse phase HPLC?
Normal phase uses a polar stationary phase and a non-polar mobile phase, so non-polar compounds elute first. Reverse phase uses a non-polar stationary phase, such as C18, and a polar mobile phase, so polar compounds elute first. Reverse phase is used for most pharmaceutical methods.
Which column is most commonly used in HPLC?
The C18 (octadecylsilane, ODS) column is the most widely used, typically 150–250 mm long and 4.6 mm in internal diameter, packed with 3–5 µm particles. Read our complete guide to the C18 column.
Which detector is most commonly used in HPLC?
The UV-Visible detector is the most common, because most drug molecules absorb UV light. A photodiode array (PDA) detector is often used when peak purity needs to be checked.
What is retention time in HPLC?
Retention time is the time a compound takes to travel from the injector to the detector. Under fixed conditions it is characteristic of the compound, so it is used to identify peaks by comparison with a reference standard.
What is the difference between HPLC and UHPLC?
UHPLC (ultra-high-performance liquid chromatography) uses columns packed with particles smaller than 2 µm and works at much higher pressures, often above 1,000 bar. This gives faster runs and sharper peaks than conventional HPLC.
What is system suitability in HPLC?
System suitability is a set of checks run before sample analysis to confirm the HPLC system is performing well enough for the method. Common parameters are resolution, tailing factor, theoretical plates and the %RSD of replicate standard injections.
References:
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- https://www.azom.com/article.aspx?ArticleID=8468
- https://www.thermofisher.com/blog/ask-a-scientist/what-is-chromatography/
- https://www.knauer.net/en/Systems-Solutions/Analytical-HPLC-UHPLC/HPLC-Basics–principles-and-parameters
- https://microbenotes.com/high-performance-liquid-chromatography-hplc
- https://laboratoryinfo.com/hplc/
- https://www.shimadzu.com/an/service-support/technical-support/analysis-basics/basic/what_is_hplc.html
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