HPLC Principle

HPLC Principle, Definition, Parts, Types, Uses, HPLC Diagram

Before starting with HPLC Principle here are some full form of HPLC and Abbreviation of HPLC.

A. Abbreviation of HPLC:

  • High-Performance Liquid Chromatography or High-Pressure Liquid Chromatography

B. Use:

  • Quantitative analysis of Drug in Formulation, Purity and Potency check, Impurities detection  

C. HPLC Principle:

  • The distribution of the analyte (Drug-sample) between a mobile phase (eluent) and a stationary phase (packing material of the column) is the basis of liquid chromatography.
  • Based on the chemical structure of the Sample (Analyte), the molecules are retarded or delayed passing through the stationary phase (Column). In overall, High-Performance Liquid Chromatography is used to separate the components of a mixed drug substance
  • The time a sample spends “on-column” is determined by the specific intermolecular interactions (also known as Polarity) between its molecules and the packing material.
  • Different elements of a Sample (Analyte) are eluted at different times intervals. As non-polar column attracts non-polar substance and delays the elution of non-polar part or sample elements where polar ingredient allowed elutes first.
  • As a result, the sample ingredients can be separated. After the analytes have left the column, a detection device or detector (such as a UV detector) detects them.
  • A data management system (computer software) converts and records the signals, which are subsequently shown in a chromatogram.
  • The mobile phase can then be exposed to additional detector units, a fraction collecting unit, or the waste after passing through the detection unit.
  • A solvent reservoir, a pump, an injection valve, a column, a detector unit, and a data processing unit are the main components of an HPLC system (as shown in Figure).
Principle of HPLC, HPLC Parts
  • The pump drives the solvent (eluent) through the system at high pressure and steady speed.
  • A continuous and pulseless flow from the pump is critical to keep the detector signal’s drift and noise as low as feasible. The injection valve delivers the analyte (sample) to the eluent.
Principle of HPLC, HPLC Working flow

Types of High-Performance Liquid Chromatography (HPLC) and principle.

  1. Normal phase:
    • Column packing material is polar in nature (For example silica) and the mobile phase is non-polar in nature. It is mainly used for chiral compounds, geometric isomers water-sensitive compounds and isomers (cis-trans) identification.
  2. Reverse phase:
    • The column packing material is non-polar in nature (e.g C18), the mobile phase is water and water-miscible solvent (For Example Methanol, Acetonitrile, Iso propyl alcohol -IPA). Mainly used for ionizable, non-polar, and polar, ionic samples.
  3. Ion exchange:
    • Ion Exchange LC: Mobile phase is buffer & Column packing contains ionic groups. It’s employed to separate cations from anions. Ion Chromatography is one of type.
  4. Size exclusion:
    • In a porous medium, molecules diffuse into the pores and are segregated based on their relative size to the pore size Large molecules elute first, followed by smaller molecules.

Related: Definition of Tailing Factors, Resolution, USP Plates, Difference Between Isocratic and Gradient Elution

D. Parts of HPLC:

1. Mobile phase Bottle Line with Degasser

  • There may be a Binary or Quarterly Pump System that comes Mobile phase line to pump via degasser.
  • The eluent used for Liquid Chromatography analysis may contain gases such as oxygen that are non-visible to our eyes.
  • When gas is existing in the eluent, it will be detected as noise and causes an unstable baseline in result.
  • Degasser removes gases using specific polymer membrane tubing.
  • The surface of the polymer tube has numerous, extremely tiny pores that permit air to pass through but do not permit liquid to do the same.

2. Pump:

  • The pump system was developed as a result of HPLC development.
  • The pump creates a flow of eluent from the solvent reservoir into the system and is positioned in the liquid chromatography system’s uppermost stream.
  • A Primary need of pumps is the ability to generate high pressure, but it should also be able to deliver a constant pressure under any circumstance as well as a predictable and repeatable flow rate.
  • The majority of pumps used in modern LC systems generate the flow by moving a motor-driven piston back and forth (reciprocating pumps).
  • This piston motion causes “pulses” to be produced.
  • Modern HPLC Technique involved different types of Pump in market:
    • Dual-Piston Pumps, Dual-Piston In-Parallel Pumps, Dual-piston In-Series Pumps, Low-Pressure Mixing Pumps, High-Pressure Mixing Pumps
HPLC Pump type

credit@ https://www.chromatographyonline.com/

3. Sample Cooler Chamber

  • The sample cooler chamber is mainly used when the solution of the sample is not stable at Room temperature and to avoid the temperature fluctuation effect in the result.
  • Sampler trays are placed in the sample cooler chamber such that easy for autosampler detection.
  • Sample cooler chamber range from 2-8°.

4. Injector (Autosampler)

  • An injector is placed very next to the pump.
  • Sample injection operation is performed by Injector from Sample via to column with help of mobile phase. Sample are mainly liquid sample.
  • The simplest method is to use a syringe, and the sample is acquaint with to the flow of eluent.
  • The most widely used injection method is based on sampling loops.
  • The use of an autosampler system is also commonly used in pharma industries that allow repeated injections in a set scheduled timing.
  • The user needs to a add sample try number, No. of Injection, Volume, and No. of time injection system will do.  

5. Column & Column Oven Chamber

  • Inside of the column is where the separation is performed.
  • Instead of glass columns, modern columns are frequently manufactured in stainless steel (SS) casing/housing (As SS is tolerant towards a large variety of solvents).
  • The column packing material is normally prepared from silica or polymer gels compared to calcium carbonate.
  • The eluent used for liquid chromatography ranging from acidic to basic solvents.
  • The column temperature frequently has a significant impact on the LC separation.
  • Maintaining constant temperature conditions is crucial for getting reproducible results.
  • At higher temperatures (50 to 60°C), improved resolutions can also be attained for specific analyses, such as sugar and organic acid.
  • In Some case if temperature is higher than external Column Heater chamber are available in market.
  • In the column oven, columns are typically retained as a result (column heater).

6. Detector

  • Analyte separation is carried out inside the column, and the detector monitor the separation.
  • When an analyte is not present, the eluent’s composition is constant. While the eluent’s composition is altered by the analyte’s presence. Measurement of these differences is what a detector performs.
  • This variation is tracked via an electrical signal. There are various detectors to choose from.

Related: HPLC Detector

7. Software or Recorder

  • The change in eluent detected by an HPLC detector is in the form of an electronic signal, and thus it is still not visible to our eyes.
  • In previous times, the pen (paper)-chart recorder mainly used. In modern HPLC technique, a computer-based data processor (integrator) is use.
  • There are various types of data processors; from a simple system consisting of the in-built printer and word processor while those with software that are specifically designed for a Chromatography system .
  • They are not only data acquisition but features like peak-fitting, molecular weight determination, Linearity check, SN Ratio, Tailing Factor, baseline correction, automatic concentration calculation, etc.

Related: Mobile Phase preparation of HPLC, Calibration of High-Performance Liquid Chromatography HPLC

Available High-Performance Liquid Chromatography (HPLC) in the market with software and Approximately Price of HPLC:

Sr. No.HPLC NameCompany NameSoftware
11. Alliance HPLC
2. Breeze HPLC
3. Arc HPLC
Waters (US)Empower
21. 1220 Infinity II LC System  
2. 1260 Infinity II LC System  
3. 1260 Infinity II Prime LC System
4.1290 Infinity II LC System  
Agilent HPLC Solutions (US)OpenLab software & Agilent ChemStation
3LC-2050 seriesShimadzu (Japan)LabSolutions
4Dionex VanquishThermoFisher (US)Chromeleon
5LC 300 HPLCPerkinElmer (US)SimplicityChrom CDS Software
6ÄKTA™ pureGE Healthcare (US)GoSilico™ Chromatography
7ChromasterHitachi (Japan)ChromAssist
8Shodex™Showa Denko (Japan)NA
9CoPrime® Biochromatography SystemMerck Millipore (Germany)NA

Read More: Difference Between Stationary Phase and Mobile Phase, Difference Between C18 and C8 HPLC Column, HPLC chromatography principle HPLC

What is the Price of High-Performance Liquid Chromatography (HPLC) price:

  • In India is Approximately 35000 USD for Vanquish Core HPLC by ThermoFisher, and 40000 USD for Waters HPLC. It may vary depending on the Software and Detector.

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