What is HPLC?
Basic principles
How HPLC works
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High-performance liquid chromatography (HPLC) is a broad analytical chemistry technique used to separate compounds in a chemical mixture. These separations utilize the pressure-driven flow of a mobile phase through a column packed with a stationary phase.

The mobile phase carries a liquid sample through the column to the detector, and compounds or analytes separate due to varying degrees of interaction with the stationary phase.

A detector measures the analytes after elution from the column, and a chromatography data system (CDS) translates the detected signal. 

The translated data output of an HPLC analysis is called a chromatogram, where the x-axis is a measure of time and the y-axis measures a specific signal generated by the detector.    

Analyte – target compound(s) of interest for detection in an HPLC analysis

Mobile phase – phase in motion and composed of solvent or eluents flowing from injection to detection 

Stationary phase – the still phase where physical separation of analytes occurs

Flow rate – how fast the mobile phase flows with respect to time

Retention time – time between sample injection and the maximum peak signal of the analyte in a chromatogram

Efficiency – given as the number of theoretical plates, a key metric for quantifying performance of a separation    

Resolution – ability to distinguish between peaks and the primary concern for any separation 

Selectivity – ability to separate two analytes

Void volume – all volume within a column not occupied by the stationary phase.

Limit of detection – the smallest quantity of an analyte which can be reliably detected

Limit of quantitation – the lower or upper quantity of an analyte which can be reliably quantified

Who invented HPLC?

Mikhail Semyonovich Tsvet gets credit for inventing liquid column chromatography. In 1901, he presented an adsorption chromatography method for separating plant pigments with petroleum ether in a narrow glass tube filled with calcium carbonate. Tsvet published his work in 1903 and tested 126 different powdered adsorbents. He was the first to use the term “chromatography” in his papers printed in 1906.

Then 40 years later, in 1941, Archer John Porter Martin and Richard Lawrence Millington Synge published a new type of partition chromatography that used silica gel in columns to keep the water stationary while chloroform flowed through the column to separate amino acids.

This experiment was the beginning of the HPLC development journey, although it took another 30 years before using pumps to push a liquid phase through the packed column.

Today, laboratories worldwide use HPLC to identify and quantify non- and semi-volatile chemical components in liquid samples.

An HPLC instrument has four major components: a pump to deliver the mobile phase, an autosampler to inject the sample, a stationary phase column to separate the sample compounds, and a detector to measure the compounds. Additional elements include connective capillaries and tubing to allow the continuous flow of the mobile phase and sample through the system and a CDS package to control the HPLC instrument, separation, detection, and result evaluation.

Every HPLC analysis includes the following steps:

  1. Mobile phase begins to flow. The pump pushes the eluents or solvents through the system at a specified flow rate.

  2. Sample injection. Once injected into the mobile phase flow path, the sample travels with the mobile phase from the injection point to the head of the column.

  3. Compound separation. Physical separation of the compounds happens on the column stationary phase. After elution from the column, the separated sample components travel to the detector.

  4. Analyte detection. Detection of target analytes based on an electrical signal generated by specific properties.

  5. Chromatogram generation. Translation of the detected analyte signal by the CDS into a chromatogram of analyte signal versus time.

Factors affecting HPLC separations

Many factors, including mobile phase composition, stationary phase chemistry, and temperature influence HPLC separations. Successful separation only occurs if the analytes have differing affinities for the stationary phase, so selecting the appropriate stationary phase for your compounds is crucial. The main factors influencing the overall separation process are: 

  • Physiochemical properties of the analyte, such as size, charge, polarity, and volatility
  • Physiochemical properties of the stationary phase, such as polarity, charge, and viscosity
  • Physiochemical properties of the mobile phase used and interaction with the analyte and stationary phases

 

Isocratic versus gradient separations

All HPLC separations are carried out in one of two modes, isocratic or gradient.  

Isocratic methods separate by using a consistent eluent composition during analysis, like 100% acetonitrile or a 50:50 mixture of acetonitrile to water.

On the other hand, gradient methods include a change in the mobile phase composition across a separation. These methods often employ two solvents, called A and B. The run will begin with a certain percentage of A to B, like 60% water to 40% acetonitrile, for instance, followed by a percentage change throughout a separation.

Gradient separations usually provide superior performance over isocratic modes but are more complex and require advanced pump hardware. 

Given the infinite number of compounds and structural diversity of potential analytes, HPLC is rarely a one-size-fits-all approach. From nano to preparative scale separations, here is a list of the most common types of HPLC techniques and when to apply each.

High-performance liquid chromatography

High-performance liquid chromatography

High-performance liquid chromatography methods are developed on stationary phase particles between 3-5 µm and run at operating pressures up to 600 bar with flow rates between 1-2 mL/min. Standard HPLC is the most widely used liquid chromatography type across various industries.

  • Routine HPLC applications include quality assurance/quality control of small and large molecules in pharmaceuticals, industrial chemicals, and food safety.
Ultra-high-performance liquid chromatography

Ultra-high-performance liquid chromatography

Ultra-high-performance liquid chromatography (UHPLC) methods are based on <2 µm stationary phase particles and run at operating pressures between 600-1200 bar with flow rates from 0.2 – 0.7 mL/min . This increased pressure range offers better resolution and sensitivity, higher throughput, and less solvent usage than standard HPLC systems. UHPLC extends the capabilities of traditional HPLC by allowing users to leverage smaller inner diameter columns and particles and achieve faster analyses.

  • Typical applications of UHPLC are prominent in research and development labs and pharma and biopharma fields for the development and characterization of small molecule drugs, peptides, and antibodies.
Liquid chromatography-mass spectrometry

Liquid chromatography-mass spectrometry

Liquid chromatography-mass spectrometry (LC-MS) utilizes a mass spectrometer instead of the traditional optical detector like a UV-Vis detector. Here the mass-to-charge ratio of the analyte is measured instead of optical properties.

  • Peptide and protein analysis are routine in LC-MS methods.
Low-flow liquid chromatography

Low-flow liquid chromatography

Low-flow liquid chromatography encompasses nano-, micro-, and capillary-flow ranges spanning from low nL/min to about 50 µL/min and provides increased sensitivity due to the associated decrease in column inner diameter, leading to less dilution of analyte bands. These analyses are usually paired with mass spectrometry due to the inverse relationship between flow rate and electrospray ionization efficiency, significantly enhancing method sensitivity.

  •  Low-flow techniques are ideal for high-sensitivity measurements of molecules in complex biological matrices where analyte concentrations can span several orders of magnitude.   
Preparative liquid chromatography

Preparative liquid chromatography

Preparative LC techniques involve  collecting fractionated eluent into discrete sample containers to isolate one or more analytes in order to purify main components or isolate impurities for further investigations. Preparative LC separations fall under three categories: Analytical, semi-preparative and preparative and the goal of the separation determines the scale,  column size and flow rate.

  • Preparative LC separations are for the large-scale purification of mass components and often utilized for manufacturing purposes. Columns usually range from 50-200 mm inner diameter and are run at flow rates of > 50 mL/min. 
  • Semi-preparative LC applications range from small-scale purification for manufacturing to characterization workflows such as isolating compounds for structural analysis. Columns are from 10-30 mm inner diameter and run at flow rates between 5-50 mL/min.
  • Analytical scale purifications find use in upstream and downstream processes, like improving product yields or isolating pure compounds for cryo-EM characterizations. Columns and flow rates fall below 4.6 mm and 2 mL/min, respectively.
Two-dimensional liquid chromatography

Two-dimensional liquid chromatography

Two-dimensional liquid chromatography (2D-LC) is an advanced separation technique using two complementary column chemistries in series for a multi-dimensional separation instead of running the sample through one column. Chromatographers can employ three unique types of 2D-LC methods to help improve sample resolution by utilizing multiple column selectivity.

  • Applications of 2D-LC can apply to complex chemical mixtures like vaccines and foods with interfering sample matrices.

  1. Comprehensive 2D-LC. The entire sample separates in the first dimension (¹D) column and subsequently separates on a second complementary second dimension (²D) column.

  2. Loop heart-cut 2D-LC. Automatically cuts out specific eluent fractions from the first-dimension (¹D) column using a sample loop for transfer to  a complementary second-dimension (²D) column.

  3. Trap heart-cut 2D-LC. Allows you to automatically cut out a single eluent fraction from the first dimension (1D) column onto a trap column. Trap methods allow pre-concentration of low-abundant analytes and address solvent incompatibility issues before the fraction is eluted onto a second dimension (2D) column to resolve difficult or co-eluting peaks.

Dual liquid chromatography

Dual liquid chromatography

Dual liquid chromatography is a multichannel HPLC method using two separate flow paths in a single system to run two analyses simultaneously. These HPLC systems have two pumps with two independent solvent paths, two dosing units inside the autosampler, and two detectors, but keep the footprint of a single HPLC system.

  • Dual LC methods are helpful for any situation when you need to increase your sample throughput, like analyzing a sample for residual pesticides and phenolic content in a single run or performing replicate analyses simultaneously.
Tandem liquid chromatography

Tandem liquid chromatography

Tandem liquid chromatography techniques use a second pump and intelligent column switching to maximize utilization of the detector by minimizing downtime associated with column reconditioning. Tandem gradient runs segment into two main parts: Pump one delivers the analytical gradient to column one while pump two reconditions. Pump one then delivers the analytical gradient to column two while pump two reconditions column one.

  • Tandem LC methods find the best use in applications like lead selection for drug discovery labs to increase sample throughput and maximize detector utilization.
Inverse gradient liquid chromatography

Inverse gradient liquid chromatography

A change in the organic composition over a gradient elution can fluctuate the analyte response for some detectors, such as charged aerosol detection, and complicate analysis. Post-column application of inverse gradient compensation eliminates this effect by ensuring the eluent entering the detector has the exact solvent composition throughout the entire gradient separation.

  • Inverse gradient separations are applied exclusively when using a charged aerosol detector and find use in the pharma field, where quantifying impurities in drugs is essential.

 
 
 
FAQ

What is the basic principle of HPLC?

The separation principle of HPLC is based on the distribution of sample compounds between a mobile phase (from the pump) and a stationary phase (in a column). When passing through the column, compound groups interact differently with the stationary phase and are retained depending on chemical properties, hence, separation takes place.

How does HPLC work?

A pump delivers the mobile phase through a column packed with a stationary phase. An autosampler injects the sample onto the column. The stationary phase separates the sample compounds or analytes. A detector measures the analytes after separation and elution from the column.

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