When HPLC was “discovered” over 50 years ago, it stirred up the field of analytical chemistry. Since then, it has become a commonplace technology in the analysis of food products, among many other applications.

Before any food product hits the market, it undergoes several quality and safety checks, one of them being chromatography. Quality control experts apply chromatography in the separation of mixtures, examination of the product for contaminants  that could lead to bacterial spoilage, chemical compounds like pesticide residues and food additives such as colorants, preservatives, antioxidants, artificial flavorings and sweeteners.

As food additives became more common, the Food Additives Amendment Act of 1958 gave the US Food and Drug Administration (FDA) the jurisdiction to regulate food additives. To this end, food companies were obligated to verify they were complying with regulations by using various technologies – one of them being chromatography.

High Performance Liquid Chromatography (HPLC) is a chromatographic technique used to separate, identify and quantify components in liquid samples. It is widely accepted as an invaluable technique for the analysis of many food components. In many instances HPLC methods have replaced laborious analyses and, in general, the chromatographic methods are more specific and precise, coupled with a significant reduction in analysis times.

The primary components in an HPLC system include the solvent reservoir, or multiple reservoirs, a high-pressure pump, a column, injector system and the detector. Each component of the sample interacts differently with the adsorbent material in the chromatography columns being used, resulting in different flow rates for each component, causing them to be separated as they flow out of the column.

Selection of an HPLC system

Once a manufacturer decides to purchase an HPLC system, the next decision is to choose between HPLC or ultra-high-performance liquid chromatography (UHPLC) as they each have their own advantages and disadvantages.

High efficiency chromatography separates the maximum number of peaks in the shortest time frame. To do that, the choice of LC column becomes important and the system hardware must be “optimized” to allow the column to deliver that kind of performance. In general, UHPLC has the ability to separate sample constituents in a shorter timeframe — it is considered to be higher efficiency chromatography, owing to the use of smaller particle LC columns (1.7 to 3 µm). Chromatographic peaks in UHPLC are narrower and sample throughput is higher.  However, there are other factors which must be considered.

A goal-oriented approach to choosing an HPLC system demands that some questions be asked about the specific performance goals like the complexity of the sample analysis, key analytes to be determined, desired sample throughput and sensitivity not forgetting versatility of the system.

Based on this information the consumer might choose UHPLC because of the efficiency of the resulting separation.

For the greenhorn user, HPLC is a more robust, rugged methodology. The robustness/ruggedness of an analytical procedure is a measure of its ability to remain unaffected by small, but deliberate variations in method parameters and further indicates its reliability during normal usage.

Because of the robustness of HPLC, a technician of limited experience could be utilized in place of the higher-experienced chemist for operation, sample preparation, and maintenance. Conversely, UHPLC demands that the highest quality solvents (UHPLC or LCMS grade) be used and that samples be religiously filtered of particulates. UHPLC, while powerful, is far less forgiving than HPLC in this regard.

Beside robustness, capital cost and operating cost for HPLC are lower – instrument cost for HPLC are approximately 20% lower than the UHPLC. The reduced operating cost include utilizing HPLC grade solvents instead of higher cost UHPLC or LCMS grade solvents. Finally, the maintenance frequency for HPLC is lower as well, resulting in a 30-50% reduction in consumables costs (e.g. seals, plungers, autosampler injection valve rotor & stator).

Types of HPLC equipment

The types of HPLC equipment generally depend on the phase system used in the process. The following types of HPLC generally are used in analysis.

Adsorption chromatography

Adsorption chromatography involves the analytical separation of a chemical mixture based on the interaction of the adsorbate with the adsorbent. An adsorbent is a substance which is generally porous in nature with a high surface area to adsorb substances on its surface by intermolecular forces. Some commonly used adsorbents are silica gel H, silica gel G, silica gel N, silica gel S, hydrated gel silica, cellulose microcrystalline, alumina, modified silica gel, etc.

The mixture of liquid gets separated when it passes over the adsorbent bed that adsorbs different compounds at different rates.

Ion pairing chromatography

Ion pair chromatography helps separate charged analytes using the popular reverse phase chromatography.

Ion pair reagents enhance peak shape and retention time when modifying mobile phase ratios or changing stationary phase is not of much help. It is an alternative to ion exchange chromatography. It is a form of chromatography in which ions in solution can be “paired” or neutralized and separated as an ion pair on a reversed-phase column.

Mixtures of acids, bases, and neutral substances are often difficult to separate by ion-exchange techniques. In these cases, ion-pairing chromatography is applied. The stationary phases used are the same reversed phases as developed for reversed-phase chromatography.

Chiral chromatography

Chiral chromatography is a collection of techniques used to separate the enantiomers of a chiral compound. It can be used to analyze the ratio of the enantiomers present in a sample or preparatively to isolate samples of the individual enantiomers.

It involves passing a mobile phase which in this case is liquid, over a chiral stationary phase in a cylindrical column (essentially a skinny pipe). A sample of analyte is injected into the mobile phase. The two enantiomers have different affinities for the chiral stationary phase. As the enantiomers equilibrate between the mobile phase and the stationary phase, the enantiomer that bonds more strongly will spend more time on average bound to the stationary phase. Therefore it will emerge from the column later.

Ion-exchange chromatography

Ion exchange chromatography involves the separation of ionizable molecules based on their total charge.

This technique enables the separation of similar types of molecules that would be difficult to separate by other techniques because the charge carried by the molecule of interest can be readily manipulated by changing buffer pH.

It can be used for almost any kind of charged molecule including large proteins, small nucleotides, and amino acids. Retention is based on the attraction between solute ions and charged sites bound to the stationary phase. Ions of the same charge are excluded. The use of resin (the stationary solid phase) is used to covalently attach anions or cations onto it. Solute ions of the opposite charge in the mobile liquid phase are attracted to the resin by electrostatic forces.

Size exclusion chromatography (SEC) or gel permeation chromatography (GPC)

Size exclusion is a chromatographic method in which molecules in solution are separated by their size, and in some cases molecular weight. It is usually applied to large molecules or macromolecular complexes such as proteins and industrial polymers.

This type of chromatography lacks an attractive interaction between the stationary phase and solute. Sample molecules small enough to enter the pore structure are retarded, while larger molecules are excluded and therefore rapidly carried through the column. Thus, size exclusion chromatography means the separation of molecules by size.

Reversed phase chromatography

Reversed phase chromatography (RPC) is the most common type of HPLC separation technique and is used for separating compounds that have hydrophobic moieties and do not have a dominant polar character. It is the reverse of the normal phase chromatography in which the stationary phase is non-polar and the mobile phase is polar. An example of the mobile phase is organic solvents (methanol, acetonitrile), buffer (phosphate buffer).

Faced with growing demands for increased throughput, improved margins and more efficient processes in the industry, PerkinElmer launched the LC 300 HPLC platform to meet these needs in 2020.

The system has five available detectors, ultraprecise gradient flows, low dispersion, and new Simplicity Chrom CDS software, providing improved workflows, throughput, and usability.

It is expected that the global HPLC market will continue its moderate growth during the next five years.



Technological advancements drive market growth

Progress in any field is expected if not inevitable, as technology advances and as new knowledge is gained.

According to Mordo Intelligence the global HPLC market is expected to witness CAGR of 4.8% over the 2022-2027 forecast period. The primary driving factors for the growth of the market are technological advancements in HPLC techniques. The increasing number of contract research organizations around the world is also anticipated to fuel market growth.

The preference for HPLC over gas chromatography thanks to its reliability and accuracy gives the technology another edge in the market.

These advancements have aided in carrying out high-quality analysis that provides more accurate results in lesser time and enhances the overall convenience for the user. HPLC instrument manufacturers are also developing innovative column designs that can withstand high pressure from smaller, superficially porous particles, which is acting as another major growth-inducing factor. Looking forward, it is expected that the global high-performance liquid chromatography (HPLC) market will continue its moderate growth during the next five years.

However, we all know that with technological advancements comes the need for more competent professionals and higher costs of purchase, factors which are likely to derail the market growth over the forecast period.

The global HPLC market comprises of the following players who have invested in strengthening their product portfolio and research and development operations include Shimadzu Corporation, PerkinElmer Inc., Bio-Rad Laboratories, Inc., Thermo Fisher Scientific, Inc., Agilent Technologies, Inc., Waters Corporation, Gilson, Inc., and Phenomax, Inc., Hitachi, Jasco, to name but a few.

Let’s dive into what some of them have come up with to simplify the analytical world.

Eradicating corrosion issues in stainless steel HPLC

HPLC separation problems caused by corroding stainless steel surfaces have been reported for the analysis of inorganic ions and proteins. Poor electrochemical detection (detection by oxidation or reduction of the sample peak) has sometimes been shown to be due to metal ion contamination found or released from stainless steel surfaces. The appearance of your HPLC’s baseline noise is one of the most useful indicators of its overall performance and cleanliness.

To avert such defects, PerkinElmer has come up with the Next-generation Speciation Analysis Ready system engineered with a completely inert and metal-free fluid path, enabling laboratories to meet low chromatographic background requirements on the most challenging speciation applications. It has an inert HPLC pump engineered to deliver pulse-free and quiet baselines, with a fluid path that is completely metal-free and composed of inert materials. Further, it has an ergonomically designed solvent tray to reduce cavitation and store solvent bottles in an inert holder, preventing leaks or spillages from damaging the rest of the system.

In addition, Shimadzu has introduced the Nexera Bio UHPLC for biomolecule analysis as some proteins can adsorb onto the stainless-steel surfaces and high salt conditions in the HPLC mobile phase resulting in corrosion. With Nexera Bio, crucial metal-free components define the wetted surfaces while maintaining a high-efficiency flow path (66 MPa). The system is unaffected by high salt or ion pairing agents.

Analyzing polar compounds using liquid chromatography has historically been a challenge. Poor retention and peak shape, complex mobile phases that may not be MS-friendly, long equilibration times, low sensitivity, and sample derivatization are all complications that reduce lab efficiency and productivity. However, the development of a novel column that is specifically designed for the analysis of a broad range of polar compounds allows scientists to avoid these problems by taking advantage of the true power of chromatography.

The Quick Polar Pesticides Method (QuPPe-PO) is a method collection for the analysis of highly polar pesticides in food of plant origin and honey. It involves extraction with acidified methanol and Liquid Chromatography with tandem mass spectrometry (LC-MS-MS) measurement.

Version 12 of the QuPPe-PO Method for products of plant origin includes an approach using Restek’s Raptor Polar X column for the analysis of a wide range of polar pesticides using LC-MS/MS.

The Raptor Polar X LC column was specifically designed to address the challenges of LC-MS/MS analyses of polar compounds. It provides excellent retention and separation of polar compounds through its unique hybrid phase chemistry, offering the best of hydrophilic interaction chromatography (HILIC) and ion-exchange retention mechanisms, while still equilibrating quickly and using MS-friendly mobile phases. The Raptor Polar X column can quickly and easily be switched between polar retention modes by simple changes in mobile phase conditions, providing an unprecedented ability to retain and separate a wide variety of polar compounds, even in the same analysis.

Automated process monitoring

The COVID 19 pandemic has accelerated the need for a more flexible work style, driven by increased automation, while attaining consistent, high-quality results for all users.

With this need in mind, Shimadzu designed its “Advanced i-Series” liquid chromatographs which consist of the Prominence-i HPLC and Nexera-i UHPLC systems featuring pressures of 50 MPa or 70 MPa respectively and can be combined with a variety of detectors. So if your need is analyzing a large number of samples or speedily re-processing data at the comfort of your living room, then the Advanced i-Series will serve you just right.

Retaining the excellent basic functions of the compact “i-Series” instruments, the Advanced i-Series boasts increased pressure resistance and additional functions to support remote work. Both the system itself and the dedicated software enable reliable data acquisition for all users, with automation using Analytical Intelligence (AI) to replicate the handling of expert operators.

Companies like ThermoFisher Scientific and ChromSword, a leader in automation development, have also made forward leaps towards HPLC automation. The two companies have tied up to launch an automated high-performance liquid chromatography (HPLC) and ultra-high-performance liquid chromatography (UHPLC) method development system, designed to enable chromatographers to deliver robust and validated methods in less time and with higher confidence.

The Vanquish method development HPLC and UHPLC system provides an integrated, network-deployable solution for automated method development and validation for diode-array, charged aerosol and mass spectrometric detection. The system combines the Vanquish HPLC and UHPLC systems and the company’s Chromeleon chromatography data system (CDS) with ChromSwordAuto and AutoRobust software.

It uses Artificial Intelligence (AI) to minimize manual interaction and enables method creation for complete compound detection with no prior sample knowledge required. The system incorporates multiple detection capabilities, while the integrated Chromeleon Data Vault is designed to reinforce data integrity and compliance.

Chromatographic retention time reproducibility

Are you looking for a rugged, reliable and modern HPLC system that can run established HPLC methods regardless of the brand of liquid chromatograph on which they were originally developed, while at the same time preserving the chromatographic retention time reproducibility of those methods? Well, Waters Corporation has got your back.

Waters Corporation has introduced the Waters Arc HPLC System, a new high-performance liquid chromatograph (HPLC) for routine testing in the food market.

Retention time (RT) is a measure of the time taken for the solute to pass through a chromatography column. It is calculated as the time from injection to detection. The RT for a compound is not fixed as many factors can influence it even if the same GC and column are used.

The system offers ultra-low analyte carryover, superb injection precision and backpressure tolerance to 9,500 psi at 5.0 ml/min. It is designed to meet all the requirements of a top-of-the-line HPLC system in a cost-competitive package that takes routine testing to the next level.

In October 2020, Jasco debuted the LC-4000 Series, the latest in a long history of innovative HPLC systems they have developed, reaching all the way back to the start of commercial HPLC in the early 1970s.

The concept of the integrated LC-4000 series HPLC provides key separation platforms at 50 MPa, 70 MPa and 130 MPa which correspond to conventional HPLC, the increasingly popular Rapid Analysis (RHPLC) and sub 2 μm UHPLC, respectively.

Each platform is supplied with a dedicated pump and autosampler matched to the operating pressure and all three platforms share common detectors optimized for high-speed 100 Hz acquisition and the narrow peak shapes common to both RHPLC and UHPLC.

When we take a step back and reflect on this technology’s journey since inception, HPLC has undeniably made momentous contributions to the methodology of food analysis and this contribution will without a doubt increase in the future.

This feature appeared in the March/April 2022 issue of Food Safety Africa. You can read the magazine HERE