New to HPLC? Tips for Avoiding Beginner Pitfalls

Operating an HPLC (High-Performance Liquid Chromatography) system can be overwhelming for a new user. There are numerous pitfalls or “gotchas.” Even the matter of which tubing and fittings are compatible, and which are not, can pose a serious impediment to successfully operating the HPLC.

In this discussion, we will cover:

  • The significance of connections
  • Considerations for the mobile phase
  • Quality considerations
  • Preventing microbial growth
  • Compatibility of sample and solvent
  • Characteristics of the column; their significance and limitations
  • The importance of sample preparation



Rita Steed
Application Engineer
Agilent Technologies, Inc.

Rita Steed began supporting the LC column line for Agilent in 1999. As a Chromatography Specialist (first with Chromatography, Inc., then directly for Agilent), Rita worked on-site with researchers in Pharmaceutical and other industries presenting technical seminars and assisting researchers with troubleshooting and method development. Rita has over 20 years of Chromatography experience in the Biotechnology, Chemical, and Pharmaceutical industries. She has held positions in Research, Sales, and Technical Service. Rita has earned degrees in Microbiology and Life Sciences/Biochemistry. In her current position, she is an inside Application Engineer supporting LC columns.

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Fingerprinting Honey to Ensure Purity

How pure is that honey in your jar?

Although there’s a rising demand for honey, the honey bee population is also under threat. Another not-so-sweet issue is the number of products labelled as honey on retail shelves that don’t meet the criteria to be classified as pure honey.

The term “adulterated honey” means any honey to which has been added honeydew, glucose, dextrose, molasses, sugar, sugar syrup, inverted sugar, or any other similar product or products other than the nectar of floral exudations of plants gathered and stored in the comb by honey bees.

Food fraud is a significant concern for consumers and producers, with research indicating that fraud accounts for up to 25% of all globally reported food safety incidents. The growing demand for food authenticity means consumers regularly pay a premium for organic and sustainably produced goods like honey. Fraudsters have been flooding markets with adulterated, low-quality, or mislabeled foodstuffs, damaging the livelihoods of legitimate businesses and potentially risking consumer health.


Increasing demand

Consumers have become quite specific in their demand for honey, focusing on unifloral honey or monofloral honey obtained predominantly from bees that feed on a single species of plant flowers. This results in a unique colour, flavour, and fragrance exclusive to each type of unifloral honey. As consumers are willing to pay more for these products, protections must ensure that they purchase what they expect.

According to data from the Food and Agriculture Organization of the United Nations, China, Mexico, Russia, Turkey, and the United States are among the major honey-producing countries accounting for approximately 55 per cent of world production. The most common form of adulteration involves extending or diluting honey with other less expensive sweeteners. Commonly identified extenders are corn, cane, and beet syrups.


Testing for authenticity to mitigate honey fraud

Global e-commerce is placing honey sales outside regulatory oversight more frequently—a trend expected to continue. This, combined with increased fraudulent activities, makes tackling the problem critical. This is why it is important to identify these substances quickly, efficiently, and consistently. The food industry requires analytical instruments and testing techniques to consistently and rapidly analyze food and identify trace chemicals.

Analytical testing is essential for assessing food authenticity, which is important to protect consumers’ health, the brand, and producers’ income. Testing is a necessary part of an overall strategy to mitigate fraud risk, and methods for authenticity testing are rapidly evolving, with innovative technologies now available for developing robust food testing techniques.

Agilent 1290 Infinity II LC System

For example, it has been demonstrated in recent years that coupling high-performance liquid chromatography with quadrupole time-of-flight (LC/Q-TOF), such as the Agilent 1290 Infinity II LC System with Agilent 6545 LC/Q-TOF, provides a sensitive method to reveal the chemical composition of honey samples. Using this method with a non-targeted approach enables the identification of new types and sources of fraud through the chemical markers in the honey, highlighting which kind of fraudulent activity is occurring. Since this technique evaluates multiple markers in honey to determine authenticity, it is very difficult for fraudsters to cheat by adding one or a few adulterants. This innovative technique is called honey fingerprinting.

Agilent 6545 LC/Q-TOF


Determining honey’s unique chemical composition

Honey fingerprinting is the practice of using a suitable technique to record as much information as possible on the chemical composition of a particular honey sample. In the same way, a human fingerprint is unique to individuals, this fingerprinting method unlocks and records the unique molecular composition of authentic honey samples. This enables the mapping of food components in an unprecedented fashion that will revolutionize how honey is regulated for quality, safety, and authenticity.

Utilizing a non-targeted workflow begins with identifying other compounds, including pesticides, molecules that indicate freshness, like a compound called HMF (which suggests thermal processing or age if present in high numbers), and phenolic compounds, which are related to the floral origin of honey. The advantage of using LC/Q-TOF for this technique is its efficiency: higher molecular/trace information levels can be obtained from just one sample in less time versus targeted methods focusing on just a few parameters.


Standardising honey fingerprinting methods

Although previous work has been done developing case studies for fingerprinting foodstuffs, including honey, the approaches among laboratories have been different regarding sample preparation and instrumental condition. There are also differences in terms of data processing and analysis. As a result, two laboratories analyzing the same sample may obtain slightly different results. Ideally, developing a standardized fingerprinting method that could be used across all LC/MS-based workflows, enabling the same testing technique to be used across multiple laboratories, would be optimal and where future work is aimed.

When addressing the issues of food safety, product quality, and authenticity, each may be governed by separate sets of regulations. For example, looking at the residues of contaminants in honey, such as pesticides, there may be differences globally. Countries may have their own restrictions for the maximum limit for specific compounds. Contaminants are a part of the picture when considering fingerprinting for honey, but permitted levels may vary between countries.

Additionally, as samples come from the field to the lab for testing, there is potential interest in reversing this and bringing the lab out into the field instead. This interesting but not yet recognised capability would enable regulators and the global food industry to respond more quickly to honey contamination and food fraud.


Taking a global approach to ensure honey purity

As the food supply chain becomes increasingly globalized, raising the opportunity for food fraud, experts predict that testing, such as those described above, will become more accessible, increasingly automated, and easier to perform. Fingerprinting methods—in which the entire molecular profile of food can be obtained—will be a feature of future fraud prevention and identification systems.

A positive step forward is the focus on building a library of authentic honey samples and making it an accessible, open database so that honey fingerprinting information is available across multiple stakeholders in the global supply chain. With increased knowledge, more scientists will be able to adopt techniques such as LC/Q-TOF and could also use this testing for other types of food—for example, maple syrup.

The ultimate goal is for food testing laboratories to confidently measure contaminants that threaten the global food chain and tackle food fraud head-on to ensure that consumers can access authentic and safe honey.

(This article has been modified from its original appearance on the Agilent website)

Preventive and Routine Maintenance for Your HPLC System

Similar to our cars needing regular oil changes, tire pressure adjustments, or washer fluid top ups, our HPLC systems also need routine maintenance in order to keep them performing at their best.  Even if you pay a mechanic to change your oil or a Field Service Engineer to change your pump seals, you should still know how frequently these preventive maintenance activities need to occur.


This presentation will look at several key issues, such as:

  • How often typical preventive maintenance (PM) tasks should be performed.
  • What signs to look for that may indicate a need for maintenance.
  • Tools for testing the LC systems before and after maintenance.
  • Basic troubleshooting and diagnostics.

The goal of this talk is to lay a strong and broad foundation from which to build a better understanding of your own HPLC systems and their specific maintenance needs.


Paul Altiero
Application Engineer
Agilent Technologies, Inc.


Paul Altiero is an Applications Engineer located at the Agilent Little Falls site in Wilmington, DE. Prior to coming to Agilent in 2009, Paul worked in the pharmaceutical industry performing LCMS bioanalysis supporting Drug Discovery DMPK. While at Agilent, Paul has been a field service engineer for LCMS as well as the integrating program manager for the RapidFire high throughput LCMS system. In his current role Paul’s responsibilities include technical support and application assistance for LCMS consumables.


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HPLC Column Care

Are you just getting started with a new column you have never used before? Are you looking for ways to help your columns last a bit longer? These are some of the most common questions asked of our HPLC columns technical support team. In this webinar, we will discuss best practices for HPLC column care, including reversed phase, SEC, HILIC and more. This will include initial equilibration, benchmarking a new column, column storage, and other tips to help make your columns last longer.


Mark Powell
Application Engineer
Agilent Technologies, Inc.


Mark Powell is an Applications Engineer located at Agilent’s Little Falls site in Wilmington, DE. Before joining Agilent in 2011, Mark worked in the pharmaceutical industry synthesizing, purifying and analyzing drug candidates. He provides applications assistance and technical support for Agilent’s HPLC columns and consumables.


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InfinityLab OnLine HPLC Solution

In modern production of small molecule pharmaceuticals, biopharmaceuticals, and, in fact, ALL production batches, the reaction has to be closely monitored and potentially even controlled by, for instance, online reaction monitoring analytics. Therefore, connecting a UHPLC instrument by a sampling device to the reaction vessel can be helpful. The 1260 Infinity II Prime Online LC offers a combined UHPLC and integrated reactor sampling interface for automated reaction sample analysis with the Agilent 1260 Infinity II Online Sample Manager. This device enables the drawing of samples from a reactor and dilution/quenching prior to an injection.

This presentation will include a look at the hardware and software used for the Agilent OnLine LC system.  We will log into a live system to view the setup possibilities and review data acquired on the system.


Bob Giuffre
Application Engineer
Agilent Technologies, Inc.


Bob Giuffre has been working with HP/Agilent for almost thirty years as an Application Scientist for the HPLC product line. During that time Bob has worked on applications ranging from small molecule pharmaceutics to large molecule polymers, to monoclonal antibodies. Currently, Bob is working in the area of ultra-high pressure two-dimensional liquid chromatography as a means to increase peak capacity for complex formulations. Bob has authored a chapter on the use of Diode-Array Detection in Pharmaceuticals. He is currently writing a chapter on Troubleshooting Peptide and Proteins Analysis using LC-MS.


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Boost your Performance with 2D-LC Separation

This scientific presentation is for analytical chemists, chromatographers, and mass spectrometrists that want to learn, how an additional chromatographic dimension can deliver deeper insights into their samples, particularly when integrated with a mass selective detection system.

Several concepts of the different types of 2D-LC will be explained to enable a comprehensive understanding of this well-established technology.

The benefits of comprehensive Multiple Heart-Cutting, High-Resolution Sampling 2D-LC and the unique technique of Active Solvent Modulation will be explained in detail. Real-life examples will be shown to illustrate the advantages that incorporating an additional separation dimension brings to LC and LC/MS analysis.


What you will learn

•    First principles of 2D-LC technology
•    How to configure a mass detector with 2D-LC
•    How 2D-LC technologies enhance the quality of MS detection
•    Where 2D-LC makes a positive difference with several application examples


Dr. Jens Trafkowski
Product Specialist HPLC
Agilent Technologies


Jens Trafkowski studied food chemistry and received his PhD at the Institute of Legal Medicine from the University of Bonn for his work on applications of HPLC-MS/MS in forensic and clinical toxicology.

Prior to joining Agilent in 2011, Jens spent more than 6 years as LC-MS as an application specialist in the industry before leaving to undertake a global HPLC product management responsibility at Agilent Technologies. Later, in 2017 Jens moved into his current role as an HPLC product specialist where he is based in Basel Switzerland.


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