HPLC/UHPLC/SFC Systems

Key principles of HPLC technology

HPLC is an analytical technique that uses liquid to separate a sample mixture into individual components so they can be determined and recorded in a chromatograph. A powerful tool in analytical chemistry, HPLC is a means to gain highly sensitive and selective quantitative and qualitative data pertaining to the molecular structure of a target compound and, as a result, is utilized frequently in the fields of forensics, pharmaceuticals, food safety and environmental testing. Some of the key components of chromatographs are devices dedicated to molecular separation, known as columns, and high-performance pumps for delivering solvent at a stable flow rate. As these technologies have progressed, greater resolution and greater precision in the analysis of complex mixtures have been afforded by the creation of HPLC, which also delivers high-speed results.

How does HPLC work?

HPLC analysis begins with preparing a sample mixture of the matter to be tested. There are two phases of HPLC analysis – the mobile phase and the stationary phase. The sample solution consists of the liquid that is miscible with sample solution containing the target compound. The stationary phase is the part of the column that interacts with the target compound. The sample solution is injected into a separation column, otherwise known as the stationary phase, and then to the detector at a stable flow rate controlled by the solvent delivery pump. The compounds contained in the sample are separated in the column before being detected by a detector. Once eluted (or removed), each compound is identified and quantified in a chromatogram.

Advanced features of Shimadzu HPLC systems

Shimadzu is a pioneer in the advancement of HPLC systems, leading with accuracy and efficiency. Shimadzu’s HPLC systems are highly reliable, with the equipment renowned for outstanding performance. Our HPLC machines are invaluable because they’re carefully designed for broad applications and easy operations. They’re also prized for ultra-low carryover, exceptional area reproducibility and superior data quality, with additional support from software, components and accessories tailored to the needs of the user.

Revert back to HPLC Applications

HPLC applications are very broad, as the level of analysis is incredibly sensitive, accurate and reliable. HPLC is invaluable in the fields of clinical research, toxicology, and forensics, as it can detect trace compounds in blood and urine. Pharmaceuticals, including the cosmetics industry, find HPLC analysis vital for testing for allergens and accurately quantifying the molecular structures of products. Similarly, food and beverage companies look to HPLC to identify allergens and maintain quality and consistency in products. And, for its ability to qualify and quantify pollutants and pesticides, HPLC analysis is a powerful tool used by environmental scientists. You’ll find Shimadzu’s range of application-specific instruments here.

Selecting the right HPLC column for your analysis

Choosing the right HPLC column for your analysis depends on your goal. The dimensions of the HPLC column you’re choosing will be better suited for certain outcomes. For example, if you’re looking for fast analysis, you’ll want a shorter column length in your HPLC instrument. Alternatively, a longer column length will optimize the resolution of the target compounds of constituents. If you want to minimize solvent consumption, choose an HPLC column with a narrower inner diameter. Considering your analysis goals, such as sample throughput, resolution and target chemistries, you can narrow down the right HPLC column for your needs.

Automated solutions with Shimadzu’s Analytical Intelligence

Shimadzu’s recent innovations have seen HPLCs incorporate cutting-edge advancements to enable higher productivity and maximum reliability. Shimadzu LC’s are now equipped with Analytical Intelligence features that allow automated support of many functions that previously required user attention. For example, automated startup and auto-purge will prepare the instrument for the operation before the analyst gets into the lab, thus, reducing the operator time required for the instrument preparation and increasing the instrument throughput. The Flow Pilot feature will gradually increase the flow rate until the complete system warm up, allowing to protect valuable columns and save costs on buying expensive lab consumables. The Auto-Diagnostic and Auto-Recovery feature detects the pressure fluctuations, stops the analysis, and performs auto-purge and system recovery, which prevents data loss, sample waste, and time-consuming reruns. In summary, Analytical Intelligence features allow Shimadzu instruments behave as if they were operated by an expert, streamlining issues and prioritizing efficiency.

Frequently Asked Questions

What is HPLC, and how does it work?

HPLC is an analytical technique that separates compounds dissolved in a liquid sample. It allows quantitative analysis of respective components that are contained in the sample.

What is the principle of separation in HPLC?

HPLC seeks to separate compounds in a sample solution based on their interactions with a mobile phase – a liquid – and a stationary phase – a solid adsorbent or a liquid-coated surface of a packing material. The sample solution is injected into a separation column in which, the stationary phase is immobilized, and then introduced to the detector at a stable flow rate controlled by the solvent delivery pump. The compounds contained in the sample are separated in the column before being detected by a detector, and once eluted from the column, each compound is quantified in the form of chromatography.

Which columns are used in HPLC?

HPLC columns are categorized into the following:

• Normal-phase columns: Used for the separation of low-polarity compounds such as steroids and lipids, which have excessive retention in reversed phase mode.
• Reversed-phase columns: Used for separating synthetic polymer and compounds ranging from small to medium-sized molecules including pharmaceuticals and peptides.
• Size-exclusion columns: Used for separating large biomolecules, including proteins and nucleic acids.
• Ion-exchange columns: Used for separating inorganic or metal ions and ionizable organic compounds based on their charge.
• HILIC columns: A kind of normal-phase column, used for separating highly polar compounds, such as sugars and nucleic acids, which are difficult to retain in reverse-phase mode because of the action of the hydrated layer on the surface.

What are reversed-phase and HILIC HPLC?

Depending on the compounds analyzed and the separation requirements of the analysis, you may need to choose between reversed-phase columns or HILIC columns. If you’re aiming to separate polar compounds such as sugars and alkaloids, you’ll look to HILIC chromatography, as polar compounds are more easily retained by polar stationary phases. Alternatively, reversed-phase chromatography is ideal if your ambition is to separate mixtures with less polarity.

What is the main difference between HPLC and UHPLC?

UHPLC offers time based greater separation efficiency than HPLC. This is because UHPLC can be operated at higher pressures thanks to smaller particle sizes of the column packing material. This also affords reduced solvent use.