top-banner

METHODS AND TECHNOLOGIES

High Pressure Liquid Chromatography

HIGH PRESSURE LIQUID CHROMATOGRAPHY

Chromatography is a technique used to separate a mixture into its individual components by exploiting their differing interactions with a mobile phase and a stationary phase. In High-Performance Liquid Chromatography (HPLC), the analyte mixture is dissolved in a solvent and separated within a column packed with solid particles. The separation process requires elevated pressure (100–300 bar) to move the sample through the column efficiently.

Device design

The HPLC system consists of the following components:

  1. Mobile phase: Storage containers hold various liquids (organic solvents, aqueous solutions, or buffers) that serve as the mobile phase.
  2. High-pressure pump: Moves the mobile phase through the column at a constant flow rate.
  3. Mixer: Solvents are combined in a mixer to achieve a desired composition, which can be adjusted during the separation process (gradient separation).
  4. Autosampler: Introduces a precise volume of the prepared sample into the system.
  5. Column: This is where the separation of analytes occurs based on their interactions.
  6. Detector: Converts the analytes chemical signals into electrical signals. Optical detectors such as UV/VIS or fluorescence are commonly used, depending on whether the analytes absorb UV light or emit fluorescence.
  7. Evaluation unit: Software generates a chromatogram for analysis.
  8. Waste container: Collects the processed samples after detection.

Separation principle

The sample is injected at one end of the chromatography column, which is packed with small, solid particles. The column is flushed with the mobile (moving phase), causing the mixture to move continuously through the column. The chemical properties of the analytes, primarily polarity, affects interactions between the analyte and the mobile or stationary phase.

Substances with the strongest interaction with the mobile phase move faster through the column in the running medium, thus reaching the detector sooner.

Substances with greater interaction with the stationary phase move more slowly, eluting later.

By altering the mobile phase composition, the separation of complex mixtures can be optimized.

Chromatogram and data analysis

The separation process is visualized in a chromatogram, which plots retention time (time analytes spend on the column) against signal intensity measured by the detector. The detected measurement signal changes in proportion to the analyte concentration. The resulting peaks have Gaussian distribution. To evaluate the data, the peak areas are integrated and compared with calibration data from known concentrations for quantification.

Advanced HPLC technology

Ultra-performance liquid chromatography (UPLC) improves upon HPLC by utilizing smaller column particles and higher total pressures (up to 1000 bar) to improve separation performance with shorter analysis times.

WAYS TO PARTNER WITH US

TarCET IVD Kits

CE-IVD kits containing reagents
for hereditary indications in
an easy-to-use kit form

Learn more

Technology Transfer

Out-of-the-box genetics workflow
from sequencing to reporting
for non-invasive prenatal testing

Learn more

Genetic Testing Laboratory

Your partner for
all genetic tests

Learn more

Contact Us

Please get in touch with us
for any questions, inquiries,
feedback, or with any
comments you might have.

Learn more

BLOG ARTICLES

February, designated as Cancer Awareness Month, marks a time to reflect on the remarkable progress made in cancer research and to inspire hope for th…

Read more

Introduction Cardiovascular disease (CVD) is a major health issue as it remains a leading cause of death worldwide [1]. CVD is responsible for mor…

Read more

Antimicrobial resistance (AMR) is one of the most pressing global health threats, and accurate identification and surveillance of multidrug-resistant…

Read more

Colorectal cancer remains a significant health concern globally. While genetic factors play a crucial role in its development, identifying the exact …

Read more

A new meta-analysis links trans-kingdom gut microbiota (bacteria, eukaryotes, viruses, archaea) to immune checkpoint inhibitor (ICI) response in canc…

Read more

Reproductive health is a fundamental aspect of human well-being, affecting individuals and communities worldwide [1]. It encompasses a wide range of …

Read more

It seems as though everyone is talking about artificial intelligence, usually referred to as AI, these days! Indeed, not only are AI tools now access…

Read more

Orphan drugs are those developed specifically for the treatment of rare diseases. Within the pharmaceutical industry, the drug development process is…

Read more

A study of 629 pregnancies with ultrasound-detected anomalies found that exome sequencing identified pathogenic variants in 14% of cases. The detecti…

Read more

Breast cancer is a type of cancer that originates in the breast cells. Genetic changes in the DNA of the healthy breast cells can lead to the formati…

Read more

OUR NETWORK