Analysis of mixed samples is a common problem in practical work. High-performance liquid chromatography (HPLC) as a good separation and analysis method, has been widely used in the analysis of mixed samples. The detector is the core component of the high-performance liquid chromatograph, which can accurately and quickly detect the components separated from the chromatographic column to achieve qualitative and quantitative analysis. The UV-Vis absorption detector is the most widely used detector in HPLC. Almost all liquid chromatographs are equipped with ultraviolet-visible absorption detectors. However, many substances (such as hydrocarbons that do not contain unsaturated bonds) have no obvious absorption in the ultraviolet-visible spectrum region, which leads to certain limitations in the application of ultraviolet detectors.
Infrared spectroscopy is a powerful means of structural identification. Almost no two substances have exactly the same infrared spectrum. Since most organics have absorption peaks in the infrared spectral region, infrared spectroscopy can be considered a detection method for liquid chromatography. Early dispersive infrared spectroscopy had a slow scanning speed and low sensitivity, which could not meet the requirements of rapid chromatographic determination. In the 1970s, the appearance of Fourier infrared spectroscopy (FTIR) greatly improved the scanning speed and sensitivity, and met the requirements of HPLC detection.
High-performance liquid chromatography-Fourier infrared spectroscopy (HPLC-FTIR) is a combination of HPLC and FTIR. The mixture is separated on a high-performance liquid chromatograph. The sample eluted by the chromatogram is tested on a Fourier Infrared Spectrometer. This technology combines the ability of HPLC to effectively separate thermally unstable components and high-boiling compounds with the diagnostic ability of FTIR to provide a large amount of molecular structure information. It is an effective means to separate and analyze complex organic mixtures.
The key to the hyphenated technology is the development of applicable interfaces. The carrier gas of gas chromatography is usually an inert gas. Inert gases have no absorption bands in the infrared region. Therefore, the interface between gas chromatography and Fourier transform infrared spectroscopy (GC-FTIR) does not need to consider the influence of the carrier gas on the absorption band of the measured fraction in the optical tube. Various forms of GC-FTIR interfaces are currently available on the market.
For samples with high boiling points and poor thermal stability, GC analysis is more difficult. HPLC is not limited by sample volatility and stability. However, the mobile phase of HPLC usually uses organic solvents. Organic solvents often have strong absorption in the infrared region, which seriously affects the infrared detection of chromatographic fractions. Therefore, the interface of HPLC-FTIR must first solve the problem of mobile phase interference. At present, the existing interface technology of HPLC-FTIR can be divided into two types: the flow cell method and the mobile phase removal method.
1. Sample Separation (HPLC stage)
The sample is injected into the HPLC system, where the mobile phase carries it through the chromatographic column. Different components are separated sequentially based on their interactions with the stationary phase, such as polarity, hydrophobicity, or ionic affinity. The column effluent, therefore, delivers distinct analyte fractions in time-resolved order.
2. Transfer and Interface
The separated effluent must be transferred to the FTIR detector. Since common solvents such as water and organic eluents strongly absorb infrared radiation and interfere with detection, specialized interface techniques are used. These include solvent elimination through evaporation or drying, and thin-layer deposition, where analytes are deposited as a film on an infrared-transparent substrate (e.g., ZnSe or CaF2 crystal) before spectral acquisition.
3. Spectral Acquisition (FTIR stage)
Once the analyte reaches the FTIR system, it is irradiated with infrared light. Molecular vibrations, such as stretching and bending of chemical bonds, cause characteristic absorptions at specific wavenumbers. The interferogram signal is then mathematically transformed using the Fourier Transform, resulting in an infrared absorption spectrum unique to each compound.
4. Data Interpretation
By combining chromatographic retention times with the FTIR fingerprint spectrum, both separation and structural information are obtained. This dual-dimensional dataset enables qualitative identification, structural elucidation, and quantitative analysis. Furthermore, spectra can be compared with reference libraries for rapid compound identification.
In the flow cell method, the chromatographic effluent is directly introduced into the flow cell in order for infrared detection. This method does not need to remove the mobile phase. The data processing system of the instrument can subtract the superimposed spectra of the mobile phase and the analyte, subtract the interference of the mobile phase, and obtain the spectrum of the measured substance. This interface is generally only suitable for normal-phase chromatography. For reversed-phase chromatography, a thin inner diameter column and a deuterated solvent are required. The use of microporous columns (inner diameter<1mm) can reduce the amount of mobile phase. Moreover, under the same injection volume, the chromatographic peak concentration of the distillate is much higher than that of a conventional column. The interface of the flow cell is simple in structure and easy to operate, but it still has major limitations. The interference of the mobile phase is difficult to completely eliminate and cannot be used for gradient elution.
The flow cell method is most suitable for relatively simple mixtures and non-aqueous mobile phases, where the analytes are generally organic-soluble small molecules such as fatty acids, esters, or simple aromatic compounds. It is ideal for normal-phase chromatography systems in which solvent absorption does not significantly overlap with the analyte spectra, and for samples that do not require gradient elution or extremely high sensitivity.
The mobile phase removal method is a method that first removes the mobile phase and then performs infrared detection. The interface of mobile phase removal method includes diffuse reflection turntable interface, buffer storage interface and particle beam interface. Various interfaces have their scope of application and limitations. For example, the direct deposition method collects the effluent onto an infrared-transparent window, such as ZnSe or CaF2, where the solvent evaporates and the analyte forms a thin film for spectral measurement. The nebulizer or spray interface, as a variant of direct deposition, atomizes the effluent into fine droplets, allowing rapid solvent evaporation and analyte deposition on a substrate. While these techniques effectively minimize solvent interference and improve spectrum clarity, they require precise control of flow, deposition, and evaporation, and often involve more complex instrumentation compared to a simple flow cell.
The mobile phase removal method is particularly suitable for more complex samples, including natural product extracts with multiple components, pharmaceutical formulations containing excipients or low-volatility active ingredients, and compounds analyzed under reversed-phase or gradient HPLC conditions where solvent interference would otherwise obscure spectral features. It is also appropriate for analytes present at low concentrations that require higher sensitivity detection.
In short, the HPLC-FTIR hyphenated technology is not yet fully mature, its price is still relatively expensive, its application is not universal enough, and it is still in the development stage. However, BOC Sciences offers professional HPLC-FTIR analytical services, combining advanced instrumentation with our experienced technical team to provide reliable and high-quality results. By leveraging our expertise, clients can access the benefits of HPLC-FTIR without the need for significant capital investment and receive tailored analytical solutions for complex samples.
Fig.1 Direct Deposition HPLC-FTIR Using ZnSe3,4.
HPLC-FTIR has emerged as a powerful analytical platform in research and development. By integrating the high separation efficiency of HPLC with the molecular structural identification capability of FTIR, this hyphenated technique allows for the analysis of complex mixtures that are difficult to resolve using traditional detection methods. Its ability to provide both qualitative and quantitative information makes it invaluable across multiple scientific and industrial domains.
In pharmaceutical and biochemical research, HPLC-FTIR is widely used for the characterization of active pharmaceutical ingredients, metabolites, and impurities. The technology is particularly advantageous for analyzing thermally labile or high-molecular-weight compounds that may degrade under traditional analytical conditions. By combining chromatographic separation with infrared structural fingerprints, researchers can elucidate chemical structures, monitor synthesis pathways, and support formulation development. This capability enhances the accuracy of drug discovery studies, quality control, and stability assessments.
HPLC-FTIR also plays a critical role in food, environmental, and chemical analysis. In food science, it enables the detection of trace contaminants, additives, and degradation products in complex matrices. Environmental applications include the identification and quantification of pollutants, while chemical analysis benefits from its capacity to characterize multi-component mixtures such as polymers, surfactants, and industrial reagents. The technique's ability to minimize solvent interference and provide clear infrared spectra ensures reliable identification, even for compounds lacking strong UV absorption.
In industrial and manufacturing contexts, HPLC-FTIR supports quality assurance and process monitoring. It is used to verify the composition and structural features of raw materials, intermediates, and final products. The method allows rapid detection of impurities and batch-to-batch variation, facilitating efficient process optimization. By providing both separation and structural information in a single workflow, HPLC-FTIR improves analytical efficiency, product consistency, and overall process reliability.
Clients seeking HPLC-FTIR services often have a variety of analytical objectives, such as identifying unknown components in complex mixtures, characterizing thermally unstable or high-boiling compounds, detecting low-concentration analytes, or monitoring intermediates in chemical synthesis. Sample types can vary widely, including natural product extracts, pharmaceutical intermediates, synthetic chemical mixtures, polymers, or multi-component environmental samples. Each sample presents unique challenges in solubility, volatility, polarity, and stability, requiring tailored analytical strategies to obtain accurate structural and compositional information. At BOC Sciences, we provide customized HPLC-FTIR solutions to address these specific needs:
By customizing workflows based on the client's sample type and analytical objectives, BOC Sciences delivers reliable, high-quality data while eliminating the need for in-house instrument investment, providing efficient and flexible analytical solutions.
Through this systematic workflow and comprehensive service coverage, BOC Sciences provides high-quality, reliable, and flexible HPLC-FTIR analytical solutions, enabling clients to efficiently and accurately achieve their analytical goals without the need for in-house investment in expensive instrumentation.
What is the difference between HPLC and FTIR?
HPLC separates components of a mixture based on their interactions with a stationary phase and a liquid mobile phase, providing quantitative and qualitative composition data. FTIR identifies molecular functional groups by measuring infrared absorption, giving structural information. HPLC-FTIR combines both techniques to separate compounds and simultaneously obtain structural fingerprints.
What is FTIR testing used for?
FTIR testing is used to identify chemical structures, verify functional groups, confirm compound identity, detect polymorphs, and monitor chemical modifications. When coupled with HPLC, it allows analysis of individual components in complex mixtures.
What does FTIR analysis tell you?
FTIR analysis provides information on the molecular vibrations of a compound, revealing functional groups, chemical bonds, and molecular conformations. It helps confirm identity, detect impurities, and assess chemical integrity.
Why combine HPLC with FTIR?
Combining HPLC with FTIR enables the separation of complex mixtures and the immediate structural characterization of each component. This integration improves analysis efficiency, reduces sample preparation, and ensures precise identification in complex matrices.
What are the applications of HPLC-FTIR in pharmaceutical research?
HPLC-FTIR is applied in drug development for impurity profiling, polymorph identification, stability studies, and verification of synthetic intermediates. It provides high-confidence structural data while analyzing individual compounds from multi-component formulations.
BOC Sciences combines high-performance liquid chromatography with Fourier infrared spectroscopy for advanced structural analysis. Our services deliver clarity in complex molecular characterization.
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