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 as 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 interference 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 point 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: flow cell method and mobile phase removal method.
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 deuterated solvent are required. The use of microporous columns (inner diameter <1mn) 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 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.
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.