Used laboratory analytical instruments

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High-performance liquid chromatography, like gas chromatography, is a type of chromatography with characteristics such as high selectivity, high separation efficiency, high sensitivity, and fast analysis speed. This article compares the application scope and instrument structure of the two chromatographic methods.

Concepts of Gas and Liquid Phases

Gas Phase

Gas chromatography is a physical separation method. It utilizes the slight differences in the distribution coefficients (solubility) of the components of the substance being tested between two different phases. When the two phases move relative to each other, these substances undergo repeated distribution between the phases, amplifying the originally slight differences to achieve separation of different components.

Liquid Phase

High-performance liquid chromatography is based on classical chromatography and incorporates the theory of gas chromatography. Technically, the mobile phase is delivered under high pressure; the chromatographic column is filled with small-particle packing using special methods, greatly increasing column efficiency compared to classical liquid chromatography (with plate numbers per meter reaching tens of thousands or even hundreds of thousands). Additionally, a highly sensitive detector connected after the column allows continuous detection of the effluent.

Application Scope

Gas Phase

Good separation ability, high sensitivity, fast analysis speed, and convenient operation.

Due to technical limitations, substances with very high boiling points or poor thermal stability are difficult to analyze using gas chromatography. Generally, for substances below 500°C that are not easily volatile or decompose upon heating, derivatization or pyrolysis methods can be partially applied.

Liquid Phase

High-performance liquid chromatography only requires the sample to be dissolved into a solution and does not require vaporization, so it is not limited by the volatility of the sample. It is suitable for organic compounds with high boiling points, poor thermal stability, and high molecular weight (greater than 400), which account for almost 75% to 80% of all organic compounds. In principle, high-performance liquid chromatography can be used for the separation and analysis of these compounds. According to statistics, about 20% of known compounds can be analyzed by gas chromatography, while about 70% to 80% can be analyzed by liquid chromatography.

Gas Chromatography Instrument Structure

Gas Phase

Consists of a carrier gas source, injection part, chromatographic column, column oven, detector, and data processing system. The temperatures of the injection part, chromatographic column, and detector are all controlled.

1. Column Oven:

The chromatographic column is the heart of the gas chromatograph. The various components in the sample are separated through repeated distribution in the chromatographic column, achieving the purpose of analysis. The column oven's function is to house the chromatographic column. Since both ends of the chromatographic column are connected to the injector and detector, the lower ends (connections) of the injector and detector are inserted into the column oven. The column oven can accommodate various packed columns and capillary columns and is easy to operate. The chromatographic column (sample) needs to work under certain temperature conditions, so a microcomputer is used to control the temperature of the column oven. Due to its reasonable design, the temperature gradient within the column oven is very small.

For samples with complex compositions and wide boiling ranges, the column oven can also perform three-stage programmed temperature control. Once the program is set, it runs automatically without manual intervention, and it can automatically open the rear door to exhaust heat during cooling.

2. Injector:

The injector's function is to introduce the sample into the chromatographic column. If the sample is liquid, the injector must also vaporize it. Therefore, a microcomputer is used to control the temperature of the injector.

Depending on the type of chromatographic column and the injection method, five types of injectors are available:

Packed column injector

Capillary non-split injector accessory

Capillary split injector accessory

Capillary split/non-split injector

Six-port valve gas injector

3. Detector:

The detector's function is to convert the chemical signals of the sample into physical signals (electrical signals).

The detector also needs to operate under certain temperature conditions, so a microcomputer is used to control its temperature.

Depending on the chemical and physical properties of various samples, five types of detectors are available:

Hydrogen flame ionization detector (FID);

Thermal conductivity detector (TCD);

Electron capture detector (ECD);

Nitrogen-phosphorus detector (NPD);

Flame photometric detector (FPD);

4. Data Processing System:

This system can perform operations such as collecting, storing, displaying, printing, and processing test data, ensuring the correct execution of sample separation, preparation, or identification.

High-Performance Liquid Chromatography Instrument Structure

Liquid Phase:

The high-performance liquid chromatograph mainly consists of: an injection system, pumping system, separation system, detection system, and data processing system.

1. Injection System

Generally, a septum injector or high-pressure injection valve is used to complete the injection operation, and the injection volume is constant. This is beneficial for improving the repeatability of sample analysis.

2. Pumping System

This system includes a high-pressure pump, mobile phase reservoir, and gradient device. The general pressure of the high-pressure pump is 1.47–4.4×10^7 Pa, with adjustable and stable flow rates. When the high-pressure mobile phase passes through the chromatographic column, it reduces the diffusion effect of the sample in the column and speeds up its movement, which is beneficial for improving resolution, recovering samples, and maintaining the biological activity of samples. The mobile phase reservoir and gradient device allow the mobile phase to change according to the properties of the stationary phase and sample, including altering the polarity, ionic strength, pH of the eluent, or switching to...

3. Separation System

This system includes: the chromatographic column, connecting tubes, and thermostat.

The chromatographic column is generally 10–50 cm in length (if two columns need to be connected, a connecting tube can be added between them) and has an inner diameter of 2–5 mm. It is made of high-quality stainless steel, thick-walled glass tubes, or titanium alloy. The column is packed with a stationary phase (composed of a matrix and stationary liquid) with a particle size of 5–10 μm. The matrix of the stationary phase is made of mechanically strong resin or silica gel, which are inert (e.g., silanol groups on the silica gel surface are largely removed), porous, and have a large specific surface area. Additionally, their surfaces are mechanically coated (as with the preparation of stationary phases in gas chromatography) or chemically coupled with various groups (e.g., phosphate, quaternary ammonium, hydroxymethyl, phenyl, amino, or alkyl groups of various carbon chain lengths) or ligands of organic compounds.

Therefore, this type of stationary phase has good selectivity for substances with different structures. For example, after coupling pea lectin (PSA) to the porous silica gel surface, a specific glycoprotein in fibroblasts can be separated.

Moreover, the small particle size of the stationary phase matrix allows the column bed to easily achieve a uniform and dense state, reducing the eddy diffusion effect. The small matrix particle size and shallow micropores shorten the mass transfer path of the sample in the micropore region. These factors are beneficial for narrowing the band width and improving resolution. According to theoretical analysis of column efficiency, smaller matrix particle sizes result in a larger number of theoretical plates (N).

This further proves that smaller matrix particle sizes improve resolution. Additionally, the thermostat in high-performance liquid chromatography can adjust the temperature from room temperature to 60°C, improving mass transfer speed, shortening analysis time, and increasing the efficiency of the chromatographic column.

4. Detection System

The commonly used detectors in high-performance liquid chromatography are ultraviolet detectors, refractive index detectors, and fluorescence detectors:

(1) Ultraviolet Detector

This detector is suitable for samples that absorb ultraviolet (or visible) light.

Its characteristics: wide applicability (e.g., proteins, nucleic acids, amino acids, nucleotides, peptides, hormones, etc., can all be used); high sensitivity (detection limit of 10^-10 g/mL); wide linear range; insensitivity to changes in temperature and flow rate; capable of detecting gradient elution samples.

(2) Refractive Index Detector

Any sample component with a refractive index different from that of the mobile phase can be detected using a refractive index detector; this detection system is used for carbohydrate compounds. This system is versatile and easy to operate, but it has low sensitivity (detection limit of 10^-7 g/mL), and changes in the mobile phase can cause changes in refractive index. Therefore, it is not suitable for trace analysis or gradient elution sample detection.

(3) Fluorescence Detector

Any fluorescent substance, under certain conditions, has fluorescence intensity proportional to its concentration. Therefore, this detector is only suitable for the determination of fluorescent organic compounds (e.g., polycyclic aromatic hydrocarbons, amino acids, amines, vitamins, and certain proteins, etc.). It has very high sensitivity (detection limit of 10^-12 to 10^-14 g/mL) and can be used for trace analysis and gradient elution sample detection.

5. Data Processing System

This system can perform operations such as collecting, storing, displaying, printing, and processing test data, ensuring the correct execution of sample separation, preparation, or identification.

Analysis of Advantages and Disadvantages of High-Performance Liquid Chromatography and Gas Chromatography

Only 20% of samples can be satisfactorily separated by gas chromatography without chemical treatment, while 80% of organic compounds require high-performance liquid chromatography for analysis.

In gas chromatography, the mobile phase is inert and does not interact with the components, serving only as a carrier. In high-performance liquid chromatography, the mobile phase not only acts as a carrier but also has a certain affinity for the components, allowing separation selectivity to be improved by changing the type and composition of the mobile phase. Additionally, there are many compounds that can be used as mobile phases, providing a wide range of choices.

Another advantage of high-performance liquid chromatography compared to gas chromatography is that sample recovery is easier. By placing an open container at the end of the column, the separated components can be easily collected. Recovery is quantitative and can be used to purify and prepare substances of sufficient purity.

The disadvantage of high-performance liquid chromatography is that, currently, the sensitivity of detectors is not as high as that in gas chromatography. Special attention must be paid to the impact of "extra-column effects" on column efficiency and chromatographic separation.

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