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Introduction of HPLC - liquid chromatography

What is liquid chromatography?

Historical overview and definition

The definition of liquid chromatography is proposed in early twentieth Century by the Russian botanist Mikhail S. tsweet. He was the first to try to use a solvent to separate the compounds extracted from the plant [leaf pigment] on a column filled with particles. Tsweet particles fill glass column opening. He found two special materials, which were very useful at the end of chalk (calcium carbonate) and alumina. He poured the sample [the solvent extracts of the leaves of the plant] into the column and let the sample flow through the grain and the grain bed. The pure solvent was then added. As the sample flows through the column from top to bottom, it can be seen that the bands of different colors are separated because some components move faster than others. He associates the separate bands of different colors with the original different compounds in the sample. According to the chemical affinity of each compound to the fillers, the law of separation of these compounds was analyzed. The compounds with strong affinity to the particle fillers moved slowly, and the compounds with strong affinity to the solvent moved faster. This process can be described in this way: the distribution of the compounds in the sample is not the same as that of the solid particles (the stationary phase) in the flowing solvent (the mobile phase). This allows each compound to move at a different speed, resulting in the separation of the compounds.

The use of tswett chromatography [chromatography comes from the Greek word chroma meaning, color, graphy means literally record color recording] to describe his color test. [curiously, Russian name tsweet means color. ] today, liquid chromatography, in various forms, has become one of the most powerful tools in analytical chemistry.

The sample of technology 1. was placed on the thin layer chromatography particles fixed on the glass plate and flowing through the thin layer [figure B]. The bottom end of the glass plate is placed in the solvent. The flow produced by capillary action diffuses the solvent [mobile phase] to the dry granular layer and moves upwards along the glass plate. This technique is known as TLC or TLC.


In figure C, in figure C, the sample is placed on a paper [fixed phase]. The solvent [mobile phase] is added to the center of the sample point to produce radiation flow to the surrounding area. This is a way of paper chromatography. [TLC with traditional paper chromatography and linear flow is a similar mode of operation. ] in the above picture, the same black FD and C dyes are placed on the paper.

Graph C: paper chromatography
When compared with thin layer chromatography, please pay attention to the difference between this special paper separation ability. Green rings indicate that the paper is unable to separate yellow and blue dyes, but it separates the red dye. In the next picture, a green sample of the same yellow and blue dye is on the paper. As you can expect, this paper can't separate the two dyes. In the picture, the purple samples made up of red and blue are placed on paper, and they are separated.
Very good.
Technology 3. in this most effective method, the sample flows into a column or a cylindrical tube filled with a proper particle [fixed phase]. These particles are called chromatographic packings. The solvent [mobile phase] flows through the device. In solid phase extraction, [SPE], the sample is loaded into the tube, and the solvent carries the sample out of the device. As in tsweet experiments, sample compounds because of flow velocity in the pipeline to separate different. The black sample was placed in the tube. Each step is separated by using different solvents.


Figure D-1: column chromatography - solid phase extraction of [SPE]
There are several ways to generate flow when the pipeline is used. Gravity or vacuum can be used on columns that cannot bear pressure. In particular, the particle size used in this experiment is larger [greater than fifty microns], so the flow resistance is very small. The opening of the glass column [tsweet] is a typical example. In addition, small plastic columns, typical of the shape of the pin, can be loaded into the filler particles for separation of samples. This method is called solid phase extraction [SPE]. This device is called tubular chromatography with solid phase extraction, usually flow in the vacuum booster, was used for further purification of complex samples. To improve the separation capacity, it is necessary to use small particle size packing particles [less than ten microns]. However, small particles have greater resistance to flow, so higher pressure is required to obtain the desired solvent flow rate. Pumps and columns that can withstand higher pressure must be designed. The method of using high pressure to flow the solvent through the chromatographic column is called HPLC.


Figure D-2: HPLC column
What is high performance liquid chromatography (HPLC) [HPLC]?
The abbreviation HPLC, the Pittsburgh Conference in 1970 after Csaba Horv from Professor th, originally referred to produce the desired velocity in the packed column in need of high voltage (high-pressure) of this fact. The early pump can only bear 500 psi [35bar]. This is called high pressure liquid chromatography ([HPLC]), which has made great progress in the early 70s. The new high pressure liquid chromatography instrument can withstand the pressure of 6000 psi [400 bar], and can also be equipped with an improved sampling device, detector and chromatographic column. High pressure liquid chromatography (HP) has indeed begun to become a general method of analysis in the middle and late 1970s. With the continuous improvement of performance during this period [smaller particles, higher pressure], the letter abbreviation remained the same, but the full name changed to high-performance [HPLC]. High performance liquid chromatography (HPLC) [HPLC] is one of the most powerful tools for analytical chemistry. It can separate, qualitative, and quantify any compound that can be dissolved in a liquid. Today, trace amounts of compounds are even as low as tens of tens of tens of tens of [ppt].



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