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High-Performance Liquid Chromatography For Separation

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Chromatography is an important physical technique that is used for separation, identification, and purification of multi-component mixtures for qualitative and quantitative analysis (Coskun, 2016). There are many types of chromatographic systems such as liquid chromatography, which began in the late 1930s (Guiochon, Felinger, Shirazi and Katti, 2006), and gas chromatography, which was developed in the 1950s (Scott, 1995). A very important type of chromatography known as High-Performance Liquid Chromatography (HPLC) has been developed as a logical improvement to overcome the inherited problems of traditional liquid chromatography (Snyder, Kirkland and Dolan, 2010). HPLC is composed of a detector, a column, a pump, and an injector. HPLC separates chemical substances depending on their relative interaction with the separation media located in the column (Holler, Skoog and Crouch, 2007). The affordability and adaptability of HPLC, have drastically led to the popularity of this separation technique in biomedical, environmental and analytical laboratories. In addition, the use of sophisticated computers provided useful means data processing (Pang, Zhu, Lu, Gu and Chen, 2016). Unfortunately, samples must undergo multi-step sample preparation procedures prior to their introduction into the HPLC system thus it might create experimental errors regardless of the efficiency of the separation technique (‘HPLC’, 2018). The HPLC has been recently used many fields such as the pharmaceutical industry, environmental and food analysis (Hassan, 2012). The near future of HPLC is dedicated to productivity, incorporation of technology and system performance enhancements to deliver greater efficiency, automation, and speed of separation (Cross, 2018).

Historical Background

Chromatography is unique in the history of analytical methodology and was born as a preparative technique at the beginning of the last century when there were no physical methods of analysis and when the acquisition of chemical data was slow and limited to a few parameters of low specificity (Guiochon, Felinger, Shirazi and Katti, 2006).

1890s to 1910s

The first chromatography known as “classical column chromatography” (see figure 1a-d) was discovered by the Russian botanist Tswett who employed a primitive form of liquid chromatography. After a condemnation of the method by other scientists, the importance of chromatography was not recognized (Scott, 1995).

LC was introduced but the progress was slow and desultory. The development of LC was slow. It was developed to simpler form called “paper chromatography” using a strip of paper instead of the column and was later modified by coating a thin layer into a glass plate (see figure 1f). On the other hand, the development of GC was very fast and caused that LC was neglected for a decade (Snyder, Kirkland and Dolan, 2010).

1960s to 1980s

HPLC, which represents the modern culmination of the development of LC, was introduced (see figure 1g) with pumps for faster separation and reusable and more effective columns (Lough and Wainer, 1996).

1990s to Present

right1606550Figure 2. The expanding importance of HPLC research and application. (a) The number of publications per year, (b) Total sales of HPLC equipment per year.

HPLC is now probably the most powerful and versatile technique available to modern analysis. Progressive improvements in HPLC causes separation times decreasing and better control for the overall process for more precise and reproducible results. The growth of HPLC following its introduction shows its importance nowadays (see figure 2) (Snyder, Kirkland and Dolan, 2010).


HPLC has two main phases: the mobile phase and stationary phase. It consists of a pump, a column, a detector, and an injector (see figure 4). Each one has its own purposes.

The injector places the sample into the flowing mobile phase. It works within a loop which rotates to control solute injecting operation. In addition to that, the injector can be used for good precision, and it is problem free. (Reuhs and Rounds, 2009).

The analytical column can be made of stainless steel, glass, silica titanium and titanium, and polyether ether ketone (PEEK) resin. It is packed with tiny molecules. The efficiency of the column varies 10-15% for equally packed columns. Usually, it becomes with Size: 5 cm × 50 cm. The most commonly used is 10, 15, or 25 cm long (Reuhs and Rounds, 2009).

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The precolumn is a short and expendable column which called guard column. It helps and protects the analytical column (Reuhs and Rounds, 2009).

The detector records the components of the solute as it leaves the column individually. After that, it sends an electrical, spectrochemical, or electrochemical signals for the concentration of each component of the solute.

Moreover, the detector has many types. One of the most widely used types is the UV-Vis absorption detector, which has some organic compounds that absorbed the UV-Vis radiation at a longer wavelength. Additionally, the electrochemical detector based on electrochemical oxidation-reduction reactions or on changes in the conductivity of the solute. The Refractive Index detector (RI) measures the changes in the RI of the mobile phase and it is a universal method of detection (Reuhs and Rounds, 2009).


A reservoir that has the solvent and is connected to a high-pressure pump is used to generate the flow rate of the mobile phase. After that, the injector is on injecting position that allows the sample to be carried with the mobile phase into the HPLC column. In the same time, the extra amount of the sample is going to waste tank. Then, the stationary phase, which is in the column, is holding the sample by reacting with its components to separate them. Finally, the detector records each component leaves the column and transmits an electrical signal to computer data system (“How Does High Performance Liquid Chromatography Work?”, 2019).


The affordability and adaptability of HPLC have drastically led to the popularity of this separation technique in many fields such as biomedical, environmental, and analytical laboratories. Nollet expresses (2010, p.156) that HPLC is flexible. This beneficial invention allows you to test great diversity samples. It is extremely quick and efficient. The time of the completed process can be roughly 10 to 30 minutes, and it delivers a high resolution. It is accurate and highly reproducible. Because HPLC is largely automated, it can be used with minimal training. HPLC is versatile and extremely precise when it comes to identifying and quantifying chemical components. According to Cserhati (2008, p.141), the HPLC techniques allow the successful separation and qualitative analysis of analytes.


Despite its advantages, HPLC can be costly, requiring large quantities of expensive organics. It also is costly when sophisticated air pumps and vacuum pumper are needed. Samples must undergo multi-step sample preparation procedures prior to their introduction into the HPLC system thus it might create experimental errors regardless of the efficiency of the separation technique. Nollet notes (2010, p.156) that the additional functionality increases the number of parameters which can be manipulated during method optimization, making the process more complicated. He also adds that the molecular diffusion is a little bit slow in liquids. Some techniques were inadequate for quantification of compounds and resolution between similar compounds. In HPLC we deal with the time-dependent process. Chromatogram produces noisy baseline sometimes because of big peaks or much higher frequency than actual chromatographic peak (see Figure 5).


It can be used in more than one field such as in the Pharmaceutical industry, environmental and food analysis.

Pharmaceutical industry

According to Hassan (2012), one of the most commonly used of HPLC is to make a test about its impact on human and to determine the raw elements inside it. In addition, The United States of America sets conditions for the sale of these products, which is to detect the quality before selling by using HPLC.

Environmental Analysis

HPLC plays an important role in the analysis of environmental samples such as soil and water after proper processing. For instance, polycyclic aromatic hydrocarbons are well known carcinogenic molecules that can be found near industrial facilities. Their monitoring via analytical methods such as HPLC is crucially important for ensuring the safety of humans and animals in close regions 474980048260000(Mezzogiorno,2010)

Food Analysis

Chromatographic systems such as HPLC are widely used in the area of analyzing pesticides and herbicides. The ability of HPLC to detect these chemicals is well founded and empowered by using advanced instrumentations to assist the quantitative and qualitative analysis of these chemicals in vegetables and fruits prior consumption (Rajput, Kumari, Arora, Kaur,2018)


Chromatography is unique in the history of analytical methodology and was born as a preparative technique at the beginning of the last century when there were no physical methods of analysis. HPLC is probably the most powerful and versatile technique available to the modern analysis with two main phases. It consists of many components which efficiently work together to separate chemical substances. Although HPLC has many disadvantages, it has a lot of advantages which make it widely used in many fields.


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