In the world of academia, forensics, and research for every question, there is an answer, and for every answer, there is a process. And that process is chromatography. Yet, chromatography would not work if it were not for the small, unassuming HPLC column.
High-performance liquid chromatography or HPLC, as its better known, helps to separate compounds that are dissolved in a solution. The idea is that specific compounds act differently when they interact with water. The HPLC process helps to separate the compounds for further study.
There are four main types of chromatography, so let’s take a look at a few and see how they work.
No matter the type of HPLC column you are using, you will need a machine to help you with your analysis. In the most basic terms, an HPLC machine is where you will place your sample, and it will then travel through the columns, which then separate the compounds for testing.
Within this machine, the compounds move through the system during what is called the mobile phase. The additional substance that is within the compound that helps to separate the element is called the stationary phase. Each one is vital to the process and, if altered, can alter the results of the analysis.
Four Types Of HPLC
When looking at the process of compound analysis, one must first understand their subject matter. The type of compound you are working with will help you to determine which method of HPLC you utilize.
Regardless of the compound and method, every lab tech uses an HPLC machine to help facilitate their study. While there are many moving parts to HPLC machines, it is the columns where we will be focusing our attention. As stated above, there are four main types:
Normal Phase HPLC Columns
When separating the compounds of the sample while in normal phase HPLC, this process uses polarity to complete its operation. This task is accomplished by the stationary phase remaining polar while the mobile stage of the process is nonpolar, with each component extracted at a different time in the process.
Normal phase HPLC is ideal for testing such things as organic acids, specific drugs, and other components. Widely used in the pharmaceutical industry, this method is crucial to our understanding of how various medications interact with one another.
Reverse Phase HPLC Columns
When it comes to utilizing reverse phase HPLC columns, the process is almost identical to the normal phase HPLC. As the process title states, the only difference in the process is that it is reversed from normal phase HPLC columns.
The Reverse Phase HPLC is the most widely used method in chromatography and typically takes anywhere from 10 to 60 minutes to complete.
Ion Exchange Phase HPLC Columns
When separating carbohydrates, proteins, and other amino acids, the Ion Exchange Phase columns is the process used. During this analysis, the catatonic ions have a positive charge, and anionic ion charges are negative, and both charges separate compounds at different rates. This process is widely used in the food and beverage industry to test for the quality of a product.
Size Exclusion Phase HPLC Columns
Another way to test carbohydrates is through the size exclusion phase with HPLC column. The simplest way to think of this process is to think of a filter. Like those who pan for gold, this method uses a filtration process where the compound under analysis is passed through a specific material that has varying sized holes.
The Testing Process
The overall process of HPLC can often be quite tedious, and the poor soul that is assigned to the task must sit there for hours on end monitoring every step of the way.
Injecting the columns is often a manual process, which is why a lab technician must always be present. If you’re one of the lucky laboratories that have in its possession a self-injecting machine, then your job just got a lot easier.
One of the reasons that the HPLC machines are preferred, has to do with the fact that the process utilizes a pump rather than gravity to obtain its results. Additionally, some devices come equipped with programming that allows the lab tech to input all sample data into its system. With this type of automated processing, there is not much to the job other than supervision to ensure the testing goes accordingly.
Uses And Applications
In High-Pressure Liquid Chromatography, the applications are most often found in the medical industry, as well as the food and beverage industry and quality assurance roles of chemistry. When working with analytical data of chemical compounds, the first and foremost goal is to produce accurate data. This is especially important in the Pharmaceutical industries.
Monitoring chemical reactions for various products helps to keep us safe, and the implications of its use in the medical industry are not to be understated. Strong knowledge and a deep understanding of chemistry are required to work in many fields where HPLC is a factor.
Results Are Key
We often hear the phrase; there are many ways up the same mountain, and the same is true in chromatography. Yet, there is one method that offers quicker results that are proven to be more accurate, and that is high-performance liquid chromatography.
Thanks to advances in technology, the HPLC columns are more reliable and provide extremely precise results. HPLC testing can be costly; however, the results, and in-depth analysis, far outweigh the cost.
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