Introduction to HPLC
High-Performance Liquid Chromatography, or HPLC, is a part of column chromatography. It is a method of separating substances that involves forcing a liquid or solid sample (also drugs or phytochemicals) that has been dissolved correctly along a column while applying a lot of pressure. The purpose of this technique, which can also be referred to as High-Performance Liquid Chromatography, is to isolate, characterize, or analyze each component that makes up a mixture. Therefore, high-performance liquid chromatography (HPLC) is essentially an advanced version of column chromatography. Instead of allowing a solvent to trickle through a column due to gravity slowly, high pressures of close to 400 atm are used to drive solvent through the column.
In high-performance liquid chromatography (HPLC), a sample is broken down into parts by using the relative affinity of different substances for the mobile and stationary phases employed in the separation process. This is the fundamental idea behind HPLC. High-performance liquid chromatography (HPLC) separates analytes between a mobile (eluent) phase and a stationary phase (which is called the packing material of the column). Depending on the analyte's chemistry, molecules enter the stationary phase slowly. Forces between sample molecules and packing material determine how long a sample stays "on-column." Consequently, the components of a sample elute at various intervals, which allows for the achievement of the desired separation. The following are examples of instruments used in HPLC:
Solvent: The term "mobile phase reservoir" refers to both the solvent reservoir and the mobile phase reservoir. It is not recommended to use a solvent with a high viscosity because it requires a higher operating pressure and takes much longer to go through the column. Because of this, peak broadening occurs; therefore, avoiding using such a solvent is best. The characteristics of the sample and the degree to which the detector is sensitive will determine which solvent is used.
Pump: The purpose of the pump is to push a liquid, also called the mobile phase, through into the liquid chromatograph at a predetermined flow rate, which is expressed as several milliliters per minute. During the chromatographic experiment, a pump can either maintain a constant composition of the mobile phase or gradually change the composition of the mobile phase (known as a gradient).
Injector: The injector is the component that is used to introduce the sample solution into the flow stream of the mobile phase. The amount of each sample can range anywhere from 5 to 20 microliters.
HPLC column: Chromatograph's column is often referred to as the instrument's "heart." The length of the column can range anywhere from 5 cm to 30 cm, and its diameter can be anywhere from 2 mm to 50 mm. For the most part, stainless steel is employed as a material for building the tube, whereas silica and alumina particles are utilized for packing. A pump draws the mobile phase from the solvent reservoir and directs it to move through the column toward the detector.
Detector: The detector is responsible for identifying individual molecules as they pass through the column. This information is then sent to a recorder or a computer, which ultimately results in a liquid chromatogram.
Data analyzer: It receives the signal from the detector and processes it in order to determine the duration of elution, which is known as retention time, of the components of the sample that does the qualitative analysis, as well as the amount of sample that does the quantitative analysis.
Figure: Process of separation of a compound from a mixture and its analysis.
The different kinds of HPLC are chiral HPLC, where geometric isomers, water-sensitive compounds, and cis-trans isomers are characterized. In the reverse phase, polar, ionizable, non-polar, and ionic samples can all be analyzed with it. Ion exchange is employed in the process of cation and anion separation. Molecules can diffuse into the pores of a porous media by a process called size exclusion. Large molecules dissolve first, and smaller compounds elute subsequently.
Why HPLC is a tricky topic for students?
1. Beginner level
As the topic is related to pure chemistry and a part of the subdivision of chromatography, it might be difficult for the beginner to understand it properly. The basic concepts of solute, solvent, buffer and solution might confuse the students. Also, the data analysis part at the end of the technique involves graph representation which comes from the knowledge of statistics, thus can be overwhelming for students to learn. Moreover, the preparation of the solution, the conversion of units and the balancing equation, all done by formulas, can also be difficult for the beginners students.
2. Theoretical Approach
Normally, the chromatography topic, its types, and its principle are governed by theoretical learning with the help of diagrams. It may create a mental picture in students' minds through a flow chart. But they find it hard to learn about the significance of each step. The progression from one concept to the next is the key contributor to how challenging HPLC is. You need to have a complete understanding of a few different topics before so that you can have that level of comprehension with regard to the HPLC process. It is essential to keep in mind that the root mechanism of each topic, which once more would be done in a theoretical setting, is not the answer here.
3. A long set of Instructions to follow
As discussed above, before coming to HPLC, there is a need to learn the basic methodology of chromatography. Because HPLC methodology consisted of many steps and a set of instructions to do things properly. HPLC is a costly procedure, and any negligence can cause the loss of the experiment and resources. Many things should be considered while doing HPLC, such as: What should be done when the backpressure becomes greater? How can you estimate the void volume in HPLC? What results can I expect if the solvent of my sample is more concentrated than the mobile phase? What results can I expect if the solvent of my sample is more concentrated than the mobile phase? What kind of arrangement should be used for the columns?
Five Ways to make HPLC an engaging topic for students
1. Building the base
The idea here is to develop the basic concept of solution, mixture, solvent, buffer, saturation, and concentration. A student must be able to differentiate between these concepts. This can be done by providing examples from everyday life. The students are given an overview of the characteristics that set mixes and solutions apart from one another. The students have the chance to try and compare the physical features of a few straightforward mixtures and solutions during a presentation that will be led by the teachers in the classroom. They talk about diverse engineering uses of mixes and solutions as well as the process of separating mixtures with solutions back into their original constituents. By that means, the idea then shifted towards chromatography and how we separated mixtures of different compounds, proteins, and drugs.
2. Hands-on practice
Then the students are advised to perform some experimentation of chromatography. These "hands-on" exercises are beneficial to the growth of students' practical abilities and aid in the formation of their comprehension of scientific ideas and occurrences. The development of knowledge of the hazard, risk, and safety working is one further benefit that comes as a direct result of participating in practical training, particularly in the process of learning HPLC.
3. Statistical Coaching
In the field of chemical research, statistical methods are employed for the purposes of data collecting and the evaluation of chemical compounds in order to facilitate more effective information flow management. They make it possible to predict the physiochemical properties of the compounds that make up chemicals. They assist in reaching more conclusive scientific results and in making better use of the resources that are already accessible. For such purposes, statistical coaching of students must be done.
4. Discussing the problems with experts
This is important when you are following a set of instructions and don't have a definite answer for every question. Experts like online platforms are available for such purposes. If you have any questions regarding chemistry, the forums are here to assist you.
5. Virtual lab simulations
As HPLC is a complex topic and its hands-on practice can be costly. So one way to learn HPLC is through virtual lab simulation. Here at Labster, students investigate the high-performance liquid chromatography (HPLC) machine by looking inside it. They find the many components, such as the sample injection station, columns, pumps, and UV detector, and they learn everything there is to know about the fundamentals of chromatography, more specifically, HPLC. The HPLC Lab was built with detail and information for pharmaceutical students. It can also be used in biology and biochemistry, which utilize chromatography methods. Pharmacists utilize HPLC and must be careful of drug stability. It mixes theory and practice. After the first phase of the HPLC lab, students will learn how to examine medicines in different settings and measure active component concentrations.