HPLC | High performance liquid chromatography

Summary

High Performance Liquid Chromatography (HPLC) is a technique used in analytical chemistry to separate, identify, and quantify each component in a mixture. Utilizing a highly pressurized column filled with a specific absorbent, HPLC can distinguish small, intricate differences in molecular size or charge, providing high resolution and efficiency in separation. Each component is detected and represented by a peak on a chromatogram, measured against known standards to determine the identity and concentration of various compounds.

Highlights

• HPLC uses high pressure to enhance separation efficiency and resolution, providing detailed analysis. 🚀
• Stainless steel columns in HPLC withstand pressures up to 50 MPa! 🏋️
• Various detectors like UV and mass spectrometers improve detection capabilities. 📈
• Different solvents are used based on the sample characteristics, ensuring versatility. 🌊
• Retention time is key to understanding what components are in your sample. ⏱️
• The diverse methods (Normal Phase, Reverse Phase, etc.) allow flexibility in separation strategies. 🔄
• Standard curves help quantify unknown samples, crucial for concentration determination. 📐

Key Takeaways

• HPLC stands for High Performance or High Pressure Liquid Chromatography and is a powerful technique for separating compounds. 🧪
• It involves a high-pressure pump, a stainless steel column, and a variety of detectors to analyze samples. 🔬
• Different types of HPLC include Normal Phase, Reverse Phase, Size Exclusion, and Ion Exchange, each offering unique separation methods. 🔍
• Retention times measured by detectors help in identifying compounds by comparing them with known standards. 🕒
• HPLC allows for both qualitative and quantitative analysis of substances, crucial for research and industry applications. 📊

Overview

High Performance Liquid Chromatography, or HPLC as it's often abbreviated, is a fascinating and powerful tool in the field of chemistry. Typically used to separate, identify, and quantify each component in a mixture, HPLC excels in providing highly detailed analysis due to the high pressure exerted on its columns. Whether you're dealing with complicated mixtures or trying to determine the concentration of a single component, HPLC offers a robust solution.

One of the defining features of HPLC is its high-pressure pump, which can create pressures up to 50 MPa. This is paired with stainless steel columns and a variety of detectors, such as UV or mass spectrometers, which work in harmony to detect even the smallest nuances in sample populations. The versatility of HPLC is further enhanced by its capacity to switch between normal phase, reverse phase, size exclusion, or ion exchange chromatography based on the nature of the samples.

At the core of HPLC's capability is its use of differing solvents and precisely controlled retention times, which, in combination with standard curves, help scientists accurately identify and quantify the contents of complex mixtures. HPLC is a cornerstone technique in both research settings and industrial applications, offering unparalleled insight into the molecular world.

Chapters

• 00:00 - 00:30: Introduction to HPLC The chapter titled 'Introduction to HPLC' introduces the concept of High Performance Liquid Chromatography (HPLC), also known as High Pressure Liquid Chromatography. It explains that HPLC is a form of modified column chromatography. In this process, a column packed with an absorbent material such as silica is used, and the mobile phase is passed down the column typically under the influence of gravity.
• 01:00 - 03:00: HPLC Components The chapter 'HPLC Components' discusses the setup and function of high-performance liquid chromatography (HPLC). A high-pressure pump, capable of generating pressures up to 40 megapascals, is integrated with the column. The column is packed with an absorbent material featuring very small particles, which offer a large surface area for interaction with sample molecules. This setup significantly enhances separation efficiency, resulting in high resolution.
• 03:00 - 04:00: HPLC Working Principle The chapter discusses the components and working principle of High-Performance Liquid Chromatography (HPLC). It focuses on the column, which is a crucial component of HPLC. The column is made of stainless steel to withstand high pressures, and its dimensions can vary, typically being 5 to 25 cm in length with an internal diameter of 4.5 mm. The flow rate of the mobile phase through the column is generally between 1 to 3 ml per minute. The chapter also touches upon the stationary phase, however, the details are not fully provided in the given transcript snippet.
• 04:00 - 05:00: HPLC Application Example The chapter "HPLC Application Example" provides insights into the components of HPLC, specifically the stationary and mobile phases. The stationary phase consists of an absorbent material with small, uniform particle sizes, often made of chemically modified silica or D-venile Benzene. The mobile phase is described as a mixture of different solvents, highlighting the versatility in its composition.
• 05:00 - 07:00: Types of HPLC The chapter discusses the types of High-Performance Liquid Chromatography (HPLC) and focuses on the role and choice of solvents used in the process. The selection between polar or non-polar solvents depends on the type of sample molecules to be separated. These solvents, comprising the mobile phase, are typically stored in a solvent reservoir. A pump, connected to the reservoir, is responsible for pushing the mobile phase through the column at high pressure. Additionally, the process includes an injector located just before the HPLC column, which is used for introducing samples into the column for separation.

HPLC | High performance liquid chromatography Transcription

• 00:00 - 00:30 hey everyone quick back chemistry Basics here let's talk about hlc hlc is also known as high performance liquid chromatography or high pressure liquid chromatography hplc is usually a modified column chromatography in column chromatography a column is specked with an absorbent material like silica and the mobile pH is passed down the column because of gravity
• 00:30 - 01:00 but in hlc a high press pump is attached with the column the high press pump can generate a high pressure up to 40 map pascals the column is filled with an absorbent material which has a very small particle size the small particle size gives a very large surface area for the sample molecules to interact as a result the efficiency of Separation increases giving high resolution
• 01:00 - 01:30 let's see the components of hlc the column the column is made up of stainless steel which can withstand a very high pressure up to 50 map pascals the length of the column can vary from 5 to 25 cm and have an internal diameter of 4.5 mm the flow rate of mobile pH through the colum is usually 1 to 3 ml per minute the stationary phase as discussed
• 01:30 - 02:00 earlier the stationary phase is made up of an absorbent material that has a very small particle size the particle size is kept uniform to obtain a better performance usually chemically modified silica D venile Benzene Etc are commonly used as a stationary phase the mobile phase the mixture of different solvents can be used as a mobile phase
• 02:00 - 02:30 the solvent use depends on the type of sample molecules which are to be separated the mobile phas solvent can be polar or non-polar the mobile phas is usually kept in the solvent Reservoir the solvent Reservoir is attached with the pump which pumps the mobile face in the column with a high pressure just before hplc column there's an injector which allows introduction of the sample in the column
• 02:30 - 03:00 the detector in order to detect the uite that is sample coming out of the column the hlc column is attached with a detector different types of detector such as UV detector IR detector fluoresence detector refractive index detector Mass spectrometer electrochemical detectors Etc can be used the detectors are connected with the computer which collects the information
• 03:00 - 03:30 now let's see the working of hlc as the sample molecules get separated they are detected by the detector and a peak is obtained on the computer this peak is plotted with respect to the retention time to identify the components we need to have standards let's understand this with an example let's see we run glucose as our sample and the peak of glucose is obtained at 5 minutes
• 03:30 - 04:00 next we run sucrose as our sample and the peak of sucrose is obtained at 8 minutes now we analyze the unknown sample for the detection of sugars present in it many Peaks are obtained in the chromatogram the peak obtained at 5 minutes indicates the presence of glucose however there's no Peak at 8 minutes which indicates that sucrose was absent in our sample
• 04:00 - 04:30 the remaining two peaks are still unknown as we don't have any standards to compare the retention time this is how the detection of sample is done in hlc and for the detection we must have the standards using hplc it is also possible to find the concentration of sample but before doing so we need to have standard curve let's say we take different conc conentration of glucose and run through
• 04:30 - 05:00 hlc the peaks of glucose are obtained at 5 minutes and with an increase in the concentration of glucose the area under the curve increases once the standard information about the area under the curl and the concentration of glucose is obtained just by measuring the area of glucose peak in the unknown sample the concentration of the glucose can be estimated
• 05:00 - 05:30 now let's see different types of hlc normal phase hlc in this type of hlc the stationary face is polar while the mobile face is non-polar usually silica is preferred as a stationary phase as silica has s ioh Group which gives its polar nature during normal phase hlc polar molecules are retained on the stationary phase while the non-polar molecues move fast
• 05:30 - 06:00 down the column with the mobile phase reverse phase hlc in this method the stationary face is nonpolar while the mobile face is polar the non-polar molecules are retained on the stationary phase while the polar molecules moves fast down the column size exclusion hlc this method is based on size exclusion graphy or gel
• 06:00 - 06:30 permeation chromatography in this method the stationary phase particles are porous as a result small molecules gets inside the pores and takes a long time to move while the large molecules easily pass down the column hence separation occurs based on the molecular size ion Exchange hlc in this method the stationary phase has an ionic charge if the molecules of Interest has a positive charge
• 06:30 - 07:00 then a stationary phase is kept negative and if the molecule of Interest has a negative charge then the stationary phase is kept positive hence separation occurs based on the molecular charge [Music]