What is HPLC/ High Performance Liquid Chromatography?

HPLC stands for High Performance Liquid Chromatography.

Its earlier name was High Pressure Liquid Chromatography because it involved use of liquid mobile phase requiring higher pressures than gases used in Gas Chromatography. The technique has found immense scope of applications in both academic and industrial laboratories requiring identification and quantification of mixtures of organic compounds.

What Is HPLC?

 

The basic question that needs a simple answer is What is HPLC? In simple terms, HPLC is an analytical technique used for the separation of components of an organic mixture of compounds when such compounds are nonvolatile, thermally unstable, and have relatively high molecular weights.

A liquid carrier stream termed as the mobile phase serves to carry the injected sample through the separation column and to the detector. In the separation column, the individual components are separated based on physicochemical interactions, and the elution order is based on such interactions. The separated components are detected by the detector based on the absorption of light or changes in refractive index, electrochemical/conductivity changes, or simply the size distribution of eluting molecules.

The detector response is in the shape of peaks having an area in direct proportion to the amount present. The output is monitored and evaluated by the operating software which not only does required calculations on the response but also supports operating parameters like injection volume, injection sequence, detection wavelength, wash cycles, etc.

Today, the advanced features of available software have made operations highly user-friendly. The majority of time spent by a user is in mobile phase preparation, preparation of buffers and standards, and making record entries.

Separation sciences have contributed immensely to the growth and understanding of analytical chemistry and better control over the quality of consumer products such as pharmaceuticals, foods and beverages, polymers, environmental monitoring, etc. Major credit goes to chromatographic techniques and HPLC in particular.

Introduction to High-Performance Liquid Chromatography and It’s Parts

High-Performance Liquid Chromatography (HPLC) is a popular and versatile technique that provides affordable solutions on separation, identification, and quantification of constituents of complex organic samples. At first sight, the assembly of complex modules and a bunch of tubings might scare you. However, after gaining an insight into the role of each component, the mystery will start unfolding and the HPLC system will turn out to be a user-friendly piece of equipment that if properly looked after will ensure the highest accuracy and reliability of results, analysis after analysis.

This PowerPoint presentation on “Introduction to High-Performance Liquid Chromatography” is a brief and concise introduction to the components of HPLC. It is important to understand the individual component parts and how each one contributes to the overall reliability of the analysis. Your increased comfort level will make you realize that HPLC is a simple-to-use system and holds the promise of unlimited applications.

In the last slide of this HPLC PPT, you will find useful links on an advanced Certificate Course on HPLC.We invite you to join the program and take the opportunity to interact with our experts through participation in the discussion forums and webinars which are held from time to time. Take a look at the HPLC chromatography PPT given below.

Different combinations of these parts are:

·         Pumps

·         Detectors

·         Injectors yield an infinite number of configurations based on the application

You need to have a good understanding of the parts of your HPLC system and the HPLC principles to generate data of the highest reliability. A conceptual understanding of the function of each component will add to your comfort level with your HPLC system, and you will ensure long term usage with high reliance on output data.

Mobile Phase

The liquid phase is pumped at a constant rate to the column packed with the stationary phase. Before entering the column, the analysis sample is injected into the carrier stream.

On reaching the column, the sample components are selectively retained based on physicochemical interactions between the analyte molecules and the stationary phase.

Mobile Phase

The mobile phase serves to transport the sample to the system. Essential criteria of the mobile phase are inertness to the sample components. Pure solvents or buffer combinations are commonly used. The mobile phase should be free of particulate impurities and degassed before use.

Mobile Phase Reservoirs

These are inert containers for mobile phase storage and transport. Generally, transparent glass bottles are used to facilitate visual inspection of mobile phase level inside the container. Stainless steel particulate filters are provided inside for the removal of particulate impurities in the mobile phase if any.

Pumps

Variations in flow rates of the mobile phase affect the elution time of sample components and result in errors. Pumps provide a constant flow of the mobile phase to the column under constant pressure.

Injectors

Injectors are used to provide constant volume injection of the sample into the mobile phase stream. Inertness and reproducibility of injection are necessary to maintain a high level of accuracy.

Column

A column is a stainless-steel tube packed with a stationary phase. It is a vital component and should be maintained properly as per supplier instructions for getting reproducibility and separation efficiency run after run.

Column Oven

Variation of temperature during the analytical run can result in changes in the retention time of the separated eluting components. A column oven maintains a constant column temperature using air circulation. This ensures a constant flow rate of the mobile phase through the column.

Detector

A detector gives a specific response for the components separated by the column and also provides the required sensitivity. It has to be independent of any changes in mobile phase composition. The majority of the applications require UV-VIS detection, though detectors based on other detection techniques are also popular these days.

Data Acquisition & Control

Modern HPLC systems are computer-based and software controls operational parameters such as mobile phase composition, temperature, flow rate, injection volume and sequence, and also acquisition and treatment of output.

Specialized Versions

Specialized HPLC systems might also have solvent selection valves, vacuum degasser, autosamplers, column switches, pre or post-column derivatization, and fraction collectors. These are all covered at length in our online certificate program on HPLC – Certificate Course on HPLC.

Infographic: What Really Goes into Running an HPLC?

Anyone who is working in a laboratory is likely to use an HPLC at some time or the other. Right from the time you complete your graduation, post-graduation, doctorate, or start working in an industry, you may have to rely on HPLC to answer some vital questions. Most of us believe that it is enough to be able to run the software and inject samples.

Far from this myth, HPLC operation requires an understanding and knowledge of a lot more than just the software and hardware. These are, in fact, a very small part of the bigger picture. On an average day, in an HPLC lab, you would spend only a fraction of your time running the system. Most of the time will be spent preparing the samples, mobile phase, buffers, filling logbooks, and various forms and formats.

The infographic is designed to guide you through a series of 18 logical steps that chart the path to achieve your learning objectives. The systematic approach will offer a complete package to assure you of value on your investment and time. Read our 18-step simplified guide to High-Performance Liquid Chromatography and get started!

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HPLC – The New Age Analytical Technique

Analytical chemistry is becoming a popular choice as a career option for students pursuing the discipline of chemistry. The obvious reason is that it applies knowledge of chemistry to gain an understanding of the chemical properties of materials. It makes a valuable contribution in establishing the quality of natural resources and synthetic industrial products for our consumption. The quality of such resources and their consistency is greatly dependent on the precise determinations carried out by analytical chemists in their laboratories.

Chromatography in general and HPLC, in particular, has made remarkable contributions in the areas of research and product development. HPLC deserves special mention as it finds numerous applications in pharmaceuticals, foods, biochemical research, synthetic polymers, studies on bio-molecules, and environmental monitoring. The scope of HPLC is expanding at an astonishing pace and many new applications are being reported in reputed research journals almost daily.

The present article highlights some of the recent developments that have contributed to the large-scale acceptance of HPLC as a popular technique in modern-day laboratories.

Scale of Operation

HPLC makes it possible to analyze a sample over a vast concentration range and molecular weights. The analysis is conventionally carried out for sample amounts ranging from picograms to gram level quantities through the use of different column dimensions and operational modes. Developments in the field of synthetic polymers and large bio-molecules have resulted from the capability of HPLC to handle samples having low molecular weights to those having molecular weights in several millions.

Advances in Stationary Phase Technologies

Silica-based columns have remained the backbone of stationary phases to this day. However, columns with monolithic phases, zircon base phases and those based on core shell-based technologies have contributed to greater speed of analysis, wider temperature and pressure range of operation, and studies over extended pH range of media.

Choice of Detectors

HPLC provides several detection options based on the characteristic properties of eluting compounds. The common options available are UV detectors, RI detectors, Photodiode array detectors, Fluorescence detectors, conductivity detectors, light scattering detectors, and mass selectivity detectors. LC-MS-MS is today widely acknowledged as a highly sensitive technique that provides analytical solutions in the analysis of complex mixtures and low limit detections in bioanalytical studies and studies on pesticide residues in agricultural products.

Choice of Mobile Phases

Reverse-phase chromatography is the most popular mode of operation which makes use of polar solvents as mobile phases. However, the use of combinations of solvents of different polarities, buffers, and isocratic/gradient modes of operation can be useful in resolving complex mixtures of molecules having closely spaced peaks.

The analysis options offered by the HPLC technique have contributed to its increasing popularity. Modern-day laboratories have several HPLC systems running round the clock. Structured learning of the technique can help contribute value addition to your career growth as an analytical chemist.

 

Glossary of HPLC terms

The glossary will help you to understand the terminology in case you aren’t already familiar with the technique.

HPLCseparation technique for components of organic mixtures involving retention of components on stationary phase packed inside column on the basis of physico – chemical interactions followed by sequential elution
Stationary phase solid bed inside column whose particles are coated with the retention phase
Mobile phase liquid carrier medium used for transporting the sample through the HPLC system
Normal phase separationseparation mode in which the retention material is polar and mobile phase is nonpolar. Retained sample components are eluted in ascending order of polarity
Reverse phase separation separation mode in which the stationary phase is nonpolar and mobile phase is polar. Elution order of components is in decreasing order of polarity.It is the most commonly used mode of HPLC separations.
Column efficiencyexpressed in terms of HETP which expresses resolution power of the HPLC column.
Columna steel tube packed with the stationary phase for separation of sample components
Autosamplera device for automated precise selection and introduction of programmed sample volume into the HPLC system
Injectormanual or automated device capable of precise sample volume injection of sample into the HPLC system
Filterfrit fitted with a screen membrane to remove solid suspensions from mobile phase of sample
Degassingprocedure for removal of dissolved air from mobile phase using vacuum filtration, helium purging or online degassing
Pumpa module of the system for metering controlled flow rate mobile phase through the system
Detectordevice for providing response of the component after separation by the HPLC column
Column Oven a housing for the HPLC column which can be maintained at a constant temperature. It permits constant flow rate of mobile phase
Ion-exchange chromatography branch of HPLC involving separation of charged sample components by attraction to the oppositely charged stationary phase
Gel permeation chromatography separation technique in which separation is based on molecular size of component molecules. Smaller molecules get trapped in pores of stationary phase and are eluted after the large molecules.
Chiral chromatography chromatographic separation technique for optically active sample components
Analytical scale separations separations in the mass range mg-ng involving use of columns packed with 3-5μm size particles
Semi-preparative HPLC HPLC separations involving mass range from mg-gm involving collection of separated fractions.
Preparative HPLC mass range gm-kg involving collection of separated fractions
UHPLCUltra-high-Pressure or Performance HPLC. Particle size is less than 2 µm. Such systems and columns are capable of handling back pressures in the range of 15,000 up to 18,000 psi
Isocratic Elution mobile phase composition does not change during the chromatographic run
Gradient Elutionmobile phase composition varies during the run and can be programmed before starting the chromatographic run
Retention timetime taken between injection and the maximum of the peak response of a component
Mobile phase reservoir a container that is filled with the mobile phase
Fraction collector a carousel packed with empty vials in which different components get collected after separation on the HPLC column
Syringehand held glass device capable of injecting selected volume of sample into the chromatograph
Bulk property detector detector based on detection of total property of mobile phase and sample component as a single system
Specific property detectordetector based on specific property of eluting molecules and is independent of changes in the mobile phase composition
HETPHeight equivalent to theoretical plate. It is a measure of column efficiency and is expressed as a numerical value without units
HETP = L/N
Where L is column length and N the number of theoretical plates.The larger the number of theoretical plates the lower is HETP and better is the column efficiency.

Join our FREE Course on HPLC

Refresh your concepts by registering for the free course which will provide you an introduction to the technique and even prepare you for an interview if you are applying for a job in the laboratory equipped with HPLC systems.Want to  read all the High Performance Liquid Chromatography free course modules right now? Here are all links to all the modules for you!
Want to learn more about HPLC? Continue with our library of articles on HPLC below –

We regularly publish articles specially to help you upgrade your laboratory skills and to expose you to new concepts and developments in the field of HPLC.

You’ll find the list is ever growing with inclusion of newer published articles. We are confident that you’ll find the article content of immense use. Continue learning more about High Performance Liquid Chromatography by clicking any of the articles that interest you.

 

Do you want to master the fundamentals and operation of HPLC? You can join our certificate course on HPLC and become an expert and get a certificate online!

HPLC certificate program is an advanced level program that is designed keeping the needs of the laboratory’s worker in mind. It covers the subject in detail and will instill in you the confidence to start working on HPLC the day you come face to face with it. The course includes self-evaluation quiz sessions in addition to several opportunities to interact with our experts in regular webinar sessions besides participation in discussion forums. It is not a time-bound program and you can complete it at your convenience. Our subscribers found it to be even more beneficial than regular hands-on training programs.

To know more about the program and joining guidelines, please go through the link to Join HPLC Certificate Program.

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