HPLC instrumentation and chromatography principle  

HPLC instrumentation and chromatography principle

This HPLC chromatography lecture explains the HPLC principle and instrumentation.
 
https://www.youtube.com/embed/pGYXd4k5F9o
 

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History Of Chromatography  

Chromatography was first employed in Russia by the Italian-born scientist Mikhail Tsvet in 1900. He continued to work with chromatography in the first decade of the 20th century, primarily for the separation of plant pigments such as chlorophyll,carotenes, and xanthophylls. Since these components have different colors (green, orange, and yellow, respectively) they gave the technique its name. New types of chromatography developed during the 1930s and 1940s made the technique useful for many separation processes.
Chromatography technique developed substantially as a result of the work of Archer John Porter Martin and Richard Laurence Millington Synge during the 1940s and 1950s, for which they won a Nobel prize.They established the principles and basic techniques of partition chromatograph

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chromatography  

Chromatography is a physical method of separation that distributes components to separate between two phases, one stationary (stationary phase), the other (the mobile phase) moving in a definite direction. The eluate is the mobile phase leaving the column. The eluent is the solvent that carries the analyte.

Chromatography

chromatography.hplchplc.com/ 
 







Chromatography is a physical method of separation that distributes components to separate between two phases, one stationary (stationary phase), the other (the mobile phase) moving in a definite direction. The eluate is the mobile phase leaving the column. The eluent is the solvent that carries the analyte.


Partition Chromatography


chromatography.hplchplc.com/page-461157.html
 
Partition chromatography was one of the first k

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HPLC (high performance liquid chromatograph ) Instrument Operation  

General components of a high performance liquid chromatograph.
HPLC solvent delivery systems.
How automatic injectors work.
Common HPLC detectors.
Downlod powerpoint file about hplc instrumentation .
source:Philadelphia University

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HPLC Electro Chemical Detector ( ECD )  

There are several different types of ECs. The detection is based on amperometry, polarography, coulometry, and conductrometry. They offer high sensitivity, simplicity, convenience, and wide-spread applicability. It is especially suitable for the use with semi-micro or capillary type system.
Introduction Of Electro Chemical Detector ( ECD )
Electrochemical detection (ECD) for HPLC or UHPLC is an extremely selective and sensitive detection technique that is applied in a number of analyses such as the neurotransmitters dopamine, serotonin and noradrenalin. In combination with the proper electronics, ECD has an enormous linear dynamic range of more then 6 orders of magnitude. This means that concentrations can be measured as low as 50 pmole/L and as high as 100 µmol/L or more.
Fig. 1. HPLC w

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Waters Breeze 2 HPLC  

The Breeze™ 2 HPLC System delivers technology and performance in an affordable, compact, and user-friendly system platform. Complete with software, pump, detector and injector, the system comes pre-configured for different levels of HPLC operational needs.
From teaching purposes to daily analytical work, the Breeze 2 HPLC System integrates simplicity, sensitivity, accuracy, and reliability. It is ideal for any organization seeking a quality HPLC platform with limited budget and chromatography experience including university laboratories, government laboratories, or start-up companies.
The Waters® Breeze 2 HPLC System delivers routine analyses and robust performance day after day, providing the  onfidence you need to get the job done. Used in laboratories worldwide, chromatograp

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Conquer Scientific Address / Phone / Work Hours  

Address: 6259 Progressive Ave #300, San Diego, CA 92154, USA


Phone: +1 619-690-7300


Hours: Open today · 8:30AM–5PM

Source:google

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principle of hplc:High Performance Liquid Chromatography (HPLC)  

High performance liquid chromatography (HPLC) has become a very versatile and powerful separation and analytical method over the years. It is an advanced form of liquid chromatography (LC).
Instead of introducing the solvent into the column and allowing it to drip down under the influence of gravity, in HPLC the sample is forced through the column under high pressures of nearly 400 atm, resulting in faster and more efficient separation.
This technique is also called high pressure liquid chromatography.

The Principle of HPLC
HPLC follows the same basic principle as chromatography. Different components in the sample have varying affinities to the adsorbent material. This causes a difference in the flow rate for each component which leads to their separation as they come out of the column.

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column chromatography principle  

When a mixture of mobile phase and sample to be separated are introduced from top of the column, the individual components of mixture move with different rates.Those with lower affinity and adsorption to stationary phase move faster and eluted out first while those with greater adsorption affinity move or travel slower and get eluted out last.
The solute molecules adsorb to the column in a reversible manner. The rate of the movement of the components is given as follows
R= Rate of movement of a component / Rate of movement of mobile phase. i.e. it is the ratio of distance moved by solute to the distance moved by solvent.
source:studyread.com/

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principle of hplc:How Does High Performance Liquid Chromatography Work?/what is Detector,Chromatogram?  

How Does High Performance Liquid Chromatography Work?

The components of a basic high-performance liquid chromatography [HPLC] system are shown in the simple diagram in Figure E.
A reservoir holds the solvent [called the mobile phase, because it moves]. A high-pressure pump [solvent delivery system or solvent manager] is used to generate and meter a specified flow rate of mobile phase, typically milliliters per minute. An injector [sample manager or autosampler] is able to introduce [inject] the sample into the continuously flowing mobile phase stream that carries the sample into the HPLC column. The column contains the chromatographic packing material needed to effect the separation. This packing material is called the stationary phase because it is held in place by the column hardware.

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Brief History and Definition :What Is HPLC (High Performance Liquid Chromatography)?  

Liquid chromatography was defined in the early 1900s by the work of the Russian botanist, Mikhail S. Tswett. His
pioneering studies focused on separating compounds [leaf pigments], extracted from plants using a solvent, in a
column packed with particles.
Tswett filled an open glass column with particles. Two specific materials that he found useful were powdered chalk
[calcium carbonate] and alumina. He poured his sample [solvent extract of homogenized plant leaves] into the
column and allowed it to pass into the particle bed. This was followed by pure solvent. As the sample passed down
through the column by gravity, different colored bands could be seen separating because some components were
moving faster than others. He related these separated, different-colored bands to the diffe

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Evaporative Light Scattering Detector (ELSD)  

An evaporative light scattering detector (ELSD) is a detector used in conjunction with high-performance liquid chromatography (HPLC). It is commonly used for analysis of compounds where UV detection might be a restriction and therefore compounds does not very efficient absorb UV radiation, such as sugars, antivirals, antibiotics, lipids, phospholipids, terpenoids, and alcohols. ELSDs fall under the category of general-purpose detectors, similar to refractive index detectors (RI).

ELSD | Evaporative Light Scattering Detector

elsd.hplchplc.com/

 

... Scattering Detector. An evaporative light scattering detector (ELSD) is a detector used in conjunction with high-performance liquid chromatography (HPLC).

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P

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Column Chromatography is the the first form of techniques developed in chromatography!  

Column chromatography is the prototype or the basic type of chromatography. It was the first form of techniques developed in chromatography. One can easily explain the principle and procedure of chromatography using it.
Other types of chromatography techniques like high performance liquid chromatography (HPLC), gas chromatography (GC), paper chromatography were developed with column chromatography as a module and making slight variations.
In-spite of many advanced methods of chromatography, still this method of chromatography is widely used in research and industry.
This chromatography is basically a type of adsorption chromatography techniques.

Here the separation of components depends upon the extent of adsorption to stationary phase. Here the stationary phase is a polar solid materia

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Principle of HPLC:High Performance Liquid Chromatography (HPLC) : Principle, Types, Instrumentation and Applications  

Chromatography is a technique to separate mixtures of substances into their components on the basis of their molecular structure and molecular composition. This involves a stationary phase (a solid, or a liquid supported on a solid) and a mobile phase (a liquid or a gas). The mobile phase flows through the stationary phase and carries the components of the mixture with it. Sample components that display stronger interactions with the stationary phase will move more slowly through the column than components with weaker interactions. This difference in rates cause the separation of variuos components. Chromatographic separations can be carried out using a variety of stationary phases, including immobilized silica on glass plates (thin-layer chromatography), volatile gases (gas chromatography

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Displacement Chromatography  

The basic principle of displacement chromatography is: A molecule with a high affinity for the chromatography matrix (the displacer) will compete effectively for binding sites, and thus displace all molecules with lesser affinities. There are distinct differences between displacement and elution chromatography. In elution mode, substances typically emerge from a column in narrow, Gaussian peaks. Wide separation of peaks, preferably to baseline, is desired in order to achieve maximum purification. The speed at which any component of a mixture travels down the column in elution mode depends on many factors. But for two substances to travel at different speeds, and thereby be resolved, there must be substantial differences in some interaction between the biomolecules and the chromatography m

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Principle Of Column Chromatography  

Column chromatography in chemistry is a method used to purify individual chemical compounds from mixtures of compounds. It is often used for preparative applications on scales from micrograms up to kilograms. The main advantage of column chromatography is the relatively low cost and disposability of the stationary phase used in the process. The latter prevents cross-contamination and stationary phase degradation due to recycling.
The classical preparative chromatography column is a glass tube with a diameter from 5 mm to 50 mm and a height of 5 cm to 1 m with a tap and some kind of a filter (a glass frit or glass wool plug – to prevent the loss of the stationary phase) at the bottom. Two methods are generally used to prepare a column: the dry method and the wet method.

For the dry

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Shimadzu Photodiode Array SPD-M20A LC Detector  

General-purpose model
The SPD-M20A is a general-purpose model incorporating a deuterium lamp. The light-source compensation function achieves a noise level of 0.6×10-5AU. Cell temperature control ensures baseline stability.
Key Benefits:
Noise level of 0.6×10-5 AU
Superior Linearity - 2.0AU
Ethernet interface & web control functions
Can be incorporated into non-Shimadzu HPLC systems
Light source: Deuterium (D2) lamp, tungsten (W) lamp
Wavelength range: 190 to 800 nm
Wavelength accuracy: 1 nm max.
Wavelength precision: 0.1 nm max
Cell temperature control range: 5°C above room temperatre to 50°C
Operating temperature 4°C to 35°C
Power requirements: 100 VAC, 150 VA, 50/60 Hz
The SPD-M20A PDA is the third member of the UV-Vis absorbance detectors. The M20A, like

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Shimadzu CDD-10AVP Conductivity LC Detector  

Handles a Wide Variety of Analysis Options
The CDD-10AVP conductivity detector achieves an even higher level of sensitivity and makes it possible to perform a wide variety of analysis scenarios with a single unit. An option card enables the simultaneous 2-channel measurement of anions and cations, and a suppressor option allows expansion to a suppressor system for ultra-high sensitivity work. Organic acids can be analyzed using Shimadzu's unique post-column pH-buffered electroconductivity method.
Low noise, low drift and wide dynamic range assure proven performance of the Shimadzu VP series HPLC. Data acquired by the CDD-10AVP is transferred to the SCL system controller via an optical fiber interface and are digitally processed by workstation. By adding the optional cell (dual kit NS

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Waters ACQUITY Arc HPLC and UHPLC System  

HPLC and UHPLC method compatibility at the flip of a switch

The ACQUITY Arc System is a quaternary-based, modern LC system for scientists working with established methods who are looking for the versatility and robustness required to bridge the gap between HPLC and UPLC while continuing to support validated assays.

True plug-and-play method compatibility and the ability to switch between HPLC and UHPLC separations at the flip of a switch with Arc Multi-flow path Technology
Get mass detection, without changing the way you work, with the ACQUITY QDa Detector
Deploy the benefits of 2.x μm particle columns to boost the chromatographic performance of your methods
On-demand mobile phase creation with Auto●Blend Plus Technology, eliminating the cumbersome and error-prone appr

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Partition Chromatography  

Partition chromatography was one of the first kinds of chromatography that chemists developed. The partition coefficient principle has been applied in paper chromatography,thin layer chromatography, gas phase and liquid–liquid separation applications. The 1952Nobel Prize in chemistry was earned by Archer John Porter Martin and Richard Laurence Millington Synge for their development of the technique, which was used for their separation of amino acids. Partition chromatography uses a retained solvent, on the surface or within the grains or fibers of an "inert" solid supporting matrix as with paper chromatography; or takes advantage of some coulombic and/or hydrogen donor interaction with the stationary phase. Analyte molecules partition between a liquid stationary phase and the eluent.

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Shimadzu Evaporative Light Scattering LC Detector LTII (ELSD-LTII)  

In the history of high-performance liquid chromatographs, which dates to the early 1960s, refractive index detectors (RI detectors) have often been used as general-purpose detectors. RI detectors enable the detection of components that do not possess UV absorbance and give a proportional relationship between the heights of detected peaks and the quantities of detected components. So, in comparison with absorbance detectors (UV detectors), they offer advantages such as the ability to ascertain unknown component quantities and obtain molecular weight distributions for macromolecules. On the other hand, they also have various disadvantages. For example, they cannot be used for gradient analysis, the baselines they produce are susceptible to the influence of fluctuations in the ambie

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Troubleshooting HPLC- Tailing Peaks  

As in the previous posts for this series, these suggestions assume that you are working with an established method and had successful results in the past. If a method is rugged, it is developed to minimize tailing by using a stationary column phase that is base-deactivated as well as appropriate modifiers and pH adjustment in the mobile phase as needed.  Keep in mind that if the method is not as rugged as it should be, change(s) in the method might be advised if tailing continues to be an ongoing problem.

With troubleshooting, as always, it is critical to note when the change occurred and how it might correlate to changes in the system.  Equally important is to change one thing at a time to identify the source(s) of difficulty. The following are examples of things that could contribute

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Reversed Phase Chromatography (RPC)  

Reversed phase HPLC (RP-HPLC) has a non-polar stationary phase and an aqueous, moderately polar mobile phase. One common stationary phase is a silica which has been surface-modified with RMe2SiCl, where R is a straight chain alkyl group such as C18H37 or C8H17. With such stationary phases, retention time is longer for molecules which are less polar, while polar molecules elute more readily (early in the analysis). An investigator can increase retention times by adding more water to the mobile phase; thereby making the affinity of the hydrophobic analyte for the hydrophobic stationary phase stronger relative to the now more hydrophilic mobile phase. Similarly, an investigator can decrease retention time by adding more organic solvent to the eluent. RP-HPLC is so commonly used that it is oft

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