Tuesday, April 24, 2018

HPLC Injector and Types of HPLC Injector

HPLC injection is a technique to inject the sample without disturbing the flow rate and pressure of the HPLC system. The working pressure of an HPLC is adequately high that we cannot inject a sample into the mobile phase by inserting a syringe, so we need an injector that gives a reproducible result and without interrupted the flow rate and system pressure. Nowadays HPLC sample injector set in the different range of volume so you can inject the specific requires a volume.
Here are certain requirements of HPLC Injector are as follows.
1. Introduce the Sample with constant pressure and flow rate of the HPLC system
2. Introduce the sample without air bubbles
3. The injection volumes of sample are in microliter so it should be accurate
4. The sample must be free from any particulate matter
There are three types of injector are available for HPLC systems.
Rheodyne Injector: The most commonly used injector is Rheodyne, easy to use with high accuracy and precision. As per requirement, the analyst can set the volume of the sample loop. Rheodyne has two positions load and inject. The load position allows loading the sample into to loop with the help of a syringe which is commonly used to sample load into the injector. After a load of the sample, manually rotates of the sample injector to the inject position allows flowing the sample onto a column without any air bubbles, without disturbing flow rate and pressure.
Septum Injector: In this system, the sample injects through the rubber septum.
Stop flow Injector: In this type of system mobile phase stopped while the sample is injected. 


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Mobile Phase of HPLC

The retention time of analytes in HPLC is managed by the polarity of the mobile phase. The mobile phase of HPLC typically uses of nonpolar and polar mixtures/combinations of miscible organic solvents and water/buffers. Selection of proper mobile phase is an important aspect in HPLC method development. Before uses mobile phase and buffer it should be filtered throughout 0.5-micron and degas all aqueous and organic solvents to remove all particulate impurities and dissolve gases. Check the compatibility, relative polarity, and miscibility of the solvents being used. Make use of only high purity solvents for mobile phase, and water should be HPLC grade or filtered and deionized, Generally transparent glass bottles use to storage of mobile phase because of glass is chemically inert and for visual inspection.

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HPLC Pumps and Types of HPLC Pumps

As the name of high-pressure liquid chromatography show need to generate the pressure, HPLC require high pressure which gives continuous and reproducible flow of mobile phase throughout the HPLC system, Pump must be able to take solvent from a single or more than one reservoir with pulse-free output and different flow rates, there are three types of pumps to provide the required pressure and flow rate as below.
The types of HPLC pumps are as follows.
Reciprocating Pump:

This is a common and widely used pump technique in modern chromatography because of his precise flow rate, generate high pressure, it should operate as an isocratic and gradient mode. It consists of a small motor-driven piston which moves quickly back and forth in a hydraulic chamber that can vary in the volume of a mobile phase. Produce pulsing flow is the major disadvantage of a reciprocating pump.
 
Syringe Pump:
This type of pump consists of a large syringe with a plunger motorized by the electronic motor to drive used to carry a constant flow rate to the stationary phase. It is inconvenient for the change of the mobile phase and also for its capacity. This is a simple technique used in the early years.
Pneumatic Pump:
In this type of pump mobile phase run into the column with the help of pressure created from a gas cylinder, this type of column provide continuous flow, this technique does not use as widely because have some limitations like the low capacity of solvents, generate low pressure, pump rate varies with viscosity.



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HPLC Detectors
HPLC Columns
Applications of HPLC
HPLC Chromatography and Types of HPLC Chromatography
HPLC Injector and Types of HPLC Injector
HPLC Principle and Types of HPLC Chromatography
HPLC Column Regeneration
Gas chromatography detectors types
How many types of pumps are there in HPLC
HPLC column care and maintenance
Role of ion-pairing reagents in HPLC
Sample Injection System in HPLC
Selection of buffer in HPLC method development
What is an ion-pairing reagent and why is used in HPLC 

What are the common buffers used in HPLC?

HPLC detectors and types of HPLC detectors

The detector is the tool used to identify the analytes eluted from the column. It is transforming the effluents into the electrical signal and recorded by the system.
There are different detectors are used in the LC analysis as below.
• UV/VIS and PDA Detector:
The UV/VIS and PDA detectors use as an absorbance detector, UV/VIS measure the ability of analytes to absorb the light at a particular wavelength in the UV/VIS range and PDA is monitoring the absorbance of analytes at several different wavelengths. It detects every compound having absorbance more than the zero this is the advantage of the PDA and the analytes with no UV activity cannot be detected.
The types of UV/VIS detectors available are as follows.
1-Fixed Wavelength Detector
Absorbance monitor at only one wavelength and which do not permit to modify/change it called fixed wavelength detector. This is a cheap and simple detector, but it detects limited types of compounds. 
2-Variable Wavelength Detector
Single wavelength monitors at any given time, but any wavelength from UV range can be selected. This type of detector is more sensitive than the fixed wavelength detector, it vagaries from 190-900 nm and used for a wide range of compounds. 
• Refractive Index Detector:
Refractive index is the universal detector in HPLC because of its detection capabilities, RI Detectors measure the refractive index of (analytes) column eluent passing through the flow cell it is based on the two principal one is deflection and another is a reflection type refractometer. 
Some advantages of a refractive index detector are as follows.
1. It Senses low to the air bubble and dirt in the cell.
2. Covers the whole range of the refractive index.
3. Detect everything with RI different from the solvent, RI has low sensitivity.
• Mass Detector (LC-MS):
It is a combined analytical chemistry technique for the physical separation of liquid chromatography with mass spectroscopy. This is an important technique to provide structural information about analytes and also use to calculate the low detection limit of molecular components. It combines the best sensitivity with the most identification ability.
• Fluorescence Detector:
The compound with fluorescence can be measured by fluorescence detectors. The fluorescence detectors have better sensitivity than the UV/VIS detectors because of its high sensitivity to the selective groups of compounds at definite lambda max. It requires a high intensity of the light to detect the analytes.

• Electrochemical Detector:
The electrochemical detector uses to measure the electrochemical properties of the analytes on the base of electrochemical oxidation. It is a simple, convenient and sensitive detector. There are three electrodes are working for the detection i.e working electrode, auxiliary electrode, and a reference electrode.
• Conductivity Detector:
This type of detector used in ion chromatography to determine the electronic resistance in proportion to the concentration of ions present in solutions.
• Light Scattering Detector:
The detection of light scattering does not require analytes to have chromophores. the method of light scattering will not work with volatile analytes. It uses full for the detection of high molecular weight analytes; it also works with the gradient method. The nebulization, mobile phase evaporation, and detection are the steps that are involved in a light scattering detector.
• Infrared Detector:
For the absorbance, there are two types of the detector are available, the wavelength scanning detector provides by semicircular filter wedges, between the wavelengths ranges 4000 to 960 cm–1. It is used to a limited extent only for non-polar lipid analysis with the specific absorbance between the 1650 to 1860 cm–1. The other type is the Fourier transform, it is more sophisticated.


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HPLC Columns
What is HPLC
Liquid Chromatography
HPLC Chromatography and Types of HPLC Chromatography
Applications of HPLC
How many types of pumps are there in HPLC
HPLC Injector and Types of HPLC Injector
HPLC Principle and Types of HPLC Chromatography
HPLC Pumps and Types of HPLC Pumps
HPLC column care and maintenance
HPLC Column Regeneration
Role of ion-pairing reagents in HPLC
Sample Injection System in HPLC
Selection of buffer in HPLC method development
What is an ion-pairing reagent and why is used in HPLC
What are the common buffers used in HPLC?

HPLC Columns

The most HPLC columns are made from stainless steel, the actual separation happen into the column usually columns are 30 to 250 mm in length, 01 to 05 mm in diameter and 03, 05 and 10 microns in pore size. The column is packed with porous particles. The Porous particles are made from polymer and surrounded by a thin equal layer of silica and polystyrene. In early years LC carried out in a glass column with diameter 01 to 05 cm and length 50 to 500 cm. Commonly HPLC have guard column in front of the analytical column to protect from contaminants and remove particulate material it helps to extend the life of analytical column, the guard column and analytical column have same stationary phase.
Here are listed some common bonded phase for HPLC columns.
SI, C1, C2, C3, C4, C5, C6, C8, C18, CN, NH2, NO2, OH, PHENYL, SCX, SAX, WCX, WAX. 

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HPLC column care and maintenance




Applications of HPLC

HPLC is an analytical technique to separate, identify, and quantify components in a mixture. It is the single leading chromatography technique essential to most laboratories worldwide.
Let’s check some applications of HPLC.
 1     Drug analysis in pharmaceutical industries
     2     Environmental analysis
     3     Food analysis
     4     Forensic applications
     5     Identify and Quantify the analytes in a sample
     6     Use in drug synthesis to check the progress of a reaction
     7     Use in scientific research for the discovery
     8     Applications in Clinical Tests

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     What is HPLC
















Liquid Chromatography

Liquid chromatography is a technique in analytical chemistry that is used to separate, quantify and identify the analytes. The small amount of sample is loaded and injected onto a tube filled with porous particles. The analytes of the sample mixture are travel along a column with a mobile phase move by high pressure or gravity. The sample components are separated on the basis of affinity for the mobile phase and the stationary phase.



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What is HPLC

What is HPLC

HPLC is an analytical technique to separate, identify, and quantify components in a mixture. It is the single major chromatography technique essential to most laboratories worldwide.

Monday, April 23, 2018

HPLC Principle and Types of HPLC Chromatography

Introduction to HPLC:
HPLC is a technique used to separate, identify, quantitative and for qualitative analysis of each molecule in the sample, depends on its interaction with the stationary phase and polarity. Pump flows a mobile phase by high pressure with the sample mixture through a column and allows for better separation, so it is also called as high-pressure liquid chromatography. Each molecule of the sample interacts slightly with the adsorbent material, the molecule that interacts strongly with the stationary phase will go slowly through the column than a molecule with low interaction. this difference causes the separation rate of the different analytes.
The types of HPLC Chromatography are as follows:
Normal Phase Chromatography:
In this type of chromatography, the moderately polar mobile phase and the polar stationary phase are used to separate the analytes which are freely soluble in moderator solvents. The use of more polar solvents in the mobile phase decreases the retention time (RT) of analytes. In NP-chromatography less polar analytes elute first than the polar analytes. The NP-Chromatography is better for the separation of analytes that differ in the number of functional groups. It is used for protein separation.
Reversed-Phase Chromatography:
This is a vital analytical technique that is commonly used, in this method analytes be separated on the base of polarity. The non-polar stationary phase and polar mobile phase uses in RP-Chromatography. Retention time is more for analytes which more non-polar, while polar analytes elute more readily. The more hydrophobic the analytes, the more strongly it will attach to the column and the higher the concentration of organic solvent that will be required to elute the analytes. The RP-Chromatography most popular because it applies to the wide range of molecules. It cannot apply for the proteins because the organic solvent causes the denaturation of proteins.

Size Exclusion Chromatography:
SE-Chromatography or Gel filtration chromatography technique applies to separate the particles on the basis of size. The large molecules flow rapidly throughout the column than the smaller molecules, SE-Chromatography is non-absorptive interaction with the samples. This is a vital analytical technique to determine the molecular weight of proteins as well as polysaccharides.
Ion Exchange Chromatography:
This method most useful for the analysis of water, protein purification. It separates the polar molecules and ions, based on similarity to the ion exchanger. It is used for any type of charged molecules. Ion exchange chromatography has two types, cation and anion chromatography. cations exchange chromatography holds the positive charged and anion exchange chromatography hold anion with the positively charged functional group.


You may also like this.

What is HPLC
Liquid Chromatography
Applications of HPLC
HPLC Columns
HPLC Detectors
HPLC Pumps and Types of HPLC Pumps
Mobile Phase of HPLC
HPLC Injector and Types of HPLC Injector
How many types of pumps are there in HPLC
Care and Maintenance of HPLC Columns
HPLC column care and maintenance
Pharmaceutical Applications of Column Chromatography
Role of pKa in HPLC method development
Selection of buffer in HPLC method development
HPLC Pumps and Types of HPLC Pumps

SAP SE

The name stands for Systems, Applications & Products, it is German-based software multinational company for making Enterprise Resource Planning, it allows to manage the business operations, customer relations. It refers to collect & integrate data from different types of business. It helps to streamline the processes with effectively and more efficiently into the competitive world. as of May 2016, SAP had over 310,000 customers worldwide as well as SAP is the world’s #1 in enterprise applications and third largest independent software manufacturer in the world.
SAP provides a different type of modules in different categories as below.

Manufacturing,
Marketing,
Sales
Service
Asset Management
Sustainability
Supply chain management
Research and Development
Resource and Procurement,
Commerce
Finance, are the modules provide by SAP