Chromatography terms / analyte / Analytical chromatography / bonded phase / chromatogram /...  

The analyte is the substance to be separated during chromatography. It is also normally what is needed from the mixture.
Analytical chromatography is used to determine the existence and possibly also the concentration of analyte(s) in asample.
A bonded phase is a stationary phase that is covalently bonded to the support particles or to the inside wall of the column tubing.
A chromatogram is the visual output of the chromatograph. In the case of an optimal separation, different peaks or patterns on the chromatogram correspond to different components of the separated mixture.
 Plotted on the x-axis is the retention time and plotted on the y-axis a signal (for example obtained by a spectrophotometer, mass spectrometer or a variety of other detectors) corresponding to the response creat

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HPLC Peak Splitting. Common Reasons For It  

True "Split" HPLC peaks can be caused by a number of chromatography problems. Here are a few examples and their solutions:

Sample overload. Sample overloading is one of the most common reasons for observing peak "splitting". Reduce the sample concentration by factors of ten to see if the peak shape improves.
 A poor quality HPLC method. Poor quality methods which do not use mobile phase solutions which are at an appropriate pH, dissolve the sample in (are fully soluble) are unstable or show sample or mobile phase precipitation can cause this effect. Always check solubility before starting.
A partially plugged or fouled column. A dirty or fouled column (from not washing down properly with a solution which is STRONGER than the mobile phase). Analysis methods should be followed by sep

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How to Prevent Mobile Phase Problems in HPLC  

Low sensitivity and rising baselines, noise, or spikes on the chromatogram can often be attributed to the mobile phase. Contaminants in the mobile phase are especially troublesome in gradient elution. The baseline may rise, and spurious peaks can appear as the level of the contaminated component increases. Water is the most common source of contamination in reversed phase analyses. You should use only high purity distilled or deionized water when formulating mobile phases. However, several common deionizers introduce organic contaminants into the water. To remove these contaminants, pass the deionized water through activated charcoal or a preparative C18 column. Use only HPLC grade solvents, salts, ion pair reagents, and base and acid modifiers. Cleaning lower quality solvents is time cons

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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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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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1290 HPLC  

The Agilent 1290 Infinity LC system provides the highest levels of speed, resolution, flexibility and sensitivity for any LC and LC/MS application. 


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1100 HPLC  

Introduced in 1995, Agilent's 1100 series HPLC system is considered the world's most popular HPLC. They are incredibly robust machines that can last a long time. Finding a used Agilent 1100 on LabX is easy and maintenance for older machines consists of changing seals, filter solvents, lamps, and the odd part in the vacuum degasser. Parts from the Agilent 1050 are often shared with the Agilent 1100 which make it easy to maintain.


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Agilent HPLC 1260  

The Agilent 1260 Infinity HPLC-Chip/MS system is a re-useable microfluidic chip-based technology for high sensitivity nanospray LC/MS. The Agilent 1260 replaced the Agilent 1200 and delivers results faster to save time and money. 

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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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HPLC Refractive-Index (RI) Detector  

RI detector measures change in reflex index. A glass cell is divided into two chambers (cells). The effluent from LC column flow through the "sample cell", while other cell called "reference cell" is filled with only mobile phase. When the effluent going through the sample cell does not contain any analyte, the solvent inside both cells are the same (Figure 1A). When a beam is irradiate on the cells, the observed beam will be straight in this case. However, in a case the effluent contains any components other than mobile phase; bending of the incident beam occurs due to the reflex index difference between the two solvents (Figure 1B). By measuring this change, the presence of components can be observed.

RI detector has lower sensitivity compared to UV detector, and that's the main reason

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Two Types Of UV Detectors Are Commonly Used Today  

Two types of UV detectors are commonly used today:

Variable-wavelength (sometimes called "spectrophotometric" detectors )
Photodiode Array (sometimes simply called "diode array" detectors.)
Variable-wavelength detectors are less expensive; they are the standard detector type for quantitative analysis and routine assays. Photodiode array detectors are more versatile, because they allow simultaneous acquisition of both chromatographic and spectral information; they are frequently used in method development.
The detector wavelength is an important characteristic of an HPLC separation. As a general rule, the wavelength is set to the absorbance maximum of the analyte. Using the wrong wavelength may result in decreased peak sizes, or even no peaks at all!
Because different compounds can h

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HPLC (high performance liquid chromatograph ) Instrumentation  

Main components in an HPLC system include the solvent reservoir, or multiple reservoirs, a high-pressure pump, a column, injector system and the detector.

The reservoir holds the solvent, which is referred to as the mobile phase because it moves. There are usually a minimum of two reservoirs in a system, with each holding up to 1000 cc of solvent and usually fitted with a gas diffuser through which helium can be bubbled. A pump is used to generate a specified flow of the mobile phase. Although manual injection of samples is still possible, most HPLCs are now fully automated and controlled by computer. The injector, or auto sampler, introduces the solvent into a phase stream that carries the sample into the high pressure (up to 400 bar) column, which contains specific packing material nee

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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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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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Agilent HPLC 1200  

The Agilent 1200 Series HPLC System was introduced in 2010 with a modular design allowing users to define a configuration ideally suited to meet their HPLC and UHPLC applications and requirements. 

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Troubleshooting HPLC- Loss in Response for Some, but Not All Analytes  

Patterns in your HPLC chromatography can exhibit telltale signs that point toward probable sources of error.  In this brief series of posts, we will look at possible scenarios that you may encounter in the laboratory and how you might approach resolving those difficulties.
When some, but not all of your peaks are low, and you are analyzing multiple analytes, the number one rule is to look for trends. As mentioned in the previous post for this series, when you have only one analyte, or a handful of very similar analytes, the following may also apply. Again in this post, the assumption is that you are using an established working method and that you see a loss in response for every sample or standard that is injected. If you are only seeing the problem with samples and not your quantitation

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What Is Column chromatography?  

For more details on this topic, see Column chromatography.
Column chromatography is a separation technique in which the stationary bed is within a tube. The particles of the solid stationary phase or the support coated with a liquid stationary phase may fill the whole inside volume of the tube (packed column) or be concentrated on or along the inside tube wall leaving an open, unrestricted path for the mobile phase in the middle part of the tube (open tubular column). Differences in rates of movement through the medium are calculated to different retention times of the sample.
In 1978, W. Clark Still introduced a modified version of column chromatography called flash column chromatography (flash).The technique is very similar to the traditional column chromatography, except for that the

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waters hplc  

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Breeze 2 HPLC Comes with a pump, detector, injector, and is pre-configured for different levels of HPLC. It includes Breeze Software, an intuitive interface that is easy to set-up, learn, and use.

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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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Ion Exchange Chromatography / Ion chromatography  

In ion-exchange chromatography (IC), retention is based on the attraction between solute ions and charged sites bound to the stationary phase. Solute ions of the same charge as the charged sites on the column are excluded from binding, while solute ions of the opposite charge of the charged sites of the column are retained on the column. Solute ions that are retained on the column can be eluted from the column by changing the solvent conditions (e.g. increasing the ion effect of the solvent system by increasing the salt concentration of the solution, increasing the column temperature, changing the pH of the solvent, etc...).
Types of ion exchangers include:

Polystyrene resins – These allow cross linkage which increases the stability of the chain. Higher cross linkage reduces swerving,

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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 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
Partition chromatography was one of the first k

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High Performance Liquid Chromatography Detectors ( HPLC Detectors)  

1. HPLC UV, VIS, and PDA Detectors
2. HPLC Refractive-Index Detector
3. HPLC Evaporative Light Scattering Detector
4. HPLC Multi-Angle Light Scattering Detector
5. HPLC Mass Spectrometer
6. HPLC Conductivity Detector
7. HPLC Fluorescence Detector
8. HPLC Chemiluminescence Detector
9. HPLC Optical Rotation Detector
10.HPLC Electro Chemical Detector
The actual separation of each component in the sample is carried inside a column; however this separation needs to be "collected" for us to be able to see it. The detectors are used for this purpose. The separated coponents are monitored and expressed electronically. There is no universal detector that can monitor all compounds and there are many detectors used for LC analysis. Some are listed below.

Common Abbreviation

Ultra Violet


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What Is Chromatography?  

Chromatography  is the collective term for a set of laboratory techniques for the separation of mixtures. The mixture is dissolved in a fluid called the mobile phase, which carries it through a structure holding another material called the stationary phase. The various constituents of the mixture travel at different speeds, causing them to separate. The separation is based on differential partitioning between the mobile and stationary phases. Subtle differences in a compound's partition coefficient result in differential retention on the stationary phase and thus changing the separation.
Chromatography may be preparative or analytical. The purpose of preparative chromatography is to separate the components of a mixture for more advanced use (and is thus a form of purification). Analytica

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HPLC Fluorescence Detector (FLD)  

The advantage of fluorescence method is its high sensitivity for selective groups of compounds at ~fg level. By using a specific wavelength, analyte atoms are excited and then emit light signal (fluorescence). The intensity of this emitted light is monitored to quantify the analyte concentration. Most pharmaceuticals, natural products, clinical samples, and petroleum products have fluorescent absorbance. For some compounds which do not have fluorescence absorbance or low absorbance, they can be treated with fluorescence derivatives such as dansylchloride. The system is easy to operate and relatively stable.
Principles Of HPLC  Fluorescence Detector (FLD)
Fluorescence detectors are probably the most sensitive among the existing modern HPLC detectors. It is possible to detect even a presenc

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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.

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HPLC Conductivity Detector (CDD)  

Solutions containing ionic components will conduct electricity. Conductivity detector measures electronic resistance and measured value is directly proportional to the concentration of ions present in the solution. Thus it is generally used for ion chromatography.

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Mobile Phase (eluent) In Column Chromatography  

The mobile phase or eluent is either a pure solvent or a mixture of different solvents. It is chosen so that the retention factor value of the compound of interest is roughly around 0.2 - 0.3 in order to minimize the time and the amount of eluent to run the chromatography. The eluent has also been chosen so that the different compounds can be separated effectively. The eluent is optimized in small scale pretests, often using thin layer chromatography (TLC) with the same stationary phase.
There is an optimum flow rate for each particular separation. A faster flow rate of the eluent minimizes the time required to run a column and thereby minimizes diffusion, resulting in a better separation. However, the maximum flow rate is limited because a finite time is required for the analyte to equi

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High performance liquid chromatography (HPLC)  

High Performance Liquid Chromatography (HPLC) is a form of column chromatography that pumps a sample mixture or analyte in a solvent (known as the mobile phase) at high pressure through a column with chromatographic packing material (stationary phase). The sample is carried by a moving carrier gas stream of helium or nitrogen. HPLC has the ability to separate, and identify compounds that are present in any sample that can be dissolved in a liquid in trace concentrations as low as parts per trillion. Because of this versatility, HPLC is used in a variety of industrial and scientific applications, such as pharmaceutical, environmental, forensics, and chemicals.

Sample retention time will vary depending on the interaction between the stationary phase, the molecules being analyzed, and the s

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Normal phase Chromatography  

Normal–phase chromatography was one of the first kinds of HPLC that chemists developed. Also known as normal-phase HPLC (NP-HPLC) this method separates analytes based on their affinity for a polar stationary surface such as silica, hence it is based on analyte ability to engage in polar interactions (such as hydrogen-bonding or dipole-dipole type of interactions) with the sorbent surface. NP-HPLC uses a non-polar, non-aqueous mobile phase (e.g. Chloroform), and works effectively for separating analytes readily soluble in non-polar solvents. The analyte associates with and is retained by the polar stationary phase. Adsorption strengths increase with increased analyte polarity. The interaction strength depends not only on the functional groups present in the structure of the analyte molecu

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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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HPLC Column | Agilent ZORBAX Eclipse XDB-CN Small Molecule Separations LC Column  

For normal-phase chromatography, the ZORBAX product line offers a choice of bonded and non-bonded silica packings.
Made from highly pure Rx-SIL
Excellent choice for normal-phase applications with basic compounds
Equilibrates more rapidly than ZORBAX Rx-Sil and is used for many of the same normal-phase applications
To compare technical information and specifications for different phases, see our phase specifications chart.

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Principles Of Operation In Evaporative light Scattering Detector (ELSD)  

ELSDs analyze solvent after elution from HPLC. As the eluent passes from an HPLC, it is mixed with an inert carrier gas and forced through a nebulizer, which separates the liquid into minute aerosolized droplets. These droplets then pass into a heated drift tube, where the mobile phase solvent is evaporated off. As the mobile phase evaporates, the droplets become smaller and smaller until all that is left is minute particles of dried analyte. These particles are pushed through the drift tube by the carrier gas to the detection region. In this region, a beam of light crosses the column of analyte and the scattering of light is measured by a photo multiplier tube./wikipedia

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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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Preventing Leaks In HPLC Analyses  

Leaks are a common problem in HPLC analyses. To minimize leaks in your system, avoid interchanging hardware and fittings from different manufacturers. Incompatible fittings can be forced to fit initially, but the separation may show problems and repeated connections may eventually cause the fitting to leak. If interchanging is absolutely necessary, use appropriate adapters and check all connections for leaks before proceeding. Highly concentrated salts (>0.2 M) and caustic mobile phases can reduce pump seal efficiency. The lifetime of injector rotor seals also depends on mobile phase conditions, particularly operation at high pH. In some cases, prolonged use of ion pair reagents has a lubricating effect on pump pistons that may produce small leaks at the seal. Some seals do not perform

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clogged column frit HPLC problem  

Leaks are a common problem in HPLC analyses. To minimize leaks in your system, avoid interchanging hardware and fittings from different manufacturers. Incompatible fittings can be forced to fit initially, but the separation may show problems and repeated connections may eventually cause the fitting to leak. If interchanging is absolutely necessary, use appropriate adapters and check all connections for leaks before proceeding. Highly concentrated salts (>0.2 M) and caustic mobile phases can reduce pump seal efficiency. The lifetime of injector rotor seals also depends on mobile phase conditions, particularly operation at high pH. In some cases, prolonged use of ion pair reagents has a lubricating effect on pump pistons that may produce small leaks at the seal. Some seals do not perform

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