How does hplc work ppt

Search for:. Deepak October 7, Introduction to High Performance Liquid Chromatography and its parts Chromatography equipment looks rather intimidating to anyone who has not handled it before, but on a closer look and as you get familiar with the equipment you realize that behind the network of wires, complex plumbing and circuitry is a simple machine with only a few major parts.

Different combinations of these parts namely : Pumps Detectors Injectors yield an infinite number of configurations based on the application. What is HPLC? Mobile Phase The liquid phase is pumped at a constant rate to the column packed with the stationary phase.

Mobile Phase Mobile phase serves to transport the sample to the system. Mobile Phase Reservoirs These are inert containers for mobile phase storage and transport. Pumps Variations in flow rates of the mobile phase effect elution time of sample components and result in errors. Injectors Injectors are used to provide constant volume injection of sample into the mobile phase stream.

Column A column is a stainless steel tube packed with stationary phase. Column Oven Variation of temperature during the analytical run can result in changes of retention time of the separated eluting components. This ensures a constant flow rate of the mobile phase through the column Detector A detector gives specific response for the components separated by the column and also provides the required sensitivity. Specialized Versions Specialized HPLC Systems might also have solvent selection valves, vacuum degasser, auto samplers, column switches, pre or post column derivatization and fraction collectors.

Keep Learning to Keep Succeeding — visit www. Need samples to be tested visit www. Categories: HPLC. Related Articles. Deepak September 13, Dr Saurabh June 28, Madhu Kumari March 18, Deepak March 1, Deepak September 1, Responses Cancel reply Connect with:. Urmila Sathe March 13, Deepak March 14, Deepak July 2, Venu July 2, Course Preview this Course.

The pKa values of compounds are given in physics and chemical dictionaries and in books that refer to acid-base equilibrium. Preparation Procedure Prepare the solution by mixing the acid with its salt. For example, to prepare acetate buffer solution, mix acetic acid in an undissociated state with its salt, sodium acetate. Sodium ions are strongly basic, and because they almost completely dissociate in aqueous solutions, adding sodium acetate is essentially equivalent to adding dissociated acetate ions.

To adjust the pH value of a buffer solution so that it is close to the pKa value, mix the acid with its salt so that they have the same molar concentration.

To separate the pH value somewhat from the pKa value, change the mixing ratio of the acid and its salt. Does not adversely affect detection. Does not damage column or equipment. Commonly Used Acids Phosphoric acid pKa 2.

One obvious requirement of a buffer solution is that it has a high buffering power in a neighborhood of the desired pH. If it is to be used as an HPLC eluent, several other properties are required.

First, it is important that it does not adversely affect detection. With UV detection, it must have no UV absorption. With fluorescence detection, it must have no fluorescence.

With electrical conductivity detection, it must either have a low equivalent conductivity or it must be difficult to ionize. It is important to use a buffer solution that does not damage the column or any other equipment. In general, the wetted parts of an HPLC system are composed of stainless steel, so it is advisable to avoid halogens or other substances that may corrode the steel. With LCMS, nonvolatile salts that may precipitate at the interface must not be used.

Phosphate buffer solution satisfies the above conditions, is relatively inexpensive, and is the most commonly used substance. Acetate and citrate buffer solutions are also commonly used. All of these solutions are used in the acidic region, for pH values in a range of approximately 2 to 7. Because the ODS column, which is the most representative separation column for HPLC, is not suited to alkalis, the use of buffer solutions with pH values in the alkaline range is relatively uncommon.

The buffering capacity increases with the concentration of the buffer salt. Therefore, it is advisable to prepare the buffer solution with as low a concentration as possible within the range in which an appropriate pH buffering capacity is attained. This may not be appropriate, however, if the salt concentration exerts a large influence on separation e. The most commonly used buffer solution in HPLC is phosphate buffer solution.

Phosphoric acid dissociates in three stages, so there are three pKa values, and it has a buffering power in the neighborhoods of the corresponding pH values. In particular, it is often used as an acidic buffer solution in the range of pH 2 to 3 and as a neutral buffer solution in a neighborhood of pH 7. Also, because it has almost no UV absorption, there is almost no background signal in UV detection, which is the most representative HPLC detection method.

This is another reason why this buffer solution is used so widely. Although phosphate buffer solution is used so widely, because it has no volatility, it is difficult to use in LCMS and evaporative light scattering detection.

The lack of volatility also makes post-processing difficult in the preparative separation and purification of peak components. In this case, use another acid that has volatility, even if it has inferior buffering capacity. Reversed phase ion pair chromatography is also briefly mentioned. Regarding the stationary phase, it is generally appropriate to use an ODS column.

Situations where another type of column must be used are quite rare. Regarding the eluent composition, if the target components are substances that do not ionize, use a mixture of water and acetonitrile or a mixture of water and methanol. Choose between acetonitrile or methanol on the basis of which better facilitates separation and which is more convenient generally. Adjust overall retention by changing the mixing ratio of the water and these organic solvents.

The above diagram shows how the retention strength for benzoic acid changes. The acid dissociation constant pKa of benzoic acid is 4. In eluents of greater acidity, it exists as undissociated benzoic acid, whereas in eluents of greater alkalinity, it exists as benzoic acid ions. In general, because ionic substances have more affinity towards networks of hydrogen bonds, the ionized form of a substance dissolves more readily in polar eluents, and is less easily retained by the stationary phase.

Therefore, for an acidic anionic substance such as benzoic acid, if the pH of the eluent is more acidic than the acid dissociation constant, the retention strength increases, whereas if it is more alkaline than the acid dissociation constant, the retention strength decreases.

Naturally, the opposite occurs with basic cationic substances. With neutral substances that do not ionize, the pH of the eluent has almost no influence on the retention strength. With substances e. The eluent is rendered acidic in order to suppress the ionization of the target components and to make it easier for them to be retained by the stationary phase.

A phosphate is used as the buffer salt. This is because it has a high pH buffering power in a neighborhood of pH 2 to 3, it has almost no UV absorption, and it is inexpensive. One possible method is to use a mixture of an alkali buffer solution and either acetonitrile or methanol in order to suppress ionization. This method is certainly effective with regard to separation, but it creates another problem and is not often used.

The problem is that the silica gel dissolves in alkalis; consequently, the packing material deteriorates rapidly. The usable pH range for a standard ODS column is approximately 2 to 8, and it is said that staying within a range of approximately 2 to 5 helps prolong the service life of the column.

In other words, the pH of the eluent must be set in the acidic-to-neutral range. However, packing material in which the surface of the silica gel has been specially treated in order to protect it from the influence of alkalis, thereby improving the alkali resistance, has recently been developed.

In addition to silica gel, there is also packing material that was produced by bonding octadecyl groups with a polymer substrate. Therefore, when using an alkali as the eluent, it is necessary to use a separation column filled with a packing material that can withstand it. Si Si O N -O-Si-O Another possible method that can be used for basic substances, as with acidic substances, is to use a mixture of an acidic buffer solution and an organic solvent.

In this case, although the target component is cationized, the decrease in retention strength is compensated for by lowering the proportion of organic solvent in the eluent. It is appropriate to set the pH of the buffer solution in the neighborhood of 2 to 3. This is to suppress the influence of residual silanol groups for which the octadecyl groups have not bonded to the surface of the stationary phase.

Although octadecyl groups are chemically bonded to silanol groups on the surface of the silica gel, because of their bulky structure, it is impossible to bond to all silanol groups. The residual silanol groups are weakly acidic and therefore attract basic substances, which causes tailing of the peaks.

Using an acidic buffer solution makes it possible to suppress dissociation of the residual silanol groups and reduce their influence to some extent. With this treatment, bonding relatively compact trimethylsilyl groups and other substances with the residual silanol groups makes it possible to reduce the influence of the silanol groups on separation.

Even if the eluent is made acidic, with packing material containing a large number of residual silanol groups, it is impossible to suppress the tailing that occurs with basic substances.

When analyzing basic substances, be sure to use a column that has been subjected to end-capping. Note that residual silanol groups are not the only cause of tailing.

For example, tailing is prone to occur if packing material containing metallic impurities is used for compounds that easily form complexes with metals. Separation columns with reduced metal content are commercially available. So, if necessary, try using one of these columns instead. Anions and cations with low charge densities form ion pairs in aqueous solutions, thereby balancing the charge. This causes the hydrophobicity and the strength of retention by the stationary phase to increase.

It also suppresses tailing. Sodium perchlorate is widely used for this purpose. To further increase the retention strength, add sodium alkylsulfonate to the eluent. Further details are provided in the section on reversed phase ion pair chromatography. Basic substances form ion pairs with perchlorate ions, thereby balancing the charge and increasing the retention strength.

Depending on the properties of the target substances, it may not be possible to suppress tailing. Tailing suppression effect is relatively high. The concentration and pH of the alkyl sulfonic acid and the concentration of the organic solvent must be optimized.

Of the above, method 3 is relatively easy to implement and trouble-free. Therefore, first try method 3 , and if there seems to be a problem, try method 4 or try using a different separation column or separation mode. Ion pair formation Ion pair formation If the target substance is highly soluble in water, and has either cationic or anionic properties, it is usually appropriate to use cation or anion exchange chromatography.

If, however, you want to simultaneously analyze the uncharged solutes in the sample, use reversed phase ion pair chromatography.

Ions that have the opposite charge to the target substance, and that contain hydrophobic functional groups, are dissolved in the eluent. The target substance and the ion pair reagent form ion pairs in the eluent. As a result, the overall charge is balanced out, the hydrophobicity increases, and the retention strength of the stationary phase increases. Furthermore, if an ion pair reagent that has hydrophobic functional groups, such as alkyl groups, is used, the ion pair reagent itself is retained in the stationary phase, and due to a type of ion exchange-like interaction, solutes with the opposite charge are also retained.

This method makes it possible for highly water-soluble ions that are not retained at all with an eluent that does not contain an ion pair reagent to be retained with an ODS column. Tetrabutylammonium salts are often used in the analysis of anionic substances. Alkylamines are also used for this purpose.

Alkyl sulfonate salts are used in the analysis of cationic substances. They come in a variety of chain lengths and use a type that is suitable for the retention and separation conditions. Prepare the eluent by adding one of the above to a pH buffer solution at an appropriate concentration. In general, increasing the concentration increases the retention strength.

Beyond a certain point, however, ion pair reagent molecules combine with each other, causing micellization, and the retention strength stops increasing. Concentration of Ion Pair Reagent In general, the retention strength increases with the ion pair concentration, but there is an upper limit. Proportion of Organic Solvent in Eluent Optimize the separation conditions by considering the type and concentration of the ion pair reagent.

In ion pair chromatography, there are many parameters, such as the type and concentration of the ion pair reagent, the pH of the eluent, and the concentration of the organic solvent, involved in the setting of the mobile phase conditions. Also, in the simultaneous analysis of ionic substances and nonionic substances, because of differences in the behavior of substances that are retained by the ion pair effect and substances that are not, setting conditions is even more complex.

Therefore, in cases where the addition of an ion pair reagent may not be absolutely necessary, it may be faster to first consider the condition setting guidelines previously given for standard reversed phase chromatography. If this is not successful, consider the use of ion pairs.

In fact, there are other separation modes, and in some cases, multiple separation modes are used simultaneously. It would be impossible to exhaustively categorize every type. For now, just remember the four types mentioned above. Liquid-solid chromatography The retention strength increases with the hydrophilicity of the solute.

Chemically unmodified silica gel is often used as the stationary phase. Because the silanol groups Si-OH on its surface are hydrophilic, polar solutes are retained by the stationary phase due to hydrogen bonding and hydrophilic interactions, such as dipolar interactions.

Substances with higher degrees of hydrophilicity undergo stronger interactions with the stationary phase; consequently, they are retained with greater strength in the column. Because the force that acts between the solutes and the stationary phase in this mode is almost the same as the one that acts in normal phase partition chromatography, which is described later, adsorption chromatography and normal phase partition chromatography are not distinguished in some cases.

Liquid-liquid chromatography In adsorption chromatography, the strength of solute retention is mainly determined by the degree of adsorption to a solid surface. In partition chromatography, the retention strength is determined by whether the solute dissolves more easily in the stationary phase, which can be thought of as a liquid, or the mobile phase. Of course, the liquids used for the stationary phase and mobile phase must not be mutually soluble.

Apparently, in early partition chromatography, the stationary phase was actually impregnated with a liquid. The impregnated liquid would gradually flow out, however, so materials created by chemically bonding functional groups to the surface of the stationary phase came into use. As a general rule, similar substances tend to be mutually soluble; so, in order to ensure mutual insolubility, substances with contrasting properties are set as the stationary and mobile phases.

Therefore, use a combination of stationary phase and mobile phase in which one has a high polarity hydrophilic and one has a low polarity hydrophobic.

Materials created by bonding polar functional groups, such as aminopropyl groups and cyanopropyl groups, to the surface of silica gel are often used as the stationary phase. Nonpolar solvents, such as hexane and chloroform, are used as the mobile phase. As with adsorption chromatography based on silica gel, the force that acts between the solutes and the stationary phase is based on hydrophilic interactions, such as hydrogen bonding and dipolar interactions.

Therefore, it has become common to not distinguish between adsorption chromatography and normal phase partition chromatography. This is said to be the start of chromatography. Additional solvents: Alcohols, ethers, etc. Stationary Phase Materials created by chemically modifying silica gel with cyanopropyl groups or aminopropyl groups are typically used as the stationary phase in normal phase chromatography. While chemically unmodified silica gel and silica gel modified with cyanopropyl groups are widely used in general applications, silica gel modified with aminopropyl groups is often used in the analysis of sugars.

Mobile Phase The mobile phase is prepared by adding an additive solvent to a basic solvent. The following are often used as the basic solvent: Hydrocarbons pentane, hexane, heptane, octane Aromatic hydrocarbons benzene, toluene, xylene Organochloride compounds chloroform, dichlormethane The following are used as the additive solvent: Alcohols 2-propanol, ethanol, methanol Ethers methyl-t-butyl ether, diethyl ether Tetrahydrofuran, dioxane, pyridine, ethyl acetate, acetonitrile, acetone In general, the basic solvent is selected first, and the additive solvent is added to adjust the retention time and separation.

Selecting solvents with little UV absorption is beneficial for UV detection. A small amount of acid is sometimes added to the mobile phase in order to suppress the ionization of solutes. For this reason, elution tends to take longer if the sample contains any of the following functional groups: -COOH: Carboxyl groups -NH2: Amino groups -OH: Hydroxyl groups On the other hand, if the sample is a hydrocarbon and there are no hydrogen bonding sites, or if there are large hydrocarbon groups around the hydrogen bonding sites that act as steric hindrances, elution tends to occur sooner.

Steric hindrance. The above diagram shows how differences in the eluent affect the chromatogram. As the polarity of the eluent increases i. In general, normal phase mode is said to be effective for the separation of structural isomers that differ in terms of the connection points of functional groups.

On the other hand, it is not effective for the separation of homologs that differ only in the length of aliphatic chain. Reversed phase mode is more effective for this application. Regarding the time taken for the stationary phase and mobile phase to reach an equilibrium, reversed phase mode is significantly faster than normal phase mode.

Reversed phase mode is also superior in terms of the stability of the stationary phase. Many of the columns used for normal phase mode have shorter service lives. In actual analysis work, the cost and safety of the eluent used cannot be ignored. Water, methanol, and acetonitrile, which are solvents mainly used in reversed phase mode, are inexpensive and relatively easy to handle.

The fact that these solvents have little UV absorption is an important advantage in HPLC, in which absorption detectors are often used.

On the other hand, many of the nonaqueous solvents that are mainly used in normal phase mode are expensive and somewhat difficult to handle due, for example, to adverse effects on health and the danger of explosions resulting from contact with flames or static electricity. In addition to the above points, reversed phase mode can be used for a wide range of target substances so, at present, the most common approach in situations where either mode can be used is to first try using reversed phase mode.

Therefore, the force that acts between the solutes and the stationary phase probably consists mainly of Coulomb forces. Ion exchange chromatography can be divided into two types according to the charge on the target solutes: cation exchange chromatography and anion exchange chromatography. Naturally, ionic substances are the target of ion exchange chromatography. Proteins, peptides, and amino acids are all ionic substances, so they can be analyzed using ion exchange chromatography.

Therefore, this separation mode is used widely in the field of biochemistry. Although these terms names are similar, try to use them correctly. Silica gel is also used. Many different types of resin, including polystyrene resin and methacrylic resin, are used. Silica is an inorganic material that is also often used, and there are many other types of materials that are being developed and sold. Strong ion exchangers have functional groups whose exchange capacity does not depend greatly on the pH of the eluent and are continuously ionizing.

More specifically, exchangers created by chemically bonding sulfonic acid groups and quaternary ammonium groups are often used.

Weak ion exchangers have functional groups whose dissociation is suppressed and whose exchange capacity decreases to an extent that depends on the pH of the eluent. More specifically, exchangers created by chemically bonding carboxyl groups and tertiary ammonium groups are often used.

Use an exchanger that is appropriate for the substance to be separated and the pH characteristics of that substance. An eluent ion is driven away and a solute ion is adsorbed.

The solute ion is driven away by an eluent ion and is adsorbed by the next exchange group. In ion exchange chromatography, the solutes and the stationary phase have the opposite charge and attract each other electrostatically. Why, then, are the solutes eluted from the column? Why are they not continuously held by the stationary phase?

This is because ions with a charge of the same sign as the solutes are added to the eluent, and these ions compete with the solute ions for exchange groups. Therefore, the overall elution rate can be increased without greatly changing the separation selectivity by increasing the overall salt concentration of the eluent. In other words, the effect that is achieved in reversed phase and normal phase chromatography by changing the mixing ratio of the solvents comprising the eluent in order to adjust the polarity and thereby increase or decrease the overall elution time is achieved in ion exchange chromatography by adjusting the salt concentration of the eluent.

Solute ions and eluent ions compete for ion exchange groups. If the salt concentration of the eluent increases, the solutes are eluted sooner.

The mechanism of operation, however, is completely different. If the solutes and the stationary phase have a charge of the same sign, a Coulomb repulsive force acts.

It is considered that the strength of this repulsive force increases with the ease with which the solutes ionize. On the other hand, ions that do not dissociate readily and substances with no charge are not subject to the same level of repulsion, so they reach the interior of the pores in the packing material. They are consequently retained by the stationary phase due to size exclusion and reversed phase interactions.

Ion exclusion chromatography, then, is a separation mode in which the retention strength is determined mainly by the degree of dissociation of the solute ions. In practice, this mode is often used for the separation of short-chain fatty acids. Strong acid ions are repelled by charge and cannot enter the pore.

Depending on the level of dissociation, some weak acid ions can enter the pore. The name varies with the application field! Size Exclusion Chromatography SEC Gel Permeation Chromatography GPC Chemical industry fields, synthetic polymers, nonaqueous systems Gel Filtration Chromatography GFC Biochemical fields, biological macromolecules, aqueous systems Whereas all of the previously described separation modes use chemical or electrostatic interactions to produce retention strength, in size exclusion chromatography, there are ideally no interactions between the solute molecules and the stationary phase.

Gel particles with a gigantic network structure are used as the stationary phase, and separation takes place according to the degree to which the solute molecules penetrate the pores of these particles. In general, it is used in the quality control and property evaluation of chemically synthesized resins. Hydrophobic resins, such as polystyrene, are used as the stationary phase and substances such as tetrahydrofuran, chloroform, and dimethylformamide are used as the eluent.

It is mainly used in relation to the analysis of biological macromolecules, such as proteins, peptides, nucleic acids, and polysaccharides. Because all of these macromolecules are soluble in water, highly hydrophilic resins are often used as the stationary phase, and aqueous solutions are often used as the eluent. Packing material When solutes are carried by the flow of the eluent into the stationary phase, which consists of macromolecules with a gigantic network structure, relatively small molecules penetrate the interior of the network whereas relatively large molecules cannot enter the network and are excluded.

In other words, the range in which the solute molecules can move around inside the column i. Solutes with a low molecular weight can move around in a large range and consequently are eluted slowly, whereas solutes with a high molecular weight are restricted and consequently are eluted quickly. Solute molecules smaller than a certain size completely permeate the interior of the stationary phase and are consequently all eluted at almost the same position.

In size exclusion mode, separation takes place between the exclusion limit and the permeability limit. These limits are determined by the packing material used, so it is necessary to estimate the molecular weight of the target substances and select an appropriate separation column.

Molecular weight markers, for which the molecular weight is already known, are commercially available, so obtain elution capacities retention times for these markers and use the results to create a calibration curve. It is difficult to cover a wide range of molecular weights with the same packing material i.

Select a column that is appropriate for the desired molecular weight range. If the molecular weight distribution of the sample covers a wide range, select multiple columns from the same series that collectively cover this range, and connect them in series.

A possible alternative approach is to use a column that has been filled with packing materials that have differing degrees of cross-linkage. Elution capacity For separation of small molecular weights. Molecular weights and molecular weight distributions are calculated using special calculation software. Chromatogram Calibration curve Special calculation software is required to calculate various average molecular weights and molecular weight distributions.

Usually, a chromatogram is sampled with a chromatogram data processor software , and special software performs molecular weight calculations using the data file obtained. Basically, the chromatogram is sliced at regular intervals, and the respective area values are obtained. At the same time, the molecular weights for the slices are calculated from a previously created calibration curve. Then, calculating the area at the position of each molecular weight makes it possible to obtain the average molecular weights.

It is said that these correlate with the various properties of macromolecular compounds, so use these values as necessary and evaluate the results. Retention time. Is there UV absorption or fluorescence? Is derivatization possible? There are many different separation modes in HPLC, so it is necessary to select a mode that is appropriate for the target substances and samples.

The following kind of information is required for the selection of the separation mode: Soluble Solvent In principle, HPLC can be used for any substance that can be dissolved in a liquid. Conversely, however, if the liquids in which the substance dissolves are not known, then appropriate analytical conditions cannot be established. First and foremost, then, it is essential to know which solvents the substance dissolves in.

Molecular Weight Size exclusion mode is used for substances of more than a certain molecular weight, whereas other separation modes are used for substances of less than this molecular weight. Size exclusion mode is the first choice for molecular weights of more than a few thousand. Even in this case, however, in order to select a column with an appropriate exclusion limit, information on the approximate molecular weight range is required.

In addition, it is desirable to have as much information as possible on the chemical properties, including the structural formulas, of the substances and samples to be analyzed. In particular, knowing if the substances ionize makes it possible to know whether or not ion exchange mode or reversed phase ion pair mode can be used, and the existence of UV absorption or fluorescence has a bearing on detector selection.

The main reason for this is its wide application range. Therefore, except in a few situations, reversed phase mode is the first choice. Some exceptions are described below: For compounds with a molecular weight of more than a few thousand, size exclusion mode is used. Also, regarding the analysis of lipophilic substances that do not dissolve in mixtures of water and organic solvents, this mode is the first choice in many cases.

Although ion chromatography is, in principle, a branch of HPLC, dedicated equipment is commercially available that is unlike standard HPLC, which is mainly used for the analysis of organic substances, so it is distinguished by the use of a different name. In addition, for sugars, amino acids, and short-chain fatty acids, the special separation columns listed below are commercially available.

It is advisable to select one these columns in accordance with the application. Monosaccharides, oligosaccharides: Reversed phase mode using column with amino group-bonded packing material Size exclusion-ligand exchange Anion exchange, etc. Amino acids: Cation exchange pre-column derivatization- reversed phase mode, etc.

Short-chain fatty acids: Ion exclusion Ion exchange, etc. Selectivity The detector must be able to detect the target substance without, if possible, detecting other substances.

Adaptability to separation conditions Operability, etc. HPLC analytical conditions can be divided broadly into separation conditions and detection conditions. There is also the category of pretreatment conditions but this is omitted here. The most important considerations with regard to the detection conditions are probably sensitivity and selectivity. Sensitivity Although high sensitivity is generally required, this may not apply to all situations.

If peaks are clipped or if the linearity of the calibration curve is poor, it may be better to use a detector of lower sensitivity. Selectivity It must be possible to detect the target substances, ideally without detecting other substances. Even if other substances are detected, this is not a problem as long as they are separated in the column. In fact, if no other substances are detected, separation may not be necessary.

As with sensitivity, a high level of selectivity may not necessarily be suitable in every situation. For example, when investigating the impurity content of samples, a low level of selectivity may be preferable.

Adaptability to Separation Conditions With some detectors, there are restrictions with regard to the setting range of separation conditions. For example, some detectors cannot be used for gradient analysis and some detectors cannot handle nonvolatile salts. Also, it is usually not permissible to add substances to the eluent that are detected by the detector. It is necessary to confirm that these restrictions do not present problems before setting the analytical conditions.

The representative detectors used in HPLC are listed above. Here, the principles and ranges of application of these detectors are described in order. It directs light of a certain wavelength through a solution, and observes the decrease of intensity in the light that passes through. Because, in accordance with the Lambert-Beer law, the absorbance is proportional to the concentration of the absorbing substance, measuring changes in the absorbance of the eluent makes it possible to calculate the concentrations of peak components.

These detectors can be used for substances that have absorption in the ultraviolet region approx. Not only do many organic compounds, which are the main target substances of HPLC, have this absorption, the detectors have a relatively high sensitivity and are not easily influenced by factors such as temperature and pulsations.

For this reason, they are the most representative HPLC detectors and are widely used. A deuterium lamp ultraviolet light or a tungsten lamp visible light is mainly used as the light source. Monochromatic light produced by a diffraction grating passes through a cell, and when it enters a silicon photodiode or some other detector, it is converted to an electrical signal. Usually, before the light reaches the cell, it is split into two beams, with one beam passing through a sample cell and the other beam passing through a reference cell which is usually just an empty space for reference.

Measurement data is obtained from the ratio of the intensities of these two beams. Whether or not an organic compound has absorption in the ultraviolet or visible regions depends on its molecular structure. In general, organic compounds with double bonds have ultraviolet absorption.

This detector is particularly suited, however, to substances with conjugated structures, such as conjugated dienes and benzene skeletons. There is a tendency for compounds with large numbers of conjugated double bonds to have absorption at longer wavelengths. The ultraviolet spectrum for caffeine is shown above.

All compounds have unique spectra like this. Usually, high sensitivity is desirable; in this case, set a wavelength that corresponds to the absorption maximum as the detection wavelength.

However, in terms of selectivity, it is advantageous to set a longer wavelength, so, if there are many co-existing substances in the sample, set a long wavelength as the detection wavelength, even if sensitivity is somewhat sacrificed. With standard absorbance detectors, however, the light is divided by a diffraction grating before it reaches the cell and monochromatic light is used, whereas with photodiode array detectors, polychromatic light is passed through the cell and guided to the detector.

This gives photodiode array detectors the important advantage of being able to obtain spectra in real time. The principle behind the optical system of this type of detector is illustrated above. After polychromatic light passes through the cell, it is divided by a diffraction grating, and guided to photodiode array elements. The photodiode array consists of a large number of elements in many cases , and these elements simultaneously convert light components corresponding to a continuous range of distinct wavelengths separated by intervals of 1 nm in many cases into electrical signals.

This allows real-time spectrum measurement. Photodiode array. For this reason, the data is processed using special analysis software instead of the standard type of chromatograph data processor. This software makes it possible to view the spectra corresponding to specified times and the chromatograms corresponding to specific wavelengths.

Wavelength Retention time. It can obtain chromatograms simultaneously at multiple wavelengths so, in addition to being able to perform the role of multiple single-wavelength absorbance detectors as a single unit, it offers the following advantages: Peak Identification Based on UV Spectra Basically, retention time is the only means of identification available in chromatography; so, in terms of qualitative capability, chromatography is a poorer analytical technique than techniques such as NMR, MS, and FTIR.

For this reason, attempts are made to offset this weakness using the selectivity and qualitative capability of the detector. With a photodiode array detector, a UV spectrum can be obtained for each peak, so these spectra can be used as a means of identification. Furthermore, many types of analysis software incorporate a library search function, making it possible to search the library for the UV spectra that most closely resemble the ones obtained.

Evaluation of Peak Purity From the detection start point to the detection end point of a pure peak, the shape of the spectrum should not change simply due to changes in absorbance. Therefore, by obtaining and comparing the spectra obtained at different times for the same peak, it is possible to evaluate the optical purity of that peak. Many software products incorporate a function that obtains and compares the spectra for three points of a given peak: the top, a point on the up-slope, and a point on the down-slope.

If such a substance is irradiated with light of a certain wavelength, light of a longer wavelength is emitted. The molecules in organic compounds are connected via shared electrons. When irradiated with light, the kinetic energy of such an electron changes, and the electron is transferred from the ground state to an excited state.

In this case, light with a wavelength longer than that of the irradiating light is emitted. The number of substances that emit fluorescence is relatively small compared to the number of substances that have absorption in the ultraviolet region, so the selectivity of fluorescence detection can be described as high.

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Permission to Dream Chris Gardner. Gundry, MD. Single On Purpose: Redefine Everything. Find Yourself First. John Kim. Ananthu Ananthu. Siddharth Salve. Lavish Kumar. Amisha Patil. Show More. Views Total views. Actions Shares. No notes for slide. Hplc 1. Sagar Kishor savale [Department of Pharmacy Pharmaceutics ] avengersagar16 gmail. Normal phase mode 2. Adsorption chromatography 2. Ion exchange chromatography 3. Partition chromatography 4. Isocratic separation 2. Analytical HPLC 2.

Qualitative analysis 2. Quantitative analysis 3 4. Separation of moderately polar analytes using adsorption onto a pure stationary phase e. This can be overcome by use of dual pump heads or elliptical cams to minimize such pulsations.

Single-piston pump with slow filling cycle B. Single-piston pump with a rapid filling cycle C. A dual-piston pump with rapid filling cycles and operate out of phase. Precolumn 1. Analytical column 2.

The solid support can be silica gel, alumina. RF Fluorescent compounds, usually with fused rings or highly conjugated planar system.