A seminar on HPLC


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ppt on hplc detectors and derivatization


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A Seminar on HPLC:

A Seminar on HPLC By Bhavana. S M. Pharmacy 1 st year, Priyadarshini college of pharmaceutical sciences

Contents :

Contents History General introduction Detectors employed in HPLC Derivatization in HPLC Advantages of HPLC Applications of HPLC

History of HPLC :

History of HPLC In 1903, a Russian biochemist TSWETT developed a separation technique that involved the passage of the mixture of the plant pigments to be separated through a column of a finely powdered adsorbent. He poured the sample into the column containing alumina and calcium carbonate along with mobile phase. As the sample moved down the column, colored bands appeared correlating to the sample components. From this the word chromatography was developed meaning color writing.

History of HPLC :

History of HPLC The fundamental principles of HPLC were laid in 1960, but the actual development of the method took place during 1970’s. in 1990, the development of micro columns, stable detectors coupled with integrated data acquisition, storage and retrieval capabilities has increased the speed and efficiency of the HPLC instruments.

Introduction :

Introduction HPLC is an improved form of column chromatography. Instead of the mobile phase moving through the column as a result of gravity, it is forced through the column under high pressure. Typical operating pressures : 500-6000psi To get improved separation smaller sized packing material is required (<10µm).

Introduction :

Introduction Among all the liquid chromatographic procedures HPLC is the most accepted method. The basic principle in this method is the separation of a mixture based on partition.

Instrumentation :


Typical HPLC :

Typical HPLC

Detectors in HPLC:

Detectors in HPLC

Detectors :

Detectors A component that emits a response due to the eluting sample component and subsequently signals on to a chromatogram is called as a detector. These are very crucial as they makes the separation visible. Tswett’s offline detections methods were wet chemical, gravimetric, optical techniques which were time consuming and in convenient. Hence on line detection methods were developed.

Characteristics of a detector :

Characteristics of a detector Ideal characteristics of an HPLC detector- High sensitivity and predictable response. Response to all solutes/specificity. Not be affected by changes in temperature and carrier flow. Response independent to the mobile phase. No contribution towards extra column peak broadening.

Characteristics of a detector :

Characteristics of a detector Reliable and convenient to use. Response proportional to the solute concentration. Non destructive to the solute. Should provide quantitative information on the detected peak. However no detector possesses all the ideal characteristics.

Types of detectors in HPLC:

Types of detectors in HPLC The detectors employed in HPLC are divided into various categories based on the method of their detection. They are: Bulk Property Detectors- bulk property measurement. Sample Specific detectors : sample specific detection. Mobile phase modification detectors : mobile phase modification. Hyphenated detectors : hyphenated techniques.

Bulk property detectors :

Bulk property detectors Universal detector. Measures the change in a property which is common to all compounds i.e. differential measurement between the mobile phase with sample and without sample. Advantage : detect all compounds. Disadvantage: lack of sensitivity. Examples : refractive index detector,

Refractive index detector:

Refractive index detector Arrangement of refractive index detector

Working of RID:

Working of RID First described by Tiselius and claesson. Measures the change in the refractive index of the mobile phase as it leaves the column. Presence of solute changes the refractive index. Methods of measuring RI are angle of deviation method Fresnel method and thermal lens method.

Pros and cons of RID:

Pros and cons of RID Pros: 1) Measures a bulk property 2) Nearly Universal (different RI than mobile phase) 3) Comparable response for different analytes. 4) Detects species with no chromophores. Cons: 1) Temperature dependent 2) Poor sensitivity (LOD ≈ 100 ng) 3) No gradient elution

Sample specific detectors :

Sample specific detectors For every sample, some characteristics are unique and the sample specific detectors responds to those unique characteristics. Little high sensitivity than the bulk property measurement detectors. Examples: UV detectors, conductivity detectors and electrochemical detectors, fluorescent detectors.

UV Detector :

UV Detector Working UV detector responds to substances that absorb light in the range of 180- 350 nm. Working is based on beer lamberts law. Commonly used detector in HPLC.

Instrumentation :


Instrumentation of UV- Vis:

Instrumentation of UV- Vis Multi wavelength UV- Vis detector

Chromatogram :

Chromatogram Example of chromatogram from multi wavelength UV detector

Pros and cons of UV- Vis detector:

Pros and cons of UV- Vis detector Pros: 1) Measures a specific property 2) Nearly Universal (must have chromophore) 3) Most common of all detectors (~75%) 4) Potential to provide qualitative info. 5) Simple, robust Cons: 1) Fair sensitivity (LOD ≈ 1 ng) 2) Expensive with PDA, limited with Hg Lamp 3) Misses some important analytes

Fluorescent detector :

Fluorescent detector It is a variant of absorption spectrophotometer. Generally not employed for usage. Convenient and highly sensitive for fluorophores.

Instrumentation :

Instrumentation Fluorescence detector

Fluorescence detector :

Fluorescence detector

Chromatogram :


Pros and cons :

Pros and cons Pros: 1) Measures a specific property 2) Highly Selective (must fluoresce) 3) Second most common of all detectors (~15%) 4) High sensitivity (LOD ≈ 0.01 ng) 5) Can interrogate very small volumes Cons: 1) Not Universal 2) Limited Applications

Electrochemical detector :

Electrochemical detector Potential is applied and the solute undergoes oxidation or reduction depending upon the potential and chemical structure of the solute. The current produced in the reaction due to the application of potential is measured. Examples of compounds are: aromatic amines and phenols.

Pros and cons :

Pros and cons Pros: 1) Measures a specific property 2) Highly Selective (depends on reduction potential) 3) High sensitivity (LOD ≈ 0.01 ng) Cons: 1) Not Universal 2) Must have electrolyte in mobile phase 3) Mobile phase must be aqueous 4) Gradients not possible

Mobile phase modification detectors :

Mobile phase modification detectors These detectors change the mobile phase after the column to produce a change in the properties of the analyte. Those changes are liquid phase chemical reactions and the gas phase chemical reactions. Example: evaporative light scattering detectors and the mass spectrometric detectors.

Evaporative light scattering detector :

Evaporative light scattering detector

Evaporative light scattering detector :

Evaporative light scattering detector Working Works by measuring the light scattered by the solid solute particles remaining after nebulization and evaporation of the mobile phase. Mostly used for un derivatized lipids.

Pros and Cons:

Pros and Cons Pros: 1) Measures a bulk property 2) Nearly Universal (must be non-volatile) 3) Does not detect liquids (gradients are ok) 4) Detects species with no chromophores Cons: 1) Signal not linear with concentration 2) Fair sensitivity (LOD ≈ 1 ng) 3) No salts or buffers in mobile phase

Hyphenated techniques :

Hyphenated techniques Coupling of an independent analytical equipment to the HPLC systems to provide efficient detection. Examples: LC-MS, LC- IR, LC- FTIR etc.

Newer advancements :

Newer advancements Capability of recording entire spectrum of a compound with the increasing use of computers to collect, store and process the detector response data. Enhancement of detection sensitivity by forming derivatives of the samples. Example: amino acids can be detected efficiently by a fluorescence detector if they are first converted into their dansyl derivatives.

Derivatization in HPLC:

Derivatization in HPLC In order to increase the detect ability of various classes of compounds ( for which sensitive detectors are not available ) derivatisation is carried out in HPLC. A good amount of work has been performed on the labeling of compounds with chromophores and flurophores for detection using UV spectrometers and fluorimeters respectively. There are 2 important types of derivatisation. These are 1. Pre column derivatisation 2. Post column derivatisation

Derivatization in HPLC:

Derivatization in HPLC PRE COLUMN DERIVATISATION: In pre column derivatisation there are no restrictions on the solvents, reagents, or reaction rates chosen and excess of reagents can be removed before the injection. However, artifact formation, if present, can be checked by positive identification of the eluted peaks. For example, in the derivatisation of a triketone with more than one functional group capable of being derivatised there is a possibility of range of derivatives being formed from one solute. It is clearly necessary to check that the derivatisation reactions are quantitative or the sample derivatisations proceed in a manner analogues to the derivatisation of standards.


PRE COLUMN DERIVATISATION Examples of pre column derivatisation to form UV chromophores include treatment of ketosteroids with 2,4, DNP and the benzoylation of hydroxy steroids or the esterification of fatty acids. Similarly, fluorophores have been introduced into amino acids, biogenic amines, and alkaloids by treatment with dansyl chloride.

Derivatization in HPLC:

Derivatization in HPLC POST COLUMN DERIVATISATION : It is carried out on the separated solutes as they emerge from the chromatographic column. In HPLC, this places serious restriction on the derivatisation reactions, because dilution of the eluent peak must be minimized. Consequently, very fast reactions must be used and the reagents and mobile phase must be compatible.


POST COLUMN DERIVATISATION Examples of post column derivatisation reactions for use with UV detectors include: Reaction of amino acids with ninhydrin and fluorescamine. Reaction of fatty acid with ortho nitro phenol. Reaction of ketones with 2, 4, DNP. Thermal or acid treatment of carbohydrates. An oxidation detector for the fluorimetric analysis of carbohydrates in body fluids using Ce (III) flourescence has also been reported.

Advantages of HPLC::

Advantages of HPLC : Separations fast and efficient (high resolution power) Continuous monitoring of the column effluent It can be applied to the separation and analysis of very complex mixtures Accurate quantitative measurements. Repetitive and reproducible analysis using the same column. Adsorption, partition, ion exchange and exclusion column separations are excellently made.

Advantages of HPLC:

Advantages of HPLC 7. HPLC is more versatile than GLC in some respects, because it has the advantage of not being restricted to volatile and thermally stable solute and the choice of mobile and stationary phases is much wider in HPLC 8. Both aqueous and non aqueous samples can be analyzed with little or no sample pre treatment 9. A variety of solvents and column packing's are available, providing a high degree of selectivity for specific analyses. 10. It provides a means for determination of multiple components in a single analysis

Applications of HPLC :

Applications of HPLC HPLC is one of the most widely applied analytical separation techniques . Pharmaceutical: Tablet dissolution of pharmaceutical dosages. Shelf life determinations of pharmaceutical products. Identification of counterfeit drug products. Pharmaceutical quality control

Applications of HPLC:

Applications of HPLC Environmental Phenols in Drinking Water. Identification of diphenhydramine in sediment samples. Bio monitoring of PAH pollution in high-altitude mountain lakes through the analysis of fish bile. Toxicity of tetracycline's and tetracycline degradation products to environmentally relevant bacteria. Assessment of TNT toxicity in sediment.

Applications of HPLC:

Applications of HPLC Forensics A mobile HPLC apparatus at dance parties - on-site identification and quantification of the drug Ecstasy. Identification of anabolic steroids in serum, urine, sweat and hair. Forensic analysis of textile dyes. Determination of cocaine and metabolites in meconium. Simultaneous quantification of psychotherapeutic drugs in human plasma.

Applications of HPLC:

Applications of HPLC Clinical Quantification of DEET in Human Urine. Analysis of antibiotics. Increased urinary excretion of aquaporin 2 in patients with liver cirrhosis. Detection of endogenous neuro peptides in brain extracellular fluids.

Applications of HPLC:

Applications of HPLC Food and Flavor Ensuring soft drink consistency and quality. Analysis of vicinal diketones in beer. Sugar analysis in fruit juices. Polycyclic aromatic hydrocarbons in Brazilian vegetables and fruits. Trace analysis of military high explosives in agricultural crops. Stability of aspartame in the presence of glucose and vanillin.

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