DERVATIZATION AND APPLICATION OF HPLC DEPARTMENT OF PHARMACEUTICAL CHEMISTRY MCOPSSUBMITTED TO SUBMITTED BYDr Suvarna G. kini Shikha TyagiAssociate. Professor 100602017
High-performance liquid chromatography (or high-pressure liquid chromatography,HPLC) is a chromatographic technique that can separate a mixture of compounds and isused in biochemistry and analytical chemistry to identify, quantify and purify theindividual components of the mixture.High performance liquid chromatography is basically a highly improved form ofcolumn chromatography.Instead of a solvent being allowed to drip through a column under gravity, it isforced through under high pressures of up to 400 atmospheres. That makes it much faster.smaller particle size for the column packing material which gives a much greatersurface area for interactions between the stationary phase and the molecules flowingpast it.This allows a much better separation of the components of the mixture.
SCHEMATIC REPRESENTATION OF HPLC
LISTS OF STEPS NEEDED BEFORE ANYONE RUNS HPLCFilter the solvents with membranes with 0.22-0.45 mmUse clean and transparent reservoirs through which precipitates andcolloids can be distinguished.Make sure that the solvents will be easily mixed with the previous solventsin the same inlets . For example methanol or water should not be placedinstead of hexane directly, or any organic solvent should not be placeddirectly instead of a buffer reservoir.Degass the solvents and purge all the tubing that lead to the pump.Connect the column carefully according to the flow direction marked on it(do not connect directly to the detector).Flow the appropriate solvents through the column at a low flow-rate (0.1-0.5 ml/min) or reach the composition gradually using the appropriategradient. Select the appropriate wavelength (or other type of setting) in thedetector and wait for stable baseline .
Prepare the set of methods in the workstation Processing method for the data processing and the Report method for thereport of final results.When the system and the methods are ready a blank run should beperformed to test the system and verify that it is clean from interferences
DERIVATIZATION Derivatization is a technique used in chemistry which transforms a chemicalcompound into a product (the reactions derivate) of similar chemical structurecalled a derivative. Generally, a specific functional group of the compound participates in the derivatizationreaction and transforms the it to a derivate of deviating reactivity, solubility, boiling point melting point,aggregate state, or chemical composition. Resulting new chemical properties can be used for quantification or separation of thecompound
RATIONALE BEHIND DERIVATIZATIONIn liquid chromatography, fluorescent derivatives can be prepared to render thesubstances specifically detectable at high sensitivity.To prepare fluorescent derivatives of phenols, and primary and secondary amines,dansyl chloride (5-dimethyl aminonaphthalene-1-sulphonyl chloride) is stronglyrecommendedTo render involatile substances volatile for GC analysis, organic acids can beesterified using boron trifluoride as a catalyst or directly with diazomethane.the polarity of a solute needs to be drastically reduced to improve itschromatographic behavior and reduce tailing. Polarity reduction can often beachieved for amino, hydroxyl and thiol groups by acylation.In LC analyses, UV chromaphores and fluorophores are often introduced intosample molecules to increase their sensitivity to UV absorption and fluorescencedetection. Benzoyl chloride, m-toluol chloride and p-nitrobenzoyl chloride arereagents that can add a benzene ring to a solute molecule and render it UVabsorbing.
SEVERAL CHARACTERISTICS ARE DESIRABLE FOR A DERIVATIZATION REACTIONThe reaction is reliable and proceeds to completion.Less unreacted starting material will simplify analysis.Also, this allows a small amount of analyte to be used.The reaction is general, allowing a wide range of substrates, yet specific to a single functional group,reducing complicating interference.The products are relatively stable, and form no degradation products within a reasonable period, facilitating analysis.
DERIVATIZATION TYPESPre columnPost column
PRE-COLUMN OFF-LINE DERIVATIZATIONWhen there is a mixture of many componds they may interfere in the separation andresolution in such case we can derivatize the compound of interest to change itsproperties so that it can be separated .Merits :(a) Requires no modification to the instrument i.e., a plus point when compared to the post-column methods(b) Imposes fewer limitations with regard to reaction-time and conditions.Demerits(a) Formation of a stable and well-defined product is an absolute necessity.(b) Presence of excess reagent or by products may invariably interfere withseparation.(c) Very often derivatization may altogether change the chromatographic propertiesof the sample which facilitated separation.
Nineteen amino acids in a standard mixture were separated using a GROM-SILOPA-1 column after precolumn derivatization with OPA /mercaptopropionic acidand detected by fluorescence spectroscopy at 330 and 450nm.Nineteen amino acids in a standard mixture were separated using a GROM-SIL OPA-1column (150 x 4mm, Part No. GSOP10308S1504) after precolumn derivatization withOPA/mercaptopropionic acid by gradient elution with25mM sodium phosphate, pH 7.2 and THF 995/525mM sodium phosphate, pH 7.2, methanol and acetonitrile 50/35/15Time - 41 minutesDetection - Fluorescence spectroscopy at 330 and 450nm.The amino acids separated were aspartic acid, glutamic acid, asparagine, serine,glutamine, glycine, threonine, histidine, citrulline, 3-methylhistidine, alanine,taurine, arginine, alpha-aminobutyric acid, tyrosine, valine, methionine, nor-valineand tryptophan.
POST-COLUMN ON-LINE DERIVATIZATIONThe following experimental parameters should be maintained(a) Derivatization performed in a special-reactor strategically positioned between the columnand the detector.(b) Reaction must be completed rapidly at moderate temperatures.(c) Derivatization reaction need not even go to completion provided it can be madereproducible.(d) No detector-response should exist due to any excess reagent present.(e) Reaction must be carried out in a medium other than the mobile-phase.Merit : The main merit of post-column-on-line derivatization is that ideally the separation anddetection processes can be optimized individually
IMPORTANT CONSIDERATIONS IN POST COLUMN DERIVATIZATIONReactants and conditions must be chosen so that conversion to the desired product(s)takes place rapidly (usually < 1 minute) and reproducibly.PCD system must ensure good mixingIf the conversion is not sufficiently rapid, it may be necessary raise the temperature,incorporate a catalyst or in some other way accelerate the rate of derivatization.completeness of reaction is not always a required result.In many qualitative applications it may be sufficient that each analyte derivative beformed in abundance great enough to generate a detectable signal.
REAGENTS FOR DERIVATIZATION Derivatization for UV-Detectors : Ninhydrin a chromatag is commonly employed to yield corresponding derivatives of amino acids that show absorption specifically at about 570 nm as shown in the following reaction :
Derivatization for Fluorescence DetectorsDansyl Chloride (a fluorotag) is invariably used to obtain fluorescent derivatives ofproteins, amines and phenolic compounds, the excitation and emission wavelengths being335 to 365 nm and 520 nm respectively.
Reagents for flouroscent derivatization
APPLICATIONDrug manufacturing control requires high level and intensive analytical and chemicalsupport of all stages to ensure the drugs quality and safetyThe pharmacopeia constitutes a collection of recommended procedures foranalysis and specifications for the determination of pharmaceutical substances,excipients, and dosage forms.
Used in Qualitative and Quantitative analysis.Used in the routine assay of new drugs as well as substitutes for the oldermoretroublesome assays for marketed drugs.Used in the studies of drug samples in biological fluids.Isolation of natural pharmaceutical active compounds.Control of microbiological process.Used to study the impurity profile of the new drugs.