Derivatization in GC andHPLCProf. RavisankarVignan Pharmacy CollegeVadlamudiGunturAndhra PradeshINDIAbanuman35@gmail.com0091 9059994000
DerivatizationDerivatization is the process of chemically modifying acompound to produce a new compound which has properties that aresuitable for analysis using a GC or HPLC.The chemical structure of the compound remains the same andjust modifies the specific functional groups of reacting compound toderivative of deviating chemical and physical properties in order to makethem detectable and analysable.Derivatization is needed in GC, HPLC, UV-Vis spectroscopy etc.,
Why derivatize in GC?• To permit analysis of compounds which are not directly amenable toanalysis due to for example, inadequate stability and volatility.• To improve chromatographic behaviour or detectability.Many compounds do not produce a useable chromatography orthe sample of interest goes undetected. As a result it may be necessary toderivatize the compound before GC analysis is done.The main reason for derivatizing is to impart volatility tootherwise non-volatile compounds.Derivatization is a useful to allowing the use of GC & GC/MS tobe done on sample that would other wise be not possible in various areasof chemistry such as medical, forensic & environmental.
Ideal characters and disadvantages ofderivatization• A derivatization reaction should be rapid,quantitative, and produceminimal by product. Excess reagent should not interfere with theanalysis and should be easily removed.• Derivatization often is a last resort when developing a method.Introduction of a reaction pre or post column increasescomplexity, chances of error, and total analysis time.• Care should be taken that the reaction is quantitative and noadditional impurities are introduced into analysis.
What Derivatization accomplish?• Increases volatility(i.e. sugars):- Eliminates the presence of polar OH, NH & SH groups- Derivatization targets O, S, N and P function groups (withhydrogens available)• Enhances sensitivity for ECD. The introduction of ECD detectablegroups, such as halogenated acyl groups, allows detection ofpreviously undetectable compounds.• Increases detectability, i.e. steriods• Increases stability(thermostability)• To reduce adsorption of polar samples on active surfaces of columnwalls and solid support.
Conditions for choosing a derivatizing agent• The derivatizing agent must be stable.• The derivatizing agent and its products formed during derivatizationshould not be detectable or must be seperable from analyte.• The analyte should be reactive with derivatizing agent underconvenient conditions.• If possible, it should be non-toxic.• The rocedure should be adaptable to automation.
Types of Derivatization• Silylation• Alkylation• Acylation• Chiral derivatization
Silylation Most prevalent method, readily volatizes the sample.Mechanism-• This process produces silyl derivatives which are more volatile, morethermally stable.• Replaces active hydrogens with TMS (trimethyl silyl groups)• Silylation occurs the nucelophilic attack (SN2). The better theleaving group, the better the silylation.
Solvents and precautions-• Silylation reagents will react with H2O & alcohols first care must betaken to ensurs that both sample & solvent are dry.• Solvent should be as pure as possible. This will eliminate excessivepeaks. Try using as little solvent as possible as this will prevent a largesolvent peak.• Pyridine is the most commonly used solvent. Atthough pyridine mayproduce peak tailing it is an acid scavenger & will drive the reactionforward.• In many cases, the need for a solvent is eliminated with silylatingreagents (if a sample readily dissolves in the reagent, it usually is asign that the derivatization is complete.
Ease of reactivity of functional group toward silylation follows the order-Alcohol > Phenol > Carboxyl > Amine > Amide > HydroxylThe order of alcohols is 1 > 2 > 3• Care needs to be taken not to inject silylating reagent onto columnwhich have active hydrogen’s in the st.phase, because they will bederivatized. Example of column not compatible with silylatingreagents are carbowax & free Fatty Acid phase.
Silylation - advantages anddisadvantagesAdvantages:• Ability to silylate a wide variety of compounds.• Large number of silylating reagents available.• Easily prepared.Disadvantages:• Silylation reagents are moisture sensitive.• Must use aprotic (no proton available) organic solvents.
Silylating agents and their mechanisms1. N,O-bis(trimethylsilyl)acetamide (BSA)
7. Dimethyldichlorosilane (DMDCS) the order of reactivities of the silylation reagents are-TSIM>BSTFA>BSA>MSTFA>TMSDMA>TMSDEA>TMCS>HMDS• MSTFA- N-methylsilyltriflouroacetamide;• TMSDMA- trimethylsilyldimethylamine;• TMSDEA- trimethylsilyldiethylamine.
AlkylationAlkylation reduces molecular polarity by replacing activehydrogens with an alkyl group. These reagents are used to modifycompounds with acidic hydrogens, such as carboxylic acids andphenols. These reagents make esters, ethers, alkyl amines and alkylamides.The principal reaction employed for preparation of thesederivatives is nucleophilic displacement.
AlkylationAdvantages• Wide range of alkylation reagents available• Reaction conditions can vary from strongly acidic to• strongly basic• Some reactions can be done in aqueous solutions• Alkylation derivatives are generally stableDisadvantages• Limited to amines and acidic hydroxyls• Reaction conditions are frequently severe• Reagents are often toxic
Alkylating agents and their mechanisms1. trimethylanilinium hydroxide (TMAH)
2. Boron trichloride in chloroethanol or methanol
AcylationAcylation reduces the polarity of amino, hydroxyl, and thiolgroups and adds halogenated functionalities for ECD. In comparisonto silylating reagents, the acylating reagents target highlypolar, multifunctional compounds, such as carbohydrates and aminoacids.• Acylation converts these compounds with active hydrogens intoesters, thioesters, and amides. They are formed with acylanhydride, acyl halide, and activated acyl amide reagents.• The anhydrides and acyl halide reagents form acid by-products, which must be removed before GC analysis.• Acylations are normally carried out in pyridine, tetrahydrofuran or
Acylation• Fluorinated acyl groups, going from trifluoracetyl toheptafluorobutyryl , can be used to increase retention times.• Acyl derivatives tend to direct the fragmentation patterns ofcompounds in MS applications, and so provide helpful informationon the structure of these materials.
Acylation – advantages and disadvantagesAdvantages-• Addition of halogenated carbons increased detectability by ECD• Derivatives are hydrolytically stable• Increased sensitivity by adding molecular weight• Acylation can be used as a first step to activate carboxylic acidsprior to esterfication (alkylation)Disadvantages-• Acylation derivatives can be difficult to prepare• Reaction products (acid by-products) often need to be removedbefore analysis• Acylation reagents are moisture sensitive• Reagents are hazardous and odorous
Acylating reagents and their mechanisms1. Acetic anhydride
Chiral derivatizationThese reagents target one specific functional group and produceindividual diastereomers of each of the enantiomers.There are two ways of separating enantiomers by chromatography:1. separation on an optically active stationary phase.2. preparation of diastereomeric derivatives that can be separatedon a non chiral stationary phase.Reagents1. TPC (N-trifluoroacetyl-L-prolyl chloride)Used for optically active amines, most notably amphetamines2. MCF ((-) menthylchloroformate)Used for optically active alcoholsIf an optically pure reagent is used to prepare diastereomericderivatives, then only two derivatives are formed. The enantiomeric ratiois reflected in the relative peak sizes.
Functional groups and their derivatization methods
Why derivatize in HPLC?• To improve detectability.• To prepare soluble derivatives of insoluble compounds for HPLCanalysis.• To change the molecular structure or polarity of the analyte forbetter chromatography.• To change the matrix for better seperation.• To stabilise a sensitive analyte.• To enhance separation.• To reduce tailing, poor peak resolution and/or asymmetrical peaks.
Types of HPLC derivatization• for UV-Vis spectrophotometric detection.• For flourimetric detection.• For chiral analysis.According to when and where the derivatizationis done• Pre-column derivatization• Post-column derivatization
For Chiral Separation• 1-fluoro-2,4-dinitrophenyl-5-l-alanine amide(FDAA or Marfey’sreagent)• 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl Isothiocyanate (TAGIT)• O,O - dibenzoyl tartaric acid anhydride• For carbohydrates,2-aminopropionitrile-fumarate-borate is used for flourscentderivatization• For guanidine,Benzoin is used for flourscent derivatization.• For α-ketocarboxyl groups,O-phenylenediamine is used for flourscent derivatization.
Chiral derivativesFunctional groups derivativeAmino groups Amides,carbamates,ureas,thioureas,sulfamidesHydroxyl groups Esters,carbonates,carbamatesCarboxy groups Esters,amidesEpoxides Isothiocyanates,olefinsthiols Thioesters
Pre- and Post-column derivatizationPre-column derivatization-• Performed before the analytical separation is attained.• Sample is derivatiszed manually or automatically and injected into the hplccolumn.• Separation of components occurs after derivatization.Advantages-• Fewer equipment and reaction chemical restrictions.• Can be performed manually or automatically.• No time constraints on the kinetics of the derivatization of derivatizationreaction.Disadvantages-• Introduction of contaminants.• Loss of analyte through adsorption.• Sample degradation and incomplete reaction.• Poorer precision due to increased complexity.
Post-column derivatization-• Performed after analytical separation of compounds but prior todetection.• Addition pump is used for addition of derivatizing agent to the elutedsample from column.Advantages-• Minimal artifact formation.• Complete reaction is not essential as long as it is reproducible and thechromatography of analyte remains unaffected.Disadvantages-• Band brodening• Added complexity for method development and routine use.
ConclusionChemical derivatization of drugs is critical for GCbecause these samples, which often contain multiple polarsubstituents, are simply not volatile or thermally stable.Chemical derivatization with HPLC to permitfluorescence or UV-Visible detection is certainly one of themore desirable methods for routine use to solve selectivity anddetectability problems for drug samples during analysis.
references1. GC Derivatization – from Regis 1998-99 Chromatography Catalogand Knapp.D.R.-HandBook of Analytical Derivatization Reactions.2. HPLC Method Development by Snyder et al.3. Basic Gas Chromatography- Harold McNair et al.4. Modern Methods of Pharmacetical Analysis- Roger.E.Schirmer(vol-2,2nd edition)5. Chemical Reagents & Derivatization Procedures in Drug Analysis-Neil.D.Danielson et al.6. GC Derivatization Reagents.pdf @ www.registech.com/gc7. Derivatives for HPLC Analysis – Mrs.laurence Coppex.