Derivatization in GC and HPLC Prof. RavisankarDerivatization is the process of chemically modifying a compound to produce a new compound which has properties that are suitable for analysis using a GC or HPLC. The chemical structure of the compound remains the same and just modifies the specific functional groups of reacting compound to derivative of deviating chemical and physical properties in order to make Derivatization is needed in GC, HPLC, UV-Vis spectroscopy etc.,+ To permit analysis of compounds which are not directly amenable to analysis due to for example, inadequate stability and volatility. + To improve chromatographic behaviour or detectability. “Many compounds do not produce a useable chromatography or ee eaten a einer "Tis ai “reason for seating is to ieietie Wenner lao lec ice ain eedece Ne CaNET be done on sample that would other wise be not possible in various areasenna + A derivatization reaction should be rapid,quantitative, and produce minimal by product. Excess reagent should not interfere with the analysis and should be easily removed. PG aes ke eee + Care should be taken that the reaction is quantitative and no additional impurities are introduced into analysis.+ Increases volatility(i.e. sugars): - Eliminates the presence of polar OH, NH & SH groups -- Derivatization targets O, S, N and P function groups (with hydrogens available) + Enhances sensitivity for ECD. The introduction of ECD detectable groups, such as halogenated acyl groups, allows detection of previously undetectable compounds. + Increases detectability, ie. steriods + Increases stability(thermostability) * To reduce adsorption of polar samples on active surfaces of column walls and solid support.+ The derivatizing agent and its products formed during derivatization should not be detectable or must be seperable from analyte. + The analyte should be reactive with derivatizing agent under convenient conditions. * If possible, it should be non-toxic. * The rocedure should be adaptable to automation.> Most prevalent metho, readily vlatizes the sample Mechanism- © This a . + pain i nc anitaken to ensurs that both sample & solvent are dry. * Solvent should be as pure as possible. This will eliminate excessive peaks. Try using as little solvent as possible as this will prevent a large * Pyridine is the most commonly used solvent. Atthough pytidine may produce peak tailing it is an acid scavenger & will drive the reaction forward. * In many cases, the need for a solvent is eliminated with silylating reagents (if a sample readily dissolves in the reagent, it usually is aThe order of aleohols is 1_>2 >3_ . Care needs to be taken not to inject silylating reagent onto column which have active hydrogen’s in the [Link], because they will be derivatized. Example of column not compatible with silylating+ Ability to silylate a wide variety of compounds. + Easily prepared. sihhateaothcesSilylating agents and their mechanisms 1. N,O-bis(trimethylsilyl)acetamide (BSA) CH, CH3— as CHa obs —0r Che 0 O-TMS | + H-Y-R ——* TMS-Y-R + HiC—C—N—TMS HsC—C=N—TMS | H TMS = Si(CHe)s Y=0, 8, NH, NRF, COO R,R'=Alk, Ar2. Trimethylchlorosilane (TMCS) CH 4 di CH, CHa H,C—-Si—C| + H-Y-R ——» H,C—Si-y-R CH, Y=0, 8, NH, NR! CHa R, R'=Alk, ArCH, 3, N-trimethylsilylimidazole (TMSI) | i‘ vai ig SI Gig, N O N— 0H CH, | /S | S HOS | + HOR —» Hy-SHO-R + HW | \eN \zN Chy RRI=Alk, Ar CHy4. N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) 8 CHs— Si—CH, CH3 CFa— C=N—Si—CH CH O—TMS + H-Y—-R ——> TMS-Y-R + FxC—C—N—TMS F¢—C=N—IMs H TMS = Si(CH,)3 Y=0,8,NH,NR', COO R,R'=Alk, Ar5. Hexamethyldisilazane (HMDS) fe CHs— Si—NH—Si—CH, CH3 CHg hf — Pe + H-Y-R — het + a HyC HCH Y=0,S,NH,NRICOO CHa CH R,RI=Alk, Ar6. N-t-Butyldimethylsilylimidazole(TBDMSIM) os CH3—C —CH, | y—N CHs — Si—N | \ CHy OH, Oy P ' ; Fo TOSI sage -b: + 0-Sx > [se 0-Si- > Sae-0-3-C + Xe NOHO | | | | | 4 Hy HGH thy7. Dimethyldichlorosilane (DMDCS) v the order of reactivities of the silylation reagents are- TSIM>BSTFA>BSA>MSTFA>TMSDMA>TMSDEA>TMCS>HMDS + MSTFA- N-methylsilyltriflouroacetamide; * TMSDMA- trimethylsilyldimethylamine; * TMSDEA- trimethylsilyldiethylamine.Alkylation reduces_molecular polarity by replacing active hydrogens with an alkyl group. These reagents are used to modify compounds with acidic hydrogens, such as carboxylic acids and phenols. These reagents make esters, ethers, alkyl amines and alkyl amides. The principal reaction employed for preparation of these derivatives is nucleophilic displacement.Some reactions can be done in aqueous solutions Disadvantages Limited to amines and acidic hydroxyls Reaction conditions are frequently severe Reagents are often toxicAlkylating agents and their mechanisms 1. trimethylanilinium hydroxide (TMAH) CHs | CHg N— oh [OH] O SS Esterification = GeneralMechanism a4 = aki f\ _f RCOOH + CHOH + R:COO-CH, + H,0 XS ree Adapted rom) vy ov KA MOA Transesterification HOH Ch fC acid we ReCOOR’ + CH,OH > R-COO-CH, + ROH phenobarbital + — methyl derivative2. Boron trichloride in chloroethanol or methanol cI I e —c! cl Esterification acid (BCI) R-COOH + CICH,CH,OH — R-COO-CH,CH,CI + H,O Transesterification acid (BCI,) R-COOR' + CICH,CH,OH — R-COO-CH,CH,CI + R'-QH3. Boron triflouride in butanol or methanol ao 4 Esterification acid (BF) R-COOH + CH,(CH,),OH —» R-COO-~(CH,),CH, + H,O Transesterification acid (BF) R-COOH + CH (CH,),OH — R-COO-~(CH,),CH,+ R'-OH4. Methanol in acid (HCI or H,SO,) H | HeeeH H Esterification acid (H,SO,) R-COOH + CH,OH —+° R-COO-CH, + HO Transesterification acid (H,SO,) R-COOR' + CH,OH —» R=COO-CH, + R'-OH5. Pentafluorobenzyl Bromide and . ¢ \ — Hexaoxacyclooctadecane \—=/ 6 > ¢ y Pentafluorobenzyl Bromide of I R—C—OH + PFRBr > R—C—O—CH2 — CoP, + HBr Extractive Alkylation (Phenols) 1,4,7,10,1,16-Hexaoxacyclooctadecane Phen -OR" + RUN? XS Phen AY =X Organic | = wooo’ © } —> AcOo Ce) potassium” (, . Phan =OH + AM* Xm Phen "Ry HX gresey i = Rcoo” (2) + A'-x—+ nooo-A’ + (2) reo Qx CHigOly + PFE + B+ PFB-OPh=n Reao £3} + KK [Prev IMC HSI” ou 8.10)Acylation reduces the polarity of amino, hydroxyl, and thiol polar, multifunctional compounds, such as carbohydrates and amino acids. " prsbetinr iat ara besamoreletns Celanslve + Acylations are normally carried out in pyridine, tetrahydrofuran or eaendtlee enfant! raw alia able apesd oe ectbinie aad dsweaidee teAcylati + Fluorinated acyl groups, going from —trifluoracetyl_ to heptafluorobutyryl , can be used to increase retention times. + Acyl derivatives tend to direct the fragmentation patterns of compounds in MS applications, and so provide helpful information on the structure of these materials.eased panes a ey rau oA ce) = Aon haloes sts need say y ECD hydrolytically stable | apes nino : s (acid by-products) often need to be removed rt ey secrieea ce are ion rane te + Reagents are hazardous and odorousAcylating reagents and their mechanisms 1. Acetic anhydride A sane anhydride | pyridine R-O > R-O-COCH,2. Trifluoroacetic Acid Anhydride : Oo I I CF3—C—c —CF3 Pentafluoropropionic Acid Anhydride F Oo Oo F ae I |0 0 he + H-Y-R ——> oor + CsF-C—0H 0 0 0 | I Ofs-C-O-C—CF, + H-Y-R —> OF-C-Y-R + C:F-C—0H i i i | F-C-O-C—CF; + HHY-R—> FC-C-Y-R + FC-C-OH Y=0, NH, NR! RR! = Alk, Ar4. N-Methyl Bis (Trifluoro Acetamide) - MBTFABS These reagents target specific and produce Poe as ete le fon There are two ways of separating enantiomers by chromatography: 2. preparation of diastereomeric derivatives that can be separated on anon chiral stationary phase. Reagents ee ee 2: MER {() mmeathylehloroformare) Used for optically active alcohols ne ees used to prepare digFunctional Group Acids: Alcohols and phenols— unhindered and moderately hindered Alcohols and phenols— highly hindered Amines (1* & 2°) Amines (3°) Amides Method Silylation Alkylation Silylation Acylation Alkylation Silylation Acylation Alkylation Silylation Acylation Alkylation Alkylation Silylation (a) Acylation (by Alkylation (ce) Derivatives RCOOSKCH,), RCOOR* R—O—Si(CH,), R—O—C—PFA R—O—R’ R—O—Si(CH,), R PFA, R—O—R* R—N—Si(CHy)3 ° RC—NHSi(CH,); (unstable) "wl RC—NH- PEA ©) 9 all wnt,Amino Acids Catecholamines Esterification/Acylation Silylation (a) Acylation + Silylation (b) Alkylation (c) Acylation + Silylation (a) Alcylation (b) (a) RCHCOOSi(CH,), 1 N-Si (CH) (6) RCHOOSI(CH,), | N—TFA (c) RCHCOOR* NHR’ H R—N—HFB (a) OSi(CH)), OSi(CH)), H R—N—HEB (b) OHFB OHFBCarbohydrates Silylation (a) (a) OSi(CH,), and sugars Acylation (b) | Alkylation (c) —CH,)y— (bt) OTFA | CH.) y— (c) OR | —{CHy)y— ac Sal a“ Carbonyls Silylation TMS—O—N=C N 5 a Alkylation CH,—O—-N=C2 To change the molecular strstr or poly of the analyte for better 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.= of (HPLC eaNo, Bs . . me + aN oe Jt ie NOY = NO, R / Reaction of FONB with primary and secondary amines cr eae OPA Reaction of OPA with — @ primary amine. fluorescent derivative (Ex. 340 nm. Em. ass nm) non-fluorescent degradation productNo. ‘Compounds Reagent(s) Derivative CHO sR" CHO A 1. R’NH) (F) RSH NR’ CN CHO 2 2: RNHp (F) +CN" CLE N-R ‘CHO OCH; OCH; 3. R3NH* (F) coo” coo OCH; OCH;HPLC flourscent derivatization-table(1) cont. RSH (F) RCOOH (E) Steroid (E) a 0 o oO nyt pon meee BrHsC CHy RSHSC 9 ns{)-oH ren-{" oH HuNHn-{")-No: win-{)-No: ‘OHHPLC UV-Vis derivatization-table(2) No. Compounds Reagent(s) Derivative 1. RNH} (A) 0 (A) Oo AQ ra. rh ‘OCH, ‘OCH ©) NCS (8) CO _snicsnur \ 2. R:NH co con" 3. RN WoO xaa a, a HyC=C, CoHs a Q 4 ocnico0n RS +0) -ocricoon 5 we Penicillin NaOH, HeCh, EDTAPre-clumn desivaization- + Performed before the analytical separation is attained. . Samp eaised my orotate in hple 6 sepa of components occurs after derivatization. . neve oi in NE KTS eon OAR Disadvantages- + Introduction of contaminants. * Loss of analyte through adsorption. + Sample degradation and incomplete reaction. + Poorer precision due to increased complexity.Bos oleh et enlists * Bape a nell ecaraion skort arora cae is used for addition of derivatizing agent to the eluted sample from column, Stree + Minimal artifact formation. + Complete reaction is not essential as es is reproducible and the chromatography of analyte remains unaffected. Digaaveniaces * Band brodening + Added complexity for method development and routine use.Conclusic ‘ on ‘Chemical derivatization of drugs is critical for GC because these samples, which often contain multiple polar ‘substituents, are simply not volatile or thermally stable. Chemical derivatization with HPLC to permit ee ee eeeuae a references . GC Derivatization ~ from Regis EE ala uciale and Knapp.D.R.-HandBook of Analytical HPLC Method Development by Snyder et ee ‘Basic Gas Chromatography- Harold MeNair et al. 2,2" edition) . Chemical Reagents & Derivatization Procedures in Drug Analysis- [Link] et al. _ GC Derivatization [Link] @ [Link]/ge