Derivatizations In Separations

1. Derivatizations In Separations

2. What is Derivatization? • Reaction • Reagents • Change chemical nature analyte • Improve analysis

3. What is Derivatization? • Typically focuses on analyte • Matrix ideally should remain unaffected • Occasionally get some components in matrix reacting – not good • Why? • Not usually first step in sample preparation • Clean-up • Concentration • Change property of analyte for separation • Change property of analyte for better detection • Can be pre- or post-

4. Desired property Reagent X is a replacing group R carries the property Analyte H-donor Derivatization • Chemical reactions • Typically replace active hydrogens (OH, COOH, NH, CONH) Y – H + R – X Y – R + HX

5. Derivatization • Chemical reactions Increased nucleophile properties

6. Derivatization • Chemical reactions • Efficiency probably most important criteria • Must be complete • Time must be reasonable • Little to no loss of analyte • Stable

7. Derivatization • Chemical reactions • Forming alkyl or aryl derivatives • Silylation • Acyl derivatives • Carbon-hetero multiple bonds • Cyclic formation

8. Derivatization • Chemical reactions • Alkylation and Arylation • Analyte acts as a nucleophile (Y:, Y:H, Y:-) • SN substitution with alkylating reagent (R – X); X is usually a leaving group and R alkyl group • Alkyl iodides and bromides very common • Catalysts may be required Y:H + R – X Y – R + X:H

9. slow R – X R+ + X- Y: - H + R+ Y – R + H+ fast Y:H + R – X Y – R + X:H Y: - H + C X Y: C X Y C + HX Derivatization • Chemical reactions • Alkylation and Arylation • 2 mechanisms (SN1) (SN2)

10. Derivatization Chemical reactions Alkylation and Arylation Strong acid present then OH group is very reactive because

11. Derivatization Chemical reactions Alkylation and Arylation Acid may also act as a catalyst

12. Derivatization Chemical reactions Alkylation and Arylation Salts of heavy metals act as catalysts

13. Derivatization • Chemical reactions

14. Derivatization Chemical reactions Alkylation and Arylation For SN2 reactions, rate depends on nature of nucleophile Nucleophile negative charge better than its conjugated acid Nucleophiles whose attacking atom in same row of periodic table – nucleophilicity parallels bascity Nucleophiles whose attacking atom is in a higher period – nucleophilicity increases Freer the nucleophile, the higher rate – both atoms have unshared pair of electrons

15. Derivatization Chemical reactions Alkylation and Arylation Order of nucleophilicity – General order NH2- > RO- >OH- > R2NH > ArO- > NH3 > Pyridine > F- > H2O Arylation Similar to SN2

16. Derivatization • Chemical reactions • Silylation • Replaces active H (OH, COOH, SH, NH, CONH, POH, SOH) with a silyl group (trimethylsilyl) • Purpose • Reduce polarity of analyte • Increase analyte stability • Improve analyte behavior for GC • Can be used with solvent to aid in extraction and detection, especially useful in analyzing crude matrix

17. Derivatization • Chemical reactions • Silylation

18. Derivatization • Chemical reactions • Silylation • Similar to SN2 • Efficiency • Nature of X (leaving group); more stable as free entity better leaving property; • Higher acidy better silyl donor ability

19. Derivatization • Chemical reactions • Silylation • Similar to SN2 OCOR leaving group better donor than OR leaving group – more stable b/c two resonance structures formed

20. Derivatization Chemical reactions Silylation

21. Derivatization Chemical reactions Silylation Nature of Y:H determines silylation efficiency

22. Derivatization Chemical reactions Silylation Solvent affect silylation - bad H2O, H2O2, HCl, HNO3, H2SO4, H2SO3, H3BO4, H3PO4, H4SiO4 H2O very important, try to eliminate or minimize to as low as possible Ammonium salts Solvent affect silylation – good Dimethylformamide, pyridine, acetonitrile

23. Derivatization Chemical reactions Acylation Replace active hydrogens Reduces polarity Improves behavior of analyte in chromatographic column Detectability – very useful here

24. Derivatization Chemical reactions Acylation Most are nucleophilic substitutions, analyte the nucleophile (Y:, Y:H, Y:-) React with acylating group that contains a leaving group X Acid generation from the reaction is a hindrance and should be removed Halogen most commonly used are fluorinated acyl groups Good reaction for analytes with weakly reactive H

25. Derivatization Chemical reactions Acylation SN2 substitution

26. Derivatization Chemical reactions Acylation Carbonyl cyanides – similar to acyl chlorides HCN weaker acid compared to HX

27. Derivatization Chemical reactions Acylation Anhydrides can be used as an alternative b/c it produces a weak organic acid – reduces undesired modifications that can result with strong acids Volatility may be lower

28. Derivatization Chemical reactions Carbon-hetero multiple bonds Representative groups – C=O, C=S, C=N, CN Two modes for derivatization Derivatize active H with a hetero-multiple bond derivatizing agent Derivatize analytes with hetero-multiple bond Catalyzed by both acid and base conditions

29. Derivatization Chemical reactions Carbon-hetero multiple bonds Elimination rxn Nucleophilic or Electrophilic - proton R’s – H, R, Ar in Aldehydes and Ketones; OH in acids, OR in esters, NH or NHR in amides

30. Derivatization Chemical reactions Carbon-hetero multiple bonds Very polar (C=O), b/c displacement of  electron toward oxygen Thus, it is about 20% ionic Nucleophilic attack Elimination rxn not likely to occur when substituents are H, alkyl, aryl Electrophilic attack usually predominates under acid In both attacks – nucleophilic attack is rate limiting

31. Derivatization Chemical reactions Carbon-hetero multiple bonds Aldehydes and ketones b/c of active methylene characteristic they can undergo condensation reactions – presence of an OH group in the condensation product is not desired in derivatization Formation of hemiacetals and acetals – unstable However, if forms cyclic acetals or ketals very stable for derivatization

32. Derivatization Chemical reactions Carbon-hetero multiple bonds N=C in isocyanates (N=C=O) and isothiocyanates Very good dervatization for attaching chromophores or fluorescent groups Unstable thermal properties make it uncommon for GC applications

33. Derivatization Chemical reactions Cyclic formation Formation of new cyclics or replacement of old cyclics Nonaromatic cyclics containing O Aromatic cyclics containing one N Azoles or related compounds Azines or related compounds Cyclic siliconides Cyclic phosphothioates Cyclic boronates

34. Derivatization Chemical reactions Cyclic formation Formation of new cyclics or replacement of old cyclics Nonaromatic cyclics containing O Peroxyacid – peracetic, perfomic, perbenzoic Epoxides

35. Derivatization Chemical reactions Cyclic formation Formation of new cyclics or replacement of old cyclics Aromatic cyclics containing one N Involve bifunctional molecules

36. Derivatization Chemical reactions Cyclic formation Formation of new cyclics or replacement of old cyclics Azoles or related compounds Aromatic five membered heterocycles with a nitrogen and some other hetero atom (N, O, S)

37. Derivatization Chemical reactions Cyclic formation Formation of new cyclics or replacement of old cyclics Azines or related compounds Six membered heterocyclics with more than 1 N or 1 N and some other heteroatom

38. Derivatization Chemical reactions Cyclic formation Formation of new cyclics or replacement of old cyclics Cyclic siliconides

39. Derivatization Chemical reactions Cyclic formation Formation of new cyclics or replacement of old cyclics Cyclic phosphothioates

40. Derivatization Chemical reactions Cyclic formation Formation of new cyclics or replacement of old cyclics Cyclic boronates

41. Derivatization Chemical reactions Other derivatizations Addition across = bond Oxidation/Reduction Ninhydrin Hydrolysis

42. Derivatization Chemical reactions Other derivatizations Aromatic substitutions Electrophilic substitution – useful for aromatic cmpds with activating groups O-, OH, OR, OCOR, NH2, NHR, NR2 Complexation