2. Mobile Phase pH Analyte pK a Shift Lecture 4 Yuri Kazakevich Seton Hall University

3. 2 Measuring pH of Hydro-organic Mixtures There are three different pH scales that are employed in pH measurement of reversed phase HPLC mobile phases. w w pH scale - the electrode system is calibrated with aqueous buffers. The pH of the aqueous portion of the mobile phase is measured prior to the addition of the organic modifier. s w pH scale - the electrode system is calibrated with aqueous buffers. The pH of the hydro-organics mobile phase is measured after the addition of the organic modifier. s s pH scale - the electrode system is calibrated with buffer-organic mixtures of the same composition as the mobile phase. The pH of the hydro-organic mobile phase is measured after the addition of the organic modifier.

4. 3 pH Shift of Hydro-Organic Eluents

5. 4 Determination of the Chromatographic pK a HPLC can be used as a powerful technique for the determination of dissociation constants. Only requires small quantity of compounds Compounds do not need to be pure Solubility is not a serious concern To properly describe the effect of pH on the retention of ionizable analytes the actual pH of the hydro-organic mobile phase must be known.

6. 5 Determination of the Chromatographic pK a pK a shift Theoretical koko k1k1 -0.2 0 0.2 0.4 0.6 0.8 1 1.2 1.4 012345678 pH k' 50%- w w pH w w pK a = 2.9 Potentiometric pK a = 4.6 50%- s s pH pH shift s s pK a = 3.9 Eluent 50% Buffer 50% MeCN k o = Retention factor of analyte in its neutral form k 1 = Retention factor of analyte in its ionized form

7. 6 Chromatographic Conditions Column: 15 cm x 0.46 cm Luna C18(2) Eluent: Aqueous/ 10, 20, 30, 40, 50% MeCN Aqueous: 15 mM K 2 HPO 4 7H 2 O adj. to pH 1 - 9 with H 3 PO 4 Flow rate: 1 ml/min Temp: 25 o C Wavelength: 220 nm Effect of pH on Aniline Retention

8. 7 Effect of Organic Content on Analyte pK a Shift A decrease of ~0.2 pK a units per 10% v/v MeCN for aniline was determined. The slope could be used to estimate sspKa of pharmaceutical compounds containing aromatic amine functionalities at a certain organic composition after adjusting for the mobile phase pH shift. y = -0.0349x + 4.6 R 2 = 0.9983 2 2.5 3 3.5 4 4.5 5 0102030405060 v/v% MeCN pK a s s pK a Aniline, pK a (titration) = 4.6 pH shift pK a shift y = -0.0146x + 4.6 R 2 = 0.9939 s w pK a w w pK a

9. 8 Example: 2-4 dimethylpyridine (base) has a pK a of 6.7 and initial eluent conditions are: 50% MeCN and 50% Buffer. What should the pH of the buffer be in order to obtain the basic analyte in its fully ionized form? pH Shift and pK a Shift The downward pK a shift for basic analytes must be accounted for. The working pH should be at least 2 pH units below the basic analyte pK a to be fully ionized. The upward pH shift of the aqueous acidic buffer upon addition of the organic must be accounted for. Downward analyte pK a shift. pH at which basic analyte would be protonated Upward pH shift of aqueous acidic buffer upon addition of organic Max pH of buffer in order to have analyte in fully ionized form. 0.201.00 1.0 2.7

10. 9 2,4 Dihydroxybenzoic acid pK a (tit.) 3.29 Chromatographic Conditions Column: 15 cm x 0.46 cm Luna C18(2) Eluent: Aqueous/ 10, 20, 30, 40, 50% MeCN Aqueous: 15 mM K 2 HPO 4 7H 2 O adj. to pH 1 - 7 with H 3 PO 4 Flow rate: 1 ml/min Temp: 25 o C Wavelength: 220 nm Effect of pH on 2,4-Dihydroxybenzoic Acid Retention

11. 10 Effect of Organic Content on Analyte Ionization: Acids 2,4 Dihydroxybenzoic acid, pK a (titration) = 3.29 An increase of 0.2 pK a units per 10% v/v MeCN for acidic compounds. Similar trend for other mono and disubstituted benzoic acids. The slope could be used to estimate pK a of pharmaceutical compounds in certain organic composition.

12. 11 Conclusion Accounting for the pH shift of the mobile phase and the analyte pK a shift upon the addition of organic modifier will allow the chemist to analyze the ionogenic samples at their desired pH values. This will lead to development of rugged methods and an accurate description of the analyte retention as a function of pH at varying organic compositions.

13. 12 pH effect on analyte UV absorption Part 2

14. 13 Effect of Conjugation of Chromophores  electrons are further delocalized by conjugation The effect of this delocalization is to lower the energy level of the  * orbital and give it less antibonding character Absorption maxima are shifted to longer wavelengths

15. 14 UV Absorption by Aromatic Systems UV spectra of aromatic hydrocarbons are characterized by 3 sets of bands that originate from  -->  * transitions. Benzene has strong absorption peaks at: E1184 nm  max ~ 60,000 E2204 nm  max = 7,900 B256 nm  max = 200 B band contains a series of sharp peaks due to the superposition of vibrational transitions upon the basic electronic transitions Polar solvents tend to reduce or eliminate this fine structure as do certain types of substitution.

16. 15 UV Absorption by Aromatic Systems

17. 16 Auxochromes Functional group that does not itself absorb in the UV region but has the effect of shifting chromophore peaks to longer wavelengths and increasing their intensity. -OH and -NH 2 have an auxochromic effect on benzene chromophore. Have at least one pair of n electrons capable of interacting with  electrons of the the ring. This stabilizes the  * state and lowers its energy Phenolate anion auxochromic effect more pronounced than for phenol since anion has extra pair of unshared electrons.

18. 17 AnilineAnilinium ion  niline has a pair of n electrons capable of interacting with the  electrons of ring. This stabilizes the  state thereby lowering its energy. With a decrease in protonation the absorption maxima are shifted to longer wavelengths and increasing intensities. A red shift occurs. Effect of Protonation on Aniline UV Response

19. 18 UV Absorbance as a Function of pH At 232 nm there is an decrease in absorbance as aniline becomes protonated. 0 5 10 15 20 25 01234567 pH of aqueous phase Absorbance, 232 nm 10% AcN pK a = 4.33 (corr pHshift)

20. 19 pH 1.5 pH 2.5 pH 4 pH 5 pH 6 pH 9 Chromatographic Conditions Column: 15 cm x 0.46 cm Luna C18(2) Eluent: 90% Aqueous:10% MeCN Aqueous: 15 mM K 2 HPO 4 7H 2 O adj. to pH 1 - 9 with H 3 PO 4 Flow rate: 1 ml/min Temp: 25 o C Wavelength: 220 nm Effect of Ionization on the Analyte Response Increased sensitivity is observed with increasing pH at this wavelength. Time (min.) mV

21. 20 Chromatographic Conditions Column: 15 cm x 0.46 cm Chromegabond WR-EX C18 Eluent: Aqueous/ 20% - 50% MeCN Aqueous: 15 mM Na 2 HPO 4 7H 2 O adj. to pH 2 with H 3 PO 4 Flow rate: 1 ml/min Temp: 25 o C Effect of pH and Organic Concentration on the Analyte UV Absorbance Chromatographic Conditions Column: 15 cm x 0.46 cm Chromegabond WR-EX C18 Eluent: 80% Aqueous/ 20%MeCN Aqueous: 15 mM Na 2 HPO 4 7H 2 O adj. to pH 2, 4, 8 with H 3 PO 4 Flow rate: 1 ml/min Temp: 25 o C Increasing conc. of organic shifts pH of mobile phase upward and changes in UV abs. may be observed.

22. 21 Effect of Organic Concentration on the Analyte UV Absorbance 20% MeCN, pH=2.0 30% MeCN, pH=2.0 40% MeCN, pH=2.0 50% MeCN, pH=2.0 80% MeCN, pH=2.0 Abs. Wavelength (nm)

23. 22 Chromatographic Conditions Column: 15 cm x 0.46 cm Chromegabond WR-EX C18 Eluent: Aqueous/ 30, 40, 50% MeCN Aqueous: 15 mM Na 2 HPO 4 7H 2 O adj. to pH=2.0 with H 3 PO 4 Flow rate: 1 ml/min Temp: 25 o C Effect of Organic Concentration on the Analyte UV Absorbance Toluene

24. 23 Conclusion Accounting for the pH shift of the mobile phase and the analyte pK a shift upon the addition of organic modifier will allow the chemist to analyze the ionogenic samples at their desired pH values. This will lead to development of rugged methods, increased analyte sensitivity and an accurate description of the analyte retention as a function of pH at varying organic compositions.