Mobile Phase pH Analyte pK a Shift

1. Mobile Phase pHAnalyte pKa Shift Lecture 4 Yuri Kazakevich Seton Hall University

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. • wwpH 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. • swpH 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. • sspH 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.

3. pH Shift of Hydro-Organic Eluents

4. Determination of the Chromatographic pKa 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.

5. 1.4 1.2 1 0.8 k' 0.6 0.4 0.2 0 -0.2 0 1 2 3 4 5 6 7 8 pH pKa shift pH shift sspKa = 3.9 wwpKa = 2.9 50%- sspH Theoretical 50%- wwpH Determination of the Chromatographic pKa ko Eluent 50% Buffer 50% MeCN PotentiometricpKa = 4.6 k1 ko= Retention factor of analyte in its neutral form k1 = Retention factor of analyte in its ionized form

6. 12 10 8 10% MeCN k' 6 20% MeCN 30% MeCN 4 40% MeCN 50% MeCN 2 0 9 1 2 3 4 5 6 7 8 pH of aqueous phase Aniline, pKa 4.6 Effect of pH on Aniline Retention Chromatographic Conditions Column: 15 cm x 0.46 cm Luna C18(2) Eluent: Aqueous/ 10, 20, 30, 40, 50% MeCN Aqueous: 15 mM K2HPO4•7H2O adj. to pH 1 - 9 with H3PO4 Flow rate: 1 ml/min Temp: 25oC Wavelength: 220 nm

7. 5 Aniline, pKa (titration) = 4.6 4.5 pKa shift 4 y = -0.0146x + 4.6 R2 = 0.9939 a pK 3.5 pH shift 3 y = -0.0349x + 4.6 R2 = 0.9983 sspKa 2.5 swpKa wwpKa 2 0 10 20 30 40 50 60 v/v% MeCN Effect of Organic Content on Analyte pKa Shift • A decrease of ~0.2 pKa 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.

8. pH Shift and pKa Shift • The downward pKa shift for basic analytes must be accounted for. • The working pH should be at least 2 pH units below the basic analyte pKa to be fully ionized. • The upward pH shift of the aqueous acidic buffer upon addition of the organic must be accounted for. Example: 2-4 dimethylpyridine (base) has a pKa 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? Downward analyte pKa 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. 0.20 1.00 1.0 2.7 1.0

9. Effect of pH on 2,4-Dihydroxybenzoic Acid Retention Chromatographic Conditions Column: 15 cm x 0.46 cm Luna C18(2) Eluent: Aqueous/ 10, 20, 30, 40, 50% MeCN Aqueous: 15 mM K2HPO4•7H2O adj. to pH 1 - 7 with H3PO4 Flow rate: 1 ml/min Temp: 25oC Wavelength: 220 nm 2,4 Dihydroxybenzoic acid pKa (tit.) 3.29

10. Effect of Organic Content on Analyte Ionization: Acids 2,4 Dihydroxybenzoic acid, pKa (titration) = 3.29 • An increase of 0.2 pKa 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 pKa of pharmaceutical compounds in certain organic composition.

11. Conclusion Accounting for the pH shift of the mobile phase and the analyte pKa 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.

12. pH effect on analyte UV absorption Part 2

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

14. UV Absorption by Aromatic Systems • UV spectra of aromatic hydrocarbons are characterized by 3 sets of bands that originate from p --> p* transitions. • Benzene has strong absorption peaks at: • E1 184 nm emax ~ 60,000 • E2 204 nm emax = 7,900 • B 256 nm emax = 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.

15. UV Absorption by Aromatic Systems

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 -NH2 have an auxochromic effect on benzene chromophore. • Have at least one pair of n electrons capable of interacting with p electrons of the the ring. • This stabilizes the p* state and lowers its energy • Phenolate anion auxochromic effect more pronounced than for phenol since anion has extra pair of unshared electrons.

17. Effect of Protonation on Aniline UV Response Aniline Anilinium ion • Aniline has a pair of n electrons capable of interacting with the p electrons of ring. • This stabilizes the p* 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.

18. 10% AcN 25 20 15 Absorbance, 232 nm pKa = 4.33 10 (corr pH shift) 5 0 0 1 2 3 4 5 6 7 pH of aqueous phase UV Absorbance as a Function of pH At 232 nm there is an decrease in absorbance as aniline becomes protonated.

19. pH 1.5 pH 2.5 pH 4 pH 5 pH 6 pH 9 Effect of Ionization on the Analyte Response mV Time (min.) Chromatographic Conditions Column: 15 cm x 0.46 cm Luna C18(2) Eluent: 90% Aqueous:10% MeCN Aqueous: 15 mM K2HPO4•7H2O adj. to pH 1 - 9 with H3PO4 Flow rate: 1 ml/min Temp: 25oC Wavelength: 220 nm Increased sensitivity is observed with increasing pH at this wavelength.

20. 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 Na2HPO4•7H2O adj. to pH 2, 4, 8 with H3PO4 Flow rate: 1 ml/min Temp: 25oC Chromatographic Conditions Column: 15 cm x 0.46 cm Chromegabond WR-EX C18 Eluent: Aqueous/ 20% - 50% MeCN Aqueous: 15 mM Na2HPO4•7H2O adj. to pH 2 with H3PO4 Flow rate: 1 ml/min Temp: 25oC Increasing conc. of organic shifts pH of mobile phase upward and changes in UV abs. may be observed.

21. 20% MeCN, pH=2.0 30% MeCN, pH=2.0 40% MeCN, pH=2.0 Norm. 50% MeCN, pH=2.0 80% MeCN, pH=2.0 800 700 600 500 400 300 200 100 0 200 225 250 275 300 325 350 375 nm Effect of Organic Concentration on the Analyte UV Absorbance Abs. Wavelength (nm)

22. Effect of Organic Concentration on the Analyte UV Absorbance Toluene Chromatographic Conditions Column: 15 cm x 0.46 cm Chromegabond WR-EX C18 Eluent: Aqueous/ 30, 40, 50% MeCN Aqueous: 15 mM Na2HPO4•7H2O adj. to pH=2.0 with H3PO4 Flow rate: 1 ml/min Temp: 25oC

23. Conclusion Accounting for the pH shift of the mobile phase and the analyte pKa 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.