Mobile Phase pHAnalyte pKa Shift

Lecture 4

Yuri KazakevichSeton 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

0

2

4

6

8

10

12

0 10 20 30 40 50 60

v/v% MeCN

ssp

H

pH 2

pH 3

pH 4

pH 5

pH 6

pH 7

pH 9

pH 2 pH 3 pH 4 pH 5 pH 6 pH 7 pH 9slope 0.019 0.021 0.021 0.021 0.021 0.022 0.020

R2 0.983 0.994 0.995 0.995 0.995 0.997 0.972

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

Determination of the Chromatographic pKa

pKa

shift

Theoretical

ko

k1-0.2

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 1 2 3 4 5 6 7 8pH

k'

50%- wwpH

wwpKa = 2.9

Potentiometric pKa = 4.6

50%- sspH

pHshift

sspKa = 3.9

Eluent50% Buffer50% MeCN

ko= Retention factor of analyte in its neutral formk1 = Retention factor of analyte in its ionized form )](3.2[

)](3.2[1

1 pHpK

pHpKo

a

a

e

ekkk

6

9

NH2

Aniline, pKa 4.6

0

2

4

6

8

10

12

1 2 3 4 5 6 7 8

pH of aqueous phase

k'

50% MeCN

40% MeCN

10% MeCN

20% MeCN

30% MeCN

Chromatographic Conditions Column: 15 cm x 0.46 cm Luna C18(2)Eluent: Aqueous/ 10, 20, 30, 40, 50% MeCNAqueous: 15 mM K2HPO4•7H2O adj. to pH 1 - 9 with H3PO4

Flow rate: 1 ml/minTemp: 25oCWavelength: 220 nm

Effect of pH on Aniline Retention

7

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.

y = -0.0349x + 4.6 R2 = 0.9983

2

2.5

3

3.5

4

4.5

5

0 10 20 30 40 50 60v/v% MeCN

pKa

sspKa

Aniline, pKa (titration) = 4.6

pH shift

pKa shifty = -0.0146x + 4.6 R2 = 0.9939

swpKa

wwpKa

8

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?

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.

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.

A B C

k1

ko

pH

k

pKa

7.5)2.0*5(7.6

2.25.17.3 5.1530.0 7.327.5

0.20 1.00

1.01.0 2.7

9

0

2

4

6

8

10

12

14

1 2 3 4 5 6 7

pH of aqueous phase

k'

10%

20%

25%

30%

35%

2,4 Dihydroxybenzoic acid pKa (tit.) 3.29

COOH

OH

OH

Chromatographic Conditions Column: 15 cm x 0.46 cm Luna C18(2)Eluent: Aqueous/ 10, 20, 30, 40, 50% MeCNAqueous: 15 mM K2HPO4•7H2O adj. to pH 1 - 7 with H3PO4

Flow rate: 1 ml/minTemp: 25oCWavelength: 220 nm

Effect of pH on 2,4-Dihydroxybenzoic Acid Retention

10

Effect of Organic Content on Analyte Ionization: Acids

y = 0.0291x + 3.1954

R2 = 0.9921

3

3.2

3.4

3.6

3.8

4

4.2

4.4

0 10 20 30 40

v/v% MeCN

pK

a (H

PL

C)

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

• 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

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:

E1 184 nm max ~ 60,000

E2 204 nm max = 7,900

B 256 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.

15

UV Absorption by Aromatic Systems

Compound MolecularFormula

E2 Band B Band

max (nm) max

max (nm) max

Benzene C6H6 204 7900 256 200Naphthalene C10H8 286 9300 312 289

Toluene C6H5CH3 207 7000 261 300Chlorobenzene C6H5Cl 210 7600 265 240

Phenol C6H5OH 211 6200 270 1450Phenolate ion C6H5O - 235 9400 287 2600Thiophenol C6H5SH 236 10000 269 700

Aniline C6H5NH2 230 8600 280 1430Anilinium ion C6H5NH3

+ 203 7500 254 160

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 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.

17

N

H H

:

+ H+

+

H

H

HN

Aniline Anilinium 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

18

UV Absorbance as a Function of pH

At 232 nm there is an decrease in absorbance as aniline becomes protonated.

N

H H

:

+

H

H

HN

0

5

10

15

20

25

0 1 2 3 4 5 6 7

pH of aqueous phase

Ab

sorb

ance

, 232

nm

10% AcN

pKa = 4.33 (corr pH shift)

19

200

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

200

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

200

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

200

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

200

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

0

200

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 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% MeCNAqueous: 15 mM K2HPO4•7H2O adj. to pH 1 - 9 with H3PO4

Flow rate: 1 ml/minTemp: 25oCWavelength: 220 nm

Effect of Ionization on the Analyte Response

Increased sensitivity is observedwith increasing pH at this wavelength.

Time (min.)

mV

20

-10

40

90

140

190

240

290

340

210 230 250 270 290 310

Wavelength (nm)

Ab

s.

pH=2

pH=4

pH=8

-5

0

5

10

15

20

25

30

35

210 230 250 270 290 310

Wavelength (nm)

Ab

s.

20% MeCN, pH=2.0

30% MeCN, pH=2.0

40% MeCN, pH=2.0

50% MeCN, pH=2.0

Chromatographic Conditions Column: 15 cm x 0.46 cm Chromegabond WR-EX C18Eluent: Aqueous/ 20% - 50% MeCNAqueous: 15 mM Na2HPO4•7H2O adj. to pH 2 with H3PO4

Flow rate: 1 ml/minTemp: 25oC

Effect of pH and Organic Concentration on the Analyte UV Absorbance

Chromatographic Conditions Column: 15 cm x 0.46 cm Chromegabond WR-EX C18Eluent: 80% Aqueous/ 20%MeCNAqueous: 15 mM Na2HPO4•7H2O adj. to pH 2, 4, 8 with H3PO4 Flow rate: 1 ml/minTemp: 25oC

Increasing conc. of organic shifts pH of mobile phase upward and changes in UV abs. may be observed.

21

Effect of Organic Concentration on the Analyte UV Absorbance

-5

0

5

10

15

20

25

30

35

210 230 250 270 290 310

Wavelength (nm)

Ab

s.

nm200 225 250 275 300 325 350 375

Norm.

0

100

200

300

400

500

600

700

800NH2

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

Ab

s.

Wavelength (nm)

22

-5

0

5

10

15

20

25

30

210 230 250 270 290 310

Wavelength (nm)

Ab

s.

30%, pH=2.0

40%, pH=2.0

50%, pH=2.0

Chromatographic Conditions Column: 15 cm x 0.46 cm Chromegabond WR-EX C18Eluent: Aqueous/ 30, 40, 50% MeCNAqueous: 15 mM Na2HPO4•7H2O adj. to pH=2.0 with H3PO4

Flow rate: 1 ml/minTemp: 25oC

Effect of Organic Concentration on the Analyte UV Absorbance

CH3

Toluene

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.