Mobile Phase pH
Download
1 / 23

Mobile Phase pH - PowerPoint PPT Presentation


  • 224 Views
  • Updated On :

Mobile Phase pH Analyte pK a Shift. Lecture 4. Yuri Kazakevich Seton Hall University. Measuring pH of Hydro-organic Mixtures. There are three different pH scales that are employed in pH measurement of reversed phase HPLC mobile phases.

Related searches for Mobile Phase pH

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
Download Presentation

PowerPoint Slideshow about 'Mobile Phase pH' - Mia_John


An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript

Mobile Phase pHAnalyte pKa Shift

Lecture 4

Yuri Kazakevich

Seton Hall University


Measuring ph of hydro organic mixtures
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.



Determination of the chromatographic pk a
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.


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


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


Effect of organic content on analyte pk a shift

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.


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


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


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.


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.



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


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.



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.


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.


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.


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.


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.


20% MeCN, pH=2.0 Absorbance

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)


Effect of Organic Concentration on the Analyte UV Absorbance 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


Conclusion Absorbance

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.


ad