Mobile Phase pH
Analyte pKa 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.
•
w
•
•
wpH
scale - the electrode system is calibrated with aqueous buffers.
s
wpH
scale -
s
spH
scale -
The pH of
the aqueous portion of the mobile phase is measured prior to the addition of the
organic modifier.
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.
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.
2
pH Shift of Hydro-Organic Eluents
12
10
pH 2
pH 3
pH 4
6
pH 5
s
s pH
8
pH 6
4
pH 7
pH 9
2
0
0
10
20
30
40
50
60
v/v% MeCN
slope
R2
pH 2
0.019
0.983
pH 3
0.021
0.994
pH 4
0.021
0.995
pH 5
0.021
0.995
pH 6
0.021
0.995
pH 7
0.022
0.997
pH 9
0.020
0.972
3
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.
4
Determination of the
Chromatographic pKa
s pK =
s
a
1.4
pH pKa
shift shift
3.9
ko
1.2
1
w
wpKa =
50%- wwpH
50%- sspH
2.9
k'
0.8
Theoretical
0.6
0.4
0.2
Potentiometric pKa = 4.6
k1
0
Eluent
50% Buffer
50% MeCN
-0.2
0
1
2
3
4
pH
5
ko= Retention factor of analyte in its neutral form
k1 = Retention factor of analyte in its ionized form
6
7
8
ko  k1  e[ 2.3( pKa  pH )]
k
1  e[ 2.3( pKa  pH )]
5
Effect of pH on Aniline Retention
NH2
12
10
k'
8
Aniline, pKa 4.6
10% MeCN
20% MeCN
30% MeCN
40% MeCN
50% MeCN
6
4
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
2
0
1
2
3
4
5
6
pH of aqueous phase
7
8
9
6
Effect of Organic Content on Analyte pKa
Shift
5
4.5
Aniline,
pKa (titration) = 4.6
4
pKa shift
pKa
y = -0.0146x + 4.6
R2 = 0.9939
3.5
pH shift
3
2.5
2
0
y = -0.0349x + 4.6
R2 = 0.9983
s pK
s
a
s pK
w
a
w pK
w
a
10
20
30
40
50
60
v/v% MeCN
•
•
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.
7
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.
A
B
C
Example:
2-4 dimethylpyridine (base) has a pKa
of 6.7 and initial eluent conditions are:
50% MeCN and 50% Buffer.
ko
k
pKa
k1
What should the pH of the buffer be in order to
obtain the basic analyte in its fully ionized
form?
pH
6.7  (5 * 0.2)  5.7
5.7  2  3.7
00.20
.30  5 1.00
1.5
3.7 1.0
1.5  2.7
2.2
Downward analyte pKa shift.
pH at which basic analyte would be protonated
Upward pH shift of aqueous acidic buffer upon addition of organic
8
Max pH of buffer in order to have analyte in fully ionized form.
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
14
12
10%
10
20%
2,4 Dihydroxybenzoic acid
pKa (tit.) 3.29
25%
k'
8
30%
6
35%
COOH
4
OH
2
0
1
2
3
4
pH of aqueous phase
5
6
7
OH
9
Effect of Organic Content on
Analyte Ionization: Acids
4.4
y = 0.0291x + 3.1954
R2 = 0.9921
pKa (HPLC)
4.2
4
3.8
2,4 Dihydroxybenzoic acid,
3.6
pKa (titration) = 3.29
3.4
3.2
3
0
10
20
30
40
v/v% MeCN
• 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
10
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.
11
pH effect on analyte UV absorption
Part 2
12
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
13
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
E2
B
184 nm emax ~ 60,000
204 nm emax = 7,900
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.
14
UV Absorption by Aromatic Systems
Compound
Benzene
Naphthalene
Toluene
Chlorobenzene
Phenol
Phenolate ion
Thiophenol
Aniline
Anilinium ion
Molecular
Formula
C6H6
C10H8
C6H5CH3
C6H5Cl
C6H5OH
C6H5O
C6H5SH
C6H5NH2
+
C6H5NH3
E2 Band
max (nm)
204
286
207
210
211
235
236
230
203
e max
7900
9300
7000
7600
6200
9400
10000
8600
7500
B Band
max (nm)
256
312
261
265
270
287
269
280
254
e max
200
289
300
240
1450
2600
700
1430
160
15
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.
16
Effect of Protonation on Aniline
UV Response
H
H
H
H
H
N:
Aniline
N+
Anilinium ion
+ H+
• 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.
17
UV Absorbance as a Function of pH
10% AcN
25
H
Absorbance, 232 nm
H
N:
20
15
H
H
H
10
pKa = 4.33
(corr pHshift)
N+
5
0
0
1
2
3
4
5
6
7
pH of aqueous phase
18
At 232 nm there is an decrease in absorbance as aniline becomes protonated.
Effect of Ionization on the
Analyte Response
pH 1.5
200
0
mV
200
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
12
13
14
15
16
17
18
19
pH 2.5
0
200
1
pH 4
0
200
1
pH 5
0
1
2
pH 6
200
0
1
2
pH 9
200
Time (min.)
0
0
1
2
3
4
5
6
7
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
8
9
10
11
Increased sensitivity is observed
with increasing pH at this wavelength.
19
Effect of pH and Organic Concentration
on the Analyte UV Absorbance
pH=2
340
pH=4
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
290
pH=8
Abs.
240
190
140
90
40
Abs.
35
30
20% MeCN, pH=2.0
25
30% MeCN, pH=2.0
20
40% MeCN, pH=2.0
230
250
270
290
310
Wavelength (nm )
50% MeCN, pH=2.0
15
10
5
0
-5
210
-10
210
230
250
270
290
310
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
Wavelength (nm )
20 .
Increasing conc. of organic shifts pH of mobile phase upward and changes in UV abs. may be observed
Effect of Organic Concentration on
the Analyte UV Absorbance
35
30
Norm.
25
800
20% MeCN, pH=2.0
700
30% MeCN, pH=2.0
600
40% MeCN, pH=2.0
500
50% MeCN, pH=2.0
80% MeCN, pH=2.0
400
Abs.
20
H2N
15
10
5
0
-5
210
230
250
300
270
290
310
Wavelength (nm )
200
100
0
200
225
250
275
300
Wavelength (nm)
325
350
375
nm
21
Effect of Organic Concentration on
the Analyte UV Absorbance
30%, pH=2.0
Toluene
30
40%, pH=2.0
25
50%, pH=2.0
Abs.
20
H3C
15
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
10
5
0
-5
210
230
250
270
290
310
Wavelength (nm )
22
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.
23