Effect of mobile phase pH and composition on HPLC

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Effect of mobile phase pH and composition on HPLC Powered By Docstoc
					            Departament de Química Analítica
                Universitat de Barcelona




Effect of mobile phase pH and composition on HPLC
        retention of weak acids and bases

                     ELISABETH BOSCH




                                           Barcelona, May 2005
                            GOAL




The variation of HPLC retention of ionizable compounds with
mobile phase composition, pH and type of buffer used, is
discussed for methanol-water and acetonitrile-water mobile
phases and whether in isocratic and gradient elution.
                       TABLE OF CONTENTS



1. Retention of ionizable analytes
2. Variation of pKa of acids with the solvent
3. Buffers and buffer capacity in methanol-water and acetonitrile
    water mobile phases
4. pH scales in non-aqueous and hydroorganic solvents
5. pH in methanol-water and acetonitrile-water mobile phases
6. RP-HPLC isocratic elution
        Effects of mobile phase pH and composition on retention
7. RP-HPLC gradient elution
   RETENTION OF IONIZABLE ANALYTES


                                                      aH aA [H][A] γ H γ A
HA ⇔ H + + A −                           Ka =              =               = K 'a γ A
                                                       aHA   [HA] γ HA


        k HA =
                  [HA ]s Vs                           kA =
                                                              [A ]s Vs
                 [HA ]M VM                                   [A ]M VM

                       tR − tM
                  k=           = αHA k HA + α A k A
                          tM

                                                  '
                         k HA + k A 10pH−pK a
                   k=                     '
                              1 + 10pH−pK a

                                                      '
                         t R(HA) + t R(A)10pH−pK a
                           '         '

                  tR =
                   '
                                              '
                               1 + 10pH−pK a

                                                      '
                         t R(HA) + t R(A)10pH−pK a
                  tR =                        '
                               1 + 10pH−pK a
RETENTION OF IONIZABLE ANALYTES

                                              '
                      t R(HA) + t R(A)10pH−pK a
                        '         '

               tR =
                '
                                         '
                             1 + 10pH−pK a


  4

                               Acidic analyte

  3



                                                     '
 ´
tR 2                                              pK a



  1




  0
       0   2             4           6             8     10
                                pH
  VARIATION OF pKa WITH MOBILE PHASE COMPOSITION

                                          HAz + S ↔ HS+ Az-1 ↔ HS+ + Az-1
                                   HAz + H2O ↔ H3O+ Az-1 ↔ H3O+ + Az-1


                                                                                   e 2 ( z − 1) ⎛ 1    1 ⎞ Σ sG 0 − Σw Gsolv
                                                                                                                         0
             s
                 pK a = w pK a −
                        w          vac
                                         pK a( HS + ) +   vac
                                                                pK a( H O + ) −                 ⎜   − w ⎟ − s solv    w
                                                                                  2.303 r k T ⎝ s ε     ε⎠
             s                     vac                    vac          3
                                                                                                               2.303 R T




                        intrinsic acidity of the
                                                                                        electrostatic                    solvation
                        protonated solvents in
                                                                                            term                           term
                        the vacuum (constants)


• Neutral acids (z = 0):                     • Anionic acids (z < 0):                                        • Cationic acids (z > 0):
    HA ⇔ H + + A −                                                                                                 HA + ⇔ H + + A
                                                            −
                                                    HA ⇔ H + + A − 2


                 ∆pK a = s pK a − w pK a > 0
                         s        w
                                                                                                                       ∆pK a < 0


                                                    s
                                                    s pK a          = a s w pK a + b s
                                                                          w
          VARIATION OF pKa WITH MOBILE PHASE COMPOSITION


      12
                                                        ∆pK a = s pK a − w pK a
                                                                s        w
      10
                                                              Neutral acids:
                                                                  (   ) Phosphoric pKa1
                                                                  (   ) Acetic
      8
                                                                  (   ) Citric pKa1
S
S
    pKa                                           ∆pKa>0
                                                                  (   ) Boric

      6                                                       Anionic acids:
                                                                  (   ) Phosphoric pKa2
                                                                  (   ) Citric pKa2
      4                                                           (   ) Citric pKa3



      2                                                       Cationic acids:
                                                  ∆pKa<0          (   ) Butylammonium
                                                                  (   ) Ammonium
           0   20      40     60       80   100
                    % Methanol (v/v)                s: hydroorganic or organic solvent
                                                    w: water
VARIATION OF pKa WITH MOBILE PHASE COMPOSITION



                                  ∆pK a = s pK a − w pK a
                                          s        w

                                        Neutral acids:

                                            (   ) Benzoic acid
                                            (   ) Acetic
                                            (   ) Citric pKa1
                            ∆pKa>0
                                        Anionic acids:

                                            ( ) Citric pKa2
                                            ( X ) Citric pKa3


                                        Cationic acids:
                            ∆pKa<0
                                            (   ) Pyridinium


                              s: hydroorganic or organic solvent
                              w: water
    Buffer Capacity, β



            dCB    dC A
       β=       =−
            dpH     pH


       [
       ⎛
β = 2.3⎜ HS + S +
       ⎜
           +
            ] [ ] [
               −          ]
                   HS + c aK a
                          [
                  (K a + HS +    ]
                                     ⎞
                                     ⎟
                                     ⎟
       ⎝                             ⎠
    VARIATION OF β WITH MOBILE PHASE COMPOSITION

Acetic/acetate buffer in acetonitrile-water mobile phases
VARIATION OF pKa AND β WITH MOBILE PHASE COMPOSITION


           Phthalic acid      Citric acid




           Organic modifier: acetonitrile
VARIATION OF β WITH pH and MOBILE PHASE COMPOSITION




         Organic modifier: acetonitrile
TERMINOLOGY FOR pH RECOMMENDED BY THE IUPAC



                     media where pH is measured



                                                       pH
                               (in practice, solvent
        where the calibration buffers are prepared)
                                   pH SCALES

                          s: hydroorganic or organic solvent
                          w: water


                    pH value of an aqueous solution
                                                               w
Calibration of the electrode system (or glass                  w pH
combined electrode) with aqueous buffers of known
pH values (usually w pH = 4.00 and w pH = 7.02)
                   w               w


          pH value of an organic or mixed solvent, s                  Universal pH scale
                                                               s      (reference state: the
Calibration of the electrode system (or glass                  w pH   proton in water at
combined electrode) with aqueous buffers of known                     infinite dilution)
pH values (usually w pH = 4.00 and w pH = 7.02)
                   w               w

                                                                      pH scale of the
          pH value of an organic or mixed solvent, s                  solvent s (reference
                                                               s
                                                               s pH
                                                                      state: the proton in
Calibration of the electrode system (or glass                         the solvent s at
combined electrode) with buffers of known pH values                   infinite dilution)
prepared in the solvent s
RELATIONSHIP BETWEEN THE pH SCALES IN METHANOL-WATER


                                                      0.5
                      s 0       s
      δ = E j − log ( w γ H ) = w pH − s pH
                                       s
                                                      0.0


                                                  -0.5
Electrode system     Medium effect
  liquid junction    (constant for the
                                              δ (M)
                                                  -1.0
                     solvent s)
                                                                  C.L. deLigny et al., 1960 (H2 electrode)
                                                  -1.5            R. Bates et al., 1963 (H2 electrode)


                                                  -2.0


                                                            0.0     0.2       0.4       0.6       0.8        1.0
                                                                                    φMeOH
RELATIONSHIP BETWEEN THE pH SCALES IN METHANOL-WATER


                                                       0.5
                       s 0       s
       δ = E j − log ( w γ H ) = w pH − s pH
                                        s
                                                       0.0


                                                   -0.5
Electrode system       Medium effect
  liquid junction      (constant for the
                                               δ (M)
                                                   -1.0
                       solvent s)
                                                                   C.L. deLigny et al., 1960 (H2 electrode)
                                                   -1.5            R. Bates et al., 1963 (H2 electrode)
                                                                   This work (combined glass electrode)
                               2
             0.09 φMeOH − 0.11φMeOH
δ=                                                 -2.0
                           2              3
     1 − 3.15 φMeOH + 3.51φMeOH   − 1.35 φMeOH

                                                             0.0     0.2       0.4       0.6       0.8        1.0
                                                                                     φMeOH

                                                                           s 0                 s      s
     Conclusion: E j ≈ 0 for the combined glass electrode, and δ = − log ( w γ H ) =           w pH − s pH
RELATIONSHIP BETWEEN pH SCALES IN ACETONITRILE-WATER




              δ = −0.27Φ ACN + 1.48Φ2 − 3.82Φ3
                                    ACN      ACN
               pH MEASUREMENT IN MOBILE PHASES


Aqueous buffer ( w pH ) + organic modifier : MOBILE PHASE ( w pH or
                 w
                                                            s                          s
                                                                                       s pH )




   1. Calibration of the combined glass electrode with aqueous buffers of known w pH
                                                                                w
                                      s
   2. Measurement of the mobile phase w pH by introduction of the electrode in the
      organic modifier-water mobile phase
   3. Calculation of the δ quantity for the volume fraction of methanol (φMeOH) or
      acetonitrile ((φACN) in the mobile phase by the empirical equations:


            0.09 ΦMeOH − 0.11ΦMeOH
                              2
δ=                                                     δ = −0.27ΦACN + 1.48Φ2 − 3.82Φ3
                                                                            ACN      ACN
   1 − 3.15 ΦMeOH + 3.51ΦMeOH − 1.35 ΦMeOH
                          2           3




   4. Calculation of s pH :
                     s
                                    s
                                    s   pH = w pH − δ
                                             s
     pH VALUES OF METHANOL-WATER MOBILE PHASES

         s
         s pH   =   s
                    w pH   − δ                           ∆pH = s pH −
                                                               s
                                                                            w
                                                                            w pH


                                                         % Methanol (v/v)
                                                 60 (δ = 0.17)         80 (δ = 0.08)
                                       w       s      s              s      s
Aqueous buffer                         w pH    w pH   s pH   ∆pH     w pH   s pH   ∆pH

0.01M H3PO4                            2.02    2.70   2.53   0.51    2.90   2.82   0.80
0.0065M H3Cit/0.0035M KH2Cit           3.01    4.03   3.86   0.85    4.22   4.14   1.13
0.0086M HAc/0.0014M NaAc               3.96    5.10   4.93   0.97    5.69   5.61   1.65
0.0035M HAc/0.0065M NaAc               4.98    6.14   5.97   0.99    6.65   6.57   1.59
0.0056M KNaHCit/0.0044M Na3Cit         6.05    7.54   7.37   1.32    8.37   8.29   2.24
0.0051M KH2PO4/0.0049M Na2HPO4         7.02    8.38   8.21   1.19    9.38   9.30   2.28
0.0095M NH4Cl/0.0005M NH3              7.93    7.40   7.23   -0.70   7.24   7.16   -0.77
0.0065M NH4Cl/0.0035M NH3              9.00    8.47   8.30   -0.70   8.19   8.11   -0.89
0.0056M H3BO3/0.0044M NaH2BO3          8.98    9.54   9.37   0.39    9.43   9.35   0.37
0.0019M BuNH3Cl/0.0081M BuNH2          10.89   10.57 10.40 -0.49     9.77   9.69   -1.20
Cit: citrate, Ac: acetate, Bu: butyl
    pH OF 50% METHANOL-WATER BUFFERED MOBILE PHASES


pH values of mobile phases in water ( w pH) and diluted to 50% methanol ( w pH , s pH )
                                      w
                                                                          s
                                                                                 s

                                     ∆pH = s pH − w pH
                                           s      w
                                                        w       s       s
                 Aqueous buffer                         w pH    w pH    s pH    ∆pH
             A   0.01M H3PO4                            2.20    2.72    2.60    0.40
             B   0.0065M H3Cit/0.0035M KH2Cit           2.97    3.76    3.64    0.67
             C   0.0086M HAc/0.0014M NaAc               3.99    4.81    4.69    0.70
             D   0.0035M HAc/0.0065M NaAc               4.99    5.93    5.81    0.82
             E   0.0056M KNaHCit/0.0044M Na3Cit         6.03    7.29    7.17    1.14
             F   0.0051M KH2PO4/0.0049M Na2HPO4         7.05    8.29    8.17    1.12
             G   0.0095M NH4Cl/0.0005M NH3              8.01    7.53    7.41    -0.60
             H   0.0065M NH4Cl/0.0035M NH3              8.97    8.58    8.46    -0.51
             I   0.0056M H3BO3/0.0044M NaH2BO3          9.02    9.28    9.16    0.14
             J   0.008M BuNH3Cl/0.002M BuNH2            9.88    9.16    9.04    -0.84
             K   0.001M H3BO3/0.009M NaH2BO3            10.00   10.20   10.08   0.08
             L   0.0019M BuNH3Cl/0.0081M BuNH2          11.02   10.46   10.34   -0.68

                 Cit: citrate, Ac: acetate, Bu: butyl
 EFFECT OF MOBILE PHASE pH ON RETENTION (ISOCRATIC HPLC)


                                     '     t R(HA ) + t R( A ) 10 pH − pK a
                                             '          '
                                    tR =
                                                  1 + 10 pH − pK a


 30          3-nitrophenol                                     30      3-nitrophenol


 20                                                            20

 ’
tR                                                         ’
                                                          tR
 10                                                            10




     0                                                         0
              anilinium ion                                               anilinium ion

         2       4      6       8      10          12                 2       4      6     8   10   12
                       s                                                            w
                       w pH                                                         w pH
                              Mobile phase: 50% methanol. Column: C18
 EFFECT OF MOBILE PHASE pH ON RETENTION (ISOCRATIC HPLC)


                                     '     t R(HA ) + t R( A ) 10 pH − pK a
                                             '          '
                                    tR =
                                                  1 + 10 pH − pK a


 30          3-nitrophenol
                                                                              s        s
                                                                              s pK a = w pK a   −δ
 20

 ’
tR                                                                               s         s         s
                                                                                 w pK a    s pK a    s pK a (lit.)
 10
                                                          3-Nitrophenol           9.21      9.09      8.94
                                                          Anilinium ion           4.32      4.20      4.23
     0
              anilinium ion

         2       4      6       8      10          12
                       s
                       w pH
                              Mobile phase: 50% methanol. Column: C18
EFFECT OF MOBILE PHASE pH ON RETENTION (ISOCRATIC HPLC)


                                   '     t R(HA ) + t R( A ) 10 pH − pK a
                                           '          '
                                  tR =
                                                1 + 10 pH − pK a


                                                             30      3-nitrophenol


                w        w             s
                w pK a   w pK a (lit.) s pK a (lit.)         20

                                                         ’
                                                        tR
3-Nitrophenol 9.22          8.43           8.94
                                                             10
Anilinium ion    3.46       4.60           4.23


                                                             0
                                                                        anilinium ion

                                                                    2       4      6     8   10   12
                                                                                  w
                                                                                  w pH
                          Mobile phase: 50% methanol. Column: C18
EFFECT OF MOBILE PHASE pH ON RETENTION (ISOCRATIC HPLC)




  Retention time of 3-nitrophenol and triethylamine in a polymeric
  column with the 40% acetonitrile mobile-phase: (○) 3-
  nitrophenol in neutral and anionic acid buffers, (●) 3-nitrophenol
  in ammonia and butylamine buffers, (□) triethylamine in neutral
  and anionic acid buffers, and (■) triethylamine in butylamine
  buffers.
  EFFECT OF MOBILE PHASE BUFFER ON SELECTIVITY




Elution of a mixture of ionizable compounds with a 60%
acetonitrile mobile phase prepared from aqueous buffers of
pH 8.0. (A) H2PO4-/HPO42- buffer; (B) NH4+/NH3 buffer.
Compounds: 2-nitrophenol (1), 2,4,6-trimethylpyridine (2),
3-bromophenol (3), and N,N-dimethylbenzylamine (4).
Chromatograms for the individual compounds in each mobile
phase are also given
VARIATION OF Ammonium Acetate BUFFERED MOBILE PHASE pH

     Variation of the pH of
     50 mM Ammonium
     Acetate buffer in
     methanol-water and
     acetonitrile-water
     mixtures




      s           w
      w pH   =    w pH         +mφ


                       w
           a 10 (b − w pH) + c
     m=                    w
                 10 (b − w pH)
                       VARIATION OF MOBILE PHASE pH
           Variation of the pH of 50 mM Ammonium
           Acetate buffer in methanol-water mixtures
          2.0

                                                       Initial wpH values:
                                                               w
          1.5
                                                              (   ) 2.67
          1.0                                                 (   ) 3.01
                                                              (   ) 4.06
s
  pH - wpH 0.5                                                (   ) 5.07
w      w
                                                              (   ) 6.07
                                                              (   ) 6.96
          0.0                                                 (   ) 7.94
                                                              (   ) 8.94
          -0.5                                                (   ) 9.95

          -1.0
                 0.0     0.3            0.6      0.9
                                φMeOH
                                                                     w
                                                            a 10 (b − w pH) + c
                         s
                         w pH   =   w
                                    w pH   +mφ         m=                w
                                                               10 (b − w pH)
ANALYTE pK AND MOBILE PHASE pH ON RETENTION


                                                 s            w                          s
                                                                                             pK a = w pK a − δ
                                                                                                    s
                                                 s pK a = a s w pK a + b s               s




 s           w
 w pH   =    w pH       +mφ


      a 10     (b − w pH)
                    w
                            +c
                                                                      s
                                                                      w   pK a = a s w pK a + b s + δ
                                                                                     w
 m=                 w
            10 (b − w pH)




                                        t R(HA)10pKa −pH + t R( A )
                                 tR =
                                             10pKa −pH + 1
                     EFFECT OF MOBILE PHASE pH ON RETENTION
                   Benzoic acid                                4-tert-butylpyridine
 1.0                                        1.0
                                                                                                                                        s      s

 0.8                                        0.8
                                                                                                                        t R(HA)10 w pKa −w pH + t R( A )
                                                                                                                 tR =                  s      s
                                                                                                                                       w pKa −w pH
 0.6                                        0.6                                                                                   10                 +1
tN                                         tN
 0.4                                        0.4


 0.2                                                                                                                         N        t R − t R( A )
                                            0.2
                                                                                                                         t       =
                                                                                                                                   t R(HA ) − t R( A )
 0.0                                        0.0
           2   3     4      5    6    7              2               3         4         5         6         7
                      w                                                        w                                                 tN: normalized tR
                      w pH                                                     w pH

                    Ephedrine                                                                                                Neutral acid:
     1.0                                                   1                                                                 tR(HA)=1 and tR(A)=0
                                                                           Benzoic acid
     0.8
                                                           0                                                                 Neutral base:
                                                w pKa - w pH


                                                                                   Ephedrine                                 tR(HA)=0 and tR(A)=1
     0.6
                                                        s




tN                                                        -1
     0.4

                                                          -2                                                                        80% MeOH
                                                s




     0.2

                                                                 4-tert-butylpyridine                                               60% MeOH
     0.0                                                  -3
           7   8      9     10   11   12                       0.0       0.2       0.4       0.6       0.8       1.0
                                                                                                                                    40% MeOH
                         w
                         w pH
                                                                                     φMeOH                                          20% MeOH
     EFFECT OF MOBILE PHASE pH ON GRADIENT RETENTION



Isocratic equation (dashed lines):                         4-tert-butylpyridine
                                                 2.8
          t R(HA)10pKa −pH + t R( A )
   tR =                                                                     Lidocaine
               10pKa −pH + 1                     2.4

                                            tR
Gradient equation (continuous lines):            2.0
                  s(pKa −pH)
                                                                                           Ephedrine
         t g(HA)10             + t g( A )
  tg =
              10s(pKa −pH) + 1                   1.6
                                                                                  Benzoic acid


    s: empirical fitting parameter               1.2
                                                       2          4         6          8         10
                                                                             w
                                                                             w pH
                        PUBLICATIONS

This work has been published in various articles with the generic
title:
“RETENTION OF IONIZABLE COMPOUNDS IN HPLC” in:
Analytical Chemistry
Journal of Chromatography A
Chromatographia

and in the following review:

M. Rosés and E. Bosch
“REVIEW: INFLUENCE OF MOBILE PHASE ACID-BASE
 EQUILIBRIA IN THE CHROMATOGRAPHIC BEHAVIOUR
 OF PROTOLYTIC COMPOUNDS”
Journal of Chromatography A, 982, (2002), 1-30
SOLUTE-SOLVENT INTERACCTIONS




 •   Dr. Martí Rosés
 •   Dra. Imma Canals
 •   Dra. Sònia Espinosa
 •   Sr. Xavier Subirats