NMR Controlled Titration

NMR controlled Titration 1
NMR-controlled Titrations – Principles and Progress:
Monitoring protonation and complex formation equilibria in aqueous solutions
Gerhard Hägelea*, Michael Grzonkaa, Jürgen Petersb, Harald Spiegla, Hans Werner
Kroppa, Johannes Olliga, Stephan Hermensa, Sven Augnera, Carsten Uhlemanna,
Christian Pfaffa, Zoltan Szakacsa, Toni Kellerc, Martin Rindlispacherc, Manfred
Spraulc.
a)
Institut für Anorganische Chemie, Heinrich Heine Universität Düsseldorf,
Universitätsstrasse 1, D-40225 Düsseldorf, Germany.
Phone: ++49-211-81-12288, ++49-211-81-13140, Fax: ++49-211-81-11854
Email: haegele@uni-duesseldorf.de
b)
Schott Geräte GmbH, Wattenbergstraße 10, D-55122 Mainz.
c)
Bruker Analytik GmbH; Silberstreifen, D-76287 Rheinstetten-Karlsruhe.
Abstract
A hyphenated technique, termed NMR controlled titration, elucidates protolytic and
complex formation equilibria. Stability constants, dissociation constants and ion-
specific NMR parameter are obtained. Parameters and spectral features are used to
derive the molecular structure of species involved in dynamic processes.
Keywords
NMR, titration, NMR-controlled titration, protolysis, complex formation, stability
constants, dissociation constants, ion-specific NMR parameters, macroscopic
dissociation, microscopic dissociation, intramolecular rotation.

1. Introduction
Multifaceted interests in structural and analytical chemistry combine with such of
medicine, pharmacy, biology, food sciences, agricultural chemistry and related fields,
to understand structures and reactions for a broad range of model systems. Many
important reactions in biorelevant fields involve protolytic equilibria and complex
formation processes. NMR spectroscopic methods assisted by classical analytical
tools in combination with experimental and theoretical methods are used to study
acids, bases and metal complexes. Within this context NMR-controlled titration
proved to be a powerful method for research and production control. While initially
performed by laborious series of single sample NMR experiments, more efficient,
PC-guided, automated procedures were developed and applied successfully to a
wide range of structural problems, as will be demonstrated with some examples in
this paper.
Our main aim is to introduce readers to the technical aspects of a modern
hyphenated technique combining aspects of potentiometric titration and NMR
spectroscopy. This technique yields two- and three-dimensional graphical
representations where NMR parameters are correlated with analytical parameters.
This method is called “NMR-controlled titration“, “titration-dependent NMR-
spectroscopy“, or just “NMR-Titration” where the choice of terms depends largely
on interests of individual users.
NMR-controlled titrations lead to characteristic dissociation and stability constants
(pKa, pKb, log) and NMR parameters such as resonance frequencies, chemical
shifts, coupling constants (, , J) for the individual species involved in macroscopic
or microscopic equilibria. While ,  and J are subject to averaging processes gover-
ned by molar fractions, the spectral half width does not follow this simple principle.
Most likely HW, and henceforth the relaxation time T 2*, is influenced by dynamic
effects, hydrogen bonding, complex formation, where such phenomena are well
observed and documented but not sufficiently well understood up to now. Future
research should be directed towards these interesting aspects.
NMR-controlled titration is an efficient tool to characterize carboxylic acids, amino
acids, peptides and related structures involving phosphorylated and/or fluorinated
analogues. Some typical structures are shown in Table 1:
Table 1
CH3 COOH CH3 P(O)(OH)2 (CH3)2P(O)OH
NH2 CH2 COOH H2N CH2 P(O)(OH)2 H2N CH2 P(CH3)(O)OH
HOOC CH2 COOH HOOC CH2 P(O)(OH)2 HOOC CH2 P(CH3)(O)OH
N(CH2COOH)3 N(CH2PO3H2)3 [NaHxN(CH2PO3)3]x-5
(CH2N(CH2COOH)2)2 (CH2N(CH2PO3H2)2)2 [CdHx(CH2N(CH2PO3)2)2)]2+x-8
CH2 CH NH2 CH2 CH P(O)(OH)2 CH2 CH NH-CO-CH2-PO3H2
COOH COOH COOH COOH COOH COOH
CH2 CHOH CH2 CH2 CH CHOH CH2 CH O CH2
COOH COOH COOH COOH COOH COOH COOH COOH COOH
F F
F P(O)(OH)2 CF3 CH P(O)(OH)2 FLUCONAZOL
NH2
F F
Table 1: Some typical structures investigated by NMR-controlled Titrations

Resonances from standard 1H-, 13C{1H}-, 19F-, 1H{31P}-, 31P{1H}-NMR experiments
are used. In favorable cases metal-NMR (e.g., 113Cd-NMR) may monitor the forma-
tion of metal complexes.
Basics, progress and a variety of applications of this technology studied in Düsseldorf
are described in [1-4] and references given herein.

2. Theory
2.1. Description of Acids and Bases – Macroscopic Protolytic Equilibria -
Dissociation and Stability Constants
Consider an amino acid where the number of acidic functions (e.g. C(O)OH, P(O)OH,
etc.) is represented by a and the number of basic functions (e.g., NH2, NHR, NR2,
etc.) by b. This description is consistent with a n-valent base L-a having a anionic
centers and b neutral base centers in (oN)b-R-(O-)a. Total protonation of this system
leads to an n-valent acid HnLn-a (n = a+b) having a neutral centers and b cationic acid
centers in (+HN)b-R-(OHo)a. This general concept may also be used conveniently to
characterize simpler units such as parent amines, carboxylic acids, organophospho-
rus acids, sulfonic acids, etc. as shown in Table 2:

Table 2
Short name Formula a b n

R-COOH 1 0 1
H2N-R 0 1 1
HOOC-R-COOH 2 0 2
HOOC-R-P(R´)(O)OH 2 0 2
HO(O)(R´)P-R-P(R´)(O)OH 2 0 2
H2N-R-COOH 1 1 2
H2N-R-CO-NH-R-COOH 1 1 2
H2N-R-P(R´)(O)OH 1 1 2
EAMPi CH3-CH2-NH-CH2-P(CH3)(O)OH 1 1 2
Citric Acid HOOC-CH2-C(OH)(COOH)-CH2-COOH 3 0 3
Isocitric Acid HOOC-CH2-CH(COOH)-CH(OH)-COOH 3 0 3
HOOC-R-P(O)(OH)2 3 0 3
H2N-R-P(O)(OH)2 2 1 3
HOOC-CH(NH2)-(CH2)n-COOH 2 1 3
Glufosinate HOOC-CH(NH2)-(CH2)2-P(CH3)(O)OH 2 1 3
HOOC-CH(NH2)-(CH2)n-NH2 1 2 3
HEDP (HO)2(O)P-C(CH3)(OH)-P(O)(OH)2 4 0 4
NTMP N[CH2P(O)(OH)2]3 6 1 7
EDTMP [(HO)2(O)PCH2]2NCH2CH2N[CH2P(O)(OH)2]2 8 2 10
TABLE 2: Classification of acid base systems.

Salts of such acids and bases are defined in a general way as H a-x+yXxLYy where X
and Y represent univalent ions bearing positive or negative charges respectively, as
shown in Table 3.
Table 3
Case Type X X Y y
1 Sodium salt Na+ 1<xa - 0
2 Hydrogenchloride salt - 0 Cl- 1<y<b
Table 3: Classification of salts.
The protonation equilibrium of a n-valent base is described by the reactions:
i H+  La  HiLi-a (i = 1, 2, ..., n) (1)
and by the brutto stability constants:
cH Lia
i  i
(i = 0-n) (2)
ci   cLa 
H
The i are connected with the stepwise dissociation constants K ai , K bi and corre-
sponding logarithmic expressions pK ai , pK bi for acids and bases by:
(a) pKai  lgn1i  lgni ; (3)

i
(b) lgi =  pKan+1- j ;
j=1
(c) pKbi  pK W  lgi  lgi1 ;
i
(d) lgi = i  pK W -  pKb j .
j=1
This paper will use stoichiometric variables (containing concentrations instead of
activities) in abbreviated forms: pKi (macroscopic acid dissociation constant), pki
(microscopic acid dissociation constant), pKW (ion product of water). pcH stands for
the concentration based pcH = 10log(cH). Glass electrodes are calibrated by blank
titration in terms of pcH (or less favorably by buffers in paH; see below). The complex
situation of activities and activity based parameters will not be discussed here.
The molar fractions  i of protolytic species are derived from:
10(lgi ipcH)
i  (4)
n
10(lgk kpcH)
k 0
Each species HiLi-a present in the equilibrium contributes "ion-specific" NMR-para-
meters  i in an exchange reaction which is rapid on the NMR time scale. Effectively
only one signal is observed when monitoring 31P{1H}-NMR during the course of titra-
tions. An averaged chemical shift    appears following:

n
    ii (5)
i 0
A gradient called the deprotonation shift i [ppm] is given by:
i  i1  i (6)
This gradient defines the change of chemical shift per proton abstracted. Signs and
magnitudes of gradients are used to elucidate the deprotonation and protonation
pathways of multifunctional acids, bases and ligands.
As deduced above the dynamically averaged chemical shift <  > is a function of
pcH. Experimentally, the pcH of solutions may be varied by two complementary
methods:
a) titration with a strong univalent acid (e.g., HCl) or
b) titration with a strong univalent base (e.g., NaOH).
While the experiment directly provides the well-known titration curve pcH = f(V2), it is
more convenient to calculate the inverse function V2 = f(pcH) with suitable computer
programs.
The variables used for volumes [mL] and concentrations [mol L -1] are listed in Table
4.
Table 4
Components Volume Concentration Type
Titrand VL1 CL1 acid/base/salt of ligand L
VA1 CA1 univalent strong acid, e.g., HCl
VB1 CB1 univalent strong base, e.g., NaOH
Vl1 Cl1 ion buffer 1:1, e.g., NaCl
VW1 water for dilution
Titrator VB2 CB2 univalent strong base, e.g., NaOH
VA2 CA2 univalent strong acid, e.g., HCl
TABLE 4: Analytical parameters used to describe acid-base titrations.
Using these quantities and the following expressions:
A 10pcH 10pcHpKw (7)
n
B  (x  y)   (i  a)  i (8)
i0
  1 for Titrations with strong univalent acids (HCl, HNO3, HClO4) (9a)
   1 for Titrations with strong univalent bases (NaOH, TMAOH) (9b)
leads to the generalized charge balance:
T T T T T
CQ  0  A  CB1  C A1  CL B   CT (10)
where the actual concentrations are indicated by C T terms and  CT

T stands for the
generalized titrator:
Solving for VT, the volume of titrator, yields the generalized titration function:
 A (VL1 + VA1 + VB1 + VI1 + VW1 )  VB1 CB1  VA1 CA1  VL1 CL1 B
VT  (11)
  CT  A
The two types of titration are described as follows:
a) Titration with a strong univalent acid, e.g., HCl, HNO3 or HClO4:
Ha x  y XxLYy  ( x  y  b) HY  HabLYb  x XY   1 (12)
A (VL1 + VA1 + VB1 + VI1 + VW1 )  VB1CB1  VA1CA1  VL1CL1B

VA2  (13)
CA2  A
b) Titration with a strong univalent base, e.g., NaOH or tetramethylammonium
hydroxide (TMAOH):
Hax  y XxLYy  (a  x  y) XOH  XaL  y XY  (a  x  y) H2O   1 (14)
- A (VL1 + VA1 + VB1 + VI1 + VW1 ) - VB1CB1  VA1CA1  VL1CL1B

VB2  (15)
CB2  A
In addition it is useful to define a parameter for the degree of titration :
( VB1 CB1  VA1 C A1)  V2 (CB2  C A 2 )

  xy (16)
VL1 CL1
where  increases with increasing deprotonation.
2.2 Complex formation equilibria
Macroscopic Conditional Stability Constants
Complex formation involving the three components Ln-, H+ and Mm+ leading to nsp
species of the i-th type Mpi HqiLri are conveniently described (neglecting the ionic
charges) by:
piM  qiH  riL  MpiHqiLri (17)
cMp Hq Lr
βpiqiri  i i i
(18)
pi qi
cM  cH  cLri
and the fundamental equations:
nsp
C  c M   pi  βpiqiri  c Mpi  c Hqi  c Lri
T
M (19)
i=1
nsp
C  c L   ri  βpiqiri  c Mpi  c Hqi  c Lri
T
L (20)
i=1
K W nsp
C  C  cH 
T
A
T
B   qi  βpiqiri  c Mpi  c Hqi  c Lri (21)
c H i=1
Eqs. (19)-(21) are solved numerically in free component concentrations c M , cH , and
T
cL for given βpiqiri and actual total concentrations CM and CLT ; CTA  CB
T
stand for
the hypothetical concentrations of univalent acid and base combined in the titration
vessel.
Several methods and programs were developed and described in the literature to
simulate titration curves for complex formation equilibria [5-7]. Restricting r = 1, p = 1
and 2, or p = 1, r = 1 and 2, with arbitrary q, simple algebraic equations are derived
for fast simulations with GENCOM [3b]. Cumulative stability constants piqiri are
obtained by iterative least-square treatment of titration data [5-7]. A menu-driven PC-
iterator ITERAX [2i] was written in TURBO PASCAL based upon BEST [6] which
accepts the data output from automated titrations. Very recently WINPROT [2p] and
WINSCORE [2p] running under WINDOWS were added to this armory of iterative
instruments. If p = 0 and r = 1, then the protonation of the free ligands will be covered
by eqs. (1), (2), and (4) (cM = 0) and the corresponding programs. This simplified
situation allows for an explicit algebraic solution of the titration problem.
Each component and all the species Mpi Hqi Lri present in the equilibrium contribute
ion-specific NMR-parameters, e. g., Mp Hq Lr . If the exchange reactions are rapid

i i i
on the NMR time scale, only one characteristic signal for each sensor spin is obser-
ved (a singlet in the absence of coupling phenomena), e.g., when monitoring 31P{1H}-
NMR during the course of titrations using phosphorus compounds. On the other
hand, discrete NMR-signals may be observed for kinetically inert complexes.
If we monitor the ligand L by NMR, an averaged chemical shift  L  appears fol-
lowing:
nsp
 L   i ri Mp Hq Lr  L L (22)
i i i
i 1
By analogy, when monitoring (in diamagnetic complexes) the metal M, an averaged
chemical shift  M  will be observed according to:
nsp
 M   i pi Mp Hq Lr  MM (23)
i i i
i 1
Obviously the dynamic chemical shifts (and this is valid for resonance frequencies
and coupling constants in more complex spin systems as well) are governed by the
actual pcH in solution. For given system parameters (stability constants, dissociation
constants, volumes and concentrations of reagents) pcH is a function of the degree
of titration, henceforth the volume of titrator added.

2.3 Three different titration methods may be designed:
a) Titration with equidistant increments V for the titrator: see Figure 1.
b) Titration with equidistant increments in pcH: see Figure 2.
c) Titration with equidistant steps along the titration curve pcH=f(V): see Figure 3.
Method a) is realized in standard procedures using motor-driven automatic burettes.
Denser data points are produced close to the regions of lesser slope. This method is
good for determining pK but less accurate for determining concentrations.
The latter obstacle is overcome by method b), but more sophisticated software is
required to calculate the increments V and the experimental time demand is higher
than in a). This method is more accurate for determining concentrations and less
suitable for accurate pK data.
The advantages of both the previous methods can be achieved by curvature equi-
distant titration in method c), where data points are concentrated only in relevant
sections of the titration curve.
These facts are demonstrated for simulated titrations of 0.1 M CH3COOH vs. 0.1 M
NaOH.
Figure 1
[pH] Volume_equidistant Titration pH = f(V)
14.0
12.0
10.0
8.0
6.0
4.0
2.0
0.0
0.0 2.0 4.0 6.0 8.0 10.0 12.0 [V]
Figure 1: Case a: titration with equidistant increments V for the titrator. Example:
CH3COOH vs. NaOH.
Figure 2
[pH] pH_equidistant Titration pH = f(V)
14.0
12.0
10.0
8.0
6.0
4.0
2.0
0.0
0.0 2.0 4.0 6.0 8.0 10.0 12.0 [V]
Figure 2: Case b: titration with equidistant increments for pcH. Example: CH3COOH
vs. NaOH.
Figure 3
[pH] Curvature_equidistant Titration pH = f(V)
14.0
12.0
10.0
8.0
6.0
4.0
2.0
0.0
0.0 2.0 4.0 6.0 8.0 10.0 12.0 [V]
Figure 3: Case c: titration with equidistant steps along the titration curve pcH=f(V).
Example: CH3COOH vs. NaOH.
2.4 NMR-controlled titrations lead to ,- and pcH,-plots
graphical representations of 2D NMR type
NMR-controlled titrations are represented by two typical correlations: the ,-plots
and the pcH,-plots resembling the familiar 2D COSY spectra. For so-called first
order acids, where pKi+1 - pKi > 3 a simple situation holds: the first derivative of <>
with respect to pcH d   shows extrema (maxima or minima) while the second
dpcH
2
derivative d   2 is zero for pcH = pKi. Henceforth pKi and logi are easily obtained
dpcH
from pcH,-plots in such situations.

Second order cases with pKi+1 - pKi < 3 are more complex and require iterative treat-
ment of titration data by suitable methods and programs [2m, 2x, 2y].
At this stage it is useful to introduce a term  r  the reduced chemical shift as
defined by:
    min
 r   (24)
max  min
where min and max correspond to the minimum and maximum values of all ion spe-
cific chemical shifts i. This reduced chemical shift  r  conveniently spans the
range from 0 to 1.
For introduction some examples are shown in Figures 4 to 6:

Figure 4
Figure 4: Experimental ,-contour plots for 50.288 MHz 13

C{1H}-NMR-controlled
titration of CH3COOH vs. NaOH. upper: COOH/COO--group, lower: CH3-group.

Figure 5
[<rdp>] reduced dynamic NMR-Parameter and 1. and 2. derivative vs. pH
184.0
183.0
182.0
181.0
180.0
179.0
0.0 2.0 4.0 6.0 8.0 10.0 12.0 [pH]
27.0
26.0
25.0
24.0
23.0
22.0
0.0 2.0 4.0 6.0 8.0 10.0 12.0 [pH]
Figure 5: simulated pcH,-plot for CH3COOH vs. NaOH. upper: COOH/COO- group,
lower: CH3 group.

Figure 6
1.00
0.80
0.60
0.40
0.20
0.00
-0.20
-0.40
-0.60
0.0 2.0 4.0 6.0 8.0 10.0 12.0 [pH]
2
Figure 6: Reduced chemical shift, first and second derivatives d   and d   2 , cal-
dpcH dpcH
culated for CH3COOH vs. NaOH.
2.5 The reverse problem:
how to extract ion specific chemical shifts from NMR-controlled titrations
This situation may be dealt with at three levels of sophistication.
For protonation equilibria the dynamical chemical shift   j  for each pcH j is
given by:
n
(lgi ipcHj )
 HiLia 10
  j   i 0 (25)
n
(lgk k pcHj )
10
k 0
For given lgi (pKi) regression analysis will lead to the ion-specific data H Li  a
i
wanted.
A root mean square criterion is applied for the best fit following:
n exp
 (  j (calc )     j (exp) )2
j 1
rms  (26)
(nexp  npar )
It is imperative to obtain lgi (pKi) in high precision experiments prior to NMR-control-
led titrations. In principle this procedure will give the most accurate results but is
strictly valid if and only if the analytical conditions are consistent for both experimen-
tal steps. This procedure will work even for a smaller number of experiments (n exp)
from eq. (26).
In many realistic cases NMR will need higher concentrations than required by stan-
dard lgi (pKi) determinations. This is true for less sensitive nuclei, e. g. 13C, where
concentrations around 0.1 M are used. Of course, this is far from Debye-Hueckel
conditions in analytical chemistry used for pK determination. Two different routes will
overcome this dilemma:
pcH data monitored during NMR-controlled titration may be subjected to iterative
treatment in order to obtain the revised actual lgi (pKi) data (which will differ (in
general slightly) from the results obtained under high precision conditions). However,
more experiments (a larger nexp) will then be required. In principle, a simultaneous
iteration on H Lia and lgi is possible.

i
Even without monitoring pcH data, H Lia and lgi will be accessible since pcH is
i
the inverse function of eq (11). Obviously an even larger number of experiments
(nexp) is required for accuracy.
After some consideration, those concepts may be expanded to handle more compli-
cated acid/base problems, mixtures and the formation of metal complexes as well.
Some details are given below.
2.6 Accuracies of observed and calculated parameters
A suitable error analysis starts off with estimating errors of recorded pcH data. The
extended Nernstian equation
E(pcH)  Eo  G  pcH  jH  10pcH  jOH  10pcH-pK w (27)
is designed to cover the full range from 0  pH  pKW. where jH and jOH take into
account the non-linearity of acid and base errors due to the genuine properties of the
glass electrode. Within a linear range spanning at least 2  pH  12, where jH and jOH
can be neglected in eq. (27), the standard Nernstian equation holds, which is rear-
ranged to:
E j  Eo
pcH j  (28)
G
Then error propagation leads to:
2 2 2
 pcH j   pcH j   pcH j 
 (pcH j )  
2    2 (E j )  

   2 (Eo )    2
 G    (G) (29)
 E j  o 
   E   
where the errors in E0 and G are obtained from electrode calibration. It is noteworthy,
that (Ej) is smaller for well-buffered regions and larger close to the inflection points:
2
2  E 
 (E j )  2exp(E)     2exp( V ) (30)
 V  E E j
where  2exp (E)  0.1 mV is the intrinsic uncertainty of the EMF measurements,
determined in repeated experiments.  2exp ( V )  0.02 mL is the standard deviation of
volume increments (data are based upon manufacturer’s specification). The first
E
derivative of the titration curve is generated by numerical calculations on experi-
V
mental data.
The standard deviation) of the j-th pcH value follows from:

2
 E 
 2exp (E j )      2exp ( Vj )   2 (Eo )   2 (G) / G2
 V  E E j
(pcH j )  (31)
G
E
The accuracy of pcH values is dominated by , a measure for the actual buffer
V
capacity during titration.
Using the auxiliary term:
n
SUM   k  cHk (32)
k 0
molar fractions i for the general n basic case are given by:
i
i  c H
i  (33)
SUM
with the usual definition 0  1.
Error propagation yields:
2 2 2
   n 
    
 (i )   i   2 (i )    i   2 (k )  i   2 (cH )
2
(34)
 i  
k 1 k   cH 
k i
Then the standard deviations of i follow from:
 
2
 i c Hi  ln10
SUM   c   
   (35)
n n
i 2
  2 (log  i )    k c Hk   2 (log  k )  i    k c Hk (i  k )    2 (pcH)
2
( i )   i H
SUM2 k 1  k 1 
k i
It is useful to note for coming interpretations of practical results: the uncertainty
decreases with increasing value of the molar fraction.
The evaluation of ion-specific chemical shifts is a laborious and challenging task,
utilizing three different, home-written programs, the algorithms of which are briefly
described and compared below.
2.7 Calculation of ion specific chemical shifts
2.7.1 The simplest method using Gaussian elimination
A n-basic acid HnL having nsp = n+1 protolytic species HiL in solution, is titrated step-
wise in nexp experiments. An abbreviated notation will be used in forthcoming consid-
erations:
b j   j  (36)
a ji  Hi 1L (37)
for the j-th experiment

i 1  Hi1L (38)
for the unknown ion specific chemical shift of HiL.
A set of linear equations results:
n sp
a j1x1  a j2 x 2  a j3 x 3  ...  a j,n sp x n sp   a jk xk  b j j = 1, 2, ..., nexp (39)
k 1
represented in matrix notation:
Ax  b (40)
nexp = nsp measurements are needed for a unique solution achieved by:
x  (A 1  A)  x  A 1  b (41)
Thus ion specific chemical shifts, but no errors are available up to now.
2.7.2 Multivariate linear regression
In order to increase the accuracy of results the number of experiments n exp should
exceed that of unknown parameters nsp considerably. In addition, the experimental
b-vector contains random experimental error. The sum of squared deviations can be
minimized in the least squares method treatment:

2
n exp n exp  n sp
U   (bexp  bexp  a ·x   (b  A·x )T ·(b  A·x )  min!
j  b calc 2
j )    j  jk k  (42)
j 1 j 1  k 1 
Here each observed <j>-value contributes with identical weight to the sum of error
squares. This minimization yields in one step the best values of unknown parame-
ters:
x  ( AT ·A)1  ( AT ·b) (43)
as well as their estimated standard deviations and the root-mean-square value of the
fit. This method is used in our program AMPLSQ9 [2m] and in most other programs
reported in the literature as well [8].
2.7.3 Weighted multivariate linear regression
A more sophisticated treatment [2y] takes into account that the individual inaccuracy
(standard deviation) of the observed chemical shifts changes significantly during the
course of the titration. This standard deviation can be defined as a spectral halfwidth
given in chemical shift units [ppm]:
1/ 2
(  )  (44)
Spectrometer
Consequently, a simple weighting scheme is introduced for each experimental point j,

1
wj  (45)
2
 (  j )
and used in the weighted least square error function:
2

n exp n sp 
Uw   w j  b j   a jk·xk   (b  A·x )T ·W·(b  A·x )  min!
 exp
(46)
j 1  k 1 
 
Thus experiments bearing larger inaccuracies will exert a lesser influence upon the
total sum of error squares, leading effectively to more realistic estimates of the x pa-
rameters:
xˆ  (AT  W  A) 1  (AT  W  b) (47)
and their standard deviations.
2.7.4 Implicit regression
The most complicated statistical tool, which was applied for the first time by us [2y] to
this problem takes into account that the molar fractions (a ji) also contain specific
errors, as derived in equation (35). Strictly speaking, this fact hampers the application
of the classical least-squares methods described above, where error free “indepen-
dent“ variables are assumed. In the implicit regression the difference of independent
and dependent variables ceases, leading to the following, composite error function:
n exp n sp (a exp  a calc )2 (b exp  bcalc )2 

 
 
ji ji j j
Uimpl   (48)
 2 exp
 (a ji )  (b j ) 
2 exp
j 1  i 1 
Besides minimizing this function, the linear set of equations (39) must be fulfilled as
well. We solved this constrained extremum problem using the Deming algorithm [9],
the mathematical details will be discussed elsewhere [2y]. This method finally leads
to the maximum likelihood estimates for the parameters wanted, to corresponding
estimated standard deviations and to the global rms value as well.
The implicit regression method, with a more rigid error treatment leads to higher and
more realistic error estimates for the individual parameters.
For all the methods described above more accurate values for the ion specific chemi-
cal shifts are obtained for species with high abundance, while larger errors are con-
nected with the less populated species. It is essential to bear this in mind when plan-
ning NMR controlled titrations. The pcH window used should comprise all relevant
species wanted, a situation rarely achieved, when dealing pK values of acids having
very small (close to 0) or very large (close to pKw) pK values. Than the ion specific
data for HnL and Ln- species are ill defined. This is true for many compounds, e.g.,
polyfunctional aminophosphonates N(CH2PO3H2)3 NTMP and (CH2N(CH2PO3H2)2)2
EDTMP and related structures, which have attracted much technical and biological
interests.
3. Experimental realization of NMR-controlled titrations
3.1 Single sample NMR
In the starting phase of the method discussed here analytical studies were performed
to determine dissociation and stability constants. Then NMR samples are prepared
according to the titration states wanted. The Programs AUTO_T [2h] and EVOTIT
[2c] were developed to speed up efficiently the design and the preparation of NMR
samples by constant volume titrations, while GENTIT is used to simulate such titra-
tions [3b].
NMR spectra are recorded consecutively for such series of single samples. It is obvi-
ous that the number of samples in a series is restricted by experimental and econo-
mic considerations: spectrometer time, manpower and material costs.
This method is certainly laborious but may yield spectra of high resolution quality. But
the wide scope of studies planned to characterize biorelevant and technically impor-
tant compounds demanded for more efficient methods.
3.2 Titration inside a rotating NMR tube
The first step towards automation was described by P. Yesinowsky, R. J. Sunberg
and J. J. Benedict [10], who positioned the titrand, glass electrode and burette tip
inside a 20 mm Ø NMR tube rotating vertically in a wide bore magnet. Electrode and
burette connections were introduced overhead through the magnet. A spectral half-
width of 3 Hz was achieved for 31P{1H}. Concentrations for titrand and titrator (3 M
KOH) were high, so that the standard Debye-Hueckel equations were not applicable
for a more sophisticated evaluation of results. Later on K. Albert, E.-L. Dreher, H.
Straub and A. Rieker [11] reported about 13C-NMR-controlled electrochemical reac-
tions inside 10 mm Ø NMR tubes and in addition introduced bypass experiments.

3.3 Bypass on line NMR
General comments
At this stage it seemed imperative to develop a more convenient way of monitoring
titration equilibria by NMR methods. This became possible by the advent of PCs in
Chemistry. In addition relevant progress was made by K. Albert and his team [12-14]
in related hyphenated technologies. “in flow” and “stopped flow” conditions may be
used to monitor NMR in bypass for analytical or synthetical procedures. For example
“in flow” bypass NMR techniques were combined with HPLC and eventually devel-
oped to powerful analytical tools. Rapid injection methods under “stopped flow” by-
pass NMR-control were applied to study fast kinetics [15]. R. Neudert, E. Ströfer and
W. Bremser [16] described the experimental set up and the of application of on line
NMR-control of Wittig-Horner reactions using 31P{1H}- and 13C{1H}-NMR under stop-
ped flow conditions. The authors developed bypass techniques using a home made
insert of normal glass for standard probe heads based on 10 mm Ø tubes. A resolu-
tion of 3.5 Hz for 31P{1H}-NMR was achieved.
Developing the instrumental design for stopped flow NMR-controlled titrations
The general design for PC-guided, automated NMR-controlled titrations is governed
by three imperative demands: a minimum of titrand and a maximum for spectral reso-
lution and signal-to-noise (S/N) resp. are wanted. This concept was developed using
materials as shown in five sections:
1. Analytical components: titration vessel, combined glass electrode, motor burette,
pH-meter and pump circulating the liquid through the bypass between titration
vessel and NMR-insert.

2. In field components: NMR-insert, probe head, NMR spectrometer.
3. NMR-console.
4. PC connected to analytical components and to NMR spectrometer, printer, plot-
ter.
5. Specifically designed software to organize simulataneously analytical and NMR
procedures, store and evaluate data, list results and produce graphical represen-
tations.
A simplified flow diagram for bypass NMR-controlled titrations is given in Figure 7:
Figure 7
i i Ji
<> <> <J> lgi pKi
, pH, ,pH V,pH , pH,
PC
NMR Pump Titration
Figure 7: The basic concept for NMR-controlled titration
flow of titrand
flow of information
flow of results
Hardware and software for NMR-controlled titrations were developed and improved
with increasing sophistication in five steps:
Step1: Contributions from M. GRZONKA [1b-1d, 2c]
The first experimental set up, designed by M. Grzonka, is shown in Figure 8:
Figure 8
Figure 8: The first apparatus used for automated NMR-controlled titrations.

The electronic titration equipment and PC were placed at least 2 m from the magnet
to avoid interference with the PC monitor and the titration equipment.
At the heart of the apparatus is a low cost insert – shown in Figure 9 - for a standard
10 mm probe head of an AM 200 spectrometer:
Figure 9
Figure 9: the home made insert used for NMR-controlled titrations
Our first insert was a suitable modification of the Neudert-Ströfer-Bremser-design
mentioned above, made by A. Greiner [17] in Düsseldorf. To improve resolution and
line shape it was imperative to retain perfect cylindrical symmetry and NMR glass
quality. Two concentric NMR tubes (10 mm Ø and 5 mm Ø resp.) were therefore
fixed with in- and outlets to the 20 mm Ø skeleton, fitting a standard probe head
tunable to 1H, 13C, 19F, 31P. 200 MHz 1H NMR of TMS dissolved in C6D6, yielded a
halfwidth of 2.1 Hz, which is satisfactory for a non-spinning sample. The correspon-
ding signal for 81.01 MHz 31P-NMR of an aqueous solution of 1 M H3PO4 under stop-
ped flow conditions yielded a good S/N and a reasonable, non-Lorentzian line shape
with an agreeable spectral halfwidth of 8 Hz as shown in Figure 10 (top).
Figure 10
upper stop flow, insert Figure 11

non spinning
center stop flow, new flow probehead

non spinning
lower standard probe head

rotating 10 mm NMR tube
Figure 10: Line shapes achieved with three different probe heads. upper: stop flow
insert from Grzonka, see Figure 9 above. Center: stop flow probe head from M.
Rindlispacher (BRUKER), see below. lower: standard probe head, spinning 10 mm
NMR tube.
Remarks: An analogous design was scaled down for a NMR tube to an outer diame-
ter of 5 mm Ø and an inner tube of about 2 mm Ø in order to minimize the titrand re-
quired. A considerable loss of sensitivity was noted when applied in combination with
the standard 5 mm 1H-31P-dual probe head. Furthermore the formation of gas bub-
bles lead to distorted signals. This 5 mm insert is not suitable for high quality spectra,
the 10 mm Ø insert proved to be superior.

A specific titration vessel was designed [2c] holding the titrand, a burette tip, a com-
bined glass electrode, a magnetic stirrer, inlet and outlet from/to NMR insert/pump.
The vessel was double jacketed to maintain constant temperature using a thermostat
– if required.
A pump circulated the solution between titration vessel and insert using standard
rubber tubings (3 mm Ø, 4.2 m).
Characteristic volumes of this system are given in Table 5:
Table 5
Volume [ml]
Insert 33
Titration vessel, Maximum 122
Tubing 30
Recommended volumes:
Total Minimum, Titrand 130
Total Maximum, Titrand+Titrator 185
Titrator Maximum 55
Table 5: Characteristic distribution of volumes.
Homogenization of the titrand was achieved using simultaneously magnetic stirring
inside the titration vessel and the circulation pump. This was checked by repeated
paH-readings until an accuracy criterion paH was reached. The time required for
homogenization varied from 1-5 minutes, being longer when close to the equivalence
points of acid-base titrations. A model to calculate the time dependence of homoge-
nization was developed [2c].

Much effort (and funds) have been expended in the search for a suitable pump. After
initial attempts using a peristaltic pump, a magnetically driven pump for chemicals
(REICHELT) was introduced.
In most cases 0.01 M solutions suffice for 31P{1H}-NMR of standard phosphorus com-
pounds, consequently the minimum requirement for sample material in 130 ml of the
titrand corresponds to 1.3 milli moles.
The glass electrode may be calibrated – prior to starting NMR-controlled titrations - in
two different ways:
a) Calibration with standard buffers using the standard Nernst equation:
E  E0  G  paH (49)
Our first NMR-controlled titrations were based upon method a) using three standard
buffers of pH 4, pH 7 and pH 10 which are commercially available (e. g., SCHOTT).
b) Blank titration is a multi point calibration based upon the extended Nernst equa-
tion:
E  E0    pcH  jH  10pcH  jOH  10pcHpK w (50)
While method a) calibrates in activity based paH values, the blank titration leads to
pcH data. Since most analytical programs calculate with concentration based stability
constants (neglecting activity) it is more meaningful, but more laborious, to use con-
sistently method b). It is less consistent to use activity based paH data in concentra-
tion based algorithms giving rise to problems with so called “operative pH” data.
Since heavy water D2O is expensive, NMR-controlled titrations using hetero nuclear
sensors are performed with normal water H2O as solvent. As a convenient conse-
quence problems with deuterium isotope effects on protonation equilibria are avoided
and the standard Debye-Hueckel theory can be applied.
However prior to titration the magnetic field must be locked. Our initial experiments
used a solution of (CH3O)3P in C6D6 but it proved more advantageous to apply a 10%
solution of D2O in H2O. After locking the field, the shim unit is deactivated, and the
locking sample is replaced by the actual solution to be studied. A good magnet will
keep its magnetic field strength for a time sufficient to perform the NMR-controlled
titration with satisfactory accuracy. The time dependence of the magnetic field
strength was tested by recording resonances during 7.75 h in 15 min intervals. The
deviation from average was better then or equal to 0.0125 ppm. Locking is therefore
not essential in standard NMR-controlled titrations.
It is difficult to maintain adequate temperature control. Obviously the NMR spectra
and underlying parameters are obtained at probe head temperature while the analy-
tical parameters, e.g., paH or pcH, are recorded inside the titration vessel. A PT 1000
sensor may be used to monitor the temperature inside the vessel.
It should be remembered that larger deviations may by induced by variations in con-
centration, temperature, solvent, ion buffers, and impurities, as will subsequently be
discussed. It is essential to note all those parameters in the titration protocol.

The interaction of hardware and software is described briefly in Figure 11 below.
Details are accessible in [2c].
Figure 11
START START
SPECTROTIT MICROPROG
initialize IEEE read

calibrate electrode parameter file
setup
w ait for PC
experimental data
w rite current
measure FID
pH  etc.
issue
store FID to disk
new experiment
w ait for
perform FT
ASPECT 3000
start pump
add titrator pick the peaks
until new 
stirr until
N
pH = const final experiment ?
stop pump
Y
N list NMR
final experiment ?
parameters
STOP STOP
SPECTROTIT MICROPROG
Figure 11: First software for automated NMR-controlled titrations

The analytical part is organized by the program SPECTROTIT, written in TURBO
PASCAL, running under DOS 3.2 using a PC. The primeval PC chosen in 1987
was a COMMODORE PC 10. Any IBM compatible PC would do as well. A motor-
burette (T100/TA20, SCHOTT) and a pH-meter (CG 804, SCHOTT) are connected to
the PC via IEEE 488 (INES) and a special interface box (TI 143, SCHOTT). The
NMR spectrometer (AM 200, BRUKER) is controlled by an ASPECT 3000 worksta-
tion (BRUKER) utilizing a combination of home made micro and PASCAL programs.
PC and ASPECT 3000 are connected via RS232 [18].
In general 16 scans, each holding 2 K data points are sufficient to define a FID. N
titration points give rise to N FIDs. It is advisable to choose N to 32, 64, 128, thus
determining the duration of the titration experiment. For analytical reasons N=128 is
recommended. The FIDs are stored, subjected to FT and resulting spectra are com-
bined as a 2D NMR data matrix using one analytical variable (ml Titrator, degree of
titration ) as the pseudo-f1 variable, while f2 records the NMR variable (resonance
frequency , chemical shift ). Subsequently standard spectrometer software may be
applied to produce stacked plots or preferably contours plots produced on the inte-
grated spectrometer plotter. Results were termed ,-plots.
NMR-controlled titrations were performed successfully with the above mentioned
procedures to characterize polyfunctional carboxylic acids, aminoacids and phospha
analogues of such compounds as: a) phospha carboxylic acids HOOC-R-P*, CHP*2-
CH(COOH)-CH2COOH, b) phospha amino acids NH2-R-P°, NH2-R-P*, NR´(R-P*)2,
NH(R-P°*)(R-P*), N(R-P*)3, c) poly phospha acids P°-R´-P°, R-P*, P*-R´-P*, P°-R´-
P*, CR´P°-CHP°2, CHP°2-CHP°2, CR´P*-CHP*2, and CHP*2-CHP*2. Phosphinic
P°=P(R)(O)OH and phosphonic acids P*=P(O)(OH)2 were characterized. For some

cases macroscopic and microscopic dissociation processes and intramolecular rota-
tion were discussed.
At this stage the basic hardware and software developed in Düsseldorf was copied
by M. Peters and transferred to the research team of T. Kaden in Basel. After some
local modifications this design served to characterize phosphorylated cyclams [19].
Step 2: Contributions from H. Spiegl and H. W. Kropp [1d, 1e, 1h, 3c, 20]
Considerable progress was made to improve resolution and S/N and to reduce the
minimum volume of titrand after introducing a novel integrated flow-NMR probehead
designed by M. Rindlispacher (BRUKER). A 5 mm Ø glass cell was fixed inside a
standard 1H, 13C, 19F, 31P probe head and connected by tubings to the titration ves-
sel. A resolution of 1.4 Hz for H3PO4 was achieved as shown above in Figure 10
(centre) :
The global hardware plan is shown in Figure 12:

Figure 12
Figure 12: Hardware for NMR-controlled titrations, second generation [1d]. 1 NMR
spectrometer AM 200. 2 PC. 3 Motor burette. 4 pH-meter. 5 Thermostat (optional). 6
Water jacket. 7 Magnetic stirrer. 8 Pump. 9 Special probe head for NMR-controlled
titrations. 10 The vessel contains a burette tip, separate reference and glass elec-
trodes, PT sensor, in- and outlets for titrand solution and protecting Nitrogen gas,
magnetic stirrer.
A revised software NMR_T was developed, adding a convenient menu driven user
interface to a combination of SPECTROTIT and MINI_T. A PC 386 was used to ope-
rate NMR_T.
This design was used successfully to characterize a variety of compounds using nu-
clei 1H, 13C, 19F, 31P. First attempts at Li- and 113Cd-NMR were made. Emphasis was
laid upon the protolytic behavior of biologically and technologically relevant amino-
phosphonic acids (including phospha complexones), aminophosphinic acids, amino
phosphine oxides, phosphono carboxylic and phosphino carboxylic acids.
A fine piece of research was presented by H. W. Kropp [3c] for 1-phenyl-ethane-
1,2,2-tris-(P-methyl-phosphinic acid), which gave rise to dynamic NMR spectra.
An interesting and much hoped for experiment was made by Spiegl and Kropp to
utilize the modern HPLC probe heads developed by Albert and BRUKER for NMR-
controlled titrations under in flow conditions. Low S/N and insufficient line shape re-
sulted. Consequentially this line of research was discontinued up to now.
Step 3: Contributions from J. Ollig [1d, 1e, 1g-1i, 1m, 2m]
Four different versions for the titration vessel I-IV were designed in order to reduce
the total volume of titrand and henceforth the amount of (costly) material to be inves-
tigated. The properties of those vessels are listed in Table 6:

Table 6
Volumes [ml] Total Titration Pump Tubing Probe- Tubing

In system Volume vessel to insert head to vessel
I 85 45 13 (RP) 11 5 11
II 55 19.5 13 (RP) 11 5 6.5
III 40 22 0 (PP) 6.5 5 6.5
IV 23-25 5-7 0 (PP) 6.5 5 6.5
Table 6: Volumes [ml] of single units in titration system. RP: magnet driven rotating
pump, flow rate 230 ml/min (system I) and 210 ml/min (system II). PP: Peristaltic
pump, flow rate 15 ml/min (systems III and IV).
The design of vessel III is shown in Figure 13:
Figure 13
2
3
6 1
4
Figure 13: Home made titration vessel III. 1 Combined glass electrode (N62,
SCHOTT). 2 Pt-thermometer (W5780, SCHOTT). 3 Burette tip (SCHOTT). 4 Inlet
titrand from magnet. 5 inlet and outlet Nitrogen. 6 Outlet titrand to pump.
In several cases the combined glass electrode was calibrated using two buffers only:
pH 4.01 and pH 6.87. (For more critical comments see above). The temperature was
stabilized via air-conditioning which is fixed to 21.60.5 °C. The internal temperature
is monitored with a Pt resistor and data are stored in the protocol parallel to titration.
Recommended concentrations for titrands were found as shown in Table 7:
Table 7
Nucleus Concentration in titrand

1
H 0.25-0.0100
13
C 0.50-0.0050
19
F 0.01-0.0005
31
P 0.01-0.0010
113
Cd 0.25-0.1000
Table 7: Recommended titrand concentrations for sensor nuclei in NMR-controlled
titrations.
The time dependency of the magnet field stability was checked using the methane
phosphonate anion CH3PO32-. A drift as small as 0.021 Hz in 13.3 h was observed.
This time effect is considerably smaller than temporary changes, made in the local
arrangement of the spectrometer room, will produce.
In addition the methane phosphonate dianion served to study the dilution effect dur-
ing titration. For 81.01 MHz 31P{1H}-NMR the resonance frequency of CH3PO32 may
be described by:
P  1689.52  79.6  c CH PO2 (correlation coefficient 0.929) (51)

3 3
with an effective shift of about 2 Hz for a change from c = 0.0025 M to c = 0.03 M.
Again the concentration dependent error is small (for anions) in comparison to the
protolytic effects observed by NMR-controlled titrations.
Additional progress was made allowing for two different NMR spectra to be recorded
31
subsequently for each titration point, e. g. P- and 31P{1H}-NMR.
The improved hardware is shown in Figure 14:

Figure 14
Figure 14: Hardware for 200 MHz NMR-controlled titrations: Model 1995.
1 NMR-spectrometer AM 200 SY (BRUKER). 2 Master-PC (IBM-compatible PC). 3
motor burette (T200, SCHOTT). 4 Sensor electrode (N62, SCHOTT) and potentiome-
ter (CG 841, SCHOTT). 5 Thermostat (optional). 6 Titration vessel, (thermo control-
led, protective gas: N2 or Ar) (Titration vessel type II, see Table 6). 7 Magnetic stirrer
(TM120, SCHOTT). 8 PC-guided pump (REICHELT CHEMIE TECHNIK, Type No.
90513). 9 Flow-NMR-probe head (5 mm QNP, for 1H, 13C, 19F and 31P, BRUKER).
All analytical equipment, pump and PC were assembled on a home made non-mag-
netic wagon (wood, bronze screws), easily to be moved for setup and shutdown of
NMR-controlled titrations.
The revised software of NMR_T and SPECTRO programs [1g] is shown in Figure 15
while useful explicit details for handling the NMR-controlled titrand are given [1f].
Figure 15
Figure 15: Software for 200 MHz NMR-controlled titrations: Model 1995 [1g]
Supported by this advanced equipment, we were able inspect even smaller amounts
of biorelevant materials obtained from micro synthesis. The formation of metal com-
plexes, mainly Ca- and Zn-complexes from phosphorus containing ligands was moni-
tored by 31P{1H}-NMR. Within this context automated NMR-controlled titrations using

1
H-, 1H{31P}- and 13C{1H}-NMR were performed for the first time and the following
classes of compounds were characterized successfully:
Mono-, di- and tricarboxylic acids (e. g. the biorelevant CMOS), aminoacids, pep-
tides, phosphate esters of hydroxy carboxylic acids (e.g., phospho citric acid), -acyl
phosphonates, phosphono carboxylic acids (e.g., the antiviral FOSCARNET, haloge-
nated phosphono acetic acids, technical phosphono polycarboxylates), phosphino
carboxylic acids and phosphono phosphine oxides. Attention was paid to macro and
micro dissociation, e.g., from phenylephrine, aminophosphinic and phosphinocar-
boxylic acids. 19F-NMR was used to characterize fluorinated phospha analogues of
amino acids and carboxylic acids. Compound specific dissociation constants in multi
component mixtures, which are not accessible by conventional potentiometry based
solely upon pcH observations, were determined in a most elegant way.
Step 4: Contributions from S. Hermens [1m, 2x]
Major progress in spectrometer design was made by BRUKER leading to the modern
AVANCE series. This required complete overhaul rewriting the software in C to be
used on the INDY workstation attached to the NMR spectrometer. The communica-
tion protocol was modified to match the new AVANCE DRX series.
Figure 16
Figure 16: Hard ware for 200 MHz NMR-controlled titrations. Model 1999.
1 INDY workstation, spectrometer AVANCE DRX 200 (BRUKER). 2 PC 486, 16 MB
RAM. 3 Motor burette (T200, SCHOTT). 4 pH-Meter ( CG841, SCHOTT). 5 Titration
vessel, glass electrode (N 6280, SCHOTT), temperature sensor (PT1000, SCHOTT).
6 Magnetic stirrer (SCHOTT). 7 Pump (Type No. 90513, REICHELT CHEMIE
TECHNIK). 8 Probehead for NMR-controlled titrations (BRUKER).

Figure 17
PC-Program Spectrometer
Borland Pascal program "C"
START START
NMR_T sf_NMR
initialize analytical read name of

parameters parameter file
calibrate electrode wait for the PC
setup exp.
read parameter-file
data
YES
NO
stirr until pH = const. measure and store FID
stop the pump to disk
write current pH, further

etc. NMR-exp.?
NO
start new NMR
experiment last
NMR-exp.?
wait for signal from

YES
spectrometer
STOP
sf_NMR
last
exp.?
YES
NO
start pump; add liquid STOP
to reach new degree of NMR_T
titration
Figure 17: Software for NMR-controlled titrations: Program NMR_T, model 1999 for
BRUKER-NMR-spectrometer AVANCE DRX.

The experimental conditions in NMR-controlled titrations are similar to those in po-
tentiometry, the temperature is kept at 22 ± 1 °C while the ligand concentrations are
around 0.005 m for 31P{1H}-NMR.
Supported by this hardware and software some advanced problems of polyfunctional
organophosphorus ligands were studied. It was shown that alkali metal cations form
complexes with a series of technologically relevant, polyfunctional compounds like
N(CH2P(O)(OH)2)3 (NTMP), geminal bisphosphonates RR´C[(P(O)(OH)2]2 (e.g.,
HEDP) [2x].
Step 5: Contributions from S. Augner and C. Uhlemann [2s, 2v]
An important breakthrough towards miniaturization, increased sensitivity by high field
spectroscopy was achieved recently by Augner and Uhlemann. This team, supported
by S. Hermens, succeeded to install NMR-controlled titrations on the AVANCE DRX
500 spectrometer.
Several technological obstacles were overcome successfully using the novel TXO -
HPLC-probe head from BRUKER (see Figure 18) tunable to 13C-, 19F- or 31P-NMR.
Figure 18
Figure 18: essential parts of the novel TXO-HPLC-probe head from BRUKER
The active cell volumes in field are reduced to the following data: in cell region 340
l; in coil region 60 l; out side coil region 80 l; tubing between probehead, titration
vessel and pump:200 l. Capillaries in cell Ø 0.5 mm; tubing 1.0 mm between capilla-
ries and inlet/outlet fittings (metal, BRUKER), PP tubings Ø 0.8 mm (i.d.) from probe-
head, to titration vessel and pump.
Much time and funds were used to find a special pump (FM 1.30, KNF FLODOS),
special fittings made from PP (UNF ¼´-28, VALCO EUROPE) to match the condi-
tions of 500 MHz NMR controlled titrations using a HPLC probe head.
Homogenization of the titrand is achieved on two levels: a) using the magnetic stir-
ring inside the titration vessel and simultaneously b) by pumping, circulating the ti-
trand along the tubings, the probehead and the titration vessel. The total process can
be handled as a dilution and mixing process. A model for this process was designed
as shown below in Figure 19:
Figure 19
Figure 19: Model used to simulate the mixing process.

The following variables are used as shown in Table 8:
Table 8
VV0 Starting volume of titrand in titration vessel

VT total volume of titrand in tubing, pump, and probehead
CV0 starting concentration of titrand in titration vessel, in tubing, in pump, and in
probehead
VD volume of titrator added to titration vessel
VV1 volume of titrand in titration vessel after addition of VD
CV1 concentration of titrand in titration vessel after addition of VD
ninc number of increments in VT for pump cycle
VP,i i-th volume increment in pump cycle (VT/nincr)
CP,i concentration of titrand in i-th unit volume increment
Npc total number of pump increments applied for mixing
Table 8: Variables used to model the mixing process.
In the starting phase a homogeneous solution with a ligand concentration of CV0 is
evenly distributed in the titration vessel, the tubing, the probehead and the pump, all
of the ninc volume increments will have CP,i = CV0. Addition of VD ml of the titrator to
the titrator vessel gives rise to an increase of volume VV1 and – in absence of pum-
ping, but presence of magnetic stirring - a decrease in concentration CV1:
VV1  VV0  VD (52)
V  VD
C V1  V 0 (53)
VV1
Now the pumping cycle is started with one increment VP leading to:
VV1,i  C V1,i  VP  C V1,i  VP  C V1,inc

C V1,i 1  (54)
VV1
All increments VP,i are shifted by one unit in flow direction. This cycle is repeated to
convergence. An example is shown in Figure 20:
Figure 20
[conc] concentration vs. number of pump cycles
0.9950
0.9940
0.9930
0.9920
0.9910
0.9900
0.9890
0.9880
0.9870
0 50 100 150 200 250 300 350 [npc]
Figure 20: Simulating the mixing process with VV0 20 ml; CV0 1 mol/liter; VP 0.25 ml;
Vtot 18 ml; ntot 72; npump 400.
For the parameters chosen here a mixing time equivalent to about 250 pumping
steps is sufficient. In practice the titrand is homogeneous after 90 s. Taking into ac-
count potential variations in viscosity during titration, a safety value of 300 s for all
titration experiments was chosen.
The pump FM 1.30 of KNF FLODOS does not produce a continues flow, it pulses
(see Figure 21) with an integrated flow rate of 15 ml per min. The KNF FLODOS
pump was superior for NMR-controlled titrations [21].
Figure 21
Figure 21: Recording the pressure as a function of time. The KNF FLODOS pump
does not produce a constant but a pulsing pressure. The internal gear box is set to
50%.
We wish to point out the importance of using the KNF FLODOS pump properly: After
each experiment the pump is washed with distilled water, opened, the membrane
part is dried (Kleenex paper), closed again and then is ready for the next experiment.
The residual amount of water inside the pump is estimated to be about 3 mL which
will acts as an unaccounted dilute without the precautions described above.

The field lock and field homogenization steps were modified: Prior to actual measure-
ments D2O is filled into the probehead via syringe, the field is locked and shimmed,
shim parameters stored, the shim unit deactivated and finally the probehead dried in
place using nitrogen gas.
The combined glass electrode was calibrated by blank titrations (see above), now
providing the pcH data needed for analytical procedures.
Basic investigations showed, that the stability of the non-locked 500 MHz Magnet is
superb, having a variation of less than 0.01 ppm within 22 h only. The standard de-
viation was 0.0022 ppm using 8196 data points, 202,46MHz 31P{1H}-NMR and 1-hy-
droxy-1,1-bisphosphonic acid HEDP as a test compound and a TBI probe head. This
astounding stability of the 500 MHz magnet is better by a factor of 2 to 3 observed for
the 200 MHz magnets from M. Grzonka and J. Ollig [2c, 2m].
Figure 22
20,767
20,766
20,765
20,764
20,763
 [ppm]
20,762
20,761
20,760
20,759
20,758
20,757
0 120 240 360 480 600 720 840 960 1080 1200 1320
t [min]
Figure 22: Time dependent 202,46 MHz 31P{1H}-NMR of 0.00016 M 1-hydroxy-1,1-
bisphosphonic acid (HEDP) in H2O using a TBI probe.

Results using the TXO-HPLC probe head, 0.00033 M HEDP in D2O and 202.46 MHz
31
P{1H}-NMR were satisfactory as well: a variation of less than 0.018 ppm within 16 h
was achieved with a standard deviation of 0.00493 ppm for 16384 data points. In ad-
dition the 500.13 MHz 1H resonances for HOD and the CH3-group of HEDP were
monitored as well.
Figure 23
0,0060
0,0040
0,0020
0,0000
 [ppm]
-0,0020
-0,0040
-0,0060
-0,0080
-0,0100
-0,0120
0 100 200 300 400 500 600 700 800 900 1000
t [min]
Figure 23: Time dependent 202,46 MHz 31P{1H}- and 500,13 MHz 1H-NMR of
0.00033 M HEDP in D2O.  31P{1H}-NMR of HEDP.  1H-NMR of CH3 –group from
HEDP. 1H-NMR of HOD.
The 1H- or the 2D-resonances of H2O, HOD, D2O are sensitive towards pH. Conse-
quently NMR-controlled titrations under locked conditions will use a magnetic field
dependent on the actual state of titration, which might give rise to systematic errors
with external referencing. As derived from observations described above NMR-con-
trolled titrations under non-locked conditions practically use an effectively stationary
field and referencing is good enough within 0.01 ppm.

In addition the chemical shift H(HOD) depends on the actual temperature inside the
sample. An equation
H  5.060  0.0122  t  0.0000211 t 2 (55)
established [22] which is represented in Figure 24 below:
Figure 24

5.00
4.80
4.60
4.40
4.20
4.00
-10 0 10 20 30 40 50 60 70 80 90 [t]
Figure 24: H  5.060  0.0122  t  0.0000211 t 2 : chemical shift H(HOD) vs. tem-
perature t [°C].
The in-field sample temperature was determined for a LC-1H/19F and a LC-TXO
probe head using 99.8% deuterated methanol (without addition of HCl). The tempe-
rature was calculated from the observed chemical-shift difference of the residual OH
and the CHD2 protons  = H(OH) – H(CHD2) [23] following eq. (56):
T  398.7  0.1347    0.0006109 2 (56)
In absence of pulse irradiation the in-field sample temperature for the LC-1H/19F
19
probe corresponds to 305.57 K which is equivalent to 32.4 °C. Doing a typical F
NMR experiment (96 scans) the in-field sample temperature increases up to 305.6 K
equivalent to 32.5°C. This leads to the conclusion that the increase in temperature
caused by the 19F pulse program is less than the accuracy of this method which was
19
given to 0.69 K [23]. Even decoupling the protons by cpd F{1H}-NMR with 16 scans
does not have an important effect on the in-field sample temperature. This increase
of 0.13 K is negligible as well. It is thus justified to assume, that the in-field tempera-
ture is virtually constant during the measurements. These facts are summed up in
Table 9:
Table 9
at the after 19F after 19F cpd 10 min later

beginning 96 scans 16 scans without pulses
T [K] 305.57 305.63 305.70 305.64
T [C] 32.42 32.48 32.55 32.49
T [K] 0.06 0.13 0.07
Table 9: Changes in temperature inside the cell as caused by typical pulse programs
used for 19F studies.
Similar results, shown in Table 10, are obtained for the LC-TXO probe head used for
1
H-, 13C- and 31P-NMR studies.
Table 10
31 1 31
start P H P{1H}-cpd 10 min later
256 scans 256 scans 192 scans without pulses
T [K] 307.84 307.94 307.99 308.57 308.15
t [C] 34.69 34.79 34.84 35.42 35.0
T [K] 0.10 0.15 0.73 0.31
Table 10: Changes in temperature inside the cell caused by irradiation of standard
pulse sequences used for 1H and 31P studies.
Taking into consideration the field distribution shown in Figure 25:
Figure 25
Figure 25: field distribution of magnet from a BRUKER AVANCE DRX 500.
The electronic titration equipment and PC was placed at least 2.5 M off the DRX 500
magnet. This avoids interactions with the technical equipment and PC.
This most advanced equipment is shown in Figure 26 below:
3
6
2
7
4
5
8
Figure 26: Hardware for 500 MHz NMR-controlled titrations. Model 2001.
1 INDY workstation, spectrometer AVANCE DRX 500 (BRUKER). 2 PC 486, 16 MB

RAM. 3 Motor burette T200 (SCHOTT). 4 pH-Meter CG841 (SCHOTT). 5 KNF Micro
membrane liquid pump FM 1.30 (KNF FLODOS). 12 ml/min, 4.3 bar. 15 ml/min, 4.9
bar. Dead volume in pump: ca. 7 ml. 6 Titration vessel, glass electrode blueline 11,
PT1000 sensor (all: SCHOTT). 7 Magnetic stirrer (SCHOTT). 8 Magnet holding TXO-
LC-probe head (BRUKER) for H1, C13 and P31, but an LC-head 1H/19F - 19F/1H
switchable (BRUKER) for F19.
All FIDs are processed using standardized conditions under the Serial Processing
13 19 31
function of WIN-NMR. This procedure holds conveniently for C, F and P-NMR
spectra, but 1H-spectra require manual corrections to overcome distortions due to
strong water signals. The Serial Processing function of WIN-NMR also provides the
deconvolution of NMR spectra leading to individual data for resonance frequencies,
(chemical shifts), intensities and spectral half widths. A program SF-SIMULATION,
written in VISUAL BASIC® by I. Reimann [2p], is able to read out those data and
form a systematic list of titration results. After export to EXCEL® further mathematical
evaluation and graphical representation of the final results are performed using Mul-
tiple NMR Graphics designed by C. Pfaff [2u] (see below).
A comparison between the 200 MHz and the 500 MHz installation was made using
HEDP. Results are shown in Table 11. For details see below in section Examples.
Table 11
DRX 200 DRX 500

31
P{1H}-NMR, 31P-Frequency [MHz] 81,01 202,46
Volume of Titrand [ml] 80 25
sample HEDP [mg] 84.3 33.3
sample HEDP [mmol] 0.409 0.160
Total time for 64 experiments [h] 8 16
Waiting time for homogenization [s] 15 300
Waiting time after FID [s] 13 480
Total time needed for set up [h] 4 16
Table 11: Comparison of 200 MHz and the 500 MHz installation using 1-hydroxy-
ethane-1,1-bis-phosphonic acid (HEDP)

Using this experimental and computational means we were able to characterize by
500.13 MHz 1H, 125.72 MHz 13C-, 13C{1H}-, 470.39 MHz 19F-, 202.46 MHz 31P-,
31
P{1H}-NMR a series of biorelevant products such as: complexing agents used in
calcium related disorders (e. g. geminal bisphosphonates, bisphosphinates, acyl-
phosphonates) and fluorinated amino acids as will be shown below.
Software development supporting NMR-controlled titrations
1. Analytical and NMR-programs
The present state of NMR-controlled titrations is supported by a variety of studies
concerning soft ware and hard ware development in the Düsseldorf group spanning a
period from 1986 to 2001. A condensed list of results is shown below in Table 12.
In addition we wish to point at three programs described in the open literature, which
are available from the authors. HYPNMR [24] was designed for applications involving
single sample NMR studies and applied for several interesting studies. HYPNMR
determines ion specific NMR parameters for several nuclei simultaneously and stabi-
lity constants. But HYPNMR can not handle larger data series e.g., 128 spectra in
our NMR controlled titrations, which is recommended for more accurate studies, as
discussed above. OPIUM [25], made by the Lukes group and LAKE [26] from the
Holmström team might be used in a similar way like HYPNMR.

Table 12
PROGRAM Authors Applications Type
AUTO_T J. Peters Titration EXP

MINI_T A. Bier Titration EXP
WIN_CAL I. Reimann Blanktitration ENE
AUTOKAL A. Bier Blanktitration ENE
GENTIT G. Hägele Simulation ABE, NMRCT
A. Hupperts
GENCOM G. Hägele Simulation CFE
A. Hupperts
MICROSIM G. Hägele Simulation ABE, NMRCT
K. Wuscher
SIMENU A. Schiefke Simulation CFE
NACOM G. Hägele Simulation CFE, NMRCT
C. Tilmann
MV3 G. Hägele Simulation NMRCT
PHSTAT G. Hägele Simulation CFE
C. Tilmann
SF_NMR_SIM G. Hägele Simulation ABE, NMRCT
MINILA U. Weber Simulation NMR
LAOTIT G. Hägele Simulation NMRCT
Z. Szakacs
ITERAX A. Bier Iteration ABE, CFE
WINPROT I. Reimann Iteration ABE, NMRCT
WINSCORE I. Reimann Iteration ABE, CFE, NMRCT
AMPLSQ G. Hägele Iteration NMRCT
J. Ollig
S. Hermens
SON4 H.W. Kropp Iteration NMRCT
J. Ollig
MULTINMRPK Z. Szakacs Iteration NMRCT
IMPLICIT Z. Szakacs Iteration NMRCT
NMRMICROPK Z. Szakacs Iteration ABE, NMRCT
CANPOD S. Hermens Iteration ABE, CFE, NMRCT
Table 12: Soft ware development for analytical Chemistry, NMR-controlled titration,
and NMR spectroscopy. Type: EXP organizing potentiometric titration; ABE acid
base equilibria; CFE complex formation equilibria; NMRCT NMR controlled titration;
NMR NMR spectral simulation. For detailed information please contact G. Hägele.
2. Graphical representation of NMR-controlled titration spectra
Data obtained from NMR-controlled titrations can be visualized by several methods.
One type of representation is given as a stacked plot diagram, with features well
known from 2D NMR spectroscopy. A series of individual traces is plotted with con-
stant increments for vertical and horizontal shifts. By erasing parts of lines which lie
under those in front, white washed spectra are produced, thus imitating a realistic
view upon a three dimensional surface [2u].
Such z = f(x,y) graphics can be obtained with commercial software, e.g., with 2D-
WinNMR, XWinNMR from BRUKER or EXCEL from MICROSOFT, but only for
those cases, where axes with equidistant variables x and y exist. This is true for V- or
τ-equidistant titrations. But the storage demand of such graphics is high. In addition
EXCEL, 2D-WinNMR and XWinNMR do not provide a routine access for data from
NMR-controlled titrations. Another problem occurs when handling non-equidistant
data for x- or y-axes, then distorted representations result. 2D-WinNMR and Xwin-
NMR are not able to accept variable titles and values for the y-axis, as is required for
analytical parameters V, τ or pcH.
An alternative to stacked plots are true representations of functional surfaces, espe-
cially those projecting the three dimensional z = f(x,y) function to the two dimensional
x,y-plane at given levels of z. Contour plots can be generated with the software men-
tioned above, but identical restrictions prevent an easy and general access of data
and graphical output.

An interesting approach was made by C. Tillmann [2o] to produce graphical repre-
sentations of spectra from simulated NMR-controlled titrations. The program SFNMR-
PLOT used the graphical tool OLECTRACHART for visualization. But again: the
axes in the x,y-plane have to be equidistant.
After having tested further commercial software we decided to develop a tailor-made
new software matching the specific demands, leading to an optimal visualization of
spectra obtained from NMR-controlled titrations. „Multiple NMR Graphics”, designed
by C. Pfaff [2u], is running under Microsoft Windows 95 or higher and Microsoft
Windows NT 4.0 or higher.
Multiple NMR Graphics produces stacked plots with or without white wash and cor-
responding contour plots. Equidistant and non-equidistant axes allow to use V, τ or
pcH as variables. The resulting graphics are displayed on the screen and can be
copied to other applications or stored as an enhanced metafile.
The metafiles contain the graphical representation as graphical objects (e.g., lines,
circles, polygons). Those metafiles can be scaled without restrictions, in contrast to
conventional bitmaps, containing the graphical object as pixels. In general, the stor-
age demand of metafiles is low.
A further reduction in file size is achieved by testing all data points if they are really
needed. Points which do not contribute significant information to the graphic will be
neglected [2u].
Consequently Multiple NMR Graphics is able to handle larger data sets from NMR-
controlled titrations while other conventional programs are not able to do so. The
program is also faster than other applications e.g., EXCELL; 2DWin-NMR.
The usage of Multiple NMR Graphics is straight forward and intuitive via a functio-
nal user interface, keeping strictly in line with conventions known from standard soft-
ware, e.g., WORD, EXCEL, and the operating system.
Multiple NMR Graphics is able to load NMR-spectra directly from the BRUKER-
specific file formats used in WinNMR and XWinNMR. In addition it accepts files in a
special text format provided from other sources, e.g., home made simulators. The
analytical data (V, τ, pcH) can be added via text files. All data imported may be saved
into a specific storage file format typical for Multiple NMR Graphics. Those files can
be accessed faster for reloading in subsequent visualizations.
All generated graphic files, stored as enhanced meta files, are accessible with the
usual applications e.g., EXCELL, WORD, POWER POINT. A copy command is also
available for fast integration of graphics into other applications.
Multiple NMR Graphics is a most versatile and convenient tool to produce graphics
of NMR-controlled titrations. Some examples are shown in Figures 27 below.

Figure 27
-2
3
-1
4
0
5
Titrationsgrad
1 6
pH
7
2
8
3
9
4 10
5 11
19,0 18,8 18,6 18,4 18,2 19,0 18,8 18,6 18,4 18,2
ppm ppm
-2
3
-1
4
0
5
Titrationsgrad
1 6
pH
7
2
8
3
9
4 10
5 11
19,0 18,8 18,6 18,4 18,2 19,0 18,8 18,6 18,4 18,2
ppm ppm
-2
3
-1
4
0
5
Titrationsgrad
1 6
pH
2 7
8
3
9
4 10
5 11
19,0 18,8 18,6 18,4 18,2 19,0 18,8 18,6 18,4 18,2
ppm ppm
Figure 27: Examples for Multiple NMR Graphics. Left: P- diagrams. Right: P-pH
diagrams. Upper: contour plots black and white. Middle: contour plots color. Lower:
stacked plots black and white. 81 MHz 31P{1H}-NMR controlled retro titration of 1-hy-
droxi-3-amino-propane-1,1-bis-phosphonicacid, NH2-CH2-CH2-CH(OH)(PO3H2)2,
PAMIDRONATE [2u].
4.1 Univalent acids HL
Consider a univalent acid of concentration CL which is directly titrated with a univalent
base of concentration CB, than three simplified relations can be deduced from eqs.
(57) to (59) describing the degree of titration  and the dynamic chemical shift
   as given below:
10-pK
- 10-pcH  10pcHpK w  CL
VB  VL  10-pK  10-pcH (57)
CB  10-pcH  10pcHpK w
10-pK
- 10-pcH  10pcHpK w  CL

CB
 10-pK  10-pcH (58)
CL CB  10-pcH  10pcHpK w
10-pK
    HL  (L  HL )  (59)
10-pK  10-pcH
leading to the typical pH,V-, pH,-, ,-, and pH,-plots as will be shown in a few ex-
amples. To plan the experimental part it proved advantageous to use a pcH,pcT-
diagram given in Figure 28:

Figure 28
14
12
8
10
8
2 5
pcH
6
8 5
4
2
2
0
0 1 2 3 4 5
pcT
Figure 28: pcH,pcT-diagram. Examples calculated for pK values of 2, 5 and 8.
The starting pcH for a solution of HL with a concentration of c T  10pcT is given by:
pcH  0.5  (pK  pcT) (60)
An analogous solution of the corresponding sodium salt NaL will follow from:
pcH  0.5  (pK  pcH  pcT) (61)
The observable window has a width of pcH  0.5 pK w  pcT units centered around
pcHc  0.5 (0.5 pK w  pK) . NMR controlled titrations outside this range do not
contribute additional information on the HL/L system.

Case 1: CH3COOH
13
C{1H}-NMR-controlled titration may be used to investigate carboxylic acids. The
most simple case is shown with acetic acid. Corresponding ion specific chemical
shifts are shown in Table 13:
Table 13
HL L- i
C (C1) 179.17 183.80 4.63
C (C2) 22.83 25.68 2.85
Table 13: Ion specific chemical shifts C [ppm] and gradients i [ppm] of CH3COOH.
Deprotonation induces a down field shift for the carboxylic unit and the methyl group
d  r  d2  r 
as well. The pH,-plot and the corresponding derivatives and directly
dpcH dpcH2
indicate the pK value having minimum- and zero-values at pH = pK. Corresponding
graphs are shown in Figures 29 a – d (see below), which were simulated using the
program GENTIT and the analytical data: pKw 13.78; pKa 4.75; Volume of titrand:
85.00 ml; concentration of titrand: 0.01000 m Hac; concentration of titrator: 0.97130
m NaOH.
Figures 29 a and b
27.0
26.0
25.0
24.0
23.0
22.0
0.0 2.0 4.0 6.0 8.0 10.0 12.0 [pH]

a
184.0
183.0
182.0
181.0
180.0
179.0
0.0 2.0 4.0 6.0 8.0 10.0 12.0 [pH]

b
Figure 29: Simulated pcH,-plots from 13C{1H}-NMR-controlled titration of acetic acid
vs. NaOH. a: CH3-group. HL 22.83, L 25.68. b: COOH/COO--group. HL 179.17, L
183.80. y-axis C [ppm].

Figures 29 c and d
1.00 1
0.80
0.60
0.40
2
0.20
0.00
-0.20
3
-0.40
-0.60
0.0 2.0 4.0 6.0 8.0 10.0 12.0 [pH]
c
1.00 L
0.80
0.60
0.40
0.20
0.00 HL
0.0 2.0 4.0 6.0 8.0 10.0 12.0 [pH]

d
Figure 29 Simulated 13C{1H}-NMR-controlled titration of acetic acid vs. NaOH. pcH,-
d  r 
plots: c: 1 reduced chemical shift <r>, 2 first and 3 second derivative, and
dpcH
d2  r 
2
resp.. Identical plots result for the CH3- and the COOH/COO--groups. y-axis
dpcH
Cred. d: Molar fractions for species HL and L- vs. pH. y-axis: molar fractions.
It is tempting to analyze the ,-plot shown in Figures 30 a – d, which were simulated
using the program GENTIT and the analytical data given above: Figures 30 a - c
exhibit the intersection of two linear functions, where the intersection point may be
used to calculate the concentration of the acid for known base concentration. But
some (time consuming) algebra yields, that in principle the ,-function is not strictly
linear but polynomial, which will not be discussed in this paper.
Figures 30 a and b
27.0
26.0
25.0
24.0
23.0
22.0
-0.10 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 [tau]
a
184.0
183.0
182.0
181.0
180.0
179.0
-0.10 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 [tau]
b
Figure 30: Simulated 13C{1H}-NMR-controlled titration of acetic acid vs. NaOH. ,-
plots: a: CH3-group. HL 22.83, L 25.68. b: COOH/COO--group: HL 179.17, L
183.80. y-axis C [ppm].

Figures 30 c and d
1.00
0.80
0.60
0.40
0.20
0.00
-0.10 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 [tau]
1.00
0.80
0.60
0.40
0.20
0.00
-0.10 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 [tau]
d
Figure 30: Simulated 13C{1H}-NMR-controlled titration of acetic acid vs. NaOH. ,-
plots. c: Reduced chemical shift <r>. Identical plots result for the CH3- and the
COOH/COO--group. y-axis Cred. d: Molar fractions for species HL and L- vs. . y-axis:
molar fractions.
Case 2: Propionic acid
1
H-NMR-Spectra of propionic acid and the corresponding propionate anion
- H+
CH3 CH2 COOH CH3 CH2 COO-
+
+H
represent first order systems of A2M3- (500 MHz) to second order A2B3- (100 MHz)
type. Chemical shifts H, C, coupling constants 3JHH were determined by HR 500 1H-
and 125 MHz 13C{1H} NMR resp. using a ca. 0.1 m solution in D2O. Obviously the
carbon chemical shifts and resonance frequencies are more sensitive towards pro-
tonation then the corresponding proton data (see Table 14). It is instructive, to simu-
late the 1H-NMR spectra with WINDAISY. The calculated 100 MHz 1H-NMR spectra
of propionic acid (HL) and propionate anion (L-) are shown in Figure 31.
Table 14
HL L- i
C COOH / COO- 182.4963 187.6332 -5.1369 ppm
C CH2 29.9234 33.3652 -3.4418 ppm
C CH3 11.0629 12.8572 -0.7943 Ppm
H CH2 2.4006 2.1669 +0.2337 Ppm
H CH3 1.0968 1.0402 +0.0466 Ppm

3
JHH CH3-CH2 7.54 7.68 -0.14 Hz
Table 14: HR 500 1H- and 125 MHz 13C{1H}-NMR data from propionic acid (HL) and
sodium propionate (L-), 0.1 m solutions in D2O.

Figure 31
2.8 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6
(ppm)
2.8 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6
(ppm)
Figure 31: calculated 100 MHz 1H-NMR spectra of propionic acid (HL) and
propionate anion (L-). Upper: HL; lower: L-.

Case 3: Dimethylphosphinic acid (CH3)2P(O)OH
31
This example shows results from P{1H}-, 13C{1H}-, 1H- and 13C-NMR controlled
titrations which yielded the ion specific chemical shift shown in Table 15 when using
the analytical data given in Table 16:
Table 15
Sensor HL L- i
H 1.576 1.298 0.278

2
JPH -14.51 -13.65 -0.86
C 17.67 19.39 1.72

1
JPC 93.51 92.93 -0.58
P 57.25 42.61 -14.61
Table 15: Ion-specific parameters and gradients i of (CH3)2P(O)OH.
Table 16
Vv C1 C2 Type pK NMR
[mol l-1]
[ml] [mol l-1]
31
85 0.01 0.9713 NaOH 3.07 P{1H}
13
85 0.20 4.5300 KOH 3.16 C{1H}
Table 16: Analytical data for (CH3)2P(O)OH.
Deprotonation of the phosphinic unit gives rise to a high field shift for P which is in
contrast to the up field shift for c. See Figures 32 a - d:
Figures 32 a and b
58.0
56.0
54.0
52.0
50.0
48.0
46.0
44.0
42.0
0.0 2.0 4.0 6.0 8.0 10.0 12.0 [pH]

a
1.00
0.80 1
0.60
0.40
2
0.20
0.00
-0.20
3
-0.40
-0.60 4
0.0 2.0 4.0 6.0 8.0 10.0 12.0 [pH]

b
Figures 32 a and b: 81 MHz 31P{1H}-NMR-controlled titration. a: Dynamic chemical
shift <P> vs. pcH. b: curve 1: molar fraction HL and reduced dynamic chemical shift
d 2  P red  d P red 
<Pred>. curve 2: molar fraction L. curve 3: . curve 4: . Simulated
dpcH2 dpcH
with: pKW 13.78, pKa 3.07, max 1.10, HL 57.25 ppm, L 42.61 ppm, titrand 85.00 ml,
0.0100 m (CH3)2POOH, titrator 0.9713 m NaOH.

Figures 32 c and d
Figures 32 c and d: ,-plots from 81 MHz 31P{1H}-NMR-controlled titrations of 0.1 m
dimethyl phosphinic acid. Left: home made probe head, titration vs. 1 m TMAOH.
Right: Rindlispacher probe head, titration vs. 0.9713 m NaOH.

4.2 Bivalent Acids H2L
4.2.1 First Order - Macroscopic Dissociation
Case 4: Methane phosphonic CH3P(O)(OH)2
pK1 pK2
CH3 PO3H2 CH3 PO3H- CH3 PO32-
Since pK = pK2-pK1 > 3 methane phosphonic acid represents a classical first order
acid where well separated consecutive deprotonation steps are observed. The cor-
responding experimental spectra from NMR controlled titrations are understood after
reflecting upon the pH,-, x,- and x,pH-diagrams shown below in Figures 33-35:
Figure 33
Figure 33: pH,-diagram - titration of methane phosphonic acid vs. NaOH.

Figure 34
Figure 34: x,-diagram - titration of methane phosphonic acid vs. NaOH.
Figure 35
Figure 35: x,-diagram - titration of methane phosphonic acid vs. NaOH.
Using exp. conditions shown in Table 17 the following ,- and pH,-plots were ob-
tained from NMR controlled titrations:

150
Table 17
V1 CA1 CB2 Type pK1 pK2 pK NMR MHz

-1 -1
[ml] [mol l ] [mol l ]
31
85 0.0120 0.9713 NaOH 2.06 7.66 5.60 P{1H} 80.015
31
85 0.0095 0.9713 NaOH 2.00 7.68 5.68 P 80.015
13
85 0.2690 4.8200 KOH 2.27 7.85 5.58 C{1H} 50.288
Table 17: Analytical and NMR data relevant for NMR-controlled titration of methane
phosphonic acid CH3P(O)(OH)2.
Characteristic 81 MHz 31P- and 31P{1H}-NMR controlled titration plots of t,d-type are
shown in Figure 36 and Figure 37:
Figure 36
Figure 36: ,-contour plot from proton-coupled 80.015 MHz 31P-NMR-controlled
titration of CH3P(O)(OH)2 vs. NaOH.

Figure 37
Figure 37: ,-plots from proton-decoupled 80.015 MHz 31P{1H}-NMR-controlled
titration of CH3P(O)(OH)2 vs. NaOH. Upper: contour plot, lower: stacked plot.
The first automated pH,-spectra ever made in our group are shown in Figure 38.
Those early plot files were rather large (12 to 20 MB) and motivated us to develop
the more efficient plotter program Multiple NMR Graphics, which will be demonstra-
ted at the end of this chapter.

Figure 38
Figure 38: pH,-plots from proton-decoupled 80.015 MHz 31P{1H}-NMR-controlled
titration of CH3P(O)(OH)2 vs. NaOH. Upper: contour plot, lower: stacked plot. x-axis:
C-resonance frequency [Hz] (not chemical shift C [ppm]).

13
Numerical results for ion specific parameters methane phosphonic acid obtained
from 81 MHz 31P{1H}- and 50 MHz 13C{1H}-NMR controlled titrations are given in
Table 18:
Table 18:
1 2
C P
31
Species Gradient JPC JPH P{1H}
H2L 14.27 135.92 33.07 -17.65 33.03
1 1.26 -2.1 -8.23 1.13 -8.24
HL- 15.53 133.82 24.84 -16.52 24.79
2 1.08 -3.87 -3.94 1.00 -3.71
L2- 16.61 129.95 20.90 -15.52 21.08
Table 18: Ion specific NMR chemical shifts C and P [ppm], coupling constants 1JPC
and 2JPH [Hz] and gradients i [ppm] / [Hz] for methane phosphonic acid.
Deprotonation leads to a characteristic upfield shift in P but gives rise to a downfield
shift in C. In addition the absolute values of couplings constants 1JPC and 2JPH
decrease with progressing deprotonation, with increasing negative charge attached
to the phosphonate unit. Inflection points for pH-correlated plots clearly indicate the
pK-data wanted.
The spectral halfwidth HW [Hz] in 31P{1H}-NMR of methane phosphonic acid clearly
depends on the state of protolytic equilibria. By single sample NMR on a more dilute
solution (0.01 m) a characteristic pattern for the HW,-diagram (see Figure 39) was
observed. A maximum of HW for  around 1.5 possibly indicates the formation of
hydrogen bridges P-O-H-O-P.

Figure 39
6
4
2
0
0 1 2 3 4
Figure 39: Spectral halfwidth HW [Hz] of the 31P{1H}-singlet for 0.01 m methane
phosphonic acid vs. 0.1 m NaOH. x axis: degree of titration . y-axis: HW [Hz].
It was tempting to simulate the pH,- and ,-plots for 50 MHz 13C{1H}-NMR control-
led titrations starting off from experimental data found as listed in Tables 17 and 18
by means of Multiple NMR Graphics. This proved to be of intrinsic didactic value
helping to understand the planning and evaluation of such experiments. Figure 40b
shows a high density of traces where pH approximates pKa1 or pKa2. This type of ex-
periment would be best to determine the dissociation constants. In Figure 40d traces
accumulate around  = 1 and 2, where species LH- and L2- are formed. Consequently
this type of experiment would be especially useful to determine selectively the con-
centrations (even in mixtures). These conclusions are valid for all the nuclei used in
NMR controlled titrations.

Figure 40
13
13
12 12
11 11
10 10
9 9
8 8
pH
pH
7 7
6 6
5 5
4 4
3 3
2 2
19 18 17 16 15 14 13 12 19 18 17 16 15 14 13 12
ppm
a ppm
b
2,8
2,8
2,6 2,6
2,4 2,4
2,2 2,2
2,0 2,0
1,8
Titrationsgrad
Titrationsgrad
1,8
1,6 1,6
1,4 1,4
1,2 1,2
1,0 1,0
0,8 0,8
0,6 0,6
0,4 0,4
0,2 0,2
19 18 17 16 15 14 13 12 19 18 17 16 15 14 13 12
ppm
c ppm
d
Figure 40: Simulated 50 MHz 13C{1H}-NMR controlled titration of methane phospho-
nic acid vs. NaOH. a and b: pH,-plots; c and d: ,-plots; a and d: titration equidis-
tant in pH; b and c: titration equidistant in . Spectra were simulated with the program
MPSCP [27] and plotted with Multiple NMR Graphics [2u].

Case 5: Leu-Ala - a peptide - 50.288 MHz 13C{1H}-NMR controlled titration
C5H3 H H O H H
H C4 C3 C2 C1 N C7 C6OOH
C5´H3 H NH2 C8H3

LEU-ALA
Analysis and iteration of the 200 MHz 1H-NMR spectrum with WINDAISY (BRUKER)
[28] supported by 13C{1H}-NMR and H,H- and C,H-COSY studies established unequi-
vocally the assignment of carbon atoms C2-C5, C7 and C8. Carbonyl carbons C1
and C6 were assigned via NMR controlled titration shown in Figure 41 and Figure
42.
Figure 40
C4
C8
C5´
C3
H O C5
COOH
6
H2N C C
2 1
NH C H
7
H C H
3
CH3
8
H C CH3
4 5´
CH3
5
Figure 41: 50.288 MHz 13C{1H}-NMR controlled titration of Leu-Ala vs. NaOH. HNO3
was added prior to titration.

Figure 42
C1 C2
C7
C6
H O
COOH
6
H2N C C
2 1
NH C H
7
H C H
3
CH3
8
H C CH3
4 5´
CH3
5
Figure 42: 50.288 MHz 13C{1H}-NMR controlled titration of Leu-Ala vs. NaOH. HNO3
was added prior to titration. Weak signals from C6 and C1 are emphasized by
auxiliary lines.
While the first deprotonation takes place on the carboxylic unit with strong influences
on C of carbons C6-C8, the ammonium cationic site is depronated in the second
step strongly affecting C of the carbons C1-C3. C4, C5 and C5´ are less sensitive
sensors for the protolytic equilibrium. The carbonyl signals C1 and C6 exhibit lower
S/N and demand a more careful inspection of experimental data. Within this context
a gradient for deprotonation was defined as i = C(HiL) – C(Hi-1L). This significant
gradients are negative for C1-C3 and C6-C8 as well for both deprotonation steps.
Corresponding data are liste in Table 19:

Table 19
Spec Grad C1 C2 C3 C4 C5 C5´ C6 C7 C8

LH2+ 172.80 54.42 42.41 26.17 24.07 23.58 178.5 51.38 18.46
2 0.75 -0.38 0.06 -0.05 -0.18 0.10 -3.45 -2.52 -1.23
LH 172.05 54.60 42.34 26.22 24.25 23.48 181.95 53.90 19.69
1 -7.83 -1.15 -3.56 0.30 -0.47 -0.31 -0.44 0.60 -0.33
L- 179.88 55.75 45.90 26.52 24.72 23.79 182.39 53.30 20.03
Table 19: Ion specific chemical shifts C [ppm] and deprotonation gradients i [ppm]
of Leu-Ala, calculated with SON4 [2c].
Analytical parameters: pK1 = 3.42 (COOH/COO-) and pK2 = 8.33 (NH3+/NH2). Titrand:
55 ml of a solution 0.063 m in Leu-Ala and 0.1649 m in HNO3. Titrator: 0.9937 m
NaOH.
The ,pH molar fraction diagram for Leu-Ala is shown in Figure 42, clearly indicating
the first order character of this acid/base system:
Figure 43
LH
LH+ L-
2
Figure 42: ,pH-diagram for Leu-Ala

pH,C-plots are simulated and given in Figure 44 below. The inflection points indicate
the pK values wanted.
Figure 44
Figure 44: 50.288 MHz 13C{1H}-NMR controlled titration of Leu-Ala vs. NaOH.
Simulated pH,C-plots. upper: C2, C7 and C3; middel: C1 and C6; lower: C5, C5,
C5´and C8.
Case 6: 2-amino-4-fluoro 2-methylpent-4-enoic Acid
The first 470.59 MHz 19F-NMR controlled titration [29]
F H 3C NH3+
H C C
C C COO-
H H H
19
This H2L problem is a typical first order case where the F sensor reflects the depro-
tonation-protonation equilibrium. The 19F sensor is separated as far as four bonds
from nitrogen and five bonds from the oxygen atoms involved in proton exchange.
The first gradient 1 in F is -0.60 ppm and -0.62 ppm respectively, while the second
gradient 2 is -1.76 ppm. Consequently the first protonation takes place at the COOH
unit (pK1 = 1.879) and the second on the NH3+ site (pK2 = 9.054), which is consistent
with standard knowledge in amino acid chemistry. Those conclusions may be drawn
from Table 20 and Figures 45 to 50 shown below.
19
The following experimental conditions were used: 470.59 MHz F-NMR. pK1 =
1.8790.094, pK2= 9.0540.016. Titrand: 25.00 ml of a solution holding 66.5 mg of
the amino acid - trifluoroacetic acid adduct (exp. stoichiometric ratio 1.000 : 0.9863).
Analytical investigations showed, that this solution contained 36.90 mg (0.251 mmol)
amino acid, C6H10FNO2, 28.20 mg (0.248 mmol) trifluoroacetic acid and 1.41 mg
(0.078 mmol) H2O, which corresponds to a content of 2.11% water. Furthermore the
solution contained 2.50 ml (0.09968 M) HCl (0.255 mmol), 5.0 ml (2.50 mmol) 0.5 M
TMACl,. Titrator: 0.1007 M TMAOH.
19
F-chemical shifts were referenced as follows: A high precision NMR tube (WILMAD
Coaxial Insert WGS-5-BL, WILMAD NMR Tube 535-PP-7) was used for a high reso-
lution 470.59 MHz 19F-NMR experiment. The inner compartment was filled with 100%
CFCl3, the outer compartment contained 0.75 ml of D2O. The position of the reso-
nance signal from the CF35Cl237Cl isotope was determined and used to calibrate the
spectra measured during the titration.
Two different methods, described in [1m, 2y], where used to determine the ion spe-
cific parameters given in Table 20.
Table 20
Gauss-elimination Implicite iteration

Species Gradients F F F F
H2L+ -94.82 0.0058 -94.81 0.0036
1 -0.62 -0.60
HL -94.20 0.0028 -94.21 0.0133
2 -1.76 -1.76
L- -92.44 0.0021 -92.45 0.2708
Table 20: Ion specific F(HiL) parameters 2-amino-4-fluoro 2-methylpent-4-enoic acid
obtained from 470.59 MHz 19F-NMR controlled titration.
The ,- and lower: the ,pH-diagrams (see Figure 45) for the 470.59 19F-NMR-con-
trolled titration of 2-amino-4-fluoro 2-methylpent-4-enoic acid vs. TMAOH clearly
show the first order character of a H2L+ cationic acid.

Figure 45
1.0
L-
0.9
0.8
0.7
H2L+
0.6
0.5

0.4 HL
0.3
0.2
0.1
0.0
-1 -0.5 0 0.5 1 1.5 2 2.5 3

1.0
L-
0.9 +
H2L
0.8
0.7
0.6
0.5

0.4
0.3 HL
0.2
0.1
0.0
1 3 5 7 9 11
pH
Figure 45: upper: the V,- and lower: the pH,-diagrams for the 470.59 19F-NMR-
controlled titration of 2-amino-4-fluoro 2-methylpent-4-enoic acid vs. TMAOH
The ,- and the ,pH-diagrams for the 470.59 19F-NMR-controlled titration of 2-
amino-4-fluoro 2-methylpent-4-enoic acid vs. TMAOH are combined in Figure 45.
The typical first order pattern is easily recognized.

Figure 46
-92,0 12
-92,5 10
-93,0 8
 [ppm]
pH
-93,5 6
-94,0 4
-94,5 2
-95,0 0
-1 -0,5 0 0,5 1 1,5 2 2,5 3

Figure 46: The ,- and the ,pH-diagram for the 470.59 19F-NMR-controlled titration
of 2-amino-4-fluoro 2-methylpent-4-enoic acid vs. TMAOH.
The following Figure 47 compares two different types of graphical representations for
,-contour plots: a) the efficient, storage saving plot made with Multi NMR Graphics
and b) the conventional EXCEL plot, which produce much larger data sets.
The corresponding ,-stacked plots are shown in Figure 48.

Figure 47
3.0
2.5
2.0
1.5

1.0
0.5
0.0
-0.5
-1.0
-92.0 -92.5 -93.0 -93.5 -94.0 -94.5 -95.0

2,87
2,22
1,58
0,94 
0,29
-0,35
-1,00
-92,2 -92,7 -93,2 -93,7 -94,1 -94,6
 [ppm]
Figure 47: ,-contour plots for the 470,59 MHz 19F-NMR controlled titration 2-amino-
4-fluoro 2-methylpent-4-enoic acid vs. TMAOH. Plots were generated with: upper:
Multi NMR Graphics (0.045 MB); lower: EXCEL (2.393 MB).

Figure 48
3.0
2.5
2.0
1.5

1.0
0.5
0.0
-0.5
-1.0
-92.0 -92.5 -93.0 -93.5 -94.0 -94.5 -95.0

2,87
2,10
1,32
-92,2
-92,7 0,55 
-93,2 -0,22
-93,7
 [ppm] -94,1 -1,00
-94,6
Figure 48: ,-stacked plots for the 470,59 MHz 19F-NMR controlled titration 2-
amino-4-fluoro 2-methylpent-4-enoic acid vs. TMAOH. Plots were generated with:
upper: Multi NMR Graphics (0.313 MB); lower: EXCEL (3,270 MB).
pH,-contour and pH,-stacked plots for the 470,59 MHz 19F-NMR controlled titration
2-amino-4-fluoro 2-methylpent-4-enoic acid vs. TMAOH, yield a visual, first hand
guess of pKa data as may be derived from Figure 49 below:
Figure 49
11
10
9
8
7
pH
6
5
4
3
2
-92.0 -92.5 -93.0 -93.5 -94.0 -94.5 -95.0

11
10
9
8
7
pH
6
5
4
3
2
-92.0 -92.5 -93.0 -93.5 -94.0 -94.5 -95.0

Figure 49: upper: pH,-contour plot, lower: pH,-stacked plot for the 470,59 MHz 19F-
NMR controlled titration 2-amino-4-fluoro 2-methylpent-4-enoic acid vs. TMAOH,
generated with Multi NMR Graphics [2u, 2v] (0.040 MB and 0.313 MB).
Clearly the fluorine chemical shift reflects the first and second deprotonation steps
and corresponding pKa data may be evaluated via the first derivative dF/dpH as
shown in Figure 50.
Figure 50
-92.0
-92.5
-93.0
 [ppm]
-93.5
-94.0
-94.5
-95.0
1 2 3 4 5 6 7 8 9 10 11 12
pH
Figure 50: 470.59 19F-NMR-controlled titration of 2-amino-4-fluoro 2-methylpent-4-
enoic acid vs. TMAOH. F (exp. And calc.) and the first derivative dF/dpH are shown
as curves (-) and (-) resp..
It is interesting to note the variation in spectral half width during titration (see Figure
51): a maximum of 6.61 Hz is connected with =1.5. This corresponds to the situa-
tion, where association may take place and a hydrogen bonded anion a [L-H-L]-1 may
be formed.
Figure 51
6.5
5.5
HW [Hz]
4.5
3.5
3
-1 0 1 2 3

6.5
5.5
HW [Hz]
4.5
3.5
3
1 2 3 4 5 6 7 8 9 10 11 12
pH
Figure 51: The spectral half width HW as functions of (upper)  or (lower) pH resp.
during the 470.59 19F-NMR-controlled titration of 2-amino-4-fluoro 2-methylpent-4-
enoic acid vs. TMAOH.

4.2.2 Second order - Macroscopic dissociation
Case 7: 1-Hydroxyethane-P,P´-dimethyl-1,1-bisphosphinic acid
31
The first 202.46 MHz P{1H}-NMR controlled titration using the AVANCE DRX 500
spectrometer - now a save standard application in our laboratories.
HO CH3
CH3 C CH3
P P
O OH HO O
In search for Ca-complexing agents resembling the well known family of geminal bis-
phosphonates the above mentioned phosphinic analogue of HEDP was synthesized
by HENKEL and given to us for NMR studies. 202.46 MHz 31P{1H}-NMR controlled
titration of 1-hydroxyethane-P,P´-dimethyl-1,1-bisphosphinic acid were performed
using analytical parameters: Titrand: pK1 = 1.7310.0094; pK2 = 3.5910.0187. 31.00
mg (0.153 mmol) of C4H12O5P2 [202.08]), 1.18 mL 0.0980 m HCl, (0.122 mmol HCl),
5.00 ml 0.50 m NaCl, (2.50 mmol NaCl), were made up to 25.00 mL. Titrator: 0.1033
m NaOH. This bifunctional acid H2L is of second order since pK2 – pK1 = 1.86.
Figure 52a shows the 202.46 MHz 31P{1H}-NMR ,P-contur-plot while Figure 52b
represents the ,P- stacked-plot of 1-hydroxyethane-P,P´-dimethyl-1,1-bisphosphinic
acid, both plots processed via Multi NMR Graphics.

Figure 52
3,0
2,5
2,0
1,5

1,0
0,5
0,0
-0,5
50 48 46 44 42

3,0
2,5
2,0
1,5
1,0

0,5
0,0
-0,5
50 48 46 44 42

Figure 52: 202.46 MHz 31P{1H}-NMR controlled titration of 1-hydroxyethane-P,P´-
dimethyl-1,1-bisphosphinic acid: a) ,-contur-plot; b) ,-stacked-plot; both produced
with Multi NMR Graphics. Please bear in mind, that the ,-function is non-linear.
A corresponding x,-molar fraction diagram is shown in Figure 53a while data for pH
and dynamic chemical shift P are plotted vs.  in Figure 53b:

Figure 53
1,0
2-
L
0,8
HL-
0,6

0,4 H2L
0,2
0,0
-0,5 0,0 0,5 1,0 1,5 2,0 2,5 3,0

a
50,0 14
12
48,0
10
46,0
 [ppm]
pH
44,0 6
4
42,0
2
40,0 0
-1 0 1 2 3

b
dimethyl-1,1-bisphosphinic acid: a: x,-molar fraction diagram: b: pH,- and ,-plots
produced with EXCEL.
The significant functions of P vs. pH together with the first derivative are shown in
Figure 54:
Figure 54
50,0 5
4
48,0 3
2
46,0 1
 [ppm]
d/dpH
0
44,0 -1
-2
42,0 -3
-4
40,0 -5
2 3 4 5 6 7 8 9 10 11 12
pH
dimethyl-1,1-bisphosphinic acid: pH,-plot produced with EXCEL.
It is interesting to see in Figure 55 an abrupt drop in spectral linewidth (from max 15
to min 3 Hz) after total deprotonation. Proton exchange in proton bridges seems to
contribute remarkably to the overall line width observed.

Figure 55
16
14
12
HWB [Hz]
10
8
6
4
2
0
-1 0 1 2 3

dimethyl-1,1-bisphosphinic acid: HWB,-plot produced with EXCEL. Spectral
halfwidth vs. .
The ion specific chemical shifts, obtained by two different, are listed in Table 21..
Parameter errors P derived from implicite iteration are more realistic.
Table 21
Gauss elimination Implicite iteration

Species P P P P
H2L 52.43 0.0219 52.31 0.5993
HL- 48.02 0.0050 47.96 0.0725
2-
L 41.17 0.0025 41.18 0.0036
Table 21: Results from 202.46 MHz 31P{1H}-NMR controlled titration of 1-hydrox-
yethane-P,P´-dimethyl-1,1-bisphosphinic acid: Ionspecific chemical shift obtained via
Gauss elemination and implicite iteration.

Case 8: Benzoylphosphonic acid and Calcium - Metal complex formation
Acylphosphonates have attracted interests in medicinal chemistry as potential lig-
ands in calcium related disorders. A series of mono- and bis-acylphosphonates was
synthesized by E. Breuer (Jerusalem) and characterized by analytical and NMR

31
studies in Düsseldorf [1i, 2m, 3d, 3g, 30]. P{1H}-NMR was used to monitor the pcH
dependent ligand/calcium equilibrium of benzoyl phosphonic acid. While the free acid
is less stable, the pure mono sodium salt was accessible in solid state.
O O
C P ONa
OH
Formation of a 1:1 calcium complex of benzoyl phosphonic acid was deduced from
the retro-titration of a) HNaL, NaOH (1:2) and b) HNaL, NaOH, Ca(NO3)2 (1:2:1) vs.
HNO3. The following constants were determined: pKa1 = 0.39; pKa2 = 5.60; log[CaL]
= 2.00. In experiment a) the first equivalent of HNO3 is spent to neutralize the surplus
of OH- from NaOH. The second step is characterized by L2-  HL-. Since HL- is a
strong anionic acid further addition of HNO3 does not protonate HL- H2L and <P>
effectively remains constant. Experiment b) involves the simultaneous titration of equi
molar amounts of sodium benzoyl phosphonate and calcium nitrate. The species ML
is detected, giving rise to a characteristic coordination shift. (FIGURE 56). The ion
specific NMR parameters are obtained with AMPLSQ9 and shown in TABLE 22. ).
The titration pH vs.  and the molar fraction diagrams x vs.  and x vs. pH are calcu-
lated with WINSCORE (FIGURE 57-59). It is evident, that benzoylphosphonic acid

forms a weak calcium complex of type CaL.
FIGURE 56
FIGURE 56: 81 MHz 31P{1H}-NMR controlled titration of: upper: sodium benzoyl
phosphonate + 2 eq. of NaOH vs. HNO3. lower: sodium benzoyl phosphonate + 2
eq. of NaOH + 1 eq. of Ca(NO3)2 added vs. HNO3. Remarks: y-axis represents
degree of titration t. negative -values imply HNO3 / H2L, while positive -values stand
for NaOH / H2L.

Table 22
P a) P b)
CaL 1.351
L2- -0.034 -0.032
HL- -0.956 -0.957
TABLE 22: Ion specific chemical shifts P [ppm] obtained from 81 MHz 31P{1H}-NMR
controlled titrations in absence (a) and in presence (b) of calcium. It is not possible to
determine a reliable value for P from H2L.
FIGURE 57
FIGURE 57: titration diagrams pH vs . blue: upper: sodium benzoyl phosphonate +
2 eq. NaOH vs. HNO3; red: sodium benzoyl phosphonate + 1 eq. of Ca(NO3)2 + 2 eq.
NaOH vs. HNO3, calculated with WINSCORE.

Figure 58
FIGURE 58: molar fraction diagrams x vs.  for: upper: sodium benzoyl phosphonate
+ 2 eq. NaOH vs. HNO3; lower: sodium benzoyl phosphonate + 1 eq. of Ca(NO3)2 +
2 eq. NaOH vs. HNO3, calculated with WINSCORE.

Figure 59
FIGURE 59: molar fraction diagrams x vs. pH for: upper: sodium benzoyl
phosphonate + 2 eq. NaOH vs. HNO3; lower: sodium benzoyl phosphonate + 1 eq.
of Ca(NO3)2 + 2 eq. NaOH vs. HNO3, calculated with WINSCORE.

4.2.2 Second order - Microscopic dissociation of bivalent acids H2L
Combining two mono functional units like CH3COOH or (CH3)2P(O)OH three different
bifunctional acids 9 to 11 may be constructed: succinic acid 9, ethane-1,2-bis-(P-me-
thyl-phosphinic acid) 10, and 2-P-methyl-phosphinopropionic acid 11:
O H H O O H H O O H H O
HO C C C C OH HO P C C P OH HO C C C P OH
H H H3C H H CH3 H H CH3
Case 9 Case 10 Case 11
Compounds 9 to 11 clearly behave as second order acids H2L with pK = pK2 - pK1 <
3 as may be seen from Table 23. 13C{1H}-NMR controlled titration yielded specific
parameters listed in Table 24 and Table 25.
Table 23
9 10 11
pK1 4.09 1.93 2.50
 -1.20 -1.29 -2.10
pK2 5.29 3.22 4.60
Table 23: pK data for compounds 9 to 11 in H2O, at 25 °C and I = 0.1 M.
For 13C-NMR studies of 2-P-methyl-phosphinopropionic acid 11 the following spin
labeling is used:
H H O
HOOC1 C2 C3 P OH
H H C4H3
Table 24
Compound Carbon H2L HL- L2-

C C  C  C
9 1 180.04 -2.45 182.49 -2.73 185.22
9 2 31.75 -2.15 33.99 -2.96 36.95
10 2 24.35 1.38 25.73 1.78 27.51
10 Me 15.69 0.92 16.61 0.83 17.44
11 1 179.39 1.31 180.70 4.45 185.15
11 2 29.38 1.2 30.58 3.12 33.7
11 3 27.43 1.66 29.09 2.05 31.14
11 Me 16.35 1.31 17.66 0.05 17.61
Table 24: Chemical shifts C [ppm] and gradients  [ppm] for compounds 9 to 11
Table 25
Species Compound 11
1 1 1
JPC(C1) JPC(C3) JPC(Me)
LH2 15.1 94.3 92.2
 0.8 -1.9 0.5
LH- 15.9 92.4 92.7
 1.3 -0.1 1.2
L2- 17.2 92.3 91.5
Table 25: Coupling constants nJPC [Hz] and gradients  [Hz] for 2-P-methyl-phosphi-
nopropionic acid 11.

At this stage of our analysis it is useful to introduce a convenient notation for acid -
base systems given in Table 26:
Table 26
Form Symbol Example Example

Acid A COOH P(CH3)(O)OH
Base B COO- P(CH3)(O)O-
Table 26: Acid - base notation used for microscopic dissociation
Using this notation H2L and L2- are assigned to AA and BB forms resp. for com-
pounds 9 to 11. The intermediate anion HL- for 9 and 10 give rise to symmetry equi-
valent anions of type AB and BA while the asymmetric 11 yields two different anions
AB and BA resp. forming a pair of tautomers. This observation leads to the concept
of microscopic dissociation which is described in Figure 60 comparing the macro-
scopic analogue.
Figure 60
Figure 60: Comparing the concepts of microscopic and macroscopic dissociation

Macroscopic (K1, K2) and microscopic dissociation constants (k1 to k4) are interrela-
ted according to the following equations:
K1  k1  k 2 (62)
K 21  k 31  k 41 (63)
K1  K 2  k1  k 3  k 2  k 4 (64)
k 2  K1  k1 (65)
k 3  K1  K 2  k11 (66)
k 4  K1  K 2  (K1  k1)1 (67)
The dynamic NMR parameter is reformulated to:
    x AA   AA  x AB   AB  xBA  BA  xBB  BB (68)
and the microscopic molar fractions are defined by:
2
cH
x AA  (69)
2
cH  c H  K1  K1  K 2
c H  k1
xBA  (70)
2
cH  c H  K1  K1  K 2
cH  k 2
x AB  (71)
2
cH  c H  K1  K1  K 2
K1  K 2
xBB  (72)
2
cH  c H  K1  K1  K 2
In general, microscopic dissociation constants are not accessible from potentiometry
with a glass electrode and NMR. But in fortunate cases NMR may be used success-
fully to determine the micro dissociation constants wanted. This is true, if and only if
one sensor nucleus reflects the protonation state for one of the two acid/base centers
only leading to  AA   AB and BA  BB . Than the dynamical shift will follow:
    ( x AA  x AB )   AA  ( xBA  xBB )  BB (73)
The combined molar fraction  is defined as follows:
c H  k1  K1  K 2
  xBA  xBB  (74)
2
cH  c H  K1  K1  K 2
    (1  )   AA    BB (75)
     AA
 (76)
BB   AA
It is immediately evident, that the dynamic chemical shift <> reflects the group-spe-
cific dissociation in this case. Henceforth the wanted microscopic dissociation con-
stants k1 is extracted from:
2
  (c H  c H  K1  K1  K 2 )  K1  K 2
k1  (77)
cH
Suitable regressional analysis involving all data points (pcH, <>) from NMR control-
led titration will lead to the optimized k1 and henceforth to k2, k3 and k4 as well.
MICRO_IT was written for such purposes. For accuracy, macroscopic dissociation
constants K1 and K2 and the limiting specific chemical shifts AA and BB may be de-
termined by separate experiments prior to NMR controlled titration. But it is essential,
to maintain identical conditions for solvents, concentrations, and temperature since
those factors influence the dissociation constants K and chemical shifts  values.
It is tempting to reflect upon the general case of microscopic dissociation equilibria
which is suitable to handle polyfunctional acid base systems. Such systems may be
described by 2^n – 1 microscopic stability constants  (given as log data) instead of
n*2^(n-1) stepwise micro dissociation constants k (given as pk data), which carry re-
dundant information. In novel iterators the iteration is based on the microscopic sta-
bility constants.
Our novel program NmrMicroPk [2y] is more advanced then described above for the
special case of bivalent acids H2L. NmrMicroPk can handle the general n-basic
cases HnL of microscopic dissociation systems and compute the hitherto unknown
Sudmeier-Reilly-coefficients [2y] for nuclei which are influenced of the dissociation at
several acidic sites.
E. g.: Micro stability constants are defined for the H2L case by Figure 61 and equa-
tions (78) to (80):

Figure 61
BB
AB BA
AB BA
AA
AA
Figure 61: The concept of micro stability constants
c AB
 AB  (78)
c BB  c H
c BA
BA  (79)
c BB  c H
c AA
 AA  (80)
2
c BB  c H
It is straight forward to intervonvert the microscopic stability constants (used for cal-
culations) and dissociation constants (generally adopted in chemical literature).
2-P-methyl-phosphinopropionic acid 3 represents a model system to illustrate micro-
scopic dissociation. 31P{1H}-NMR is not suitable, since P is influenced by protonation
equilibria situated at the phosphinic and the carboxylic unit as well. This is true as
well for C from the skeleton carbons P-C-C-C. But the P-Methyl group is sufficiently
far away from the protolytic center COOH/COO- and henceforth 13C{1H}-NMR is used
to determine the micro pki: pk1 = 2.51, pk2 = 5.14, pk3 = 4.49, and pk4 = 1.85. Macro
pKi are: pK1 = 2.499 and pK2 = 4.491. Obviously the dominating dissociation route
uses AABABB, since the phosphinic unit is more acidic than the carboxylic
group:
AA O H H O O H H O BA
-
HO P C C C OH O P C C C OH
H3C H H H3C H H
AB O H H O O H H O BB
HO P C C C O- -
O P C C C O-
H3C H H H3C H H
These findings are demonstrated in Figures 62 to 67 together with a microscopic
x,pcH-diagram.
Figure 62
1.0
BB
AA
AB
0.8
Mole fraction
0.6
0.4
0.2
BA
0.0
1 2 3 4 5 6 7 8
pH
Figure 62: microscopic x,pcH-diagram for 2-P-methylphosphinopropionic acid 11.

Figure 63
H 4
O 1 H
C 2 C CH3
C1 H O C 3 P
H H O O H
Figure 63: 50.228 MHz 13C{1H}-NMR-controlled titration of P-methylphosphinopro-
pionic acid 11 vs. NaOH. COOH/COO- group.
Figure 64
C3 H 4
O 1 H
C 2 C CH3
H O C 3 P
H H O O H
C2
pionic acid 11 vs. NaOH. P-C-C carbons.

Figure 65
H 4
O 1 H
C4 CH3
C 2 C
H O C 3 P
H H O O H
pionic acid 11 vs. NaOH. P-CH3 group.
It is tempting to reflect upon the conformation of 2-P-methyl-phosphinopropionic acid
and corresponding anions. The 3JPC for the skeleton P-C-C-C does not vary much in
a sequence of 94.3, 92.4 and 92.3 Hz for H2L, HL- and L2- species indicating a domi-
nant trans conformation in a rapid intramolecular exchange process.
An equivalent conclusion may be drawn from analysis and iteration of [AB] 2M3X sys-
tems in 500 MHz 1H-NMR spectra [2y].
200 MHz 1H-NMR controlled titrations of 2-(methylphosphino)propionic acid 11
yielded the following spectra shown in Figure 66:

Figure 66
11
2,0
10
1,5
9
1,0
8
0,5
7
0,0
pH

6
-0,5
-1,0 5
-1,5 4
-2,0 3
-2,5 2
3,0 2,5 2,0 1,5 3,0 2,5 2,0 1,5


a 
b
11
2,0
10
1,5
9
1,0
8
0,5
7
0,0
pH

6
-0,5
5
-1,0
4
-1,5
3
-2,0
2
-2,5
3,0 2,5 2,0 1,5 3,0 2,5 2,0 1,5

c 
d
Figure 66: 200 MHz 1H-NMR controlled titrations of 2-(methylphosphino)propionic
acid 11: a and b: ,-plots; c and d: pH,-plots.
Using the following spin notation:
H H O A M 1 3
HOOC C C P OH X 6
H H CH3 A´ M´ Q3 2 4 5
chemical shifts  [ppm] and coupling constants J [Hz] of 2-(methylphosphino)propio
nic acid 11 obtained from high resolution spectra are listed in Table 27:
Table 27
NMR parameters at pH* 1 2 12

H A, A H1, H2 2.6560 2.6473 2.3222
H M, M H3, H4 2.1248 2.0985 1.7528
H Q H5 1.5521 1.5257 1.2182
6
P X P 57.5774 56.4124 44.4442
2
JHH JAA J12 -17.0 (n.i.) -19.0 (n.i.) -17.0 (n.i.)
3
JHH JAM = JAM J13 = J24 9.68 (1) 9.18 (1) 12.50 (1)
3
JHH JAM = JAM J14 = J23 6.12 (1) 6.67 (1) 4.84 (1)
2
JHH JMM J34 -15.23 (n.i.) -19.0 (n.i.) -17.0 (n.i.)
3
JPH JAX = JAX J16 = J26 12.24 (2) 12.48 (1) 8.73 (1)
2
JPH JMX = JMX J36 = J46 -14.40 (2) -14.36 (1) -14.23 (1)
2
JPH JQX J56 -14.168 (5) -14.114 (3) -13.327 (4)
Table 27: Results from 500.13 MHz 1H- und 202.46 MHz 31P{1H}-NMR-spectra of 3-
(methylphosphino)propionic acid 11 (0.05 M in D2O), iterated with WINDAISY. Che-
mical shifts  [ppm] (standard deviations < 10–4 ppm); coupling constants J [Hz]
(standard deviations in brackets); n.i.: not iterated.
For completeness sake, corresponding results from 80.015 MHz 31P{1H}-NMR-con-
trolled titration of P-methylphosphinopropionic acid 11 vs. NaOH are shown in Figure
67:
Figure 67
56
54
52
 P [ppm]
50
48
46
44
-2,5 -2,0 -1,5 -1,0 -0,5 0,0 0,5 1,0 1,5 2,0 2,5

Titrationsgrad
56
54
52
 P [ppm]
50
48
46
44
1 2 3 4 5 6 7 8 9 10 11 12
c
pH
Figure 67: upper: P vs ; lower: P vs pH from 80.015 MHz 31P{1H}-NMR-controlled
titration of P-methylphosphinopropionic acid 11 vs. NaOH.  experimental,  calcu-
lated data.
The reader is invited, to focus attention towards the accuracy and consistency of two
different concepts for evaluation of dissociation constants: a) iterations based upon
the potentiometric data (E [mV] vs. V [ml]; using WinScore), and b) iterations based
upon the NMR-titration curve ( [ppm] vs. pH; with MultiNmrPK). The following mac-
roscopic dissociation constants were obtained for P-methylphosphinopropionic acid
11:
Program pK1 pK2

WinScore 2.46 ± 0.01 4.592 ± 0.009
MultiNmrPK 2.4638 ± 0.0007 4.571 ± 0.003
Clearly indicating the excellent accuracy of this NMR-controlled titration method.
Furthermore, it was shown, that all the protolytic species derived from P-methylphos-
phinopropionic acid 11 prefer a trans conformation with respect to the C-C-C-P skele-
ton. From iteration of HR 1H- and 13C-NMR spectra of 11 coupling constants nJHH,
n
JPH, and nJPC, were extracted and several models of rotational analysis were perfor-
med leading to rotamer specific (submicroscopic) dissociation constants. This fine
piece of research is due to meticulous studies by Z. Szakacs [2y] and beyond the
scope of our review presentation.

4.3 Trivalent Acids H3L
4.3.1 First order - Macroscopic Dissociation
Case 12: LiOOC-CH2-P(O)(OLi)2 - a retro titration study
Phosphonoacetic acids representing tribasic acids of H3L type,
O X
HO P C COOH
HO Y
X, Y = H, F, Cl
have attracted considerable interests in medicinal chemistry for anti viral activities.
We have characterized a series of such compounds by 13C{1H}-, 31P{1H}- and 19F-
NMR techniques. The method of retro titration is demonstrated for the re-protonation
of Trilithiumphosphonoacetate with perchloric acid:
-
OOC-CH2-PO32- -
OOC-CH2-PO3H- HOOC-CH2-PO3H- HOOC-CH2-PO3H2
Corresponding 80.015 MHz 31P{1H}-NMR-controlled titrations are shown in Figure 68
below:
Figure 68
-1
-2
-3
Figure 68: upper: ,-contour plot, lower; ,-stacked plot from 80.015 MHz 31P{1H}-
NMR- controlled titration of (LiO)2(O)P-CH2-COOLi vs. HClO4.

Reprotonation of a P-O- unit to P-OH gives rise to a positive gradient , while repro-
tonation of COO- to COOH has the opposite effect. Those effects are typical for all
phosphonocarboxylic acids (HO)2(O)P-(CH2)n-COOH studied so far, but will decrease
with increasing chain length n. From the stacked plot clearly visible is an increase of
spectral halfwidth with increasing re-protonation. Numerical results for phosphono-
acetic acid are shown in Table 29.
Table 29
P  P  P  P
L3- LH2
-
LH2- LH3
14.58 2.89 17.47 -4.15 13.32 4.372 17.04
Table 29: Ion specific chemical shifts P and re-protonation gradients. Experimental
data and results: Titrand: 85 ml of 0.01 m (LiO)2(O)P-CH2-COOLi, pK1 = 1.44, pK2 =
4.98, pK3 = 7.99. Titrator: 0.3928 m HClO4.

4.3.2 First order and Second order
Combining Macroscopic and Microscopic Dissociation in Subsystems.
Case 13: Phosphinothricine - Glufosinate - a Phosphaglutaminic acid
1-Amino-3-(P-methylphosphino)-butyric acid
1-Amino-3-(P-methylphosphino)-butyric acid, known commercially as Phosphino-
thricine or Glufosinate, represents a phospha glutaminic acid and gave rise to an
interesting study of macroscopic and microscopic dissociation following Figure 69:
FIGURE 69
FIGURE 69: Complete ionization scheme of Glufosinate.
The macroscopic dissociation behavior of the molecule has been studied by potentio-
metric titrations [2y]. The most basic pK value of this molecule, pK 3=9.42 characte-
rize the acidity of the ammonium group. The other two values, pK2=2.69 and pK1=1.7
quantitate the overlapping dissociation of the carboxylic and phosphinic groups, but
they cannot be assigned directly to these individual sites.
Denoting the carboxylic function by 1 and the phosphinic acid site by 2, the protona-
tion scheme (Figure 69) shows their overlapping equilibria at a submolecular level.
(The protonation state of the amino group remains unaltered at pH < 6, thus glufosi-
nate can be treated as a dibasic acid H2L in this range). The methylprotons are report
selectively the phosphinic acid deprotonation, while the -proton is supposed to indi-
cate the dissociation state of the carboxyl moiety only. The NMR titration curves of
these two nuclei, for this case obtained from a series of single NMR samples, are
shown in Figure 70. The titration medium was 90% H2O / 10% D2O. The total ionic
strength of each sample was accurately adjusted to 1 M by addition of NaNO 3.
FIGURE 70
FIGURE 70: 200 MHz 1H NMR-pH titration curves of selected protons of Glufosinate.
(CL=0.02m, I=1, NaNO3, HNO3, NaOH).

In the pH-range between 7 and 11 the titration curve for the CH 3 group only indicates
a minor titration shift of 0.03 ppm, in contrast to the 0.53 ppm deprotonation shift at
the proximate CH proton. This evidences, that inductive effects, accompanying de-
protonations at -functional groups hardly influence the chemical shift of the consti-
tutionally remote methylprotons. This confirms our initial assumption of selectivity for
the methyl sensor.
The microconstants were calculated from experimental data <> in a range pH < 6.
From the total of four microconstants three are independent only and the fourth one
is redundant [7b,25].
Knowing the terminal ion-specific chemical shifts for the diprotonated and non-proto-
CH CH CH
nated species, H L3 = 4.226 ± 0.006 ppm, H = 1.573 ± 0.006 ppm, L 3 = 3.812
2 2L
± 0.004 ppm and LCH = 1.263 ± 0.004 ppm, it is possible to calculate the combined
molar fractions (1 and 2) for the carboxylic (1) and phosphinic acid (2) moieties:
  CH   LCH k1 ·[H  ]  k 2 k 4
1   (81)
 HCH2L   LCH [H  ]2  (k1  k 2 )·[H  ]  k 2 k 4
  CH3   LCH3 k 2 ·[H  ]  k 2 k 4

2   (82)
 HCH2L3   LCH3 [H  ]2  (k1  k 2 )·[H  ]  k 2 k 4
The three unknown microconstants were obtained by simultaneous fitting of equa-
tions (81) and (82) to the experimental (pH) data (see Figure 70). The resulting
microconstants (given as pki) are listed in Table 30.

Table 30
Microscopic dissociation constants

COOH P(CH3)(O)OH
pk1 2.20 pk2 2.17
pk4 2.37 pk3 2.35
Table 30: Microscopic dissociation constants of glufosinate, determined by 1H NMR
titration.
The phosphinic acid group is only slightly more acidic than the carboxylic moiety,
hence the microspecies AB and BA are almost equally populated for any given pH.
(see Figure 71).
Figure 71
Figure 71: x,pH-diagram: distribution of microscopic species of Glufosinate.

An intriguing piece of NMR spectral analysis opens up when inspecting the titration
dependent HR 1H-NMR and the 31P{1H}-NMR spectra of glufosinate. AFGMNQ3X
spectra are observed, where the single protons correspond to AFGMN, the P-methyl
protons to Q3 and the phosphorus is represented by X. The following spin notation
and the stereochemical assignment given below is used to indicate pH-dependent
chemical shifts shown in Figure 72.
COOH H5
HA HF HM O O
2
HOOC C C C PX OH H1
H
H2N H4 P
NH2 HG HN Q
CH3 OH
H3 CH3
Figure 72
4,4 H1 60
4,0
P 55
3,6
50
3,2
 H [ppm]
 P [ppm]
2,8 45
2,4 H2
40
2,0
5
H3
H H 4
35
1,6
H6
1,2 30
0 1 2 3 4 5 6 7 8 9 10 11 12
c ,D
pH
Figure 72: 200.13 MHz 1H- und 81.02 MHz 31P{1H}-NMR controlled titration of Glufo-
sinate (0,02 M in H2O/D2O 9/1, I = 1 M, T = 22 °C)

Corresponding ion specific chemical shifts are listed in Table 31.
Table 31
 
Parameter H3L+ HH32LL H2L HHL2 L HL HL
L2
L2 
H1 4.225 (1) -0.20 (2) 4.02 (2) -0.21 (2) 3.811 (1) -0.532 (1) 3.279 (1)
H2 2.259 (1) -0.10 (1) 2.16 (1) -0.07 (1) 2.090 (1) -0.303 (1) 1.786 (1)
H3 2.211 (1) -0.07 (1) 2.14 (1) -0.08 (1) 2.056 (1) -0.330 (1) 1.726 (1)
H4 2.051 (1) -0.20 (2) 1.85 (2) -0.19 (2) 1.654 (1) -0.140 (1) 1.514 (1)
H5 1.949 (1) -0.19 (2) 1.75 (2) -0.17 (2) 1.581 (1) -0.066 (1) 1.515 (1)
H6 1.572 (1) -0.16 (2) 1.41 (2) -0.15 (2) 1.262 (6) -0.030 (1) 1.233 (1)
P7 56.33 (6) -6.6 (8) 49.7 (8) -6.5 (8) 43.26 (3) 1.90 (6) 45.16 (5)
Table 31: Ionspecific chemical shifts H and P [ppm] of Glufosinate (in H2O/D2O
9/1. T = 22 °C. I = 1 M NaNO3)
These results were extracted from experimental 200 MHz 1H-NMR AFGMNQ3X-
spectra of glufosinate shown in Figure 73 while the regions for the FGMN protons
(the ethylene skeleton) are enhanced in Figure 74.
Corresponding simulated spectra were obtained using the novel program LAOTIT.
This combines the concepts of NMR parameters, dynamically averaged by protolytic
equilibria, and classical simulation of NMR spectra using an efficient LAOCOON type
procedure called MINILA. LAOTIT is described briefly by Figure 75.

Figure 73
5,5
5,0
4,5
4,0
3,5
pH
3,0
2,5
2,0
1,5
1,0
4,0 3,5 3,0 2,5 2,0 1,5


exp
5,5
5,0
4,5
4,0
3,5
pH
3,0
2,5
2,0
1,5
1,0
4,0 3,5 3,0 2,5 2,0 1,5


sim
Figure 73: upper: experimental, lower: simulated AFGMNQ3-part of AFGMNQ3X
system in Glufosinate. 200 MHz spectra simulated with LAOTIT.

Figure 743
5,5
5,0
4,5
4,0
3,5
3,0
2,5
2,0
1,5
1,0
2,3 2,2 2,1 2,0 1,9 1,8 1,7
exp
5,5
5,0
4,5
4,0
3,5
3,0
2,5
2,0
1,5
1,0
2,3 2,2 2,1 2,0 1,9 1,8 1,7 1,6
sim
Figure 74: upper: experimental, lower: simulated FGMN-part of AFGMNQ3X system
in Glufosinate. 200 MHz spectra simulated with LAOTIT.

Excellent agreement is achieved between experimental and simulated spectra!
Figure 75
LAO_TIT - a combination of LAOCOON and NMR-Titration

LAOTIT generates pH,-, ,-, and V,-plots
by simulation of NMR-controlled titrations
pH  j (HiL) TrFrMin TrFrMax

pHmin pHmax npH J jk (HiL) TrIntMin
V1 C1L C1A C1IP C2B HWB j (HiL) HWB
log i (HiL) pKW j = 1 to nspins LoFrMin LoFrMax
i = 0 to nH k = j to nspins npoints
<j >
< J jk >
 i (HiL) < HWB j >
AB-Simulator Mixing MINILA
pH V2 
LoFr
LoInt
pH, V, ,

SPECTRA MNMRG 2D STORE
Figure 75: Basic principles of LAOTIT. Analytical data produce molar fractions used
to weight spin parameters to yield titrations dependent dynamical spin parameters.
Transition frequencies and intensities are calculated in a LAOCOON type procedure
MINILA and converted to Lorentzian line shape. The titration dependent spectra are
stored in a two-dimensional matrix and plotted with Multiple NMR Graphics to yield
the ,- and pH,-plots wanted.

4.4 Second order NMR spectra of hetero nuclei by NMR controlled titrations -
50.228 MHz 13C{1H}-studies on 1,2-Ethanebisphosphonic acid – H4L case
Case 14: 1,2-Ethanebisphosphonic acid
50.228 MHz 13C{1H}-NMR spectra of 1,2-ethanebisphosphonic acid
O H H O
HO P C C P OH
OH H H OH
reveal the typical AA´X pattern for the 31P-13C-12C-31P three spin system.
Figure 76
IN / 2
N
I
i
S = 0
i
Io / 2
So
1300 1250 1200 1150 1100 1050

   (Hz)  
1 2 3 4 5
Figure 76: 13C{1H}-NMR-spectrum of 1,2-ethanebisphosphonic acid ( = 0).

Lorentzian deconvolution was performed with WINNMR to extract optimized frequen-
cies and intensities yielding the absolute value for the 3JPP coupling via eq. (83)
shown below. Corresponding definitions are shown in Figure 76 above:
SO Ii
JXX   (83)
2 Io  Ii 
A ,-stacked plot from 50.228 MHz 13C{1H}-NMR-controlled titration of 1,2-ethane-
bisphosphonic acid vs. NaOH is shown in Figure 77, while experimental conditions
are listed as exp. 2 in Table 32.
Figure 77
3

2
29.0 28.0 27.0 26.0 25.0 24.0 23.0 22.0 21.0 0

ppm
FIGURE 77: ,-stacked plot from 50.228 MHz 13C{1H}-NMR-controlled titration of
1,2-ethanebisphosphonic acid vs. NaOH. For data see exp. 2 in Table 32.
Figure 78 demonstrates the characteristic changes in 3JPP as a function of :
Figure 78
85
80 Jxx´ (D2O) Jxx´ (H2O)
75
70
65
60
55
50
0 1 2 3 4 5 6
Figure 78: ,3JPP-plot obtained from 50.228 MHz 13

C{1H}-NMR-controlled titration of
1,2-ethanebisphosphonic acid vs. NaOH in H2O (green) and NaOD in D2O (red).
x-axis: . y-axis: 3JPP [Hz].
Table 32
Experiment 1 2 3
Method Potentiometry NMR NMR
Volume 55 ml H2O 55 ml H2O 55 ml D2O
Ligand 0.005 m 0.257 m 0.247 m
Ion buffer 0.1 m NaNO3 - -
Titrator 0.0998m NaOH 3.99 m NaOH 3.32 m NaOD
pK1 1.37 1.13 1.54 (D2O)
pK2 2.47 2.85 3.04 (D2O)
pK3 7.01 7.59 8.17 (D2O)
pK4 8.58 9.37 9.95 (D2O)
TABLE 32: Potentiometric titrations of 1,2-Ethanebisohosphonic acid. Calculations
with ITERAX (case 1) and GENOPT (case 2 and 3). pH-readings in D2O were con-
verted using pD = pH + 0.41 (25°C, [31]).

A minimum in 3JPP is found for  = 3 possibly indicating the increased population of a
hydrogen bond supported gauche rotamer symbolized by Figure 79:
Figure 79
O O
O O O O H
P P O
O
H H H P
O
C C
H H H H
P H
O O
O
Figure 79: Rotamers of 1,2-ethanbisphosphonic acid species L4- and HL3-
Using Grossmann´s results [32] for trans 3JPP = 67 Hz and gauche 3JPP = 3 Hz at  =
4 it is tempting to guess a population of 60% trans- and 40% gauche-rotamers at  =
3. H4L, H3L-, H2L2- and L4- prefer trans conformations. X-ray diffraction studies
confirmed this for solid H4L (Peterson et. al. [33]) and L4- (Becker [34]). A solvent
isotope effect is observed: 3JPP is larger in D2O than in H2O leading to the conclusion
that proton bridges might be more stable than deuteron bridges.
Numerical results for ion specific 3JPP are listed in Table 33:
Table 33
H2O D2O
H4L 82.9 Hz 85.5 Hz
H3L- 69.9 Hz 71.3 Hz
H2L2- 69.2 Hz 71.2 Hz
HL3- 46.2 Hz 49.0 Hz
L4- 68.5 Hz 68.3 Hz
Table 33: Ion specific 3JPP [Hz] from 13C{1H}-NMR-controlled titrations of 1,2-ethane-
bisphosphonic acid vs. NaOH in H2O and NaOD in D2O, iterated with SON4 [3c].
These date were obtained for experimental conditions listed in Table 32 and calcula-
ted molar fractions shown in Figure 80:
Figure 80
1,2
0,8
LH4
LH3 -
0,6
LH2 2-
0,4 LH3-
L4-
0,2
0
0 1 2 3 4 5 6
Figure 80: Molar fraction x,-diagram for 50.228 MHz 13C{1H}-NMR-controlled titra-
tion of 1,2-ethanebisphosphonic acid vs. NaOH in H2O. x-axis: degree of titration .
y-axis: molar fraction x. Calculations with GENTIT.

4.5 Analysis of mixtures by NMR controlled titrations
Case 15: 1-Phosphonopropane-1,2,3-tricarboxylic acid (PPTC)
COOH COOH COOH

H C C C H
H H P(O)(OH)2
a mixture of threo and erythro diastereomers.
An example for second order acids of type H5L.

31
P{1H}-, 13C{1H}-, 1H- and 1H{31P }-NMR studies.
In search of powerful complexing agents 1-phosphonopropane-1,2,3-tricarboxylic
acid was synthesized as a mixture of threo and erythro forms:
H H H H
(HO)2(O)P C COOH HOOC C P(O)(OH)2 HOOC C P(O)(OH)2 (HO)2(O)P C COOH
H C COOH HOOC C H H C COOH HOOC C H
H C COOH HOOC C H H C COOH HOOC C H
H H H H
RS/SR / erythro SS/RR / threo
a fact, which was discovered by three different methods: ion chromatography, routine
31
P{1H}- and 13C{1H}-NMR spectroscopy [2m, 2n, 3c, 35]. Two components in a molar
ratio of 69:31, 66:34 and 70:30 resp. were found but no stereo specific assignments
were made. Conventional high precision titration of the technical product and iteration
of the standard pH,V-data lead to averaged dissociation and stability constants. No
access to reliable diastereo specific parameters was possible in the beginning phase
of such investigations.
But combining the armory of 1D and 2D NMR together with NMR controlled titrations
and classical high precision titrimetry lead to an unequivocal assignment of diastere-
omers, diastereo specific pKi data, ion specific NMR parameters and an educated
guess for rotameric equilibria for individual dissociation species HiLi-5. Some details
of these intriguing problems and solutions are shown below. More comprehensive
descriptions may be found in [2n, 2m, 3c].
Both diastereomers have individual sets of NMR parameters. 81 MHz 31P{1H}-NMR
controlled titrations of PPTC clearly show the individual signals for two diastereome-
ric forms as shown in Figures 81 a and b:

Figure 81a
19
18
17
16

[ppm]
15
14
13
12
0 1 2 3 4 5 6
Figure 81a: 80.015 MHz 31P{1H}-NMR controlled titrations of PPTC. ,-plot.
Figure 81b
19
18
17
16

[ppm]
15
14
13
12
0 2 4 6 8 10 12 14
pH
Figure 81b: 80.015 MHz 31P{1H}-NMR controlled titrations of PPTC. pH,-plot.

Based upon 200 MHz 1H-integrals (not shown) the ratio of components was deter-
mined to be 64:36. This result was used to iterate the high precision pH,V-titration
curve of a mixture of two pentavalent acids H5L and H5L´ to yield two individual data
sets for pKi (i = 1-5) listed in Table 34. (Exp. data: 600 data points, 50 ml titrand hol-
ding 4.65632 mmol PPTC, I = 1, NaNO3, titrator 0.100313 m NaOH, max. 30 ml,
increments 0.05 ml/step, temperature 25°C, calculations by ITERAX).
Table 34
pKi RS/SR / 64% RR/SS / 36%

1 1.12  0.02 1.02  0.07
2 3.75  0.01 3.37  0.03
3 4.78  0.01 4.87  0.03
4 6.63  0.01 6.30  0.02
5 9.13  0.01 9.19  0.02
Table 34: pKi-data for RS/SR and RR/SS diastereomers of PPTC.
50.228 MHz-13C{1H}-NMR controlled titrations, shown in Figure 82 and Figure 83, in
combination with 200 MHz 1H-NMR spectral analysis and iteration lead to an unequi-
vocal assignment of type and concentration for each diastereomer.
Ion specific NMR parameters and dissociation constants specific for each
diastereomer were calculated and listed in Table 35 and Table 36:

Figure 82
Figure 82: 50.228 MHz-13C{1H}-NMR controlled titrations of PPTC + 6.2 eq. of NaOH
vs. HNO3. Carboxylic groups C1* to C3*.
Figure 83
Figure 83: 50.228 MHz-13C{1H}-NMR controlled titrations of PPTC + 6.2 eq. of NaOH
vs. HNO3. Skeleton carbons from C1 to C3.

Table 35.
1 3
Species C JPC C C C JPC2* C C
C1 C1* C2 C2* C3 C3*
H5L 49.52 127.1 175.86 42.74 179.28 14.2 36.58 178.38
1 1.46 11.7 1.48 0.87 1.0 -0.7 0.98 0.14
H4L- 50.98 115.4 177.34 43.61 180.28 13.5 37.56 178.52
2 2.42 1.2 1.36 2.36 2.69 -5.4 2.83 2.30
H3L2- 53.40 114.2 178.70 45.93 182.97 8.1 40.39 180.82
3 0.83 0.6 0.89 0.66 0.54 -1.6 2.38 1.98
H2L3- 54.23 113.6 179.59 46.59 183.51 6.5 42.77 182.81
4 1.98 1.1 1.05 0.99 1.00 2.7 0.69 1.13
HL4- 56.21 112.5 180.64 47.58 184.51 9.2 43.46 183.94
5 1.61 0.0 2.96 2.84 1.86 7.4 0.27 1.04
L5- 57.82 112.5 183.6 50.42 186.37 16.6 43.73 184.98
Table 35: Ion specific chemical shifts C [ppm], coupling constants nJPC [Hz] and
gradients i [ppm or Hz] for RS/SR of PPTC. 3JPC3 not resolved and not iterated.
Table 36.
1 3 3
Species C JPC C C C JPC2* C JPC3 C
C1 C1* C2 C2* C3 C3*
H5L 50.12 124.5 174.67 42.56 179.19 13.4 36.38 6.2 178.81
1 0.87 -5.9 1.73 0.55 1.04 -0.9 0.16 -1.0 0.46
H4L- 50.99 118.6 176.40 43.09 180.23 12.5 36.54 5.2 179.27
2 2.54 -0.6 2.54 1.52 1.97 -3.0 2.98 -0.7 2.46
H3L2- 53.53 118.0 179.14 44.61 182.20 9.5 39.52 4.5 181.73
3 1.32 -0.1 1.23 1.69 2.01 -3.2 2.11 3.4 2.23
H2L3- 54.85 117.9 180.37 46.30 184.21 6.3 41.63 7.9 184.00
4 0.82 0.2 0.15 1.53 0.89 1.9 0.54 2.6 0.12
HL4- 55.67 118.1 180.52 47.83 185.10 8.2 42.17 10.5 184.12
5 0.93 -1.1 2.28 0.43 1.01 1.6 -0.44 -3.3 1.22
L5- 56.60 117.0 182.80 48.26 186.11 9.8 41.73 7.2 185.34
Table 36: Ion specific chemical shifts C [ppm], coupling constants nJPC [Hz] and
gradients i [ppm or Hz] for RR/SS of PPTC.

The first and the final deprotonation steps for both diastereomers are P-OH  P-O-.
This follows from 1JPC. (and from . 80.015 MHz 31

P{1H}-NMR controlled titrations
shown above). Deprotonation steps 2-4 are determined by C-OH  C-O-. 13

C{1H}-
NMR controlled titrations indicate a dominating deprotonation sequence, C2* - C3* -
C1* for diastereomer 1, but for diastereomer 2: C1* - C3* - C2* is deduced.
At this stage it appeared inevitable to perform the full cycle of HR 1H-NMR analysis
and iteration in order to define unequivocally constitution and conformation of the
diastereomers discussed hitherto in a more global sense above.
In principle each diastereomeric pair of PPTC (RS/SR or RR/SS) gives rise to one
specific ABCDX system contributing with specific statistic weight to the total experi-
mental spectrum. PPTC was adjusted to  = 3 with NaOH in a solvent mixture of 10
% D2O and 90 % H2O. 1H-, 31P,- 31P{1H}-, 13C{1H}, HH-COSY and HC-COSY-NMR-
spectra were performed. Automated analysis and iteration of a corresponding 500
MHz 1H-NMR-spectrum (see Figure 84) was performed using the fragmentation
technique available in WINDAISY. Subsequent stereo specific interpretation of 3JHH
coupling constants identified the higher populated pair of enantiomers as the RS/SR
form.
Figure 84
3.2 3.1 3.0 2.9 2.8 2.7 2.6 2.5

(ppm)
Figure 84: 500.133 MHz-1H-NMR of the genuine diastereomeric mixture of PPTC at
 = 3. Upper: experimental, lower: simulated spectrum.
After understanding the stereo specific NMR parameters (see Table 37) for the titra-
tion status of  = 3 it was possible to analyze and iterate the more complex spectra
for all relevant  = 0 to  = 5. Taking into account the molar fractions for each proto-
lytic species HiLi-5 of each diastereomer ion specific NMR parameters were extracted
and listed in Table 38 and Table 39 [2n].

Table 37
RS/SR H H   HW
H1 2.915 1457.844 (5) 2.11
H2 3.139 1569.706 (8) 2.11
H3 2.549 1274.634 (6) 2.22
H4 2.504 1252.259 (6) 1.95
3
JH1H2 6.143 (9)
3
JH2H3 6.461 (10)
3
JH2H4 8.227 (10)
2
JH3H4 -15.145 (12)
2
JPH1 -23.374 (20)
3
JPH2 7.706 (18)
RR/SS H H   HW
H1 2.977 1488.858 (8) 2.16
H2 3.127 1563.879 (15) 2.34
H3 2.616 1308.502 (8) 2.20
H4 2.504 1252.362 (10) 2.14
3
JH1H2 6.479 (12)
3
JH2H3 10.459 (15)
3
JH2H4 4.006 (10)
2
JH3H4 -16.095 (11)
2
JPH1 -21.166 (20)
3
JPH2 14.414 (23)
Table 37: 500.133 MHz-1H-NMR parameters for diastereomers RSSR and RRSS of
PPTC at  = 3, iterated with WINDAISY. JH1H3, JH1H4, JPH3 and JPH4 not resolved. Final
Sum of Squares : 8.585, Number of points: 2110, R-Factor: 0.261852. HW: Halfwidth
[Hz]., H [ppm].
Table 38
Parameter H5L H4L- H3L2- H2L3- HL4- L5-

H1 3.449 3.275 3.068 2.979 2.811 2.609
H2 3.470 3.416 3.266 3.184 3.220 3.060
H3 3.069 3.085 2.779 2.570 2.723 2.956
H4 2.955 2.955 2.738 2.544 2.513 2.251
H1 1724.789 1637.972 1534.629 1489.825 1405.754 1304.639

H2 1735.253 1708.654 1633.480 1592.486 1610.234 1530.257
H3 1534.873 1542.710 1389.958 1285.346 1361.933 1478.457
H4 1477.787 1478.038 1369.433 1272.444 1256.963 1125.584
JH1H2 11.079 9.152 7.546 5.723 8.337 11.043
JH2H3 3.844 3.925 5.435 6.807 4.636 3.546
JH2H4 7.751 8.182 8.883 7.873 10.649 12.083
JH3H4 -17.350 -17.233 -15.660 -15.072 -15.403 -14.484
JH1P -22.507 -21.444 -22.684 -23.705 -21.511 -18.356
JH2P 12.030 10.167 8.362 7.416 9.581 8.435
Table 38: Ion specific NMR parameters for each protolytic species HiLi-5 of RS/SR
PPTC.
Table 39
Parameter H5L H4L- H3L2- H2L3- HL4- L5-

H1 3.622 3.459 3.271 3.035 2.889 2.855
H1 3.453 3.388 3.302 3.203 3.037 2.997
H1 2.992 2.999 2.946 2.681 2.478 2.534
H1 2.901 2.969 2.809 2.543 2.483 2.627
H1 1811.654 1730.116 1635.625 1517.819 1444.746 1427.851

H2 1726.951 1694.365 1651.450 1601.908 1519.068 1498.900
H3 1496.507 1499.911 1473.565 1340.809 1239.199 1267.134
H4 1451.071 1484.759 1404.637 1271.758 1241.816 1313.833

JH1H2 5.884 5.688 5.149 6.258 8.183 6.743
JH2H3 10.146 10.913 10.503 10.438 10.574 11.995
JH2H4 3.404 2.795 3.379 4.084 4.085 3.029
JH3H4 -17.424 -17.152 -16.655 -16.114 -15.780 -15.679
JH1P -24.281 -23.568 -22.961 -21.499 -18.895 -19.322
JH2P 9.000 7.485 13.303 15.602 10.410 10.116
Table 39: Ion specific NMR parameters for each protolytic species HiLi-5 of RR/SS
PPTC.
Experimental comments
Chemicals: Standard Chemicals were obtained from commercial sources in analy-
tical qualities. Some model systems were home made, others were obtained from
laboratories in universities and in industry as shown in Table 40:
Table 40
Case Compound Ref

1 CH3-COOH Merck
2 CH3-CH2-COOH Merck
3 (CH3)2P(O)OH Hoechst a
4 CH3-PO3H2 Hoechst a
5 Leu-Ala Aldrich
6 CH2=CF-CH2-C(CH3)(NH2)-COOH Haufe b
7 CH3C(H)(PO3H2)2 Hoechst a
8 C6H5-CO-PO3H2 Breuer c
9 (CH2-COOH)2 Merck
10 (CH2-P(CH3)(O)OH)2 Henkel d
11 HOOC-CH2-CH2-P(CH3)(O)OH Hoechst
12 LiOOC-CH2-P(O)(OLi)2 Breuer c
13 Glufosinate, Phosphinothricine Hoechst a
14 (CH2-P(O)(OH)2)2 Hoechst a
15 PPTC Henkel d
Table 40: Model compounds 1 to 15 used in this study. Thanks are due to: a: Dr. H.
J. Kleiner, Dr. W. Klose, Hoechst AG, Frankfurt; b: Prof. Dr. G. Haufe, Münster; c:
Prof. Dr. E. Breuer, Jerusalem; d: Dr. K. H. Worms, Dr. P. Christophliemk, Dr. Blum,
Dr. Kranz, Henkel KGaA, Düsseldorf.

Procedures: Dissociation and stability constants, given as pK and log values have
been determined by potentiometric titrations under standard experimental conditions:
25.0 ± 0.1 °C temperature and 0.1 M ionic strength (adjusted with addition of NaNO 3,
NaCl or tetramethylammonium chloride TMACl). The ligand concentration varied typi-
cally between 0.002 and 0.008 M. Iterative calculations used programs GENOPT,
ITERAX or WINSCORE. For details see references in examples given above.
References
1. Publications Heinrich-Heine-University Düsseldorf:
a) G. Hägele; UNI-MOSAIK 4 57 (1988) Universität Düsseldorf
b) M. Grzonka und G. Hägele; Workshop "Computer in der Chemie", Software-
Entwicklung in der Chemie 2 229 (1988), Springer Verlag, Berlin. (Edit. J.
Gasteiger).
c) G. Hägele und M. Grzonka; Workshop "Computer in der Chemie", Software-
Entwicklung in der Chemie 3 181 (1989). Springer-Verlag, Berlin (Hrsg. J.
Gasteiger)
d) G. Hägele, M. Grzonka, H.-W. Kropp, J. Ollig und H. Spiegl; Phosph., Sulf.
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2. Ph. D. Thesis, Heinrich-Heine-University Düsseldorf:
a) U. Fischer, 1986; b) A. Gaedcke, 1986; c) M. Grzonka, 1989; d) M. Batz,
1989; e) H. Papadopoulos, 1990; f) U. Prior, 1992; g) E. Wilke, 1992; h) J.
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3. Diploma Thesis, Heinrich-Heine-University Düsseldorf:
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1995; e) G. Sievers; 1996; f) S. Hermens; 1996; g) C. Tillmann; 1997. i) C.
Pfaff; 1998; j) C. Uhlemann; 1999.

4. DER Thesis, Université de Nantes / Heinrich-Heine-University Düsseldorf,
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17. Thanks are due to A. Greiner (Düsseldorf) and his skills in glassblowing using
advice given by W. Bremser (BASF).
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Lindner and J. Ollig; to be published.
-----------------------------------
Editorial comments:
This review attempts to compress results from a variety of research projects, on dif-
ferent levels, performed by the Düsseldorf, spanning a period of 16 years. The lan-
guage for handling the object has undergone changes and henceforth some indivi-
dual NMR and analytical parameters may appear to bear several symbols (e. g.: x or
 for molar fraction, HW or HWB for spectral halfwidth). The reader is asked to neg-
lect kindly those inconsistencies.

NMR Controlled Titration

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NMR Controlled Titration

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NMR controlled Titration 1

NMR-controlled Titrations – Principles and Progress:

Monitoring protonation and complex formation equilibria in aqueous solutions

Kroppa, Johannes Olliga, Stephan Hermensa, Sven Augnera, Carsten Uhlemanna,

Christian Pfaffa, Zoltan Szakacsa, Toni Kellerc, Martin Rindlispacherc, Manfred

Universitätsstrasse 1, D-40225 Düsseldorf, Germany.

Phone: ++49-211-81-12288, ++49-211-81-13140, Fax: ++49-211-81-11854

A hyphenated technique, termed NMR controlled titration, elucidates protolytic and

complex formation equilibria. Stability constants, dissociation constants and ion-

derive the molecular structure of species involved in dynamic processes.

NMR, titration, NMR-controlled titration, protolysis, complex formation, stability

constants, dissociation constants, ion-specific NMR parameters, macroscopic

dissociation, microscopic dissociation, intramolecular rotation.

Multifaceted interests in structural and analytical chemistry combine with such of

important reactions in biorelevant fields involve protolytic equilibria and complex

formation processes. NMR spectroscopic methods assisted by classical analytical

performed by laborious series of single sample NMR experiments, more efficient,

PC-guided, automated procedures were developed and applied successfully to a

wide range of structural problems, as will be demonstrated with some examples in

Our main aim is to introduce readers to the technical aspects of a modern

hyphenated technique combining aspects of potentiometric titration and NMR

spectroscopy. This technique yields two- and three-dimensional graphical

representations where NMR parameters are correlated with analytical parameters.

This method is called “NMR-controlled titration“, “titration-dependent NMR-

spectroscopy“, or just “NMR-Titration” where the choice of terms depends largely

on interests of individual users.

NMR-controlled titrations lead to characteristic dissociation and stability constants

or microscopic equilibria. While ,  and J are subject to averaging processes gover-

research should be directed towards these interesting aspects.

NMR-controlled titration is an efficient tool to characterize carboxylic acids, amino

acids, peptides and related structures involving phosphorylated and/or fluorinated

analogues. Some typical structures are shown in Table 1:

CH3 COOH CH3 P(O)(OH)2 (CH3)2P(O)OH

NH2 CH2 COOH H2N CH2 P(O)(OH)2 H2N CH2 P(CH3)(O)OH

HOOC CH2 COOH HOOC CH2 P(O)(OH)2 HOOC CH2 P(CH3)(O)OH

N(CH2COOH)3 N(CH2PO3H2)3 [NaHxN(CH2PO3)3]x-5

(CH2N(CH2COOH)2)2 (CH2N(CH2PO3H2)2)2 [CdHx(CH2N(CH2PO3)2)2)]2+x-8

CH2 CH NH2 CH2 CH P(O)(OH)2 CH2 CH NH-CO-CH2-PO3H2

COOH COOH COOH COOH COOH COOH

CH2 CHOH CH2 CH2 CH CHOH CH2 CH O CH2

COOH COOH COOH COOH COOH COOH COOH COOH COOH

Table 1: Some typical structures investigated by NMR-controlled Titrations

Resonances from standard 1H-, 13C{1H}-, 19F-, 1H{31P}-, 31P{1H}-NMR experiments

tion of metal complexes.

Basics, progress and a variety of applications of this technology studied in Düsseldorf

are described in [1-4] and references given herein.

2.1. Description of Acids and Bases – Macroscopic Protolytic Equilibria -

Dissociation and Stability Constants

centers in (+HN)b-R-(OHo)a. This general concept may also be used conveniently to

characterize simpler units such as parent amines, carboxylic acids, organophospho-

rus acids, sulfonic acids, etc. as shown in Table 2:

Short name Formula a b n

TABLE 2: Classification of acid base systems.

and Y represent univalent ions bearing positive or negative charges respectively, as

1 Sodium salt Na+ 1<xa - 0

2 Hydrogenchloride salt - 0 Cl- 1<y<b

Table 3: Classification of salts.

The protonation equilibrium of a n-valent base is described by the reactions:

i H+  La  HiLi-a (i = 1, 2, ..., n) (1)

and by the brutto stability constants:

sponding logarithmic expressions pK ai , pK bi for acids and bases by:

(a) pKai  lgn1i  lgni ; (3)

(c) pKbi  pK W  lgi  lgi1 ;