Direct Separations of the Six Positional Isomers of Disubstituted Benzoic
Acids Using ACQUITY UPC 2 Torus Columns
John P. McCauley
Waters Corporation, New Castle, Delaware, USA
A P P L I C AT I O N B E N E F I T S
■■
Six different isomers of both
dimethoxybenzoic scid and
dimethylbenzoic acid are separated.
■■
Direct separation is achieved, without
derivatization of the acids, using the
ACQUITY UPC.2 ®
■■
Waters ® ACQUITY UPC 2 Torus™ phases,
DIOL and 2-PIC, are able to accomplish
these challenging separations quickly
and efficiently.
■■
Dimethylbenzoic acid (DMBA) is a
clinical marker for industrial exposure to
trimethybenzene (TMB). The separation of
all of the six positional isomers is required.
■■
Current technology for the DMB isomer
separations and measurements use GC
techniques, which require the extra step
of pre-formation of silyl derivatives.
INT RODUC T ION
Positional isomers are compounds that only differ in the location of the
substituent groups. Each one of these isomers differs only slightly in physical
properties from the others. For this reason, positional isomer mixtures can
often pose serious challenges in attempts to achieve their separations. This
study illustrates the rapid and efficient separation of all six of the possible
positional isomers of both the six disubstituted dimethoxybenzoic acids and
also the six dimethylbenzoic acids (DMBA) (Figure 1). This latter example is
particularly interesting, as DMBA is a metabolite of trimethylbenzene (TMB)
and its appearance in human urine can be used as a clinical marker for
measuring industrial exposure to TMB.1
Figure 1. Dimethoxybenzoic Acids and Dimethylbenzoic Acids (DMBA).
WAT E R S S O LU T I O N S
ACQUITY UPC 2 System
ACQUITY UPC 2 Torus Columns
Waters Vials
MassLynx ® Software
KEY WORDS
UPC,2 Torus DIOL and 2-PIC columns,
positional isomers, dimethoxybenzoic
acid, dimethylbenzoic acid, DMBA,
trimethylbenzene, TMB, metabolite,
clinical marker
1
E X P E R IM E N TA L
Sample description
The six isomer standards for the disubstituted
dimethoxybenzoic acids and the dimethylbenzoic
acids were obtained from Sigma-Aldrich and from
TCI Americas respectively. Samples were dissolved
in methanol at a concentration of 0.1 mg/mL.
Dimethoxybenzoic
Acid Method 1
Dimethoxybenzoic
Acid Method 2
Dimethylbenzoic
Acid Method 3
ACQUITY UPC 2
Torus DIOL 1.7 μm,
3.0 x 100 mm
(p/n 186007611)
ACQUITY UPC 2
Torus 2-PIC 1.7 μm,
3.0 x 100 mm
(p/n 186007602)
ACQUITY UPC 2
Torus 2-PIC 1.7 μm,
3.0 x 100 mm
(p/n 186007602)
1.2 mL/min
1.2 mL/min
1.5 mL/Min
Gradient %B
4–20%
9–40%
3.3–35%
Run time
3.5 min
6.0 min
8.0 min
ACQUITY UPC 2
Column
Flow rate
UPC 2 conditions
Temperature
60 °C
60 °C
60 °C
See Table 1
Backpressure
2175 psi
2175 psi
2175 psi
Mobile phase A: Supercritical CO2
Mobile phase B: Methanol containing
0.2% formic acid
Vials: 12 x 32 mm glass screw
neck vials with Quick
Thread Lectra Bond caps
with preslit PTFE/Silicone
septa (p/n 186000307C)
PDA detector:
240 nm wavelength
compensated
(340–410 nm)
MS conditions
System:
Waters TQD
Ionization mode:
ESI-
Acquisition range: 150–650 amu
Capillary voltage: 3.58 kV
Cone voltage:
20 V, SIR:
DMBA 149.1 amu,
dwell 0.1
Chromatography and
MS Software: MassLynx
Table 1. UPC 2 method conditions.
R E S U LT S A N D D I S C U S S I O N
Due to the very similar physical properties of positional isomers, their separations
have often proven to be quite challenging. Substitution patterns, such as groups
located in the ortho, meta, and para positions of the aromatic ring do have
effects on the electronic contributions to aromatic resonance. Ortho substitutions
also contribute to the level of the steric hindrance in the carboxylic acid group
environment, which affects its ability to maintain coplanarity with the aromatic
ring. All of these subtle differences contribute to small differences in pi cloud
effects in the ring and to the pK a of the acid itself. These small steric and electronic
differences are also manifested in the different extinction coefficients for each of
the positional isomers. Such subtle property differences are also the basis upon
which separation of the individual positional isomers is possible. Isomer separations
are often required for starting material qualifications, reaction monitoring and
in an example which will be shown here, for following a particular isomer which
is a known metabolite resulting from exposure to a toxic material and which is
used as a clinical marker for industrial exposure.1 The ACQUITY UPC 2 System
utilizing ACQUITY UPC 2 Torus Column chemistries is capable of such separations,
as several examples presented here can demonstrate. The Torus Column family is a
new generation of stationary phases designed specifically for UPC 2 ® separations.
Torus Technology is built upon proven BEH (ethylene bridged hybrid) particles
as a proprietary two stage bonding process. The first stage of the bonding controls
the retention characteristics of the phase and the second imparts the observed
selectivity. In the first example shown separation of six isomers of dimethoxybenzoic
acids is accomplished using the ACQUITY UPC 2 Torus DIOL Column (Figure 2).
Separations with a diol phase are based on polar interactions and hydrogen
bonding interactions. This same set of isomers can also be separated with the
ACQUITY UPC 2 Torus 2-PIC Column chemistry (Figure 3).
Direct Separations of the Six Positional Isomers of Disubstituted Benzoic Acids Using ACQUITY UPC2 Torus Columns
2
Isomer
Elution
Time
Peak
Order
2,3
1.88
2
2,4
2.19
4
2,5
1.78
1
2,6
2.94
6
3,4
2.30
5
3,5
1.97
3
4.8e-1
4.6e-1
4.4e-1
4.2e-1
4.0e-1
3.8e-1
3.6e-1
3.4e-1
3.2e-1
3.0e-1
2.8e-1
2.4e-1
2.2e-1
2.0e-1
1.8e-1
1.6e-1
1.4e-1
1.2e-1
1.0e-1
8.0e-2
6.0e-2
4.0e-2
2.0e-2
0.0
-0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90 2.00 2.10 2.20 2.30 2.40 2.50 2.60 2.70 2.80 2.90 3.00 3.10 3.20 3.30
Isomer
4.2e-1
4.0e-1
3.8e-1
3.6e-1
3.4e-1
3.2e-1
3.0e-1
2.8e-1
2.6e-1
Elution
Time
Peak
Order
2,3
3.96
5
2,4
2.27
1
2,5
3.39
3
2,6
5.65
6
3,4
3.21
2
3,5
3.84
4
Time
Figure 2. Separation of the 6
isomers of dimethoxybenzoic
acid using the ACQUITY UPC 2
Torus DIOL Column.
2.4e-1
AU
AU
2.6e-1
2.2e-1
2.0e-1
1.8e-1
1.6e-1
1.4e-1
1.2e-1
1.0e-1
8.0e-2
6.0e-2
4.0e-2
2.0e-2
0.0
-0.00 0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
2.00
2.20
2.40
2.60
2.80
3.00
3.20
3.40
3.60
3.80
4.00
4.20
4.40
4.60
4.80
5.00
5.20
5.40
5.60
5.80
Time
6.00
Figure 3. Separation of the 6
isomers of dimethoxybenzoic
acid using the ACQUITY UPC2
Torus 2-PIC Column.
Direct Separations of the Six Positional Isomers of Disubstituted Benzoic Acids Using ACQUITY UPC2 Torus Columns
3
The ACQUITY UPC 2 2-PIC phase also has the possibility of separating with acid-base and pi-pi interactions,
and in fact the isomer elution orders are different on the two different phases. The ACQUITY UPC 2 Torus 2-PIC
Column is also capable of separating all six of the positional isomers of the dimethylbenzoic acids (DMBA)
which as mentioned before, has been used as a metabolic clinical marker (Figure 4). The 3,4 dimethyl isomer,
peak 4 in Figure 4, which is of the greatest clinical interest is well separated from the other isomers. The
chromatography figures shown are all wavelength compensated UV traces, but it is also possible to monitor
these peaks using negative ion mass spectrometry. While all the peaks are isobaric, MS monitoring does allow
for gains in sensitivity and specificity, which is especially important in clinical metabolite monitoring. In fact,
using SIR (selective ion recording) methods it is possible to reach much higher sensitivities which are well
below the LOQ for UV monitoring, while at the same time reducing interferences from many matrix effects.
The SIR recording of a chromatogram from a more dilute DMBA sample mix, using the same chromatographic
method as before, is shown over layed with a standard for the 3,4 DMBA isomer alone (Figure 5). Actual
clinical applications are beyond the scope of this study, although it is clearly shown that it is possible to
employ ACQUITY UPC 2 methods, working with the ACQUITY UPC 2 Torus Columns, using techniques which are
derivatization free, to monitor these positional isomers using SIR MS detection. This is compared to the current
GC methods, which do require a silylation derivative forming reaction step, the efficiency of which is another
possible source for error.1
Isomer
Elution
Time
Peak
Order
2,3
6.37
5
2,4
4.17
1
2,5
4.62
2
2,6
6.97
6
3,4
5.30
4
3,5
4.93
3
2.7e-1
2.6e-1
2.5e-1
2.4e-1
2.3e-1
2.2e-1
2.1e-1
2.0e-1
1.9e-1
1.8e-1
1.7e-1
1.6e-1
AU
1.5e-1
1.4e-1
1.3e-1
1.2e-1
1.1e-1
1.0e-1
9.0e-2
8.0e-2
7.0e-2
6.0e-2
5.0e-2
4.0e-2
3.0e-2
2.0e-2
1.0e-2
0.0
Time
-0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 6.20 6.40 6.60 6.80 7.00 7.20 7.40 7.60 7.80 8.00
Figure 4. Separation of the 6
isomers of dimethylbenzoic
acid using the ACQUITY UPC 2
Torus 2-PIC Column.
Direct Separations of the Six Positional Isomers of Disubstituted Benzoic Acids Using ACQUITY UPC2 Torus Columns
4
%
3,4
0.40
0.80
1.20
1.60
2.00
2.40
2.80
3.20
3.60
4.00
4.40
4.80
5.20
5.60
6.00
6.40
6.80
7.20
7.60
8.00
%
1
-0.00
3,4
2
-0.00
0.40
0.80
1.20
1.60
2.00
2.40
2.80
3.20
3.60
4.00
4.40
4.80
5.20
5.60
6.00
6.40
6.80
7.20
7.60
Time
8.00
Figure 5. Separation of the 6
isomers of dimethylbenzoic
acid using the ACQUITY UPC 2
Torus 2-PIC Column. MS SIR
monitoring of DMBA employed
at sensitivity levels below
the UV LOQ. Overlay with
3,4 standard.
C O N C LU S I O N S
Reference
The positional isomers of two examples of disubstituted benzoic
acids are rapidly and efficiently separated into their individual
components with ACQUITY UPC 2 Torus Column chemistries using
the ACQUITY UPC 2 System. This separation is accomplished
without any need for prior derivatization reactions, as is necessary
in the competing GC methods.1 In one example, for DMBA on the
ACQUITY UPC 2 Torus 2-PIC Column, this separation is
demonstrated to be highly sensitive and selective with the
use of MS SIR monitoring detection techniques.
1. Schettgen, T. and Alt, A. Dimethylbenzoic acids-metabolites of
trimethylbenzene [Biomonitorong Methods, 2010]. The MAK Collection
for Occupational health and Safety. 237–259.
Waters, T he Science of W hat’s Possible, ACQUITY UPC,2 UPC,2 and MassLynx are registered trademarks of Waters Corporation.
Torus is a trademark of Waters Corporation. All other trademarks are the property of their respective owners.
©2014 Waters Corporation. Produced in the U.S.A. November 2014 720005221EN AG-PDF
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