Journal of Chromatographic Science 2013;51:893– 898
doi:10.1093/chromsci/bms186 Advance Access publication November 23, 2012
Article
Analysis of Glyoxal and Related Substances by Means of High-Performance Liquid
Chromatography with Refractive Index Detection
Zhiyong Zhang1*, Dishun Zhao2 and Baoyun Xu3
1
Tianjin Key Laboratory of Applied Catalysis Science and Technology and State Key Laboratory for Chemical Engineering (Tianjin
University), School of Chemical Engineering, Tianjin University, Tianjin, 300072, China, 2College of Chemistry and Pharmaceutical
Engineering, Hebei University of Science and Technology, Shijiazhuang 050018, China, and 3Shanghai Research Institute of
Chemical Industry, Shanghai 200062, China
*Author to whom correspondence should be addressed. Email: [email protected]
Received 14 December 2011; revised 24 October 2012
A simple and rapid method is described for the analysis of glyoxal
and related substances by high-performance liquid chromatography
with a refractive index detector. The following chromatographic
conditions were adopted: Aminex HPX-87H column, mobile phase
consisting of 0.01N H2SO4, flow rate of 0.8 mL/min and temperature of 6588 C. The application of the analytical technique developed
in this study demonstrated that the aqueous reaction mixture produced by the oxidation of acetaldehyde with HNO3 was composed
of glyoxal, acetaldehyde, acetic acid, formic acid, glyoxylic acid,
oxalic acid, butanedione and glycolic acid. The method was validated by evaluating analytical parameters such as linearity, limits
of detection and quantification, precision, recovery and robustness.
The proposed methodology was successfully applied to the production of glyoxal.
Introduction
Glyoxal (Chemical Abstracts Service number: 107-22-2;
International Union of Pure and Applied Chemistry name: oxaldehyde) is an organic compound with the formula OCHCHO.
Anhydrous glyoxal has a melting point of approximately 158C.
However, it is typically supplied as an aqueous solution (usually
containing 30 –50% glyoxal) in which hydrated oligomers are
present (1). Glyoxal is used as a chemical intermediate in the
production of pharmaceuticals and dyestuffs, as a nonformaldehyde cross-linking agent in the production of a range
of different polymers, as a biocide and as a disinfecting agent.
A novel process for producing high-quality glyoxal by the
liquid phase oxidation of acetaldehyde with dilute nitric acid
was developed in the authors’ laboratory. A theoretical analysis
of the reaction system showed that some acetic acid, glyoxylic
acid, oxalic acid and glycolic acid would be produced as
by-products during the oxidation; that is, these substances
coexist in the reaction mixture. Because the system is complex
and the structures of some of these substances are similar,
qualitative and quantitative analysis is a substantial problem.
Many existing methods and techniques are not satisfactory
for the analysis of glyoxal. Because glyoxal molecules have
weak or no absorption in the ultraviolet (UV) and visible light
regions, they cannot be directly detected by spectrophotometry.
Therefore, the measurement is always conducted after derivation and the use of high-performance liquid chromatography
(HPLC) or another instrument. However, this leads to low precision for the analysis of glyoxal. Several papers have reported
the instrumental analysis of glyoxal. Edelkraut and Brockmann
(2) detected and quantified glyoxal in water samples by using
the typical 2,4-DNPH derivatization followed by HPLC with
diode array detection at 360 nm. They reported a detection
limit of 295 ng/L. Glaze et al. (3) employed aqueous-phase
PFBHA derivatization, yielding the corresponding pentafluorobenzyl oxime, followed by hexane extraction and detection by
gas chromatography (GC) –electron capture detection (ECD)
or GC –mass spectrometry (MS). A minimum detection limit of
5.1 mg/L was obtained using GC–ECD, whereas GC– MS detection resulted in a minimum detection limit of 7.7 mg/L. As a
viable alternative, derivatization using o-phenylenediamine to
measure the corresponding quinoxaline before HPLC –UV detection has been described (4). Schramm and Rinderer (5)
investigated the determination of cotton-bound glyoxal via an
internal Cannizzaro reaction by means of HPLC. Chen et al. (6)
developed a new method that combines ion chromatography
with a conductivity detector to separate and analyze mixtures
of glyoxal, glycolic acid, oxalic acid and glyoxylic acid. Both of
these methods analyzed glyoxal by converting it to glycolic
acid in the presence of a strong base. Due to the long reaction
time for glyoxal derivatization, the analysis process was timeconsuming and always led to large errors. Furthermore, the
methods mentioned previously present other disadvantages,
such as the requirement of many chemical reagents and low
sensitivity.
Most of the instrumental analysis methods have been used
for the qualitative and quantitative determination of glyoxal
only; they cannot be used to analyze the other by-products
that are produced during the reaction. The chemical analysis
method has been the primary method used in the commercial
production of glyoxal. Total aldehydes have been estimated by
the modified hydroxylamine hydrochloride method. Glyoxal
has been estimated by using sodium periodate. Nitric and
organic acids have been estimated by titrating against alkali by
using different indicators (7). However, the chemical analysis
method cannot offer the possibility of qualitative evaluation
of all components. The concentrations of acetic, glyoxylic,
formic, glycolic and oxalic acids cannot be obtained; therefore,
it is not possible to evaluate the degree of oxidation of acetaldehyde needed to improve the process. Furthermore, the
chemical analysis method has the following inherent disadvan-
# The Author [2012]. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
tages: (i) sensitivity of operating steps to a variety of factors,
(ii) time-consuming detection, and (iii) interference. It is important to develop a fast and reliable analytical method that
can simultaneously determine glyoxal and other reaction
by-products.
To date, no analytical method exists that allows the qualitative and quantitative determination of all components in the
aqueous reaction mixture produced by the oxidation of acetaldehyde with nitric acid. Beyond simple technical details, little
is known about the reaction itself. Therefore, the present study
is focused on the quantitative analysis of the glyoxal reaction
solution by HPLC using a differential refractive index detector
(RID). This method is important because the complete analysis
of the oxidation mixture is required to evaluate reaction selectivity, kinetic analysis and choice of appropriate process parameters for the optimal product yield. It will also allow
improved understanding of the oxidation reaction mechanism.
Experimental
Chemicals
All chemicals were of analytical or HPLC grade and all solutions
were prepared from deionized water. Glyoxal (40%, w/w),
oxalic acid (99.5%, w/w), formic acid (97%, w/w), glyoxylic
acid monohydrate (97%, w/w) and glycolic acid (98%, w/w)
were purchased from Merck (Darmstadt, Germany). Acetic acid
(99.5%, w/w), acetaldehyde (98.5%, w/w) and H2SO4 (98%, w/w)
were obtained from Ke Wei Co. (Tianjin, China).
Instrumentation
The HPLC analyses were performed on an LC-20A (Shimadzu,
Kyoto, Japan) instrument equipped with a degasser, LC-20AB
pump, autosampler and Aminex HPX-87H column, which had
dimensions of 300 7.5 mm and was packed with strong cationic exchange resin (Bio-Rad Labs, Richmond, CA). The
column was thermostated in a Shimadzu CTO-20A oven compartment. The system included a 20 mL loop and an RID-10A
(Shimadzu) refractive index detector. Data acquisition was conducted with LC Solution 1.22 sp1 chromatography software
(Shimadzu).
Selection of detector and chromatography column
The sample contained both organic and inorganic acids, including acetic acid, glyoxylic acid, oxalic acid and nitric acid.
Ion-exclusion chromatography was recognized as a useful technique for the separation of organic and inorganic weak acids
(8,9). Glyoxylic acid and glyoxal are thermo-sensitive materials
that have weak or no absorption in the UV or visible light
regions. Furthermore, the inorganic anions in the solution
interfered with conductivity detection. By contrast, RID detectors have been widely used as general purpose detectors. They
are especially suitable for routinely analyzing weak or non
UV-absorbing substances. Therefore, an RID detector was
selected for the research presented in this article.
The selection of a chromatographic column was another important aspect in developing an effective HPLC method. The
Aminex HPLC column packed with polystyrene divinyl
894 Zhang et al.
benzene resin has good properties, such as pH stability and
high pressure resistance, column efficiency and selectivity.
Resin-based HPLC columns can separate materials based on the
mechanisms of ion exclusion, ion exchange, ligand exchange,
size exclusion and reversed-phase and normal-phase partitioning. These multiple interaction modes offer a unique ability to
separate compounds. The Aminex column is commonly used in
the industry to analyze carbohydrates, organic acids, organic
bases and other small organic molecules in standard products
such as peptides and nucleic acids (10 –12). Many different
Aminex columns are dedicated to different classes of compounds. In this work, an Aminex HPX-87H column was
selected to conduct the investigation. In this case, two primary
kinds of separation mechanisms operated during the separation
process: ion exclusion and reversed-phase partitioning. The
two types of solutes should be reported as (i) polar ionizable
compounds whose ionization degrees depend on the pH of the
mobile phase, (ii) neutral molecules that are more or less polar.
Solutes that are partially ionized had an intermediate behavior.
Ions bearing a charge of the same sign as the functional groups
of the stationary phase (sulfonate groups) are excluded by electrostatic repulsion, whereas neutral molecules are not. Therefore,
a key to the retention mechanism for compounds that are ionizable is their ionization degree (or effective charge), and their
retention time results from the action between repulsion and
partition, because at a given pH, the ionized fraction cannot be
separated from the molecular fraction. This is why the retention characteristics and the resolution depend on the sulfuric
acid concentration. Therefore, organic acids are eluted according to their constant acidity values. For neutral molecules, only
their hydrophobicity plays a role, which shows that reversedphase partitioning is involved in the separation. In this case,
the more hydrophobic molecules eluted later than the less
hydrophobic molecules, as manifested by the increasing retention time. At the same time, nitrate ions were totally excluded
and eluted at the void volume. Experimental results showed
that the Aminex HPX-87H column could effectively separate
the sample components.
Selection of diluting solvent
Generally, the negative peak caused by water has little effect
on analysis; in this system, however, water may affect oxalic
acid analysis. When using water as a diluent, the large negative
peak will impede the oxalic acid analysis because they have
similar retention times. During this study, all standard and
sample solutions were diluted in the mobile phase (dilute sulfuric acid) to eliminate the disturbance caused by the negative
peak of water.
Determination of chromatographic conditions
To establish the operating conditions that provide the
maximum component peak resolution and minimum analysis
time (i.e., elution time), the effects of parameters that influence
resolution and analysis time must be investigated. With Aminex
columns, several chromatographic variables have an impact on
resolution and retention times and help to control the speed of
analysis. These variables included eluent composition, eluent
pH, column temperature and flow rate. After the proper
column was selected, each of these variables had to be optimized to achieve the best possible separation.
In this paper, each parameter was varied over a wide range
of values (mobile phase concentration: 0.005– 0.15N; column
temperature: 55– 658C; eluent flow rate: 0.6 –1.0 mL/min),
while keeping all other parameters constant. The value of the
parameter under study that provided the optimum resolution
and analysis time was then used in the study of the next parameter, and so on, until all parameters were adjusted to their
optimum values. Therefore, the optimum values used in further
work were chosen by a compromise between resolution and
analysis time. The analyses by HPLC were performed at 658C
under isocratic conditions. The mobile phase consisted of a
0.01N H2SO4 solution that was filtered through a 0.45 mm
Millipore membrane (Milford, MA) and degassed by sonication
for 10 min before use. The flow rate was 0.8 mL/min and the
injection volume was 20 mL.
Preparation of sample solutions
Preparation of mobile phase
Sulfuric acid (0.01N) was used as the HPLC mobile phase. In a
2 L volumetric flask, 2.00 mL standardized 10N sulfuric acid
was added and the volume was brought to 2 L with HPLC
grade water. The solution was filtered through a 0.45 mm membrane filter and degassed before use.
Figure 1. HPLC chromatogram chart of the real oxidation mixture. Peaks: 1, oxalic
acid; 2, butanedione; 3, glyoxalic acid; 4, glyoxal; 5, glycolic acid; 6, formic acid; 7,
acetic acid; 8, acetaldehyde.
Preparation of standard solution
One gram of pure substance was weighed accurately and added
to a 50 mL volumetric flask, where it was dissolved and diluted
with mobile phase. A certain volume of the solution was
diluted to 250 mL with mobile phase, mixed well and filtered
through a 0.45 mm membrane filter.
Preparation of sample solution
Samples were prepared by reacting 40% nitric acid and 40%
acetaldehyde at a molar ratio of 1:0.8 at 408C for 4 h. Then, 1 mL
of sample solution was withdrawn, diluted to 250 mL with
mobile phase and filtered through a 0.45 mm membrane filter.
Results and Discussion
Qualitative test
The system under investigation was an aqueous solution of the
reaction mixture produced by oxidation of acetaldehyde with
nitric acid. There were eight primary peaks in the chromatogram. A theoretical analysis of the reaction system showed that
acetic acid, glyoxylic acid, oxalic acid and glycolic acid were
produced during the oxidation, in addition to glyoxal (13–15).
To associate the corresponding material with each chromatographic peak, HPLC –MS and GC–MS analysis methods were
adopted. The HPLC –MS analysis results show that there are
acetic acid, glyoxylic acid, oxalic acid, glycolic acid and glyoxal
in the oxidation solution. The GC –MS analysis shows that butanedione exists in the reaction solution. Figure 1 shows a representative chromatogram of the sample. In the chromatogram,
the peak before the oxalic acid peak was a system peak due to
the variations in the concentration of H2SO4.
Figure 2. HPLC chromatogram chart of the synthetic standard solution. Peaks: 1,
oxalic acid; 2, butanedione; 3, glyoxalic acid; 4, glyoxal; 5, glycolic acid; 6, formic
acid; 7, acetic acid; 8, acetaldehyde.
The chromatogram was compared with those of solutions of
pure oxalic acid, glyoxylic acid, glyoxal, glycolic acid, acetic
acid, acetaldehyde, formic acid and butanedione. Figures 1 and
2 show that, under the selected conditions, the retention times
of most peaks corresponded to those of the pure compound(s). To illustrate this point, Figure 1 shows the chromatogram of the real reaction mixture produced by oxidation of
acetaldehyde with nitric acid, and Figure 2 shows the chromatogram of a synthetic standard solution prepared from the
eight pure components.
Calibration, detection limits and quantification limit
Quantification was conducted by the use of external standard
calibration curves. Five aqueous solutions, covering a broad
range of concentrations, were used to plot the calibration
Analysis of Glyoxal and Related Substances by Means of High-Performance Liquid Chromatography with Refractive Index Detection 895
Table I
Calibration Curve Characteristics, LOD and LOQ
Analyte
Number of points
Range of peak area (105)
Concentration range (mg/L)
Linear regression equation
R2
LOD (mg/L)
LOQ (mg/L)
Oxalic acid
Glyoxylic acid
Glyoxal
Glycolic acid
Formic acid
Acetic acid
Acetaldehyde
5
5
5
5
5
5
5
[0.13; 1.28]
[0.13; 1.38]
[0.14; 1.42]
[0.09; 1.01]
[0.08; 0.77]
[0.10; 0.99]
[0.071; 0.75]
[97.54; 980.76]
[86.12; 874.77]
[37.25; 389.04]
[72.49; 725.13]
[102.32; 1027.63]
[104.82; 1050.66]
[77.63; 783.25]
y ¼ 130.55x þ 407.51
y ¼ 158.45x – 56.99
y ¼ 364.39x þ 979.57
y ¼ 142.171x – 1197.4
y ¼ 75.00x þ 174.6
y ¼ 94.13x – 47.26
y ¼ 96.66x – 735.41
0.9999
0.9998
0.9991
0.998
0.9999
0.9978
0.9998
0.10
0.12
0.06
0.18
0.33
0.34
0.34
0.35
0.39
0.21
0.61
1.09
1.13
1.12
For the analytical method established in this paper, the LOD
and LOQ for each substance are shown in Table I.
Table II
Recovery Investigation Results of Each Component
Name of
compound
Initial amount
(mg/mL)
Added amount
(mg/mL)
Detected amount
(mg/mL)
Recovery
(%)
Oxalic acid
Glyoxylic acid
Glyoxal
Glycollic acid
Formic acid
Acetic acid
Acetaldehyde
0.85
11.70
76.47
8.45
64.22
38.88
47.70
0.42
6.02
35.82
4.23
35.0
20.15
25.18
1.30
17.87
112.18
12.87
103.20
58.58
72.46
103.5
101.3
99.85
102.3
106.2
98.85
99.12
curve for each compound. Duplicate injections were made for
each standard solution. The standard sample used in this study
was prepared from a mixture of pure oxalic acid, glyoxylic
acid, glyoxal, glycolic acid, acetic acid, acetaldehyde and formic
acid. Because the butanedione concentration in the oxidation
mixture was very low, butanedione was omitted from the
standard sample. Standard calibration solutions were prepared
from this standard solution and diluted with mobile phase by
factors of 10, 30, 50, 80 and 100. HPLC analysis of each calibration solution was performed, and the calibration curves of
every component were established using the peak area (y)
versus the component concentration (x). Linearity was determined by constructing seven calibration curves (these plots are
shown in Supplementary Figures 1– 7), each using the external
standard method at five concentration levels, as shown in
Table I. The calculated correlation coefficients (R 2) were
0.9992 or better for every compound under the established
chromatographic conditions.
In qualitative analysis, the limit of detection (LOD) is an important parameter. The LOD represents the smallest concentration that can be detected, but not necessarily exactly
quantified. In chromatography, the LOD is the injected amount
that results in a peak at a signal-to-noise ratio (S/N) of 3.
The limit of quantitation (LOQ) of an individual analytical
procedure is the lowest amount of the analyte in a sample that
can be quantitatively determined with suitable precision and
accuracy. It is generally determined by analyzing samples with
known concentrations of the analyte and establishing the
minimum level at which the analyte can be quantified with acceptable accuracy and precision. For chromatographic
methods, the LOQ can also be determined by comparing the
measured signals from samples with known low concentrations
of the analyte with those of blank samples. This establishes the
minimum concentration at which the analyte can be reliably
quantified. A typical S/N ratio is 10:1.
896 Zhang et al.
Precision and recovery
To judge the described method, the precision and recovery
were determined by experiment. The precision was investigated
six times on the same day and under the same experimental
conditions for each standard calibration solution. The relative
standard deviation (RSD) was in the range of 0.69– 3.68% (the
data are listed in Supplementary Table I). These data revealed
that the method had an acceptable degree of precision.
Recovery tests were conducted to further investigate the
accuracy of the method. Recovery was investigated by
the standard additions method. The resultant samples were
then analyzed with the method established in this paper. With
the results, the component mass detected was compared with
the known mass added. The recovery of the method was in the
range of 98.85 –106.5%, as shown in Table II.
Robustness
The robustness of an analytical procedure has been defined by
the International Conference on Harmonization (ICH) as a
“measure of its capacity to remain unaffected by small, but deliberate variations in method parameters” (16). The most important aspect of robustness is to develop methods that allow
for expected variations in method parameters (17). For the
determination of a method’s robustness, many method parameters, such as pH, flow rate and column temperature, were
varied within a realistic range and the quantitative influence of
each variable was determined. If the influence of the parameter
was within a previously specified tolerance, the parameter was
said to be within the robustness range of the method.
In this work, the robustness of the method was determined
by making slight, deliberate changes to chromatographic conditions. Table III shows the experiments performed for the
robustness evaluation and the results obtained, relative to the
percentage of recovery. As shown in the table, the recovery
remained within the range of 94 –108%. These data were subjected to an analysis of variance (ANOVA) test to determine
any significant difference between the data sets. No significant
( p , 0.05) difference in mean assay was found, because the
calculated value of F was lower than the critical value of F.
Therefore, small variations in the chromatographic parameters
did not cause significant changes in the recovery values.
In addition, the inter-day robustness evaluation was conducted and the data are listed in Table IV. No marked changes
Table III
Intra-Day Robustness Evaluation Results Calculated as the Percentage of Recovery
Operating conditions
Oxalic acid
Glyoxylic acid
Glyoxal
Glycolic acid
Acetic acid
Acetaldehyde
Formic acid
Normal*
Variation: low†
Variation: high‡
Variation: low§
Variation: high**
Variation: low††
Variation: high‡‡
Mean + SD
106.5
98.25
108.32
105.52
107.48
97.65
103.46
103.9 + 4.3
101.3
100.34
98.26
105.62
103.42
92.5
95.35
99.5 + 4.6
99.85
96.25
96.36
92.26
105.25
108.24
95.34
99.1 + 5
102.3
95.87
100.26
99.34
105.28
108.33
94.87
100.9 + 4.8
106.2
92.35
102.78
107.32
108.23
95.68
101.46
102 + 6.0
98.85
102.23
96.86
106.22
108.12
96.75
98.35
101.5 + 4.6
99.12
98.25
105.34
93.98
106.75
107.54
92.34
100.5 + 6.2
*Mobile phase 0.01N H2SO4, flow rate 0.8 mL/min, temperature 658C.
Mobile phase 0.008N H2SO4, flow rate 0.8 mL/min, temperature 658C.
Mobile phase 0.012N H2SO4, flow rate 0.8 mL/min, temperature 658C.
§
Mobile phase 0.01N H2SO4, flow rate 0.75 mL/min, temperature 658C.
**Mobile phase 0.01N H2SO4, flow rate 0.85 mL/min, temperature 658C.
††
Mobile phase 0.01N H2SO4, flow rate 0.8 mL/min, temperature 628C.
‡‡
Mobile phase 0.01N H2SO4, flow rate 0.8 mL/min, temperature 688C.
†
‡
Table IV
Inter-Day Robustness Evaluation Results Calculated as the Percentage of Recovery
Operating conditions
Oxalic acid
Glyoxylic acid
Glyoxal
Glycolic acid
Acetic acid
Acetaldehyde
Formic acid
Day 1
Day 2
Day 3
Mean + SD
106.5
99.25
105.2
103.65 + 3.2
101.3
103.15
97.25
100.57 + 2.5
99.85
97.35
100.35
99.2 + 1.3
102.3
95.33
103.22
100.3 + 3.5
106.2
95.65
100.36
100.8 + 4.3
98.85
103.33
94.56
98.9 + 3.6
99.12
97.36
106.65
101.0 + 4.0
were observed in the chromatogram, which suggests that the
developed method is robust.
Conclusions
In the production of glyoxal, the qualitative and quantitative
analysis of the reaction mixture has been a long-standing
problem. This paper describes a simple and rapid method for
analyzing the composition of the aqueous reaction mixture
produced by the liquid phase oxidation of acetaldehyde using
HPLC with RID. Quantitative analysis showed that the reaction
mixture produced by the liquid phase oxidation of acetaldehyde was composed of glyoxal, acetaldehyde, acetic acid,
formic acid, glyoxylic acid, oxalic acid, butanedione and glycolic acid; this is very important for reaction mechanism analysis
and optimization of process conditions. Compared with the
conventional chemical analysis methods, the method established in this paper can considerably shorten the analysis time
and improve the efficiency and accuracy of the analysis. It is especially suitable for industrial laboratories, where large sets of
samples of similar origin have to be analyzed on a routine basis.
In addition, this method can be applied to glyoxal production
by means of other synthetic routes.
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