Quantitative RP-HPLC determination of some aldehydes and hydroxyaldehydes as their 2,4-dinitrophenylhydrazone derivatives

Article abstract of DOI:10.1021/ac980699f

A high-performance liquid chromatographic (HPLC) method is described for the quantitative determination of some aliphatic aldehydes and β- hydroxyaldehydes as their 2,4-dinitrophenylhydrazone derivatives. A method is described for the preparation of deriv

Full text of DOI:10.1021/ac980699f

Anal. Chem. 1999, 71, 86-91
Quantitative RP-HPLC Determination of Some
Aldehydes and Hydroxyaldehydes as Their
2,4-Dinitrophenylhydrazone Derivatives
Eija Koivusalmi,* Elisa Haatainen, and Andrew Root
Analytical Research PB 310, Corporate Technology, NESTE OY, FIN-06101 Porvoo, Finland
using a UV/ visible-detector at a wavelength between 340 and 380
nm, depending on the absorption maximums of the relevant
hydrazone.15 The qualitative analysis without derivatization has
been described for formaldehyde, propionaldehyde, and 2,2-
bis(hydroxymethyl)butyraldehyde (BHBAL) among other com-
pounds in synthesis mixtures by reversed-phase HPLC using a
refractive index detector.16
This paper deals with the HPLC analysis of the aldehydes and
â-hydroxyaldehydes as their 2,4-DNPH derivatives using an
ultraviolet detector. The samples were obtained from reaction
mixtures of FA with different aliphatic aldehydes that produced
â-hydroxyaldehydes. The DNPH derivatization of DHPAL has
been used earlier for elemental analysis for compound identifica-
tion17 and for polarographic analysis,18 but no applications for
HPLC are known to us. No references were found in the literature
either concerning derivatization or HPLC analysis of â-hydroxy-
aldehydes BHBAL and 2,2-bis(hydroxymethyl)propionaldehyde
(BHPAL) as their DNPH derivatives.
The determination of hydroxyaldehydes as their DNPH deriva-
tives is not as straightforward as for other aldehydes, because of
the numerous side reactions in derivatization. The acid- or base-
catalyzed dehydration reactions19 do not interfere with the deter-
mination of the â-hydroxyaldehydes used in this study, due to
the absence of R-hydrogen. The formation of cyclic or oligomeric
acetals typical for â-hydroxyaldehydes20,21 is minimized in the
acidic derivatization conditions. The acid- or base-catalyzed
dehydration reactions, as well as the reported rearrangement
reactions of the derivatives of R-hydroxyketones or unsaturated
aldehydes,14,22 may interfere with the determination of the side
reaction products of BHPAL and BHBAL: 2-(hydroxymethyl)pro-
pionaldehyde (HPAL), 2-(hydroxymethyl)butyraldehyde (HBAL),
2-methyl-2-propenal (R-methylacrolein, AMA), and 2-ethyl-2-pro-
penal (R-ethylacrolein, AEA).
A high-perform ance liquid chrom atographic (HP LC)
method is described for the quantitative determination of
some aliphatic aldehydes and â-hydroxyaldehydes as their
2 ,4 -dinitrophenylhydrazone derivatives. A method is de-
scribed for the preparation of derivatives for those â-hy-
droxyaldehydes where no reference compounds of known
purity are available. The detection limit of the method was
4 .3 -2 1 .0 µg/ L, depending on the aldehyde.
Aldehydes such as formaldehyde (FA), propionaldehyde
(PAL), n-butyraldehyde (nBAL), isobutyraldehyde (iBAL), and
2-ethylhexanal (EHAL) are widely used in different processes in
the chemical industry. Identification of different compounds and
their quantitative analysis are essential for the optimization of
synthesis experiments.
Several gas chromatographic (GC) methods have been de-
scribed for the determination of aldehydes in water or air.
Classically 2,4-dinitrophenylhydrazine (DNPH) has been used for
derivatization despite the low volatility of these derivatives.1,2
Another GC method involves oxime derivatization and detection
of the highly volatile derivatives by a nitrogen-selective detector.3-6
In one paper, the different forms of 2,2-dimethyl-3-hydroxypro-
pionaldehyde (DHPAL) were determined by GC as oxime deriva-
tives. After trimethylsilylation of the oxime, the total amount of
DHPAL was determined.7
The most common method in recent years has been reversed-
phase HPLC where aldehydes, ketones, and hydroxycarbonyl
compounds in exhaust gases and environmental samples are
determined as their 2,4-dinitrophenylhydrazone derivatives by UV
detection.8-14 Hydrazones are separated by HPLC and monitored
(1) Papa, L. J.; Turner, L. P. J. Chromatogr. 1 9 7 2 , 10, 747-750.
(2) Darlene, M.; Persson, P.; Skarping, G. J. Chromatogr. 1 9 9 2 , 626, 284-
288.
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53, 805-809.
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1 9 9 5 , 41, 61-65.
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1 9 8 1 , 216, 402-405.
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Chem. 1 9 8 5 , 321, 553-555.
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(18) Tikhomirova, G. P.; Belous, G. C. Zh. Anal. Khim. 1 9 7 2 , 27, 173-177.
(19) Shine, H. J. J. Org. Chem. 1 9 5 9 , 24, 1790-1791.
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Acta 1 9 8 5 , 169, 419.
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(21) Cairati, L.; Cannizzaro, M.; Comini, A.; Gatti, G.; Pasquon, I.; Vita-Finzi, P.
Chim. Ind. (Milan) 1 9 8 1 , 63, 723-725.
(11) Chiavari, G.; Bergamini, C. J. Chromatogr. 1 9 8 5 , 318, 427-432.
(12) Barnes, A. R. Pharm. Acta Helv. 1 9 9 3 , 68, 113.
(13) Edelkraut, F.; Brockmann, U. Chromatographia 1 9 9 0 , 30, 432-435.
(22) Braddock, L. I.; Garlow, K. Y.; Grim, A. F.; Kirkpatrick, A. F.; Pease, S. W.;
Pollard, A. J.; Price, E. F.; Reissmann, T. L.; Rose, H. A.; Willard, M. L.
Anal. Chem. 1 9 5 3 , 25, 301-306.
86 Analytical Chemistry, Vol. 71, No. 1, January 1, 1999
10.1021/ac980699f CCC: $18.00 © 1998 American Chemical Society
Published on Web 12/01/1998

In the method developed here, the chromatographic conditions
are selected so that the cis and trans isomers of DNPH deriva-
tives23,24 are not separated. This is more sensitive to small aldehyde
concentrations than the direct HPLC determination with RI
detector described by Cairati et al.16 for similar sample matrixes.
The main advantage of the DNPH method is that the derivatization
minimizes the volatility of the low-molar-mass compounds. Also
different aldehydes and ketones can be analyzed simultaneously
in complex mixtures since the derivatization reaction enhances
the detector response and the detection limit of the chromato-
graphic analysis for aldehydes, but the other, otherwise interfering
compounds, cannot be detected.
2-methyl-2-propenal was 95% practical grade from Fluka, and
2-ethyl-2-propenal was 85% technical grade from Aldrich. Hydroxy-
aldehydes were in-house synthesis products for analytical pur-
poses.
P reparation of 2 ,4 -DNP H Solution and Carbonyl-DNP H
Derivatives for Calibration Standards. The derivatizing agent
for samples was prepared by dissolving 7.6 g of DNPH to 500 mL
of ACN in a 500-mL volumetric flask. The solution was kept in an
ultrasonic bath for 20 min to obtain a saturated solution.
Hydrazone standards of all the other aldehydes but hydroxy-
aldehydes were prepared by the method described by Shriner et
al.26 In this method, 4 g of DNPH was dissolved in 20 mL of
concentrated sulfuric acid and then added to a solution of 28 mL
of water in 100 mL of absolute ethanol. About 1.5 g of each
aldehyde was dissolved in 60 mL of 95% ethanol and then 50 mL
of the above prepared acidic DNPH solution was added. In the
case of DHPAL, ∼1.0 g was dissolved in 60 mL of 95% ethanol
and 2 mL of concentrated sulfuric acid and then 50 mL of the
above prepared DNPH solution in sulfuric acid was added. Less
aldehyde and an addition of sulfuric acid in ethanol was used for
DHPAL in order to ensure that the molecule was mainly in the
monomeric form before the addition of the DNPH reagent. The
prepicitated DNPH derivatives were filtered off by suction on black
ribbon filter paper and aldehydes were recrystallized from ethanol.
The standard method described above was not suitable for the
preparation of derivatives of BHPAL and BHBAL even though it
worked for DHPAL, since in addition to the hydroxyaldehyde-
DNPH derivative, an insoluble precipitate was also formed. Thus,
a new derivatization procedure was developed. The synthesis
product of BHPAL or BHBAL was concentrated in a rotary
evaporator and FA, IBAL, or PAL were steam distilled. The
hydroxyaldehyde concentration was then evaluated to be ∼10%.
About 12 g of steam-distilled hydroxyaldehyde solution, 2 mL of
concentrated phosphoric acid, and 200 mL of 99.9% EtOH were
mixed and heated to 55 °C. Approximately 3 g of DNPH was added
slowly over 2 h to the hot solution with magnetic stirring. The
obtained solution was put under nitrogen purge and evaporated
to 100 mL. About 50 mL of water was added, and a bright yellow
precipitate was allowed to settle in the refrigerator over the
weekend. The precipitated DNPH derivative was filtered off by
suction on black ribbon filter paper and the aldehyde derivative
was recrystallized from a 2/ 1 water/ ethanol mixture. The deriva-
tives were dried in a vacuum desiccator.
EXPERIMENTAL SECTION
Apparatus. In the study, a Hewlett-Packard model 1090 HPLC
with a diode array detector (6-mm flow cell, 4-µm slit) was used.
The column was Waters Nova-Pak C18, 150 × 4 mm, 60-Å pore
size, 4-µm particle size at 40 °C. The detection wavelength was
360 nm and reference wavelength 550 nm. The injection volume
was 5 µL. The eluents were (A) ultrapure water and (B)
acetonitrile (ACN). The flow rate of the eluent was 1 mL/ min
and the gradient program as follows:
time/ min
% B
0
40
2
40
10
98
15
98
16
40
The melting points of the solid DNPH standards were determined
by a Mettler DSC 30 differential scanning calorimetry under
nitrogen atmosphere. The sample amount was 5 mg and the
heating rate 10 °C/ min.
The structures of the BHBAL-DNPH derivatives were also
characterized with solid-state NMR using a Chemagnetics CMX
Infinity NMR spectrometer operating at 270 MHz. The samples
were placed in 6-mm zirconia rotors and spun at 5 kHz. The cross-
polarization (CP) experiment was carried out using 65-kHz rf fields
on both channels, and a contact time of 2 ms was used. The
recycle delay was 5 s and around 1000-2000 transients were
acquired. The dipolar dephasing experiments were carried out
with a dipolar dephasing delay of 50 µs. This experiment was the
same as the CPMAS experiment except that acquisition of the
FID was carried out after a 50-µs window after the contact pulse
during which the decoupler was turned off.25 The acquired FIDs
were Fourier transformed without any weighting function.
Reagents. The water used in HPLC and sample preparation
was deionized and further purified via a Milli-Q Water System
(Millipore). ACN was HPLC grade from Rathburn (Walkerburn,
Scotland), 2,4-DNPH (H2O = 50%) was p.a. grade from Fluka
(Buchs, Switzerland), H2SO4 was 95-97% p.a. from Merck (Darm-
stadt, Germany), H2SO4 solution was 1.0 M from FF Chemicals
(Yli-Ii, Finland), H3PO4 was 85% p.a. from J. T. Baker (Deventer,
Holland), ethanol was 95% from Primalco (Rajama¨ki, Finland),
formaldehyde was 37-38% solution in water from Merck, propi-
onaldehyde 97%, butyraldehyde 99%, isobutyraldehyde 98%, and
2-ethylhexanal 98% were from Aldrich (Steinheim, Germany),
The standard solutions for HPLC analysis of the solid DNPH
derivatives were prepared by weighing 25 mg of each standard
into a 100-mL volumetric flask and filled to the mark with ACN.
The stock solutions were diluted further to four different concen-
trations with a 50/ 50 water/ ACN mixture just prior to use. The
aldehyde concentration of the standard solutions were calculated
using eq 1 and molar masses from Table 1.
Mw(aldehyde)
aldehyde (mg/ L) ) purity (%) ×
×
Mw(aldehyde-DNPH)
aldehyde-DNPH (mg/ L) (1)
(23) Kiebler, D. J.; Mopper, K. Mar. Chem. 1 9 8 7 , 21, 135-149.
(24) Terada, H.; Hayashi, T.; Kawai, S.; Ohno, T. J. Chromatogr. 1 9 7 7 , 130, 281-
286.
(26) Shriner, R. L.; Fuson, R. C.; Curtin, D. Y.; Morrill, T. C. The Systematic
Identification of Organic Compounds, 6th ed, Wiley: New York, 1980.
(25) Opella, S. J.; Frey, M. H. J. Am. Chem. Soc. 1 9 7 9 , 101, 5856.
Analytical Chemistry, Vol. 71, No. 1, January 1, 1999 87

Table 1. Melting Points of the Recrystallized DNPH
Derivatives, Molar Masses, and Limits of Detection of
the Compounds
limit of
Tm (°C)
detnd
lit.32
165.8 166
Mw (g/ mol)
detection
standard
aldehyde ald-DNPH (µg/ L)
Figure 2. Structures of BHBAL, its dimer, and oligomer.
formaldehyde
30.026 210.166
58.080 238.220
72.107 252.247
72.091 252.247
70.091 250.231
84.118 264.258
128.214 308.354
4.3
6.6
8.4
7.7
6.6
10.0
14.0
11.1
13.6
21.0
propionaldehyde 155.0 155
n-butyraldehyde 120.6 123
isobutyraldehyde 185.3 187
R-methylacrolein
R-ethylacrolein
2-ethylhexanal
DHPAL
BHPAL
BHBAL
237-238
237-238
189.5 191-19217 102.133 282.273
118.133 298.273
132.159 312.299
DNPH
(198.14)
Figure 1. Gradient run of a standard mixture with 60/40 water/ACN
to 2/98 water ACN. Concentrations in mg/L: BHPAL 7.21, BHBAL
8.13, FA 3.37, DHPAL 7.99, PAL 4.27, AMA 4.62, BAL 7.38, AEA
5.44, and EHAL 9.06.
P reparation of Samples. Samples were prepared by weighing
50-100 mg of reaction mixture into weighed solution of 10 mL
of 0.01 M H2SO4 and 2 mL of MeOH. Out of this diluted sample
solution, a 100-mg portion was weighed into a 100-mL volumetric
flask with 20-30 mL of DNPH solution and 2 mL of concentrated
phosphoric acid. After a derivatization reaction of 1 h, the flask
was filled to the mark with 50/ 50 water/ ACN mixture. A 2-mL
aliquot of the sample solution was diluted further to 100 mL with
50/ 50 water/ ACN mixture and filtered through a 0.45-µm Millex
HV filter for analysis.
Figure 3. 13C CPMAS and dipolar dephased spectra of insoluble
component in BHBAL-DNPH derivatization with sulfuric acid.
eluent gave the best resolution for early-eluting hydroxyaldehydes
and for late-eluting aliphatic aldehydes as shown in Figure 1. The
method was not optimized to separate the cis and trans isomers
from each other, but they are determined as one peak. The
resolution for isobutyraldehyde and butyraldehyde was not
optimized, since they do not exist in the same synthesis sample.
Standard P reparation and Identification. The precipitate
obtained with a Shriner’s26 method for BHBAL contained an
insoluble part which was separated from ACN solution and dried.
It was postulated that the structure was a DNPH derivative of the
BHBAL-dimer shown in Figure 2. The precipitate was analyzed
by solid-state NMR and the CPMAS and dipolar dephased NMR
spectra are shown in Figure 3. The dipolar dephased spectrum
reveals only those carbons that do not have Hs directly bonded
to them, or methyl groups. Details of this experiment can be found
elsewhere.25 It is clear from the CPMAS NMR spectrum that there
are very few aromatic carbons present in this sample so a dimer
DNPH structure is discounted. From the dipolar dephased
spectrum, it can be deduced that the peak at 40 ppm is probably
due to a quaternary carbon while that at 10 ppm is due to a methyl
RESULTS AND DISCUSSION
Optimization of the HP LC Method. The retention times of
different aldehydes were determined at first with isocratic condi-
tions using 35/ 65 water/ ACN as eluent. Under these conditions,
the polar aldehyde and ketone hydrazones, which contain ad-
ditional hydroxy groups, eluted with even lower retention times
than the formaldehyde derivative as described in the literature.11
Actually, the most polar compound, the 2,4-DNPH derivative of
BHPAL, eluted even before DNPH itself. No separation was
obtained for DHPAL and formaldehyde. Using 60/ 40 water/ ACN
a better resolution was obtained, but the retention times of the
other aldehydes were quite long and the peaks broad. The
gradient method using 60/ 40 water/ ACN to 2/ 98 water/ ACN as
88 Analytical Chemistry, Vol. 71, No. 1, January 1, 1999

Figure 5. Derivatization reaction of an aldehyde and DNPH.
Figure 4. 13C CPMAS and dipolar dephased spectra of soluble
product in BHBAL-DNPH derivatization with phosphoric acid.
group. The peak at 103 ppm is characteristic for OCH2O type
groups. Therefore, it seems likely that the main component in
the sample is like BHBAL, and since it is insoluble it is tentatively
assigned to the long-chain polymer as shown in Figure 2. Peak
assignments for this structure are shown in Figure 3.
Figure 6. DSC melting curves of DHPAL-DNPH derivatives pre-
pared by the method of (A) Shriner et al. (B) by the developed method.
In the developed method for preparation of BHPAL and
BHBAL hydrazone derivatives, it was expected that an equilibrium
reaction between monomer and dimers obeys the same rules as
in the case of DHPAL.20 Thus, at elevated temperature and in
dilute acidic conditions, the amount of monomer is high compared
to the amount of acetals. Hydroxyaldehydes can be derivatized
quantitatively because the reaction equilibrium lies on the side
of the monomer, which is continuously consumed by the added
DNPH. The CPMAS and dipolar dephased spectra of the BHBAL
derivative obtained with mild phosphoric acid conditions are
shown in Figure 4. Since there are basically five peaks in the
aliphatic area and seven peaks in the aromatic area, the structure
of BHBAL-DNPH is assumed. The peak assignments are as shown
in Figure 4. Characteristic is the peak at 160 ppm due to the
CHdN group. The absence of a peak at 90-100 ppm shows that
there are no O-CH2-O structures in the molecule. Therefore
the dimer-DNPH structure is discounted. Some of the peaks in
the aromatic area are split. This can be due to inequivalent atoms,
due to crystallographic inequivalences in the solid state or, for
the case of atom 4, due to the dipolar coupling with a nitrogen
nucleus close by. This is because the 13C-14N dipolar interaction
cannot be completely averaged out by MAS since the quantization
axis of the 14N nucleus is not aligned along the static magnetic
field.27
Sample P reparation. The samples from synthesis were
collected into an acidic solution. Methanol was added to keep the
sample soluble during the time of storage before analysis. A total
of 50% water was used in dilutions of standards and samples to
enhance the peak resolution and peak height in a chromatogram.
The water content of 60% that was used in the eluent was not
used in sample and standard preparation, since some DNPH
derivatives tend to precipitate. The derivatization reaction of an
aldehyde and DNPH is an addition reaction followed by dehydra-
tion and needs an acid catalyst, as shown in Figure 5. The reaction
rate increases with decreasing pH, and a large excess of the
DNPH reagent is needed to shift the equilibrium of the reaction
to the side of the derivative.28 Thus, a high concentration of
(27) Groombridge, C. L.; Harris, R. K.; Packer, K. J.; Say, B. J.; Tanner, S. F. J.
Chem. Soc., Chem. Commun. 1 9 8 0 , 4, 174.
Analytical Chemistry, Vol. 71, No. 1, January 1, 1999 89

Table 2. Precision of the Method for Samples with a
Low and a High Concentration of Hydroxyaldehyde
Sample from BHPAL Synthesis (wt %)
no.
BHPAL
FA
PAL
0.03
0.03
0.03
0.03
0.03
0.0000
0.03
0.0000
1
2
3
4
5
SD
av
13.17
12.52
13.17
13.02
12.72
0.2894
12.92
2.239
20.14
20.04
20.77
20.71
21.25
0.4968
20.58
2.413
RSD (%)
Sample from BHBAL Synthesis, Distillated (wt %)
no.
BHBAL
FA
BAL
AEA
0.24
0.28
0.35
0.26
0.24
0.0456
0.27
16.64
1
2
3
4
5
SD
av
63.72
61.50
61.54
62.52
61.00
1.0811
62.06
1.742
19.84
18.53
18.93
18.70
18.36
0.5807
18.87
3.077
0.92
0.87
0.85
0.89
0.87
0.0265
0.88
RSD (%)
3.006
impurities present in a sample cause melting point depression.
The results are collected in Table 1, except for unsaturated
aldehydes which after one recrystallization degrade or undergo
several rearrangement reactions before melting.22,30 The results
are in good agreement with the values obtained from the literature.
The melting behavior of DHPAL-DNPH derivatives prepared by
Shriner’s26 method and the method with preadded sulfuric acid
are compared in Figure 6. No sharp melting point can be detected
in the derivative prepared by the conventional method (Figure
6a). Instead, the derivative prepared by the developed method
melts sharply after first recrystallization at 189.5 °C (Figure 6b).
Both the DHPAL derivatives showed similar behavior in HPLC
runs.
The purity of all prepared hydrazone derivatives was checked
by HPLC. Water/ ACN solutions of each derivative as well as
reagent blanks were analyzed. The purities were determined by
subtracting the peak area percentages of impurities from 100%
since the UV responses of the aldehyde-DNPH derivatives are
quite equal.
Compound Identification in HP LC Run. The UV spectra
of the standard derivatives were collected and stored in the library
of the HPLC instrument. By comparing the UV/ visible spectra,
one can separate the peaks eluted in the samples into five
groups: formaldehyde, aliphatic aldehyde, aromatic aldehyde,
aldehyde with conjugated double bonds, and aldehyde with
conjugated triple bonds.31 The aliphatic and hydroxyaldehydes
gave identical spectra giving absorption maximums at 275 and
365 nm and an absorption minimum at 295 nm as shown in Figure
7a. Formaldehyde-DNPH gave absorption maximums at 265 and
355 nm and an absorption minimum at 295 nm. The unsaturated
aldehydes gave absorption maximums at 255 and 373 nm, an
absorption minimum at 310 nm, and an extra shoulder at 285 nm.
The retention times increased with increasing chain length. The
unsaturated aldehyde eluted slightly earlier than a saturated
Figure 7. (A) Typical UV spectra of FA (- - -), hydroxyaldehyde
(- - -), saturated aldehyde (- -), and unsaturated aldehyde (s). (B)
Chromatogram of a BHPAL synthesis sample. (C) Chromatogram of
2-methyl-2-pentenal.
phosphoric acid was used in the sample preparation and the
presence of free DNPH was required in the chromatograms to
ensure these requirements. The time needed for the derivatization
reaction varies from 30 min to 1 h, depending on the pH and the
aldehyde.28,29 In this study, a reaction time of 60 min was used,
since the presence of a hydroxy group may sterically hinder the
reaction of 2,4-DNPH with the aldehyde carbonyl.
The stability of DNPH derivatives is good when prepared in
ACN solution and stored in the refrigerator. Addition of water
either in dilution of the stock standard solution or in the sample
preparation causes the derivative to decompose relatively quickly,
because the higher water content shifts the equilibrium of the
derivatization reaction to the side of the reactants. For example,
the peak height of the FA standard after 12 h is 5-7% smaller
than that of the freshly diluted and analyzed FA standard.
P urity Determination. The purity of the dried, solid deriva-
tives was tested by determining the melting points with DSC. The
(28) Lowe, D. C.; Schmidt, U.; Ehhalt, D. H.; Frischkorn, C. G. B.; Nurnberg, H.
W. Environ. Sci. Technol. 1 9 8 1 , 15, 819-823.
(29) Cotsaris, E.; Nicholson, B. C. Analyst 1 9 9 3 , 118, 265-268.
(30) Behforouz, M.; Bolan, L.; Flynt, M. S. J. Org. Chem. 1 9 8 5 , 50, 1186-1189.
(31) Po¨ tter, W.; Karst, U. Anal. Chem. 1 9 9 6 , 68, 3354-3358.
90 Analytical Chemistry, Vol. 71, No. 1, January 1, 1999

aldehyde with the same carbon number. The highly polar
hydroxyaldehydes eluted very fast as shown in Figure 1. So, by
comparing the retention time of an unknown compound in a
chromatogram to those of known aldehydes, and by comparing a
spectrum to those in the library, one can estimate the structure
of an unknown compound.
hydrazone derivatives on the basis of the smallest calibration
standards and a system noise of 0.06 mAU multiplied by 2. The
limits of detection are collected in Table 1. The linearity of the
DNPH calibration lines of aliphatic aldehydes is excellent even
for very intensive peaks near 1000 mAU, if diluted with a water/
ACN 50/ 50 mixture. The calibration lines of hydroxyaldehydes
tend to be curved at higher concentrations. The precision of the
method was tested by analyzing a BHPAL synthesis sample and
a concentrated BHBAL synthesis sample five times. The original
values and the results are collected in Table 2. The method is
quite precise for the main components and for the other side
compounds except AEA. The spread of the AEA results is very
large, indicating poor precision. The obviously different value of
AEA, 0.35 wt %, was retained on the basis of an outlier test, since
the calculated Dixons’s Q ) 0.636 does not exeed the critical value
Q ) 0.717 for sample size 5 (P ) 0.05).33
For example, the peak in a BHPAL sample chromatogram in
Figure 7b at retention time 8.3 min elutes in the time range of C6
aldehydes and its spectrum is similar to unsaturated aldehydes.
Thus, the structure of a compound from an aldol reaction between
two propionaldehydes is supposed, which was confirmed by a
synthesized reference compound 2-methyl-2-pentenal in Figure 7c.
A peak at sample chromatogram at 3.7 min, which elutes very
fast before FA and gives a spectrum similar to a hydroxyaldehyde,
is quite obviously HPAL. In the case of BHBAL, the retention
time of HBAL is 4.8 min and the retention time of 2-ethyl-2-hexenal
the same as for 2-ethyl-hexanal, 9.5 min. The last two mentioned
compounds can be identified from their UV spectra despite the
same retention time, since they do not exist in the same sample.
Detection Limit, Linearity, and P recision. The detection
limit of the HPLC method was calculated for the aldehyde
ACKNOWLEDGMENT
The authors thank Liisa Seitvuo and Jaana Niemi for analyzing
melting points by DSC.
Received for review June 30, 1998. Accepted October 15,
1998.
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AC980699F
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