Comprehensive determination of chlorophyll derivatives by chromic acid oxidation

Article abstract of DOI:10.1246/cl.2012.571

It is difficult to measure the total amount of chlorophyll derivatives due to their variety. We applied a new method for comprehensive determination of all derivatives by ethylmethylmaleimide (EMMi) derived from chlorin which is a ring structure of chloropigments. Relationships between the amount of applied chloropigments and the yield of EMMi through the chromic acid oxidation were examined for three species of chloropigments. The yield of EMMi from chloropigments was almost constant regardless of the amount or pigment species, indicating the applicability of this method for comprehensive deterimination of chlorophyll derivatives.

Full text of DOI:10.1246/cl.2012.571

doi:10.1246/cl.2012.571
Published on the web May 12, 2012
571
Comprehensive Determination of Chlorophyll Derivatives by Chromic Acid Oxidation
1
2
3
Yuhi Satoh, Shinya Nomoto, and Takeo Hama*
1
Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572
2
Faculty of Pure and Applied Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8571
3
Faculty of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572
(
Received February 2, 2012; CL-120086; E-mail: thama@biol.tsukuba.ac.jp)
It is difficult to measure the total amount of chlorophyll
derivatives due to their variety. We applied a new method for
comprehensive determination of all derivatives by ethylmethyl-
maleimide (EMMi) derived from chlorin which is a ring
structure of chloropigments. Relationships between the amount
of applied chloropigments and the yield of EMMi through the
chromic acid oxidation were examined for three species of
chloropigments. The yield of EMMi from chloropigments was
almost constant regardless of the amount or pigment species,
indicating the applicability of this method for comprehensive
deterimination of chlorophyll derivatives.
In the present study, the method to determine the concen-
tration of B-ring using chromic acid was examined. In the acidic
condition, the B-ring is oxidized to ethylmethylmaleimide
(EMMi) by chromic acid oxidation (Scheme 1). The concen-
tration of EMMi was analyzed by gas chromatography/mass
spectrometry (GC/MS), and the quantitative evaluation of
EMMi produced by chromic oxidation was examined using
authentic chlorophyll derivatives.
Three authentic derivatives were prepared (Chl a, pheophy-
tin a: Pheo a, and pyropheoforbide a: Pyrobide a; Wako Pure
Chemical Industries, Ltd., Chart 2), and EMMi was synthesized
4
in our laboratory. These derivatives were dissolved in acetone
(
0.01­10 nM), and the oxidizing reagent (5 mL) mixed with
Accumulation of chlorophyll and its degradative derivatives
is well known to reflect the past dynamics of various biological
and crustal thermodynamic phenomena, especially in aquatic
chromium trioxide (10% aq, w/v) and sulfuric acid (25% aq,
w/v) at a ratio of 1:1 (v/v) was simultaneously added to the
derivative solution. At this time, the volume of the oxidizing
agent was at least 10-fold larger than acetone to maintain a
sufficient oxidative effect. The solutions were left to stand for
2 h in ice and then for 2 h at room temperature in the dark for
oxidation. Na SO was added to the mixture to stop the reaction.
The reaction mixture was then extracted with dichlorometh-
ane:acetone (9:1, v/v, 3 mL) three times and the combined
extract was passed through silica gel (10 mL, particle size 125­
1
environments. Chloropigment contents in marine water column
and sediments have attracted considerable interest. High-
performance liquid chromatograph (HPLC) equipped with
photodiode array or fluorescence detector has been most
commonly used for quantitative analysis of chlorophyll deriv-
atives. However, the presence of over 70 species of chlorophyll
2
3
2
derivatives in the environment makes their accurate determi-
nation difficult using a photometric detector because of the
variability in photometric properties of each pigment.
The tetrapyrrole ring, chlorin, gives a specific structure to a
chlorophyll derivative. The structure of chlorin of chlorophyll
a (Chl a), which is the foremost chloropigment of the plant
kingdom, has already been elucidated (Chart 1). One of the
pyrrole rings in chlorin, the B-ring, is of interest since it contains
no reactive moieties but methyl and ethyl groups. This implies
that the B-ring is likely conserved through diagenetic processes,
suggesting that the amount of B-ring allows one to estimate the
total chlorophyll derivatives existing in a particular environ-
ment. Although some pigments such as chlorophyll c3 or
phycobilin are likewise known to contain the B-ring, their
3
production is significantly lower than Chl a. Thus, the amount
Scheme 1.
and the distribution of B-ring likely reflect the dynamics of
chlorophylls.
Chart 1.
Chart 2.
Chem. Lett. 2012, 41, 571­573
© 2012 The Chemical Society of Japan
www.csj.jp/journals/chem-lett/

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Table 1. Summary of oxidation examination of authentic
derivatives
Correlation Yield constanta
Slope Intercept
2
coefficient (r )
/%
Chl a
Pheo a
Pyrobide a 0.38
0.42
0.52
0.03
0.08
0.16
0.99
0.97
0.92
37.6
39.7
36.1
aYield constants were estimated by each linear regression
in Figure 2 and the value of authentic EMMi detected by
GC/MS.
chloropigments by measurement of the authentic EMMi. The
slopes of the regression lines of the molar ratio of EMMi/IS and
the area ratio of EMMi/IS were obtained from the oxidation of
chloropigments (Figure 2) by the slopes obtained from the
analysis using the authentic EMMi. The resultant yield constants
are shown in Table 1.
The yield constants were almost the same for the three
species of chloropigments, suggesting that the difference of
physicochemical characteristics such as molecular weight (MW,
Figure 1. Total ion monitor mass chromatogram (A) and
fragmentation pattern (B) of EMMi and IS analyzed by GC/MS.
Scan range was from m/z 100 to 145.
(
A) Chl a
(B) Pheo a
(C) Pyrobide a
y = 0.42x – 0.03
y = 0.52x – 0.08
y = 0.38x – 0.16
2
2
2
r
= 099
r
= 097
r
= 092
n = 28
n = 30
n = 29
5
34.7­893.5 in Chart 2), hydrophobicity (the presence or
absence of phytol chain), and chelated state (the presence or
absence of Mg) in chloropigments has little effect on the
efficiency of the oxidation. Moreover, this relation was
applicable to a comparatively wide range (0.01­10 nM). The
yield constants reported by previous study varied over a rather
wide range; the yield producing ketone by chromic acid
oxidation using various low molecular compounds ranged from
Figure 2. Relationships between the amount of molar ratio of
authentic samples to IS and the area ratio of derived EMMi to IS.
6
,7
8
2
5 to 75%, while EMMi using etioporphyrin from 10 to 20%.
3
4
3
50 ¯m , Wako Pure Chemical Industries, Ltd.; packed into a
0 mmº © 120 mm column) to remove remaining water and
This variability in the yield probably reflects the difference
in the organic chemicals applied and in oxidation reaction
procedure. Since the conditions were strictly controlled in the
present study, such as use of reducing agent, Na2SO3, to stop the
oxidation, constant yields were likely obtained. A quantitative
loss of EMMi during the refining process through silica gel
column is likely possible but it is included in the yield constant
estimated. Thus, the mean yield constant (37.8%) was applied to
estimate the concentration of chloropigments from the measure-
ment of EMMi in the samples obtained from aquatic environ-
ments.
washed with the same solvent (30 mL). The eluates were added
with henicosanoic acid methyl ester (purchased from Wako Pure
Chemical Industries, Ltd.) as internal standard (IS, 2­4 nM) and
subsequently concentrated to about 100 ¯L. Care was taken not
to allow it to dry because the recovery ratio declined when the
dried residue was dissolved. A GC/MS (Varian, 300 series)
system equipped with a capillary column (VF-5 ms, VARIAN,
5
3
0 m © 0.25 mm i.d.) was used for analysis. Identification of
EMMi and IS was done by comparison of the retention time with
authentic samples and the fragment pattern (Figure 1). Selected
ion monitor (SIM) mode was applied for the detection of EMMi
and IS at m/z 139 and 143. Blank test of EMMi was done by
measurements of the samples composed of acetone containing
no authentic derivatives. A little peak was measured for the
blank and was included in Figure 2. The accuracy of repeated
measurements through the entire operation was within 10%.
Table 1 and Figure 2 show the proportional relationship
between the applied amount of molar ratio of authentic samples
to IS and the area of EMMi derived by chromic acid oxidation to
IS. All regression lines showed strong correlations, suggesting
the constancy of this oxidative reaction regardless of chloropig-
ment species. Although the same molar quantity of EMMi with
the applied chloropigment should be produced on chemical
reaction (Scheme 1), the amount of EMMi derived from
chloropigments by chromic acid oxidation was lower than the
theoretical value, probably due to oxidative degradation. There-
fore, we estimated the yield constant of EMMi derived from
We examined the reproducibility of the analysis of natural
particulate organic matter (POM) collected from coastal sea-
water. POM on the glass fiber filter (Whatman GF/F, 0.7 ¯m
pore size) was soaked in the oxidizing agent with sonication for
2 min in ice-cold water, then left to stand for 2 h in ice-cold
water followed by 2 h at room temperature. Table 2 summarized
the results of Chl a, particulate organic carbon (POC), and
EMMi for POM obtained from Tokyo Bay and Oarai Coast.
The existence of free EMMi in natural water samples
produced in natural environments should be considered since it
possibly causes the overestimation of EMMi produced by
chromic acid oxidation. The amount of free EMMi should be
subtracted from the concentration determined by GC/MS to
obtain that of chlorophyll derivatives.
Chlorohyll derivatives ¼ ðCrEMMiÞ=Y ꢀ RðfEMMiÞ ð1Þ
where, Y is the yield constant of EMMi produced by chromic
acid oxidation (37.8%), (CrEMMi) is a measured value of
Chem. Lett. 2012, 41, 571­573
© 2012 The Chemical Society of Japan
www.csj.jp/journals/chem-lett/

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Table 2. Concentration of Chl a, POC, EMMi, and chlorophyll
derivatives in particulate matter collected from Tokyo Bay and
Oarai Coast
POM, such as the contribution of living phytoplankton and the
physiological state, differ between the two stations.
We hereby describe a new method to determine the
concentration of chlorophyll derivatives using the oxidation
reaction. The concentration of EMMi was determined quantita-
tively by conchromic acid oxidation and it is applicable to
chlorophyll derivatives determination. This method can afford
significant biogeochemical information on the diagenetic proc-
esses of plant organic matter, which has not been elucidated by
ordinary methods such as HPLC.
Chl aa POCb EMMic Variation coeffecient
/nM
/¯M
/nM
of EMMi
Tokyo bay
Oarai coast
a
15.0
2.0
217
17.9
19.2
1.3
12.0% (n = 7)
6.8% (n = 5)
Chl a was measured by a fluorometer (Turner design) as an
b
indicator of biomass. POC (particulate organic carbon) was
measured by an elemental analyzer (Fisons EA 1108). These
c
values were estimated from a calibration line using a
relationship between the applied amount of mole ratio of
authentic EMMi to IS and the area of EMMi to IS.
This study was financially supported by the Sasakawa
Scientific Research Grant from The Japan Science Society
(research number: 21-747M).
References
EMMi after chromic acid oxidation, R is the residual ratio of
free EMMi through chromic acid oxidation (we obtained the
value of 55.7% for R by chromic oxidation of authentic EMMi),
and (fEMMi) is a measured value of free EMMi. (fEMMi),
which can be determined using a nonoxidized sample. The
concentrations of fEMMi were 0.2 and 0.1 nM for the samples
from Tokyo Bay and Oarai Coast, respectively. These account
for only 0.4 and 2.9% of chlorophyll derivatives in both
samples, showing that a small amount of free EMMi exists in
the water column. This low contribution of free EMMi to
chlorophyll derivatives suggests that a very small fraction of
chlorin derived from Chl a is decomposed to monomer of
pyrrole within the water column. It is expected that the free
EMMi concentrations are likely high in the samples mainly
composed of degraded organic matter like sediment.9
1
S. Nomoto, M. Satou, T. Yoshida, H. Mita, G. Kumagai, K.
Nomoto, H. Kigoshi, Y. Kashiyama, Chem. Lett. 2008, 37,
490, and references cited therein.
R. J. Porra, E. E. Pfundel, N. Engel, in Phytoplankton
Pigments in Oceanography: Guidelines to Modern Methods,
ed. by S. W. Jeffrey, R. F. C. Mantoura, S. W. Wright,
UNESCO, Paris, 1997, pp. 85­127.
S. W. Jeffrey, M. Vesk, in Phytoplankton Pigments in
Oceanography: Guidelines to Modern Methods, ed. by
S. W. Jeffrey, R. F. C. Mantoura, S. W. Wright, UNESCO,
Paris, 1997, pp. 37­84.
S. Nomoto, M. Kozono, H. Mita, A. Shimoyama, Bull.
Chem. Soc. Jpn. 2001, 74, 1975.
M. Kozono, S. Nomoto, H. Mita, A. Shimoyama, Geochem.
J. 2001, 35, 225.
J. Rocek, A. E. Radkowsky, J. Am. Chem. Soc. 1973, 95,
7123.
R. Rangarajan, E. J. Eisenbraun, J. Org. Chem. 1985, 50,
2435.
S. Nomoto, M. Kozono, H. Mita, A. Shimoyama, Chem.
Lett. 2001, 1174.
2
3
4
5
6
7
8
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The concentrations of chlorophyll derivatives which are
corrected by the concentration of free EMMi in Tokyo Bay and
along the Oarai Coast were 50.7 and 3.4 nM, respectively. The
contribution of Chl a to chlorophyll derivatives in both samples
(29.6 and 58.5%) suggests that substantial amounts of chloro-
phyll derivatives exist in the water column. The contribution of
Chl a to total chlorophyll derivatives was higher in eutrophic
Tokyo Bay, indicating that the biogeochemical properties of
M. Kozono, S. Nomoto, H. Mita, R. Ishiwatari, A.
Shimoyama, Biosci., Biotechnol., Biochem. 2002, 66, 1844.
Chem. Lett. 2012, 41, 571­573
© 2012 The Chemical Society of Japan
www.csj.jp/journals/chem-lett/