1(1) (2012), 16-24
Advanced technologies
THE IDENTIFICATION OF CHLOROPHYLL AND ITS DERIVATIVES IN THE PIGMENT MIXTURES:
HPLC-CHROMATOGRAPHY, VISIBLE AND MASS SPECTROSCOPY STUDIES
Sanja M. Milenković1*, Jelena B. Zvezdanović1, Tatjana D. Anđelković2,
Dejan Z. Marković1
1Faculty of Technology, University of Niš, Leskovac, Serbia
2 Faculty of Sciences, Department of Chemistry, University of Niš, Niš, Serbia
This work represents an application of the Visible Spectrometry, High Pressure Liquid Cromatography and Electronspray Ionisation-Mass Spectrometry
analysis for the identification of chlorophyll and its derivatives, pheophytin
and chlorophyllide, in their purified mixtures (the chlorophyll, pheophytin and
chlorophyllide fraction, respectively). The chlorophyll, pheophytin and chlorophyllide fractions were obtained by using a pigment extraction from the plant
material (Spinacia Oleracea L), coupled with the column chromatography. The
main components in the chlorophyll, pheophytin and chlorophyllide fractions
were identified as chlorophyll a & b, pheophytin a & b and chlorophyllide a, respectively. Spectrophotometrically determined concentration ratios of the main
identified pigments, chlorophyll a/b (in the chlorophyll fraction), pheophytin a/b
(in the pheophytin fraction) and chlorophyllide a/b (in the chlorophyllide fraction) were: 4.95/1, 1.05/1 and 6.89/1, respectively.
(ORIGINAL SCIENTIFIC PAPER)
UDC 547.979.7:543.422
Keywords: chlorophyll, pheophytin, chlorophyllide, VIS, HPLC, ESI-MS.
Introduction
Chlorophylls belong to the group of the most important
bioorganic molecules; they are the principal pigments in
photosynthesis, capable of light energy absorbing and its
conversion to “chemical energy” by the formation of chemical compounds rich in energy (needed for the biosynthesis
of carbohydrates and other compounds in photosynthetic
organisms such as plants, algae and photosynthetic bacteria [1, 2]).
*Author address: Sanja Milenkovic, Faculty of Technology, 16000
Leskovac, Bulevar oslobođenja 124, Serbia
e-mail: milenkovic_sanja@yahoo.com
The manucsript received: April, 03, 2012.
Paper accepted: Jun, 18, 2012.
16 The chlorophylls comprise a group of more than 50
tetrapyrrolic pigments with common structural elements
and function [3]. In such a way, the major photosynthesis
pigment - chlorophyll (Chl) is chlorin (porphyrin derivative)
in chemical terms, a cyclic tetrapyrrole with an isocyclic
cyclopentanone ring, fused at the edge of the right-bottom pyrrole ring [1- 2], as shown in Figure 1. The central
Mg-atom plays a coordinating role in Chl-molecule: it is
Figure 1. Chlorophyll (A), pheophytin (B) and chlorophyllide (C) structures. C-atoms are numerated according to IUPAC
nomenclature rules. In the position C-7, -R corresponding to:
-CH3 & –CHO in the cases of Chl a & Chl b, Pheo a & Pheo b, and
Chlid a & Chlid b, respectively.
1(1) (2012), 16-24
Advanced technologies
bonded to N-atoms (N(21-24)) of the four pyrrole rings by atom [8-10], as shown in Figure 1B. The involvement of
two covalent and two coordinative bonds (Figure 1A). The pheophytin a (Pheo a) as a primary electron acceptor in
5-membered isocyclic ring (E) is connected to the chlorin the PSII reaction center (of the photosynthetic apparatus)
in C(13-15) positions, while the phytol -esterified residue has been demonstrated by Klimov et al. [5], and largely
of propionic acid is attached to the chlorin C-17 position strengthened by numerous biophysical measures [5]. On
(Figure 1A). The groups: -CH3 and –CHO (in a position the other hand, pheophytin can be easily obtained during
C-7), in the case of chlorophyll a (Chl a) and chlorophyll b the extraction processes of the plant material, due to the
(Chl b), respectively (Figure 1A), make the only difference liability of magnesium in the Chl-molecule [11].
between those two chlorophylls [2, 4]. The major function
Chlorophyllide (Chlid) is a derivative of chlorophyll,
of Chl a in photosynthesis is connected to light-harvesting namely, chlorophyll without phytil-chain in C-173 position
in the antennas and light conversion processes in the Pho- as shown in Figure 1C. It is formed in the last steps of
tosystems I and II (PSI and PSII), inside the photosynthet- Chl biosynthesis [5, 12], or in degradation processes like
ic apparatus [5-7].
senescence of leaves [3-14]; the degradation processes
In various photosynthesis “subjects” (plants, algae, include the loss of phytol (C-173) and modification of side
bacteria), chlorophyll is often accompanied by the pres- chains of the isocyclic ring (E) of Chl (Figure 1), in the reence of its various derivatives, such as pheophytins (Pheo), actions catalyzed by several enzymes [13].
chlorophyllides (Chlid), and many others [8-9].
Table 1 gives a short preview of the natural occurrence
Pheophytin (Pheo) is a chlorophyll derivative - demeta- of chlorophyll a and some of its main derivatives (Pheo a
lated chlorophyll or simply chlorophyll without central Mg- & Chld a).
Table 1: Natural occurrence of the investigated chlorophylls` pigments.
Chlorophyll a
All photosynthetic algae (except prochlorophytes) and higher plants [1,2]
Pheophytin a
Photosynthetic reaction canters of higher plants, plant and algal detritus [2]
Chlorophyllide a
Senescent tissue, damaged centric diatoms, zooplankton faecal pellets [1,2]
Extraction of plant pigments. Extraction of plant pigments from spinach leaves, Spinacia oleracea L (found in
the local market), were performed by using the already
published method [15]. Fresh spinach leaves were firstly
depleted from the mid ribs and washed with cold water.
The extraction and re-extraction mixtures were methanol
and petroleum ether in a 2:1 ratio, and petroleum ether
and diethyl ether (1:1), respectively. The methanol removes water from the plant material and the petroleum
ether picks up the pigments before they undergo secondary reactions [21]. The diethyl ether increases the pigments solubility in the organic phase. The final extract was
a mixture of pigments containing large amounts of various
chlorophyll forms, as well as accessory pigments, carotenoids (carotenes and xanthophylls) [21].
Chlorophyll fraction. The chlorophyll fraction – the purified mixture of various chlorophyll forms (e.g. Chl a and
Chl b) – was isolated from the pigment extract by using
column chromatography with silica gel as the adsorbent
(silica gel 60, Merck, 0.063-0.200 mm) and n-hexane/acetone mixture as the eluent [15, 18]. The n-hexane/acetone
ratio was changed from the initial 1:0 to final 1:1, to allow
an easier elution of the polar fractions. Chlorophyll fraction
was eluted at the eluent composition 1: 0.1 (n-hexane/acExperimental
etone, respectively), and then transferred in acetone.
Pheophytin fraction. The pheophytin fraction – the mixAll experiments were performed under dim light as long
ture of various pheophytin forms (e.g. Pheo a and Pheo
as possible, and inside the vessels and equipment covb) – was made from the collected chlorophyll fraction by
ered with aluminum foil or black cloth, preventing the pigdrop-wise addition of 1.0 M HCl [10]. The conversion of
ment exposure to light [8].
chlorophyll to pheophytin was completed in approximately
The isolation, separation and identification of chlorophyll and its derivatives from the plants and the other
photosynthetic organisms have been studied intensively
during the years [1, 3, 7]. The decomposition of chlorophylls and the analysis of chlorophyll derivatives in foods
have also been investigated because of their importance
for the quality of fruit and vegetable products [3, 7]. The
extraction and preparation (by open column chromatography) of photosynthetic pigments from different sources
(in many different variations of these methods), are largely
used for the isolation and separation of chlorophyll and
its derivatives [13-16]. On the other hand, RP-HPLC
chromatography (Reversed Phase-High Pressure Liquid
Chromatography), as well as the absorbance (visible, VIS)
spectrometry and MS (Mass Spectrometry) are well used
methods for the analysis and identification of Chls [17-20].
The objective of this study is to represent one of the
methods for fast and simple isolation, preparation and
identification of chlorophyll and its derivatives (pheophytin
and chlorophyllide) from the plant material (such as spinach from the local market) by using the extraction method
and column chromatography as well as VIS, HPLC and
E(electron) S(Spray) I(Ionization) – MS measurements.
17
1(1) (2012), 16-24
Advanced technologies
2 h in the dark, observed as a change of color from green
to olive brown. Freshly made Pheo-stuff was then extracted by n-hexane and then dissolved in acetone.
Extraction of chlorophyllides. The chlorophyllides extract was obtained from dried spinach leaves (firstly depleted from the mid ribs). The chlorophyllides extraction
was performed by the action of the endogenous enzyme
chlorophyllase on chlorophylls, by incubating 5 g of dehydrated spinach with 100 mL acetone/0.2 M Tris–HCl buffer
(pH 8.0, 1:1 v:v) for 2 h in the dark at 40 0C [3]. The final
chlorophyllide extract was a mixture of pigments containing large amounts of various chlorophyllide forms, as well
as their derivatives, in smaller amounts.
Chlorophyllide fraction. The chlorophyllide fraction – the
purified mixture of chlorophyllide forms (e.g. Chlid a and
Chlid b) – was isolated from the chlorophyllide extract by
column chromatography with silica gel as the adsorbent
(silica gel 60, Merck, 0.063-0.200 mm) and the n-hexane/
acetone mixture as the eluent. The n-hexane/acetone ratio was changed from the initial 1:0 to final 1:10, to allow
an easier elution of the polar fractions. The chlorophyllide
fraction was eluted at the eluent composition 1:3.3 (n-hexane/acetone, respectively).
VIS spectroscopy. The spectrophotometric measurements were made on a Varian Cary-100 spectrophotometer equipped with 1.0 cm quartz cells. All spectra were
recorded from 350 to 800 nm with 1.0 bandwidth. The
spectra of all compounds were recorded in acetone. The
contents of the investigated pigments in the chlorophyll,
pheophytin and chlorophyllide fractions were determined
as reported [22, 23], in the acetone. The concentrations of
pigments in acetone were in the range between 10-4 and
10–5 mol/dm3.The molar concentration ratios of the investigated pigments: cchl a / cChl b (in the chlorophyll fraction),
cPheo a / cPheo b (in the pheophytin fraction) and cChli a / cChlid b (in the
chlorophyllide fraction) were: 4.95/1, 1.05/1 and 6.89/1, respectively.
HPLC analysis.The high-pressure liquid chromatography analysis of isolated chlorophyll, chlorophyllide and
pheophytin fraction was performed under isocratic conditions on the apparatus: Agilent 1100 Series, Waldborn,
Germany, column: Zorbax Eclipse XDB-C18, by using a
iode array detector set at detection wavelengths (λdet.): 430
and 660 nm; the isocratic conditions were: mobile phase
– acetonitrile/methanol/ethyl-acetate in the ratio 60:20:20,
respectively, flow rate - 0.5 cm3/min, and temperature: 25 0C.
The concentrations of the pigments were in range between
10-4 and 10–5 mol/dm3.
ESI-MS spectrometry.The pigment fractions were analyzed by the flow-injection ESI-MS/MS method (Electronspray ionisation- mass spectrometry), using methanol as
the carrier phase. The ESI-MS/MS investigation was performed on LCQ Deca Ion Trap Mass Spectrometer (Thermo Finnigan, USA) with auxiliary equipment, by using the
possibility for automatic tuning of every molecular ion in
the mass spectrum to make MS/MS experiment. Mass
spectra for all compounds was recorded in the most suit 18 able mode; the value of the collision energy was increasing from 20 to 35 % until the noticeable fragmentation of
smaller values of m/z, so that the initial “Parent” ion could
still be seen in the spectrum. The optimization of ESIsource parameters was carried out to admit the detector
light aspiration achieved by the mobilizing gas flow (10 L/min),
transported under the pressure of 30 psi. The lowest voltage (50 V) was applied to ensure the efficient transport of
generated ions to the analyzing module.
Results and discussion
Absorption (VIS) spectra of chlorophyll, pheophytin
and chlorophyllide fractions. The absorption spectra of
chlorophyll (A), chlorophyllide (B) and pheophytin (C) fraction in acetone are shown in Figure 2.
Chlorophylls as the porphyrin derivatives have two major absorption bands in the visible range, due to extended
π-delocalization at the edge of cyclic tetrapyrrole (porphyrin) skeleton (Figure 1): ‘‘red’’ (Q-) band and ‘‘blue’’ (Soret
or B-) band [9, 24].
The “red” and “blue” bands of Chl a (assigned as Qyand Soret-) are located at 662 and 430 nm for acetone
solutions, respectively [8-9], similar to the ones shown in
Figure 2A for the chlorophyll fraction (consisting of Chl a
and Chl b in the ratio 4.95/1) in acetone. The corresponding bands (Soret- and Qy-) for Chl b in acetone are located
at 645.5 and 456.9 nm, respectively [8]. The small “shoulder” at 457 nm (probably due to the presence of Chl b in
the mixture) is present in the absorption spectrum of the
chlorophyll fraction in acetone (Figure 2A). On the other
hand, the ratios of absorbance intensities for the Soretand Qy-bands (ASoret/AQy) are 1.23 for Chl a, and 2.82 for
Chl b [8]; the corresponding ASoret/AQy ratio for the chlorophyll fraction in acetone is 1.30 (Figure 2A). The observed
small bands located in the spectral range between the Soret- and Qy- bands for the chlorophyll fraction in acetone
(at 534 nm, 581 nm and 616 nm – Figure 2A) are in accordance with the corresponding literature data reported
for Chl a dissolved in acetone [8].
Absorption maximums of the Soret- and Qy-bands are
located at 409.5 and 665.5 nm for Pheo a in acetone [8],
similar to the one shown in Figure 2B for the pheophytin
fraction (consisting of Pheo a and Pheo b in ratio 1.05/1)
in acetone. As compared to chlorophylls, the “red” absorption maximum of pheophytins in acetone is shifted to
longer wavelengths [9]. However, the maxima in the “blue”
spectral region are shifted to shorter wavelengths [8]. The
ratios of the Soret- and Qy- band absorptions (ASoret/AQy)
are much higher than those to chlorophylls [22]. For Pheo
a and Pheo b in acetone, ASoret/AQy = 2.26 and 5.30, respectively [8] and for the pheophytin fraction in acetone, the
corresponding ASoret/AQy ratio is 2.33 (Figure 2B). The observed small bands located in the spectral range between
the Soret- and Qy- bands for the pheophytin fraction in
acetone (at 505 nm, 535 nm and 606 nm – Figure 2B) are
in accordance with the corresponding literature data for
Pheo a dissolved in acetone [8].
Advanced technologies
Figure 2: The absorption spectra of chlorophyll (A), pheophytin (B) and chlorophyllide (C) fraction in acetone. The positions
of the absorption maximums (Soret and Qy) are displayed within
the corresponding spectra. The positions of several observed
small absorption bands (between the Soret- and Qy-) are also
displayed within the corresponding spectra. The concentrations
of Chl and its derivatives in the corresponding fractions are in the
range of 10–4–10–5 mol/dm3. Spectrophotometrically determined
concentration ratios of the main pigments in the fractions (Chl a &
b in the Chl-, Pheo a & b in the Pheo- and Chlid a & b in the Chlidfractions are also displayed in Figure 2 (A, B and C, respectively).
Chlorophyllide a also has a chlorin system characterized by the absorption bands around 440 and 660 nm (in
organic solvents) of almost equal intensities (ε ≈ 100.000)
[3, 5-6, 8], similar to the one shown in Figure 2C for the
1(1) (2012), 16-24
chlorophyllide fraction (consisting of Chlid a and Chlid b
in ratio 6.89/1) in acetone. Since the phytil tail – a main
structural factor that differentiates Chl a and Chlid a – has
a very negligible absorption contribution compared to the
chlorine structure, the absorption maximum positions (AQy
and ASoret) are almost the same – 662.0 - 664.0 nm and
431 nm in acetone, for the “red” Qy- and the “blue” Soret- bands, respectively, for both Chl a & Chlid a [8, 22].
The corresponding bands (Soret- and Qy-) for Chlid b in
acetone are located at 457.5 and 645.7 nm, respectively
[8]. Small “shoulder” at 460 nm (probably due to the presence of Chlid b in the mixture) is present in the absorption
spectrum of the chlorophyllide fraction in acetone (Figure 2C).
The ratios of absorbance intensities for the Soret- and Qybands (ASoret/AQy) are 1.14 for Chlid a in acetone [8] and
1.46 for the chlorophyllide fraction in acetone (Figure 2C).
The observed small bands located in the spectral range
between the Soret- and Qy- bands for the chlorophyllide
fraction in acetone (at 581 nm and 614 nm – Figure 2C)
are in accordance with the corresponding literature data
for Chlid a dissolved in acetone [8].
HPLC-VIS analysis of chlorophyll, chlorophyllide and
pheophytin fractions. This part illustrates the possibilities
of applying the silica-based HPLC (with octadecyl silica, or
C-18 column) coupled with absorption measurements, for
the separation and identification, Chl, Pheo and Chlid, in
the corresponding mixtures, chlorophyll, pheophytin and
chlorophyllide fractions, respectively.
HPLC-chromatograms of the chlorophyll, pheophytin
and chlorophyllide fractions are shown in Figure 3 (A, B
and C), respectively. The absorption spectra of the main
compounds in the eluent mixture (mobile phase) observed
in the HPLC-chromatograms at the retention times, tret= 13.6
& 9 min (assigned as Chl a & b), 30.5 & 19 min (assigned
as Pheo a & b) and 2.7 min (assigned as Chlid a), were
shown in the increments of Figure 3 (A, B and C), respectively. The spectra shown in increments were taken from
the HPLC-VIS system (photodiode array data). The main
observed peaks in this work presented HPLC-chromatograms were identified by the comparison of the absorption
spectra from the photodiode array detection data (from
the HPLC-VIS system) with the corresponding absorption
spectra given in literature [8, 25-26]. A parallel review of
the most important spectral data obtained from the corresponding absorption spectra of the observed compounds
(Chl a & b, Pheo a & b and Chlid a) from the HPLC-system
and the corresponding literature data [7, 27] for the same
compounds in acetone (as well as in different solvents),
was given in Table 2 providing the possibilities for their
comparison.
High-pressure liquid chromatography (HPLC) has already been considered to be the quickest, simplest and
most reproducible method for analyzing complex mixtures
of pigments in food and other sources [17]. For example,
the separation of chlorophylls and their derivatives by reversed-phase HPLC using an octadecyl silica (C-18) column
and 100 % methanol as the eluting solvent was proposed
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1(1) (2012), 16-24
Advanced technologies
by Canjura and Schwartz [28]. Leeuwe and co-workers
(2006) have also proposed an optimized method for the
analysis of algal pigments by using HPLC equipped with
C-18 column by the gradient eluting regime [29]. Since
the analysis of this type of compounds (such as chlorophylls and derivatives) predominantly uses C-18 column
[30-33], the retention time always decreases in the same
order (pheophytin a > chlorophyll a > pheophytin b >chlorophyll b > chlorophyllide a > chlorophyllide b), and predominantly depends on the polarity of the mobile phase
[26, 30-31, 34-36]. The isocratic method that was used in
this work allowed the successful separation and identification of chlorophyll and its derivatives, pheophytin and
chlorophyllide.
Table 2. The parallel review of the most important spectral data obtained from the corresponding absorption spectra of the observed
compounds (Chl a & b, Pheo a & b and Chlid a) - from the HPLC-VIS system (left) and the corresponding literature data reported for
the same compounds (right).
Experimental data
Compounds in
HPLC-mobile phase
Absorption maxima positions/nm
Chl a
Chl b
Pheo a
Pheo b
Chlid a
Chlid b
tret./min
Soretband
Qyband
13.6
9.0
30.5
19.0
2.7
431.0
461.0
409.0
434.0
431.0
662.5
648.0
665.9
653.0
663.5
411.0
532.0
581.0
Literature data [7]
Compounds in acetone
617.0
598.0
505.0
412.0
535.9
608.5
529.0
598.5
580.0
616.5
Band
ratio
ASoret/AQy
1.21
2.85
2.39
5.27
1.20
Absorption maxima positions/nm
Soretband
Qyband
430.3
456.9
409.5
434.5
431.0
662.1
645.5
665.5
653.5
664.0
410.7
534.1
580.3
616.5
596.7
505.3
534.7
559.5
527.7
412.0
608.9
599.9
580.0
617.0
Band
ratio
ASoret/AQy
Band
ratio*
ASoret/AQy
1.23
2.82 2
2.26
5.30 4
1.14 1
0.95-1.30
.77-2.83
2.07-2.34
.92-5.30
.1-1.2
not
595.9
457.5 645.7
2.88
found
*Data are given in the value range for the compounds dissolved in different solvents, e.g. methanol, acetone, diethyl-ether, HPLC-mobile phase given in the literature.
2.83-2.88
HPLC-chromatogram of the chlorophyll fraction shows of the pheophytin fraction also consisted of four good refour good resolved peaks at retention times, 9.0, 9.8, 13.6 solved peaks (but at longer retention times than the ones
and 16.0 min (Figure 3A). Two major peaks at tret. 9.0 and observed for Chl-fraction) at 19.0, 21.7, 30.5 and 34.0 min, as13,6 min correspond to Chl b and Chl a, respectively. The signed as Pheo b, Pheo b’, Pheo a and Pheo a’, respecreplacement of-CH3 group (in Chl a) with -CHO group (in tively (Figure 3B). Since pheophytins (the demetalated
Chl b) at the position C-7 (Figure 1A) increases the polarity chlorophylls) are less polar than the corresponding chloroof the b chlorophylls [26]; since more polar than Chl a, Chl b phylls, their longer tret is expected in non-polar C-18 type of
appears on the shorter retention time, bearing in mind the the column, as shown in Figure 3(A,B). Two major peaks
predominantly non-polar C-18 type of the column [26]. The at tret. 19.0 and 30,5 min correspond to Pheo b and Pheo a,
absorption spectra of the corresponding compounds in the respectively. The absorption spectra of the corresponding
mobile phase (taken from the peaks at the tret = 9.0 and compounds in the mobile phase (taken from the peaks at
13.6 min by using HPLC-VIS – photodiode ray measure- the tret = 19.0 and 30.5 min by using HPLC-VIS - photodiments – increment of Figure 3A) showed good agreement ode ray measurements – increment of Figure 3B) showed
with the literature data for the Chl b and Chl a, respectively good agreement with the literature data for the Pheo b and
[30-31, 34-35] as well as with the literature data given in Pheo a, respectively [30-31, 34-35] as well as with the litTable 2. The other two observed components at retention erature data given in Table 2. The other two observed comtimes 9.8 and 16 min are C-132 epimers of Chl b and a ponents at retention times 21.7 min and 34.0 min (Figure 3B)
– Chl b’ and a’, respectively1. Chl-epimers have identical are C-132 epimers of Pheo b and a – Phe b’ and a’, respecabsorption spectra with the “regular” Chls (spectra not tively; Pheo a’ and b’ usually accompany the corresponding
shown), but different chromatographic abilities [2]. Chl a Pheo a and b in chlorophyll and its derivatives preparations
eluted before Chl a’ (Figure 3A) because the –CHOOCH3 [2, 22, 37].
substituent at the C-132 position in the Chl a molecule (FigHPLC-chromatogram of the chlorophyllide fraction
ure 1A) is not on the same plane of the C-173 phytil group shows two good resolved peaks at retention times 2.7 min
and is therefore less hindered and thus more polar than and 3.0 min (Figure 3C). The first peak (at 2.7 min) is Chlid a
Chl a’ [26]. Similarly, Chl b eluted before Chl b’ (Figure 3A). and the second (at 3.0) could be Chlid a’ – the absorpEpimers of chlorophylls (Chls’) are almost always present tion spectra of both compounds in the mobile phase (taken
in the chlorophyll and its derivatives preparations: they are from the HPLC-VIS system) are the same, which is charnaturally present in small amounts in photosynthetic or- acteristic of epimers [2].
ganisms [2, 22]; on the other hand, chlorophylls can be, in
small amount, converted to the 132-epimers (Chls’) during 1Chl a’ is 132(S)-epimer of Chl a, while, Chl a is the 132(R)-epimer; the same
thing is with other epimers of Chl and its’ derivatives, e.g. Chl b’ & Chl b,
the extraction processes2 [2, 37].
Pheo a’ & Pheo a, Pheo b’ & Pheo b and etc.
Similarly to the observed HPLC-chromatogram of the 2Chl’ (132(S)-epimer) is less stable than Chl itself [2].
chlorophyll fraction (Figure 3A), HPLC- chromatogram
20 Advanced technologies
1(1) (2012), 16-24
group at C-17 - Figure 1C are much more polar than the
corresponding chlorophylls; they were the first eluted from
the non-polar C-18 column (Figure 3). Chlorophyllide a
(peak at 2.7 min) is dominant in the chlorophyllide fraction,
as shown in Figure 3C. Spectral data (from the absorption
spectrum of the compound in the mobile phase at the tret. =
2.7 min) have shown good agreement with the spectral
data for Chlid a given in literature, as shown in the Table
2. On the other hand, a detectable peak (which could be
assigned to Chlid b) at tret. < 2.7 min is not found in HPLCchromatogram of the chlorophyllide fraction (Figure 3C):
probably due to Chlid b small amounts determined spectrophotometrically – almost 7 times lower Chlid b than Chlid a
concentration, and/or simply, Chlid b high polarity has as a
consequence its getting around the non-polar (C-18) column
together with the solvent used for the sample (acetone).
MS spectra of chlorophyll, pheophytin and chlorophyllide fractions. Electronspray-ionization mass spectrometry, ESI-MS is used for the detection and characterization of one or more ionic species in the solution, and is
confirmed as very useful in studies of various systems [3840]. ESI-MS allows the acquisition of mass spectra directly
from a liquid sample [38, 41]. Depending on the charge of
ions in the solution, positive or negative ion mode can be
applied (ESI+ or ESI-, respectively) in order to obtain optimum results [39]. If there is the ambiguity in the interpretation of spectra, a high resolution mass spectrometry and
MS/MS analysis leads to almost unequivocal identification
[39]. On the other hand, the composition of the solutions
subjected to the analysis is conditioned by significant limitations: not to apply high ionic strength because even relatively low concentrations of nonvolatile components (e.g.
Na+ ions) can be disturbing [40].
In the MS spectra of the chlorophyll, pheophytin and
chlorophyllide fractions (not shown), molecular peaks at
the m/z values which correspond to the molecular weights
of the found compounds from the HPLC-VIS results were
observed: Chl a (893.8), Pheo a (871.7), Chlid a (614.2)
among the Chl b, Pheo b and Chlid b. Molecular ion radicals, [M]+, for all pigment mixtures used in this work (Chl-,
Pheo-, and Chlid- fractions) were observed in the positive
ion mass spectra. The MS/MS spectra of Chl a, Pheo a and
Chlid a from the chlorophyll, pheophytin and chlorophyllide
fractions are shown in Figure 4 (A, B and C), respectively.
Figure 3. HPLC profile of chlorophyll (A), pheophytin (B) and
The most abundant fragment ions in positive ion mass
chlorophyllide (C) fractions. The pigment contents expressed as spectra of chlorophyll and its derivatives usually correconcentration ratios of the main compounds in the fractions, cchl a / spond to the fragmentation with the loss of groups from
cChl b (in the chlorophyll fraction), cPheo a / cPheo b (in the pheophytin
the C-17 and C-132 positions (Figure 1) [26, 30, 39, 42fraction) and cChli a / cChlid b (in the chlorophyllide fraction) were:
43]. The former ones can be the result of the loss of the
4.95/1, 1.05/1 and 6.89/1, respectively. The detection was carried out at 660 nm. Chromatographic conditions are described in phytil chain (as the phytadiene, C20H38) or CH3COOC20H39
the Experimental part. The corresponding absorption spectra of group – (C-17 position in Figure 1) – which appeared in the mass
the Chl a & b, Pheo a & b and Chlid a in the mobile phase, taken spectrum at m/z values corresponding to [M-C20H38]+ = [Mfrom the peaks at the retention times 9.0 & 13.6, 19.0 & 30.5 and 278]+ and [M-CH3COOC20H39]+ = [M-338]+ , respectively
2.7 min (from the photodiode array data), were displayed in the [26]. The last ones can be the result of the loss of CH3OH
increments of Figure 3(A, B and C), respectively.
(in C-133 position) or the whole ester-group (COOCH3 in
C-132 position) – (Figure 1) – which appeared in the mass
The chlorophyllides with an unesterified propionic acid spectrum at m/z values corresponding to [M-CH3OH]+ =
21
1(1) (2012), 16-24
Advanced technologies
[M-32]+ and [M- COOCH3]+ = [M-59]+, respectively [18].
tion is not an unusual phenomenon [26, 40]. In addition, a
peak observed at m/z 861.1 (Figure 4A) is agreeable with
literature [18] and corresponds to [M-CH3OH]+ ion typical
for chlorophylls fragmentation in C-133 position (Figure 1).
The first MS/MS study of pheophytin a was carried out
by Jacson 30 years ago [42]. A complete removal of Mgatom from chlorophyll in Chl-fraction obtained in this work
was confirmed by the presence of the peak at m/z 871.7
(assigned to molecular ion of Pheo a) and the absence
of m/z 893.8 (for Chl a) in the mass spectrum of pheophytin fraction (not shown). The second fragmentation of
the same peak found at m/z 871.7 gave MS/MS spectrum
typical for the Pheo a, as shown in Figure 4B. In the corresponding mass spectrum of Pheo a two most abundant
fragmentation-ions were clearly observed: at m/z 533.5
(corresponding to [M-CH3COOC20H39]+ = [M-338]+) and
593.3 (corresponding to [M-C20H38]+ = [M-278]+) among
the most abundant molecular ion-peak found at 871.7
(Figure 4B) which is in agreement with the literature data [26].
In the mass spectrum of the chlorophyllide fraction,
the observed peak at m/z 614.2 was assigned as the
molecular ion- peak of Chlid a (not shown). The second
fragmentation of the same peak found at m/z 614.2 gave
MS/MS spectrum typical for the Chlid a, as shown in Figure
4C. Fragment ions corresponding to [M - CH30H]+ and
[M - COOCH3]+, at 583.1 and 555.2 m/z respectively were
found in the mass spectrum of the Chlid a (Figure 4C) and
they are in agreement with the literature data [38-39, 43].
Conclusion
Figure 4. Full scan MS/MS spectra of the corresponding [M]+
ions of chlorophyll a (A), pheophytin a (B) and chlorophyllide a
(C) taken from the MS spectra of chlorophyll, pheophytin and
chlorophyllide fraction, respectively.
In the mass spectrum of the chlorophyll fraction, the
observed peak at m/z 893.8 was assigned as a molecular ion-peak of Chl a (not shown). The second fragmentation of the same peak found at m/z 893.8 gave MS/MS
spectrum typical for the Chl a, as shown in Figure 4A. In
the corresponding mass spectrum of Chl a, two most important fragmentation-ions were clearly observed: at m/z
555.7 and 639.0, among the most abundant, molecular
ion-peak at 893.8 (Figure 4A). The peak at m/z 555.7 corresponds to the fragmentation of Chl a molecule in C-17
position (Figure 1A) with the loss of the CH3COOC20H39
([M- 338]+) that is in agreement with the literature [26]. The
second, more abundant fragment ion in the mass spectrum of Chl a was observed at m/z 639.0, corresponding to
[M – (C20H38) + Na]+: the presence of Na+ in ESI-MS fragmenta 22 This work represents an application of VIS spectrometry, HPLC-VIS and ESI-MS/MS analysis used for the
identification of chlorophyll and its derivatives pheophytin
and chlorophyllide in their purified mixtures (the chlorophyll, pheophytin and chlorophyllide fraction, respectively). As expected, the main components in the chlorophyll,
pheophytin and chlorophyllide fractions were identified as
chlorophyll a & b, pheophytin a & b and chlorophyllide a,
respectively.
Acknowledgements
This work was supported by the Ministry of Education
and Science of the Republic of Serbia under the project
numbers TR-34012 and OI-172044.
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Izvod
IDENTIFIKACIJA HLOROFILA I NJEGOVIH DERIVATA U RASTVORIMA PIGMENATA: HPLC
HROMATOGRAFSKA, VIS I MASENA SPEKTROMETRIJSKA ANALIZA
Sanja M. Milenković1*, Jelena B. Zvezdanović1, Tatjana D. Anđelković2,
Dejan Z. Marković1
1Tehnološki fakultet, Univerzitet u Nišu, Leskovac, Srbija
2 Prirodno Matematički Fakultet, Univerzitet u Nišu, Niš, Srbija
Ova studija se bavi primenom VIS-spektrometrije, HPLC-hromatografije i Elektron-sprej jonizacione masene spektrometrije, u analizi i identifikaciji hlorofila
i nekih njegovih derivata kao što su feofitin i hlorofilid, u njihovim pročišćenim
smešama (hlorofilna, feofitinska i hlorofilidna frakcija, respektivno). Hlorofilna, feofitinska i hlorofilidna frakcija su dobijene iz pigmenatnih ekstrakata iz
biljnog materijala (Spinacia Oleracea L), njihovim prečišćavanjem na koloni.
Najvažnije komponente hlorofilne, feofitinske i hlorofilidne frakcije su identifikovane kao hlorofil a & b, feofitin a & b i hlorofilid a, respektivno. Odnos
koncentracija datih pigmenata (hlorofil a/b, feofitin a/b, hlorofilid a/b) je određen
spektrofotometrijski i iznosi, 4.95/1 za hlorofilnu, 1.05/1 za feofitinsku, i 6.89/1,
za hlorofilidnu frakciju.
24 (ORIGINALAN NAUČNI RAD)
UDK 547.979.7:543.422
Ključne reči: hlorofil, feofitin, hlorofilid, VIS,
HPLC, ESI-MS.