Annals of West University of Timisoara
Series of Chemistry 21 (2) (2012) 37-46
UROBILINOGENIC CHLOROPHYLL CATABOLITES
S E P A R A T I O N O N R E V E R S E D -P H A S E C O L U M N S
N i n a D ja p ic
University of Novi Sad. Technical Faculty “Mihajlo Pupin”, Djure Djakovica bb, 23000
Zrenjanin, SERBIA
Received: 10 November 2012
Modified: 15 November 2012
Accepted:18 November 2012
SUMMARY
The reversed-phase liquid chromatography tandem mass spectrometry analysis of
chlorophyll catabolites is still in development. The reversed-phase analytical columns: C-2,
C-4, C-8 and C-18 Pyramid were used in the separation of the urobilinogenic chlorophyll
catabolites. The packing of the reversed-phase stationary phase influences the retention,
peak shape and separation of the urobilinogenic chlorophyll catabolites. The capacity
factor, the plate number, the plate height, the reduced plate height and the total porosity
of the analytical columns were investigated on four commercial available reversed-phase
analytical columns: C-2, C-4, C-8 and C-18 Pyramid. The reduced analysis time was
achieved on the C-4 column. The highest capacity factor was obtained on the C-18
Pyramid column. The plate number and the total porosity of the column were the highest
on the C-8 column. The results obtained are useful for choosing a column packing in
separation of urobilinogenic chlorophyll catabolites. The results obtained gave insight in
the selection of the reversed phase column packing for the separation of the urobilinogenic
chlorophyll catabolites.
Keywords:
urobilinogenic
chromatography.
chlorophyll
catabolites;
reversed-phase
liquid
INTRODUCTION
The most widely used technique in separation of compounds is carried out by the
reversed-phase liquid chromatography (RPLC) [1]. Much effort has been done to
37
DJAPIC N.
understand the retention in RPLC in order to predict the chromatographic properties [2].
The RPLC stationary phases show different chromatographic properties [3, 4, 5]. The
selection of the RPLC column is important step in the analysis of the urobilinogenic
chlorophyll catabolites. For the separation of the chlorophyll catabolites the octadecylsilane
(ODS or C-18) column was used [6]. In the separation of urobilinogenic chlorophyll
catabolites (1) (Figure 1) the RPLC C-8 and C-4 columns were used [7, 8, 9]. The RPLC
column packings have different chromatographic properties regarding the retention and
separation of the urobilinogenic chlorophyll catabolites. The recorded retention data were
used to calculate the retention factor k’ since this parameter reflects the chemical nature of
the chromatographic packing. The plate number N, the plate height H, the reduced plate
height h and the total porosity of the column єT allowed the comparison of the columns. The
relative retention α was calculated in order to determine the chemistry of the urobilinogenic
chlorophyll catabolites separation. The paper describes a continuing study on urobilinogenic
chlorophyll catabolites (1) retentivity involving commercially available RPLC packings.
OH
O
NH
NH
O
HN
HN
MeOOC
COOH
O
1
Figure 1. The structure of the urobilinogenic chlorophyll catabolite isomers (1)
MATERIALS
AND
METHODS
Analytical methods
Four RPLC columns were used: RP EC 250x4 mm Nucleosil® 100-7 C-2, RP EC
250x4 mm Nucleosil® 120-5 C-4, RP EC 250x4 mm Nucleosil® 100-5 C-8 and RP EC
250x4 mm Nucleodur® 110-5 C-18 Pyramid (Macherey-Nagel, Oesingen, Switzeland). The
solvents used were HPLC grade (Acros Organics, Geel, Belgium) and trifluoroacetic acid
38
UROBILINOGENIC CHLOROPHYLL CATABOLITES SEPARATION ON REVERSED-PHASE COLUMNS
(TFA) was reagent grade (Fluka, Buch, Switzerland).
The injection volume was 10 μl via autosampler injection. Mobile phase consisted
of 0.1 % TFA (modifier) in water and methanol. The proportion of methanol was increased
linearly from 10% to 100% in 70 minutes and in next 20 minutes elution was continued
with methanol. The flow rate was 0.2 ml/min. After each separation the column was
reequlibrated linearly from 100 % methanol to 90% water (0.1% TFA):10% methanol in 10
minutes and additional 5 minutes at 90% water (0.1% TFA): 10% methanol. The on-line
LC/UV/ESI – MS analysis were performed on Waters 2695 Separations Module (Milford,
MA, USA, 2006) coupled to a Waters 2996 PDA UV-Vis detector and connected to Bruker
Daltonics esquire HCT (Bruker Daltonik, GmbH, Bremen, Germany, 2006) equipped with
an electrospray ionization (ESI) source. Nitrogen produced by nitrogen generator (Domnick
Hunter Group plc, Durham, England, 2006) was used as nebulizer (20 psi) and drying gas (9
l/min at 320ºC) in ESI experiments. The ESI detection was done in positive mode with the
target mass of 700 m/z. The capillary voltage in a ramp ranged from 4.5 to 1.5 kV. The PDA
detection was in range of 200-800 nm and the chromatograms were extracted at λ=244 nm.
The temperature of the column oven was 220C for all measurements. Data were acquired by
HyStarTM and processed by Bruker Daltonics Data Analysis running under Windows NTTM
(Microsoft, Redmond, USA).
Sample preparation
The analytes were urobilinogenic chlorophyll catabolites (1) extracted from
Hamamelis virginiana (Hvir) induced leaf senescence leaves. Hvir branches with green
leaves were cut during summer time, placed in a 1000 ml beakers and left in permanent
darkness. After two weeks, 5 g of “fresh” weight (3.65 g dry weight) leaves were collected,
frozen with liquid nitrogen, grinded and extracted with methanol at room temperature. After
centrifugation, the methanol extract was filtered and partitioned between hexane and
methanol. Water was added to the methanol phase. Urobilinogenic chlorophyll catabolites
(1) were extracted with dichloromethane from the water-methanol phase. Evaporation of
dichloromethane (t<400C) yielded 5.1 mg of the Hvir crude extract. The crude extract
obtained was dissolved in 1.5 ml methanol-water (2:1) solvent mixture. In every sample 10
μl of uracil (0.01 mg ml-1) was added. The crude extract was subject to the analysis on
different RPLC columns by LC/UV/ESI – MS analysis. The detection of urobilinogenic
chlorophyll catabolites was done by the retention time and mass spectra determined
previously [8].
Equations used
The following parameters were calculated [10]:
1. retention factor k’ using uracil as non-retained compound:
k’=(tR – t0)/t0 (1)
39
DJAPIC N.
where tR is the retention time of the urobilinogenic chlorophyll catabolite peak and t0 is
retention time of non-retained compound (uracil).
2. the plate number N, a mathematical concept, was calculated using the equation:
N=5.545 (tR/wh)2(2)
where the N is number of theoretical plates, tR is the retention time of the urobilinogenic
chlorophyll catabolite and wh is peak width at the half height (in units of time).
3. the plate height H:
H=L/N(3)
where L is the length of the column
4. the reduced plate height h:
h=H/dp(4)
where dp is the particle diameter
5. the total porosity of the column єT:
єT=F/urc2 π = Ft0/Lrc2 π(5)
where u is linear velocity (L/t0), F is the flow-rate and rc is the radius of the column.
6. the relative retention α
α=k2/k1(6)
was used to describe whether the chemistry of separation of urobilinogenic chlorophyll
catabolites remains invariant on transfer of the separation from one column to another.
RESULTS
The detection of the urobilinogenic chlorophyll catabolites was done by the RPLC
– MS analysis of the crude Hvir leaf extract, where the chlorophyll biodegration was
induced by leaving the green leaves in permanent darkness. The extract was subjected on
RPLC C-2, RPLC C-4, RPLC C-8 and RPLC C-18 Pyramide analytical columns under the
same acquisition parameters and elution solvent mixture, at 295 K, as described previously
[6, 7]. In all the chromatograms obtained revealed was the presence of the urobilinogenic
chlorophyll catabolites (Figure 2, 3, 4).
40
UROBILINOGENIC CHLOROPHYLL CATABOLITES SEPARATION ON REVERSED-PHASE COLUMNS
Figure 2. Chromatograms of urobilinogenic chlorophyll catabolites on RP C-2 and C-18 Pyramid
columns. Conditions: mobile phase: water (0.1 % TFA): methanol, gradient elution, column temperature
0
22 C, flow rate 0.2 ml/min. Detection: UV 244 nm, injection: 10 μl.
Figure 3. Chromatograms of urobilinogenic chlorophyll catabolites on RP C-4 and C-18 Pyramid
columns. Conditions: mobile phase: water (0.1 % TFA): methanol, gradient elution, column temperature
0
22 C, flow rate 0.2 ml/min. Detection: UV 244 nm, injection: 10 μl.
Figure 4. Chromatograms of urobilinogenic chlorophyll catabolites on RP C-8 and C-18 Pyramid
columns. Conditions: mobile phase: water (0.1 % TFA): methanol, gradient elution, column temperature
0
22 C, flow rate 0.2 ml/min. Detection: UV 244 nm, injection: 10 μl.
41
DJAPIC N.
The Parrotia persica (Pp) urobilinogenic chlorophyll catabolite was detected at
58.1 min., with the the m/z 633, the major urobilinogenic chlorophyll catabolite isomer
present in Hvir autumnal leaves at 59.3 minutes, was the molecular ion [M+H]+
corresponding to m/z 633 and the, up to now, unkown urobilinogenic chlorophyll catabolite
isomer was detected at 60.9 min. with the m/z 633 on the RPLC C-8 column (Figure 5). On
the RPLC C-2, RPLC C-4 and RPLC C-18 Pyramide analytical columns the Pp, Hvir and
the unknown urobilinogenic chlorophyll catabolite isomer were detected and had the
molecular ion of m/z 633. The data obtained from the chromatograms recorded were used in
further calculations.
Figure 5. The ESIMS of the urobilinogenic chlorophyll cataboltes. From left to right the ESIMS of the Pp,
Hvir and the unknown isomer, respectively. Molecular ions were extracted from the Total Ion
Chromatogram (TIC), during the separation on the RP C-8 analytical column
The proposed mechanism on separation of nonpolar compounds by RPLC involves
the interaction between the nonpolar part of the solute and the hydrocarbon part of the
column packing. Interactions in retention of semipolar compounds are more complex than
those of nonpolar compounds due to residual nonderivatized silanol groups on the packings
surface which participate in the separation. A general rule is that more hydrophobic column
packing has greater retentivity of nonpolar compounds. It is expected that a nonpolar
compound is more retentive on the C-18 column than on the C-8 packing. It is also expected
that the nonpolar compounds are more retentive on the C-8 packing than on the short alkyl
chain bonded phases like the C-4 and C-2. In this study, on the three urobilinogenic
chlorophyll catabolite isomers (1), the retention factor (k’) of the Pp isomer, along with the
plate number N, the plate height H and the reduced plate height h, on the four RPLC
analytical columns used, is depicted in Table I.
42
UROBILINOGENIC CHLOROPHYLL CATABOLITES SEPARATION ON REVERSED-PHASE COLUMNS
Table I. Properties and characteristics of the columns investigated
for the Pp isomer
C-2
k’
2.73
N
14038
H [mm]
0.018
h
2.57
єT
0.82
C-4
2.13
44915
0.006
1.20
0.88
C-8
2.42
81240
0.003
0.60
1.08
C-18
6.14
65327
0.004
0.80
0.47
The retentivity of the Pp isomer on four RPLC analytical columns used is depicted
in Figures 2, 3 and 4. The Hvir urobilinogenic chlorophyll catabolite isomer (1), exhibited
the strongest retention (high k’ value) on C-18 Pyramid column (Table II).
Table II. Properties and characteristics of the columns
investigated for the Hvir isomer
C-2
k’
2.80
N
40477
H [mm]
0.006
h
0.86
єT
0.82
C-4
2.20
46593
0.005
1.00
0.88
C-8
2.49
84631
0.003
0.60
1.08
C-18
6.37
69660
0.004
0.80
0.47
The k’ value was higher on the short alkyl chain bonded phase C-2 than on the
packings C-4 and C-8 for the Hvir isomer. In the chromatogram obtained during the
separation on the C-2 analytical column no peak – shape symmetry of urobilinogenic
chlorophyll catabolite isomers was observed. The chromatogram obtained had no Gaussian
peak resolution and had no uniform peak – shape and therefore were completely distorted. It
can be concluded that C-2 analytical column is least suitable for the separation of
urobilinogenic chlorophyll catabolites (Figure 2). The C-18 Pyramide packing has exhibited
high retentivity toward the urobilinogenic chlorophyll catabolites (1) (Table I, II and III).
43
DJAPIC N.
Table III. Properties and characteristics of the columns
investigated for the unknown isomer-1
C-2
k’
-
N
-
H [mm]
-
h
-
єT
-
C-4
2.27
48952
0.003
0.60
0.88
C-8
2.58
89259
0.002
0.40
1.08
C-18
6.60
74132
0.002
0.40
0.47
DISCUSSION
The high retention value for the three urobilinogenic chlorophyll catabolite isomers
(1), indicated that the separation mechanism can involve the interaction of the
urobilinogenic chlorophyll catabolites aromatic rings’ π-electrons with the C-18 Pyramide
ligand chain length or the different penetration of the urobilinogenic chlorophyll catabolites
in between the surface ligands of the C-18 Pyramide packing compared to the other
packings investigated [11, 12]. The k’ values were rather similar for the C-4 and C-8
packings indicating that the separation mechanism is nearly similar on those two RPLC
analytical columns tested. It can be observed that the silanol activity of the C-4 and C-8
columns during the separation of the urobilinogenic chlorophyll catabolites comprises
similar types of interactions, most probably ion-ion and hydrogen bonding activity [13]. The
lowest capacity factor was on the C-4 column indicating the lowest analysis time in
separation of urobilinogenic chlorophyll catabolites (1) (Figure 3).
The migration velocity of the peak under the gradient separation, neglecting some
second order effects, used in this study, was independent in the case of C-4, C-8 and C-18
Pyramid analytical columns. The efficiency of the analytical column and therefore the
sharpness of the peaks was the highest in the case of the C-8 packing. The lowest plate
number was in case of the C-2 packing indicating the lowest efficiency of the analytical
column.
The height equivalent to theoretical plate, reported in millimeters, showed that the
shortest theoretical plates are in case of the C-8 and C-18 Pyramid analytical columns. The
least plates are contained, in any length of the column, in case of the C-2 column indicating
the lowest column efficiency. The reduced plate height h was the same in case of the C-8
and C-18 Pyramid analytical columns. The highest h value was in case of the C-2 column.
The total porosity of the column was the highest in the case of the C-8 and it
44
UROBILINOGENIC CHLOROPHYLL CATABOLITES SEPARATION ON REVERSED-PHASE COLUMNS
decreased from the C-4 to the C-2 column and was the lowest in the case of the C-18
Pyramid analytical column.
The chemistry and the pore structure of the stationary phase affected the peak
shape in the case of the C-2 analytical column where the peaks had no Gaussian peak shape.
The relative retention demonstrated that the chemistry of the separation remains invariant
on transfer of the separation from one column to another in case of C-4, C-8 and C-18
Pyramide analytical columns (Table IV, Figure 6).
Table IV. Relative retention of: kHvir-isomer/ kPp-isomer and kunknown isomer-1 /
kHvir-isomer on the C-2, C-4, C-8 and C-18 Pyramide RPLC analytical
columns
C-2
kHvir-isomer/kPp-isomer
1.03
kunknown isomer-1/kHvir-isomer
-
C-4
1.03
1.03
C-8
1.03
1.04
C-18
1.04
1.04
Figure 6. Relative retention of: kHvir-isomer/ kPp-isomer and kunknown isomer-1 / kHvir-isomer on the C-2, C-4, C-8 and C18 Pyramide RPLC analytical columns
CONCLUSION
Gradient elution with aqueous (0.1% TFA): methanol mobile phase on different
RPLC stationary phases provided a separation of urobilinogenic chlorophyll catabolites.
The lowest analysis time was on the C-4 analytical column. The structure of the stationary
45
DJAPIC N.
phase affected differently the efficiency (the plate height) in case of the Hvir urobilinogenic
chlorophyll catabolite. The C-8 and C-18 Pyramid analytical columns had the lowest
reduced plate height at the flow rate of 0.2 ml/min., gradient elution. The C-18 Pyramide
had the highest retention of the urobilinogenic chlorophyll catabolites and the retention
increased levels off compared to the C-4 and C-8 RPLC analytical columns.
In case of C-4 and C-8, the chain length of the packing most probably does not
influence the penetration of the urobilinogenic chlorophyll catabolites into the bonded
phase. The size of urobilinogenic chlorophyll catabolites, their steric characteristics or
solvation environment of the functional groups can be the explanation for their retentivity
on those two packings. The methods described, in this paper, offer a solution in choosing
the RPLC analytical column for the separation of urobilinogenic chlorophyll catabolites.
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