2. Materials and methods 2.1. Plant

2. Materials and methods
2.1. Plant materials
Leenawaty Limantara et al. / Procedia Chemistry 14 ( 2015 ) 225 – 231
227
Green leafy vegetables were collected from three different supermarkets in Malang area (East Java, Indonesia).
All samples were purchased early in the morning, transported into laboratory and directly measured by in vivo
procedure for SPAD value and % N. Then, the samples were stored at -18 °C deep freezer for further in vitro
analysis. The whole experiment was accomplished during May 2014 until June 2014.
2.2. In vivo assay
The relative chlorophyll content (SPAD value) and % N were determined by means of SPAD-502 chlorophyll
meter (Konica Minolta, Japan) and nitrogen meter Agriexpert CCN 6000 (Satake, Japan), respectively. The adaxial
side of the leaves was always placed toward the emitting window of the instrument and major veins were avoided.
Each leaf, based on its total leaf area, was marked in five up to 14 representative points for in vivo measurements,
and
the average value was calculated. As for basil, due to its small size, seven leaves were used with two up to three
spots of measurement each. This method was i
ntended to overcome the influence of non-uniform distribution of
chlorophyll in the leaf, and hence producing more representative data.
2.3. In vitro assay
Liquid nitrogen was poured to accurately weighted 0.2 g of leaf samples, and then the frozen leaf was ground in a
porcelain
mortar and added with 3 mL methanol (Merck, Germany). The extraction was continued by using Vortex
f
or 10 s with inert atmosphere. After that, the extract was filtered (0.22 μm

–1
and detection was performed over the range
400 nm to 700 nm.
2.4. Data analysis
The figures were created from numeric data and plotted using Origin 7 Software (OriginLab) and Microsoft
Ex
cel 2013. The data were expressed as the mean ± standard error of the mean (SEM).
3. Results and discussion
Ten green leafy vegetables were successfully collected from three different supermarkets in Malang (Table 1),
ex
cept basil and sword-leaf lettuce which were not available in the third supermarket during this time of research.
Af
ter purchasing, samples were subsequently subjected to in vivo and in vitro measurement. All measurements were
co
nducted under the same light condition in order to eliminate data variation caused by difference of light
in
terference which may lead to pigment degradation or any disturbance in instrumentation. Identical in vitro
extraction and HPLC procedure was similarly employed for all samples.
Fig. 1 shows the SPAD values of ten green leafy vegetables purchased from three supermarkets in Malang. The
average o
f SPAD values ranges from the lowest 2.7 ± 0.8 of head cabbage (Kbs) up to the highest 52.4 ± 3.7 of
C
hinese cabbage pak-choi (Pch). The SPAD value is proportional to the relative amount of chlorophyll content and
co
nsistent with their colour appearance, in which the head cabbage has light green leaves, whereas Chinese cabbage
pak-choi has darker green foliage. Higher SPAD value signi
fies higher chlorophyll content. Three vegetables
showed low SPAD value (below 30), i.e. head cabbage (Kbs), green leaf lettuce (Sel), and sword-leaf lettuce (Sak).