922  Nowak et al.: Journal of AOAC International Vol. 100, No.
4, 2017
SPECIAL GUEST EDITOR SECTION
The Thin-Layer Microchromatography (μTLC) and
TLC–FID Technique as a New Methodology in the Study of
Lubricating Oils
Paulina Nowak, Judyta Kosińska, Marta Glinka, and Marian Kamiński1
Gdańsk University of Technology, Faculty of Chemistry, Department of Chemical and Process Engineering, Narutowicza 11/12,
80-233 Gdańsk, Poland
This paper concerns the possibility of using TLC
coupled with a flame ionization detector (FID) and
micro-TLC (μTLC) as precursors for microfluidized
devices of analytical techniques to identify
and determine the presence and content of the
petroleum/vegetable oil base in the lubricating oils
applied in cutting devices (chainsaws). This research
is related to the problem of ensuring, in compliance
with the requirements of environmental protection,
a sufficient level of biodegradability of lubricating
oils emitted to the environment during operation
of equipment lubricated with these oils. Such oils
include those mainly used in cutting devices and
emitted in the form of a mist into the environment
during the operation of those devices. When oil
components are eco-toxic, contamination of the
environment occurs. New methodologies for the
identification and determination of the petroleum oil
base, which is very difficult to biodegrade, as well
as the easily biodegradable ingredients of vegetable
origin in the lubricating oils, are presented. The
described procedures indicate in an indisputable
way whether the oil contains the oil base originating
from crude oil and whether it contains adequate
enriching additives. The procedures also allow the
assessment of the content of particular groups of
constituents (μTLC) or the determination of the
group composition (TLC–FID).
T
he analysis of the consumption of lubricant oils for
cutting chainsaws indicates an amount of thousands of
tons per year in Europe (1). This group of lubricant oils
includes those primarily used for cutting devices in forestry and
household applications for wood cutting, trimming hedges, or
similar applications. Such oil, as a consequence of working in
an open system, is emitted to the environment in the form of
Guest edited as a special report on “Detection and Analysis of
Microbes, Bioanalytes, and Micropollutants, Focusing on Food
and Environmental Samples, Using Nanoparticle-Based Detection
Systems, Microfluidic Analytical Devices, and Related Techniques” by
Paweł K. Zarzycki.
1
Corresponding author’s e-mail address: markamin@pg.gda.pl
This work is supported by the National Science Centre, Poland,
and is a continuation of the research in the project “The use of size
exclusion chromatography and adsorption for the separation and
determination of a group of lipids and conversion products in edible
fats” (grant No. N N312 417237, contract No. 4172/B/PO1/2009/3).
DOI: 10.5740/jaoacint.17-0167
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mist during the application of the cutting devices. It is known
that the ingredients of oils containing an oil base and enriching
additives based on crude oil are ecologically toxic and,
especially in the form of mist, are harmful to the health. This
presents a real problem during the operation of cutting devices,
not only for the natural environment but also for workers’ health
in the absence of appropriate personal protective equipment.
The only, but also very economic, solution for the problem is
the application of lubricant oils manufactured using a vegetable
oil base and the same type of additives, i.e., oil not containing
any ingredients derived directly from petroleum. An alternative
is to use a polyester oil base; however, this oil is more expensive
and has a longer biodegradation time in the environment as
compared to vegetable oil. However, such requirements are not
currently proposed outside of Nordic countries. This is because
regulations of the Organization for Economic Cooperation and
Development and the European Union countries require the
biodegradability of oil for cutting devices in a specified period
of time, e.g., 75% in 28 days. The regulations are framed in
such a way that even lubricating oil with a 50% petroleum oil
base content meets the requirements of the biodegradability
test (2). The biodegradability study of materials released into
the environment, including lubricating oil for chainsaws,
is certainly advisable. However, when the released oil is
emitted into the environment, it should be required to achieve
total biodegradability (100%). Such studies are lengthy and
expensive. Much easier and far less expensive is the study of the
group composition of the lubricating oil, i.e., the examination of
its chemical composition.
The study of the chemical composition of oil products is
based on determining the content of individual groups of
chemical compounds with similar properties (i.e., examining
the composition of the group). For the determination of
petroleum product group composition, elution chromatography
and the methods of sorption/desorption in normal phase (NP)
systems are mainly applied. The standardized methods are
applied with a valid validation. These standards are mainly
methods developed by the American Society for Testing and
Materials (ASTM), e.g., ASTM D2007 “Standard Test Method
for Characteristic Groups in Rubber Extender and Processing
Oils and Other Petroleum-Derived Oils by the Clay-Gel
Absorption Chromatographic Method” (3), the most frequently
recommended method ASTM D2549 “Standard Test Method
for Separation of Representative Aromatics and Nonaromatics
Fractions of High-Boiling Oils by Elution Chromatography”
(4), and ASTM D4124 “Standard Test Method for Separation of
Asphalt into Four Fractions” (5). The International Organization
for Standardization (ISO) standards are based on the ASTM
 Nowak et al.: Journal of AOAC International Vol. 100, No. 4, 2017  923
standards and are often transformed into European (EN)
standards. Among the former, applicable Polish standards can
be distinguished, e.g., PN-72/C-04025 “Petroleum Products  –
Determination of Hydrocarbon Group Composition by Liquid
Chromatography” (6). For the more volatile compounds in
diesel fuel, the standard PN-EN 12916:2016-03 “Petroleum
Products – Determination of Aromatic Hydrocarbon Groups in
Middle Distillates – High Performance Liquid Chromatography
Method with Refractive Index Detector” (7) applies.
The principle of the procedure involves separation of the
groups on glass columns. The components of the low-volatile
product test sample are separated by adsorption on the surface
of the active adsorbent and the gradual desorption of individual
groups of components using a step elution with rising elution.
Specific fractions are collected and, after eluent evaporation,
their contents are gravimetrically determined. A freshly packed
column is used each time the method is applied. These methods
are extremely time-consuming and laborious and associated
with significant consumption of pure and anhydrous eluents. As
a result, they have little in common with currently recommended
green analytical chemistry methods. To obtain repeatable and
reproducible results, considerable experience of the analyst is
required, both in terms of column packing in a way that ensures
piston flow profile, and sorbent activation so as to maintain the
active sorbent.
An alternative to these methods for the study of oil group
composition or other nonvolatile compound mixtures is the
application of TLC or TLC with a flame ionization detector
(FID), which provides detection of low-volatility organic
compounds during movement of the rod in the detector.
Compared to the methodologies based on column separation
of compounds, TLC methods, both the “classic” option and
TLC–FID, are characterized by a much lower consumption of
organic solvents and a shorter duration of analysis. In addition,
several different samples can be examined at the same time to
obtain better reproducibility of test results.
TLC has a large number of applications, including
micropreparation applications that are used particularly in
analytics, health care, drug research, plant extracts, food, and
in many areas of modern analysis (8–23). With the use of
imaging scanners and appropriate software, TLC becomes an
analytical technique, albeit with a lower precision than HPLC.
The largest group of applications relates to the analytical
content of certain chemical compounds or chemical groups
in the separated mixtures, and a large amount of literature
describes this methodology and its applications (23). Fewer
applications of the TLC technique are found for the separation
of groups. In this case, it is used primarily as fingerprinting
methodology, e.g., in studies designed to identify the low-
volatility petroleum products in environmental monitoring
and to diagnose leaks in oil refinery process installations (24).
The TLC technique can also be applied as a tool to identify
the sources of environmental contamination and can quickly
distinguish the environmental contamination of low-volatile
petroleum products from coal, food, or different matrixes
(25,  26). Specifically, a comparative analysis of the image
spots on the TLC plate is done, taking into account the different
visualization methods of the spots on the TLC chromatograms,
primarily under visible (Vis) light at UV 254 nm (plates coated
with luminescence, i.e., F254, fluorescing in the Vis light range
under UV light at 254 nm) or at 365 nm (using fluorescence
in the Vis range of separated chemical compounds/chemical
compound groups). After iodine vapor exposure, chemical
derivatives present in the spots develop colors upon spraying
the plate with an aerosol solution of a particular reagent (27).
The TLC–FID technique is also used to separate and study the
group composition of multicompound, eluent-soluble, low-
volatile, or nonvolatile organic materials, including fats and
their conversion products, and in analytics and QC (28–33).
It is also widely applied in other analytical materials (34–36),
including petroleum products. The TLC–FID technique is
most commonly used to determine the group composition
of the base oils and similar petroleum materials (37–39),
and the fractions of the bitumen and heavy oil fraction
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(40–48), without prior isolation of asphaltenes (49, 50). The
application of TLC–FID techniques allows the determination
of the group composition of oil products divided into saturated
and aromatic hydrocarbons, resins, and asphaltenes [i.e.,
saturates, aromatics, resins, asphaltenes (SARA; 38, 51,  52)].
The undeniable advantage of this technique is the small
consumption of solvent, the possibility of 10 determinations
at the same time, and the good repeatability of assays; thus,
it can be applied for the routine determination of the group
composition of the above-mentioned analytes.
In this study, a new methodology was characterized to test
lubricating oils for the presence and content of components
derived from petroleum products, as well as components
derived from vegetable oils. Micro-TLC (μTLC) and TLC–FID
were applied. The aim was to obtain uncontested information
regarding additives. The semiquantitative examination was
performed by applying the described work conditions and
μTLC technique. It is also worth noting that the TLC technique
can be applied under different phase systems: NP, reversed
phase, and hydrophilic interaction LC (HILIC; 5–18). In
addition, different TLC plates are available: SiO2, C18, C8,
CN, and NH2.
However, the TLC–FID technique, the variation of TLC
that combines the separation ability with the versatility and
sensitivity of an FID, allows one or several separations to be
performed according to TLC principles, but only in the NP and
HILIC systems. The determination of organic low-volatility
components or groups of components of the tested mixture
in the form of chromatographic peaks, takes place by putting
the dried TLC rod with separate factions of the components
of the tested mixture “immobilized” on the sorption surface,
through the air-hydrogen flame inside the detector head of the
FID (28,  53–55). In this paper, the TLC–FID technique was
applied to estimate the determination of the composition of the
tested oil, because accurate determination requires a calibration
method by an external standard. The TLC–FID technique should
be used to determine the group composition of the tested oil.
It has undeniable advantages compared to other methods (e.g.,
SARA column fractionating in ASTM D4124). It consumes
far less organic solvents and requires less time and effort. This
paper presents the developed procedures and an assessment of
the results and their characteristics.
Previous studies of the application of TLC and TLC–
FID techniques in NP systems for group separation have
made observations regarding the development of optimal
conditions for  the analysis of groups (14, 24, 26). The most
important of these observations is to use short-length TLC plates
(30–60  mm), hence the term μTLC. These studies have also
924  Nowak et al.: Journal of AOAC International Vol. 100, No. 4, 2017
shown the efficiency of using gradual elution, i.e., the elution
of the least polar groups by using the lowest polar eluent over
a distance of approximately 90–95% of the elution distance on
a TLC plate, the medium polar distance of 60% using an eluent
of medium polarity, and the most polar at a distance of 30%
elution path by using the most polar eluent. The same procedure
was applied in the case of TLC–FID technology. It is also worth
noting that the selection of the eluent and subsequent elution
steps are important in order to enable all components of the
analytes to be soluble in the eluent used in the first elution
step (24).
Experimental
Materials: Reagents, Samples, and Standards
Components of the eluents:
(a)  Toluene, pure for analysis.—POCH (Poland).
(b)  n-Hexane (nC6), HPLC grade.—Merck (Germany).
(c)  Dichloromethane (DCM), pure for analysis.—Chempur
(Poland).
(d)  Methanol (MeOH), pure for analysis.—POCH.
Oil samples and standards:
(a)  Rapeseed vegetable oil (20 mg dissolved in 1 mL DCM).
(b)  Lubricating oil based on vegetable oil with additives
(20 mg dissolved in 1 mL DCM).
(c)  Lubricating oil consisting of a mixture of vegetable oil
and petroleum oil (20 mg dissolved in 1 mL DCM).
(d)  Lubricating oil based on petroleum oil with additives
(20 mg dissolved in 1 mL DCM).
(e)  Waste oil from oleochemical plants from the refining
process of vegetable oil (20 mg dissolved in 1 mL DCM).
(f)  Lubricant oil with petroleum oil base [Society of
Automotive Engineers (SAE) 10 (20 mg dissolved in 1 mL
DCM)].
(g)  Squalane (20 mg dissolved in 1 mL DCM).
(h)  Standard solution.—Solution containing 27.8 mg free
fatty acid (FFA), 10.2 mg monoacylglycerol (MAG), 12.7 mg
diacylglycerol (DAG), 27.8 mg triacylglycerol (TAG), 24.8 mg
fatty acid methyl ester (FAME), and 2.5 mg glycerol, dissolved
in 1 mL DCM–MeOH (95 + 5, v/v).
(i)  Lubricant oil with poly-α-olefinic synthetic base
(PAO6;20 mg dissolved in 1 mL DCM).
(j)  Lubricant oil based on low-volatile alkylate (20 mg
dissolved in 1 mL DCM).
Apparatus
Apparatus used in the μTLC technique:
(a)  TLC plates.—5 × 5 cm.
(b)  TLC Silica Gel 60 F254.—Merck (Germany).
(c)  Glass chromatographic chambers with a cellulose lid for
saturation.
(d)  UV lamp.—254 and 365 nm, Telbid TB 02 (Poland), or
equivalent.
(e)  Digital camera with 14-megapixel minimum resolution.
(f)  Microchromatography syringe with a capacity of 10 μL.
(g)  Dryer.—Capable of drying TLC plates at 105°C
(WAMED, Poland).
(h)  Glass desiccator with iodine.
Apparatus used in the TLC–FID technique:
(a)  TLC–FID IATROSCAN analyzer.—Iatro Laboratories
(Japan).
(b)  Automatic sample dispenser.—SES 3200/IS-01 (SES,
Germany).
(c)  Rod dryer.—TLC TK-8 (Iatro Laboratories).
(d)  Analog-to-digital converter with software for data
processing (“Chomik”).
(e)  Quartz rods.—Chromarod S III coated with silica gel
with special preparation (Iatro Laboratories).
Methodology and Chromatographic Conditions
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The following sample preparation was used for the μTLC and
TLC–FID techniques: A 20.00 ± 0.01 mg sample of each material
or chemical (see “Oil samples and standards” in the Materials:
Reagents, Samples, and Standards section) was weighed into
a hermetically sealed 5  mL vial. A 1.000  ±  0.001  mL portion
of DCM was added to each vial. For the standard mixture
containing FFA, MAG, DAG, TAG, FAME, and glycerol, the
solvent was 1 mL DCM-MeOH (95 + 5, v/v). The same samples
were applied in both tests.
(a) TLC elution conditions.—TLC plates were developed
using a three-step elution procedure carried out in two directions
according to two different principles with regard to the eluent
force increase in subsequent separation steps:
Elution I: increase in the polarity (elution strength) of the
eluent in the next stages of elution.
(1)  30% of the plate height in a mixture of DCM–MeOH
(95 + 5, v/v), the eluent with the highest polarity (approximately
1 min).
(2)  60% of the plate height in toluene, the eluent with
medium polarity (approximately 3 min).
(3)  100% of the plate height in nC6, the eluent with the
lowest polarity (approximately 4 min).
Elution II: decrease in polarity (elution strength) of the eluent
in the next stages of elution.
(1)  100% of the plate height in nC6, the eluent with the
lowest polarity (approximately 4 min).
(2)  60% of the plate height in toluene, the eluent with
medium polarity (approximately 3 min).
(3)  30% of the plate height in a mixture of DCM–MeOH
(95 + 5, v/v), the eluent with the highest polarity (approximately
1 min).
(b) μTLC.—Using a microsyringe, a 3 μL portion of the oil
samples and standards was applied to the TLC plates. During
the dosing of samples, blowing air at room temperature was
applied.
After evaporation of the solvent, the plate was placed in a
TLC chamber containing a particular mobile phase saturated
by eluent atmosphere. Upon reaching a predetermined height
of the eluent, the plate was removed from the chamber and
dried in an oven (105°C) for 10 min to evaporate the solvent.
Next, photographs were taken using a UV lamp at two
wavelengths of light (254 and 365  nm). After the last stage
of development, the plates were placed in a desiccator with
iodine for approximately 15  min (the desiccator was heated
to a temperature of approximately 35–40°C before and during
the test). The plate was removed from the desiccator and
photographed in Vis light. The above-mentioned steps were
done under the exhauster.
 Nowak et al.: Journal of AOAC International Vol. 100, No. 4, 2017  925
The elution was performed with regard to increasing or
decreasing the elution strength of the mobile phase according
to the procedures described in the TLC elution conditions
section.
(c) TLC–FID.—During studies using the TLC–FID technique,
the IATROSCAN analyzer and quartz rods (Chromarod S III
coated with silica gel) were applied. Immediately before each
separation, the frame with rods was activated three times for
35, 50, and 50 s in the hydrogen flame of the FID. Activated
frames with TLC–FID rods were stored for 10  min in the
desiccator to cool down. The examined samples were applied
onto rods using an automatic applicator, each rod receiving
a 1  μL dose of the test mixture solution containing 20  mg
sample per 1 mL DCM [or, in the case of the standard mixture
containing FFA, MAG, DAG, TAG, FAME, and glycerol,
1 mL DCM–MeOH (95 + 5, v/v); see the Materials: Reagents,
Samples, and Standards section]. After application of the spots,
rods were dried for 2 min in the oven at 70°C and placed for
10 min in the desiccator. After this time, the frame with rods
was hung over the eluent on the chromatography chamber and
subjected to development. The TLC rods were again dried
for 2  min in the drier at 70°C and placed for 10  min in the
desiccator. The schematic diagram of the TLC–FID station
is shown in Figure 1. The elution was performed with regard
to the order of eluent strength in the subsequent steps of the
chromatogram development by the procedure described in the
TLC elution conditions section.
The detection of the TLC–FID technique was carried
out during the mechanical passage of each 0.9  ×  100  mm
Chromarod S III rod through the hydrogen flame of the FID
detector for 35  s, with simultaneous digital signal recording
from the electrometer. The hydrogen flow rate was 150 mL/min,
and the air flow rate was 1.8 L/min.
The separation and determination of each sample was
performed twice, i.e., each sample was dosed on two rods
separately. The elution time at the height of 100% in nC6 was
approximately 20  min; for toluene at the height of 60%, the
elution time was approximately 15 min; and for DCM–MeOH
(95 + 5, v/v) at a height of 30%, it was approximately 5 min.
Figure 1.  The scheme of TLC–FID IATROSCAN measurement
site and the procedures applied while determining the group
composition.
Results and Discussion
This study involved the development of new assessment
methodologies for the identification and determination of
any type of oil and vegetable ingredients in lubricating oils.
The study  included chainsaw lubricant oils emitted to the
environment that either meet fully, meet in part, or do not fulfill
the requirements for biodegradability. The methodology applied
in this work was, to some extent, related to microfluidic dosing
techniques, as well as microfluidic detection during movement
of the rods (Chromarod S III) in the TLC–FID system.
Representative examples of the results of selected studies and
our conclusions are described below.
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Results Based on the μTLC Technique
Figure  2 shows representative photographs of μTLC
chromatograms obtained after successive elution steps and
under different visualization conditions for all tested samples on
TLC Silica Gel 60 F254 plates in the elution normal order (with
an increase in the polarity of the eluent; elution I). Alternatively,
all tested samples were also developed on TLC Silica Gel 60
F254 plates in the reverse elution order (with a decrease in the
polarity of the eluent; elution II; Figure 2).
To identify the components or groups of components in the
tested samples, the separation of standards was performed
using the elution order according to the Methodology and
Chromatographic Conditions section, as shown in Figure 4.
The study has shown that the evaluation of group
composition methodology of the oil by NP-TLC systems
allows the presence and approximate content of components
derived from petroleum, and vegetable oil or its derivatives, to
be determined without separation. On the TLC chromatogram
using SiO2 F254 plates, the presence and approximate content
of individual groups is evaluated by observing (1) the decrease
in the degree of plate luminescence under the surface of the
spots originating from the chemical compounds absorbing
at 254  nm; (2) the blue fluorescence of substituted aliphatic/
alicyclic aromatic hydrocarbons at UV 365  nm; and (3) the
development (in iodine vapors) of the spot color responsible for
chemical compounds that biodegrade well on the plate after the
three-stage development of TLC chromatogram.
In Figures  2 and 3, on the chromatograms with a subscript
365  nm, blue fluorescence spots appear, confirming the
presence of aromatic hydrocarbons. This can be observed for
sample 3 (lubricant oil consisting of a mixture of vegetable
and petroleum oil), sample 4 (lubricant oil with petroleum
oil base with enriching additives), sample 6 [lubricant oil
with petroleum oil base (SAE 10)], and sample 10 (lubricant
oil based on low-volatile alkylate) in the form of dots (A1)
eluted with nC6 (weak), and in the form of spots eluted with
toluene (two-ring and higher aromatic compounds; A2+). The
development of the chromatograms in iodine vapor was aimed
at showing good biodegradability of chemical compounds
(such as TAGs, MAGs, and some fatty acids that have double
bonds). In sample 1 (food vegetable oil), sample 2 (lubricant oil
based on vegetable rapeseed oil with additives), and sample 8
[standard mixture containing FFA (oleic acid), MAG (1-oleoyl-
rac-glycerol), DAG (dioleoyl-rac-glycerol), TAG (glycerol
trioleate), and FAME], highly biodegradable chemicals are
noticeable. This test allows for the unambiguous separation
926  Nowak et al.: Journal of AOAC International Vol. 100, No. 4, 2017

of the oil fraction based on origin (the vegetable fraction
elutes lower, whereas the petroleum fraction elutes higher on all
NP-TLC chromatograms).
The study compared two methods for evaluating the
components of oils in normal versus reversed elution order.
The optimal order of elution is considered to be elution II, in
which step 1 is 100% of the plate height in nC6 with elution to
3 cm; step 2 is 60% of the plate height in toluene with elution
to 2 cm; and step 3 is 30% of the plate height in DCM–MeOH
(95  +  5,  v/v) with elongation at 1  cm because the procedure
provides better separation factor.
Results Based on the TLC–FID Technique
Representative TLC–FID chromatograms of the examined
sample group separation obtained using Chromarod S III and
IATROSCAN FID are presented in Figures  5–7. Tables  1
and 2 summarize the chromatographic parameters obtained
for each sample related to peak number, area, and percentage
area, as well as the Rf and retention factor. The chromatograms
obtained for the elution carried out in the direction of increasing
elution strength (elution II) are presented in Figure 6, whereas
the chromatograms obtained for the elution carried out toward

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Figure 2.  Sets of chromatograms obtained in the normal order of development for NP-TLC plate Silica Gel F254 (5 × 5 cm; Merck, Germany).
Samples (20 mg dissolved in 1 mL DCM unless otherwise noted): 1, rapeseed vegetable oil; 2, lubricant oil with vegetable oil base with
enriching additives; 3, lubricant oil consisting of a mixture of vegetable and petroleum oil bases; 4, lubricant oil with petroleum oil base with
enriching additives; 5, waste oil from oleochemical plants from the refining process of vegetable oil; 6, lubricant oil with petroleum oil base
(SAE 10); 7, squalane; 8, standard solution containing 27.8 mg FFA, 10.2 mg MAG, 12.7 mg DAG, 27.8 mg TAG, 24.8 mg FAME, and 2.5 mg
glycerol dissolved in 1 mL DCM–MeOH (95 + 5, v/v); 9, lubricant oil with PAO6; and 10, lubricant oil based on low-volatile alkylate. Visualization
is designated by the superscripts 254 and 365 nm, Vis, and J2, with the latter showing development after exposure to iodine fumes. Elution
order is designated by the subscripts 100% (height of plate in nC6, 3 cm), 60% (height of plate in toluene, 2 cm) and 30% [height of plate in
DCM–MeOH (95 + 5, v/v), 1 cm].
 Nowak et al.: Journal of AOAC International Vol. 100, No. 4, 2017  927

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Figure 3.  Sets of chromatograms obtained in inverted order of development for NP-TLC plate Silica Gel F254 (5 × 5 cm; Merck, Germany).
Samples (20 mg dissolved in 1 mL DCM unless otherwise noted): 1- rapeseed vegetable oil; 2, lubricant oil with vegetable oil base with
enriching additives; 3, lubricant oil consisting of a mixture of vegetable and petroleum oil bases; 4, lubricant oil with petroleum oil base
with enriching additives; 5, waste oil from oleochemical plants from the refining process of vegetable oil; 6, lubricant oil with petroleum
oil base (SAE 10); 7, squalane; 8, standard solution containing 27.8 mg FFA, 10.2 mg MAG, 12.7 mg DAG, 27.8 mg TAG, 24.8 mg FAME, and
2.5 mg glycerol dissolved in 1 mL DCM–MeOH (95 + 5, v/v); 9, lubricant oil with PAO6; and 10, lubricant oil based on low-volatile alkylate.
Visualization is designated by the superscripts 254 or 365 nm, Vis, and J2, the latter showing development after exposure to iodine fumes.
Elution order is designated by the subscripts 30% [height of plate in DCM–MeOH (95 + 5, v/v), 1 cm], 60% (height of plate in toluene, 2 cm), and
100% (height of plate in nC6, 3 cm).

decreasing elution strength of the mobile phase (elution II) are
shown in Figure 5.
The group separation of the examined samples can be easily
observed on the obtained chromatograms. The dominant peaks
presented in the TLC–FID chromatograms can be seen in
Figures 5–7 as the following:
• The fraction of aromatic hydrocarbons (A1).
• Petroleum base oil components (samples 3, 4, 6–8 in
Figures 5 and 6).
• TAGs (samples 1–3 in Figures 5 and 6, and samples 2 and 2′
in Figure 7).
Comparing the TLC–FID chromatograms of waste oil
(separation results depicted in sample 5 in Figures  5 and 6)
with the chromatograms obtained for vegetable oil (sample 1
in Figures  5 and 6), peaks are seen that denote the presence
of the incomplete acylglycerols (MAG and DAG) and free
fatty acids (FFA), as well as their oxidized forms, indicating
that the oxidative and thermo-oxidative degradation of fats
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Figure 4.  Sets of chromatograms obtained in normal (bottom) and inverted (top) order of development for NP-TLC plate Silica Gel F254
(5 × 5 cm; Merck, Germany). Samples (20 mg dissolved in 1 mL DCM unless otherwise noted): 1- rapeseed vegetable oil; 2, lubricant oil with
vegetable oil base with enriching additives; 3, lubricant oil consisting of a mixture of vegetable and petroleum oil bases; 4, lubricant oil with
petroleum oil base with enriching additives; 5, waste oil from oleochemical plants from the refining process of vegetable oil; 6, lubricant oil
with petroleum oil base (SAE 10); 7, squalane; 8, standard solution containing 27.8 mg FFA, 10.2 mg MAG, 12.7 mg DAG, 27.8 mg TAG, 24.8 mg
FAME, and 2.5 mg glycerol dissolved in 1 mL DCM–MeOH (95 + 5, v/v); 9, lubricant oil with PAO6; and 10, lubricant oil based on low-volatile
alkylate. Visualization after the last stage of development and subsequent development in iodine fumes. Spots: Ar, aromatic hydrocarbons; G,
glycerols; A, additives; D, dyes/vitamins contained in the vegetable edible oil; Op, products of oxidation; S, squalane; and P, poly-α-olefins.

takes place during refining processes or heat treatment (e.g.,
during cooking). Generated during those processes can be a
number of new chemical compounds that differ in polarity,
ranging from low-MW volatile compounds such as esters,
ethers, aldehydes, ketones, lactones, alcohols, and short-chain
FFAs (absent on the TLC–FID chromatograms due to their
volatility) to conjugated dienes, incomplete TAGs, oxidized
TAGs, TAG polymers, oxidized acylglycerol derivatives,
and FFAs. Analyzing the chromatograms of samples of
vegetable oil (sample 1 in Figures  5 and 6), oil produced on
the basis of vegetable oil (sample 2 in Figures  5 and 6), and
oil consisting of a mixture of vegetable and petroleum oil
(sample 3 in Figures  5 and 6), it can be noticed that these
samples are characterized by a high content of vegetable oil,
which is compatible with the specification provided by the
manufacturer. In the chromatograms presented in sample 2 in
Figures  5 and 6, additional peaks are visible, indicating the
presence of enriching additives in the examined oil sample.
However, further characterization of these additives is not
the aim of this work. In contrast to the previously discussed
chromatograms, those shown in sample 3 in Figures 5 and 6,
obtained for lubricating oil consisting of a mixture of petroleum
and vegetable oil bases, illustrate additional peaks, revealing
the presence of not only the mentioned signal from TAG (tR
approximately 0.325 min), but also peaks corresponding to the
petroleum fraction (tR approximately 0.18 min):
• P+N fraction (0.18 min).
• A1 fraction (0.275 min).
• A2+ fraction (0.3 min).
On the basis of the obtained results, it can be clearly ascertained
that the examined oil sample is a mixture of petroleum and
vegetable oil base. The chromatogram obtained for the oil
containing mineral oil base and enriching additives (sample 4
in Figures  5 and 6) shows the dominant peak corresponding
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Figure 5.  Representative TLC–FID chromatograms of samples 1–8 (20 mg each dissolved in 1 mL DCM). Samples: 1, vegetable oil;
2, oil containing vegetable oil base and enriching additives; 3, oil containing both vegetable and petroleum oil bases; 4, oil containing
petroleum oil base; 5, waste oil; 6, base oil (SAE 10); 7, PAO6; and 8, low-volatile alkylate–based oil. The separation conditions: stationary
phase, Chromarod S III rods; volume of the sample applied onto the rod: 1 μL; three-step elution: (1) nC6, 10 cm; (2) toluene, 6 cm; and
(3) DCM–MeOH (95 + 5, v/v), 3 cm.

to the P+N fraction, as well as the A1 and A2+ fractions. The
peaks of enriching additives are also visible (tR = 0.375 min).
The samples of SAE 10 base oil, PAO6, low-volatile alkylate–
based oil and squalene (respective samples 6, 7, 8, and in
Figures 5 and 6; and samples 1 and 1′ in Figure 7); the refined
vegetable oil (rapeseed oil; sample 1 in Figures 5 and 6); and the
standard solution containing FFA, MAG, DAG, TAG, FAME,
and glycerol (samples 2 and 2′ in Figure  7) were treated as
reference materials.
To compare the results and effects of group separation of
all the examined samples obtained for elution I and elution
II, chromatographic parameters are listed in Tables  1 and 2,
respectively. Those data include the retention time (tR), peak
area (A), and Rf and retention factor (k) values calculated for all
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Figure 6.  Representative TLC–FID chromatograms of samples 1–8 (20 mg each dissolved in 1 mL DCM. Samples: 1, vegetable oil; 2, oil
containing vegetable oil base and enriching additives; 3, oil containing both vegetable and petroleum oil bases; 4, oil containing petroleum
oil base; 5, waste oil; 6, base oil (SAE 10); 7, PAO6; and 8, low-volatile alkylate–based oil. The separation conditions: stationary phase,
Chromarod S III rods; volume of the sample applied onto the rod: 1 μL; three-step elution: (1) DCM–MeOH (95 + 5, v/v), 3 cm; (2) toluene, 6 cm;
(3) nC6, 10 cm.

individual peaks obtained during the TLC–FID studies, as well
as the percentage contribution of each fraction to the total area of
all peaks in the TLC–FID chromatograms from Figures 5 and 6.
Taking into account all the data presented in the tables and all
the obtained chromatograms, it can be concluded that the three-
step elution carried out toward increasing elution strength of the
mobile phase results in a better group separation of oil samples
containing petroleum oil base. However, the elution carried out
toward decreasing mobile phase strength appears to be slightly
more favorable for conducting the group separation of the
vegetable oil samples. The explanation of this difference appears
to be the incomplete solubility in nC6 of such components as
MAG, glycerin, and FFA in the examined oil samples.
To summarize, the TLC–FID technique proved to be a very
useful tool in group composition studies concerning lubricating
oil samples applied in cutting devices. At the same time, it
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Figure 7.  Representative TLC–FID chromatograms of samples 1 and 1′ (20 mg squalene dissolved in 1 mL DCM) and samples 2 and 2′
(standard solution containing 27.8 mg FFA, 10.2 mg MAG, 12.7 mg DAG, 27.8 mg TAG, 24.8 mg FAME, and 2.5 mg glycerol dissolved in 1 mL
DCM–MeOH (95 + 5, v/v). Separation conditions: stationary phase, Chromarod S III rods; volume of the sample applied onto the rod: 1 μL;
three-step elution: for samples 1 and 2, (1) DCM–MeOH (95 + 5, v/v), 3 cm; (2) toluene, 6 cm; and (3) nC6, 10 cm; for samples 1′ and 2′, (1) nC6,
10 cm; (2) toluene, 6 cm; and (3) DCM–MeOH (95 + 5, v/v), 3 cm.

was found advantageous to conduct the selection of preferable Conclusions

separation conditions using a μTLC technique.
The μTLC technique, performed with three-step elution,
can be successfully used as a pilot technique for the selection
of optimal separation conditions for TLC–FID and HPLC
studies. It is also useful for group composition evaluation of
lubricating oils, including those applied in mechanical cutting
devices. However, the μTLC technique does not allow for the
identification of the P+N fraction and the assessment of its
content in oils. Studies concerning the presence and content
of the A1 fraction performed on SiO2 F254 TLC plates are
characterized by rather low sensitivity when TLC plates are
observed at 365 or 254 nm and when they are stained with iodine
vapor. Moreover, the μTLC technique does not allow for simple
evaluation of group composition in a quantitative manner. As
was proved during our research (and which will be the subject
of further work), the impregnation of TLC plates (which do
not contain fluorescein) with berberine sulfate or berberine
hydrochloride allows for the identification of lubricating oil
group composition (27). Our further studies will be aimed at
the development of such separation conditions that will lead to
achieving the full usefulness of the μTLC technique in order to
study the group composition of various types of lubricating oils.
The results of our research conducted with the application of
μTLC and TLC–FID show that these techniques provide a fast
and indisputable method (mainly TLC–FID) for the detection of
the presence of petroleum oil base in the lubricating oils samples
used in chainsaw cutting systems. The composition (TLC and
TLC–FID) of the lubricating oils can, therefore, be estimated
and determined using the methods described in this work. The
results of these studies were applied to develop procedures
applicable for the “technical” control of the lubricating oils and
evaluation of their group composition.
In the case of NP-TLC (μTLC and TLC–FID), the optimal
elution was the three-step elution carried out toward increasing
strength of the mobile phase (elution II), i.e., step 1: the elution
up to 100% of the plate’s height in nC6; step 2: the elution up to
60% of the plate’s height in toluene; and step 3: the elution up
to 30% of the plate’s height in DCM–MeOH (95 + 5, v/v). This
procedure provides favorable elution conditions, but only in
the case when all sample components are soluble in the mobile
phase characterized by the lowest polarity.
From the technical point of view, the described
methodology is considerably less costly and easier than other
chromatographic techniques; therefore, it can be applied even
932  Nowak et al.: Journal of AOAC International Vol. 100, No. 4, 2017

Table 1.  The summary of chromatographic parameters (TLC–FID) with elution Ia

Percentage of peak
Sample name Peak No. tR, min Peak area (A), V⋅min area [(Ai/ΣA)⋅100], % Rf Retention factor (k)

1 0.325 (0.001) 1.7584 (0.0011) 96.30 (0.42) 0.435 (0.001) 1.300 (0.005)
Rapeseed vegetable oil
2 0.438 (0.004) 0.0673 (0.0076) 3.70 (0.42) 0.238 (0.009) 3.197 (0.087)

1 0.300 (0.001) 0.1513 (0.0011) 8.10 (0.42) 0.479 (0.001) 1.087 (0.005)

2 0.327 (0.002) 1.3462 (0.0190) 71.60 (1.84) 0.432 (0.004) 1.314 (0.020)

Oil containing vegetable 3 0.364 (0.000) 0.1696 (0.0344) 9.00 (1.41) 0.367 (0.000) 1.725 (0.000)
oil base and enriching
additives 4 0.414 (0.000) 0.0620 (0.0119) 3.30 (0.85) 0.280 (0.000) 2.571 (0.000)

5 0.453 (0.001) 0.1327 (0.0334) 7.05 (1.48) 0.213 (0.001) 3.694 (0.027)

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6 0.529 (0.002) 0.0176 (0.0042) 0.95 (0.21) 0.081 (0.004) 11.378 (0.565)

1 0.157 (0.032) 0.9117 (0.0389) 49.45 (0.21) 0.728 (0.21) 0.378 (0.105)
Oil containing both
­vegetable and 2 0.206 (0.037) 0.1415 (0.0099) 7.70 (0.85) 0.642 (0.85) 0.566 (0.156)
­petroleum oil bases
3 0.321 (0.008) 0.7899 (0.0558) 42.85 (1.06) 0.443 (1.06) 1.260 (0.069)

Oil containing petroleum 1 0.175 (0.000) 1.9759 (0.1319) 91.05 (0.35) 0.696 (0.000) 0.438 (0.000)
oil base with additives 2 0.326 (0.000) 0.1938 (0.0052) 8.95 (0.35) 0.433 (0.000) 1.309 (0.000)

1 0.310 (0.001) 0.3637 (0.0199) 28.95 (0.78) 0.462 (0.001) 1.166 (0.006)

2 0.317 (0.002) 0.2550 (0.0201) 20.25 (0.07) 0.450 (0.010) 1.224 (0.018)

3 0.343 (0.007) 0.5001 (0.0551) 39.65 (1.20) 0.403 (0.012) 1.480 (0.076)
Waste oil
4 0.384 (0.006) 0.0267 (0.0076) 2.10 (0.42) 0.332 (0.010) 2.012 (0.089)

5 0.438 (0.000) 0.0854 (0.0012) 6.80 (0.71) 0.238 (0.000) 3.197 (0.000)

6 0.529 (0.004) 0.0284 (0.0007) 2.25 (0.07) 0.081 (0.006) 11.401 (0.943)

1 0.158 (0.004) 1.5817 (0.2196) 93.60 (0.71) 0.726 (0.006) 0.377 (0.012)
Base oil (SAE 10)
2 0.219 (0.000) 0.1067 (0.0025) 6.40 (0.71) 0.619 (0.000) 0.615 (0.000)

Squalane 1 0.161 (0.000) 1.7989 (0.000) 100.00 (0.000) 0.720 (0.000) 0.389 (0.000)

1 0.268 (0.003) 1.4146 (0.2812) 27.25 (1.06) 0.534 (0.005) 0.873 (0.017)

Standard solution 2 0.329 (0.000) 2.2091 (0.5005) 42.40 (0.42) 0.428 (0.000) 1.337 (0.000)
containing FFA, MAG,
3 0.423 (0.001) 0.8030 (0.2362) 15.20 (0.85) 0.264 (0.002) 2.783 (0.035)
DAG, TAG, FAME, and
glycerol 4 0.460 (0.004) 0.6555 (0.1949) 12.45 (0.78) 0.200 (0.007) 4.003 (0.185

5 0.524 (0.003) 0.1382 (0.0243) 2.70 (0.14) 0.089 (0.005) 10.292 (0.626)

PAO6 1 0.181 (0.001) 2.1011 (0.0353) 100.00 (0.00) 0.685 (0.002) 0.459 (0.005)

1 0.097 (0.013) 0.0029 (0.0001) 0.20 (0.00) 0.831 (0.022) 0.203 (0.032)
Low-volatile ­
2 0.173 (0.004) 1.9607 (0.2885) 99.10 (0.07) 0.699 (0.007) 0.430 (0.015)
alkylate–based oil
3 0.255 (0.007) 0.0137 (0.0005) 0.70 (0.14) 0.557 (0.012) 0.797 (0.040)
a
  Elution steps: (1) DCM–MeOH (95 + 5, v/v), 3 cm; (2) toluene, 6 cm; and (3) nC6, 10 cm. Values in parentheses are SDs.

in insufficiently equipped laboratories. This methodology can
be successfully  applied as an alternative method, replacing
expensive NP-HPLC coupled with UV-Vis diode-array, refractive
index, and evaporative light scattering detection systems for the
determination of oil and grease group composition, particularly
those containing both petroleum and vegetable oil bases. The
results of NP-TLC studies show relatively low sensitivity for
hydrocarbons A1 and a lack of sensitivity for the P+N fraction.
A very clear signal is obtained only for hydrocarbons A2+. Low
sensitivity was also observed for saturated fatty acids present
in the examined oil samples. This problem does not occur in
the case of the TLC–FID technique. As was proved during our
research (and which will be the subject of further work), the
impregnation of TLC plates with a berberine salt should solve
the problems with the low sensitivity observed during NP-TLC
studies (27).
The methodologies presented in this paper are also applicable
in the analysis of various types of edible fats.
 Nowak et al.: Journal of AOAC International Vol. 100, No. 4, 2017  933

Table 2.  The summary of chromatographic parameters (TLC–FID) with elution IIa

Percentage of peak
Sample name Peak No. tR, min Peak area (A), V⋅min area [(Ai/ΣA)⋅100], % Rf Retention factor (k)

Rapeseed vegetable oil 1 0.394 (0.004) 2.4289 (0.2466) 100.00 (0.00) 0.315 (0.007) 2.178 (0.074)

1 0.349 (0.002) 0.0253 (0.0130) 1.20 (0.20) 0.393 (0.001) 1.544 (0.004)

2 0.370 (0.001) 0.0550 (0.0160) 2.70 (0.24) 0.357 (0.002) 1.805 (0.002)

Oil containing vegetable 3 0.388 (0.001) 0.3429 (0.0090) 16.70 (0.09) 0.325 (0.001) 2.075 (0.001)
oil base and enriching
additives 4 0.414 (0.002) 1.5601 (0.0100) 76.00 (0.20) 0.280 (0.001) 2.571 (0.001)

5 0.440 (0.001) 0.0215 (0.0135) 1.00 (0.23) 0.235 (0.002) 3.259 (0.001)

6 0.520 (0.002) 0.0484 (0.0170) 2.40 (0.27) 0.096 (0.002) 9.455 (0.004)

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1 0.229 (0.002) 0.8686 (0.0936) 43.95 (1.2) 0.603 (0.004) 0.659 (0.010)

Oil containing both 2 0.288 (0.000) 0.2452 (0.0400) 12.35 (1.1) 0.499 (0.000) 1.003 (0.000)
­vegetable and
­petroleum oil bases 3 0.319 (0.001) 0.0304 (0.0004) 1.50 (0.1) 0.446 (0.001) 1.242 (0.006)

4 0.423 (0.002) 0.8319 (0.0256) 42.20 (2.1) 0.265 (0.004) 2.771 (0.052)

1 0.210 (0.001) 1.3988 (0.0150) 65.05 (0.21) 0.635 (0.002) 0.575 (0.006)
Oil containing petroleum
2 0.280 (0.001) 0.6522 (0.0170) 30.30 (0.28) 0.514 (0.001) 0.946 (0.005)
oil base with additives
3 0.312 (0.001) 0.1011 (0.0001) 4.65 (0.07) 0.457 (0.002) 1.186 (0.012)

1 0.396 (0.001) 1.0320 (0.0017) 83.50 (0.0017) 0.311 (0.001) 2.212 (0.003)

Waste oil 2 0.445 (0.004) 0.0505 (0.0357) 4.10 (0.00) 0.226 (0.002) 3.423 (0.006)

3 0.524 (0.002) 0.1528 (0.0252) 12.40 (2.1) 0.089 (0.004) 10.275 (0.052)

1 0.206 (0.001) 1.4453 (0.1215) 87.00 (0.42) 0.642 (0.002) 0.558 (0.006)
Base oil (SAE 10)
2 0.285 (0.001) 0.2167 (0.0268) 13.00 (0.42) 0.505 (0.001) 0.979 (0.005)

Squalane 1 0.220 (0.001) 1.8197 (0.0017) 100.00 (0.00) 0.618 (0.001) 0.617 (0.003)

1 0.317 (0.003) 1.1902 (0.0693) 21.90 (2.5) 0.449 (0.005) 1.229 (0.024)

Standard solution 2 0.348 (0.001) 0.8890 (0.1822) 16.25 (2.3) 0.396 (0.001) 1.527 (0.008)
containing FFA, MAG,
3 0.401 (0.001) 2.6826 (0.1525) 49.20 (0.3) 0.303 (0.001) 2.305 (0.027)
DAG, TAG, FAME, and
glycerol 4 0.438 (0.001) 0.4972 (0.0484) 9.10 (0.3) 0.239 (0.001) 3.182 (0.022)

5 0.515 (0.003) 0.1935 (0.0259) 3.55 (0.02) 0.104 (0.003) 8.594 (0.452)

PAO6 1 0.212 (0.001) 1.9290 (0.0367) 100.00 (0.00) 0.631 (0.002) 0.584 (0.006)

Low-volatile 1 0.200 (0.001) 1.2296 (0.0215) 98.40 (0.32) 0.652 (0.002) 0.533 (0.006)
alkylate–based oil 2 0.248 (0.001) 0.0200 (0.0110) 1.60 (0.35) 0.569 (0.001) 0.758 (0.005)
a
  Elution steps: (1) nC6, 10 cm; (2) toluene, 6 cm; and (3) DCM–MeOH (95 + 5, v/v), 3 cm. Values in parentheses are SDs.

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