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Optimization of TLC detection by phosphomolybdic acid staining for robust

quantification of cholesterol and bile acids

Article  in  JPC - Journal of Planar Chromatography - Modern TLC · February 2006

DOI: 10.1556/JPC.19.2006.1.9

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Optimization of TLC Detection by Phosphomolybdic Acid
Staining for Robust Quantification of Cholesterol
and Bile Acids
Paweł K. Zarzycki*, Małgorzata A. Bartoszuk, and Aneta I. Radziwon
Key Words:
Spots visualization
Detection reagent
Densitometry
Temperature
Phosphomolybdic acid (PMA)
Cholesterol
Bile Acids
Steroids
Summary
In this paper we describe a robust and sensitive detection procedure
for cholesterol and selected bile acids (cholic acid, lithocholic acid,
and taurodeoxycholic acid sodium salt) using the common derivati-
zation reagent phosphomolybdic acid (PMA). Visualization condi-
tions were studied and optimized for steroids separated on glass
TLC and HPTLC plates coated with silica gel (K60WF254S) and
octadecylsilane (RP-18W) stationary phases. Spot intensities on the
plates were quantified after spraying with PMA in methanol (10%
w/v) and heating at temperatures from 40 to 120°C for times rang-
ing from 2 to 40 min. The best conditions for high signal intensity
were determined by using 3D temperature (X)–time (Y)–analytical
signal (Z) maps generated from the raw experimental data. In con-
trast with the number of “universal procedures” described in the
literature our study indicated that for robust and sensitive quantifi-
cation of our components of interest heating should be performed at
relatively low temperatures (below 100°C) and for heating times in
excess of 10 min. Particularly robust and sensitive detection of
steroids separated on glass plates coated with both stationary phas-
es was observed for temperatures ranging between 50 and 80°C and
heating for at least 20 min in a simple gravity convection oven.
1 Introduction
Phosphomolybdic acid (PMA) is extensively used as a dye for
sensitive detection of low-molecular-mass compounds and
visualization of complex biological structures. The principle of
PMA application is based on the observation that many inor-
ganic and organic substances form intensely blue mixed oxides
P.K. Zarzycki, Laboratory of Toxicology, Department of Environmental Biology,
Technical University of Koszalin, Koszalin, Poland; and P.K. Zarzycki, M.A. Bar-
toszuk, and A.I. Radziwon, Department of Pharmaceutical Chemistry, Faculty of
Pharmacy, The Medical University of Gdañsk, Gdañsk, Poland. E-mail:
pawel_k_z@hotmail.com
52 VOL. 19. JANUARY/FEBRUARY 2006
DOI: 10.1556/JPC.19.2006.1.9
in which the initial Mo(VI) is reduced to Mo(IV). Such reaction
products can be easily viewed by light and electron microscopy,
photographed, or measured by spectroscopic techniques (λmax is
usually in the range 600–900 nm).
The main advantages of staining procedures involving PMA are
simplicity and sensitivity and the stability of the reaction prod-
ucts and the reagent itself. The process is, moreover, usually
quantitative. The main applications of PMA in chemistry and
pharmacy include colorimetric, spectrophotometric, and gra-
vimetric assays of inorganic and organic phosphates [1],
amines [2], alkaloids [3], ethoxylated mono and diglyc-
erides [4], phenothiazine antipsychotics [5], lipids [6], and pro-
teins [7]. In biology and medicine PMA-based staining has been
extensively used for, for example, determination of serum phos-
phate without sample deproteinization [8], localization of con-
nective tissue [9], visualization of the kinetoplast of try-
panosomes [10], localization of acetylcholine in the cholinergic
nerve terminals [11], visualization of cell structures [12], cate-
gorization of tumor types [13], displaying mitochondria in root
tip cells [14], studying micro-anatomy of different vertebrate
tissues [15], morphological demonstration of phospholipids in
the lungs [16], or preparation of skeletal muscle tissue for
image-analysis systems [17].
Because phosphomolybdic acid can give intensely colored reac-
tion products with many substances that are transparent to
UV–visible light, it is the most commonly employed detection
reagent in planar chromatography. PMA is regarded as a multi-
purpose stain which is sensitive to small amounts of compo-
nents of interest (nanogram per spot range) and may be
employed on silica gel, aluminum oxide, polyamide, RP-2, RP-
18, and cellulose stationary phases [18]. As is apparent from the
data presented in Table 1 the applications of PMA in TLC
include visualization of neutral lipids, phospholipids,
aminophospholipids and related compounds, particularly those
Journal of Planar Chromatography
 TLC Quantification of Cholesterol and Bile Acids

Table 1

Common applications of phosphomolybdic acid in planar chromatography.

Compounds/samples PMA concentration Temperature Stationary phase Ref.

Lipids 5% in methanol 115°C for 15 min Silica gel HPTLC 19

Neutral lipids and cholesterol 5% in ethanol 110–120°C for 5–10 min Silica gel HPTLC 20
Cholesterol esters 10% in ethanol 100°C for 2 min Silica gel HPTLC 21
Bile acids and cholesterol 10% in 2-propanol 120°C for 5–10 min HPTLC RP18W 22
Conjugated bile acids 3.5% in ethanol 70–80°C for 10 min Silica gel HPTLC 23
Saponins 5% in ethanol 110°C Silica gel TLC 24
Prostaglandins 10% in ethanol 110°C for 3–6 min HPTLC RP18W 24
Peroxides and ketodienes from linoleic acid 5% in ethanol 110°C Silica gel TLC 26
Olive oil components 20% in water 175°C for 60 min AgNO3-impregnated silica gel TLC 27
Mammalian feces 5% in ethanol 120°C for 20 min Silica gel TLC 28
Aminophospholipids 5% in ethanol 60°C for 5 min Silica gel TLC 29
Triacylglycerols and phospholipids 5% in ethanol 110°C for 10 min Silica gel HPTLC 30
Terpenes 20% in ethanol 105°C for 15 min Silica gel TLC 31
Sesquiterpene lactones 10% in ethanol 100°C for 2 min Silica gel OPTLC 32
Common sterols 10% in methanol 110°C for 10 min Whatman No. 1 filter paper 33
or ethanol

containing long carbon chains, for example prostaglandins, cho-

lesterol, bile acids, and saponins [19–30]. PMA has also been
used for the detection of terpenes, sesquiterpenes, lactones, and
several common steroids including estrogens, androgens, prog-
estagens, and phytosteroids [31–33].
It is well known that quantification procedures based on planar
chromatographic techniques can be very sensitive; they are,
however, not usually robust. One of the main sources of uncer-
tainty and poor reproducibility in quantitative TLC is the detec-
tion step [34]. In particular, the problem arises as a result of the
procedures used for post-run derivatization, for example PMA
staining. These procedures involve several highly empirical
conditions, for example dipping, heating, and cooling times,
and heating temperature. For robust quantification, all these
processes must be properly controlled, optimized and validated.
Bile acids are well-known metabolites of cholesterol. In living Figure 1
organisms they play a major role in the cholesterol balance and
) and calculated average temperature (
Measured heating trajectory (
) for a
fat digestion or absorption, and determination of cholesterol, glass TLC plate (0.1 cm × 10 cm × 10 cm) in a gravity convection oven set at
100°C.
bile acids, and their metabolites in biological samples is becom-
ing increasingly important for diagnosis of several diseases and
disorders [35–37]. Although different separation techniques, for
example gas chromatography, liquid chromatography, and cap-
illary electrophoresis, are commonly used for determination of
bile steroids, planar chromatography has several practical worth noting that separation and quantification of such steroids
advantages including simplicity, the inexpensive equipment in biological fluids can be performed directly without prior
needed, and the ease of operation [38]. In addition, a large num- sample purification [39–41].
ber of samples can be analyzed simultaneously and within short The aim of our work was systematic study and optimization of
periods of time. Because of the poor absorption of cholesterol two critical conditions, temperature and heating time, that
and the bile acids in the UV region, they cannot be detected by strongly affect the final visualization results for quantitative and
UV detection in HPLC; TLC with PMA staining is, therefore, a robust detection with PMA of cholesterol and selected bile
very attractive method for quantification of these compounds in steroids separated on glass silica gel and octadecylsilane TLC
pharmaceutical formulations and in biological samples. It is plates.

Journal of Planar Chromatography VOL. 19. JANUARY/FEBRUARY 2006 53

TLC Quantification of Cholesterol and Bile Acids

Table 2 Table 3

Steroid spot intensity raw data measured for K60W254S TLC plates Steroid spot intensity raw data measured for RP-18W HPTLC plates
after heating at different temperatures for different times. after heating at different temperatures for different times.

Temperature [°C] 2 min 5 min 10 min 20 min 40 min Temperature [°C] 2 min 5 min 10 min 20 min 40 min

Cholesterol Cholesterol
40 8 52 83 92 102 40 32 101 128 126 124
60 27 118 119 106 107 60 83 126 117 113 94
80 115 91 80 65 48 80 102 77 67 45 36
100 114 100 88 47 37 100 110 64 56 36 28
120 116 76 67 45 42 120 93 66 38 28 24
Lithocholic acid Lithocholic acid
40 0 0 0 0 0 40 0 0 0 0 12
60 0 9 81 102 122 60 0 9 70 92 81
80 0 97 87 69 42 80 0 42 39 27 24
100 51 110 91 62 57 100 33 54 44 23 11
120 90 74 56 44 37 120 60 50 25 20 12
Cholic acid Cholic acid
40 0 0 0 0 30 40 0 0 11 37 81
60 2 42 84 89 110 60 0 26 85 91 75
80 16 105 96 80 56 80 0 32 29 17 17
100 62 95 82 45 38 100 74 55 44 23 9
120 80 68 50 45 27 120 61 45 23 17 11
Taurodeoxycholic acid (sodium salt) Taurodeoxycholic acid (sodium salt)
40 0 0 0 0 0 40 0 0 0 0 13
60 0 6 31 46 51 60 0 4 32 43 37
80 8 63 61 60 41 80 0 7 12 9 7
100 36 60 48 33 30 100 31 26 21 9 0
120 45 41 33 27 17 120 27 19 8 7 0

2 Experimental spotted with 1 µg (1 µL) of the steroid standards. After evapo-

2.1 Chemicals
Steroids (cholesterol, cholic and lithocholic acids, and tau-
rodeoxycholic acid sodium salt) were products of Sigma, St
Louis, MO, USA. Steroid stock solutions were prepared in pure
methanol at a concentration of 1 mg mL–1. The organic compo-
nents of the mobile phase – dichloromethane (99.5% pro analy-
sis, stabilized with 20 ppm 2-methyl-2-butene, #1.06050.1000)
and methanol (99.8%, HPLC grade) – were purchased from
Merck (Darmstadt Germany) and from Polskie Odczynniki
Chemiczne (Gliwice, Poland), respectively. The detection mix-
ture was a 10% (w/v) solution of phosphomolybdic acid (Fluka
Chemie, Buchs, Switzerland) in methanol.
ration of the methanol the plates were placed in developing
chambers (210 mm × 40 mm) lined with filter paper. Each
chromatographic development was performed at room tem-
perature without additional chamber saturation with mobile
phase vapor.
The steroid spots were visualized by spraying the plates with
phosphomolybdic acid solution then placing them in a gravity
convection oven (ED/EED 53; WTB Binder Labortechnik, Tut-
tlingen, Germany). The plates were heated to different tempera-
tures from 40 to 120°C (±0.5°C) for different periods of time
(from 2 to 40 min). After heating the steroids were apparent as
the blue spots on yellow or gray background.
2.3 Acquisition of Quantitative Data

After visualization the planar chromatograms were scanned

2.2 Separation and Detection
Chromatography was performed on 100 mm × 30 mm glass
silica gel 60WF254S TLC plates (Merck, Darmstadt, Germany;
#16485) and glass RP18W HPTLC plates (Merck; #14296).
The mobile phases were methanol–dichloromethane, 2 + 8
(v/v), and methanol–water, 8 + 2 (v/v), for normal and
reversed-phase chromatography, respectively. The plates were
within 1 h by means of a Hewlett–Packard Scanjet 7450C scan-
ner. The raw images were recorded with 300 DPI optical resolu-
tion and 8 bits per RGB channel mode and saved as TIFF files
without compression.
Steroid spot intensities were quantitatively measured using
Scion Image freeware (ver. beta 3b) downloaded from the Scion
Image Corporation WebPage (http://www.scioncorp.com/).

54 VOL. 19. JANUARY/FEBRUARY 2006 Journal of Planar Chromatography

 TLC Quantification of Cholesterol and Bile Acids

Figure 2

Contour maps of the signal intensity of steroids calculated using raw data obtained on silica gel (left) and octadecylsilane (right) stationary phases. Time (X axis) and
temperature (Y axis) ranged from 0 to 40 min and from 40 to 120°C, respectively. The components of interests are (from the top): cholesterol, lithocholic acid, cholic acid
and taurodeoxycholic acid.

Journal of Planar Chromatography VOL. 19. JANUARY/FEBRUARY 2006 55

TLC Quantification of Cholesterol and Bile Acids

Appropriate time–temperature contour maps were generated by

use of Statistica V.6.0 (StatSoft) software.

3 Results and Discussion

Data presented in the introduction of this paper (Table 1) indi-

cate that the best conditions for visualization of cholesterol and
bile acids using PMA reagent are heating of the TLC plates for
short time (typically less than 10 min) at temperatures in excess
of 100°C. In common practice, if the separation process is per-
formed on plates coated with octadecylsilica materials, the
MDA also reacts with some of the C18 groups and other organic
residues present on the stationary phase. The consequence of
this is a dark background and, therefore, poorly sensitive solute
detection. To explore this problem quantitatively we studied
changes of steroid spot intensities using a wide range of tem-
peratures from 40 to 120°C and different heating times from 2
to 40 min. In our experiments the temperature of the NP and RP
glass plates was controlled with an accuracy of ±0.5°C using a
simple gravity convection oven. Developed plates sprayed with
10% PMA were placed on an aluminum support to enable
undisturbed airflow on both sides of the heated plates. Such an
approach results in a steady heating rate for the TLC plate mate-
rial and avoidance of the hot-spot phenomena commonly
encountered if the plates are heated by placing them directly on
the surface of a hot-plate.
Because we expected the heating rate to be an important factor
affecting the final quantification results, we measured changes
of temperature (from room temperature to 100°C) of a glass
TLC plate heated under the conditions described. The results of
this assessment are presented in the Figure 1 where the curve
composed of black dots corresponds to the real temperature of
the plate surface. The white circles show the average tempera-
ture of the plate calculated for the given heating time. It is clear-
ly apparent that during the first 10 min the temperature of the
plate changes very quickly. Such unsteady, nonlinear, and, in Figure 3
fact, uncontrolled conditions can be a significant source of poor Typical chromatograms and densitograms obtained from the analytes (1, cho-
precision in quantitative TLC. The average temperature within lesterol; 2, lithocholic acid; 3, cholic acid; 4, taurodeoxycholic acid; and 5, impu-
rities) on silica gel (top) and octadecylsilane (bottom) stationary phases under
first 20 min of heating is, moreover, substantially different from optimized visualization conditions (temperature 60°C and heating time 20 min).
the destination temperature, which was set at 100°C. This prob-
lem may be regarded as another source of the error that signifi-
cantly affects the reproducibility of solute signals, particularly
when heating times are relatively short. investigated. It is worth noting that for each experiment the
maximum signal intensity is located in the regions of low tem-
Raw data from measurement of steroid peak intensities on the perature and long heating time, although the results also con-
silica gel and octadecylsilane-coated glass TLC plates are listed firm the steroids can also be detected after treatment at high
in Tables 2 and 3. The data were used to generate 3D profiles temperature for a short time, as reported in the references listed
for each of the solutes with the X, Y, and Z axes corresponding in Table 1. Such detection cannot, however, be robust because it
to the temperature, time, and analytical signal, respectively. The is affected by the strong signal intensity gradient across the time
results, in the form of signal intensity maps, are presented in axis. This phenomenon is easy to observe for steroids that are
Figure 2, in which the red areas correspond to the maximum detected on the silica stationary phase (left side of Figure 2).
values of the analytical signal expected. From that point of view more stable conditions are observed on
According to the raw data (Table 2) and the signal intensity the reversed-phase adsorbent (right side of Figure 2) where the
maps presented in Figure 2, PMA staining is a very efficient analytical signal forms flat peninsulas and islands. Unfortunate-
means of detection of the analytes on both stationary phases ly, in terms of sensitivity, the relative intensity of the chromato-

56 VOL. 19. JANUARY/FEBRUARY 2006 Journal of Planar Chromatography


graphic peaks detected in the high-temperature region is very
low, particularly for the bile acids investigated.
Considering the whole range of conditions investigated the best
detection sensitivity was observed for cholesterol and cholic
and lithocholic acids. This is well illustrated by the chro-
matograms presented in Figure 3, which were obtained under
optimum visualization conditions (60°C and heating for 20 min)
on the basis of the data presented in Figure 2. It is apparent cho-
lesterol and lithocholic and cholic acids generate the tallest
peaks despite their relative positions in the chromatograms on
both stationary phases.
4 Conclusion
Optimization of the temperature and time of heating is neces-
sary to minimize uncertainty and maximize reproducibility in
detection and quantification based on staining with PMA. In
contrast with results reported in the literature, our results indi-
cate that robust quantification of cholesterol and selected bile
steroids is possible, particularly if derivatization is performed at
relatively low temperatures (close to 60°C) and for longer heat-
ing times (more than 15 min). Under such conditions the sensi-
tive and robust detection of our components of interest was
observed on glass plates coated with silica gel and C18 stationary
phases.
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Ms received: March 18, 2005
Accepted by SN: September 28, 2005
VOL. 19. JANUARY/FEBRUARY 2006 57