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824 II / CHROMATOGRAPHY: THIN-LAYER (PLANAR) / Densitometry and Image Analysis

Rlled by injector or detector or detector volume: Table 1 Sample loadability ratios for different distribution coef-

 N n 1/2
" i [21] KD Lp
c N k Lc

From eqn [21], an expression has been derived 1 31

linking fractional loss of resolution, Rs, due to the 10 101
100 373
injector or detector volume to column and retention
characteristics: Packed column: Lf"20 cm; dp"5 m; dpc"1 mm; i"0.5;
e"0.4; T"0.7. Open tubular column: Lc10 m; dc"50 m;

1 dF"0.2 m.
vi"0.866dc2(LH)1/2 !1 (1#k)
Increasing the column diameter is the simplest ap-
where vi is the injection volume and L is column proach to increasing the sample capacity of packed
length. Since H is a function of dc, vi is proportional columns, since the surface area of stationary-phase
support particles is generally maximized. However,
c and for a 10 m;50 m column operated under
to d5/2
practical conditions, a 1% loss of resolution requires for capillary columns, sample capacity can be in-
an injection volume of only 50 nL. creased by increasing the stationary phase Rlm thick-
For equal distribution coefRcients, KD, an equation ness, dF. This should also lead, in theory, to a reduction
relating the maximum loadabilities, Lp and Lc, for in column efRciency but in practice such a reduction
packed and open tubular columns has been derived: is only small, with Rlm thickness up to 1 m.

KDi(1!e) Further Reading

Lp dpc 2
T Lp 1/2
dp 1/2
"1.24 Anton K and Berger C (eds) (1998) Supercritical Fluid
Lc dc 4KDd Lc dc Chromatography with Packed Columns. New York:
1# $
dc Marcel Dekker.
[23] Berger TA (1995) Packed Column SFC. Cambridge: Royal
Society of Chemistry.
where d is the diameter of the packed column;  ,  Heaton DM, Bartle KD, Clifford AA, Klee MS and Berger
  TA (1994) Retention prediction based on molecular
and  are intraparticle, interstitial and total poros-
2 interactions in packed-column supercritical Suid
ities, respectively; and Lp and Lc are the lengths of the chromatography. Analytical Chemistry 66: 4253.
packed and open tubular columns, respectively. Lee ML and Markides KE (1990) Analytical Supercritical
Table 1 lists the maximum sample loadability ra- Fluid Chromatography. Provo, Utah: Chromatography
tios for different KD values for typical packed and Conferences.
open tubular columns; as might be expected, sample Smith RM (ed.) (1988) Supercritical Fluid Chromato-
loadabilities are much greater on packed columns. graphy. London: Royal Society of Chemistry.

Densitometry and Image Densitometry is a means of measuring the concentra-
Analysis tion of chromatographic zones on the developed thin-
layer chromatography high performance thin-layer
chromatography (TLC in HPTLC) layer. The instru-
P. E. Wall, Merck Limited, Poole, Dorset, UK ment does this without disturbing the substance in the
chromatogram. The method and computer-control-
Copyright ^ 2000 Academic Press led instrumentation produces results that are not only
II / CHROMATOGRAPHY: THIN-LAYER (PLANAR) / Densitometry and Image Analysis 825

reproducible, but also highly accurate (&1% stan- eliminated. Scanning densitometry dates back to the
dard deviation). Scanning is a fast process (up to 1950s when it was used to scan thin strips of paper
a scan speed of 100 mm s\1) with a spatial resolution chromatograms containing separated amino acids.
in steps from 25 to 200 m. Full UV/visible spectra Since then these primitive instruments have under-
(190}800 nm wavelength) of separated analytes can gone considerable change, to the extent that they are
be recorded at high speed and peaks can be checked now advanced analytical tools of similar capabilities
for purity by obtaining and comparing spectra from to modern HPLC instrumentation.
the start, middle and end of the peaks. With the use of Today’s scanning densitometer measures reSec-
highly sensitive charged coupled device (CCD) tance, quenched Suorescence or Suorescence induced
cameras, the photographic image of the developed by electromagnetic radiation. For this reason, the
TLC/HPTLC plate can be stored as a video image. instrument is now described as a spectrodensito-
This can be video-scanned to determine the concentra- meter. Although all three detection modes are com-
tion of separated components or can be printed when monly used, Suorescence is limited by the fact that
required as part of a document for a permanent re- fewer substances can be induced to Suoresce. Many
cord of the results. Many images can be stored on the spectrodensitometers also have an attachment for
computer hard drive and archived whenever required. scanning electrophoresis gels by transmission.
The principle of operation is based on light of
The Development of Modern Scanning a predetermined beam size and wavelength striking
the thin-layer surface perpendicularly whilst the TLC
Densitometry plate moves at a set speed under the stationary beam,
The results of a developed TLC/HPTLC plate or sheet or alternatively the beam traverses the stationary
can be quantiRed in a number of ways. Visually, an plate. Some of the electromagnetic radiation passes
estimate of concentration can be made. Many related into and through the layer (transmitted light) whilst
substance tests in the pharmacopoeias rely on the the remainder, due to the opaqueness of the layer, is
concentration of the sample impurities being less than reSected back from the surface. When the light beam
the standard concentration as seen visually. These are passes over an absorbing chromatographic zone, there
limit tests which depend on the eye of the observer is a difference in optical response and less of the light is
determining that the concentration of the unknown is reSected (or transmitted). A photoelectric cell is used
less than that of the standard. It has been estimated to measure the reSected light. When this receives a re-
that the human eye can detect down to about 1 g of duced amount of reSected light due to the presence of
a coloured spot on a TLC plate with a reproducibility an absorbing chromatographic zone, a means is pro-
of about 10}30%. vided of detecting and quantifying the analyte.
Better quantiRcation can be obtained by eluting the Fluorescence quenching mode is really a variation
relevant chromatographic zone from the adsorbent on absorption methods. An inorganic phosphorescent
followed by spectrophotometry. The position of the indicator or organic Suorescent indicator is incorpor-
zone can be marked out with a sharp bradawl and ated into the adsorbent layer. The inorganic phos-
a microspatula used to scrape away the zone. These phors give either a bright green or pale blue phospho-
scrapings are then transferred to a container where rescence depending on the compound used. The
a suitable solvent can be used to dissolve the com- phosphorescence is very short-lived. Hence it is best
pounds of interest from the particles of the adsorbent. observed by continual exposure to UV light at
The mixture is Rltered and the concentration of the 254 nm. Most HPTLC plates contain the indicator
analyte in solution determined by transmittance/ab- which exhibits the pale blue phosphorescence. This
sorption spectrophotometry. There is little to recom- indicator is acid-resistant and allows higher sensitiv-
mend in this procedure as it is both tedious and ity of detection of separated analytes due to less
time-consuming. It also requires meticulous care as intense and less ‘noisy’ backgrounds. TLC plates con-
errors can easily creep into the procedure. It is difR- taining organic Suorescers which give a bright blue
cult to ensure that all the sample is completely re- Suorescence when excited by UV light of 366 nm are
moved from the TLC layer and is transferred from the not as popular. Following chromatographic develop-
chromatographic zone for further work-up if it is not ment, the plates incorporating the Suorophore are
easily seen in the visible or UV parts of the spectrum. scanned at 254 or 366 nm in the absorbance mode.
The technique of scanning densitometry deter- As the sample components absorb the excited radi-
mines the concentration in situ. It scans at set spectral ation, the intensity of the phosphorescence or Suores-
wavelengths and does not rely on removal of any of cence is diminished. Consequently a variation occurs
the chromatographic zones from the TLC/HPTLC in the reSected light detected by the photomultiplier
plate. Hence the previous problems and errors are (or photoelectric cell).
826 II / CHROMATOGRAPHY: THIN-LAYER (PLANAR) / Densitometry and Image Analysis

When separated analytes naturally Suoresce under where R is the reSectance for an inRnitely thick

UV light, the spectrodensitometer can be used to scan in opaque layer, am is the molar absorptivity of the sam-
the Suorescence mode. The UV light provides the en- ple, c is the molar concentration of the sample and S is
ergy in these instances to excite electrons in molecules of the coefRcient of scatter per unit thickness.
the analytes from a ground state to an excited singlet This equation is clearly less than ideal as the layer
state. As the excited electrons return to the ground state, has a Rnite thickness. More meaningful expressions
energy is emitted as radiation at a longer wavelength, for the intensity of the reSected light, IR, and the
usually in the visible range. For best results in using this transmitted light, IT, for a layer of thickness (l) are
technique, it is important to use TLC/HPTLC plates given by the following hyperbolic solutions:
which do not contain a phosphorescent of Suorescent
indicator to minimize background inteference. sinh(b ) S ) l)
IR" [2]
a ) sinh(b ) S ) l)#b ) cosh(b ) S ) l)
Theory of Spectrodensitometry
In spectrophotometric measurements where the ab- IT" [3]
a ) sinh(b ) S ) l)#b ) cosh(b ) S ) l)
sorbance is measured as a result of a beam of light of
set wavelength passing through a Rxed pathlength
of solution, a direct relationship exists between the
observed absorbance and the concentration of the
S ) l#KA ) l
solution. This is known as Beer’s law. However, it a"
should be pointed out that this relationship is not S)l
linear over the whole range of concentration, and it and
depends on the sample solution being transparent.
As TLC/HPTLC plates are opaque, a somewhat b"(a2!1)1/2
different approach is required. In the 1930s Kubelka
and Munk investigated the relationship between ab- KA is the coefRcient of absorption per unit thickness.
sorbance, transmission and reSectance, deriving math- The application of the equations to quantitative
ematical expressions to explain the effects of absorb- analysis in TLC is quite complex, but it can be
ance and reSectance. When a ray of incident light greatly simpliRed by making a number of reasonable
comes into contact with the surface of the opaque TLC assumptions that would hold true for TLC. One
layer, some light is transmitted, some is reSected in all thing that eqn [2] immediately reveals is that
directions at the surface and some rays are propagated the relationship between the reSected light and the
in all directions inside the adsorbent. The theory which concentration of the chromatographic zone is nonlin-
explains to a large degree what is happening in this ear. This is what is found in practice over the full
process is known as the Kubelka}Munk theory. Cer- range of concentrations. The data when graphically
tain assumptions can be made which simplify the displayed Rt a polynomial curve (eqn [4]).
mathematical equations derived. The theory assumes
that both the transmitted and reSected components of y"a0#a1 ) x#a2 ) x2 [4]
incident light are made up only of rays propagated
inside the sorbent in a direction perpendicular to the However, over a narrow concentration range the
plane of the surface. All other directions will lead to relationship is seen to be linear. This means that if
longer pathways and hence stronger absorption. These it is necessary to have a calibration curve over the
rays therefore contribute little to either the transmitted whole range of concentrations, at least four but no
or reSected light and their contribution can be treated more than six standards will be required for the
as negligible. When light exits from the sorbent at the determination of one separated analyte. Of course,
layer}air boundary, light scattering occurs, and it is only two standards may be needed if the concentra-
distributed over all possible angles with the surface. tions are close to that of the analyte, because it
The coefRcient of light scatter (S), can therefore be can be assumed that the curve is linear over a small
proposed; this depends on the layer thickness. If we range.
assume that this is unchanged in the presence of Although it may seem that errors could easily creep
a chromatographic zone, the following equation can into the determination procedure, this is not the
be derived for an inRnitely thick opaque layer: case. The assumptions made have only a negligible
effect on the Rnal result. Hence, even including
(1!R )2 2.303 any errors which may originate from the scanning
 " ) am ) C [1] spectrodensitometer, the percentage relative standard
2R S

II / CHROMATOGRAPHY: THIN-LAYER (PLANAR) / Densitometry and Image Analysis 827

deviation is normally below 2% and quite often well data Rt is not compromised when as few as three
below 1%. standards are used over the whole concentration
For a wide concentration range, the Michaelis} range.
Menten regression curve can be used. The calibration The mathematical treatment of the data for Suores-
is calculated as a saturation curve: cence intensity can be expressed according to the
well-known Beer}Lambert law. The Suorescence

a1 ) x emission (F) is given by the equation:
y" [5]
F" ) I0(1!e\am  l  c) [12]
and is theoretically only permitted within the calib-
ration range (between the largest and the smallest where F is the Suorescence emission and  is the
standard amounts applied). This regression always quantum yield.
passes through the origin. For low sample concentrations the following as-
In some cases there is a better curve Rt to the data if sumption can be made:
the Michaelis}Menten regression does not pass
through the origin. Better resolution is therefore ob- e\am  l  c"1!am ) l ) c [13]
tained if the data produce a function that does not Therefore:
tend towards zero:
F" ) I0 ) am ) l ) c [14]

a1 ) x
y"a0# [6] It follows that, for low concentrations, the Suores-
cence emission is linearly dependent on the sample
concentration. In practice this proves to be the case
As before, this is theoretically admissible only within
even though this equation was derived without taking
the calibration range.
into consideration the inSuence of absorption or scatter.
It is also possible to linearize the data graphically.
The simplest transformation procedures involve con-
verting the data on reSectance and concentration into Pre-Scanning Considerations
reciprocals, logarithms or squared terms. The follow-
For quantiRcation by reSectance scanning, there is no
ing equations can thus be proposed:
limitation on the backing used for the chromato-
graphic layer, whether it be glass, aluminium or plas-
log Re"a0#a1 ) log c [7]
tic. However, it must be said that quality of the
scanned results, reproducibility and quantitative ac-

1 1 curacy mainly depend on the quality of the spot or
"a0#a1 [8]
Re c band application of the sample and the choice of
developing solvents. The use of automated spot and
R2e"a0#a1 ) c [9] band application equipment results in a noticeable
improvement in relative standard deviation. In practi-
where Re is the reSectance signal and c is the sample cal terms, band application gives even greater accu-
concentration. racy than spot application. This is to be expected
Eqns [7] and [9] result in linearization over the since a scanning slit length on the spectroden-
middle of the concentration range, whereas eqn [8] sitometer has to be chosen for a spot such that it
showed better linearization, but even this fails at very covers the whole length, as the concentration of the
low concentration. None of these methods is able to analyte will vary across the spot, with the highest
linearize the data over the whole concentration range. concentration being in the centre. The slit length also
A solution to the above is to use nonlinear regres- has to allow for any variation caused by migration of
sion analysis based on second-order polynomials. spots not occurring in a precisely vertical direction
These can be described by the following equations: (usually due to solvent vapour not being saturated in
the developing chamber). For band development, the
ln Re"a0#a1 ) ln c#a2 ) (ln c)2 [10] concentration of the analyte is the same across the
length of the band. Hence, there is more latitude on
Re"a0#a1 ) c#a2 ) c2 [11] the choice of slit length. Small slit lengths can be
chosen which tend to higher sensitivity. Under these
Over the whole concentration range, eqn [10] gives conditions with many separations, coefRcient of vari-
the best results. In fact, it has been shown that the ation (CV) below 1% can be achieved.
828 II / CHROMATOGRAPHY: THIN-LAYER (PLANAR) / Densitometry and Image Analysis

Instrumentation 1. single wavelength, single beam

2. single wavelength, double beam
A number of different types of scanning spectroden-
3. dual wavelength, single beam
sitometers are available. Most are now either par-
tially or fully computer-controlled. The parameters Construction 1 requires little explanation and is the
such as track length, number of tracks, distance be- type manufactured by most commercial TLC com-
tween tracks, slit length and width, scanning wave- panies. Construction 2 divides the single beam into
length and speed can all be programmed into the two by means of a beam splitter, so that one half
computer. Some spectrodensitometers can perform scans over the chromatographic zone whilst the other
a pre-scanning run to determine the position of max- scans over the background. Both beams are detected
imum absorption for the separated components on by matched photomultipliers and the difference in the
the track: this is particularly useful where spot ap- signal measured. In construction 3, two wavelengths
plication has been used. After scanning, the spectro- as close together as possible are chosen, such that
densitometer generates massive amounts of data from Suctuations caused by light scattering at the light-
all the tracks, including peak height and area and absorbing wavelength are compensated for by sub-
position of zones (start, middle and end), for every tracting the Suctuations at the different wavelength at
component. Usually a chromatogram can be dis- which there is no absorption by the chromatographic
played for all tracks. This can be baseline-adjusted zone.
and excess noise from the background of the layer can In Rxed-beam spectrodensitometers, the stage hold-
be subtracted. All peaks can be integrated, ready for ing the TLC plate under the light beam moves at
possible quantiRcation. Although a number of scann- a constant rate, propelled by stepping motors. Where
ing modes are available, such as linear, radial (scann- the light beam moves, it does so in a zigzag fashion
ing from the centre for circular chromatograms) and over the surface of the stationary plate. Usually the
circular scanning around a ring (circular develop- zigzag scanners incorporate a curve linearization
ment), by far the most popular is the linear mode, as technique for absorption measurements. This uses the
shown in Figure 1. hyperbolic solution in eqn [2].
Normally, three light sources are used in scanning
densitometry: a deuterium lamp (190}400 nm),
a tungsten halogen lamp (350}800 nm), and a high
pressure mercury vapour or xenon lamp for intense As the chromatogram is permanently or semiperma-
line spectra (254}578 nm), usually required for Su- nently held in the layer after development is complete,
orescence determinations. a number of useful techniques can be used with
Three optical methods (Figure 2) have been used in a scanning spectrodensitometer both to improve the
the construction of scanning densitometers: evaluation of the chromatogram and to collect more
important data on the separated analytes.
1. The TLC plate can be scanned at a range of
different wavelengths. The optimum wavelength
can therefore be chosen for maximum absorption
of individual sample components. Of course,
if two analytes are not completely resolved,
but absorb at different wavelengths, then it is
possible to quantify the results without further
2. UV/visible absorption spectra can be obtained for
each separated component. Some commercial
software then allows the comparison of such
spectra with a library of spectra in order to ident-
ify unknowns.
3. Spectra can also be obtained for different parts of
the chromatographic zone. Hence, spectra can be
obtained for the upslope, apex and downslope of
the peak to assist the analyst in looking for any
Figure 1 Linear scan of individual tracks using a scanning den- peak impurities. Any changes in the spectrum as
sitometer. Slit length and width, track length and speed of scan the light beam traverses the zone would indicate
are all pre-selected. nonhomogeneity.
II / CHROMATOGRAPHY: THIN-LAYER (PLANAR) / Densitometry and Image Analysis 829

Figure 2 Scanning modes: (A) single beam; (B) single wavelength, double beam in space; (C) dual wavelength, single beam in time.
PM, photomultiplier.

4. Background subtraction is another useful feature 4. Forensic: drugs of abuse, poisons, alkaloids, inks
of most spectrodensitometers. Some background 5. Clinical: therapeutic drug monitoring, identiRca-
noise will always be present, hence the scanner tion of metabolic drug disorders
software can subtract a background scan of the 6. Environmental: pesticide residues in crops, crop
TLC plate before quantiRcation. protection agents in drinking water, industrial
5. Some instruments can scan and image an entire hygiene
plate, enabling two-dimensional chromatograms 7. Industrial: product uniformity, impurity proRle,
to be evaluated (scan time less than 5 min). surfactants, synthetic dyes
The widespread use of planar chromatography
To give a Savour of the capability of the technique,
means that the applications of spectrodensitometry
the following examples can be considered. Figure 3
are almost limitless. Hence, there are extensive publi-
shows the scan obtained from the separation of
cations on the use of scanning densitometry in all
a number of sulfonamides and antibiotics from
types of industry and research. Many of the instru-
a complex animal feed matrix on an HPTLC silica gel
ment and plate manufacturers also provide applica-
plate. Scanning at Rve different wavelengths allows
tion methods and extensive bibliographies. For
each of the components to be quantiRed by measure-
example, in all of the following areas scanning den-
ment at its absorption maximum. The three-dimen-
sitometry has been used for quantiRcation.
sional presentation also allows the minor impurities
1. Biomedical: organic acids, lipids, steroids, carbo- to be more clearly identiRed. Multi-wavelength
hydrates, amino acids scanning is also illustrated in Figure 4 with an auto-
2. Pharmaceutical: stability and impurities of syn- mated multiple development (AMD) separation of
thetic drugs, antibiotics, drug monitoring, alkaloids pesticides in tap water on HPTLC silica gel plates.
3. Food science: mycotoxins (including aSatoxins), Figure 5 illustrates the Suorescence scan of a range of
drug residues, antioxidants, preservatives, natural saturated fatty acids from C6 to C24, an important
pigments, food colours, spices, Savonoids food application in fats and vegetable oils. The fatty
830 II / CHROMATOGRAPHY: THIN-LAYER (PLANAR) / Densitometry and Image Analysis

Figure 3 (See Colour Plate 26). Separation of sulfonamides in a complex animal feed matrix on an HPTLC silica gel plate. The plate
has been scanned at five different wavelengths and the chromatogram overlaid in a three-dimensional presentation. Reprinted from
Camag literature, CAMAG, Muttenz, Switzerland.

acids were derivatized before separation by a unique obtained for the unknown. This example illustrates
on-layer technique. The acids are resolved as their the need for the analyst not only to search for the best
dansylcadaverine derivatives on an HPTLC RP18 Rt, but also to check the correlation with the RF value.
layer. Had the search been limited to the spectrum library,
The use of spectral identiRcation of an unknown is ethylmorphine could well have been chosen as the
demonstrated in Figure 6. The unknown was event- unknown.
ually identiRed as morphine, but because ethylmor-
phine and codeine have such similar spectra (as
shown in the overlay), it was necessary to search the
Video Densitometry
spectrum library for the best-Rt recorded spectra, and Video densitometry has been developed in the last
also to check the correlation with the RF value. This few years and is now being deployed throughout
enabled a correlation with morphine of 98.4% to be industry and research. Such instruments use an imag-

Figure 4 (See Colour Plate 27). Separation of pesticides in tap water on an HPTLC silica gel plate by AMD. Multi-wavelength (six
wavelengths) evaluation permits resolution by optical means of fractions insufficiently separated. Reprinted from Camag literature,
CAMAG, Muttenz, Switzerland.
II / CHROMATOGRAPHY: THIN-LAYER (PLANAR) / Densitometry and Image Analysis 831

Figure 5 Fluorescence scan of dansylcadaverine derivatized fatty acids separated on an HPTLC silica RP18 plate.

ing system consisting of a high resolution CCD cam- obtain the concentration of analytes using the math-
era with a zoom attachment to focus and enlarge the ematical procedures used in scanning densitometry.
chromatogram, if required and a suitable illumina- As in spectrodensitometric scanning, the software
tion system. The camera is linked to a computer does all the necessary calculations to determine the
(usually a PC) and a video printer. The software concentration of analytes. For weakly Suorescing
controls the camera, as well as all parameters such as analytes, a small camera aperture (F : 22) can be
brightness, contrast, colour balance and intensity. used with long time integration. This enables the
These can be saved for future use or kept as a record imagining of Suorescing compounds which are often
of the results. The chromatogram can be presented as invisible to the human eye. The images can be
an image on the video printer and can be quantiRed to annotated and that annotation stored separately for

Correlation of spectra without RF value Correlation of spectra with RF value

Substance Correlation Substance Correlation

Ethylmorphine 0.9924 Morphine 0.9839

Codeine 0.9905 Atenolol 0.9223
Nalorphine 0.9848 Salbutamol 0.9174
Morphine 0.9839 Sotalol 0.8939

Figure 6 (See Colour Plate 28). UV spectra of codeine, ethylmorphine and unknown (morphine) overlaid. Spectra of codeine and
ethylmorphine taken from spectral library. Spectrum of morphine taken from chromatogram. Reprinted from Camag literature, CAMAG,
Muttenz, Switzerland.
832 II / CHROMATOGRAPHY: THIN-LAYER (PLANAR) / Densitometry and Image Analysis

Figure 7 Video scan of separation of corticosteroids on an

HPTLC silica gel plate. Detection reagent: blue tetrazolium solu-
tion. Spot application with automatic equipment.
Figure 9 Video scan of separation of pre-derivatized saturated
fatty acids on an HPTLC RP18 plate. The plate was scanned at
366 nm to produce fluorescent zones. Band application with auto-
mated equipment.

Track 3 Sample a

Peak Start Maximum End Area Subst

no name
RF H RF H [%] RF H A [%]

1 0.016 0.0 0.065 606.4 10.76 0.097 50.9 5076.4 9.53 8

2 0.097 50.9 0.126 488.3 8.67 0.146 87.9 3432.6 6.44 7
3 0.146 87.9 0.174 780.2 13.85 0.206 12.1 5353.3 10.05 6
4 0.227 0.0 0.271 1094.6 19.42 0.316 13.8 9440.8 17.72 5
5 0.324 0.0 0.368 844.7 14.99 0.397 240.7 7487.0 14.05 4
6 0.397 240.7 0.417 555.2 9.85 0.462 6.1 4914.6 9.22 3
7 0.543 26.1 0.587 509.7 9.05 0.640 10.6 6065.0 11.38 2
8 0.794 70.3 0.854 755.8 13.41 0.915 46.6 11519.2 21.62 1

Total height, 5634.96; total area, 53288.8.

Figure 8 Video scan of separation of corticosteroids on an HPTLC silica gel plate.
II / CHROMATOGRAPHY: THIN-LAYER (PLANAR) / Densitometry and Image Analysis 833

Figure 10 Separation of dye mixture developed on an HPTLC silica gel plate with toluene as mobile phase. Comparison of
spectrodensitometric scan with video scanning.

readiness in annotating further images. Such images separation of derivatized saturated fatty acids from
can be stored in a variety of Rles which can then be C6 to C24 (conditions as in Figure 5).
used in a number of well-known ofRce programs, Although it is possible to quantify results from the
such as Word, and PowerPoint. video scan, they are not as accurate as those obtained
The illumination system needs a number of features from a spectrodensitometer. Figure 10 and Table 1
in order to get the best results from the CCD camera show a comparison of the CV for a six-component
unit. Illumination from above is necessary, both vis- dye test mixture separated on an HPTLC layer.
ible light and UV light at 254 and 366 nm (depending Whereas the CVs for spectrodensitometric scan are
on the chromatogram). However, it is essential that below 2%, those for the video imaging system are
the light Rttings do not interfere with the camera’s typically from 2 to 4%. As most USP (United States
Reld of view. Lighting from below the plate can in Pharmacopoeia) and EP (European Pharmacopoeia)
some cases also prove advantageous in giving a bright monographs accept CVs of $6% in most, if not all,
image. cases, the use of video densitometry is acceptable.
Figure 7 illustrates a video print of a separation of However, it should be remembered that for Suores-
corticosteroids developed on an HPTLC silica gel cence quenching and absorption measurements below
plate. The steroids were detected with blue tetra- 254 nm, video densitometry will not show any detec-
zolium reagent. Figure 8 shows the scan taken using tion. This is one of the present limitations of the
the software option available. RF data is recorded in technique. Some substances do require shorter
the table below. Figure 9 illustrates a further video wavelength UV light for their detection. In these in-
print, this time of Suorescent chromatographic zones, stances spectrodensitometry is presently the only
photographed under UV light (366 nm). This is a solution.

Table 1 Comparison of coefficient of variance (CV) with video scanning and spectro-
densitometric scanning. Separation of dyes on an HPTLC silica gel plate using toluene as
mobile phase

Dye RF Video scan with white light Spectrodensitometric scan at 592 nm

Mean value (%) CV (%) Mean value (%) CV (%)

Black 0.04
Grey 0.10 99.4 3.50 101.5 0.83
Red 0.17 102.8 3.10 97.8 0.31
Blue 0.23 103.0 3.52 101.2 1.90
Pink 0.36 99.7 3.46 98.3 0.96
Yellow 0.51 98.6 1.30 98.8 0.56
834 II / CHROMATOGRAPHY: THIN-LAYER (PLANAR) / Historical Development

Future Trends See also: III/In-Depth Distribution in Quantitative

Thin-layer Chromatography.
It seems unlikely that video densitometry will ever
replace spectrodensitometry as both techniques have Further Reading
unique advantages. On the one hand spectroden-
sitometry allows the scanning of TLC/HPTLC plates Frei MP and Zeiloff K (1992) Qualitativ und Quantitativ
at selectable wavelengths, the acquisition of UV/vis- Du( nnschicht-Chromatographie. Weinheim: VCH.
ible spectra, the determination of peak purity and Geiss F (1987) Fundamentals of Thin Layer Chromatogra-
phy. Heidelberg: Alfred HuK thig Verlag.
high accuracy of results. On the other hand, video
Jork H, Funk W, Fischer W and Wimmer H (1989, 1994)
scanning provides a computer or printed image that Thin-layer Chromatography, Reagents and Detection
can serve as a permanent record of the results ob- Methods, vols 1a and 1b. Weinhein: VCH.
tained which can be documented at any time in a re- Poole CF and Poole SK (1992) Chromatography Today.
port. Also, for some requirements the accuracy of Amsterdam: Elsevier.
scanning is sufRcient for quantitative evaluation. Sherma J and Fried B (1994) Thin-layer Chromatography,
With improved software, both densitometric and Techniques and Applications, 3rd edn. Chromatographic
video scanners are likely to become still more user- Science Series, vol. 66. New York: Marcel Dekker.
friendly. However, more dramatic improvement in Touchstone JC (1992) Practice in Layer Chromatography,
the accuracy and reliability of results is more likely to 3rd edn. New York: Wiley-Interscience.
come from the continual improvements taking place Touchstone JC and Sherma J (1979) Densitometry in Thin
Layer Chromatography Practice and Applications. New
in the quality of adsorbents making up the layer. With
York: Wiley-Interscience.
the introduction of smaller (4 m) spherical particle Wall PE and Wilson ID (1995) Thin-layer chromatogra-
sizes, the quality of separation will improve, hence phy}techniques. In: Encyclopedia of Analytical Science.
this will be reSected in the scans and quantitative London: Academic Press.
results obtained with both spectrodensitometry and Zlatkis A and Kaiser RE (1977) HPTLC High Performance
video scanning. Thin-layer Chromatography. New York: Elsevier.

Historical Development
E. Reich, CAMAG, Muttenz, Switzerland focused on a faster microchromatographic method,
Copyright ^ 2000 Academic Press which allowed the exact identiRcation of adsorbed
substances. This situation encouraged the transition
from a regular column to an open column, a thin
History layer of adsorbent.
Today the term planar chromatography is commonly Izmailov and Shraiber are regarded as the inventors
used as a synonym for high performance thin-layer of TLC (Table 1). In 1938 they described a method in
chromatography (HPLTC) and conventional thin- which microscopic slides were coated with 2 mm
layer chromatography (TLC). Originally it referred layers of a slurry made of chalk, talc, magnesium
more generally to a family of techniques including oxide, lime aluminium oxide or other adsorbents and
TLC, some types of electrophoresis and paper water. On drying, a thin adsorbent layer was formed.
chromatography, which all have in common a sta- The authors investigated belladonna and other plant
tionary phase in the form of a Sat thin layer rather extracts by placing a drop of the extract on to the
than packed into a column. Modern planar chrom- layer. This resulted in the so-called ultra chromato-
atography is a form of liquid chromatography and its gram that was visualized under ultraviolet light.
history is closely linked to the development of The chromatogram was then developed with several
chromatography as an analytical tool. drops of solvent. The most important advantage
Early roots go back to Beyrinck who in 1889 separ- of the new method in comparison to column
ated hydrochloric and sulfuric acid by diffusion chromatography was the short time of analysis and
through a thin layer of gelatine on a glass plate. With the low consumption of adsorbents, solvents and
the same technique, Wijsman in 1898 was able to samples.
demonstrate the presence of two enzymes in malt Crowe reported in 1941 the use of a micro-
diastase. When at the end of the 1930s Tswett’s chromatographic method to select suitable solvents
column chromatography became successful, research for column chromatography. The procedure was