Encyclopedia of chromatography by jack cazes 2

Optical Activity Detectors
Hassan Y. Aboul-Enein
Ibrahim A. Al-Duraibi
Pharmaceutical Analysis Laboratory, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia

Introduction
Optical activity detectors are capable of specifically
detecting chiral compounds, taking advantage of their
unique interactions with polarized light. Much of the
work on the development of prisms and other devices
for the production of polarized light was done in the
early part of the nineteenth century. However, the
measurement of optical activity is often used for enantiomeric purity determination of chiral compounds,
which by definition have either a center or plane of
asymmetry. Enantiomers rotate the plane of polarized
light in opposite directions, although in equal amounts.
The isomer that rotates the plane to the left (counterclockwise) is called the levo isomer and is designated
(Ϫ), whereas the one that rotates the plane to the right
(clockwise) is called the dextro isomer and is designated (ϩ). Questions of optical activity are of extreme
importance in the field of asymmetric chemical synthesis and in the pharmaceutical industry.
Detection Principle
Figure 1 shows the basic optimal system of the optical
rotation detector, which is based on the nonmodulated
polarized beam-splitting method. The light radiated
from the light source is straightened by the plane polarizer, then to the lens for beam formation and concentration, and then to the flow cell.
The plane-polarized light which goes through the
flow cell is rotated by optically active substances (chiral compounds) according to their specific optical rotations and concentrations. The light then enters the polarized beam splitter and is divided into two beams
according to the polarized beam directions. These
beams are detected by two photodiodes as shown.
The angle of the plane polarizer is adjusted so that
the two photodiodes may receive the same beam intensity when no optically active substance is present in

the flow cell. When optically active substances are
present in the flow cell, the difference between the
beam intensities received by the two photodiodes is
not zero. Therefore, the difference has a linear relation

Fig. 1

Optical rotation detector.

with specific optical rotation and concentration of the
optically active substance and can be expressed by
V0 ϭ K3a 4C

where V0 is the difference of beam intensities received
by the two photodiodes (i.e., output of signal level), K
is a constant determined by cell structure and light intensity of the light source, [a] is the specific optical rotation of the chiral compound, and C is the concentration of the chiral compound.
Polarimetry Theory
Most forms of optical spectroscopy are usually concerned with the measurement of the absorption or
emission of electromagnetic radiation. Ordinary,
natural, unreflected light behaves as though it consists of a large number of electromagnetic waves vibrating in all possible orientations around the direction of propagation. If, by some means, we sort out
from the natural conglomeration only those rays vibrating in one particular plane, we say that we have
plane-polarized light. Of course, because a light
wave consists of an electric and a magnetic component vibrating at right angles to each other, the term
“plane” may not be quite descriptive, but the ray can
be considered planar if we restrict ourselves to noting the direction of the electrical component. Circu-

Encyclopedia of Chromatography
DOI: 10.1081/E-Echr 120005246
Copyright © 2002 by Marcel Dekker, Inc. All rights reserved.

1


2
lar polarized light represents a wave in which the
electrical component (and, therefore, the magnetic
component also) spirals around the direction of
propagation of the ray, either clockwise (“righthanded” or dextrorotatory) or counterclockwise
(“left-handed” or levorotatory). If, following the
passage of the plane-polarized ray through some
material, one of the circularly polarized components, say the left circularly polarized ray, has been
slowed down, then the resultant would be a planepolarized ray rotated somewhat to the right from its
original position. In addition, lasers have been incorporated into two optical rotation methods to
date: polarimetry and circular dichroism.
Optical Rotation and Optical Rotatory Dispersion
A polarimeter measures the direction of rotation of
plane-polarized light caused by an optically active substance. The specific optical activity of an asymmetrical
molecule varies with the wavelength of the light used
for its determination. This variation is called optical
rotatory dispersion (ORD). In ORD, rotations are
measured over a range of wavelengths rather than at a
single wavelength, usually covering the ultraviolet
(UV) as well as the visible region.
Circular Dichroism
In this technique, the molecular extinction coefficients
of a compound are measured with both left and right
circularly polarized light, and the difference between
these values is plotted against the wavelength of the
light used. The phase angle between the projections of
the two circularly polarized components is altered by

passage through the chiral medium, but their amplitudes will be modified by the degree of absorption experienced by each component. This differential absorption of left- and right-circularly polarized light is
termed circular dichroism (CD). So, circular dichroism
measurements provide both absorbance and optical
rotation information simultaneously.
Circularly Polarized Luminescence Spectroscopy
Circularly polarized luminescence spectroscopy
(CPLS) is a measure of the chirality of a luminescent
excited state. The excitation source can be either a
laser or an arc lamp, but it is important that the source
of excitation be unpolarized to avoid possible photoselection artifacts. The CPLS experiment produces two

Optical Activity Detectors
measurable quantities, which are obtained in arbitrary
units and related to the circular polarization condition
of the luminescence. It is appropriate to consider
CPLS spectroscopy as a technique that combines the
selectivity of CD with the sensitivity of luminescence.
The major limitation associated with CPLS spectroscopy is that it is confined to emissive molecules only.
Vibrational Optical Activity
The optical activity of vibrational transitions has been
conducted. The infrared (IR) bands of a small molecule can easily be assigned with the performance of a
normal coordinate analysis, and these can usually be
well resolved. One of the problems associated with vibrational optical activity is the weakness of the effect.
Instrumental limitations of infrared sources and detectors create additional experimental constraints on the
signal-to-noise ratios.
Two methods suitable for the study of vibrational
optical activity have been developed:
Vibrational Circular Dichroism: Vibrational circular dichroism (VCD) could be measured at good
signal-to-noise levels. Vibrational optical activity
is observed in the classic method of Grosjean and

Legrand.
Raman Optical Activity: The Raman optical activity (ROA) effect is the differential scattering of
left- or right-circularly polarized light by a chiral
substrate where chirality is studied through Raman spectroscopy.
Fluorescence-Detected Circular Dichroism
Fluorescence-detected circular dichroism (FDCD) is a
chiroptical technique in which the spectrum is obtained by measuring the difference in total luminescence obtained after the sample is excited by left- and
right-circularly polarized light. For the FDCD spectrum of a given molecular species to match its CD spectrum, the luminescence excitation spectrum must be
identical to the absorption spectrum.
Factors Affecting the Measurement
of Optical Rotation
The rotation exhibited by an optically active substance
depends on the thickness of the layer traversed by the
light, the wavelength of the light used for the measurement, and the temperature of the system. In addition,
if the substance being measured is a solution, then the


Optical Activity Detectors
concentration of the optically active material is also involved and the nature of the solvent may also be important. There are certain substances that change their
rotation with time. Some are substances that change
from one structure to another with a different rotatory
power and are said to show mutarotation. Mutarotation is common among the sugars. Other substances,
owing to enolization within the molecules, may rotate
so as to become symmetrical and, thus, lose their rotatory power. These substances are said to show racemization. Mutarotation and racemization are influenced
not only by time, but also by pH, temperature, and
other factors. Of course, rotations that determined for
the same compound under the same conditions are
identical. Therefore, in expressing the results of any
polarimetric measurement, it is, therefore, very important to include all experimental conditions.
Temperature

Temperature changes have several effects on the rotation of a solution or liquid. An increase in temperature
increases the length of the tube; it also decreases the
density, thus reducing the number of molecules involved in the measurement. It causes changes in the rotatory power of the molecules themselves, due to association or dissociation and increased mobility of the
atoms, and affects other properties. In addition, temperature changes cause expansion and contraction of
the liquid and a consequent change in the number of
active molecules in the path of the light.
The unique ability of the optical rotation detector
to respond to the sign of rotation allows precise
enantiomeric purity determination even if the enantiomers are only partially resolved. The sign of rotation is also useful in establishing enantiomer elution
order.
Because the optical rotation detectors only respond
to optically active compounds, enantiomeric purity determination to precisions of better than 0.5% can be
achieved and is possible in even the complex mixtures.
The detection can also be used as part of a flow injection analysis system to determine amount and enantiomeric purity of a drug in dosage form.
The applications using optical rotation detectors include the following:

3
1.

2.
3.
4.
5.

Qualitative analysis of chiral compounds, including drugs, pesticides, carbohydrates, amino
acids, liquid crystals, and other biochemicals
Determination of enantiomeric purity of chiral
compounds
Monitoring an enzymatic reaction
Qualitative analysis of proteins

Use as a conventional polarimeter

However, the disadvantages of optical rotation detectors may be limited by shot or flicker noise, which are dependent on the optical and mechanical properties of the
system or by noise in the detector electronics. Generally,
the usefulness of this technique has been limited by the
lack of sensitivity of commercially available instruments.
Suggested Further Reading
Allenmark, S., Techniques used for studies of optically
active compounds, in Chromatographic Enantioseparation: Methods and Application, 2nd ed., Ellis
Horwood Ltd., London, 1991.
Beesley, T. E. and R. P. W. Scott, An introduction to
chiral chromatography, in Chiral Chromatography,
John Wiley & Sons, Inc., New York, 1998, pp. 1–11.
Dodziuk, H., Physical methods as a source of information on the spatial structure of organic molecules, in
Modern Conformational Analysis, Elucidating
Novel Exciting Molecular Structures, VCH, New
York, 1995, pp. 48 –54.
Edkins, T. J. and D. C. Shelly, Measurement concepts
and laser-based detection in high-performance micro separation, in HPLC Detection: Newer Methods
(G. Patonay, ed.), VCH, New York, 1992, pp. 1–15.
Goodall, D. M. and D. K. Lloyd, A note on an optical
rotation detector for high-performance liquid chromatography, in Chiral Separations (D. Stevenson
and D. Wilson, eds.), Plenum Press, New York,
1988, pp. 131–133.
Sheldon, R. A., Introduction to optical isomersion, in
Chirotechnology: Industrial Synthesis of Optically
Active Compounds, Marcel Dekker, Inc., New
York, 1993, pp. 25 –27.
Weston, A. and P. R. Brown, HPLC and CE Principles
and Practice, Academic Press, San Diego, CA, 1997.

Yeung, E. S., Polarimetric detectors, in Detectors for
Liquid Chromatography (E. S. Yeung, ed.), John
Wiley & Sons, New York, 1986, pp. 204 –228.


Optical Quantification (Densitometry) in TLC
Joseph Sherma
Lafayette College, Easton, Pennsylvania, U.S.A.

Introduction
Quantitative evaluation of thin-layer chromatograms
can be performed by direct, in situ visual, and indirect
elution techniques. Visual evaluation involves comparison of the sizes and intensities of color or fluorescence
between sample and standard zones spotted, developed, and detected on the same layer. The series of
standards is chosen to have concentrations or weights
that bracket those of the sample zones. After matching
a sample with its closest standard, accuracy and precision are improved by respotting a more restricted series of bracketing standards with a separate sample
spot between each of two standard zones. Accuracy no
greater than 5 –10% is possible for trained personnel
using visual evaluation. The determination of mycotoxins in food samples is an example of a practical application of visual comparison of fluorescent zones.
The elution method involves scraping off the separated zones of samples and standards and elution of
the substances from the layer material with a strong,
volatile solvent. The eluates are concentrated and analyzed by use of a sensitive spectrometric method, gas
or liquid column chromatography, or electroanalysis.
Scraping and elution must be performed manually because the only commercial automatic micropreparative elution instrument has been discontinued by its
manufacturer. The elution method is tedious and timeconsuming and prone to errors caused by the incorrect
choice of the sizes of the areas to scrape, incomplete
collection of sorbent, and incomplete or inconsistent
elution recovery of the analyte from the sorbent. However, the elution method is being rather widely used
(e.g., some assay methods for pharmaceuticals and

drugs in the USP Pharmacopoeia).
Introduction to Densitometry
In order to achieve the optimum accuracy, precision,
and sensitivity, most quantitative analyses are performed by using high-performance thin-layer chromatography (TLC) plates and direct quantification by
means of a modern optical densitometic scanner with

a fixed sample light beam in the form of a rectangular
slit that is variable in height (e.g., 0.4 –10 mm) and
width (20 mm to 2 mm). Densitometers measure the
difference in absorbance or fluorescence signal between a TLC zone and the empty plate background
and relate the measured signals from a series of standards to those of unknown samples through a calibration curve. Modern computer-controlled densitometers can produce linear or polynomial calibration
curves relating absorbance or fluorescence versus
weight or concentration of the standards and determine bracketed unknowns by automatic interpolation
from the curve. Samples and standards are best applied
using an automated instrument such as the one shown
in Fig. 1. Use of manual spotting and less efficient TLC
plates results in greater errors and poorer reproducibility in quantitative results.
Instrumental Design and Scanning Modes
A commercial densitometer and a schematic diagram
of the light-path arrangement used in scanning are
shown in Fig. 2. The plate is mounted on a moveable
stage controlled by a stepping motor drive that allows
each chromatogram track to be scanned in or against
the direction of development. A tungsten or halogen
lamp is used as the source for scanning colored zones
in the 400 – 800-nm range (visible absorption) and a
deuterium lamp for scanning ultraviolet (UV)-absorbing zones directly or as quenched zones on phosphorcontaining layers (F-layers) in the 190 – 450-nm range.
The monochromator used with these continuouswavelength sources can be a quartz prism or, more often, a grating. The detector is a photomultiplier or
photodiode placed above the layer to measure
reflected radiation. [Some scanners (e.g., Fig. 2) make

use of a reference photomultiplier in addition to the
measuring photomultiplier in the single-beam mode;
the reference photomultiplier puts out a constant signal that is compared to the signal from the measuring
photomultiplier to produce a difference signal that is
more accurate than a direct signal from a single measuring photomultiplier would be.]

Encyclopedia of Chromatography
DOI: 10.1081/E-Echr 120005247
Copyright © 2002 by Marcel Dekker, Inc. All rights reserved.

1


2

Optical Quantification (Densitometry) in TLC

Fig. 2 Photograph of the DESAGA Densitometer CD 60
with a superimposed schematic diagram of the light path
including (right to left) the source lamp, two mirrors, grating
monochromator, mirror, beam splitter, plate with chromatograms to be scanned, reference and measuring detectors
(reflection) above the plate and detector (transmission) below the plate. (Courtesy of DESAGA GmbH, Weisloch,
Germany.)

Fig. 1 Automatic TLC sampler (ATS 3) used for computercontrolled application of precisely controlled volumes of
samples and standards between 10 nL and 50 mL from a rack
of vials as spots or bands to preselected origins on a plate.
(Courtesy of Camag Scientific Inc., Wilmington, NC.)

For normal fluorescence scanning, a high-intensity

xenon continuum source or a mercury vapor line
source is used, and a cutoff filter is placed between the
plate and detector to block the exciting UV radiation
and transmit the visible emitted fluorescence. For
fluorescence measurement in the reversed-beam
mode, a monochromatic filter is placed between the
source and plate and the monochromator between the
plate and detector. In this mode, the monochromator
selects the emission wavelength, rather than the excitation wavelength as in the normal mode.
Simultaneous measurement of reflection and transmission, or transmission alone, can be carried out by
means of a detector positioned on the opposite side of
the plate (Fig. 2). Ratio-recording double-beam densitometers, which can correct for background disturbances
and drift caused by fluctuations in the source and detec-

tor, were designed earlier with two photomultiplier detectors simultaneously recording the two beams (double
beam in space), but, today, such densiotmeters are
equipped with a chopper and one detector (double
beam in time). For dual-wavelength, single-beam scanning, which will correct for scattering of the absorbed
light by subtracting out the (presumably equal) scattering at a nonabsorbed wavelength, a light beam is selected by a mirror and passes through two separate
monochromators to isolate the two different wavelengths. The two beams are alternated by a chopper and
recombined into a single beam representing a difference
signal at the detector. Zigzag or meandering scanning
with a small point or spot of light is possible with densitometers having two independent stepping motors to
move the plate in the X and Y axes. Computer algorithms integrate the maximum absorbance measurements from each swing to produce a distribution profile
of zones having any shape. The potential advantages of
scanning with a moving light spot are offset by problems
with lower spatial resolution and errors in data processing, and the method is not as widely used as conventional scanning of chromatographic tracks with a fixed
slit. Some densitometers have the ability to rotate the
plate while scanning for measurement of circular and
anticircular chromatograms.

Single-wavelength, single-beam, fixed-slit scanning
is most often used and can produce excellent results


Optical Quantification (Densitometry) in TLC
when high-quality plates and analytical techniques are
employed.
Spectral Measurement
Many modern scanners have a computer-controlled motor-driven monochromator that allows automatic
recording of in situ absorption and fluorescence excitation spectra. These spectra can aid compound
identification by comparison with stored standard spectra, test for identity by superimposition of spectra from
different zones on a plate, and check zone purity by superimposition of spectra from different areas of a single
zone. The spectral maximum determined from the in situ
spectrum is usually the optimal wavelength for scanning
standard and sample areas for quantitative analysis.
Data Handling
The densitometer is connected to a recorder, integrator,
or computer. A personal computer with software designed specifically for TLC is most common for data handling and automation of the scanning process in modern
instruments. With a fully automated system, the computer can carry out the following functions: data acquisition by scanning a complete plate following a preselected
geometric pattern with control of all scanning parameters; automated peak searching and optimization of
scanning for each fraction located; multiple-wavelength
scanning to find, if possible, a common wavelength for all
substances to be quantified, to optically resolve fractions
incompletely separated by TLC, and to identify fractions
by comparison of spectra with standards cochromatographed on the same plate or stored in a spectrum library through pattern recognition techniques; baseline
location and correction; computation of peak areas
and/or heights of samples and codeveloped standards
and processing of the analog raw data to quantitative digital results, including calculation of calibration curves by
linear or polynomial regression, interpolation of sample
concentrations, statistical analysis of reproducibility, and

presentation of a complete analysis report; and storage
of raw data on disk for later reintegration, calibration,
and evaluation with different parameters.
Calibration Curves
Densitometric calibration curves relating absorption
signal and concentration or weight of standards on
the layer are usually nonlinear, especially for higher

3
amounts of standards, and do not pass through the
origin. Fluorescence calibration curves are generally
linear and pass through the origin, and analyses
based on fluorescence are more specific and 10 –1000
times more sensitive. The advantages of fluorescence
measurement may be realized for nonfluorescent
compounds by prechromatographic or postchromatographic derivatization reactions with suitable
fluorogenic reagents.
Because the incident monochromatic light is absorbed, reflected, and scattered by the opaque layer
material, the theoretical relationship between amount
of absorption and amount of substance does not follow
the simple Beer–Lambert law that is valid for solutions. The Kubelka–Munk equation is the most accepted theoretical relationship for TLC, but its use is
not necessary because of the ability of densitometer
software to handle empirical nonlinear regression
functions.
Image Analysis (Videodensitometry)
Video camera systems are available from several
manufacturers for documentation and densitometric quantification of TLC plates. As an example, the
Camag VideoScan instrument consists of a lighting
module with short- and long-wave UV and visible
sources upon which the layer is placed, a chargecoupled device (CCD) camera with zoom and longtime integration capability, and a PC under MSWindows control with frame grabber, monitor, and

printer. The available software for quantitative evaluation allows the display of the tracks of the chromatogram image acquired with the video camera as
analog curves and calculation of their peak properties (Rf , height, area, height percent, and area percent). For quantification, the computer creates a
standard curve from the areas or heights of the standards and interpolates unknown values from the
curve.
Video scanners have potential advantages, including rapid data collection, simple design with virtually
no moving parts, and ability to quantify two-dimensional chromatograms, but they have not yet been
shown to have the required capabilities, such as
sufficient spectral discrimination or the ability to illuminate the plate uniformly with monochromatic
light of selected wavelength, to replace slit-scanning
densitometers. Current video scanners can measure
spots in the visible range in transmittance, reflectance, or fluorescence modes, but they cannot perform spectral analysis.


4

Optical Quantification (Densitometry) in TLC

Applications and Practical Aspects
of Densitometry
Densitometric quantification has been applied to virtually every type of analyte and sample. For example, the
greatest number of applications is for the analysis of
drug and pharmaceutical compounds, most of which
have structures including chromophores that cause
strong UV absorption. These compounds are readily
quantified in the fluorescence quenching mode on Flayers or in the direct UV absorption mode on unimpregnated layers. Lipids are compounds that are not
easily analyzed by gas chromatography (GC) or highperformance liquid chromatography (HPLC) because
they lack volatility and the presence of a chromophore
leading to UV absorption. The most successful way to
quantify lipids is by densitometry after separation and
detection on the layer with a chromogenic reagent, most

notably phosphomolybdic acid. The quantification of
amino acids after detection with ninhydrin is another
example of densitometry in the visible absorption
mode. Fluorescence densitometry has been applied to
the determination of naturally fluorescent compounds
(e.g., quinine in tonic water) or compounds derivatized
with a fluorogenic reagent pre-TLC or post-TLC (e.g.,
amino acids reacted with fluorescamine, or carbamate
pesticides with dansyl chloride after hydrolysis).
The steps in a typical densitometric quantitative
analysis, regardless of analyte type, are the following:
1.
2.

Prepare a standard reference solution.
Prepare a sample solution in which the analyte
is completely dissolved and impurities have
been reduced to a level at which they do not interfere with scanning of the analyte.
3. Choose a layer and mobile-phase combination
that will separate the analyte as a compact
zone with an Rf value in the range 0.2 – 0.8.
4. Apply the standard and sample aliquots to the
layer using an instrument (Fig. 1) or manually
with a micropipette, onto preadsorbent, laned
plates. Generally, three or four standard zones
are applied in constant volumes from a series
of standard solutions with increasing concentrations, or in a series of increasing volumes
from a single standard solution. The sample
volume applied must provide an amount of analyte zone with a weight or concentration that
is bracketed by the standard amounts.

5. Develop the plate in an appropriate chamber
and dry the mobile phase under in a fume
hood or oven.

6. Apply a detection reagent, if necessary, by
spraying or dipping. The reagent should produce a stable colored, UV-absorbing, or fluorescent zone having high contrast with the layer
background.
7. Scan the natural or induced absorption or fluorescence of the standard and sample zones on
the plate using a densitometer with optimized
parameters.
8. Generate a calibration curve by linear or polynomial regression of the scan areas and
weights of the standards and interpolate the
weights in the sample zones from the curve.
9. Calculate the concentration of analyte in the
sample from the original weight of the sample,
the original total volume of the sample test solution, the aliquot volume of the test solution
that is spotted, the interpolated analyte weight
in that spotted volume from the calibration
curve, and any numerical factor required because of dilution or concentration steps
needed for the test solution to produce a
bracketed scan area for the analyte zone in the
sample chromatogram.
10. Validate the precision of the TLC analysis by
replicated determination of the sample and accuracy by comparison of the results to those
obtained from analysis of the same sample by
an established independent method or calculation of recovery from analysis of a spiked preanalyzed sample or spiked blank sample.
The following are some advantages of TLC densitometry compared to HPLC:
1.

2.

3.

The simultaneous analysis of multiple samples
on a single plate leads to higher sample
throughput (lower analysis time) and less cost
per sample. Up to 36 tracks are available for
samples and standards on a 10-cm ϫ 20-cm
high-performance TLC plate.
The ability to generate a unique calibration
curve using standards developed under the
same conditions as samples on each plate (insystem calibration) leads to statistical improvement in data handling and better analytical precision and accuracy and eliminates the need for
an internal standard for most analyses.
Detection is versatile and flexible because the
mobile phase is removed prior to detection. Because the detection process is static (the zones
are stored on the layer), multiple, complementary detection methods can be used.


Optical Quantification (Densitometry) in TLC
4.

5.

6.

Storage of the chromatogram also allows scanning to be repeated with various parameters
without time constraints and assures that the
entire sample is available for detection and
scanning.
Less sample cleanup is often required because

plates are not reused. Every sample is analyzed
on a fresh layer without sample carryover or
cross-contamination.
Solvent use is very low for TLC, both on an
absolute and per-sample basis, leading to reduced purchase and disposal costs and safety
concerns.

Suggested Further Reading
Fried, B. and J. Sherma, Thin Layer Chromatography— Techniques and Applications, 4th ed., Marcel
Dekker, Inc., New York, 1999, pp. 197–222.

5
Jaenchen, D. E., Instrumental thin layer chromatography, in Handbook of Thin Layer Chromatography,
2nd ed. (J. Sherma and B. Fried, eds.), Marcel
Dekker, Inc., New York, 1996, pp. 129 –148.
Petrovic, M., M. Kastelan-Macan, K. Lazaric, and S.
Babic, Validation of thin layer chromatography
quantitation with CCD camera and slit-scanning
densitometer, J. AOAC Int. 82: 25 –39 (1999).
Pollak, V. A., Theoretical foundations of optical quantitation, in Handbook of Thin Layer Chromatography (J. Sherma and B. Fried, eds.), Marcel Dekker,
Inc., New York, 1991, pp. 249 –281.
Poole, C. F. and S. K. Poole, Chromatography Today,
Elsevier, New York, 1991, pp. 649 –734.
Prosek, M. and M. Pukl, Basic principles of optical quantitation in TLC, in Handbook of Thin Layer Chromatography, 2nd ed. (J. Sherma and B. Fried, eds.), Marcel Dekker, Inc., New York, 1996, pp. 273–306.
Robards, K., P. R. Haddad, and P. E. Jackson, Principles
and Practice of Modern Chromatographic Methods,
Academic Press, San Diego, CA, 1994, pp. 180 –226.


Optimization of Thin-Layer Chromatography

Wojciech Prus
Technical University of jód´z, Bieisko-Biaia, Poland

Teresa Kowalska
Institute of Chemistry, Silesian University, Katowice, Poland

Introduction
The principal task of chromatography is the separation
of mixtures of substances. By “optimization” of the
chromatographic process, we mean enhancement of
the quality of the separation by changing one or more
parameters of the chromatographic system. An ability
to foresee, correctly, the direction and scope of these
changes is the most important goal of each optimization procedure.
Use of chemometrics to devise procedures suitable for the most crucial stage of optimization, optimization of selectivity, is generally performed in
three steps:
1.

Selection of the experimental method which
best suits the analytical problem considered. At
this stage, a chromatographic technique is chosen that ensures that the best possible range of
retention parameters is obtained for each individual component of the separated mixture.
2. Establishing the experimental conditions that
enable quantification of the influence of the optimized parameters of a chromatographic system on solute retention.
3. Fixing the experimental conditions at values
that provide the optimum separation selectivity.
Chemometric optimization of the chromatographic
system consists, in fact, in predicting local maxima in
multiparametric space and, then, in further deciding
which of these parameters is global with regard to the

overall efficiency of a given chromatographic system.
Quality of Chromatographic Separations
Elementary Criteria

ference between their respective retention parameters;
that is, the difference between their RF values,
¢RF ϭ RF2 Ϫ RF1

(1)

or between their RM values,
¢RM ϭ RM1 Ϫ RM2 ϭ log

k1
ϭ log a
k2

(2)

where k1 and k2 are the capacity (retention) factors of the
chromatographic bands and a is the separation factor.
The terms most frequently used to characterize the
separation of two chromatographic bands are the separation factor, a,
aϭ

k1
k2

(3)

where k1 Ͼ k2 , and the resolution, RS [1],
RS ϭ

21z2 Ϫ z1 2

ϭ

w1 ϩ w2

2l¢RF
w1 ϩ w2

(4)

where z1 and z2 are the distances of the geometric centers
of two chromatographic bands, 1 and 2, from the origin,
l is the distance from the origin to the mobile phase front,
and w1 and w2 are the diameters of the two chromatographic bands, measured in the direction of eluent flow.
Other elementary criteria include the separation
factor, S [2],
Sϭ

k2 Ϫ k1
k1 ϩ k2 ϩ 2

(5)

the peak-to-valley ratio of the bands, P [3],
Pϭ

f
g

(6)

(where f and g are, respectively, the average peak
height and valley depth, characteristic of a given pair of
neighboring solutes on a chromatogram), the fractional peak overlap, FO [4],

The simplest way of quantifying the separation of two
chromatographic bands, 1 and 2, is to calculate the dif-

Encyclopedia of Chromatography
DOI: 10.1081/E-Echr 120005248
Copyright © 2002 by Marcel Dekker, Inc. All rights reserved.

FO ϭ

An Ϫ An, nϪ1 Ϫ An, nϩ1
An

(7)

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2

Optimization of Thin-Layer Chromatography

(where An is the surface area of the part of the band
originating from the pure single compound, An, nϪ1 is
the surface area of the fractional overlap of the nth and
(n Ϫ 1)th bands, and An, nϩ1 is the surface area of the
fractional overlap of the nth and (n ϩ 1)th bands), and
the selectivity parameter, RR [5],
RR ϭ

RF1
RF2

(8)

where RF1 Ͼ RF2 .

nϪ1 R
nϪ1 S
Si, iϩ1
i, iϩ1
rϭ q
ϭ q
RS
S
iϭ1
iϭ1

(10)

where n is the number of the chromatographic bands,

Criteria for the Quality of Chromatograms

RS ϭ

One method which can be used to establish the optimum conditions for the separation of a complex mixture (i.e., not only a pair) of compounds consists in
searching for the maximum of a function denoted the
chromatogram quality criterion. The evaluation of
separation selectivity can be conducted with the aid of
different criteria of chromatogram quality such as the
sum of resolution, ͚ RS [6], the sum of separation factors, ͚ S [2], and other sums and products of elementary criteria, selected examples of which are the resolution product, ß RS [7],
q RS ϭ exp a a ln RS b ,

the product of the separation factors, ß S [8], the
product of the fractional peak overlap, ß FO [9], and
the product of the peak-to-valley ratio of the bands,
ß P [10].
There are also other, more complex criteria, including the normalized resolution product, r [11],

(9)

1 nϪ1
RSi, iϩ1
nϪ1 a
iϭ1

and
Sϭ

1 nϪ1
Si, iϩ1

nϪ1 a
iϭ1

and the minimum RS [8],
RS, min Ն x

or max RS, min

(11)

The minimum of a is used as a criterion of the quality
of chromatograms in liquid chromatography [12].
Other criteria are the total peak overlap, w [13],
w ϭ a exp1Ϫ2RS 2,

(12)

the informing power, Pinf [14],
n

Pinf ϭ a log2 Si

(13)

iϭ1

and the chromatographic response function, CRF [10],
n

CRF ϭ a ln Pi

(14)

iϭ1

(where Pi is the peak-to-valley ratio for the ith pair of
chromatographic bands).
Performance of the Chromatographic System
One measure of the performance of a given chromatographic system is the number of the theoretical plates
per chromatographic band (N). In its simplest form,
this can be defined as
Nϭ

Fig. 1 Graphical interpretation of the selected elementary criteria: (a) resolution, Rs ; (b) the peak-to-valley ratio, P; (c) the
fractional peak overlap, FO.

l
H

(15)

where l is the distance from the origin to the eluent
front and H is the height equivalent to one theoretical
plate (H is sometimes also denoted HETP).
The average height equivalent to one theoretical
plate (H ) can be calculated from the relationship [15]


Optimization of Thin-Layer Chromatography
Hϭ

1si 2 2

1zr Ϫ z0 2RF

ϭ

1wi 2 2
16zx

3
(16)

where si is the standard deviation, which characterizes
the width of the chromatographic spot, or the band
width on the densitogram, wi is the spot width (or the
width of the peak base on the densitogram), zr Ϫ z0 is
the distance from the origin to the eluent front, and zx
is the distance from the origin to the geometric center
of the chromatographic spot.
The relationship between the height equivalent to
one theoretical plate (H) and the velocity of the mobile-phase flow is given by the simplified van Deemter
equation [16]
HϭAϩ

B
ϩ Cu
u

(17)

where u is the linear velocity of the mobile phase, A is
a constant characterizing eddy diffusion, B is a constant
characterizing molecular diffusion, and C is a constant
characterizing resistance to interphase mass transfer.
This particular issue is of considerable significance in
planar chromatographic separations, during the course
of which the velocity of the mobile phase changes.
The concept of separation number (SN) in planar
chromatography is a practical approach to the task of
quantification of chromatographic system performance. According to this concept, such performance
can simply be evaluated by calculating how many components of the separated mixture can be comfortably
accommodated (i.e., without any overlap of adjacent
components) along the direction of migration of the
eluent. A convenient relationship proposed in Ref. 17
enables easy calculation of the numerical value of SN:
SN ϭ a log

Simultaneous Strategy
In this strategy, one must accomplish all the experiments according to a plan devised earlier. All the results
obtained must then be carefully evaluated, the optimum
experimental conditions being chosen on this basis.
Sequential Strategy
In this strategy, the optimum experimental conditions
are approached in a series of consecutive steps. The
choice of any step results strictly from the outcome of
all those accomplished previously. One example of a
relevant algorithm is the simplex method [18]; the
PRISMA [19] geometrical method is a suitable example of the overall optimization approach.
Interpretative Strategy

This method enables prediction of the quality of a separation on the basis of a relatively limited number of the
experimental data, collected in previous experiments.
According to this approach, the chromatographic results are interpreted in terms of the retention functions,
valid for each individual solute separately. Some good
examples of the interpretative strategy are the so-called
“window diagrams” approach [20] and the search for
the extremum of the multiparameter response function
with the aid of the genetical algorithm [21].
References
1.

b0
1 Ϫ b1 ϩ b0 Ϫ1
b a log
b ,
b1
1 ϩ b1 Ϫ b0

or, simplified,
1
SN Ϸ
b0 ϩ b1

2.

(18)

where b0 is the width at half-height of a spot at the origin and b1 is the width at half-height of a spot at RF ϭ
1 (extrapolated) (b0 and b1 are in RF units).
Semiempirical Optimization Strategies

Strategies used for optimization of selectivity can basically be divided into three separate groups: (a) the simultaneous strategy, (b) the sequential strategy, and
(c) the interpretative strategy.

3.
4.

5.
6.
7.
8.

T. Kowalska, Theory and mechanism of thin-layer chromatography, in Handbook of Thin-Layer Chromatography (J. Sherma and B. Fried, eds.), Chromatographic
Science Series. Vol. 55, Marcel Dekker, Inc., New York,
1991, p. 50.
P. Jones and C. A. Wellington, J. Chromatogr. 213:
357–361 (1981).
R. Kaiser, Gas-Chromatographie, Geest und Portig,
Leipzig, 1960.
P. J. Schoenmakers, Optimization of Chromatographic
Selectivity. A Guide to Method Development, Journal of
Chromatography Library, Vol. 35, Elsevier, Amsterdam, 1986, pp. 123 –125.
W. Prus and T. Kowalska, J. Planar Chromatogr. 8:
205 –215 (1995).
J. C. Berridge, J. Chromatogr. 244: 1–14 (1982).
J. L. Glajch, J. J. Kirkland, K. M. Squire, and J. M. Minor, J. Chromatogr. 199: 57–79 (1980).
P. J. Schoenmakers, A. C. J. H. Drouen, H. A. H. Billiet,
and L. de Galan, Chromatographia 15: 688 – 696 (1982).


4

9.
10.
11.
12.
13.
14.
15.

Optimization of Thin-Layer Chromatography
R. Smits, C. Vanroelen, and D. L. Massart, Fresenius
Zeitschr. Anal. Chem. 273: 1–5 (1975).
S. L. Morgan and S. N. Deming, J. Chromatogr. 112:
267–285 (1975).
A. C. J. H. Drouen, P. J. Schoenmakers, H. A. H. Billiet,
and L. de Galan, Chromatographia 16: 48 –52 (1982).
S. N. Deming and M. L. H. Turoff, Anal. Chem. 50:
546 –548 (1978).
J. C. Giddings, Anal. Chem. 32: 1707–1711 (1960).
D. L. Massart and R. Smits, Anal. Chem. 46: 283 –286
(1974).
G. Guiochon and A. M. Siouffi, J. Chromatogr. 245:
1–20 (1982).

16.

J. J. Van Deemter, F. J. Zuiderweg, and A. Klinkenberg,
Chem. Eng. Sci. 5: 271 (1965).
17. F. Geiss, Fundamentals of TLC (Planar Chromatography), Hüthig, Heidelberg, 1987.
18. W. Spendley, G. R. Hext, and F. R. Hinsworth, Technometrics 4: 441 (1962).
19. Sz. Nyiredy, B. Meier, C. A. J. Erdelmeier, and

O. Sticher, J. High Resolut. Chromatogr. Chromatogr.
Commun. 8: 186 –188 (1985).
20. R. J. Laub and J. H. Purnell, J. Chromatogr. 112: 71–79
(1975).
21. J. H. Holland, Adaptation in Natural and Artificial Systems, University of Michigan Press, Ann Arbor, 1975.


Organic Acids, Analysis by Thin Layer Chromatography
Natasˇa Brajenovic´
Rud¯ er Bosˇkovic´ Institute, Zagreb, Croatia

INTRODUCTION

GENERAL CONSIDERATIONS

A very large number of papers report new research works
on analyses of organic acids by thin layer chromatography, paper chromatography, high-performance liquid
chromatography, and other analytical techniques. One of
the most popular and widely used separation techniques
for qualitative and quantitative analyses in the laboratory
is thin layer chromatography.
The reason for using thin layer chromatography is its
wide applicability to a great number of different types of
samples, high sensitivity, and speed of separation with
relatively low cost. This technique is very fast, and many
separations can be accomplished in less than an hour.
The development of different methods of analysis in
thin layer chromatography is a very important area of
organic chemistry and biochemistry. Analysis of organic
acids by thin layer chromatography is widely applied in

different fields of environmental, pharmaceutical, industry, industrial foods, organic chemistry, cosmetics,
clinical, and biochemical assays.

Stationary Phase

OVERVIEW

Mobile Phase

Here, we present scientific activity in the analysis of
organic acids by thin layer chromatography in a period
from 1993 to 2004. The review is based on a search of
Current Contents and Science Citation Index, using
different combinations of key words relevant for thin
layer chromatography, organic acids, and different kinds
of organic acids, such as amino acids, carboxylic acids,
humic acids, aromatic carboxylic acids, and fatty acids. In
addition, the journals publishing papers covering specific
topics related to the analysis of organic acids by thin layer
chromatography were searched directly: Analytica Chimica Acta, Analytical Chemistry, Journal of Microbiological Methods, Journal of Liquid Chromatography and
Related Technologies, Journal of Chromatography (Parts
A and B), Chromatographia, and Journal of Pharmaceutical and Biochemical Analysis.

Many different solvents and mixtures of solvents are used
as mobile phases for the analysis of organic acids by thin
layer chromatography, such as chloroform, ethyl acetate,
methanol, benzene, etc.

Encyclopedia of Chromatography
DOI: 10.1081/E-ECHR-120041125

Copyright D 2004 by Marcel Dekker, Inc. All rights reserved.

Typical thin layer separations are performed on flat glass or
plastic plates that are coated with a thin and adherent layer
of particles, which constitute the stationary phase.[1] Commercial plates come in two categories: conventional [thick
layers (200–250 mm) of particles having sizes of 20 mm or
greater] and high-performance plates (film thickness of
100 mm and particles whose diameters are 5 mm or less).
Silica gel is the most extensively used adsorbent in thin
layer chromatography because it leads to excellent, uncomplicated separations. It can be successfully employed
for both qualitative and quantitative thin layer chromatographic analyses. It is usually used as a stationary phase
in separations and analysis of alkaloids, various organic
acids (especially amino acids and their derivatives), steroids, lipids, vitamins, plant pigments, pesticides, drugs,
carbohydrates, phenolic substances, etc.
Besides silica gel, cellulose, and aluminum oxide,
various other impregnated plates are also frequently used
as stationary phases.[1]

Identification
For identification in qualitative thin layer chromatography,
a great number of visualization reagents are used.[1] If
compounds are not colored, a UV lamp may be used to
visualize the plates. The quantitative determination of sample
components is performed according to the following:
1. Extracting the stained spot with solvent and analyzing
it spectrophotometrically; or
2. Scanning the plate densitometrically.[1]

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Organic Acids, Analysis by Thin Layer Chromatography

APPLICATIONS

Table 2 Rf values (Â100) of urinary phenolic acids
BzAca

Pharmaceutical Industry, Medicine,
Biochemistry, and Biology

Compound

Thin layer chromatography is used for the analysis of free
amino acids from sanguine plasma in different progression states in maladies: diabetes, renal syndrome, and
hepatic cirrhosis.[2] Elution was performed on cellulose
plates and the densitometry was achieved with a photodensitometer (Shimadzu CS-9000) at 575 nm. In the case
of hepatic cirrhosis, a better resolution was obtained. A
mixture of n-butanol–acetone–acetic acid–water (35:35:
7:23 vol/vol/vol/vol) was used as the mobile phase.
A simple and fast method for identification of
bifidobacteria using thin layer chromatographic analysis
of short chain fatty acids in a culture broth is proposed
(Table 1).[3] This approach has many advantages: the

total time required to analyze organic acids is approximately 50 min; and the identification protocol is simpler,
quicker, and more economical than conventional identification methods.
Mycolic acids analysis by thin layer chromatography
has been employed by several laboratories worldwide as a
method for fast identification of Mycobacterium.[4]
Mycobacterium tuberculosis strains identified by classical
methods were confirmed by their mycolic acid content.
Using aminopropyl-modified silica gel plates in a
normal phase system, the retention behavior of 12 acidic
drugs and biologically active aromatic acids was investigated by high-performance thin layer chromatography.[5]
The metabolism of aromatic amino acids (phenylalanine and tyrosine) can be studied following the excretion
of their characteristic phenolic acid metabolites in urine
using chromatographic methods. These apply acids to the
investigations of amino acids themselves in diagnostics.

Table 1 Retention time (Rf) and detection color of standard
organic acids
Organic acids
Lactic acid
Acetic acid
Propionic acid
Butyric acid
Succinic acid
Citric acid
Source: Ref. [3].

Rf value

Methyl red +
bromophenol blue

0.53 (Upper spot)
0.37 (Lower spot)
0.44
0.54
0.61
0.12
0.03
0.52

Red
Red
Blue
Blue
Blue
Yellow
Dark yellow
Red

Phenyllactic acid
Phenylpyruvic acid
p-OH cinnamic acid
Hippuric acid
p-OH mandelic acid
p-OH
phenylacetic acid
Vanillic acid

IprBuAmb

BuEc

Celd

SGe

Celd

SGe

SGe

88
86
83
74
11
63

68
68
60
30
6
61

65
80
36
59

28
39

62
80
52
57
40
43

—
87
88
35
34
81

96

77

19

36

84

a

BzAc = benzene–acetic acid (glacial)–water (70:29:1).

IprBuAm = isopropanol–n-butanol–t-butanol–ammonia–water (40:20:
20:10:20).
c
BuE = n-butanol–ethanol–water (100:5:10).
d
Cellulose.
e
Silica gel.
Source: Ref. [6].
b

Thin layers of cellulose or silica gel on aluminum foil
were used as stationary phases.[6] The retention factor (Rf)
of clinically important compounds in the three solvent
systems is presented in Table 2.
Thin layer chromatography is also used for direct
enantiomeric resolution of D,L-arginine, D,L-histidine, D,Llysine, D,L-valine, and D,L-leucine on silica gel plates
impregnated with optically pure (1R, 3R, 5R)-2-azabicyclo[3,3,0]octan-3-carboxylic acid, which serves as a chiral
selector in the pharmaceutical industry.[7] To successfully
resolve D,L-amino acids, various combinations of acetonitrile–methanol–water were proposed. The spot was
detected by ninhydrin (0.2% in acetone).
Thin layer chromatography is often applied as an
industrial control procedure in the synthesis of 2-hydroxy3-naphthalenecarboxylic acids, on silica gel plates with
chloroform–methanol–acetic acid (50:20:1) as developer.[8]
2-Hydroxy-3-naphthalenecarboxylic acid is an important
intermediate in the synthesis of dyestuffs and drugs.
Separation of amino acids and their identification in
different mixtures are frequent tasks encountered in
biochemistry. Thin layer chromatography is a fast, simple,
and inexpensive approach to attain this goal. Because

some of the components are UV-inactive, other methods,
such as vibrational spectroscopy, should be applied for
detection and identification. Comparative study based on
Raman spectroscopy of thin layer chromatography spots
of some weak Raman scatterers (essential amino acids)
was carried out using four different visible and nearinfrared laser radiation wavelengths: 532, 633,785, and
1064 nm.[9] The best results were obtained with simple
silica gel plates.


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Table 3 Thin layer chromatographic procedures for separation of organic acids
Standards/simple

Stationary phase

Mixture, organic acids

Mobile phase

Merck silica gel
Cellulose powder or silica gel

2-Hydroxybenzoic acid (salicylic acid)

Silica gel or Fe(III)-impregnated
silica gel or aluminum oxide
Silica gel or Fe(III)-impregnated
silica gel or aluminum oxide
Silica gel or Fe(III)-impregnated
silica gel or aluminum oxide
Silica gel or Fe(III)-impregnated
silica gel or aluminum oxide
Silica gel or Fe(III)-impregnated
silica gel or aluminum oxide
Silica gel or Fe(III)-impregnated
silica gel or aluminum oxide

4-Hydroxybenzoic acid
3,4,5-Trimethoxybenzoic acid
3,4,5-Trihydroxybenzoic acid (gallic acid)
4-Hydroxy-3,5-dimethoxybenzoic acid
1,2-Benzenedicarboxylic acid

n-Butylformate/90% formic acids/H2O
(7:2:1, vol/vol)
1-Propanol concentrated in ammonium
hydroxide (7:3 or 3:2)
Tap water
Tap water
Tap water
Tap water
Tap water

Tap water

Source: Refs. [11], [14], and [15].

Environmental, Water, Plant,
and Soil Applications

metals also react with organic acids and have a harmful
influence on the environment.
Many plants contain a variety of free acids such as
acetic acid, citric acid, fumaric acid, malic acid, succinic
acid, oxalic acid, glycolic acid, etc.[11–13] They are components of citric cycle, whereas the others are intermediates in the pathway from carbohydrates to aromatic compounds.[11] Following extraction, organic acids can be
separated and detected with a variety of techniques. Thin
layer chromatographic methods have been also employed
to separate certain organic acids,[11,14,15] as presented in
Table 3.
Thin layer chromatography of some plant phenolics,
which play an important role in plant metabolism, was

The occurrence of chlorinated organic compounds in fish
from polluted waters is rather frequent.[10] Chlorinated
carboxylic acids of fatty acid character have also been
shown to account for up to 90% of the extractable
organically bound chloride (EOCl) in fish. Purification by
thin layer chromatography of methyl esters of dichlorotetradecanoic, dichlorohexadecanoic, and dichlorooctadecanoic acids was used. They were detected at 1200 ppm of
EOCl in fish.
Organic acid complexes with metal ions significantly
affect the mobility of metal ions in plants and soils. Toxic

Table 4 Chromatographic behavior of some plant phenolic acids

Rf
Compound
o-Coumaric acid
m-Coumaric acid
p-Coumaric acid
o-Hydroxybenzoic acid (salicylic acid)
m-Hydroxybenzoic acid
p-Hydroxybenzoic acid
Gallic acid

Detection

A

B

C

D

UV

DSA

DNA

0.34
0.43
0.34
0.70

0.37
0.27
0.09

0.39
0.26
0.33
0.57
0.63
0.59
0.33

0.29
0.33
0.28
0.44
0.58
0.52
0.31

0.51
0.52
0.47
0.62
0.70
0.70
0.46

F
F

Q
F
F
Q
F

Orange
Orange
Red
Yellow
Yellow
Yellow
Green-brown

Violet
Violet
Blue
Red
Rose
Red
Tan

F = fluorescence; Q = quenching; DSA = diazotized sulfanilic acid; DNA = diazotized p-nitroaniline.
Developers: A = 2% formic acid; B = 20% potassium chloride; C = isopropyl alcohol–ammonium hydroxide–water (8:1:1); D = 10% acetic acid.
Source: Ref. [12].


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Organic Acids, Analysis by Thin Layer Chromatography

Fig. 1 Chemical structure of simple phenolics and some components of lignin. (From Refs. [13] and [14].)

carried out on cellulose.[12] The solvents were: 2% formic
acid, 20% potassium chloride, isopropyl alcohol–ammonium hydroxide–water (8:1:1), and 10% acetic acid. The
plates were examined under UV light after development.
The results of the chromatographic analysis of some
phenolic acids are shown in Table 4.
The composition of lignin (an important component of
plant cell walls) is very complex.[13] p-Coumaric acid,
cinnamic acid, ferulic acid, and others are among the
products of lignin biodegradation (Fig. 1). Thin layer
chromatography is a very rapid method for their
separation and is usually completed in a very short time.
Six different solvents were used as developer (Table 5),
with silica gel being used as a stationary phase.
Humic acids are also products of lignin biodegradation.
The characterization of humic acids by thin layer
chromatography on Fe(III)-impregnated silica gel with
tap water as developer has been presented.[14] During the
chromatographic process, complexes between Fe(III)
from the support group and the active functional group
from humic acid are formed, causing successive attaching
and detaching of Fe(III) from the support of Fe(III)
hydroxy/oxide. These results could partially answer
how the process of metal migration in soils and sediments progresses.

Some carboxy and benzene derivatives related to
humic materials were also examined by thin layer

chromatography.[15] Aluminum oxide, silica gel, and
Fe(III)-impregnated silica gel plates were used as
supports, whereas the mobile phase was water. The results
are presented in Table 3. It was concluded that the
hydroxy/oxide layer of iron and aluminum can affect the
mobility of simple organic molecules in soil. On the other
side, organic molecules having carboxy and hydroxy
groups can improve the dissolution of hydroxy/oxides of
iron or aluminum in soils and sediments.

Table 5 Different solvent systems were used as developers for
thin layer chromatographic separation for simple phenolic and
related compounds as cinnamic acid, p-coumaric acid, ferulic
acid, and tannic acid
Solvent systems were used as developers for simple
phenolic compounds and some acids, which are products
of lignin biodegradation
1) Chloroform–methanol–acetic acid (90:10:1)
2) Petroleum ether (60–80°C)–ethyl acetate–formic acid
(40:60:1)
3) Benzene–dioxane–acetic acid (85:15:1)
4) Chloroform–ethyl acetate–acetic acid (50:50:1)
5) Toluene–acetonitrile–formic acid (70:30:1)
6) Petroleum ether (60–80°C)–methanol–acetic acid (90:10:1)
Source: Ref. [13].

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Food Analysis, Agriculture, and Industry
There are several acids that are widely used in industries.
For example, synthetic carboxylic acid esters are used in the
perfume industry. Benzoic acids are used as sodium salts in
the food industry as inhibitors of microorganism growth.[16]
Benzoic acid derivatives often contain amino, hydroxy,
carboxy, and nitro groups. Analysis of substituted benzoic
acids by thin layer chromatography was performed on silica
gel, polyamide, and cellulose containing UF254 fluorescent
indicator.[17] For the mobile phase, different mixtures were
used: hexane–acetic acid; hexane–ethyl acetate–formic acid;
chloroform–methanol–phosphoric acid; cyclohexane–acetic
acid; benzene–ethanol; etc. Because benzoic acid derivatives have similar retention parameters, their separation
requires a thorough optimization of conditions (the nature of
the stationary phase, the composition of the mobile phase,
and the pH of the solutions).
For the separation of benzoic acids, planar electrochromatography was used.[18] In this approach, an
electroosmotic flow is used to drive the mobile phase
in thin layer chromatography. Planar electrochromatography has several advantages over classical thin layer
chromatography, especially substantially faster separation. For example, separation by planar electrochromatography can be 10 times faster than that using ordinary
thin layer chromatography.
Thin layer chromatographic analysis of agricultural
products, foods, beverages, and plant constituents is
described by Sherma[19] in a review paper. In laboratories

throughout the world, thin layer chromatography is widely
used for food analysis and qualitative control. Numerous
applications of thin layer chromatography have been
reported in the area of food composition, involving
determinations of compounds such as lipids, sugars,
amines, vitamins, and organic acids such as amino acids
and fatty acids.

VARIOUS ORGANIC COMPOUNDS
Thin layer chromatographic analysis is also highly
applicable to the determination of aromatic organic
acids.[20] In human organisms, aromatic acids are synthesized as metabolites in intoxication by toluene, xylene,
and ethyl benzene.[16] These compounds are easily absorbed through the skin or respiratory system, and are
oxidized to aromatic acids. The separation, identification, and quantitative analyses of aromatic acids are also
necessary because they appear as semiproducts of the
biosynthesis of aromatic amino acids in plants (phenolic
acids), and metabolites of numerous toxic substances,
drugs, and catecholamines. Polar adsorbents and polar-

5

bonded stationary phases are also widely used in
carboxylic separation by thin layer chromatography, often
coupled with densitometry.
Dansyl chloride (DNS-Cl; 5-dimethylaminonaphthalene-1-sulfonyl chloride) is used in analytical chemistry to
fluorescently label substances. This process of dansylation
creates fluorescent derivatives, which can be detected
with great sensitivity. The method for the dansylation of
hydroxyl (–OH) and carboxylic acid (–COOH) functional
groups has been described.[21] Fluorescent labeling by

dansyl chloride has applications in liquid chromatography, high-performance liquid chromatography, thin layer
chromatography, and mass spectrometry. Fast thin layer
chromatography was accomplished using acetone as
the resolving solvent, and resulted in good differentiation
of analytes.
Many thin layer chromatography systems for the
separation of amino acids have been described.[22–24]
Copper sulphate and polyamide were tried as impregnants for improving the separation of 20 amino acids on
silica gel layers. MeOH–BuOAc–AcOH–pyridine (20:20:
10:5) was used as the solvent system.[22]
D-enantiomers of amino acids have been frequently
reported in various tissues of diverse organisms. A simple
and rapid method of separating optical isomers of
amino acids on a reverse-phase thin layer chromatography plate is described.[23] Amino acids, derivatized with
1-fluoro-2,4-dinitrophenyl-5-L-alanine amide, were spotted onto a reverse-phase thin layer chromatography plate.
Acetonitrile in triethylamine–phosphate buffer was used
as the developer.
For the evaluation of protein structure, identification of
amino acids is extremely important. Thin layer chromatography is an appropriate method in this field. A
variety of spray reagents are used, among which ninhydrin is the most popular one due to its high sensitivity.[24]
Ninhydrin produces a purple/violet color with most amino
acids. A typical experimental setup includes chromatographic plates prepared from silica gel; n-propanol–water
(70:30) mixture was used as a mobile phase. For complex mixtures of substances, two-dimensional chromatography is preferred, using n-propanol–water (70:30)
mixture and methanol–chloroform (3:1) mixture as the
two developers.

CONCLUSION
Thin layer chromatography is a very widely used chromatographic technique in research activities of analytical
chemists in many laboratories in the world. Many articles
dealing with the development of new analytical methods

for the analysis and separation of different organic acids in


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Organic Acids, Analysis by Thin Layer Chromatography

various fields have been published. There is a constant
need for qualitative and quantitative analyses of organic
acids in the pharmaceutical, cosmetic, and food industries;
in medicine, biology, organic chemistry, and biochemical
analysis; and in environmental studies. The important
reasons for frequent applications of thin layer chromatography in qualitative and quantitative analyses of organic
acids are its high sensitivity, fast separation of components, and relatively low cost.

REFERENCES
1. Skoog, D.A.; Holler, J.F.; Nieman, T.A. High-Performance
Liquid Chromatography. In Principles of Instrumental
Analysis, 5th Ed.; Vondeling, J., Sherman, M., Bortel, J.,
Messina, F., Pecuilis, V., Eds.; M. Brooks/Cole, Thomson
Learning, Inc.: London, 1998; 725 – 766.
2. Gocan, S.; Ghizdavu, L.; Ghizdavu, L. TLC of some free
amino acids from sanguine plasma. J. Pharm. Biomed.
Anal. 2001, 26, 681 – 685.
3. Ki-Yong, L.; Jae-Seong, S.; Tae-Ryeon, H. Thin layer
chromatographic determination of organic acids for rapid

identification of bifidobacteria at genus level. J. Microbiol.
Methods 2001, 45, 1 – 6.
4. Leite, C.Q.F.; Desouza, C.W.O.; Leite, S.R.D. Identification of mycobacteria by thin layer chromatographic
analysis of mycolic acids and conventional biochemical
method—Four years of experience. Mem. Inst. Oswaldo
Cruz 1998, 93 (6), 801 – 805.
5. Bieganovska, M.L. Retention behaviour of some acids
drugs and biologically active compounds on silica and
aminopropyl silica layers. Chem. Anal. 1995, 40 (6), 859 –
867.
6. Ersser, R.S.; Oakley, S.E.; Seakins, J.W.T. Urinary
phenolic acids by thin-layer chromatography. Clin. Chim.
Acta 1970, 30, 243 – 249.
7. Bhushan, R.; Martens, J.; Thuku Thiongo, G. Direct thin
layer chromatography enantioresolution of some basic
DL-amino acids using a pharmaceutical industry waste as
chiral impregnating reagent. J. Pharm. Biomed. Anal.
2000, 21, 1143 – 1147.
8. Revilla, A.L.; Havel, J.; Borovcova´, J.; Vrchlabsky, M.
Capillary zone electrophoresis of hydroxynaphthalenecarboxylic acids. Purity monitoring of b-hydroxynaphthoic
acid in industry. J. Chromatogr., A 1997, 772, 397 – 402.
9. Istva´n, K.; Keresztury, G.; Sze´p, A. Normal Raman and
surface enhanced Raman spectroscopic experiments with
thin layer chromatography spots of essential amino acids
using different laser excitation sources. Spectrochim. Acta,
Part A: Mol. Biomol. Spectrosc. 2003, 59, 1709 – 1723.

10.

Mu, H.; Wese´n, C.; Nova´k, T.; Sundin, P.; Skramstad, J.;

Odham, G. Enrichment of chlorinated fatty acids in fish
lipids prior to analysis by capillary gas chromatography
with electrolytic conductivity detection and mass spectrometry. J. Chromatogr., A 1996, 731, 225 – 236.
11. Dashek, W.V; Micales, J.A. Isolation, Separation, and
Characterization of Organic Acids. In Methods in Plant
Biochemistry and Molecular Biology; Dashek, W.V., Ed.;
CRC Press: Boca Raton, FL, 1997; 107 – 113.
12. Jangaard, N.O. Thin-layer chromatography of some plant
phenolics. J. Chromatogr. 1970, 50, 146 – 148.
13. Sharma, O.P.; Bhat, T.K.; Singh, B. Thin-layer chromatography of gallic acid, methyl gallate, pyrogallol,
phloroglucinol, catechol, resorcinol, hydroquinone, catechin, epicatechin, cinnamic acid, p-coumaric acid, ferulic
acid and tannic acid. J. Chromatogr., A 1998, 822, 167 –
171.
14. Iskric´, S.; Hadzˇija, O.; Kveder, S. Behaviour of humic
acids on Fe(III)-impregnated silica gel compared with
model substances. J. Liq. Chromatogr. 1994, 17 (7), 1653 –
1657.
15. Kveder, S.; Iskrc´, S.; Zambeli, N.; Hadzˇija, O. The
behaviour of some benzene derivatives on thin layers of
aluminum oxide—Comparison with plain and Fe(III)
impregnated silica gel. J. Liq. Chromatogr. 1991, 14
(18), 3277 – 3282.
16. Waksmundzka-Hajnos, M. Chromatographic separation of
aromatic carboxylic acids. J. Chromatogr., B 1998, 717,
93 – 118.
17. Sumina, E.G.; Shtykov, S.N.; Dorofeeva, S.V. Ion-pair
reversed-phase thin-layer chromatography and high-performance liquid chromatography of benzoic acids. J. Anal.
Chem. 2002, 57 (3), 210 – 214.
18. Nurok, D.; Koers, J.M.; Carmichael, A. Role of buffer
concentration and applied voltage in obtaining a good

separation in planar electrochromatography. J. Chromatogr., A 2003, 983, 247 – 253.
19. Sherma, J. Thin-layer chromatography in food and agricultural analysis. J. Chromatogr., A 2000, 880, 129 – 147.
20. Sherma, J. Planar chromatography. Anal. Chem. 1992, 64,
134R – 147R.
21. Bartzatt, R. Dansylation of hydroxyl and carboxylic acid
functional groups. J. Biochem. Biophys. Methods 2001,
47, 189 – 195.
22. Srivastava, S.P.; Bhushan, R.; Chauhan, R.S. TLC
separations of amino acids on silica gel impregnated
layers. J. Liq. Chromatogr. 1984, 7 (7), 1359 – 1365.
23. Nagata, Y.; Iida, T.; Sakai, M. Enantiomeric resolution of
amino acids by thin-layer chromatography. J. Mol. Catal.,
B Enzym. 2001, 12, 105 – 108.
24. Laskar, S.; Sinhababu, A.; Hazra, K.M. A modified spray
reagent for the detection of amino acids on thin layer
chromatography plates. Amino Acids 2001, 21, 201 – 204.


Organic Extractables from Packaging Materials:
Chromatographic Methods Used for Identification
and Quantification
Dennis Jenke
Baxter Healthcare Corporation, Round Lake, Illinois, U.S.A.

INTRODUCTION
Plastic materials are widely used in medical items, such
as solution containers, transfusion sets, transfer tubing,
devices, and packaging systems. The physiochemical nature of these materials provides medical products with
their necessary, desirable performance characteristics.
While an important performance characteristic of plastics used in medical application is chemical inertness,

interactions between a plastic material and a contacted
pharmaceutical product are well documented. Such interactions may include sorption, the uptake of product
components by the plastic material, or leaching, i.e., the
release of plastic material components into the product. In
the case of leaching, both the identities of the leached
substances and their accumulation levels may impact the
ultimate utility of the product.
A review, related to the chromatographic methods used
to assess the accumulation of leachable substances from
packaging materials used for pharmaceutical products, is
provided. This considers methods used to identify and/or
quantify such leachables in actual products or productsimulating solvent systems.

DISCUSSION
The assessment of the impact of the accumulation of
leached substances in pharmaceutical products contacted
by a plastic material during their manufacture, storage,
and/or use is a multifaceted undertaking involving disciplines within the applied physical, chemical, and biological sciences. While numerous strategies can be envisioned, and have been utilized to perform such an
assessment, considerations include the identification of
the leached substances and the measurement of the actual
or probable accumulation levels of the identified substances. The identification process is an extensive investigation that utilizes sensitive and information-rich
scouting of analytical methods for the dual purposes of
Encyclopedia of Chromatography
DOI: 10.1081/E-ECHR 120018659
Copyright D 2003 by Marcel Dekker, Inc. All rights reserved.

first revealing the leachables and then providing relevant information (e.g., formula and structure) that leads
to their identification. In the worst-case scenario, such
an analytical investigation is conducted blind; that is,
the analytical team is faced with the unenviable challenge of finding an unknown number of unknown compounds, many of which accumulate in the product at

levels much lower than its other constituents. These constituents may include both additives and nonmaterialrelated contaminants such as ingredient impurities and
degradation products. This search for material-derived
leachables in pharmaceutical products is greatly facilitated if it is conducted with information-rich analytical
methodologies that exhibit a comprehensive ability to
respond to a large population of analytes in both a universal, but very specific, manner. The dual performance
requirements of universality and specificity are the primary reasons why chromatographic methods are almost
exclusively used in investigations specifically associated
with organic leachables.
Given the variety of packaging materials used in
pharmaceutical applications, the population of potential
primary and secondary organic leachables is large and
compositionally diverse. While an analytical chemist has
a multitude of chromatographic tools with which to perform a leachables assessment, some guidance in terms of
successfully applied strategies and methods can greatly
facilitate the assessment. Thus this article contains a general compilation of published chromatographic methods
and strategies that have been successfully applied to the
identification and quantification of packaging material
leachables. Examples are provided for each major separation strategy [e.g., high-performance liquid chromatography (HPLC), gas chromatography (GC), thin-layer
chromatography (TLC), and supercritical fluid chromatography (SFC)] and for most commonly employed detection methods [e.g., ultraviolet (UV), mass spectrometry
(MS), and flame ionization detection (FID)]. While the
compilation in Tables 1 –4 is by no means exhaustive, it is
sufficiently broad in scope to provide the investigator
with a general overview of the ways in which chromato1


Organic solvents

Oils

Oils

Solvent extracts
(hexane, ethyl
acetate, diethyl
ether)
Dissolution with
mobile phase

PVC, PE,
PS

PET

PET

PP

Laminated
polyolefin

Drug product

Organic solvents

PVC, PE,
PS

HDPE

Saline extracts

Sample matrix

PVC

Material

280

MEOH/water (70:30)

SPE concentration,
residue
reconstituted in
ethanol

20 mL

20 mL,
1 mL/min
280

Spherisorb
C18 ODS-2,
150 Â 4.6 mm,
3 mm
LiChrosorb
C18 ODS-2,
250 Â 4.6 mm,
5 mm

MEOH/water/AA
(66.5:32.5:1)

Direct injection

35°C,
2 mL/min

271

15 – 40% ethyl acetate
gradient in
75:25 MEOH/water

Spherisorb ODS-1,
50 Â 4.6 mm,
5 mm

20 mL,
1.5 mL/min

Solvent evaporation,
reconstituted in
chloroform/MEOH

254

A = water/ACN
(85:15),

B = ACN/water
(85:15)b

20 mL,
1 mL/min

20 mL,
1.5 mL/min

280

ACN/tetrahydrofuran
(THF) (95:5)

254

225

MEOH/phosphate
buffer, pH 5.5 (1:1)

20 mL,
0.8 or
0.17 mL/min
20 mL,
1 mL/min

Other

A = water/MEOH/AA

(85:15:0.25),
B = ACN/water
(85:15)c

270

L (nm)

ACN/MeOH (9:1)

Mobile phase

Microsorb C8,
250 Â 4.6 mm,
5 mm

Shandon ODS C18,
200 Â 4.6 mm,
200 Â 2.1 mm
Spherisorb
C18 ODS-2,
250 Â 4.6 mm,
5 mm
Spherisorb
C18 ODS-2,
250 Â 4.6 mm,
5 mm
Microsorb C8,
250 Â 4.6 mm,
5 mm

Column

Solvent partitioning
(ACN/hexane) with
evaporative
concentration

Solvent partitioning
(ACN/hexane)
with evaporative
concentration

Direct injection

Direct injection

Extraction into
hexane

Sample preparation

Table 1 Examples of HPLC methods used to identify and/or quantify packaging system extractables

Caprolactam,
butylhydroxytoluene,
phthalic acid derivative

Antioxidants
[Butylated hydroxy toluene

(BHT), Irganox 1010,
Irganox 1076]
PET oligomers
(trimer-octamer),
plasticizers
(diethylene glycol
dibenzoate)
PET oligomers,
terephthalic acid,
dimethylterephthalate,
bis(2-hydroxyethyl)
terephthalate
Antioxidants
(BHT, Irganox 1010,
Irganox 1076,
Irgafos 168)
Phenolic antioxidantsd;
propyl p-hydroxybenzoate

Monomers, caprolactam,
aminocaproic acid

Di-ethylhexyl-phthalate

Extractables

7

6a

5a

4a

3

2

2

1a

Ref.

2
Organic Extractables from Packaging Materials: Chromatographic Methods Used for Identification and Quantification


Water or ethanol
extracts

Methylene chloride
extract

Solvent extraction

Pharmaceutical
products

Pharmaceutical

products

Leaching of
administration
sets

Extraction with
water, 50°C,
3 days

Filter
cartridges

PC

PET, PVC

Polyolefin

PVC

PVC

Adhesives

Econosphere C18,
150 Â 4.6 mm,
5 mm
NovaPak C18,
150 Â 2.1 mm

Direct injection

Nucleosil ODS,
200 Â 4.6 mm,
5 mm

Spherisorb LC-SI,
250 Â 4.6 mm,
5 mm

Shandon Hypercarb
S, 150 Â 4.6 mm,
7 mmg
Spherisorb ODS-2g

Nucleosil 100-5,
RP18, 250 cm

Direct injection

Direct injection after
polymer ppt with
methanol
Solvent evaporation
reconstituted
in IPA
Extract with
chloroform,
reconstituted in

2-propanol
Direct injection

Direct injection or
after evaporative
concentration

1 mL/min,
50 mL,
35°C
0.25 mL/min

254

220k

A = 1% acetic acid
B = ACN
50% B for 3.5 min,
change to 65% B
40-min gradient from
5 – 85% ACN in
0.1% acetic acid

1 mL

j

11

10

9

8f

Phthalic acid, phenol
12
Cyclohexanone
Phthalide, benzoic acid
Benzaldehyde, bispenol A
Butyl hydroxyanisol
Mono-(2-ethylhexyl)
phthalate
Dibutyl phthalate
Di(2-ethylhexyl)
phthalate
13a
Phthalide
Monobutyl phthalate
Mono-(ethylhexyl)
phthalate
14
1,3-Benzenedicarboxylic
acid, mono(7,12dioxo-3,6,13,16tetraoxaoctadec-17en-1-yl) ester
Hexanedioic acid,
2-[2-[(5-carboxy-1oxopentyl)oxy] ethoxy]
ethyl 2-(ethenyloxy)
ethyl ester
1,3-Benzenecarboxylic

acid, 2-[2-[(carboxy
benzoyl)oxy]ethoxy]
ethyl 2-(ethyloxy)ethyl
ester
(Continued )

Caprolactam

25 mL,
1.5 mL/min

ACN/water (pH 2.7)i

Erucamide,
PET oligomers

Phthalates, fatty acids,
phenols, siloxanes,
acrylates, aliphatics,
amidese
Bisphenol A

Various
flow rates

20 mL
0.5 mL/min

20 mL,
1 mL/min

210

254

h

220

n-hexane/2-propanol
(9:1)

Various binary mixtures

MEOH/water/ACN
(25.0:26.2:48.8)

MEOH/water
(90:10),
ACN/water (60:40)

Organic Extractables from Packaging Materials: Chromatographic Methods Used for Identification and Quantification
3


Extraction with
acetonitrile

Sample matrix

Direct injection
after filtration

Sample preparation
Waters Symmetry
C8, 150 Â 3.9 mm

Column
Start at 30:70 ACN/water,
to 100% ACN at 10 min,
to 30% ACN at 30 min,
hold at 30% ACN for
10 min

Mobile phase

Extractables
Naugard XL,
Irganox 1076
1-Octadecanol, NC-4
3-(3,5-Di-tert-butyl-4hydroxyphenyl)
propanoic acid
7,9-Di-tert-butyl-1oxaspiro-[4,5deca-6,
9-diene-2,8-dione

Other
0.4 mL/min,
10 mL

l (nm)

MS
EI
Atmospheric
pressure chemical
ionization (APCI)
UV

Examples of HPLC methods used to identify and/or quantify packaging system extractables (Continued )

Solvent abbreviations include MeOH = methanol; ACN = acetonitrile; AA = acetic acid.
Material abbreviations include PP = polypropylene; PVC = poly(vinyl chloride); HDPE = high-density polyethylene; PC = polycarbonate; PS = polystyrene; PET = polyethylene terephthalate.
a
Method performance data provided in this reference.
b
Gradient was as follows: 0.0 min, 70% A; 18.0 min, 0% A; 22.0 min, 0% A; 23.0 min, 70% A.
c
Gradient was as follows: 0.0 min, 95% A; 8.0 min, 40% A; 16.0 min, 30% A; 17.0 min, 0% A; 21.0 min, 0%A; 22.0 min, 95% A.
d
Specific compounds detected included propyl-3,4,5 trihydroxy benzoate, 2-tert-butyl-4-methoxy phenol, 2-tert-butylphenol, 2-tert-butyl-4-methylphenol, and octyl-3,4,5-trihydroxybenzoate.
e
This cited reference documents numerous compounds in these and other general compound classes.
f
Fourier transform-infrared spectroscopy analysis of collected peaks used to confirm analyte identification.
g
Various column sizes used.
h
Fluorescence detection.
i
Gradient was as follows: 5% ACN for 2 min; 5 – 50% ACN in 28 min; 50 – 98% ACN in 15 min.
j

Multiple wavelengths are used.
k
Compound identification performed via MS and MS/MS using both APCI and electrospray ionization (ESI) in the positive ion mode.

PP

Material

Table 1

15

Ref.

4
Organic Extractables from Packaging Materials: Chromatographic Methods Used for Identification and Quantification


Sample matrix

Water extract,
121°C, 2 hr

Solvent extracts,
40°C, 10 days

Water extract

Products

Water or ethanol
extracts

Methylene
chloride extract

Pharmaceutical
products

Rubber

PET

PVC

PVC

Filter
cartridges

PC

PVC

Solid phase
extraction (SPE)
with evaporative
concentration
Direct injection
after polymer

ppt with
methanol
Solvent extraction,
evaporative
concentration

Acidification,
solvent
extraction,
evaporative
concentration

Evaporative
concentration

Evaporative
concentration

Evaporative
concentration

Sample preparation

2 mL, split,
T = 280°C
MS m/z 213,
T = 290°C

Restek RTx-5
FSOT,

30 m  0.25 mm,
1.0-mm film
3% QF-1 or 3%
SE-30
on Supelcoport

T = 230°C

1 mL, split,
T = 250°C

MS

60°C for 2 min,
to 280°C
at 10°C/min,
hold
100°C, to 280°C
at 10°C/min,
hold for 3 min

J&W DB-5,
60 m

MS EI + ,
T = 230°C

Cold on-column
injection

FID 350°C

32°C, 1.5 min;
to 75 °C
at 6°C/min;
to 250°C
at 30°C/min,
hold for 2 min
30°C for 0.5 min,
to 225°C at
6°C/min

J&W DB-5,
15 m  0.53 mm,
15 mm film

120°C for 1 min,
to 225°C at
4°C/min

3 mL (splitless),
T = 210°C

FID 350°

200 – 280°C at
8°C/min

HP1, 50 Â 0.25 mm

J&W DB-5,
30 m  0.32 mm,
0.25-mm film

Split injection
T = 250°C

FID 280°C

34°C for 1 min,
ramp at 6°C/min
to 200°C

Splitless injection,
T = 250°C

Other

DB-Wax-30N,
0.25 mm id

FID 325°Ca

Detection

30°C for 1 min,
ramp at 8°C/min
to 250°C

Oven program

Cross-linked
methyl silicone
25 m  0.3 mm id

Column

Examples of GC methods used to identify and/or quantify packaging system extractables

Material

Table 2

9,10-Epoxystearate
ester

20

9

8

19b

18

17

16

Ref.

(Continued )

Di(ethylhexyl) phthalate
Dibutyl phthalate
cyclohexanone,
phthalide
2-Ethyl-1-hexanol
2,6-Di-tert-butyl-p-cresol
Phthalates, fatty acids,
phenols, siloxanes,
acrylates, aliphatics,
amidesc
Bisphenol A

2-Butoxyethanol
Cyclohexanone
Diphenylamine
9,10-Dihydro-9,9dimethyl-acridine
Dibutylformamide
1,1,2,2-Tetrachloroethane
Acetophenone
2-Phenyl-2-propanol
Benzothiazole
2,2,5,5-Tetramethyltetrahydrofuran
Isophthalic acid
Terephthalic acid
PET oligomers
Cyclohexanone

Extractables

Organic Extractables from Packaging Materials: Chromatographic Methods Used for Identification and Quantification
5


Sample matrix

Methylene
chloride extracts

Soxhlet and
n-heptane
extracts

Soxhlet
extraction

Material

Rubber
stoppers

Laminated
polyolefind

PET

Evaporative

concentration,
with and without
silylation

Evaporative
concentration
with and without
silylation

Direct injection

Sample preparation

T = 220°C

T = 280°C

MSe

MS SIM mode,
also FID

From 100°C to
280°C at
10°C/min

50°C, hold for
10 min, to
280°C at
10°C/min

J&W DB-1,
15 m  0.53 mm,
1-mm film

Tributoxyethylphosphate
BHT, diphenylamine
4-(2,2,3,3Tetramethylbutyl)
phenol
2,2’-Methylenebis
[6,(1,1 dimethylethyl)4-ethyl] phenol
Penta- to octa-decane
Phthalic acid esters
Mono- to hepta-cosane
13-diocosenoic acid
Alkyl esters of
nonanoic acid
Ethylene glycol, BHT
Phthalic acid esters
Plamitic, stearic,
oleic acid
Terephthalic acid
Alkyl terephthalic
acid esters
2,6-bis-(1,1-methylethyl)4-ethyl phenol
Pyrogallol

1 mL, splitless,
200°C

MS EI + ,
T = 200°C,
50 – 650 amu

50°C for 5 min,
to 275°C at
10°C/min,
hold for
20 min

Extractables

Other

Detection

Oven program

J&W DB-5,
30 m  0.32 mm,
0.25-mL film

J&W DB-5MS,
30 m  0.25 mm,
0.25-mm film

Column

Table 2 Examples of GC methods used to identify and/or quantify packaging system extractables (Continued )

24f

22,23f

21

Ref.

6
Organic Extractables from Packaging Materials: Chromatographic Methods Used for Identification and Quantification


Solvent
extraction

Water extraction

PEi

Rubberk

Evaporative
concentration

Direct injection

Evaporative
concentration

3% OV17 on

Cas-Chrom Q,
1.5 m  2 mm

DB-1,
30 m  0.25 mm,
0.32-mm film

TRB-5,
60 m  0.25 mm,
0.5-mm film

140 – 200°C at
10°C/min

40°C for 1 min,
to 300°C at
20°C/min,
hold for
26 min
50°C for 2 min,
to 340°C at
5°C/min,
hold for
10 min

MS EI + ,
T = 250°C

MS EI + ,
40 – 700 Da,

T = 270°C

MS EI +

2 mL,
T = 250°C

Splitless,
T = 250°C

1 mL, splitless,
T = 300°C

1,3-Di-tert-butyl
benzene
Oligomers
2,4-Di-tert-butylphenol
Oxidized antioxidants
Butanoic acid
vinylester
Benzothiazole
derivativesl

Aliphatic hydrocarbons,
straight-chained,
branched and cyclicf,g

27

26f,j

25h

See legend of Table 1 for a list of material abbreviations.
a
Mass spectrometry was used in compound identification.
b
Method performance data are provided in this reference.
c
This reference documents numerous extracted compounds in these general categories.
d
Laminated film consisting of glycol-modified polyethylene terephthalate (PETG), polyvinylidene chloride (PVDG), and polyethylene (PE) with a polyurethane adhesive.
e
Gas chromatography/infrared spectroscopy analysis was also used under differing operating parameters to aid in compound identification.
f
These cited references document numerous compounds in these and other general compound classes.
g
Straight chained = C12 – C25; branched = C19 – C30; cyclic = C24 – C35.
h
Similar methods were used to identify compounds from polystyrene (styrene, styrene derivatives, glycolic esters of C16 – C25 fatty acids, trans-1,2-diphenylcyclobutane).
i
Material was gamma-irradiated prior to analysis.
j
A similar method was used to identify compounds from PP, PVC, PS, PET, and polyamide.
k
Components of disposable syringes.
l
Identified compounds include 2-hydroxybenzothiazole, 2-mercaptobenzothiazole, 2-(methylmercapto)benzothiazole, 2-(2-hydroxyethoxy)benzothiazole, and 2-(2-hydroxyethylmercapto)benzothiazole.

Solvent

extraction

Polyolefin

Organic Extractables from Packaging Materials: Chromatographic Methods Used for Identification and Quantification
7