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Method
Prepare the sample as directed in the individual tests of the monographs.
Pass the sample solution through a cation-exchange column with water at a
rate of about 5 mL/min., and collect about 250 mL of the eluate in a 500-mL
titration flask. Wash the resin in the column at a rate of about 10 mL/min
into the same titration flask, and titrate with 0.1 N sodium hydroxide. Continue the elution until 50 mL of the eluate requires no further titration.

CHROMATOGRAPHY
Chromatography is an analytical technique used in quantitative determination of
purity of most organic and an increasing number of inorganic reagent chemicals
and standard-grade reference materials. The broad scope of chromatography
allows it to be used in the separation, identification, and assay of diverse chemical
species, ranging from simple metal ions to compounds of complex molecular
structure, such as proteins.
In chromatography, the separation of individual components in a mixture is
achieved when a mobile phase is passed over a stationary phase. Differences in
affinities of various substances for these phases result in their separation.
Chromatography can be divided into two main branches, depending on
whether the mobile phase is a gas or a liquid. Gas chromatography is principally
used for analysis of volatile, thermally stable materials. Liquid chromatography is
particularly useful for analysis of nonvolatile or thermally unstable organic substances. Ion chromatography, a technique in which anions and cations can be
determined by using the principles of ion exchange, is a form of liquid chromatography. Thin-layer chromatography, often called planar chromatography, is also
a form of liquid chromatography.

Gas Chromatography
Gas chromatography (GC) is used in this monograph to determine the assay and/
or the trace impurities in both organic reagents and standards. Gas chromatography may be subdivided into gasliquid and gassolid chromatography. Gasliquid chromatography is by far the most widely used form of gas chromatography.
The heart of a GC system is the column, which is contained in an oven operated in either the isothermal or the temperature-programmed mode. Columns
packed with a nonvolatile liquid phase coated on a porous solid support were
used extensively in early editions of this monograph. In this edition, only capillary columns, constructed of fused silica onto which is bonded the liquid phase,
are used because of their greater resolving power and chemical inertness.
Conventional capillary gas chromatography uses long, narrow-bore columns
with an inside diameter (i.d.) from 0.22 to 0.32 mm, coated or bonded with a thin
film of the liquid phase. This arrangement results in high resolution but low sam-

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ple capacity. Special injection techniques and hardware are employed. The introduction of wide-bore capillary columns, which have an i.d. of typically 0.53 mm
and a relatively thick film of the liquid phase (1 mm to 5 mm), can allow a laboratory to use a standard packed-column instrument and conditions while gaining
the advantages of capillary technology.
Direct flash vaporization or on-column injection of the sample with standard-gauge needles can be used with capillary columns. Two types of flash vaporization injection techniques can be employed: a split mode of operation, in which
part of the sample is vented from the injector, or a splitless mode, in which a
smaller volume is injected and no portion of the sample is vented from the injector. The splitless mode, using a 0.1-mL sample size, is recommended for the assay
of most reagent solvents, while the split mode often is recommended for analysis
of most of the standard-grade reference materials.
The conventional split injector is a flash vaporization device. The liquid plug,
introduced with a syringe, is immediately volatilized, and a small fraction of the
resultant vapor enters the column while the major portion is vented to waste. To
perform a GC analysis using a split injection technique, the GC instrument
should be configured such that a preheated carrier gas, controlled by a pressure
regulator or a combination of a flow controller and a back pressure regulator,
enters the injector. The flow is divided into two streams. One stream of carrier gas
flows upward and purges the septum. The septum purge flow is controlled by a
needle valve. Septum purge flow rates are usually between 3 and 5 mL/min. A
high flow of carrier gas enters the vaporization chamber, which is a glass or quartz
liner, where the vaporized sample is mixed with the carrier gas. The mixed stream
is split at the column inlet, and only a small fraction enters the column. A needle
valve or flow controller regulates the split ratio.
Split ratios (measured column flow/measured inlet flow) typically range
from 1:50 to 1:500 for conventional capillary columns (0.22 mm to 0.32 mm i.d.).
For high sample capacity columns, such as wide bore columns and/or thick film
columns, low split ratios (1:5 to 1:50) are commonly used.
The method most commonly recommended for analysis of pure chemical
components (for example, matrices with a narrow constant boiling range) is the
hot needle, fast sample introduction. In this method, the sample is taken into the
syringe barrel (typically 25 mL in a 10-mL syringe) without leaving an air plug
between the sample and plunger. After insertion into the injection zone, the needle is allowed to heat up for 3 to 5 s. This period of time is sufficient for the needle
to be heated to the injector temperature. Then, the sample is injected by rapidly
pushing the plunger down (fast injection), after which the needle is withdrawn
from the injector within one second. Either manual or automatic sample introductions can be used. The measurement reproducibility will be enhanced by not
varying the injected volume, which typically should be 0.5 mL to 2.0 mL. The use
of an automatic injection system can significantly enhance measurement precision. Also, loosely packing the injection liner with deactivated glass wool or glass
beads can provide thorough mixing between sample and carrier gas, yielding less

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sample discrimination and better measurement precision. However, analysts

should be aware of adsorption and decomposition.
In conjunction with wide-bore columns, on-column injection can minimize
sample degradation while increasing the reproducibility of results. On-column
injection allows the injection of a liquid sample directly into the inlet of the column. Excellent quantitative precision and accuracy for thermally labile compounds and wide volatility range samples have been reported. Sample sizes range
from 0.5 mL to 2 mL. The injection should be performed as fast as possible, with
the column oven temperature below or equal to the boiling point of the solvent.
After injection, the liquid is allowed to form a stable film (flooded zone).
This takes several seconds. If the solutes to be analyzed differ much in boiling
points, in comparison to the solvent, ballistic heating to high temperature is
allowed. On the other hand, if the solute boiling points do not differ too much
compared to the solvent, temperature programming is applied to fully exploit the
solvent effect. When peak splitting and/or peak distortion is observed, the connection of a retention gap can provide the solution.
If the composition of the sample is not that complex, the use of megabore columns is recommended, particularly since automated injection becomes easier. If
high resolution with automated injection is needed, a deactivated but uncoated
wide bore precolumn (20 cm to 50 cm in length) should be connected to a conventional analytical narrow bore column. Hydrogen is the carrier gas of choice. If H2
cannot be used for safety reasons, helium may be substituted. High carrier-gas
velocities (5080 cm/sec H2, 3050 cm/sec He) ensure negligible band broadening.
In comparison to vaporizing injectors (that is, split and splitless), there are
some disadvantages to on-column injections. The two primary disadvantages
pertain to sample pretreatment. First, because the sample is introduced directly
onto the column, relatively clean samples must be prepared. Nonvolatile and
less-volatile materials collect at the head of the column, causing a loss of separation efficiency; therefore, sample cleanup is a prerequisite. Second, many samples
may be too concentrated for on-column injection and will need to be diluted.
Wide-bore columns can be used either in a high-resolution mode (carriergas flow rates less than 10 mL/min) to obtain optimum resolution of sample components or at higher flow rates (1030 mL/min), which will generate packed column-quality separations in a shorter time. The increased length of capillary columns allows for better separation of components, with the result that as few as
three columns of high, moderate, and low polarity can handle the majority of
analytical requirements.
A wide variety of detectors are used to quantify and/or identify the components in the eluent from the column. Thermal conductivity detectors (TCD) and
flame ionization detectors (FID) are examples of general detectors that provide a
linear response to most organic compounds. Electron capture detectors (ECD)
and photoionization are examples of class-specific detectors often used in trace
environmental analysis. Mass spectrometric-type detectors are used to provide
positive component identification. The detector output after amplification is used

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to produce the chromatogram, a plot of component response vs. elution time.

Modern systems convert the analog output to digital form, which allows for further manipulation and interpretation of the data.
Results from a chromatogram, when used to determine the assay of a reagent
or standard, are often expressed as area percent. A response factor correction for
each component is required for the most accurate results, especially when the
sample components differ markedly in their detector response. An internal standard reduces error due to variations in injection quantities, column conditions,
and detector conditions. Use of a TCD or FID minimizes the need to correct for
response and is usually sufficient for determining reagent or standard assay.
The use of control charts to aid the analyst in visualizing chromatographic
variability is suggested. To certify that assay results are valid, use of a system suitability test as described in a later section is recommended.

Gas ChromatographyMass Spectrometry

Gas chromatographymass spectrometry (GCMS) is one of the techniques used
in the identity requirement for standard-grade reference materials. Mass spectrometers are used for many kinds of chemical analysis, especially those where
identity or proof of chemical structure is critical. Examples range from environmental analysis to the analysis of petroleum products and biological materials,
including the products of genetic engineering. Mass spectrometers use the difference in mass-to-charge ratio (m/e) of ionized atoms or molecules to separate
them from each other. Mass spectrometry is therefore useful for quantitation of
atoms or molecules and also for determining chemical and structural information about molecules. Molecules have distinctive fragmentation patterns that provide structural information to identify structural components. The largest peak in
a mass spectrum is called the base peak and is assigned an arbitrary height of 100.
The remaining peaks are then normalized to the base peak.
The general operation of a mass spectrometer is:
1.
2.
3.

creating gas-phase ions;

separating the ions in space or time based on their mass-to-charge ratio;
and
measuring the quantity of ions of each mass-to-charge ratio.

The ion separation power of a mass spectrometer is described by the resolution, which is defined as R = m/Dm, where m is the ion mass and Dm is the difference in mass between two resolvable peaks in a mass spectrum with similar mass
values. For example, a mass specrometer with a resolution of 1000 can resolve an
ion with a m/e of 100.0 from an ion with an m/e of 100.1.
In general, a mass spectrometer consists of an ion source, a mass-selective
analyzer, and an ion detector. Since mass spectrometers create and manipulate
gas-phase ions, they operate in a high-vacuum system. The magnetic-sector, qua-

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drupole, and time-of-flight designs also require extraction and acceleration ion
optics to transfer ions from the source region into the mass analyzer.

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Liquid Chromatography
Many reagent chemicals and standard-grade reference materials described in this
monograph can be assayed by and/or tested for suitability for use in liquid chromatography (LC). Liquid chromatography is a technique in which the sample
interacts with both the liquid mobile phase and the stationary phase to effect a separation. An LC system consists of a pump that delivers a liquid, usually a solvent
for the sample, at a constant flow rate through a sample injector, a column, and a
detector. The solvent is called the mobile phase. Typically, the flow rate is 1 to 10
mL/min for conventional liquid chromatography. In isocratic operation, the composition of the mobile phase is kept constant, while in gradient elution, it is varied
during the analysis. Gradient elution is required when the sample mixture contains components with a wide range of affinity for the stationary phase. In the isocratic mode, the purity of the solvents is less critical, as the impurities are
adsorbeddesorbed at a constant rate, whereas in gradient elution impurities may
result in extraneous peaks and/or shifts in the baseline. This characteristic necessitates the incorporation of a gradient elution test in this monograph to verify the
quality of a solvent when used in this most stringent LC system mode of operation.
In liquid chromatography, a dilute solution of the sample to be analyzed is
introduced into the mobile phase via the sample injector and enters the column,
where it is separated into its individual components. The columns are usually
steel or glass, densely packed with semirigid organic gels or rigid inorganic silica
microspheres, to which a variety of substrates can be chemically bonded and
whose typical particle size is 3 mm to 10 mm.
Several mechanisms of separation are possible in liquid chromatography. In
adsorption chromatography, separation is based on adsorptiondesorption kinetics, whereas in partition chromatography, the separation is based on partitioning
of the components between the mobile and stationary phases. Ion exchange is the
dominant mechanism in ion chromatography. In practice, a successful separation
may involve a combination of separation mechanisms.
A commonly used term, coined by early chromatographers for describing
separations dominated by adsorptiondesorption, is normal phase. In normal
phase chromatography, the stationary phase is strongly polar (for instance, silica
or aminopropyl), and the mobile phase is less polar (for instance, hexane). Polar
components are thus retained on the column longer than less-polar materials.
A second term of historical origin is reverse phase. Reverse phase generally
applies to separations dominated by partition chromatography, and the elution of
components in a reverse phase separation are more or less reversed from the
order that would be obtained in a normal phase separation. Reverse phase, the
more widely used mode of chromatography, uses a nonpolar (hydrophobic) sta-

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tionary phase, such as C-18 (octadecyl) chemically bonded to silica, while the
mobile phase is a polar liquid such as acetonitrilewater. Hydrophobic (nonpolar) components are retained longer than hydrophilic (polar) components. The
elution properties of the mobile phase can be adjusted to modify a separation by
addition of appropriate ionic modifiers. These modifiers can be chosen for their
ability to either suppress or enhance ion formation in the sample. When enhancement is chosen, the separation mechanism may involve both ion exchange and
partition.
The wide range of available stationary phases in combination with changes in
mobile-phase composition makes liquid chromatography a very flexible separation-assay technique. The back pressure on the pump is several hundred to several thousand psi, depending upon the particle size of the packing material,
mobile-phase flow rate, and viscosity. The column eluent is continuously monitored by a sensitive detector chosen to respond either to the sample component
alone (for instance, an ultraviolet photometer) or to a change in some physical
property of the mobile phase due to the presence of the solute (for example, a differential refractometer). Other detectors such as electrochemical, fluorescence,
etc., are also used for more specialized applications. When photometric detectors
are used, solvents are often specified in absorbance units at a specific wavelength.
The response of the detector is related to the concentration or weight of the solute
and is displayed on a recorder. A computer is usually interfaced to the LC system
to control the method and to collect and analyze data.

Ion Chromatography
Ion chromatography (IC) is a subset of liquid chromatography. Whereas conventional liquid chromatography is mainly used for the analysis of nonionic organic
compounds, ion chromatography separates and determines ionic species or ionizable compounds, both organic and inorganic. Under ideal conditions, quantitation to the sub-part-per-billion level is attainable. For trace analysis, water and
reagents of the highest quality need to be used. Deionized water of >18 MW cm
resistivity is recommended.
For the testing of reagent chemicals, ion chromatography is ordinarily
applied to the determination of anions present as minor impurities. Generally,
the major component should be eliminated or greatly reduced in concentration
to avoid overloading the anion-exchange column used for separation.
IC columns are usually 525 cm in length and 210 mm in diameter. Columns may be either metallic or plastic. Packing materials can be either anionic or
cationic resins, depending on the separation desired, and there are several variations within each type. Typical particle sizes range from 5 to 20 mm.
In many anion applications of ion chromatography, buffered, aqueous,
mobile phases (eluents) of approximately millimole strength are used. In most
cases, IC analyses are carried out isocratically, and gradient elutions are used only
for the more complex separations. Typical eluent flow rates for ion chromatogra-

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phy are 12 mL/min, with system operating pressures at 1000 psi or less.
Microbore columns are operated at lower flow rates of 0.20.5 mL/min.
Standard and sample injections are normally made by using a loop injection
system. The most widely used detection method for ion chromatography is conductivity. However, other detection methods, including ultraviolet-visible spectroscopy, electrochemistry, and fluorescence can be used. Conductivity is a universal detection mode for ions, whereas the other detectors provide selective,
analyte-specific detection.
In conductimetric IC analysis, the impact of the significant background conductivity of the eluent on analyte determinations is minimized by chemical or
electronic suppression. Chemical suppression involves notably reducing the eluent conductivity, as well as enhancing analyte response by means of a chemical
reaction. Several innovative techniques have been developed to achieve these
changes, including packed-column and membrane-based devices. Electronic suppression minimizes eluent background noise via the design of the electronic circuitry in the conductivity detector, although the actual background eluent conductivity is not reduced, as it is with chemical suppression.
Because of the two different modes of suppression, the columns and eluents
used also fall into two categories. Columns for chemical suppression typically
have higher ion-exchange capacities than those used for electronic suppression.
Eluents used in chemical suppression also differ from those used in electronic
suppression in that they are defined by the chemical reactions that occur in the
suppression of the eluent.

Thin-Layer Chromatography
Thin-layer chromatography (TLC) is a very simple form of solidliquid adsorption chromatography. It is probably the quickest, easiest, and most frequently
applied technique for determining purity of organic compounds. As in other
forms of liquid chromatography, the sample interacts with a liquid mobile phase
and a stationary phase to effect partitioning of components based on their affinity
to the solid and liquid phase. Thin-layer chromatography serves many purposes
in the laboratory because of its simplicity. It is commonly used in organic synthesis to monitor chemical reactions. Starting materials, intermediates, and products
often elute differently, and product formation or starting-material disappearance
may be observed.
Another very common use is in purity determinations of organic compounds. Typically, it is not used as a quantitative technique, but it is useful for
looking for impurities. In many cases, thin-layer chromatography is very sensitive
and can be used to detect impurities of less than 1% in the sample. A single spot
on a TLC plate is a good indication of purity. Thin-layer chromatography is also
applied in selected requirements for standard-grade reference materials as a technique for purity confirmation. The technique is used with other complementary
purity assays to screen standard-grade reference materials for impurities. Stan-

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dard-grade reference materials have passed the purity test when, having used the
specific conditions in the method, an analyst can observe a single spot.
In thin-layer chromatography, the stationary phase is spread as a thin layer
over glass or plastic. Calcium sulfate or an organic polymer (such as starch) is
added to the solid phase to bind the solid to the glass or plastic. Often, fluorescent
material is added to the solid phase to aid in detection of analytes. TLC plates are
commercially available or can be prepared in the laboratory. Glass or liquid plates
are available commercially as sheets that are cut into strips for use. Plates can be
prepared in the laboratory by preparing a slurry of the solid phase with a solvent,
such as chloroform or methanol, and applying a thin coat over a glass plate. Plates
are most effective when dried in an oven before use. The most common stationary phases used in thin-layer chromatography are silica gel and alumina. Reversephase TLC plates are also available for elution of polar compounds. It is common
to perform TLC analysis with both silica and alumina to maximize the effectiveness of the technique.
Performing TLC analysis requires minimal equipment. The plates are cut
into strips approximately 10 cm in height. The sample is dissolved in a volatile
solvent and applied to the bottom of the plate. This is accomplished by applying
or spotting the solution about 0.5 cm from the bottom of the plate using a thin
capillary tube. The plate is placed into a development chamber that contains
enough elution solvent to come just below the sample spot. The solvent then travels up the plate and moves the components of the sample at different rates according to their affinity. When the solvent is about 1 cm from the upper end of the
plate, the plate is removed, the solvent line is marked and the plate is allowed to
dry.
Detection of spots on the TLC plate is accomplished in a number of ways.
The most common method is to view the plate under ultraviolet (UV) light.
Compounds that fluoresce will be detected. Alternatively, the plates can contain a
fluorescent material within the solid phase. In this case, the plate will fluoresce
and compounds that do not fluoresce will appear as black spots on the plate.
Another common method is to use iodine as a complexing agent. Many organic
compounds form charge-transfer complexes with iodine, resulting in a dark-colored species. The TLC plate is placed in a chamber containing iodine and developed for several minutes. Upon removal from the chamber, the plates will contain dark spots; these spots should be immediately circled with a pencil, since the
complex is reversible and the spots will fade away. In other detection methods, the
plate is sprayed with a solution to stain the analytes. Common reagents are sulfuric acid solution, which will char many organic compounds, and potassium
permanganate solution, which will oxidize many organic compounds. Combinations of two or more methods may be used to detect a broader range of analytes.
It is often useful to determine the distance that a particular compound has
moved up the plate in relation to the solvent front. This value is called the retention factor and designated Rf. The Rf value is calculated by measuring the distance
that the analyte moved from the origin and dividing that by the distance the sol-

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vent moved from the origin. For purity assays, it is desirable to have an Rf value in
the range of 0.30.5.

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RECOMMENDED CHROMATOGRAPHY
PROCEDURES
Assay Methods
Procedures published in this monograph list the parameters and values established to give satisfactory results. These procedures should be considered adequate only for determining the constituents specified in the individual tests, and
they may not necessarily separate other components in a given sample. An
attempt is made to keep each procedure for reagents as simple as possible, while
still retaining the desired sensitivity, because simplicity may lead to wider usage.
The procedures used for standard-grade reference materials are, in general, more
specific, since it is necessary to separate similar eluting impurities.
The list of parameters for gas chromatography includes such variables as column type, diameter and length of column, carrier-gas flow rate, detector type,
and operating parameters. The list for liquid chromatography includes such variables as diameter and length of column, packing type and particle size, mobilephase composition and flow rate, detector type, and operating parameters. For
reagents, the parameters represent one set of conditions that result in reproducible analyses. Because of differences among instruments, one or more of the
stated parameters may require adjustment for any given instrument. Therefore
these parameters, except for the column and type of detector, should be regarded
as guides to aid the analyst in establishing the optimum conditions for a particular instrument. Relative retention times are given as an aid in peak identification.
Exact reproduction of those times is not essential. Parameters given for the standard-grade reference materials section, however, must be adhered to exactly as
stated in the test.

Gas Chromatography
The volatile organic reagents in this monograph can be assayed by gas chromatography. A single set of instrument conditions shown here has been chosen and
found to give satisfactory results for the assay of these reagents. Three columns of
varying polarity can be used to achieve the desired separation: type I, low polarity, methyl silicone, 5 mm; type II, moderate polarity, mixed cyano, phenylmethyl
silicone, 1.5 mm; and type III, high polarity, polyethylene glycol, 1 mm. The recommended column type and reagent-specific conditions can be found listed
alphabetically under the individual reagents. The retention times and relative
retention times vs. methanol of the organic reagents are listed alphabetically
(Table X) and in increasing order (Table XI).

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