Procedure

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Chromatography
Part 2, Analytical Procedures and General Directions
eISBN: 9780841230460
Tom Tyner Chair, ACS Committee on Analytical Reagents
James Francis Secretary, ACS Committee on Analytical Reagents

ABSTRACT
Chromatography is an analytical technique used in the quantitative determination of the purity of most organic and an
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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 gas–liquid and gas–solid chromatography. Gas–liquid
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
earlier editions of this book. Now, 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 sample capacity.
Special injection techniques and hardware are used. Wide-bore capillary columns, which have an i.d. of typically 0.53 mm and
a relatively thick film of the liquid phase (1–5 µm), 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 used: 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 0.1–0.2 µL sample size, is recommended for the assay of most reagent
solvents; the split mode often is recommended for analysis of most of the standard-grade reference or pure chemical
materials.

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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
(0.22–0.32 mm i.d.). For high sample capacity columns, such as wide-bore columns and/or thick-film columns, low split ratios
(1:5–1:50) are commonly used.

The split mode hot needle fast sample introduction method is most commonly recommended for analysis of standard-grade
reference or pure chemical components (for example, matrices with a narrow constant boiling range). In this method, the
sample is taken into the syringe barrel (typically 2–5 µL in a 10 µL syringe) without leaving an air plug between the sample and
the plunger. After insertion into the injection zone, the needle is allowed to heat for 3–5 s. This period of time is sufficient for
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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 1 s. 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
0.5–2.0 µL. 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 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. Splitless 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 to 2 µL. 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), which 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 or
deactivated precolumn can provide the solution.

If the composition of the sample is not that complex, the use of wide-bore columns is recommended, particularly because
automated injection becomes easier. If high resolution with automated injection is needed, a deactivated but uncoated wide-
bore precolumn (20–50 cm in length) should be connected to a conventional analytical narrow-bore column. Hydrogen is
becoming the carrier gas of choice. If H2 cannot be used for safety reasons, helium may be substituted. High carrier gas
velocities (3–5 mL/min H2, 2–5 mL/min He) ensure negligible band broadening. High-purity grades of carrier gases should
always be used.

There are some disadvantages of on-column injections compared to vaporizing injections (that is, split and splitless). 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 (carrier gas flow rates ≤10 mL/min) to obtain optimum
resolution of sample components or at higher flow rates (10–30 mL/min), which will generate packed column quality
separations in a shorter time. The increased length of capillary columns provides better separation of components than
traditional packed columns. Improved resolution reduces the types of columns needed for most analytical requirements to as
few as three: high, moderate, and low polarity.

A wide variety of detectors are used to quantify and/or identify the components in the eluent from the column. Thermal
conductivity detectors (TCDs) and flame ionization detectors (FIDs) are examples of general detectors that provide a linear
response to most organic compounds. Electron capture detectors (ECDs) and photoionization are examples of class-specific

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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 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, analysts should use a system suitability test as described below.

Recommended gas chromatography procedures begin in [Part 2: Chromatography; Recommended Procedures; Gas
Chromatography].

GAS CHROMATOGRAPHY−MASS SPECTROMETRY

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Gas chromatography–mass spectrometry (GC–MS) 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 as follows:

1. creating gas-phase ions

2. separating the ions in space or time based on their mass-to-charge ratio

3. 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/Δm, where m is the
ion mass and Δm is the difference in mass between two resolvable peaks in a mass spectrum with similar mass values. For
example, a mass spectrometer with a resolution of 1000 can resolve an ion with an 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. Because mass
spectrometers create and manipulate gas-phase ions, they operate in a high-vacuum system. The magnetic sector,
quadrupole, and time-of-flight designs also require extraction and acceleration ion optics to transfer ions from the source
region into the mass analyzer.

GC–MS is used in this book to verify the identity of various standard-grade reference materials. The specific procedure is given
under the individual standards.

HEADSPACE GAS CHROMATOGRAPHY

Static headspace gas chromatography (HS–GC) is a technique used for separation, concentration, and analysis of volatile
organic compounds (VOCs) in complex sample matrices. Examples include analysis of residual solvents in pharmaceutical
products, alcohol in blood, monomers in polymer materials, and VOCs in cosmetics and food samples. This clean and easy
sample preparation is an excellent alternative to the costly and time-consuming classic extraction procedures.

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The HS methodology is quite simple. A sample is dissolved in a suitable solvent and sealed in a headspace vial. The vial is
thermostated for a certain amount of time to allow the analytes to volatilize, enter the gas phase, and equilibrate with the
sample phase. The gas phase is commonly referred to as headspace.

The most important parameters of the HS analysis are the partition coefficient, K, defined as the ratio of the concentration of
the analyte in the sample phase (Cs) to the concentration of the analyte in the gas phase (Cg) [K = (Cs/Cg)] and the phase ratio,
β, defined as the ratio of the volume of the gas phase to the volume of the sample phase [β = (Vg/Vs)]. Compounds with low K
values provide high vapor concentrations, high responses, and low detection limits. Lower β values generally increase the
sensitivity. The dissolution solvents should have high partition coefficients and low vapor pressure; therefore, high boiling
point solvents, such as DMSO, DMF, NMP, or water, are preferred.

Several techniques are used to inject the sample from the vial to the GC system: gas-tight syringe injection, balance-pressure
system, and pressure-loop system. To optimize the chromatographic performance, transfer lines should be made of inert
materials, dead volume should be minimized, and the system should operate under leak-free conditions.

LIQUID CHROMATOGRAPHY
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Many reagent chemicals and standard-grade reference materials described in this book can be assayed by and/or tested for
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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 produce 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, column, and detector. The solvent is
called the mobile phase. Typically, the flow rate is 1–10 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 adsorbed–desorbed 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 book 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 semi-rigid organic gels or rigid inorganic silica microspheres to which a variety of substrates can be
chemically bonded and whose typical particle size is 3–10 µm.

Several mechanisms of separation are possible in liquid chromatography. In adsorption chromatography, separation is based on
adsorption–desorption 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 adsorption–desorption, 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 liquid chromatography, uses a
nonpolar (hydrophobic) stationary phase, such as C-18 (octadecyl) chemically bonded to silica, while the mobile phase is a
polar liquid such as acetonitrile–water. 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 the 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 diode array detector (DAD), or to a change in some physical property of the mobile

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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.

Recommended liquid chromatography procedures begin in [Part 2: Chromatography; Recommended Procedures; Liquid
Chromatography; Solvent Suitability for Liquid Chromatography; Procedure for Liquid Chromatography].

LIQUID CHROMATOGRAPHY−MASS SPECTROMETRY

Liquid chromatography–mass spectrometry (LC–MS) is the combination of high performance liquid chromatography (HPLC) and
mass spectrometry (MS). This technique is used to separate and identify compounds in standard-grade reference materials,
test solvents, and additives. It is a complementary technique to gas chromatography–mass spectrometry (GC–MS). Compounds
that are thermally unstable in GC–MS will likely be good candidates for analysis by LC–MS. In addition, the broad variety of
stationary phases available in LC allow for the separation of compounds with differing polarities. Depending on the polarity of
the analytes, different modes of chromatography can be selected for the separation. Examples of common modes of
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chromatography used in LC–MS are reverse phase, normal phase, and hydrophobic interaction liquid chromatography (HILIC).

A crucial point for a liquid chromatograph interfaced to a mass spectrometer is the ionization process of the sample molecules
and removal of the mobile-phase solvent. Depending on the polarity of the solvent and/or analyte, the following three
methods may be used for the production of ions for introduction into the MS source:

Electrospray Ionization (ESI). A soft, sensitive, and most popular ionization technique based on evaporation of the mobile
phase by nitrogen and ionization by proton ([M+H]+, [M–H]–) or ion transfer ([M+Na]+, [M+HCOO]–). ESI is suitable for small,
polar molecules as well as large peptides and proteins.

Atmospheric Pressure Chemical Ionization (APCI). The mobile phase is evaporated with heated nitrogen. In this technique,
the gas flows through a corona needle charged with high voltage causing ionization of the nitrogen, which in turn ionizes
other molecules including the analytes of interest (a corona discharge). The ionization process can be controlled by the
needle voltage and current and is more selective for nonpolar compounds. This technique is also suitable for highly polar or
charged molecules in nonpolar mobile phases, for example, in normal-phase chromatography, where solvents like heptane
generally do not support the protonation of analytes in situ. This technique typically generates more fragments relative to
the parent ion than electrospray ionization.

Atmospheric Pressure Photo/LASER Ionization (APPI/APLI) The designs of APCI and APPI sources differ only in one aspect. In
APPI, the needle is replaced by a UV lamp or UV-LASER window to allow UV radiation to pass through the gas flow and ionize
UV-absorbing molecules directly or via charge-transfer of a UV-absorbing additive (“dopant”-induced ionization). The
energy of the UV radiation is high enough to ionize most of the compounds of interest but does not interact with the solvent
molecules. Even ([M+H]+, [M]+) and odd electron configuration [M]+• can be detected in mass spectra.

All typical mass analyzers, for example, sector, quadrupole, ion trap or time-of-flight mass spectrometers, depend on charged
atoms, molecules, or fragments of molecules. These fragments are separated based on their mass/charge (m/z) ratios in a
static or alternating electromagnetic field. A high vacuum within the mass spectrometer is needed to insure that no
ion–molecule reactions, scattering, and neutralization of the ions occur. LC–MS systems are equipped with one high vacuum
pump and one roughing pump to remove major parts of the nebulizer gas, neutral sample, and solvent molecules.

Additional ion optics within the mass analyzer region, for example, skimmers, ion lenses, ion funnels, lens systems with
alternating polarity, or bending multipoles, help to focus the ion beam and to remove minor residues of neutral molecules. The
different types of mass analyzers are similar to those used in GC–MS.

Generally, LC principals can also be applied to LC–MS. In most cases, LC–MS systems are equipped with a UV detector to monitor
both UV and MS signals. This dual detection option is also useful for trouble-shooting a method and for detecting compounds
that may only be seen in a single mode (either MS or UV). There are, however, some major differences in method development
compared to UV detection. These differences are usually determined by the design or limitation of the ion source. Mobile
phases can only be prepared with volatile buffers, additives, or ion pair reagents. This limits the choice of salts and may cause
changes in the retention and separation of compounds. Nearly all suitable reagents, for example, formic acid, acetic acid,
ammonium formate, TFA, ammonium hydroxide, dihexylamine acetate, ammonium bicarbonate, and acetate, are volatile.

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This volatility property is important in MS because there is no plugging of the orifice of the MS and no negative effects on ion
transfer. On the other hand, most of these chemicals absorb UV radiation and cause a dramatic baseline drift in a UV trace.

ESI is limited to reverse phase, HILIC, or other chromatographic techniques with polar and protic mobile-phase compositions.
Best sensitivities are obtained with water, acetonitrile, methanol, 2-propanol, or ethanol using formic or acetic acid and/or
volatile buffer salts, for example, ammonium acetate, ammonium formate, as additives. Higher concentrations of organic
phase are preferred as ionization efficiency will be greatly enhanced and mobile-phase removal (especially high aqueous
containing mobile phases that are more difficult to evaporate) will be easier. The flow rate should not exceed 1 mL/min. A flow
splitter between the LC and the mass spectrometer can help to reduce the mobile-phase flow into the ion source, especially in
those cases when a standard HPLC column (e.g., 4.6 mm × 250 mm, 5 µm) is needed and a higher flow rate (>1 mL/min) has to
be applied for optimum column performance. Generally, a lower flow rate increases the sensitivity of ESI that is often contrary
to the HPLC flow rate settings.

APCI and APPI/APLI ion sources do not depend on the properties of the mobile phases. They are also suitable for nonpolar
mobile phases. Unfortunately, the number of compounds that are ionizable by these sources is limited, and the sensitivity,
especially of APCI, is lower compared to ESI. On the other hand, APPI/APLI sources have emerged as a sensitive, direct
detection method for the analysis of steroids or PAHs.
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Solutions of standards can also be infused or injected directly into the mass spectrometer using a syringe pump or injection
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valve. A direct analysis of solutions can also be infused as mass calibration or concentration calibration standards. Typical flow
rates are 180–240 µL/h for ESI sources.

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 subpart per billion level is attainable. For
trace analysis, water and reagents of the highest quality need to be used. Deionized water greater than 18 MΩ 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 5–25 cm long and 2–10 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 µm.

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 chromatography are 1–2 mL/min, with system operating pressures at
1000 psi or less. Microbore columns are operated at lower flow rates of 0.2–0.5 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.

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A discussion of ion chromatography procedures begins in [Part 2: Chromatography; Recommended Procedures; Ion
Chromatography].

THIN-LAYER CHROMATOGRAPHY
Thin-layer chromatography (TLC) is a very simple form of solid–liquid adsorption chromatography. It is probably the quickest,
easiest, and most frequently applied technique for determining the 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 migrate 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
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confirmation. The technique is used with other complementary purity assays to screen standard-grade reference materials for
impurities. Standard-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. Reverse-phase 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 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 because 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 is 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 solvent moved from the origin. For purity assays, it is
desirable to have an Rf value in the range of 0.3–0.5.

Recommended thin-layer chromatography procedures begin on in [Part 2: Chromatography; Thin-Layer Chromatography].

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RECOMMENDED PROCEDURES
Assay Methods
The reagent chemicals procedures published in this book 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
reagent monographs, 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 because 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, mobile-phase 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
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aid the analyst in establishing the optimum conditions for a particular instrument. Relative retention times are given as an
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aid in peak identification. Exact reproduction of those times is not essential. However, parameters given for the standard-
grade reference materials (Part 5) must adhere exactly as stated in the test.

Gas Chromatography
The volatile organic reagents in this book 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 the reagents listed in Tables 2-14 and 2-
15. Three columns of varying polarity can be used to achieve the desired separation: type I, low polarity, methyl silicone, 5
µm; type II, moderate polarity, mixed cyano, phenylmethyl silicone, 1.5 µm; and type III, high polarity, polyethylene glycol,
1 µm. 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 2-14, and in increasing order, Table 2-15).

Table 2-14. GC Retention Time of Reagents in Alphabetical Order

Compounds Column of Type I Type I Type II Type II Type III Type III
Choice Nonpolar RT Nonpolar RR Med. Med. High High
Polar RT Polar RR Polar RT Polar RR
Acetaldehyde I 2.9 0.97 2.2 0.89 2.8 0.4
Acetic acid I 8.8 2.9 – – – –
Acetic anhydride I 11.4 3.8 – – – –
Acetone I 4.9 1.6 3.4 1.4 4.1 0.6
Acetonitrile I 4.7 1.6 4.4 1.8 8.7 1.4
Acetyl chloride I 5.2 1.7 – – – –
2–Aminoethanol I 10.6 3.5 9.9 4.0 – –
Aniline I 19.5 6.5 17.3 7.0 21.9 3.4
Benzene I 11.2 3.7 6.7 2.7 6.9 1.1
Benzyl alcohol I 20.6 6.8 18.6 7.5 23.5 3.7
2–Butanone I 8.5 2.8 6.0 2.4 6.0 1.0
Butyl acetate I 15.3 5.1 11.7 4.7 10.6 1.7
Butyl alcohol I 11.1 3.7 9.0 3.6 12.3 1.9
t–Butyl alcohol I 6.3 2.1 4.1 1.7 6.5 1.0
Carbon disulfide I 7.4 2.5 3.0 1.2 3.0 0.5
Carbon tetrachloride I 11.2 3.7 6.0 2.4 5.3 0.8

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Table 2-14. GC Retention Time of Reagents in Alphabetical Order (continued)

Compounds Column of Type I Type I Type II Type II Type III Type III
Choice Nonpolar RT Nonpolar RR Med. Med. High High
Polar RT Polar RR Polar RT Polar RR
Chlorobenzene I 16.6 5.5 12.5 5.0 16.5 2.6
Chloroform I 9.2 3.1 6.0 2.4 8.9 1.4
Cyclohexane I 11.4 3.8 5.4 2.2 3.0 0.5
Cyclohexanone I 17.5 5.8 14.9 6.0 15.4 2.4
1,2–Dichloroethane I 10.3 3.4 7.4 3.0 10.0 1.6
Dichloromethane I 6.2 2.0 3.5 1.4 6.4 1.0
Diethanolamine I 20.6 6.9 20.0 8.0 – –
Diethylamine I 11.2 3.0 5.0 2.5 – –
N,N–Dimethylformamide I 14.2 4.7 13.6 5.5 16.2 2.5
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Dimethylsulfoxide III 15.7 5.2 16.0 6.4 20.5 3.2

Dioxane I 12.2 4.1 9.1 3.6 10.3 1.6
Ethyl acetate I 9.2 3.1 5.8 2.3 5.6 0.9
Ethyl alcohol I 4.3 1.4 3.2 1.3 7.4 1.2
Ethyl ether I 5.6 1.9 2.7 1.1 2.4 0.4
Ethylbenzene I 17.0 5.6 12.5 5.0 11.8 1.8
Formamide III – – – – 23.0 3.7
2–Furancarboxyaldehyde I 15.8 5.2 14.2 5.7 17.9 2.8
Glycerol II 19.8 6.6 19.7 7.9 – –
2-Hexane I 8.6 2.9 3.2 1.3 2.3 0.4
n-Hexane I 9.2 3.1 3.5 1.4 2.3 0.4
Isobutyl alcohol I 10.0 3.3 7.9 3.2 11.2 1.7
Isopentyl alcohol I 13.2 4.4 11.0 4.4 13.5 2.1
Isopropyl alcohol I 5.4 1.8 3.8 1.5 7.3 1.1
Isopropyl ether I 9.1 3.0 2.6 1.0 2.6 0.4
Methanol I 3.0 1.0 2.5 1.0 6.4 1.0
2-Methoxyethanol III 10.2 3.4 8.4 3.4 13.2 2.1
Methyl-tert-butyl ether I 11.2 3.0 5.9 2.0 2.8 0.4
4-Methyl-2-pentanone I 13.3 4.4 10.2 4.1 8.9 1.4
1-Methyl-2-pyrrolidone I 20.6 6.8 19.3 7.7 21.8 3.4
Nitrobenzene I 22.0 7.3 19.4 7.8 22.1 3.5
Nitromethane III 7.2 2.4 7.0 2.8 12.2 1.9
1-Octanol I 21.2 7.0 17.8 7.2 19.2 3.0
2-Octanol I 20.2 6.7 16.0 6.4 18.2 2.9
1-Pentanol I 14.2 4.7 11.8 4.7 14.3 2.2
Perchloroethylene I 15.8 5.2 10.7 4.3 9.4 1.5
1,2-Propanediol III 13.4 4.5 13.4 5.4 19.8 3.1
Perchloroethylene I 15.8 5.2 10.7 4.3 9.4 1.5
Propionic acid III 14.0 4.6 12.5 5.0 18.5 2.9

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Table 2-14. GC Retention Time of Reagents in Alphabetical Order (continued)

Compounds Column of Type I Type I Type II Type II Type III Type III
Choice Nonpolar RT Nonpolar RR Med. Med. High High
Polar RT Polar RR Polar RT Polar RR
'll-Propyl alcohol III 7.6 2.5 5.9 2.4 10.1 1.6
Pyridine I 13.4 4.5 10.6 4.2 13.2 2.1
Quinoline II – – 121.6 8.7 25.0 3.9
Tetrahydrofuran I 9.8 3.3 5.8 2.3 5.1 0.8
Toluene I 14.4 4.8 10.0 4.0 9.7 1.5
1,1,1-Trichloroethane I 10.6 3.5 6.0 2.4 5.4 0.9
Trichloroethylene I 12.3 4.1 7.8 3.1 8.4 1.3
1,1,2-Trichlorotrifluoroethane I 6.7 2.2 2.7 1.1 2.4 0.4
2,2,4-Trimethylpentane I 12.4 4.1 5.9 2.4 2.6 0.4
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1,2-Xylene I 17.8 5.9 13.3 5.4 12.8 2.0

1,3-Xylene I 17.2 5.7 12.6 5.1 12.0 1.9
1,4-Xylene I 16.9 5.6 12.4 5.0 11.6 1.8
Note: RT is retention time; RR is relative retention (methanol 1.0); and “–” indicates data not available.

Table 2-15. GC Retention Time of Reagents by Chronological Order on Type I Column

Compounds Column of Type I Type I Type II Type II Type III Type III
Choice Nonpolar RT Nonpolar RR Med. Med. High High
Polar RT Polar RR Polar RT Polar RR
Acetaldehyde I 2.9 0.97 2.2 0.89 2.8 0.4
Methanol I 3.0 1.0 2.5 1.0 6.4 1.0
Ethyl alcohol I 4.3 1.4 3.2 1.3 7.4 1.2
Acetonitrile I 4.7 1.6 4.4 1.8 8.7 1.4
Acetone I 4.9 1.6 3.4 1.4 4.1 0.6
Acetyl chloride I 5.2 1.7 – – – –
Isopropyl alcohol I 5.4 1.8 3.8 1.5 7.3 1.1
Ethyl ether I 5.6 1.9 2.7 1.1 2.4 0.4
Dichloromethane I 6.2 2.0 3.5 1.4 6.4 1.0
t-Butyl alcohol I 6.3 2.1 4.1 1.7 6.5 1.0
1,1,2-Trichlorotrifluoroethane I 6.7 2.2 2.7 1.1 2.4 0.4
Nitromethane III 7.2 2.4 7.0 2.8 12.2 1.9
Carbon disulfide I 7.4 2.5 3.0 1.2 3.0 0.5
'll-Propyl alcohol III 7.6 2.5 5.9 2.4 10.1 1.6
2-Butanone I 8.5 2.8 6.0 2.4 6.0 1.0
2-Hexane I 8.6 2.9 3.2 1.3 2.3 0.4
Acetic acid I 8.8 2.9 – – – –
Isopropyl ether I 9.1 3.0 2.6 1.0 2.6 0.4
Chloroform I 9.2 3.1 6.0 2.4 8.9 1.4
Ethyl acetate I 9.2 3.1 5.8 2.3 5.6 0.9
n-Hexane I 9.2 3.1 3.5 1.4 2.3 0.4

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Table 2-15. GC Retention Time of Reagents by Chronological Order on Type I Column (continued)
Compounds Column of Type I Type I Type II Type II Type III Type III
Choice Nonpolar RT Nonpolar RR Med. Med. High High
Polar RT Polar RR Polar RT Polar RR
Tetrahydrofuran I 9.8 3.3 5.8 2.3 5.1 0.8
Isobutyl alcohol I 10.0 3.3 7.9 3.2 11.2 1.7
2-Methoxyethanol III 10.2 3.4 8.4 3.4 13.2 2.1
1,2-Dichloroethane I 10.3 3.4 7.4 3.0 10.0 1.6
2-Aminoethanol I 10.6 3.5 9.9 4.0 – –
1,1,1-Trichloroethane I 10.6 3.5 6.0 2.4 5.4 0.9
Butyl alcohol I 11.1 3.7 9.0 3.6 12.3 1.9
Benzene I 11.2 3.7 6.7 2.7 6.9 1.1
Carbon tetrachloride I 11.2 3.7 6.0 2.4 5.3 0.8
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Diethylamine I 11.2 3.0 5.0 2.5 – –

Methyl-tert-butyl ether I 11.2 3.0 5.9 2.0 2.8 0.4
Acetic anhydride I 11.4 3.8 – – – –
Cyclohexane I 11.4 3.8 5.4 2.2 3.0 0.5
Dioxane I 12.2 4.1 9.1 3.6 10.3 1.6
Trichloroethylene I 12.3 4.1 7.8 3.1 8.4 1.3
2,2,4-Trimethylpentane I 12.4 4.1 5.9 2.4 2.6 0.4
Isopentyl alcohol I 13.2 4.4 11.0 4.4 13.5 2.1
4-Methyl-2-pentanone I 13.3 4.4 10.2 4.1 8.9 1.4
1,2-Propanediol III 13.4 4.5 13.4 5.4 19.8 3.1
Pyridine I 13.4 4.5 10.6 4.2 13.2 2.1
Propionic acid III 14.0 4.6 12.5 5.0 18.5 2.9
1-Pentanol I 14.2 4.7 11.8 4.7 14.3 2.2
N,N-Dimethylformamide I 14.2 4.7 13.6 5.5 16.2 2.5
Toluene I 14.4 4.8 10.0 4.0 9.7 1.5
Butyl acetate I 15.3 5.1 11.7 4.7 10.6 1.7
Dimethylsulfoxide III 15.7 5.2 16.0 6.4 20.5 3.2
2-Furancarboxyaldehyde I 15.8 5.2 14.2 5.7 17.9 2.8
Perchloroethylene I 15.8 5.2 10.7 4.3 9.4 1.5
Chlorobenzene I 16.6 6.6 12.5 5.0 16.5 2.6
1,4-Xylene I 16.9 5.6 12.4 5.0 11.6 1.8
Ethylbenzene I 17.0 5.6 12.5 5.0 11.8 1.8
1,3-Xylene I 17.2 5.7 12.6 5.1 12.0 1.9
Cyclohexanone I 17.5 5.8 14.9 6.0 15.4 2.4
1,2-Xylene I 17.8 5.9 13.3 5.4 12.8 2.0
Aniline I 19.5 6.5 17.3 7.0 21.9 3.4
Glycerol II 19.8 6.6 19.7 7.9 – –
2-Octanol I 20.2 6.7 16.0 6.4 18.2 2.9
Benzyl alcohol I 20.6 6.8 18.6 7.5 23.5 3.7

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Table 2-15. GC Retention Time of Reagents by Chronological Order on Type I Column (continued)
Compounds Column of Type I Type I Type II Type II Type III Type III
Choice Nonpolar RT Nonpolar RR Med. Med. High High
Polar RT Polar RR Polar RT Polar RR
Diethanolamine I 20.6 6.9 20.0 8.0 – –
1-Methyl-2-pyrrolidone I 20.6 6.8 19.3 7.7 21.8 3.4
1-Octanol I 21.2 7.0 17.8 7.2 19.2 3.0
Nitrobenzene I 22.0 7.3 19.4 7.8 22.1 3.5
Quinoline II – – 21.6 8.7 25.0 3.9
Formamide III – – – – 23.0 3.7
Note: RT is retention time; RR is relative retention (methanol 1.0); and “–” indicates data not available.

Procedure for Gas Chromatography

Column . . . . . . . . . . . . . . . . . . . . . . . . . . 30 m × 0.53 mm i.d. fused silica capillary, coated with a film that has been
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surface-bonded and cross-linked

Column Temperature . . . . . . . . . . . . . . . . . 40 °C isothermal for 5 min, then programmed to 220 °C at 10 °C/min, hold
2 min
Injector Temperature . . . . . . . . . . . . . . . . . 150–220 °C
Detector Temperature . . . . . . . . . . . . . . . . 250 °C
Sample Size . . . . . . . . . . . . . . . . . . . . . . . 0.2 µL splitless
Carrier gas. . . . . . . . . . . . . . . . . . . . . . . . Helium at 3 mL/min
Detector . . . . . . . . . . . . . . . . . . . . . . . . . Thermal conductivity or flame ionization

Standard-grade reference materials (see Part 5) must be assayed by multiple methods. Specific GC methods are given under
most classes of standard-grade reference materials. Methods differ mainly in the type of stationary phase employed and the
physical dimensions of the capillary column.

Correction for water (when required) = Area % for the assay peak × (100% water by KF or any other method)/100

Identity by Gas Chromatography−Mass Spectrometry

Standard-grade reference materials (see Part 5) must be identified by multiple methods. GC–MS is one of the primary
identity methods used. Specific conditions are found under each class of standard-grade reference materials.

Carbonyl Compounds by GC−MS

Any standard GC–MS instrument may be used. The column below was used to validate the method, but any column that
provides separation of the analytes of interest is acceptable.

GC Parameters
Column . . . . . . . . . . . . . . . . . . . . . . . . . . DB1 column, 105 m × 0.32 mm i.d., 3µm phase thickness
Injector temperature and pressure . . . . . . . . Specified for each method
Carrier gas. . . . . . . . . . . . . . . . . . . . . . . . Helium
Sample size . . . . . . . . . . . . . . . . . . . . . . . Specified for each method
Oven temperature program . . . . . . . . . . . . . Specified for each method

Mass spectrometer detector parameters

Ion source and interface temperature . . . . . . 230 °C

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Ion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . Any convenient ion may be used to monitor the concentration of each
analyte
Acquisition mode . . . . . . . . . . . . . . . . . . . Scan, 0.2 s interval, speed 2500 m/z/s, mass range 15–450 m/z

Because of some system requirements, it may be necessary to turn off the filament as the solvent elutes.

Liquid Chromatography
Reagents and standard-grade reference materials can be analyzed by various modes of liquid chromatography. In this book,
reagents not suitable for assay by gas chromatography are analyzed by liquid chromatography. Some standard-grade
reference materials are used for LC applications. Specific tests for LC suitability for these materials are described in the
class specifications. Thin-layer chromatography is used to confirm the purity of standard-grade reference materials. Ion
chromatography is used to determine the anion level of some reagents.

Solvent Suitability for Liquid Chromatography

Solvents used in liquid chromatography are tested for suitability in gradient elution analysis. The objective is to assess the
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performance suitability of the solvents under routine LC operating conditions. The operating parameters found under the
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specific reagent were selected to simulate a typical gradient run. The reverse gradient portion of the chromatogram is of
little practical interest in actual sample analysis. Thus, only the peaks eluted in the “up-gradient” portion of the program
are recorded (e.g., water to acetonitrile or water to methanol). Commercially available reverse-phase columns come in a
variety of supports and stationary phases. Thus, parameters such as monomeric vs polymeric phase, endcapped vs
nonendcapped, particle size, and %C loading are specified.

Procedure for Liquid Chromatography

Column: Octadecyl (C-18 polymeric phase), 250 mm × 4.6 mm i.d., 5 µm, 12% C loading, endcapped
Mobile Phase:
A. Solvent to be tested

B. LC reagent-grade water

Conditions:
Detector: Ultraviolet at 254 nm

Sensitivity: 0.02 AUFS (absorbance units full scale)

Gradient Elution:
1. Program the LC system to develop a solvent–water gradient as follows:

Time (min) Flow (mL/min) %A (Solvent) %B (Water)

0 2.0 20 80
30 2.0 20 80
50 2.0 100 0
60 2.0 100 0

2. Start the program after achieving a stable baseline.

3. Disregard the results of the first program. Repeat the program, and use these results in the Step 4 evaluation.

4. No peak should be greater than 25% of full scale (0.005 absorbance units).

Reagent-specific conditions can be found under the individual reagents.

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Extraction−Concentration
Solvent Suitability for Extraction−Concentration
Trace-level analysis, such as for pollutants in the environment and toxic chemicals in the workplace, frequently requires
the use of solvent-extraction procedures to isolate the analyte. In some cases, the solvent is then removed to concentrate
the analyte for detection and quantification. It is critical that the solvent be of the highest purity so that potential
interferences during subsequent chromatographic analysis are minimized by a small or narrow solvent “front” and the
absence of extraneous peaks under the temperature-programmed conditions commonly used for these analyses.

In this book, the purity of the solvent is specified as suitable for use with FID (universal) and ECD (class-specific) detectors.
For analysis of solvents that are not compatible with a given detector, as in the case of halogenated solvents and ECD, a
solvent-exchange step is used before GC analysis. In LC analysis using gradient elution and UV detection, a pure solvent
would not show extraneous peaks or cause unusual baseline drift when analyzed as a sample. In this book, this suitability is
specified by measuring the absorbance through a particular spectral region for each solvent. The solvent should give a
smooth curve and contain no extraneous absorptions throughout the specified region.

Procedure for Headspace Analysis Suitability

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Parameters for GC
Column . . . . . . . . . . . . . . . . . . . . . . . . . . SPB™-624, 30 m × 0.32 mm i.d. × 1.8 µm film thickness
Column Temperature . . . . . . . . . . . . . . . . . 40 °C for 4 min, 8 °C/min to 60 °C, 5 °C/min to 85 °C, hold 2 min, 30°C/
min to 220 °C, hold 2 min
Carrier Gas . . . . . . . . . . . . . . . . . . . . . . . Helium at 1.5 mL/min
Injector Temperature . . . . . . . . . . . . . . . . . 225 °C
Injection . . . . . . . . . . . . . . . . . . . . . . . . . Headspace, split 5:1; 2 mm i.d. liner used
Detector Temperature . . . . . . . . . . . . . . . . 270 °C (FID)

Parameters for HS
Oven Temperature. . . . . . . . . . . . . . . . . . . 100 °C
Loop Temperature . . . . . . . . . . . . . . . . . . . 110 °C
Transfer Line Temperature . . . . . . . . . . . . . 150 °C
Vial Equilibration Time . . . . . . . . . . . . . . . . 10 min
Pressurization Time . . . . . . . . . . . . . . . . . . 0.2 min
Loop Fill Time . . . . . . . . . . . . . . . . . . . . . 0.2 min
Loop Equilibration time . . . . . . . . . . . . . . .3 s
Vial Pressure . . . . . . . . . . . . . . . . . . . . . . 15 psi
Transfer Line Pressure . . . . . . . . . . . . . . . . 25 psi
Loop volume . . . . . . . . . . . . . . . . . . . . . . 1.0 mL
Inject Time . . . . . . . . . . . . . . . . . . . . . . . 1 min

Results
The assay is given by the ratio of the major peak (the solvent) to all the peaks in the chromatogram.

Procedure for ECD and FID Suitability

Preconcentration
Preconcentration of the solvent is required to reach the specification levels set for ECD and FID. Preconcentration can be
accomplished with a conventional Kuderna–Danish evaporator concentrator or a vacuum rotary evaporator. The solvent
should never be evaporated to dryness.
Pr o c e d u r e . Accurately transfer 500 mL of test solvent to a 1 L evaporator. Concentrate (100×) to approximately 5 mL. If
solvent exchange is necessary, add two separate 50 mL portions and evaporate to 5 mL each time.

Analysis
Analyze the concentrated solvent and an external standard by using a gas chromatograph equipped with wide-bore capillary
columns and ECD and FID detectors. The parameters cited here have given satisfactory results.

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Parameters for GC−ECD

Column . . . . . . . . . . . . . . . . . . . . . . . . . . 30 m × 0.53 mm i.d. fused silica capillary, coated with 1.5 µm film of 5%
diphenyl, 94% dimethyl, 1% vinyl polysiloxane that has been surface-
bonded and cross-linked
Column Temperature . . . . . . . . . . . . . . . . . 40–250 °C at 15 °C/min, hold at 250 °C for 15 min
Injector Temperature . . . . . . . . . . . . . . . . . 250 °C
Detector Temperature . . . . . . . . . . . . . . . . 320 °C
Carrier Gas . . . . . . . . . . . . . . . . . . . . . . . Helium at 2–5 mL/min
Sample Size . . . . . . . . . . . . . . . . . . . . . . . 0.5 µL split/splitless (100:1 split)
Standard . . . . . . . . . . . . . . . . . . . . . . . . . 1 µg/L of heptachlor epoxide in hexane
Standard Size . . . . . . . . . . . . . . . . . . . . . . 0.5 µL split/splitless (100:1 split)

Parameters for GC−FID

Column . . . . . . . . . . . . . . . . . . . . . . . . . . 30 m × 0.53 mm i.d. fused silica capillary, coated with 1.5 µm film of 5%
diphenyl, 94% dimethyl, 1% vinyl that has been surface-bonded and cross-
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linked
Column Temperature . . . . . . . . . . . . . . . . . 40–250 °C at 15 °C/min, hold at 250 °C for 15 min
Injector Temperature . . . . . . . . . . . . . . . . . 250 °C
Detector Temperature . . . . . . . . . . . . . . . . 250 °C
Carrier Gas . . . . . . . . . . . . . . . . . . . . . . . Helium at 2–5 mL/min
Sample Size . . . . . . . . . . . . . . . . . . . . . . . 0.5 µL split/splitless (100:1 split)
Standard . . . . . . . . . . . . . . . . . . . . . . . . . 1 mg/L of 2-octanol in hexane
Standard Size . . . . . . . . . . . . . . . . . . . . . . 0.5 µL split/splitless (100:1 split)

Results
The purity of the solvents is expressed in micrograms per liter for FID and nanograms per liter for ECD, measured against an
external standard appropriate for the analysis being performed. In this book, 2-octanol is used for FID, and heptachlor
epoxide is used for ECD. Measure the peak area height for all peaks inside a window of 0.5–2 times the retention time of 2-
octanol for GC–FID suitability. The sum of the peaks must be no greater than 10 µg/L, with no single peak greater than 5.0
µg/L. Measure the peaks for all peaks inside a window of 0.2–2 times the retention time of heptachlor epoxide for GC–ECD
suitability. The sum of the peaks must be no greater than 10 ng/L, with no single peak greater than 5.0 ng/L. For pesticide
residue analysis, the standards might also be a series of pesticides, or for drinking water, a series of halomethanes. Detailed
procedures for such analysis can be found in many excellent references.

LC Suitability
Procedure for Absorbance
Measure the absorbance of the sample in a 1 cm cell against water (as the reference liquid) in a matched cell from 400 nm
to the cutoff of the reagent solvent. The absorbance should be less than 1.00 at the cutoff wavelength and not exceed the
absorbances at each wavelength specified in the requirements of the reagent.

Thin-Layer Chromatography
Procedure for Thin-Layer Chromatography
Prepare a solvent system as specified in the individual reagent monograph. Prepare also a developing chamber that can be
sealed, and fill it to a depth of approximately 3 mm with the solvent system. Cut a piece of filter paper to the appropriate
size, place it inside the chamber, and then close the chamber. Shake the chamber gently to ensure saturation of the filter
paper and chamber. Prepare a sample solution in a volatile solvent, such as methanol or dichloromethane, at a
concentration of about 1–10%. Dry a TLC plate approximately 10 cm in height in an oven at 110 °C, and store it in a
desiccator before use.

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Place a spot of the sample solution onto the plate approximately 0.5 cm from the bottom using a capillary tube. The
diameter of the spot should be 1–2 mm. Allow the solvent to evaporate, and repeat the spotting procedure one or two more
times, each time allowing the solvent to dry (the amount of sample applied will vary depending on sample concentration).
Excess sample will result in loss of resolution because the sample will “streak” up the plate or form a broad and undefined
spot. Carefully place the plate into the developing chamber on the opposite side from the filter paper. Make sure that the
sample spot is above the solvent level. Allow the solvent to climb to about 1 cm from the top of the plate. Remove the
plate, and mark the solvent front. Detect the analytes as specified in the method. The Rf value should be in the range of
0.3–0.5.

Ion Chromatography
Procedure for Ion Chromatography
Ion chromatography is used in this book to determine the anion content of various reagents. The specific procedure is given
under the individual reagent monograph. The methods are based on the initial removal of any anionic matrix, when one is
present. The use of a chemical suppression method is recommended. The water used in the preparation of the standards
and as a diluent should be on the order of 0.06 micromho cm–1. The use of a preconcentration column can dramatically
lower the concentration at which anions can be determined.
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Calibration for GC and LC Systems

Because the response of a detector is not always the same for equal concentrations of all components, an instrument must
be calibrated to obtain concentrations in terms of weight percentages. Correction of area values to weight values is
accomplished by the use of calibration factors (also referred to as response factors) that may be obtained in one of the
following ways.

Calibration Mixture
A mixture is prepared to correspond as closely as possible in composition to the sample to be analyzed. For the preparation
of a calibration mixture, it is desirable, but not always possible, to start with components of negligible impurity. When the
impurity content cannot be ignored, it may be established by an independent means. The mixture is analyzed by the
prescribed procedure, and the area of each peak is measured. The calibration factor for each component is the ratio of its
weight or concentration to its area. For an unidentified component, the calibration factor cannot be determined and is
usually assumed to be the same as that of a known component in the calibration mixture. The frequency of calibration is
left to the analyst and should follow normal good laboratory practices.

Standard Addition Technique

The sample is analyzed by the prescribed procedure before and after the addition of a measured amount of the component
of interest. The ratio of the area obtained for that component, before and after the addition, combined with the weight of
added component can be used to calculate the calibration factor.

System Suitability Tests for GC and LC Systems

To determine whether a chromatographic system can provide acceptable, repeatable results, it can be subjected to a
suitability test at the time of use. Underlying such a test is the concept that electronics, equipment, column, and standard
or samples, coupled with sample handling, constitute a single system. Specific data are collected from repeated injections
of the assay preparation or standard preparation. The results are compared with limiting values for key parameters, such as
peak symmetry, efficiency, precision, and resolution.

The most useful test parameter is the precision of replicate injections of the analytical reference solution, prepared as
directed under the individual reagent. The precision of replicate injections is expressed as the relative standard deviation
as follows:

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where SR is the relative standard deviation in percent, X is the mean of the set of N measurements, and Xi is an individual
measurement. When an internal standard is used, the measurement Xi usually refers to the measurement of the relative
area, AS:

where aR is the peak area of the substance being analyzed, and ai is the peak area of the internal standard.

The peak asymmetry value is useful and can be limited by specifying an asymmetry factor. The asymmetry factor, T, is
defined as the ratio of the distance from the leading edge to the trailing edge divided by twice the distance from the
leading edge to the peak perpendicular measured at 5% of the peak height (Figure 2-7). Therefore,
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Figure 2-7. Chromatogram showing peak asymmetry factor

This asymmetry factor is unity for a symmetrical peak and increases in value as the asymmetry becomes more pronounced.

Resolution, R, can be specified to ensure separation of closely eluting components or to establish the general separation
efficiency of the system. R can be calculated as follows:

where t2 and t1 are the retention times of the two component peaks, and W(h/2)1 and W(h/2)2 are the half-height widths of
the two peaks in units of time. An R value of 1.0 means that the resolution is 98% complete. This condition is sufficient, in
most cases, for peak area calculations. An R value of 1.5 or greater represents baseline or complete separation of the
peaks.

DOI:10.1021/acsreagents.2007
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ACS Reagent Chemicals; American Chemical Society: Washington, DC, 2017.