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Chromatography 57 Reagent ChemicalsNinth Edition

ACS SpecificationsOfficial from January 1, 20002000 American Chemical Society

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

CHROMATOGRAPHYChromatography is an analytical technique used in quantitative determination ofpurity of most organic and an increasing number of inorganic reagent chemicalsand standard-grade reference materials. The broad scope of chromatographyallows it to be used in the separation, identification, and assay of diverse chemicalspecies, ranging from simple metal ions to compounds of complex molecularstructure, such as proteins.

In chromatography, the separation of individual components in a mixture isachieved when a mobile phase is passed over a stationary phase. Differences inaffinities of various substances for these phases result in their separation.

Chromatography can be divided into two main branches, depending onwhether the mobile phase is a gas or a liquid. Gas chromatography is principallyused for analysis of volatile, thermally stable materials. Liquid chromatography isparticularly useful for analysis of nonvolatile or thermally unstable organic sub-stances. Ion chromatography, a technique in which anions and cations can bedetermined by using the principles of ion exchange, is a form of liquid chroma-tography. Thin-layer chromatography, often called planar chromatography, is alsoa 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 chromatogra-phy may be subdivided into gasliquid and gassolid chromatography. Gasliq-uid 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 oper-ated in either the isothermal or the temperature-programmed mode. Columnspacked with a nonvolatile liquid phase coated on a porous solid support wereused extensively in early editions of this monograph. In this edition, only capil-lary 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 columnswith an inside diameter (i.d.) from 0.22 to 0.32 mm, coated or bonded with a thinfilm of the liquid phase. This arrangement results in high resolution but low sam-

58 Chromatography Reagent ChemicalsNinth Edition


ple capacity. Special injection techniques and hardware are employed. The intro-duction of wide-bore capillary columns, which have an i.d. of typically 0.53 mmand a relatively thick film of the liquid phase (1 mm to 5 mm), can allow a labora-tory to use a standard packed-column instrument and conditions while gainingthe advantages of capillary technology.

Direct flash vaporization or on-column injection of the sample with stan-dard-gauge needles can be used with capillary columns. Two types of flash vapor-ization injection techniques can be employed: a split mode of operation, in whichpart of the sample is vented from the injector, or a splitless mode, in which asmaller volume is injected and no portion of the sample is vented from the injec-tor. The splitless mode, using a 0.1-mL sample size, is recommended for the assayof most reagent solvents, while the split mode often is recommended for analysisof 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 theresultant vapor enters the column while the major portion is vented to waste. Toperform a GC analysis using a split injection technique, the GC instrumentshould be configured such that a preheated carrier gas, controlled by a pressureregulator 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 gasflows upward and purges the septum. The septum purge flow is controlled by aneedle valve. Septum purge flow rates are usually between 3 and 5 mL/min. Ahigh flow of carrier gas enters the vaporization chamber, which is a glass or quartzliner, where the vaporized sample is mixed with the carrier gas. The mixed streamis split at the column inlet, and only a small fraction enters the column. A needlevalve or flow controller regulates the split ratio.

Split ratios (measured column flow/measured inlet flow) typically rangefrom 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 filmcolumns, low split ratios (1:5 to 1:50) are commonly used.

The method most commonly recommended for analysis of pure chemicalcomponents (for example, matrices with a narrow constant boiling range) is thehot needle, fast sample introduction. In this method, the sample is taken into thesyringe barrel (typically 25 mL in a 10-mL syringe) without leaving an air plugbetween the sample and plunger. After insertion into the injection zone, the nee-dle is allowed to heat up for 3 to 5 s. This period of time is sufficient for the needleto be heated to the injector temperature. Then, the sample is injected by rapidlypushing the plunger down (fast injection), after which the needle is withdrawnfrom the injector within one second. Either manual or automatic sample intro-ductions can be used. The measurement reproducibility will be enhanced by notvarying the injected volume, which typically should be 0.5 mL to 2.0 mL. The useof an automatic injection system can significantly enhance measurement preci-sion. Also, loosely packing the injection liner with deactivated glass wool or glassbeads can provide thorough mixing between sample and carrier gas, yielding less



sample discrimination and better measurement precision. However, analystsshould be aware of adsorption and decomposition.

In conjunction with wide-bore columns, on-column injection can minimizesample degradation while increasing the reproducibility of results. On-columninjection allows the injection of a liquid sample directly into the inlet of the col-umn. Excellent quantitative precision and accuracy for thermally labile com-pounds and wide volatility range samples have been reported. Sample sizes rangefrom 0.5 mL to 2 mL. The injection should be performed as fast as possible, withthe 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 boilingpoints, in comparison to the solvent, ballistic heating to high temperature isallowed. On the other hand, if the solute boiling points do not differ too muchcompared to the solvent, temperature programming is applied to fully exploit thesolvent effect. When peak splitting and/or peak distortion is observed, the con-nection of a retention gap can provide the solution.

If the composition of the sample is not that complex, the use of megabore col-umns is recommended, particularly since automated injection becomes easier. Ifhigh resolution with automated injection is needed, a deactivated but uncoatedwide bore precolumn (20 cm to 50 cm in length) should be connected to a conven-tional analytical narrow bore column. Hydrogen is the carrier gas of choice. If H2cannot be used for safety reasons, helium may be substituted. High carrier-gasvelocities (5080 cm/sec H2, 3050 cm/sec He) ensure negligible band broadening.

In comparison to vaporizing injectors (that is, split and splitless), there aresome disadvantages to on-column injections. The two primary disadvantagespertain to sample pretreatment. First, because the sample is introduced directlyonto the column, relatively clean samples must be prepared. Nonvolatile andless-volatile materials collect at the head of the column, causing a loss of separa-tion efficiency; therefore, sample cleanup is a prerequisite. Second, many samplesmay 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 less than 10 mL/min) to obtain optimum resolution of sample com-ponents or at higher flow rates (1030 mL/min), which will generate packed col-umn-quality separations in a shorter time. The increased length of capillary col-umns allows for better separation of components, with the result that as few asthree columns of high, moderate, and low polarity can handle the majority ofanalytical requirements.

A wide variety of detectors are used to quantify and/or identify the compo-nents in the eluent from the column. Thermal conductivity detectors (TCD) andflame ionization detectors (FID) are examples of general detectors that provide alinear response to most organic compounds. Electron capture detectors (ECD)and photoionization are examples of class-specific detectors often used in traceenvironmental analysis. Mass spectrometric-type detectors are used to providepositive 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 fur-ther manipulation and interpretation of the data.

Results from a chromatogram, when used to determine the assay of a reagentor standard, are often expressed as area percent. A response factor correction foreach component is required for the most accurate results, especially when thesample components differ markedly in their detector response. An internal stan-dard reduces error due to variations in injection quantities, column conditions,and detector conditions. Use of a TCD or FID minimizes the need to correct forresponse and is usually sufficient for determining reagent or standard assay.

The use of control charts to aid the analyst in visualizing chromatographicvariability is suggested. To certify that assay results are valid, use of a system suit-ability test as described in a later section is recommended.

Gas ChromatographyMass Spectrometry

Gas chromatographymass spectrometry (GCMS) is one of the techniques usedin the identity requirement for standard-grade reference materials. Mass spec-trometers are used for many kinds of chemical analysis, especially those whereidentity or proof of chemical structure is critical. Examples range from environ-mental analysis to the analysis of petroleum products and biological materials,including the products of genetic engineering. Mass spectrometers use the differ-ence in mass-to-charge ratio (m/e) of ionized atoms or molecules to separatethem from each other. Mass spectrometry is therefore useful for quantitation ofatoms or molecules and also for determining chemical and structural informa-tion about molecules. Molecules have distinctive fragmentation patterns that pro-vide structural information to identify structural components. The largest peak ina 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. creating gas-phase ions;2. separating the ions in space or time based on their mass-to-charge ratio;

and3. measuring the quantity of ions of each mass-to-charge ratio.

The ion separation power of a mass spectrometer is described by the resolu-tion, which is defined as R = m/Dm, where m is the ion mass and Dm is the differ-ence in mass between two resolvable peaks in a mass spectrum with similar massvalues. For example, a mass specrometer with a resolution of 1000 can resolve anion 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-selectiveanalyzer, and an ion detector. Since mass spectrometers create and manipulategas-phase ions, they operate in a high-vacuum system. The magnetic-sector, qua-



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

Liquid ChromatographyMany reagent chemicals and standard-grade reference materials described in thismonograph can be assayed by and/or tested for suitability for use in liquid chro-matography (LC). Liquid chromatography is a technique in which the sampleinteracts with both the liquid mobile phase and the stationary phase to effect a sep-aration. An LC system consists of a pump that delivers a liquid, usually a solventfor the sample, at a constant flow rate through a sample injector, a column, and adetector. The solvent is called the mobile phase. Typically, the flow rate is 1 to 10mL/min for conventional liquid chromatography. In isocratic operation, the com-position of the mobile phase is kept constant, while in gradient elution, it is variedduring the analysis. Gradient elution is required when the sample mixture con-tains components with a wide range of affinity for the stationary phase. In the iso-cratic mode, the purity of the solvents is less critical, as the impurities areadsorbeddesorbed at a constant rate, whereas in gradient elution impurities mayresult in extraneous peaks and/or shifts in the baseline. This characteristic necessi-tates the incorporation of a gradient elution test in this monograph to verify thequality 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 isintroduced into the mobile phase via the sample injector and enters the column,where it is separated into its individual components. The columns are usuallysteel or glass, densely packed with semirigid organic gels or rigid inorganic silicamicrospheres, to which a variety of substrates can be chemically bonded andwhose typical particle size is 3 mm to 10 mm.

Several mechanisms of separation are possible in liquid chromatography. Inadsorption chromatography, separation is based on adsorptiondesorption kinet-ics, whereas in partition chromatography, the separation is based on partitioningof the components between the mobile and stationary phases. Ion exchange is thedominant mechanism in ion chromatography. In practice, a successful separationmay involve a combination of separation mechanisms.

A commonly used term, coined by early chromatographers for describingseparations dominated by adsorptiondesorption, is normal phase. In normalphase chromatography, the stationary phase is strongly polar (for instance, silicaor aminopropyl), and the mobile phase is less polar (for instance, hexane). Polarcomponents are thus retained on the column longer than less-polar materials.

A second term of historical origin is reverse phase. Reverse phase generallyapplies to separations dominated by partition chromatography, and the elution ofcomponents in a reverse phase separation are more or less reversed from theorder that would be obtained in a normal phase separation. Reverse phase, themore widely used mode of chromatography, uses a nonpolar (hydrophobic) sta-



tionary phase, such as C-18 (octadecyl) chemically bonded to silica, while themobile phase is a polar liquid such as acetonitrilewater. Hydrophobic (non-polar) components are retained longer than hydrophilic (polar) components. Theelution properties of the mobile phase can be adjusted to modify a separation byaddition of appropriate ionic modifiers. These modifiers can be chosen for theirability to either suppress or enhance ion formation in the sample. When enhance-ment is chosen, the separation mechanism may involve both ion exchange andpartition.

The wide range of available stationary phases in combination with changes inmobile-phase composition makes liquid chromatography a very flexible separa-tion-assay technique. The back pressure on the pump is several hundred to sev-eral thousand psi, depending upon the particle size of the packing material,mobile-phase flow rate, and viscosity. The column eluent is continuously moni-tored by a sensitive detector chosen to respond either to the sample componentalone (for instance, an ultraviolet photometer) or to a change in some physicalproperty of the mobile phase due to the presence of the solute (for example, a dif-ferential refractometer). Other detectors such as electrochemical, fluorescence,etc., are also used for more specialized applications. When photometric detectorsare 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 soluteand is displayed on a recorder. A computer is usually interfaced to the LC systemto control the method and to collect and analyze data.

Ion Chromatography

Ion chromatography (IC) is a subset of liquid chromatography. Whereas conven-tional liquid chromatography is mainly used for the analysis of nonionic organiccompounds, ion chromatography separates and determines ionic species or ion-izable compounds, both organic and inorganic. Under ideal conditions, quantita-tion to the sub-part-per-billion level is attainable. For trace analysis, water andreagents of the highest quality need to be used. Deionized water of >18 MW cmresistivity is recommended.

For the testing of reagent chemicals, ion chromatography is ordinarilyapplied to the determination of anions present as minor impurities. Generally,the major component should be eliminated or greatly reduced in concentrationto avoid overloading the anion-exchange column used for separation.

IC columns are usually 525 cm in length and 210 mm in diameter. Col-umns may be either metallic or plastic. Packing materials can be either anionic orcationic resins, depending on the separation desired, and there are several varia-tions 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 mostcases, IC analyses are carried out isocratically, and gradient elutions are used onlyfor the more complex separations. Typical eluent flow rates for ion chromatogra-



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 injectionsystem. The most widely used detection method for ion chromatography is con-ductivity. However, other detection methods, including ultraviolet-visible spec-troscopy, electrochemistry, and fluorescence can be used. Conductivity is a uni-versal detection mode for ions, whereas the other detectors provide selective,analyte-specific detection.

In conductimetric IC analysis, the impact of the significant background con-ductivity of the eluent on analyte determinations is minimized by chemical orelectronic suppression. Chemical suppression involves notably reducing the elu-ent conductivity, as well as enhancing analyte response by means of a chemicalreaction. Several innovative techniques have been developed to achieve thesechanges, including packed-column and membrane-based devices. Electronic sup-pression minimizes eluent background noise via the design of the electronic cir-cuitry in the conductivity detector, although the actual background eluent con-ductivity is not reduced, as it is with chemical suppression.

Because of the two different modes of suppression, the columns and eluentsused also fall into two categories. Columns for chemical suppression typicallyhave higher ion-exchange capacities than those used for electronic suppression.Eluents used in chemical suppression also differ from those used in electronicsuppression in that they are defined by the chemical reactions that occur in thesuppression of the eluent.

Thin-Layer Chromatography

Thin-layer chromatography (TLC) is a very simple form of solidliquid adsorp-tion chromatography. It is probably the quickest, easiest, and most frequentlyapplied technique for determining purity of organic compounds. As in otherforms of liquid chromatography, the sample interacts with a liquid mobile phaseand a stationary phase to effect partitioning of components based on their affinityto the solid and liquid phase. Thin-layer chromatography serves many purposesin the laboratory because of its simplicity. It is commonly used in organic synthe-sis to monitor chemical reactions. Starting materials, intermediates, and productsoften elute differently, and product formation or starting-material disappearancemay be observed.

Another very common use is in purity determinations of organic com-pounds. Typically, it is not used as a quantitative technique, but it is useful forlooking for impurities. In many cases, thin-layer chromatography is very sensitiveand can be used to detect impurities of less than 1% in the sample. A single spoton a TLC plate is a good indication of purity. Thin-layer chromatography is alsoapplied in selected requirements for standard-grade reference materials as a tech-nique for purity confirmation. The technique is used with other complementarypurity assays to screen standard-grade reference materials for impurities. Stan-



dard-grade reference materials have passed the purity test when, having used thespecific conditions in the method, an analyst can observe a single spot.

In thin-layer chromatography, the stationary phase is spread as a thin layerover glass or plastic. Calcium sulfate or an organic polymer (such as starch) isadded to the solid phase to bind the solid to the glass or plastic. Often, fluorescentmaterial is added to the solid phase to aid in detection of analytes. TLC plates arecommercially available or can be prepared in the laboratory. Glass or liquid platesare available commercially as sheets that are cut into strips for use. Plates can beprepared 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. Platesare most effective when dried in an oven before use. The most common station-ary 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 commonto perform TLC analysis with both silica and alumina to maximize the effective-ness of the technique.

Performing TLC analysis requires minimal equipment. The plates are cutinto strips approximately 10 cm in height. The sample is dissolved in a volatilesolvent and applied to the bottom of the plate. This is accomplished by applyingor spotting the solution about 0.5 cm from the bottom of the plate using a thincapillary tube. The plate is placed into a development chamber that containsenough elution solvent to come just below the sample spot. The solvent then trav-els up the plate and moves the components of the sample at different rates accord-ing to their affinity. When the solvent is about 1 cm from the upper end of theplate, the plate is removed, the solvent line is marked and the plate is allowed todry.

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 afluorescent material within the solid phase. In this case, the plate will fluoresceand 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 organiccompounds form charge-transfer complexes with iodine, resulting in a dark-col-ored species. The TLC plate is placed in a chamber containing iodine and devel-oped for several minutes. Upon removal from the chamber, the plates will con-tain dark spots; these spots should be immediately circled with a pencil, since thecomplex is reversible and the spots will fade away. In other detection methods, theplate is sprayed with a solution to stain the analytes. Common reagents are sul-furic acid solution, which will char many organic compounds, and potassiumpermanganate solution, which will oxidize many organic compounds. Combina-tions 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 hasmoved up the plate in relation to the solvent front. This value is called the reten-tion factor and designated Rf. The Rf value is calculated by measuring the distancethat the analyte moved from the origin and dividing that by the distance the sol-

Recommended Chromatography Procedures 65 Reagent ChemicalsNinth Edition


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

RECOMMENDED CHROMATOGRAPHY PROCEDURES

Assay Methods

Procedures published in this monograph list the parameters and values estab-lished to give satisfactory results. These procedures should be considered ade-quate only for determining the constituents specified in the individual tests, andthey may not necessarily separate other components in a given sample. Anattempt is made to keep each procedure for reagents as simple as possible, whilestill retaining the desired sensitivity, because simplicity may lead to wider usage.The procedures used for standard-grade reference materials are, in general, morespecific, since it is necessary to separate similar eluting impurities.

The list of parameters for gas chromatography includes such variables as col-umn type, diameter and length of column, carrier-gas flow rate, detector type,and operating parameters. The list for liquid chromatography includes such vari-ables as diameter and length of column, packing type and particle size, mobile-phase composition and flow rate, detector type, and operating parameters. Forreagents, the parameters represent one set of conditions that result in reproduc-ible analyses. Because of differences among instruments, one or more of thestated parameters may require adjustment for any given instrument. Thereforethese parameters, except for the column and type of detector, should be regardedas guides to aid the analyst in establishing the optimum conditions for a particu-lar instrument. Relative retention times are given as an aid in peak identification.Exact reproduction of those times is not essential. Parameters given for the stan-dard-grade reference materials section, however, must be adhered to exactly asstated in the test.

Gas Chromatography

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

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