1586
`
`(731} Loss on Drying / Physical Tests
`
`centration at the interface, producing a greater voltage drop at
`the interface, which in tum causes the glycinate to catch up to
`the chloride. Under these conditions, a very sharp interface is
`maintained, and as it moves through the sample and spacer layers,
`the proteins in the sample tend to stack themselves at the inter(cid:173)
`face in very thin layers m order of mobility. The process is called
`stacking and is the source of the disks which arc separated.
`When the stacked proteins reach the high-density separating
`gel, they are slowed down by a molecular sieving process. The
`higher pH encountered in the running gel also causes the glycin(cid:173)
`ate to migrate faster, so that the discontinuous buffer interface
`overtakes the proteins and eveniually reaches the bottom of the
`separating gel. During this period, the dfaks of protein continue
`to separate by electrophoresis and molecular sieving in the sep(cid:173)
`arating gel. At the end of the run, the pH of the separating gel
`will have risen above its original value of 8.9 to a value of about
`pH 9.5.
`.
`.
`Relative Mobi/ity--Bromophcnol blue is often used as a stan(cid:173)
`dard for calculating the relative mobility or separated zones and
`to judge visually the progress of a run. It may be added to one
`of the sample wells, or mixed with the sample itself, or simply
`added lo the buffer in the upper sample reservoir.
`Relative mobility, M8, is calculated as
`M _
`distance from origin to sample zone
`8
`- distance from origin to bromophenol blue zone
`· .
`Visualization of Zones-Since po!yacrylamide is transparent,
`protein bands may be located by scanning in a densitometer with
`ultraviolet light. The zones may be fixed by immersing in protein
`precipitants such as phosphotungstic acid or 10% trichloroacetic
`acid. A variety of staining reagents including naphthalene black
`(amido black) and Coomassie brilliant blue R250 may be used.
`The fixed or stained zones may be conveniently viewed and ph~
`tographed with transmitted light from an X-ray film illuminator.
`
`SAFETY PRECAUTIONS
`Voltages used in electrophoresis can readily deliver a lethal
`shock. The hazard is increased by the use of aqueous · buffer
`solutions and the possibility of working in damp environments.
`The equipment, with the possible exception of the power sup(cid:173)
`ply, should be enclosed in either a grounded metal case or a case
`made of insulating material. The case should have an interlock
`that deene~izes the power supply when the case is opened, after
`which reactivation should be prevented until activation of a teset
`switch is carried out.
`· High-voltage cables from the power supply to the apparatus
`should preferably be a type in which a braided metal shield com(cid:173)
`pletely encloses. the insulated central conductor, and the shield
`should be grounded. The base of the apparatus should be grounded
`metal or contai1fa grounded metal rim which is constructed in
`suc.h a way that any leakage of electrolyte will produce a short
`which wilJ deenergize the power supply before the electrolyte can
`flow beyond the protective enclosure.
`If the power supply contains capacitors as part of a filter cir(cid:173)
`cuit, it should also contain a bleeder resistor to ensure discharge
`of the capacitors before the protective case is opened. A shorting
`bar that is activated by opening the case may be considered as
`an added precaution.
`·
`Because of the potentiaJ hazard associated with electrophoresis,
`laboratory personnel should be completely familiar with electro·
`phoresis equipment.befo~ using it.
`
`(731) LOSS .ON DRYING
`The p.roccdure set forth in this chapter determines the amount
`of volatile matter of any kind that ii; driven off under the con(cid:173)
`ditions specified . . for substances appearing to contain water as
`the only volatile constituent, the procedure given in the chapter,
`Water Determination {921 }, is appropriate, and is specified in
`the individual monograph.
`Mix and accurately weigh the substance to be tested, aod,
`unless otherwise directed in the individual monograph, conduct
`the determination on 1 to 2 g. If the lest specimen is in the form
`of large crystals, reduce the particle size to about 2 mm by quickly
`crushing. Tare. a glass-stoppered, shallow weighing bottle that
`has been dried for 30 minutes under the same conditions to be
`
`USP XXII
`
`employed in the determination. Pul the test specimen in the
`bottle, replace the cover, and accurately weigh the bottle and the
`contents. By gentle, sidewise shaking, distribute the lest speci(cid:173)
`men as evenly as practicable to a depth of about 5 mm generally,
`and not more than 10 mm in the case of bulky materials. Place
`the loaded bottle in the drying chamber, removing the stopper
`and leaving it also in the chamber. Dry the test specimen at the
`temperature and for the time specified in the monograph.
`[NOTE-The temperature specified in the monograph is to be
`regarded as being within the range of ±2° of the staled figure.]
`Upon opening the chamber, close the bottle promptly, and allow
`it to come to room temperature in a desiccator before weighing.
`If the substance melts at a lower lemperafilre than that spec(cid:173)
`ified for the determination of Loss on drying, maintain .the. bottle
`with its contents for l to 2 hours at a temperature 5° to lb 0
`below the melting temperature, then dry at the s~ified tem-
`·
`perature.
`Where the specimen under test is Capsules, use a portion· !)f
`the mixed contents of not less than 4 capsules. .
`·
`Where the specimen under test is Tablets, use powder from
`not less than 4 tablets ground to a fine powder.
`· Where the individual monOb'TllPh directs that loss on drying be
`detennined by thermogravimetric analysis, a sensitive electr~
`balance is to be used. ·
`·
`·
`Where drying in vacuum over a desiccant is directed in the
`individual monograph, a vacuum desiccator or a vacuum drying
`pistol, or other suitable vacuum drying apparatus, is to be used.
`. Where drying in a desiccator is specified, exercise particular
`care to ensure that the desiccant is kept fully effective by frequent
`replacement.
`Where drying in a capiUary-l!toppered bottle in vacuum is di(cid:173)
`rected in t.be individual monograph, use a bottle or tube fitted
`with a stopper having a 225 ± 25 I'm diameter capillary, and
`maintain the heating chamber at a pressure of 5 mm or less of
`mercury. At the end of the heating period, admit dry air to the
`heating chamber, remove the bottle, and with the capillary stop(cid:173)
`per stil! in piace aiiow it to cooi L'l a <iesiccator before weighing.
`(733) LOSS ON IGNITION
`This procedure is provided for the purpose of determining the
`percentage of test material that is volatilized and driven off under
`the conditions specified. The procedure, as generally applied, is
`nondestructive to the substance under test; however, the sub(cid:173)
`stance may be converted to another form such a.s an anhydride.
`Perform the test on finely powdered materiaJ, and break up
`lumps, if necessary, with the aid of a mortar and pestle before
`weighing the specimen. Weigh the specimen to be tested.without
`further treatment, unless a preliminary drying at a lower tem(cid:173)
`perature, or other special pretreatment, is specified in the indi(cid:173)
`vidual monograph. Unless· other equipment is designated in the
`individual monograph, conduct the ignition in a suitable muffle
`furnace or oven that is capable of maintaining a temperature
`within 25° of that required for the test, and use a suitable cru(cid:173)
`cible, complete with cover, previously ignited for l hour at the
`temperature s~cified for the test, cooled in a desiccator, and
`·
`accurately weighed.
`Unless otherwise directed in the individual monograph, transfer
`to the tared crucible an accurately weighed quantity, in g, of the
`substance to be tested, about equal to that calculated by the
`formula:
`
`10/L,
`in which L is the limit (or th.e mean value of the limits) for Loss
`on ignition, in percentage. Ignite the loaded uncovered crucible,
`and cover at the temperature ( ± 25°) and for the period of time
`designated in the individual monograph. Ignite for successive I(cid:173)
`hour periods where ignition to constant weight is indicated. Ucon
`completion of each ignition, cover the crucible, and allow it to
`cool in a desiccator to room temperature before weighing.
`
`(736) MASS SPECTROMETRY
`Mass spectrometers can be used for the measurement of ionic
`mass-to-charge ratio, for the determination of ionic abundance,
`and for the study of the ionization process. In addition, studies
`of ionic reactions in the gas phase such as unimolecular decom-
`
`FRESENIUS EXHIBIT 1068
`Page 121 of 158
`
`

`

`USPXXII
`
`Physical Tests / Mass Spectrometry
`
`(736}
`
`1587
`
`position processes, and ion molecule reactions, are also possible.
`A mass spectrometer is an instrument that produces a beam
`of ions from a substance under investigation, sorts these ions into
`a spectrum according to their mass-to-charge ratio (m/z), and
`records the relative abundance of each ionic species present. Tra(cid:173)
`ditionally only lhe positive ions have been studied, principally
`because the negative ion yield from electron impact (EI) sources
`is normally low. With the introduction of the chemical ionization
`(CI) and fast atom bombardment (F AB) techniques, both of which
`can produce a high negative ion yield, interest in the analysis of
`negative ions has increased. : ·.
`In general, a mass spectrometer consists of three major com(cid:173)
`ponents, as shown in the accompanying figure: an ion source for
`producing gaseous ions from the substance(s) being studied; .an
`analyzer for resolving the ions into their characteristic mass com(cid:173)
`ponents according to the mass-to-charge.ratios of the ions present;
`and a detector system for recording the relative abundance or
`intensity of each of the resolved ionic species present. In addition.
`a sample introduction system is necessary in order to admit the
`samples to be studied to the ion source while still maintaining
`the high vacuum requirements ( ~ 10-6 to 10- s torr) of the tech(cid:173)
`nique. · As tbe accompanying figure indicates, most commercial
`instruments include a computer for conveniently handling the
`large amounts of data produced by these instruments.
`
`Sample Introduction
`Sy$tem
`
`ionSOurce
`
`..._ ___ __
`
`Detector
`
`;-------
`'
`
`I
`Computer J
`
`Analyzer
`
`Analyzers-The mass analyzer sorts the different masses pres(cid:173)
`ent in the ionized sample, and this allows one to dete.rmine the
`inass and ultimately the abundance or reJativ.c intensity of each
`ionic species present. Four of the several methods that are com(cid:173)
`monly used for analysis are (l) the quadrupole, (2) the magnetic
`analyzer, (3) the time-of-flight analyzer, and (4) the Courier trans(cid:173)
`form analyzer. Electrostatic analyzers are often used in con(cid:173)
`junction with other mass analyzers.
`In the quadrupole, mass separation may be achieved in an
`instrument composed of four coaxial rods; ideally each rod pos•
`sesses a hyperbolic cross section, but in practice circular rods are
`commonly used. Two opposite rods have a fixed electric potential
`(U), the other two have a radio-frequency alternating potential
`(V,!!). · Under the action of these electric fields, aJl of the ions
`(except one selected mass) in the ion beam are deflected to the
`sides and lost;.the selected mass (determined by the settings of
`U,V,O) is allowed through the rods. Thus, because all other m/
`z values but the selected one are rejected, the analyzer is some(cid:173)
`times called a mass filter. The theory is that as high mass ions
`take longer to traverse the analyzer and consequentfy have more
`opportunity both to fragment and t-0 collide with residual gas
`molecules, sensitivity decreases with increasing mass: this phe(cid:173)
`nomenon is called mass discrimination, and it is a fun~mental
`characteristic of quadrupole analyzers:
`In the presence of a magnetic field perpendicular to the motion
`of the positive ion beam, each ion experiences a force at right
`angles to both its direction. of motion and the direction of the
`magnetic field, thereby deflecting the beam of ions. The follow(cid:173)
`ing equation of motion applier.:
`mjz ""' lfl,J. /2V,
`in which m is the mass in atomic mass units; z is the number of
`electronic charges; H is the magnetic field strength in gauss; r
`is the ion trajectory radius in centimeters; and .Vis the acceler(cid:173)
`ating voltage. The mass spectrum is scanned by varying the
`strength of the magnetic field and detecting those ions passing
`through the exit slit as they come into "focus."
`In the time-of,flight analyzer, separation of ions of different
`masses is based on all ions being given equal energy; therefore,
`
`ions of different masses have different velocities. If there is a
`flxed distance for the ions to travel, the time of their travel will
`vary with their mass, the lighter masses traveling more rapidly
`and thereby reaching the detector in a shorter period of time.
`The time of flight is given by:
`. · t(f) .., k v'm/z,
`in which t(j) is th.e time of flight in seconds, and m and z are
`the same as defined previously. Thus, the time-of-flight of the
`various ions is simply proportional to the square root of the mass·
`to-charge ratio of the ions.
`Fourier transform mass spectrometry (Ff-MS) is a technique
`based on the cyclotron motion of ions in a uniform magnetic field.
`In such a field of flux density B, ions are constrained to move in
`circular (cyclotron) orbits. The angular frequency, w, of the cy(cid:173)
`clotron motion is given by the equation:
`z X B
`w = - - -
`m
`In the cyclotron resonance mass s_pectrometer, the cyclotron or(cid:173)
`bits can be expanded by subjectmg the ions to an alternating
`electric field. When the frequency of the signal generator matches
`the cyclotron frequency, the ions are steadily accelerated to larger
`and larger radii leading to a coherent motion (of an ensemble of
`ions) corresponding to a significant amount of kinetic energy.
`After excitation is turned off, the cyclotronic ions give rise to an
`alternating image current on the electrodes, which is amplified.
`A frequency analysis of the corresponding receiver signal yields
`the mass of the ions involved with high precision. Thus, the
`Fourier transform of the time domain transient signal yields the
`corresponding frequency spectrum from which the mass spectrum
`is computed.
`Ionization Techniques-Positive ions may be produced by
`passing a beam of electrons through a gas at pressures of about
`10-• to 10-6 mm (Hg). Pressures other than these may be em(cid:173)
`oloyed, but lbis range is the mo.qt common. l'hP, energy t.>f the
`electron beam is usually controlled. If the energy is greater than
`the ioniz.ation potential of the gas, the electrons m.ay cause ion(cid:173)
`ization and/or fragmentation of the gas moleculc:s, represented
`as follows:
`·
`·
`e- + M-M+· + 2e-.
`Sources of this type are called electron bombardment (or elec(cid:173)
`tron impact, EI) sources. The electrons are usually emitted from
`a 4eated tungsten or rhenium filament.
`Ions formed in the ionization chamber are accelerated through
`the source exit slit toward the analyzer region by a repeller/draw
`out field determined partly by field penetration through the source
`exit slit, and partly by a small potential applied to an ion repeller
`plate in the ionization chamber. The ions are further accelerated
`by the much larger field existing between the ionization chamber
`and the source exit ~lits, the final slits being _at gro~nd potential.
`Conver$ion of a mass spectrometer to the negative 10n mode
`is straightforward, and modern equipment is designed to execute
`the procedure automatically on selection of a single parameter.
`All that is required, in theory, is to reverse all operating voltages
`and fields. While ne~tive ions are aJso formed in the various
`ionization processes discussed, the introduction of a sample with
`a high electron capture cross section leads to the formation of
`abundant negative ions. For this reason, multi-haJide derivatives
`of compounds to be studied are often prepared.
`Netative ion MS studies l;lave been successfully applied to
`pestietde analyses, since their structures are favorable f01: the
`technique. ·
`·
`'
`In the field ionization (Fl) source ions are formed in the strong
`elec~tatic field set up at the tip of a WJre electrode to which
`a bigh voltage is applied. · fons fonned from molecules present
`.on the tip of the wire are almost all parent ions. The source is
`not widely used but is of cons1dera~le value in ~udying very un~ta(cid:173)
`ble molecules or very complex mixtures and in surface reaction
`studies.
`·
`·
`Field desorption (1-1)) may be considered as an extension of
`field ionization; the ma.in difference is that the sample is coated
`on the field ion emitter tip and ionization occurs from the solid
`phase. The technique requires experience to obtain reliable re(cid:173)
`sults. Mass spectra consisting mamly of molecular ions may be
`recorded from highly nonvolatile and thermally labile compounds.
`
`FRESENIUS EXHIBIT 1068
`Page 122 of 158
`
`

`

`1588
`
`(741) Melting Range or Temperature / Physical Tests
`
`USP XXII
`
`Chemical ionization (CI) is a popular secondary ionization
`technique, and most new instruments are • purchased with this
`capability.
`In chemical ioni1..ation, a reagent gas at a pressure of about
`0.1 to 10 torr is admitted to the source and ionized. At this
`pressure, ion-molecule reactions occur and the primary reagent
`gas ions react further. The most commonly used reagent gases
`are methane, isobutane, and ammonia. Typical reactions for
`methane are shown in the following equations:
`CH.+ e--. CH.+.+ CH3+. + CH2+.
`CH4+. +CH•-+ CHs+ + CH,•
`CH3+ + CH4 ..... CaHs+ + H2
`The species CH5 + is a strong Bronsted acid and can tramfer
`a proton to most organic compounds, as follows:
`CHs + + M-+ MH + + CH4
`With methane, the protonated molecule ion (MH)+ formed
`initially may be sufficiently ener~etic to dissociate further.
`The fast atom bombardment {FAB) method uses a beam of
`fast (neutral) atoms to bombard the sample. Thus,.the first re(cid:173)
`quirement .of this technique is a beam of fast-moving atoms, prop(cid:173)
`erly aimed at the target sample, which is dissolved in a nonvolatile
`liquid matrix. This is relatively easy to achieve, and methods for
`producing such beams are well developed. Essentially a fast atom
`gun consists of an ion gun with a collision cell in front of it. The
`ion gun is used to produce a xenon ion beam, which is then charge
`exchanged in ,the collision cell with xenon gas to produce the
`required beam of fast xenon atoms. The process is summarized
`in the following equations:
`Xe -xe+ + e(cid:173)
`--
`Xe+ + Xe ·-. Xe + Xe+
`
`-
`
`in which the subscript arrow indicates the fast-moving particle.
`$ince FAl:J Ill a surface analysis techruque, the preparation of
`the sample, in 9rder to optimize the surface conditions, is of
`paramount importance. When the sample is coated on the probe
`by evaporation of a solution, the resultant sample ion .. beam is
`often transitory. Adduct ions are frequently produced. The pref(cid:173)
`erential formation of (M + Na) and (M + K) adduct ions has
`some parallels in FD, especially in the ioniza~on. of su~a~. lp.is
`phenomenon can be used to good effect to assist m the_1omzauon
`of these classes of compounds. Frcq_uently, the sample surface
`is treated with sodium chloride solution to enhance the yield of
`the adduct ions. Heating the sample during analysis can some(cid:173)
`times increase the ion yield;
`The suppression of sample ion is probably due to destruction
`of the sample surface, and a means of continuously replenishing
`the sample surface during the analysis is required. Dissolving
`the sample in a suitable, nonvolatile liquid and coating this mix~
`ture onto the probe tip achieves this. Using this approach, sample
`lifetimes of greater than I hour have been realized in .. the 10n
`source, and the range of compounds amenable to FAB has ex(cid:173)
`panded dramatically. · These long sa_mple lifetimes and higher
`sensitivity make FAB an important mass spectral technique for
`producing mass spectra from novel, difficult-to-band.le, biochem-
`1ca!s, and also aUow unequivocal identification of the elemental
`formula of the material through accuni.te·mass determination. A
`further advantage of FAB, useful in structural determination. is
`tb.e presence of fragment ions within the spectra.
`•
`Sampie Introduction-The sample is to be admitted to the
`ionization chamber in the gaseous or vapor state. Since in.any
`samples are gases or liquids at room temperature i,.nd atmospheric
`pressure, a. sample handling system and a leak arrangement to
`tbe ion source are all that are requir~.
`.
`To pl'Oduce from solids I\ mo1er.11lAr ™".Am. directly within the
`vacuum SY.Stem can be il simple matter of heating, a splid sample
`in a cruc1blc to a sufficiently high .temperature. Several com(cid:173)
`mercially available probes, or cartridges, are used, depending
`upon the particular mstrumcnt and applications involved.
`Other analytical instruments are used as inlets into the mass
`spectrometer. The most popular and most successful early de(cid:173)
`velopment in . mass spectrometry was the co.mbination of a gas
`chromatograph and .mass spectrometer (GC/MS). This com(cid:173)
`bined instrument was a ready success, since the effluent of the
`
`gas chromatograph was in the vapor state and the primary prob(cid:173)
`lem or the combined instrument was the task of selectively ~
`moving the unwanted carrier gas.
`•
`Combining the liquid chromatograph with the mass spectrom(cid:173)
`eter (LC/MS) was a far more challenging problem. While the
`li9uid chromatograph is a powerful separative imtrument, the
`w1de!y used eluti~ solvents are often quite P-O!ar, compJex, a!1d
`relatively nonvolatile. Nevertbel~, the coupling of the two tn•
`struments has been achieved and LC/MS instruments are com(cid:173)
`mercially available.
`Finally, nearly all of the various combinations of one mass
`spectrometer being an inlet system with another mass spec;trom(cid:173)
`eter (MS/MS) have been developed and studied (e.g., TOF with
`Magnetic sector, two magnetic sectors, quadrupole with a mag(cid:173)
`netic sector, etc.). Application of this technique has been most
`successful for mixture analysis. It has been applicable also to
`structure analysis where it was necessary to ionize the molecule
`of interest by a techni<\ue yielding mostly parent ions, then in(cid:173)
`troducing these parent ions into a second mass spectrometer in
`order to study fragmentation patt.crns.
`Data Analysis and lnterpretation-Altbou~ molecules are
`normally electrically neutral, if one electron ,s taken away or
`added, a molecular ion results. The mass of this ion is the mo(cid:173)
`lecular weight of the molecule under study. Furthermore, it is
`often possible to determine the acc1irate mass of this ion with
`sufficient precision to enable the calculation of the empirical
`formula of the compo1ind. Accurate masses may be determined
`at high resolution by either scanning or by peak-matching mea(cid:173)
`surements.
`Fragment ions are those produced from the molecular ion by
`various bond cleavage processes. Numerous papers in the liter(cid:173)
`ature relate the bond cleavage panems (fragmentation patterns)
`to molecular structure.. Correlations of mass spectra and molec(cid:173)
`ular structure are discussed for steroids, aromatics, aliphatics,
`and, recently, complex compounds arising from biotechnology.
`The mass spectrum is often very complex and not.all of the
`ions may be separated by the mass spectrometer. The limit of
`the 11bility of the instrument to separate two ions very close in
`mass is called the resolving power of the instrument. The most
`common definition of the resolving power of a mass spectrometer
`is the "10% valley" definition. This states that the resolving
`power of a mass spectrometer is the highest mass number at which
`peaks of adjacent molecular weight and equal heights have a
`valley between them of 10% of the peak height. In mass spec(cid:173)
`trometry, low resolution covers the range of about 100 to 2000,
`medium resolution 2000 to 10,000, and high resolution greater
`than 10,000.
`. · Quantitative analysis in mass spectrometry is usually per(cid:173)
`formed in one of two ways. The first is selective ion monitormg.
`In this technique the ions, or group of ions of interest, are indi(cid:173)
`viduaJJy focused on the detector and measured. Both sensitivity
`and selectivity are enhanced by this technique.
`·
`The second most popular quantitative technique is isotope di•
`Jution. This method may be applied through the use of either
`radioactive or stable isotopes, the latter is most popular for mass
`spectrometry. The isotope dilution technique has the unique ad(cid:173)
`vanta(:e that it is not necessary to recover all of the original
`matenal being analp,ed to obtain the quaotitative information
`desired. The technique has been successfully applied in biological
`studies, often in combination with GC/MS or LC/MS.
`.: .
`·. (741) MELTING RANGE OR
`·TEMPERATURE
`For Pharmacopeial purposes, the melting range or temperature
`of a solid is defined as those points of temperature within which,
`or the point at which, the solid coalesces. and is completely melted,
`1:rr.,ent 'IR tlr.finP.ci otherwi.!lf: for. Classes l( Mrl ITr below. Any
`apparatus or method capable of equal accuracy may be used:
`The accuracy should pc checked frequently by the use of one or
`more of the six USP Melting Point Reference Standards, pref(cid:173)
`erably the one that melts nearest the melting temperat111e of the
`com.JlOund to be: tested (see USP Reference Standards { 11) ).
`· Five procedures for the determination of melting range or tem(cid:173)
`perature are given herein, varyin;in accordance with the nature
`of the substance. When no class ts designated in the monograph,
`use the procedure {or Class Ia.
`
`FRESENIUS EXHIBIT 1068
`Page 123 of 158
`
`

`

`USP XXII
`
`Physical Tests / Minimum Fill {755}
`
`1589
`
`The procedure known as the mixed-melting point detennina•
`tion, whereby the melting range of a solid under test is compared
`with that of an intimate mixture of equal parts of the solid.and
`an authentic specimen of it, e.g., the corresponding USP Ref•
`erence Standard, if available, may be used as a confirmatory
`identification test. A$reement of the observations on the original
`and the mixture const1ll1tes reliable evidence of chemical identity.
`ApparatlLS--An example of a suitable melting range apparatus
`consists of a glass container for a bath of transparent fluid, a
`suitable stirr.ing device, an accurate thermometer (see Thermom(cid:173)
`eters {21)),* and a controlled source of heat. The bath fluid is
`selected with a view to the temperature required, but light par(cid:173)
`affm is used generally and certain liquid silicones are well adapted
`to the higher temperature ranges. The fluid is deep enough to
`permit immersion of the thermometer to its specified immersion
`depth so that the bulb is still about 2 cm above the bottom of
`the bath. The heat may be supplied by an open f1ame or elec(cid:173)
`trically. The capillary tube is about 10 cm long and 0.8 to 1.2
`mm in internal diameter with walls 0.2 to 0.3 ni.m in thickness.
`Procedure for Ous I-Reduce the substance under test to a
`very fine pow~er, and! unless ot~crwise ~irccted, render it. an(cid:173)
`hydrous when 1t contains water, of hydration by drying it at the
`temperature specified in the monograph, or, when the substance
`contains no water of hydration, dry it over a suitable desiccant
`for not less than 16 hours.
`Charge a capillary glass tube, one end of which is sealed, with
`sufficient of the dry powder to form a column in the bottom·of
`the tube 2.5 to 3.S mm high when packed down ·as ·closely as
`pos.5ible by moderate tapping on a solid surface. · · · ·
`Heat fhe bath until the temperature is about 30° ·below the
`expected melting point. Remove the thermometer, and quickly
`attach the capillary tube to the thermometer by wettin~ both
`with a drop of the liquid of the path or otherwise, and adJust its
`height so that the material in the capillafy is level with the ther•
`mometer bulb. Replace the thermometer, and continue the heat(cid:173)
`ing, with constant stirring, sufficiently to cause the temptrature
`to rist', at 11. r&tc: of about 3° per minute. When the temperature
`is about 3° below the lower limit of the expected melting range,
`reduce the heating so that the temperature rises at a rate of about
`la to 2° per minute. Continue heating until melting is complete.
`The temperature at which the ·column of the substance under
`test is observed to collapse definitely against the side of the tube
`et any point is defined as·the beginning of melting, and the tem(cid:173)
`perature at which the test substance becomes liquid throughout
`1s defined as the end of melting or the "meltio~ point." The two
`temperatures fall within the limits of the mcltmg range. ·
`Procedure for Class l.a- PreP.3re the test substance and chasge
`the capillary as ·d~ted for Class l. Heat the bath until the
`temperature is about 10° below the expected melting point and
`is rjsing at a rate of 1 ± 0.5° per minute. insert the capillary
`BB dire<;ted under Class I when the temperature is aQOut 5 ° below
`the lower limit of the expected melting range, and continue heat·
`ing until melting is complete. Record the melting range as dj.
`rccted for Class I.
`Procedure ror Class lb-Place the test substance in a closed
`container and cool to 10°, or lower, for at least 2 hours. Without
`previous powdering, charge the cooled material into the capillary
`tube as directed for Class l, then immediately place the charged
`tube in a vacuum desiccator and dry at a pressure not exceeding
`20 mm of mercury for J hours. Immediately upon removal from
`the desiccator, fue-seal the open end of the tube, and as soon as
`practicable proceed with the determination of the meltinl range
`as follows: Heat the bath until a temperature 10 ± l below
`the expected melting range is reached, then introduce the charged
`tube, and beat at a rate of rise of 3 ± O.S 0 per minute until
`melting is complete. Record the melting range as directed for
`·
`·
`Class I.
`Jf the particle i.ize of the material is too .lar~e for the capillary,
`pre,.cool the test substance as above directed, then with as little
`pressure as possible genlly crush the particles to fit the capillary,
`and immediately charge the tube.
`.
`Procedure for Ous II-Carefully melt the material to be tested
`at as low a temperature as. possible, (Ind draw it into a capilla,ry
`tube, which is left open at both ends, to a depth of about 10 mm.
`Cool the charged tube-at I 0°, or lower, for 24 hours, or in contact
`* ASTM Method E77 deal with "Verificatio11 and Galibration
`of Liquid-in-glass Thermometers."
`
`with ice for et least 2 hours. Then attach the tube to the ther(cid:173)
`mometer by suitable means, adjust it in a water bath so th·at the
`upper edge of the material is 10 mm below the water level, and
`heat as directed for Class l except, within 5° of the expected
`melting tem11erature, to regulate the rate of rise of temperature
`to 0.5 ° to 1.0° per minute. The temperature at which the material
`is observed to ·rise in the capillary lube is the melting temperature.
`Procedure fpr Class Ill-Melt a quantity of the lest substance
`slowly, while stirring, until it reaches a temperature of 90° to
`92°. Remove the source of the heat and allow the molten sub(cid:173)
`stance to cool to a temperature of 8° to 10° above the expected
`melting point. Chill the bulb of a suitable thermometer (sec
`Thu111()meters (21)) to 5°, wipe it dry, and while it is still cold
`dip it into the molten substance so that approximately the lower
`half of the bulb is submerged. ,Withdraw it immediately, and
`hold it vertically away from the heat until the WaJ{ surface dulls,
`the~ ~ip it for 5 minutes into a water bath having a temperature
`not higher than 16°.
`Fix the thermometer securely in a test tube so that the lower
`point is 15 mm above the b9ttom of the test tube. Suspend the
`test tube in a water bath adjusted to about 16°, and raise the
`temperature of the l>ath at the rate of 2° per minute to 30°, then
`change to a rate of l O per minute, and note the temperature at
`which the rus