`
`
`
`Pesticide Analytical Manual Vol. I
`
`
`
`SECTION 601
`
`601: GENERAL INFORMATION
`
`In recent years, high performance liquid chromatography (HPLC) has grown in
`
`
`
`
`
`
`
`
`
`
`popularity as a determinative step for residue analysis, until today it is accepted as
`(GLC). HPLC
`
`
`
`complementary to the more traditional gas liquid chromatography
`
`
`provides capabilities not possible with GLC, most importantly the ability to sepa­
`
`
`
`
`
`
`
`rate and quantitate residues of polar, nonvolatile, and heat-labile chemicals. These
`
`
`characteristics make HPLC the determinative step of choice for many residues
`
`
`
`previously beyond the applicability of multiresidue methodology.
`
`601 A: PRINCIPLES
`
`Chromatography
`
`�
`
`Gas Liquid
`
`Chromatography comprises a family of sepa­
`
`
`Figure 601-a
`
`ration techniques (Fi
`
`gu re 601-a), all of which
`
`Chromatographic Separation
`
`share common characteristics. A narrow ini­
`Techniques
`
`
`tial zone of mixture is applied to a sorptive
`
`stationary phase having a large surface area.
`
`
`Development with mobile phase causes com­
`
`
`ponents of a mixture to move through the
`
`
`stationary phase at different rates and to
`
`
`
`separate from one another. Differential mi­
`
`
`gration occurs because of differences in dis­
`
`
`tribution between the two phases. The mo­
`Column Planar
`
`
`bile phase can be a gas or a liquid. Liquid
`I
`
`
`chromatography is divided into two main
`I
`I
`HPLC
`Classical
`
`types, planar ( thin layer and paper chroma­
`
`
`
`tography) and column. Column liquid chro­
`I
`
`
`matography, both the classical (low pressure)
`
`
`version and the high performance version
`
`
`discussed here, is further subdivided accord­
`IEC
`SEC
`LLC
`
`
`ing to the mechanism of separation into five
`BPC
`
`major types: liquid-solid (adsorption) chromatography, LSC; liquid-liquid (parti­
`
`
`
`
`tion) chromatography, LLC; bonded phase chromatography, BPC; ion exchange
`
`
`
`chromatography, IEC; and size exclusion chromatography, SEC.
`
`�
`
`I
`
`LSC
`
`HPLC developed steadily during the late 1960s as high efficiency, small particle
`
`
`
`
`
`
`
`
`packings and improved instrumentation were produced. In contrast to classical
`
`
`
`column liquid chromatography, HPLC uses high pressure pumps; short, narrow
`
`
`
`
`columns packed with microparticulate phases; and a detector that continuously
`
`
`records the concentration of the sample.
`
`HPLC systems use the principles of classical column chromatography in an analyti­
`
`
`
`
`
`
`
`
`
`cal instrument. Development of HPLC has been directly related to availability of
`etc.)
`
`
`
`
`
`suitable hardware (columns, pumps, inlet systems, low dead volume fittings,
`
`
`that allows precise flow control under the elevated pressures needed, as well as the
`
`
`
`
`
`ability to manufacture a wide variety of column packing materials in particle sizes
`
`of exacting micron (µm) dimensions.
`
`In contrast to GLC, where the gas mobile phase is inert and does not affect
`
`
`
`
`
`
`separation of analytes from one another, the HPLC mobile phase is critical to this
`
`
`
`Chapter 6 is revised from a chapter on HPLC written for FDA in 1989-90 by Joseph Sherma,
`
`
`
`
`
`
`
`
`Ph.D., Lafayette College, Easton, PA.
`
`Transmittal No. 94-1 (1/94)
`
`Form FDA 2905a (6/92)
`
`601-1
`
`
`
`
`
`Regeneron Exhibit 1053.001
`
`

`

`
`
`SECTION 601
`
`
`
`
`
`Pesticide Analytical Manual Vol. I
`
`resolution. Choice of mobile phase is second only to the choice of operating
`
`
`
`
`
`mode in determining the suitability of the system to produce the desired separa­
`tions.
`
`HPLC had limited use for routine trace multiresidue analysis in the absence of
`
`
`
`
`
`
`
`
`
`
`sensitive element-selective detectors. Early development work relied primarily on
`
`
`
`
`
`refractive index (RI) or fixed wavelength UV absorbance detectors. Neither detec­
`
`
`
`
`
`tor demonstrated sufficient sensitivity or selectivity for use in trace residue analysis.
`
`
`In the mid-l 970s, the fluorescence detector was shown to provide the needed
`
`
`
`
`
`sensitivity and specificity for pesticides that are naturally fluorescent or can
`
`
`
`
`
`be chemically labeled with a fluorophore. This resulted in the first practical appli­
`
`
`
`cation of HPLC to multiresidue pesticide determination (see method for
`
`
`N-methylcarbamates, Section 40 I).
`
`More recently, scientists have investigated photoconductivity and electrochemical
`
`
`
`
`
`
`
`
`detectors and certain applications of the newer multiwavelength UV detectors.
`
`
`
`
`
`
`This research indicates that these detectors can also fulfill the sensitivity and
`
`
`
`
`
`selectivity requirements for determination of certain pesticides at residue levels.
`
`601 B: MODES OF OPERATION
`
`HPLC
`I
`I
`
`� I SAX
`
`sex
`SEC
`I
`
`Figure 601-b
`HPLC Modes of Operation
`Separations by HPLC are achieved
`
`
`using the five basic operational modes
`
`
`(Figure 601-b). The mode chosen for
`
`
`a particular application will depend on
`
`
`the properties of the analyte(s) to be
`LLC
`
`
`separated and determined. For residue
`LSC
`
`determination, as for HPLC analyses
`
`in general, BPC is the most widely
`used.
`
`BPC
`
`I
`
`Ion
`Ion
`pair
`suppression
`
`GPC GFC
`
`There are two variations within the five
`
`
`
`operational modes ofHPLC operation;
`
`
`
`phases:
`
`
`
`these distinctions are based on the relative polarities of stationary and mobile
`
`1)normal phase (NP) chromatography: stationary phase is more polar than
`
`
`
`
`
`the mobile phase; the least polar analytes elute first; analyte retention is
`
`
`increased by decreasing mobile phase polarity.
`
`2)reverse phase (RP) chromatography: stationary phase is less polar than
`
`
`
`
`
`
`
`
`the mobile phase; the most polar analytes elute first; analyte retention is
`
`
`increased by increasing mobile phase polarity.
`
`
`
`Liquid-Solid Chromatography
`
`LSC, also called adsorption chromatography, uses an adsorbent, usually uncoated
`
`
`
`
`
`
`
`
`
`silica gel. The basis for separation is the selective adsorption of polar compounds,
`
`
`presumably by hydrogen bonding, to active silanol (SiOH) groups by orientation
`
`
`
`
`
`and on the surface of the silica gel. Analytes that are more polar will be attracted
`
`
`
`
`
`
`
`more strongly to the active silica gel sites. The solvent strength of the mobile phase
`
`
`determines the rate at which adsorbed analytes are desorbed and eluted.
`
`601-2
`
`Transmittal No. 94-1 ( 1 /94)
`
`
`Form FDA 2905a (6/92)
`
`1053.002
`Regeneron Exhibit
`
`

`

`
`
`
`
`Pesticide Analytical Manual Vol. I
`
`
`
`SECTION 601
`
`LSC is useful for separation of isomers and classes of compounds differing in polarity
`
`
`
`
`
`
`
`
`
`
`
`and number of functional groups. It works best with compounds that have relatively
`
`
`
`
`low or intermediate polarity. Highly polar compounds may irreversibly adsorb on the
`
`
`
`
`
`
`column. Poor LSC separations are usually obtained for chemicals containing only
`
`
`nonpolar aliphatic substituents.
`
`
`
`Liquid-Liquid Chromatography
`
`LLC, also called partition chromatography, involves a solid support, usually silica
`
`
`
`
`
`
`
`
`
`gel or kieselguhr, mechanically coated with a film of an organic liquid. A typical
`
`system for NP LLC is a column coated with B,B'-oxy dipropionitrile and a nonpolar
`
`
`
`
`solvent like hexane as the mobile phase. Analytes are separated by partitioning
`
`
`
`
`
`
`between the two phases as in solvent extraction. Components more soluble in the
`
`
`
`stationary liquid move more slowly and elute later. LLC has now been replaced by
`
`
`BPC for most applications.
`
`Bonded Phase Chromatography
`
`BPC uses a stationary phase that is chemically bonded to silica gel by reaction of
`
`
`
`
`
`
`
`silanol groups with a substituted organosilane. Unlike LLC, the stationary phase is
`
`
`
`
`
`
`not altered by mobile phase development or temperature change. All solvents can
`
`
`
`
`be used, presaturation of the mobile phase with the stationary phase is not re­
`
`quired, and gradient elution can be used to improve resolution.
`
`Specialized applications of BPC have been developed for ionized compounds,
`
`
`
`
`
`
`which are highly water soluble and generally not well retained on RP BPC col­
`
`
`
`
`
`
`umns. Retention and separation can be increased by adding an appropriate pH
`
`
`
`
`buffer to suppress ionization (ion suppression chromatography) or by forming a
`
`
`
`
`lipophilic ion pair (ion pair chromatography) between the analyte and a counter
`
`
`
`
`
`
`
`ion of opposite charge. The resultant nonionic species are separated by the same
`
`
`
`
`column techniques used for naturally nonionic organic molecules.
`
`Ion suppression is the preferred method for separation of weak acids and bases,
`
`
`
`
`
`for which the pH of the mobile phase can be adjusted to eliminate analyte ioniza­
`
`
`
`
`tion while remaining within the pH 2-8 stability range of bonded silica phases. The
`
`
`
`analyte is chromatographed by RP HPLC, usually on a C-18 column, using metha­
`
`
`nol or acetonitrile plus a buffer as the mobile phase. The technique is often
`
`
`preferred over IEC (see below) because C-18 columns have higher efficiency,
`
`
`
`
`equilibrate faster, and are generally easier to use reproducibly compared to ion
`
`
`
`
`
`exchange phases. Strong acids and bases are usually separated on an ion exchange
`column or by ion pair chromatography.
`
`Ion pair chromatography is used to separate weak or strong acids or bases as well
`
`
`
`
`
`
`as other types of organic ionic compounds. The method involves use of a C-18
`
`
`column and a mobile phase buffered to a pH value at which the analyte is com­
`
`
`
`
`
`pletely ionized (acid pH for bases, basic pH for acids) and containing an appro­
`
`
`
`
`
`
`priate ion pairing reagent of opposite charge. Trialkylammonium salts are com­
`
`
`
`monly used for complex acidic analytes and alkylsulfonic acids for basic analytes.
`
`
`
`
`The ion pairs separate as if they are neutral polar molecules, but the exact mecha­
`
`
`
`nism of ion pair chromatography is unclear. Retention and selectivity are affected
`
`
`
`
`by the chain length and concentration of the pairing reagent, the concentration
`
`
`
`
`
`of organic solvent in the mobile phase, and its pH. Retention increases up to a
`
`
`
`
`point as the chain length of the pairing reagent or its concentration increases,
`off [I] .
`
`then decreases or levels
`
`Transmittal No. 94-1 (1/94)
`
`Form FDA 2905a (6/92)
`
`601-3
`
`
`
`
`
`Regeneron Exhibit 1053.003
`
`

`

`
`
`SECTION 601
`
`
`
`
`
`Pesticide Analytical Manual Vol. I
`
`Compounds not ionized at the operative pH will not pair with the reagent, but
`
`
`
`
`
`
`
`
`they may still be strongly retained by a C-18 column depending on their alkyl
`
`
`
`
`
`structure. In this case, however, retention will not increase with the addition of an
`
`
`
`
`
`ion pairing reagent, and some decrease in retention may occur, probably due to
`
`
`
`reagent competition for the stationary phase [I].
`
`
`
`Ion Exchange Chromatography
`
`IEC is used to separate ionic compounds. Microparticulate insoluble organic poly­
`
`
`
`
`
`
`
`
`
`
`
`mer resin or silica gel is used as the support. Negatively charged sulfonic acid
`
`
`groups chemically bound to the support produce strong acid cation exchange
`
`
`
`
`
`(SCX) phases. Positively charged quaternary ammonium ions bound to the sup­
`
`
`
`port produce strong base anion exchange (SAX) phases. The most widely used
`
`
`
`
`
`resin support is cross-linked copolymer prepared from styrene and divinylbenzene.
`
`Mobile phases are aqueous buffers.
`
`Separations in IEC result from competition between the analytes and mobile phase
`
`
`
`
`
`
`
`
`ions for sites of opposite charge on the stationary phase. Important factors control­
`
`
`
`
`ling retention and selectivity include the size and charge of the analyte ions, the
`
`
`
`type and concentration of other ions in the buffer system, pH, temperature, and
`
`
`the presence of organic solvents.
`
`Ion chromatography, a subcategory of IEC, has been used primarily for separa­
`
`
`
`
`
`
`
`
`
`
`tions of inorganic cations or anions. Because a conductivity detector is usually
`
`
`
`employed, some means is required to reduce the ionic concentration and, hence,
`
`
`
`the background conductance of the mobile phase. A second ion exchange sup­
`
`
`
`pressor column to convert mobile phase ions to a nonconducting compound may
`
`
`
`
`be used. Alternatively, a stationary phase with very low exchange capacity may be
`
`
`
`used with a dilute, low conductance mobile phase containing ions that interact
`
`strongly with the column.
`
`
`
`Size Exclusion Chromatography
`
`SEC separates molecules based on differences in their size and shape in solution.
`
`
`
`
`
`
`
`
`
`SEC cannot separate isomers. SEC is carried out on silica gel or polymer packings
`
`
`
`having open structures with solvent-filled pores of limited size range. Small analyte
`
`
`molecules can enter the pores and spend a longer amount of time passing through
`
`
`the column than large molecules, which are excluded from the pores. Ideally,
`
`
`
`there should be no interaction between the analytes and the surface of the station­
`ary phase.
`
`(GPC)
`Two important subdivisions of SEC are gel permeation chromatography
`
`
`
`
`
`
`
`
`and gel filtration chromatography (GFC). GPC uses organic solvents for organic
`
`
`
`
`
`
`polymers and other analytes in organic solvents. GFC uses aqueous systems to
`
`
`
`
`
`
`separate and characterize biopolymers such as proteins and nucleic acids.
`
`The chemist developing an HPLC method must first consider the properties of
`
`
`
`
`
`
`the analytes of interest and choose an HPLC separation method that best takes
`
`
`
`
`
`
`advantage of those properties. Many of the references in the bibliography (Section
`
`
`
`
`608)offer guidance to making these choices. A general, simplified guide for
`selecting an HPLC
`
`
`
`
`mode according to the properties of the analyte (s) is illustrated
`
`
`
`[2].of Snyder and Kirkland in Figure 601-c; the guide is based on the principles
`
`601-4
`
`Transmittal No. 94-1 ( 1 /94)
`
`
`Form FDA 2905a (6/92)
`
`1053.004
`Regeneron Exhibit
`
`

`

`
`
`Pesticide Analytical Manual Vol. I
`
`
`
`
`
`SECTION 601
`
`Figure 601-c
`
`Guide to Selection of HPLC Mode
`
`(based on analyte characteristics)
`
`GPC
`
`SAX
`
`Molecular we�
`
`□:EC
`Organic
`No ► GFC
`soluble? Yesl
`,.
`No�
`
`Molecular sizes
`very different?
`Yes
`No
`lonizable?
`BPC
`
`Ion Suppression _____. RP
`Chromatography org-aq
`solvent Anionic
`Ion Anionic?
`_Y_e_sl�_N_o_► Counter Ion
`Pairing
`Yes
`RP [C-8, C-18)
`org-aq solvent
`No
`Cationic
`Counter Ion
`
`RP [C-8, C-18)
`org-aq solvent
`No ► SCX
`IEC Anionic?
`Yesl
`aq solvent
`Strongly lipophilic?
`
`aq solvent
`RP, C-18, polar
`org solvent
`
`silica; polar
`org solvent
`
`silica; polar normal phase
`RP, C-8
`org-aq
`org solvent [CN, NH 2, dial)
`solvent
`org solvent
`
`No
`
`BPC
`
`LSC
`
`BPC
`
`LSC
`
`BPC
`
`This scheme categorizes analytes as either ionic/ionizable (and therefore water
`
`
`
`
`
`
`
`soluble) or nonionic/nonionizable (not water soluble). Based on these distinc­
`
`
`
`
`
`
`tions, and on the polarity of the analytes, the diagram provides general rules for
`
`
`
`
`the analytes. likely to separate choosing an HPLC mode of operation
`
`
`
`601 C: INSTRUMENTATION ANO APPARATUS
`
`Basic Components
`
`The following basic components are typically included in an HPLC system (Fig­
`
`
`
`
`
`
`
`ure 601-d): solvent reservoir(s); optional gradient-forming device; one or more
`
`
`
`
`
`precision solvent delivery pumps; injector; analytical column and optional
`
`
`
`
`precolumn and guard column; column oven; detector; recorder, integrator, or
`
`
`
`
`
`
`computerized digital signal processing device; and associated plumbing and wir­
`ing.
`
`Transmittal No. 94-1 (1/94)
`
`Form FDA 2905a (6/92)
`
`601-5
`
`
`
`Regeneron Exhibit 1053.005
`
`

`

`
`
`SECTION 601
`
`
`
`
`
`Pesticide Analytical Manual Vol. I
`
`Gradient
`device
`
`Sample
`1-----1: ,11--injection
`system
`
`Figure 601-d
`Thermostatted
`Block Diagram of
`column oven
`HPLC System
`
`Column
`
`[Reprinted with permission of
`
`
`McGraw-Hill Book Company, from
`
`West, C.D. ( 1987) Essentials of
`
`
`Quantitative Analysis, Figure 14.1,
`.----r---,_ _ _ Th ermostatted
`page 346.]
`
`detector oven
`
`Pump A-
`
`
`
`Solvent A-
`
`Recorder or
`
`readout device
`
`by pumps For analytical HPLC, typical flow rates of 0.5-5 mL/min are produced
`
`
`
`
`
`
`
`operating at 300-6000 psi. Although pumps are capable of high pressure opera­
`
`
`
`tion, state-of-the-art 25 cm X 4 mm id columns with 5 µm packings typically pro­
`
`
`
`
`
`duce 1000-2000 psi at I mL/min. High pressures should be avoided because they
`
`
`contribute to limited column life expectancies.
`
`Sample extract is applied to the column from an injector valve containing a loop
`
`
`
`
`
`
`
`
`that has been filled with sample solution from a syringe. After passing through the
`
`
`
`
`column, the separated analytes are sensed by visible/UV absorption, fluorescence,
`
`
`
`
`extra-column minimize electrochemical, photoconductivity, or RI detectors. To
`
`
`
`peak spreading, the instrument components must be connected using low dead
`
`
`
`volume (ldv) fittings and valves and tubing as short and narrow in bore as possible.
`
`Isocratic methods. or gradient elution Analytical HPLC may use either isocratic
`
`
`
`
`
`
`
`
`
`
`elution uses a mobile phase of constant composition, whereas the strength of the
`
`
`mobile phase in gradient elution is made to increase continually in some prede­
`
`
`
`termined manner during the separation. Gradient elution, which requires an
`
`
`
`
`automatic electronic programmer that pumps solvent from two or more reservoirs,
`
`
`
`
`
`reduces analysis time and increases resolution for complex mixtures in a manner
`
`
`
`
`
`
`similar to temperature programming in GLC. Gradient elution capability is highly
`
`
`
`
`
`recommended for systems to be used for residue determination. However, it is not
`
`
`always possible to employ gradient elution because some HPLC column/solvent
`
`
`
`
`
`systems and detectors are not amenable to the rapid solvent and pressure changes
`involved.
`
`Stationary phases are uniform, spherical, or irregular porous particles having
`
`
`
`
`
`
`
`nominal diameters of 10, 5, or 3 µm. Bonded phases produced by chemically
`
`
`
`bonding different functional groups to the surface of silica gel are most widely
`
`
`
`
`
`used, along with unmodified silica gel and size exclusion gels. Columns are usually
`
`
`
`stainless steel, 3-25 cm long and 4.6 mm id, prepacked by commercial manufac­
`
`
`
`
`turers. There has been increasing use of microbore columns having diameters ::;;2
`
`
`
`
`tempera­out at ambient can be carried mm.Although many HPLC separations
`
`
`
`ture, column operation in a thermostatted column oven is necessary for reproduc­
`
`
`
`
`
`
`ible, quantitative results, because distribution coefficients and solubilities are tem­
`
`perature dependent.
`
`Depending on the nature of the analyte (s), certain additional equipment may be
`
`
`
`
`
`
`
`
`
`required. For example, apparatus and reagents for performing post-column
`
`601-6
`
`Transmittal No. 94-1 ( 1 /94)
`
`Form FDA 2905a (6/92)
`
`1053.006
`Regeneron Exhibit
`
`
`

`

`
`
`
`
`Pesticide Analytical Manual Vol. I
`
`
`
`SECTION 601
`
`derivatization, as used in Section 401 ror 1\-methylcarbamates, may be needed to
`
`
`
`
`
`
`
`
`convert analytes to compounds Lhat can be detected wiL11 L11e reqt1irecl sensitivity
`
`and/ or sclecti,; ty.
`
`HPLC System Plumbing
`
`Rand broadening can occur not only in the analytical and guard columns, but also
`
`
`
`
`
`
`
`in dead volume in the i�jector, detector, or plumbing connecting the various
`
`
`componenL,;; of Lhe HPLC sysLem. This
`Figure 601-e
`
`
`effect, called extra-column dispersion,
`Column Outlet Fittings
`
`musL be minimized for high efficiency.
`The proper choice and use of tubing
`1/4"�
`stainless
`
`
`and fittings are critical in this regard.
`steel
`tubing
`
`Stainless
`steel frit,
`2 µm
`
`Fittings . Figure 601-e ill usu·ates three
`
`
`types of column outlet fittings. The
`
`conventional fitting (i) used in GLC
`
`
`
`and general laboratory plumbing has
`
`excessive dead volume. Tt has been
`modified to produce a zero dead vol­
`
`ume (zdv) fitting· (ii) in which Lhe
`metal column and the tubing are
`
`
`buued up direCLly against the stainless
`[i) Conventional reducing union [dead volume is
`
`
`
`steel frit. There is evidence that the
`
`
`
`shaded]; [ii) zdv union; (iii) ldv union.
`I Reprinted with permission of John Wiley and Sons. Inc .. from
`
`
`
`
`
`nattu-e of the tubing connection in the
`
`
`Lindsay. S. (19871 High Performance Liquid Chromatography
`
`zdv litting may lead to some loss in
`Figure 2.3a. page 28.]
`
`
`efficiency, especially if the connection
`
`is not made carefully. The ldv filling
`(iii)improves efficiency by use of a cone-shaped dislribuLor connecting the gauze
`
`
`
`
`
`
`or frit at the end of the column with the tubing. A typical dead volume for the ldv
`
`filling· is O. l µL
`
`(i)
`
`[ii]
`
`[iii)
`
`Figure 601-f
`Low Dead Volume Fitting
`
`Columns are usually received from
`
`
`manufacturers with a 1/4-1/Hi" zdv or ldv
`outleL fitting and a 1/4" nuL and cap or
`
`
`a reducing union at the inlet (i.e., not
`[{;�'·':ci) ?sJif'
`
`1/4" in size, but suitable for 1/4" tubing).
`
`Figure 601-f shows a complete lclv fitting
`
`connect.ion between a column and a
`Afi&' y
`� v�Ferrule
`
`deLecLor. The column fiL� snugly inside
`�'9 +-2 11m porous frit
`
`
`the stainless steel end fitting and is scaled
`
`
`by a high compression ferrule. A 2 µm
`�'tlf§"y �1 /4" end f1tt1ng
`porous frit is firmly seated between the
`�-<·· 0.01" id
`
`column and end fitting. The column and
`
`
`detector are connected by a short length
`�
`steel (or polym er) tubing.
`of' stainless
`p
`The column is also connected
`to the
`¥1a detector
`
`ir�jection valve using a zdv or ldv fiuing
`
`and a short length of' stainless steel tub­
`!Reprinted with permission of Howard Sloane. Savant.
`
`
`
`from LC-102 audiovisual program.]
`mg.
`
`Transmittal No. 94-1 (1/94)
`
`Form FDA 2905a (6/92)
`
`601-7
`
`Regeneron Exhibit 1053.007
`
`

`

`
`
`SECTION 601
`
`
`
`Pesticide Analytical Manual Vol. I
`
`External column end fittings (Figures 601-e and 601-
`
`
`
`
`Figure 601-g
`
`f), which were formerly popular, are not durable
`
`Standard Internal Fitting
`
`
`during repeated attachments and removals. Thus,
`
`
`
`
`the internal fitting is practically standard today. This
`
`uses female threads in the fitting body and a male
`
`nut (Figure 601-g).
`
`1/16" tubing___,.
`
`Unions are fittings that connect two pieces
`
`
`Unions.
`
`
`of tubing. The most commonly used type is the
`
`
`
`internal thread ldv type (Figure 601-h). The union
`
`
`is not drilled through completely, but a short (0.02")
`web of metal is left between the two pieces of tub­
`
`
`ing with a small diameter (approximately 0.02 or
`
`
`0.01") hole drilled through. Even though the tub­
`
`
`ing ends do not butt against each other as in early
`
`
`
`zdv unions, there is essentially no dead volume added
`
`to the system through their use. For this reason,
`
`
`they are commonly classified as zdv unions. This
`
`
`
`type of union has fewer assembly, re-assembly, and
`
`
`tubing interchange problems than the early butt­
`[Reprinted with permission of John Wiley
`
`
`
`together zdv type.
`and Sons, Inc., from Meyer, V.R. (1988)
`
`
`Practical High Performance Liquid
`Chromatography,
`Figure 6.18, page 80.]
`Fittings consist of four parts:
`
`Assembly of Fittings.
`
`
`the body, tubing, ferrule, and nut. The nut and
`
`
`
`ferrule are slid onto the tube end, the tube is pushed all the way into the fitting
`
`
`
`
`
`body and held there securely, the nut is finger-tightened, and then another three­
`
`
`
`
`quarter turn is made with a wrench. This procedure should assure that the ferrule
`
`
`is pressed ("swaged") onto the tub-
`
`
`ing. To replace the ferrule, the tub­
`Figure 601-h
`
`ing must be cut and the fitting re­
`
`Internal Thread Low Dead Volume Fitting
`
`made. When using fittings to con­
`
`nect system components, the nut
`
`should be finger-tightened and then
`
`
`tightened a one-half turn more with
`
`a wrench. If leaking is observed,
`
`
`slightly more tightening should be
`
`
`sufficient to complete the seal. Over­
`
`tightening of nuts can lead to fit-
`
`ting distortion and leaks.
`
`[Reprinted with permission of Aster Publishing Corporation,
`
`
`
`P. (1988) LC-GCS,
`from Dolan, J.W., and Upchurch,
`Figure 3, page 788.]
`Fitting components from different
`
`
`
`
`manufacturers have dissimilar de-
`signs, sizes, and thread types and are usually not interchangeable. Ferrules from
`
`
`
`
`
`
`
`
`different manufacturers have unique shapes, but they are usually interchangeable
`
`
`
`
`because the front edge is deformed when pressed onto the tubing. However, as a
`
`
`
`
`general rule, it is best to purchase all fittings and spare parts from one manufac­
`
`
`
`
`
`
`turer. Even fittings from a given manufacturer differ slightly because of manufac­
`
`
`
`
`turing tolerances. However, this is of concern only with microbore columns, for
`
`
`
`
`which dead volume is a greater consideration. For these columns, it is best to not
`
`
`even interchange fittings from the same manufacturer.
`
`A variety of fittings are available that can be finger-tightened to the degree nec­
`
`
`
`
`
`
`
`essary to seal stainless steel tubing at 2000-6000 psi. All of these are based on the
`
`
`
`
`use of polymeric ferrules, but some have a steel nut, whereas others are all plastic.
`
`601-8
`
`Transmittal No. 94-1 ( 1 /94)
`
`Form FDA 2905a (6/92)
`
`1053.008
`Regeneron Exhibit
`
`
`

`

`
`
`
`
`Pesticide Analytical Manual Vol. I
`
`
`
`SECTION 601
`
`They are used mostly on frequently attached and detached high pressure connec­
`
`
`
`
`
`
`
`tions, such as between the injector and column or column and detector, and for
`
`
`
`polymer tubing waste lines from the injector or detector.
`
`Fittings must be kept free of silica particles, which may scratch surfaces between
`
`
`
`
`
`
`
`the ferrule and union and cause leaks.
`
`Stainless steel tubing is available commercially that is supposedly ready for
`
`
`
`
`
`Tubing.
`
`
`
`
`immediate use in HPLC systems. It is machine cut, polished, and deburred to
`
`
`
`
`provide perfectly square ends. It is also cleaned by sonication, passivated, washed,
`
`
`
`
`and rinsed with a solvent such as isopropanol to eliminate residual dirt or oils.
`
`
`
`
`Despite this careful preparation, it is a wise precaution to rinse new tubing with
`
`
`
`mobile phase under operating pressure before using it as part of the HPLC system.
`
`The most commonly used tubing for connecting components of the chromato­
`
`
`
`
`
`
`Tubing with 1/16" od, with different inside diameters.
`
`
`graph is 316 stainless steel,
`
`0.01" (0.25 mm) id is commonly used in areas where dead volume must be mini­
`
`
`
`
`
`precolumn and column, the injector mized to maximize efficiency, e.g., between
`
`
`
`
`and column, columns in series, and column and detector, and for preparing pulse
`damping spirals.
`
`Typical lengths of tubing connections are 3-6 cm. Tubing with 0.005 or 0.007" id
`
`
`
`
`
`
`
`is used to connect microbore or short 3 µm particle size columns to detectors and
`
`
`
`
`
`injectors. Filtering of samples and solvents is especially critical to prevent clogging
`
`
`
`of this narrow bore tubing. Tubing with 0.02-0.05" id is available when ldv is not
`
`
`
`
`important and low resistance to flow and pressure drop is desirable. For example,
`
`
`1 mm (0.04") tubing is often used between the pump and sample injector.
`
`Tubing can be cut to any required length in the laboratory, but it is important not
`
`
`
`
`
`
`
`
`to distort the interior or exterior during the process. The simplest method is to
`
`
`score the tubing completely around the outside with a file and then bend it back
`
`
`
`and forth while holding it on either side of the score with two smooth-:iawed pliers.
`
`
`
`The ends are filed smooth and deburred, and the tubing is thoroughly washed
`
`
`
`
`with solvent. If the bore should become closed by the bending and filing, the tube
`
`
`
`can be reamed out with an appropriate drill bit before final smoothing and wash­
`
`
`
`ing. A number of types of manual and motorized tubing cutters are available from
`
`
`
`chromatography accessory suppliers. Proper cutting of tubing to make leak-free
`
`connections is an art that requires considerable practice.
`
`Although stainless steel tubing and fittings are standard for systems using organic
`
`
`
`
`
`
`
`
`
`and salt-free aqueous solvents, corrosion becomes a problem with buffers contain­
`
`
`
`
`a have available ing salts, particularly halide salts at low pH. HPLC companies
`
`
`
`
`variety of accessories that can solve this problem. These include titanium high
`
`
`pressure system components, for use in the flow stream at all points of mobile
`
`
`
`
`
`phase contact, and titanium or polymeric fluorocarbon tubing with id values simi­
`
`
`lar to stainless steel. One such polymer is Tefzel (ethylene-tetrafluoroethylene
`
`
`
`
`copolymer), which can withstand pressures of 5000 psi or higher. (Teflon is lim­
`
`
`
`
`ited to pressures <1000 psi.) Titanium and polymeric plumbing components are
`
`
`chromatography. especially valuable for biochemical HPLC and ion
`
`
`Reference 3 is a valuable source of information to help avoid many tubing instal­
`
`
`
`
`
`lation problems.
`
`Transmittal No. 94-1 (1/94)
`
`Form FDA 2905a (6/92)
`
`601-9
`
`1053.009
`Regeneron Exhibit
`
`
`

`

`
`
`SECTION 601
`
`
`
`
`
`Pesticide Analytical Manual Vol. I
`
`because liquid System Leaks. Leaks are relatively easy to detect in LC instruments
`
`
`
`
`
`
`
`will be visible around a loose fitting. A loss of system pressure when using a
`
`
`
`
`constant volume pump is a common sign that a leak may be present. If this occurs,
`
`
`
`all fittings, especially sample valve and column fittings, should be checked and
`
`
`
`
`tightened if necessary with two open-ended wrenches. Care must be taken not to
`
`
`
`overtighten. If leaking does not stop, the faulty fitting must be replaced.
`
`601 0: SOL VENTS ANO REAGENTS
`
`a particu­The mobile phase in HPLC is chosen for its ability, in combination with
`
`
`
`
`
`
`
`
`lar column, to provide the required separation of the analyte(s). The solvents used
`
`to prepare the mobile phase must be of high purity, most often HPLC grade,
`
`
`
`
`spectrophotometric grade, or distilled from all-glass apparatus. Other factors of
`
`
`
`
`
`
`
`importance include cost, viscosity, toxicity, boiling point, compressibility, UV trans­
`
`
`
`parency (if a UV detector is used), RI (if an RI detector is used), vapor pressure,
`
`
`
`
`
`
`flash point, odor, inertness with respect to sample compounds, and ability to cause
`
`
`
`
`
`corrosion. Choices of solvents and reagents cannot be made without careful con­
`
`
`
`sideration of the effect their presence can have on the entire HPLC system.
`
`prepa­step and in sample Solvents and reagents used in the HPLC determinative
`
`
`
`
`
`
`
`ration procedures preceding HPLC should not:
`
`
`
`
`
`
`
`
`
`1) cause degradation or unintended reaction of the analyte(s);
`
`
`
`2) cause the solvent delivery system to malfunction;
`
`
`
`
`
`
`
`3)cause damage to the analytical column;
`
`
`
`
`
`
`
`4) cause damage to the detector; or
`
`
`
`5)contribute noise or increased or decreased detector response for the
`
`
`
`
`
`
`
`analyte.
`
`
`
`Potential Problems
`
`Many of the problems with mobile phases arise because of the presence of impu­
`
`
`
`
`
`
`
`
`
`rities, additives, dust or other particulate matter, or dissolved air. Examples of
`
`
`
`
`
`some specific potential problems with solvents and reagents and suggested solu­
`tions follow.
`
`Analytes can be degraded by solvents and reagents used in the ex­
`
`
`
`
`Degradation.
`
`
`
`
`Analyte traction and cleanup steps of the analysis, or in the HPLC step itself.
`
`
`
`
`
`
`chemistry is usually known in advance, and reagents likely to cause degradation
`
`
`
`
`
`can be avoided. Unexpected reaction of the analyte(s) will usually be demon­
`
`
`
`strated by poor or no recovery of the compound(s) through the method, or by
`
`
`
`
`
`detection of additional reaction products in the determinative step.
`
`The presence of impurities in solvents or reagents is often the cause of such
`
`
`
`
`
`
`
`
`
`
`unexpected reactions. For example, traces of oxidizing agents in solvents have
`
`
`
`been found to degrade N-methylcarbamates prior to their determination by HPLC.
`
`
`
`
`Purity of all reagents used in trace-level determinations should always be as high
`as possible.
`
`601-10
`
`Transmittal No. 94-1 ( 1 /94)
`
`
`
`Form FDA 2905a (6/92)
`
`
`
`
`
`Regeneron Exhibit 1053.010
`
`

`

`
`
`
`
`Pesticide Analytical Manual Vol. I
`
`
`
`SECTION 601
`
`The presence of dissolved gases in solvents composing the mo­
`
`
`
`
`Dissolved Gases.
`
`
`
`
`
`bile phase is a major cause of practical problems in HPLC. Gas bubbles can collect
`
`
`
`in pumps, the detector cell, or other locations in the HPLC system. This can affect
`
`
`
`the reproducibility of the volume delivered by the pump, or large bubbles may
`
`
`
`
`completely stop the pump from working. Detection can be affected in various
`
`
`
`ways. With the UV detector, air in the detector cell can cause seriously increased
`
`
`
`
`detector noise or high absorbance. Dissolved oxygen can interfere with detection
`
`
`
`
`at short wavelengths, as oxygen absorbs radiation at <200 nm. Solvents must be
`
`
`
`"degassed," a topic covered in Section 603 B, Mobile Phase Preparation.
`
`to replace. damaged and expensive are easily Damage to Columns. HPLC columns
`
`
`
`
`Therefore, Bases can remove the functional groups from bonded HPLC phases.
`
`
`
`
`
`prior to their removal bases should not be used in analyses involving BPC unless
`
`
`
`
`
`chromatography can be assured. B