l E
`
`Chapter 6
`HPLC
`
`Pesticide Analytical Manual Vol.
`
`
`.
`
`Table of Contents
`
`601:
`
`General Information
`
`601 A:
`
`Principles
`
`601 B: Modes of Operation
`Liquid—Solid Chromatography
`Liquid-Liquid Chromatography
`Bonded Phase Chromatography
`Ion Exchange Chromatography
`Size Exclusion Chromatography
`
`601 C:
`
`Instrumentation and Apparatus
`Basic Components
`HPLC System Plumbing
`
`601 D:
`
`Solvents and Reagents
`Potential Problems
`
`Specific Solvents
`
`page
`
`date
`
`601—1
`
`601—2
`601—2
`601—3
`601—3
`601—4
`601—4
`
`601—5
`601—5
`601—7
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`601—10
`601-10
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`601-12
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`1/94
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`1/94
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`lgmzagtaszigje/QB]
`
`5004
`
`Regeneron Exhibit 1053.001
`
`

`

`Pesticide Analytical Manual Vol.
`
`I
`
`601 E:
`
`Reagent Blanks
`Safety Precautions
`
`Sample Preparation
`Sample Cleanup
`Sample Filtration
`Sample Solvent Degassing
`Choice of Sample Solvent
`
`601 F:
`
`Reference Standards
`
`Stock Solutions
`
`Working Standard Solutions
`Storage
`
`References
`
`602:
`
`Columns
`
`602 A:
`
`Column Selection
`
`602 B:
`
`Analytical Columns
`Liquid—Solid Chromatography
`Bonded Phases
`
`Ion Exchange
`Ion Pair
`
`Size Exclusion
`
`602 C:
`
`Column Evaluation
`
`602 D:
`
`Column Specifications
`
`602 E:
`
`Analytical Column Protection
`Filters
`
`Precolumns
`
`Guard Columns
`
`602 F:
`
`Column Maintenance and Troubleshooting
`Column Care
`
`Column Evaluation by Injection of
`Test Mixtures
`
`Column Storage
`Column Regeneration
`
`References
`
`page
`601-14
`601-14
`
`601-14
`601-14
`601—14
`601-15
`601—15
`
`601—15
`
`601-15
`
`601—16
`601—16
`
`601—16
`
`602—1
`
`602—1
`602—2
`602—2
`
`602—3
`602—4
`
`602—4
`
`602—5
`
`602—6
`
`602—8
`602—8
`
`602—9
`
`602—9
`
`602—9
`602—9
`
`602—10
`
`602—11
`602—11
`
`602—1 2
`
`603:
`
`Mobile Phase Selection, Preparation, and Delivery
`
`603 A: Mobile Phase Selection
`
`Normal Phase Chromatography
`Reverse Phase Chromatography
`Ion Exchange Chromatography
`Ion Pair Chromatography
`Size Exclusion Chromatography
`Gradient Elution in HPLC
`
`603 B: Mobile Phase Preparation
`Filtering Solvents
`Degassing Solvents
`
`603—1
`
`603—1
`603—3
`603—4
`603—5
`603—5
`603—5
`
`603—6
`603—6
`603—6
`
`date
`1/94
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`600—2
`
`“mfifil'iialgi'agg‘ééllgfiil
`
`Regeneron Exhibit 1053.002
`
`

`

`Pesticide Analytical Manual Vol.
`
`I
`
`Preparation of Multisolvent Mobile Phases
`Solvent Reservoirs
`
`603 C: Mobile Phase Delivery Systems
`Pumps
`Gradient Programming Systems
`
`603 D: Maintenance and Troubleshooting
`Problems with Pumps
`
`References
`
`604:
`
`Injection Systems
`
`604 A:
`
`Injection Valves
`
`604 B: Automatic Injectors
`
`604 C: Operation, Maintenance, Troubleshooting,
`and Repair of Injection Valves
`
`References
`
`605:
`
`Detectors
`
`605 A:
`
`UV/VIS Absorbance Detectors
`
`Fixed Wavelength UV Detectors
`Variable Wavelength UV Detectors
`Solvents
`
`Performance Characteristics
`
`page
`603—7
`603—7
`
`603—8
`603—8
`603—10
`
`603—1 0
`603—10
`
`603—1 3
`
`604—1
`
`604—3
`
`604—3
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`date
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`1 / 94
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`604-4
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`605—2
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`605—3
`605—4
`605—4
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`Multichannel or Photodiode Array Detectors 605—5
`Applications
`605—5
`Problems, Maintenance, and
`605—5
`Troubleshooting
`
`605 B:
`
`Fluorescence Detectors
`
`Detector Design
`Solvents
`
`Performance Characteristics
`
`Parameter Adjustments
`Applications
`Detector Maintenance
`
`605 C:
`
`Electrochemical Detectors
`
`Conductivity Detectors
`Amperometric and Coulometric Detectors
`Performance Characteristics
`
`Applications
`
`605 D:
`
`Photoconductivity Detectors
`Apparatus
`Performance Characteristics
`
`Applications
`
`605 E: Mass Spectrometric Detectors
`
`605 F:
`
`Derivatization for Detection Enhancement
`
`Comparison of Pre— and Post—Column
`Derivatization
`
`605—6
`
`605—6
`605—7
`
`605—7
`
`605—8
`605—8
`605—8
`
`605—8
`
`605—9
`605—9
`605—10
`
`605-11
`
`605—11
`605—11
`605—11
`
`605—1 3
`
`605—1 3
`
`605-14
`
`605-14
`
`12:2??328'22852‘15331/93
`
`BOO—3
`
`Regeneron Exhibit 1053.003
`
`

`

`I
`
`Pesticide Analytical Manual Vol.
`
`Post—Column Reactor Design
`References
`
`606:
`
`606 A:
`
`Residue Identification and Quantitation
`Residue identification
`
`Co—chromatography
`Use of Alternative Columns
`
`Spectrometric Confirmation
`
`Quantitation
`Reference
`
`Quality Assurance and Troubleshooting
`
`Liquid Chromatograph Monitoring and
`Performance Testing
`
`606 B:
`
`607:
`
`607 A:
`
`607 B:
`
`608:
`
`page
`605—14
`
`605—16
`
`606—1
`
`606—1
`
`606—2
`
`606—2
`
`606—3
`
`606—3
`
`date
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`608—1
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`608—1
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`608—2
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`608—2
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`608—2
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`601—1
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`601—2
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`601—5
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`Troubleshooting from Chromatograms
`
`607—2
`
`1/ 94
`
`Bibliography
`General Texts
`
`Columns
`
`Detectors
`
`Troubleshooting
`
`Application to Pesticides
`
`601—a
`
`601—b
`
`601—c
`
`Figures
`Chromatographic Separation Techniques
`HPLC Modes of Operation
`Guide to Selection of HPLC Mode
`
`601—d
`
`601—e
`
`601-f
`
`601—g
`601-h
`
`602—a
`
`603-a
`
`603-b
`
`603-c
`
`603-d
`
`604—a
`
`604—b
`
`605—a
`
`605—b
`
`605—c
`
`605—d
`
`605—e
`
`Block Diagram of HPLC System
`Column Outlet Fittings
`
`Low Dead Volume Fitting
`Standard Internal Fitting
`Internal Thread Low Dead Volume Fitting
`
`Calculation of Column Performance Parameters
`
`Reciprocating Pump
`Gradient System I
`Gradient System 11
`Gradient System 111
`
`External Loop Injector: Six—Port Injection Valve
`Internal Loop Injector
`
`UV/VIS Detector
`
`Variable Wavelength UV/VIS Detector
`Fluorescence HPLC Detector
`
`Three Electrode Electrochemical Detector
`
`Post—Column Reactors
`
`601—6
`
`601—7
`
`601—7
`
`601—8
`
`601—8
`
`602—5
`
`603—8
`
`603—9
`
`603—9
`
`603—10
`
`604—1
`
`604—2
`
`605—3
`
`605—4
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`605—6
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`605—10
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`605—15
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`9/96
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`1 / 94
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`1 / 94
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`600—4
`
`Transmittal No. ElB-E’l [El/QB]
`Form FDA 2905a [8/92]
`
`Regeneron Exhibit 1053.004
`
`

`

`Pesticide Analytical Manual Vol.
`
`I
`
`Tables
`
`HPLC Column Specification Elements
`602—a:
`602—b: Minimum Efficiency Values
`
`(SOS—a:
`
`Properties of Common HPLC Solvents with
`Alumina Columns
`
`page
`
`date
`
`602—7
`602—8
`
`603—2
`
`1/94
`1/94
`
`1/94
`
`603—b:
`
`Classification of Solvent Selectivity
`
`603—2
`
`1/94
`
`Transmittal No. 9B-E’l [9/98]
`Form FDA 2905a [8/92]
`
`BOO—5
`
`Regeneron Exhibit 1053.005
`
`

`

`Pesticide Analytical Manual Vol.
`
`I
`
`BOO—B
`
`Transmittal N0. 9B-E’l [9/98]
`Form FDA 2905a [8/92]
`
`Regeneron Exhibit 1053.006
`
`

`

`SECTION 60"
`I
`Pesticide Analytical Manual Vol.
`
`
`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
`complementary to the more traditional gas liquid chromatography (GLC). HPLC
`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.
`
`507 A: PRINCIPLES
`
`Figure 601-5:
`Chromatographic Separation
`Techniques
`Chromatography
`
`Gl—k—l
`L'qu'd
`l—k—l
`
`as
`
`Chromatography comprises a family of sepa—
`ration techniques (Figure 601—a), all of which
`share common characteristics. A narrow ini-
`tial'zone of mixture'is applied to a sorptive
`stationary phase hav1ng a large surface area.
`
`Developmentwith 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—
`
`Planar
`
`Column
`|—I—|
`Classical
`HPLC
`
`tribution between the two phases. The mo-
`bile phase can be a gas or a liquid. Liquid
`chromatography is divided into two main
`types, planar (thin layer and paper chroma—
`tography) and column. Column liquid chro-
`matography, both the classical (low pressure)
`version and the high performance version
`discussed here, is further subdivided. accord—
`ing to the mechanism of separation into five
`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.
`
`LSC
`
`LLC
`
`BPC
`
`IEC
`
`SEC
`
`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
`suitable hardware (columns, pumps, inlet systems, low dead volume fittings, etc.)
`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 (um) 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 revisedfrom a chapter on HPLC writtenfor FDA in 1989-90 byjoseph Sherma,
`Ph.D., Lafayette College, Easton, PA.
`
`Transmittal No. 94-1 [’l/El4]
`Form FDA 2905a [8/92]
`
`601—1
`
`Regeneron Exhibit 1053.007
`
`

`

`Pesticide Analytical Manual Vol.
`
`I
`
`SECTION 601
`
`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 (R1) or fixed wavelength UV absorbance detectors. Neither detec—
`tor demonstrated sufficient sensitivity or selectivity for use in trace residue analysis.
`In the mid—1970s, 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 fiuorophore. This resulted in the first practical appli—
`cation of HPLC to multiresidue pesticide determination (see method for
`N—methylcarbamates, Section 401).
`
`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.
`
`501 B: 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
`separated and determined. For residue
`determination, as for HPLC analyses
`in general, BPC is the most widely
`used.
`
`There are two variations within the five
`
`Figure 60“,
`HPLC Modes of Operation
`
`HPLC
`
`LSC
`
`LLC
`
`BPC
`SAX
`|—|—|
`Ion
`Ion
`suppression
`pair
`
`IEC
`
`SCX SEC
`.—|—.
`GPC
`GFC
`
`operational modes of HPLC operation;
`these distinctions are based on the relative polarities of stationary and mobile
`phases:
`
`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 [’l/El4]
`Form FDA 2905a [8/92]
`
`Regeneron Exhibit 1053.008
`
`

`

`SECTION 60"
`I
`Pesticide Analytical Manual Vol.
`
`
`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—l8 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,
`then decreases or levels off [1].
`
`Transmittal No. 94-1 [’l/El4]
`Form FDA 2905a [8/92]
`
`60" —3
`
`Regeneron Exhibit 1053.009
`
`

`

`Pesticide Analytical Manual Vol.
`
`I
`
`SECTION 601
`
`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 [1].
`
`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.
`
`Two important subdivisions of SEC are gel permeation chromatography (GPC)
`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
`in Figure bOl—c; the guide is based on the principles of Snyder and Kirkland [2].
`
`601—4
`
`Transmittal No. 94-1 [’l/El4]
`Form FDA 2905a [8/92]
`
`Regeneron Exhibit 1053.010
`
`

`

`SECTION 60"
`I
`Pesticide Analytical Manual Vol.
`
`
`Figure 601-0
`Guide to Selection of HPLC Mode
`
`[based on analyte characteristics]
`
`Molecular weight >20009
`Y
`
`BS
`
`No
`
`.
`SEC Organic
`soluble?
`
`Molecular sizes
`
`very different?
` No
`
`lonizable?
`
`Ion Suppression
`Chromatography
`
`No
`
`GFC
`
`Yes
`
`(3pc
`
`ch
`
`’ RP
`org—aq
`solvent
`
`BS
`
`Ion
`-
`
`Pa'r'“9
`
`Anionic?
`
`Yele RP [,08 C18]
`
`Anionic
`Counter Ion
`
`org—aq solvent
`
`Cationic
`Counter Ion
`RP [C—8, C—’l 8]
`org—aq solvent
`
`Strongly lipophilic?
`
`“83‘le
`
`aq solvent
`
`aq solvent
`
`lEC Anionic?
`
`BPC
`RP, C—’|8, polar
`org solvent
`
`LSC
`
`silica; polar
`org solvent
`
`BPC
`
`BPC
`’
`RP 08
`org—aq
`solvent
`
`silica;polar
`org solvent
`
`normal phase
`[CN, NHg, diol]
`org solvent
`
`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
`choosing an HPLC mode of operation likely to separate the analytes.
`
`507 C: INSTRUMENTATION AND APPAFI’A TUB
`
`Basic Components
`
`The following basic components are typically included in an HPLC system (Fig-
`ure bOl—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 [’l/El4]
`Form FDA 2905a [8/92]
`
`601—5
`
`Regeneron Exhibit 1053.011
`
`

`

`Pesticide Analytical Manual Vol.
`
`I
`
`SECTION 601
`
`Gradient
`device
`
`
`
`
`Pump A—»' I
`
`Sample
`injection
`system
`Thermostatted
`column oven
`
`Column
`
`
`
`
`Recorder or
`readout device
`
`
`Thermostatted
`detector oven
`Detector
`
`Amplifier
`
`.
`Figure 601 _d
`Block Diagram of
`HPLC System
`
`[Reprinted with permission of
`McGraw—Hill Book Company, from
`West, CD. [1987] Essentials of
`Quantitative Analysis, Figure ’l4.’l,
`page 348.]
`
`For analytical HPLC, typical flow rates of 0.5—5 mL/ min are produced by pumps
`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 pm packings typically pro-
`duce 1000—2000 psi at 1 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,
`electrochemical, photoconductivity, or R1 detectors. To minimize extra-column
`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.
`
`Analytical HPLC may use either isocratic or gradient elution methods. 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 pm. 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
`mm. Although many HPLC separations can be carried out at ambient tempera—
`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 [’l/El4]
`Form FDA 2905a [8/92]
`
`Regeneron Exhibit 1053.012
`
`

`

`
`
`1/4"—>
`stainless
`
`steel
`tubing
`
`
`
`
`
`
`Stainless
`stgelfrit,
`Hm
`
`Fittings. Figure 601-e illustrates three
`types of column outlet fittings. The
`conventional fitting (i) used in GLC
`and general laboratory plumbing has
`excessive dead volume. It has been
`modified to produce a zero dead vol-
`ume (zdv) fitting (ii)
`in which the
`metal column and the tubing are
`butted up directly against the stainless
`steel frit. There is evidence that the
`
`SECTION 60"
`I
`Pesticide Analytical Manual Vol.
`
`
`derivatization, as used in Section 401 for N—methylcarbamates, may be needed to
`convert analytes to compounds that can be detected with the required sensitivity
`and/ or selectivity.
`
`HPLC System Plumbing
`
`Figure SCH-e
`Column Outlet Fittings
`
`Band broadening can occur not only in the analytical and guard columns, but also
`in dead volume in the injector, detector, or plumbing connecting the various
`components of the HPLC system. This
`effect, called extra—column dispersion,
`must be minimized for high efficiency.
`The proper choice and use of tubing
`and fittings are critical in this regard.
`
`,,
`(—llflgé
`[ii]
`[iii]
`
`[i]
`
`[i] Conventional reducing-union [dead volume is
`Shaded]? [”1 ZdV union; ““1 'dV union.
`
`nature Of thC tubing connection in thC [Reprinted with permission of John Wiley and Sons, Inc., from
`ZdV fitting may 163d t0 SOIHC 1055 in
`Lindsay, 9. [’l987] High Performance Liquid Chromatography,
`.
`.
`.
`.
`Figure 2.3a, page 28.]
`effic1ency, espec1ally 1f the connection
`is not made carefully. The ldv fitting
`(iii) improves efficiency by use of a cone—shaped distributor connecting the gauze
`or frit at the end of the column with the tubing. A typical dead volume for the ldv
`fitting is 0.1 uL.
`
`Figure SCH-f
`Low Dead Volume Fitting
`
`Column
`[’| /4" od]
`
`
`
`
`..
`'
`
`Ferrule
`(—2 um porous frit
`
`KTo detector
`
`[Fleprinted with permission of Howard Sloane, Savant,
`from LEI—102 audiovisual program.]
`
`Columns are usually received from
`manufacturers with a 1/4—1/16" zdv or ldv
`
`outlet fitting and a 1/4" nut and cap or
`a reducing union at the inlet (i.€., not
`1/4" in size, but suitable for 1/4" tubing).
`Figure 601-f shows a complete ldv fitting
`connection between a column and a
`
`detector. The column fits snugly inside
`the stainless steel end fitting and is sealed
`by a high compression ferrule. A 2 um
`porous frit is firmly seated between the
`column and end fitting. The column and
`detector are connected by a short length
`of stainless steel (or polymer)
`tubing.
`The column is also connected to the
`
`injection valve using a zdv or ldv fitting
`and a short length of stainless steel tub—
`111g.
`
`Transmittal No. 94-’| [’l/94]
`Form FDA 2905a [8/92]
`
`601—7
`
`Regeneron Exhibit 1053.013
`
`

`

`SECTION 601
`
`Pesticide Analytical Manual Vol.
`
`I
`
`Figure 601-9
`Standard Internal Fitting
`
`
`
`’I/1B" tubing —>
`
`_
`_
`_
`_
`_
`[Fleprinted With permissmn of John Wiley
`and Sons, Inc., from Meyer, VP. [1888]
`Practical High Performance Liquid
`Chromatography, Figure 8.18, page 80.]
`
`External column end fittings (Figures 601—e and 601—
`f), which were formerly popular, are not durable
`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).
`
`Unions. Unions are fittings that connect two pieces
`of tubing. The most commonly used type is the
`internal thread ldv type (Figure GOl—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—
`together ZdV type‘
`
`Figure 601-h
`Internal Thread Low Dead Volume Fitting
`
`
`
`Assembly of Fittings. Fittings consist of four parts:
`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—
`ing must be cut and the fitting re—
`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,
`from Dolan, J.W., and Upchurch, P. [1888] LC—GCB,
`
`Fitting components from different
`manufacturers have dissimilar de—
`
`Figure 3' Page 788-]
`
`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. 84-1 [1/84]
`Form FDA 2805a [8/82]
`
`Regeneron Exhibit 1053.014
`
`

`

`SECTION 60"
`I
`Pesticide Analytical Manual Vol.
`
`
`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.
`
`Tubing. Stainless steel tubing is available commercially that is supposedly ready for
`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—
`graph is 316 stainless steel, 1/16" od, with different inside diameters. Tubing with
`0.01" (0.25 mm) id is commonly used in areas where dead volume must be mini-
`mized to maximize efficiency, 6g, between the injector and column, precolumn
`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 um 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-jawed 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-
`ing salts, particularly halide salts at low pH. HPLC companies have available a
`variety of accessories that can solve this problem. These include titani