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Text of Sample Preparation and GPC/SEC/APC Method Development · Sample Analysis Flow Diagram Weighing...

  • Miroslav JANČO

    Sample Preparation and GPC/SEC/APC

    Method Development

    1 The Dow Chemical Company, 400 Arcola Road, Collegeville, PA 19426

  • Page 2

    Overview

    A. Sample Preparation for GPC/SEC/APC Analysis

    1. Sample Types

    2. Solvents

    3. Filters

    B. GPC/SEC/APC Method Development

    1. Eluent(s) / Mobile Phase(s)

    2. Additives

    3. Column(s)

    4. Sample Loading

    5. Flow Rate

    6. Temperature

    7. Standards and Calibration

    C. Safety Considerations

    D. Literature

  • Page 3

    Sample Analysis Flow Diagram

    Weighing

    Dilution

    Filtration

    Eluents

    Columns

    Standards

    Detectors

    Data Collection /

    ProcessingSample PreparationMethod Development /

    Sample Analysis

    Report Generation

    Information

    to CustomerSample Production

    Collection / Sampling

    Sample

    Transport

    Archiving

    Modified from Ronald E. Majors, HPLC 2016 San Francisco

  • Page 4

    Representative Sample Types

    A. Solids:

    B. Liquids:

    Powders Pellets Pads Tapes Foams

    Solutions, Oils Emulsions / Slurries Paints

    C. Pastes, Waxes, and Creams:

  • Page 5

    Myriad of Sample Prep Techniques

    *Doug Raynie, Sample Prep Perspectives, LCGC N. America 34(3) 174–188 (2016)

  • Page 6

    Representative List of Common GPC/SEC Solvents

    A. Organic

    1. Ambient T

    Tetrahydrofuran (THF)

    Chloroform (CHCl3)

    2. Elevated T

    Toluene

    N,N-Dimethylacetamide (DMAc)

    3. High Temperature (~140 - 210 oC)

    Trichlorobenzene (TCB)

    4. Specialty

    Hexafloroisopropanol (HFIP)

    B. Aqueous

    Water

    Aqueous buffers at different pH

    Aq. buffers / Methanol mixed

    solvents (up to 50% MeOH)

    Polymer Handbook, 4th ed.; Editors: Brandrup, E. H. Immergut, and E. A. Grulke, Willey-Interscience, 1999

  • Page 7

    Solvent Selection Guide: Polymers Soluble in Organic Solvents

  • Page 8

    Solvent Selection Guide for Aqueous Soluble Polymers:

  • Filtration Step

    The life of an HPLC column can be extended up to 46x by filtering samples prior to injection

    www.pall.com/lab

    A

    B

    C

    Page 9

  • Page 10

    Filtration Step

    A. Organic solvents

    1. PTFE

    2. Nylon

    B. Aqueous

    1. PVDF

    2. Cellulose

    C. High Temperature

    1. SS Frits

    Diameter:

    4, 13, 17, 25 and 30

    mm ID

    Pore Sizes:

    0.1 µm

    0.2 µm (APC)

    0.45 µm (SEC)

    1, 3.1 and 5 µm

  • Page 11

    Membrane Chemical Compatibility Guide

  • Page 12

    Automatization in Sample Preparation

    Chemspeed Technologies

    http://www.chemspeed.com

    CTC Analytics AG

    http://www.ctc.ch

    Weighing station

    Solvent selector (up to 5 solvents)

    Temperature controlled shaker

    Centrifuge

    Filtration

  • Page 13

    Steps in SEC/APC Method Development

    Step 1:

    Defining the Analysis Goal

    Step 2:

    Method Development Factors

    Step 3:

    Data Processing and Reporting

  • Page 14

    Step #1: Defining the Analysis Goal

    What is the driving analytical request?

    A. High resolution

    Optimize column porosity for target polymer

    Maximize column length based upon flow rate, solvent

    viscosity and system pressure

    B. High throughput

    Optimize column porosity for target polymer

    Evaluate short columns taking full advantage of low

    dispersion system

    C. What is the polymer type?

    Polymer composition

    Single polymer, mixture, blend…

    This will impact the method conditions and the system

    configuration

  • Page 15

    Step #2: Method Development Parameters

    Column Selection

    Sample Loading

    Flow Rate

    CalibrationEluent Selection

    The eluent is selected with the following considerations:

    Sample Solubility

    Column Packing Compatibility

    Viscosity

    Additives

    SEC System / Detection Compatibility

    Cost

  • Page 16

    Eluent / Mobile Phase Selection Criteria

    Sample Solubility Need an eluent that can entirely dissolve the polymer sample and allow

    complete sample elution (100% sample recovery) from the column set

    Column Packing Compatibility No swelling/shrinking of column bed (stationary phase)

    Viscosity What is the impact on the separation conditions?

    Additives How to identify and eliminate surface interactions to get purely SEC

    mode separations?

    System/Detection Considerations The solvent is compatible with your polymer, but what about the

    chromatographic system?

  • APC Conditions:

    Column set: 3BEH 45+125+450Å columns

    (150x4.6 mm ID each) packed with 2.5 m

    particles

    Eluent: High Viscosity Solvent

    Flow rate: 0.5 mL/min

    Injection volume: 25L

    Inj. C: 2.5mg/mL

    Detection: UV @ 254nm

    Page 17

    Considering Eluent Viscosity for SEC/APC Separations

    Solvent Viscosity (cP)

    Acetone 0.32

    Hexane 0.33

    Acetonitrile 0.34

    Heptane 0.39

    Dichloromethane 0.39

    Methyl ethyl Ketone 0.40

    Ethyl Acetate 0.45

    Isooctane 0.50

    Tetrahydrofuran 0.55

    Chloroform 0.57

    Toluene 0.59

    Methanol 0.60

    Dimethylacetamide 0.77

    Dimethylformamide 0.90

    Water 1.00

    Ethanol 1.00

    Hexafloroisopropanol 1.02

    Dimethyl sulfoxide 1.10

    N-methylpyrrolidone 1.65

    Isopropanol 2.30

    High viscosity solvents will lead to:

    High column backpressures

    Lower mass transport and

    May need higher column temperatures

    14 600 psi

  • Page 18

    Considering Eluent UV Cut Off Values

    Solvent UV Cutoff (nm)

    Acetonitrile UV 190

    Pentane 190

    Water 190

    Hexane UV 195

    Cyclopentane 198

    Cyclohexane 200

    Heptane 200

    Isopropyl Alcohol 205

    Methanol 205

    Ethanol 210

    2-Metoxyethanol 210

    Methyl t-Butyl Ether 210

    n-propyl Alcohol 210

    Trifluoroacetic Acid 210

    Tetrahydrofuran UV 212

    n-Butyl Alcohol 215

    1,4- Dioxane 215

    Ethyl Ether 215

    Solvent UV Cutoff (nm)

    Ethylene Dichloride 228

    1,1,2-Trichlorotrifluoroethane 231

    Dichloromethane 233

    Chloroform 245

    n-Butyl Acetate 254

    Ethyl Acetate 256

    N,N-Dimethyl Acetamide 268

    N,N-Dimethylformamide 268

    Dimethyl Sulfoxide 268

    Toluene 284

    N-Methylpyrrolidone 285

    Chlorobenzene 287

    o-Xylene 288

    o-Dichlorobenzene 295

    1,2,4-Trichlorobenzene 308

    Methyl Ethyl Ketone 329

    Acetone 330

    Methyl Isobutyl Ketone 334

  • Page 19

    Considering Eluent Refractive Index Values

    Solvent Refractive Index

    Hexafloroisopropanol 1.275Methanol 1.329

    Water 1.33Acetonitrile 1.344Acetone 1.359Ethanol 1.361Ethyl Acetate 1.37Hexane 1.373

    Methyl ethyl Ketone 1.379Isopropanol 1.38Heptane 1.387Isooctane 1.404Tetrahydrofuran 1.408Dichloromethane 1.424Dimethylformamide 1.428Dimethylacetamide 1.438Chloroform 1.443N-methylpyrrolidone 1.468Dimethyl sulfoxide * 1.477Toluene 1.4961, 2,4 -Trichlorobenzene 1.571

    Polystyrene– Blue

    Polypropylene (Red)

    Eluent:

    TCB @ 140 C

    THF

    Toluene

    Polydimethylsiloxane (PDMS)

  • Page 20

    Additives: Why To Use Them?

    To eliminate / suppress:

    Polymer / surface (column packing)

    Polymer chain interactions

    Typical additives: LiBr, Triethylamine, Formic Acid, Acetic

    Acid, Trifluoroacetic Acid, Ammonium Acetate…

    Courtesy of PSSCourtesy of Agilent

  • Page 21

    Step #2: Method Development Parameters

    Column Selection

    Sample Loading

    Flow Rate

    CalibrationEluent Selection

    After information is gathered in Step 1, columns are

    selected based on:

    Surface chemistry

    Particle size

    Pore size

    Column length

  • Page 22

    GPC/SEC/APC Column Requirements

    Modern GPC/SEC/APC columns must fulfill a variety of properties regarding:

    • Physical requirements

    •Mechanical stability: (pressure, temperature, pH...)

    •Large pore volume

    •Small interstitial volume

    • Chemical requirements

    • Well defind surface (homogeneity)

    • Mo adsorbtion or repulsion

    • Excellent solvent compatibility (no swelling/shrinking)

    • Technical requirements

    • Column hardware: solvent / corrosion resistant

    • Clearly labeled, easy to handle

    • Clear documentation

  • Page 23

    Analytical Column Selection Criteria

    Column Packing/Surface Chemistry:

    Inorganic (bare and surface modified Si based)

    Organic (PS-DVB, DVB, PMMA, PVOH, etc.)

    Hybrid (BEH)

    Particle size:

    (1.7 – 2.5 – 3 – 5 – 10 – 20 µm)

    Smaller particles for higher resolution

    Larger particles to avoid shear degradation of very high MW components

    Pore size:

    Depends on molecular weight range of sample

    Avoid exclusion of sample

    Maximize pore volume in required separation region

    Column Length/ID (Column Format):

    Guard (30-50 x7.5-7.8 mm ID

    Analytical (300 x 7.5-7.8 mm ID)

    Preparative (300 x 25 mm ID)

  • 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6

    102

    103

    104

    105

    106

    Vt= 1.9mL

    Vp=1.05mL V

    g=0.6mL

    Vc= 2.5mL

    log M

    Retention time, [min]

    Vo=0.85mL

    VR = Vo + KDVp, KD=0-1

    ~3K – 80K

    Page 24

    SEC Column (150x4.6 mm ID) Characteristics

  • Page 25

    Particle Size Effect

    Smaller Particle Size :

    Higher Resolution

    Lower band dispersion

    Higher number of theoretical plates

    Higher column back pressure Risk of shear induced elongation and degradation

    Lower mechanical stability (large pore size) of the beads

    Particle Size Typ. Applications

    20µm Ultra High Molar Mass Polymers (106 -107 Da)

    10µm High viscosity eluents

    5µm Low viscosity eluents

    3µm Oligomers, Proteins

    1.7-2.7µm APC Applications (dimer-2M Da)

  • Particle Size Impact on Ɖ of PS 11.6K standard

    1K

    30K

    Useful separation range

    Page 26

    As particle size decreases, the

    calculated dispersity becomes

    closer to the reported Ɖ value,

    and thus provides greater

    accuracy of M data.

    PS 11.6K

    BHT

    10 µm

    5 µm

    3.5 µm

    1.7 µm

    Particle

    size

    Average M data of PS 11.6K standard

    Mw Mn Ɖ

    10 11 100 9 700 1.14

    5 11 400 10 900 1.05

    3.5 11 500 11 200 1.03

    1.7 11 400 11 100 1.03

  • Page 27

    SEC Conditions:

    Columns: (a) BEH 200 SEC

    column (150x4.6 mm ID)

    BEH packed with 1.7 m

    particles and (b)

    conventional SEC column

    (250x4.6 mm ID) packed

    with 5µm PS-DVB particles

    Eluent: THF, Flow rate: 1.0

    (a) and 0.5 mL/min (b)

    Injection volume: 5L

    Inj. C: 250ppm/component

    Detection: UV @ 254nm

    580

    3K

    11

    .6+

    7K

    30

    +2

    2K

    10

    0+

    66.3

    K

    a) APC

    b) Conventional SEC

    APC and Conventional SEC of 8 Component PS MixtureU

    V d

    ete

    cto

    r re

    sp

    on

    se

    , [A

    U]

    Retention time, min

    0 2 64

  • Page 28

    Individual Pore Size vs Mixed Bed Columns

    Individual Pore Size Columns Mixed Bed / Linear columns

    Deliver:

    Larger pore volume

    High(er) resolution

    Narrower/Limited

    separation range

    Deliver:

    Wide separation range

    Low(er) resolutionIdeal for use as scouting columns when MW of the

    sample is unknown or for analysis of samples with

    broad dispersity

    4 5 6 7 8 9 10 11 1210

    2

    103

    104

    105

    106

    107

    108

    102

    103

    104

    105

    106

    107

    108

    Linear S

    Linear M

    LInear XL

    SDV

    Mola

    r M

    ass [

    Da]

    Elution Volume [ml]

    4 5 6 7 8 9 10 11 12

    102

    103

    104

    105

    106

    107

    108

    102

    103

    104

    105

    106

    107

    108

    10 000 Å

    100 000 Å

    1 000 000 Å

    10 000 000Å

    50 Å

    100 Å

    500 Å

    1 000 Å

    SDV

    Mola

    r M

    ass [

    Da]

    Elution Volume [ml]

  • Page 29

    Combination of the SEC/APC Columns

    Page 29

    Use Column Combination to Enhance: RESOLUTION

    Add column of identical pore size (Increase pore volume)

    Doubling column length increases resolution by factor of 1.4

    PSS SUPREMA 5µm, 100Å

    (8x300 mm)

    Sample: Protein (Monomer / Dimer)

    PSS SUPREMA 5µm

    2x 100Å (8x300 mm each)

    Courtesy of PSS

  • Page 30

    Combination of the SEC/APC Columns

    Page 30

    Use Column Combination to Enhance: SEPARATION RANGE

    Add column of different pore size

    Avoid column missmatch

    Courtesy of Waters

    µRIU

    0.00

    15.00

    30.00

    Minutes

    0.00 1.50 3.00 4.50 6.00

    3 Columns in series

    45A + 125A + 450A

    2 Columns in series

    45A + 125A

  • Altering the Resolution of SEC separation

    SEC Conditions:

    Column set: 3PLgel (300x7.5 mm ID) columns packed

    with PS-DVB gel, Pore Size: 102+103+104 A, Particle

    Size: 5 m, Tc= 40 oC

    Eluent: THF (HPLC grade fro J.T. Baker), Flow rate: 1

    mL/min

    Injection volume: 100 L, Inj. C: ~1 mg/mL/component

    Detector: RI, TD = 40 oC

    SEC Conditions:

    Column set: 2PL Oligopore (300x7.5 mm ID)

    columns packed with PS-DVB gel, Pore Size:

    Oligopore, Particle Size: 6 m

    Eluent: THF (HPLC grade), Flow rate: 1 mL/min

    Injection volume: 100 L, Inj. C: ~2

    mg/mL/component

    Detector: ELSD, TNEB = 40 oC, TECH=110

    oC

    Polymer

    Mw~700K, Mn~100K

    Oligomer

    Mw~1200, Mn~600

    Page 31

  • Page 32

    SEC/GPC Column Selection Guide (Waters)

    http://www.psscolumnselector.com/

    http://navigator.chem.agilent.com/

    https://www.phenomenex.com/size-exclusion-

    chromatography-column

    http://www.psscolumnselector.com/http://navigator.chem.agilent.com/https://www.phenomenex.com/size-exclusion-chromatography-column

  • Page 33

    SEC/GPC Column Selection Guide (PSS):

    The Magic Triangle from PSS...

    (or the importance of the polarity of the packing material)

    http://www.psscolumnselector.com/

    http://www.psscolumnselector.com/

  • Page 34

    Column Choices Guide (Agilent)

  • Page 35

    Care and Use of GPC columns

    Operate column(s) in column packing compatible solvent only and do not exceedrecommended backpressure and temperature limits

    Change solvents by flushing the column at 0.1ml/min (overnight) in the new solvent

    Never use methanol or acetonitrile with PS-DVB gels

    Polar solvents shrink the gel packing, causing voids

    Note: Flush the complete system with solvent to be used before connecting

    the columns to the system

    Always keep the flow through the columns at low flow, 0.1mL/minute; do not letsystem stand idle

    Increase flow at 0.1 mL/minute to the specified flow rates of the columns

    For columns operated at high temperatures (140 - 150 oC) - purge the columns at0.1 mL/min overnight at ~80 oC and then ramp up to operating temperature overseveral hours

    Store the column bank together in the solvent used

    Keep end fittings on the column tight to keep the column packing from drying out

    Be careful not to drop the columns (they are fragile)

    Filter sample solutions!

  • Page 36

    Step #2: Method Development Parameters

    Solvent Selection

    Sample Loading

    Flow Rate

    CalibrationColumn Selection

    The amount of polymer injected in the column can

    impact the fidelity of the separation. The two factors

    that contribute to this are:

    Concentration

    Injection volume

  • Page 37

    Sample Loading Criteria

    Polymer Concentration

    Mass overload on column will lead to a loss of resolution

    Need to balance this with sensitivity, particularly if looking

    for lower level contributors

    Sample Molar

    Mass

    g/mol

    Typical

    Concentration,

    weight to volume

    [mg/mL], (%)

    < 10 000 2 (0.2%)

    10 000 – 1 000 000 2-1 (0.2-0.1%)

    > 1 000 000 0.2-0.5 (0.02-0.05%)

    Courtesy of PSS

  • Page 38

    Sample Loading Criteria

    Injection Volume

    Too large of an injection volume can also lead to mass

    overload

    Large sample volumes increase the peak volumes which

    contributes to a loss in resolution

    20µL

    200µL

    Number of Analytical

    Columns

    Guideline Injection

    Volume

    [µL]

    4, 5 200

    3 100

    2 50

    1 20

  • Page 39

    Step #2: Method Development Parameters

    Solvent Selection

    Sample Loading

    Flow Rate

    CalibrationColumn Selection

    Flow rate is often not evaluated in GPC method development.

    However, it is an important method variable that impacts:

    Speed of analysis

    Separation efficiency

  • High Speed SEC Conditions:

    Column: PSS High Speed SEC column (50x25 mm ID) packed with 5 m PS-DVB particles, pore size: linear M

    Eluent: THF

    Flow rate: 6.25 mL/min

    Injection volume: 100 L

    Inj. C: 1mg/mL

    Detection: RI

    Disadvantages:

    High solvent consumption, compromised resolution of separations

    Fast SEC

    Page 40

    Courtesy of PSS

  • Page 41

    Rapid SEC

    Rapid SEC Conditions:

    Column: PL Rapid SEC column (100x10 mm ID) packed with 5 m PS-DVB particles, pore size: linear M or L

    Eluent: THF

    Flow rate: 1, 2 or 3mL/min

    Injection volume: 100 L

    Inj. C: 1mg/mL

    Detection: [email protected]

    Disadvantages:

    Compromised resolution of separation Courtesy of Agilent

  • Step #2: Flow Rate

    Courtesy of Waters

    AU

    0.000

    0.010

    0.020

    0.030

    0.040

    AU

    0.000

    0.010

    0.020

    0.030

    0.040

    AU

    0.000

    0.010

    0.020

    0.030

    0.040

    Minutes

    0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00 10.50 11.00 11.50 12.00 12.50

    8.4K

    8.4K

    8.4K

    67K

    67K

    67K

    579K

    579K

    579K

    7.52M

    7.52M

    7.52M

    200 µL/min

    400 µL/min

    600 µL/min

    Poly(styrene) standard

    7.52 M distorts with

    increased flow rate –

    Slalom Chromatography

    Page 42

  • Page 43

    Step #2: Method Development Parameters

    Solvent Selection

    Sample Loading

    Flow Rate

    CalibrationColumn Selection

    Ensuring that you are adequately calibrating your bank of columns

    is an important variable in method development. You need to

    consider:

    • Which standards?

    • How many standards?

    • Calibration type

    • Frequency of calibration

  • Page 44

    Readily Available GPC/SEC Standards

    A. Organic

    1. Ambient T

    Poly(styrene) (PS)#

    Poly(methyl methacrylate)#

    (PMMA)

    Poly(vinyl chloride)

    Poly(dimethyl siloxane)

    Poly(tetrahydrofuran) (PTHF)

    2. High Temperature (~140 - 210 oC)

    Polyethylene (PE)

    A. Aqueous

    Polyacrylic acid (PAA)

    Polymethacrylic acid

    (PMAA)

    Polyethylene glycol/oxide

    (PEG/PEO)%,#

    Polystyrene sulphonate

    Dextran

    Pullulan

    Poly(vinyl Pyridine)

    % - can be used for both organic and aqueous systems

    # - as individual standards and mixes

  • SEC Charts of PS Standards and Calibration Curve

    SEC Conditions:

    Column set: 3PLgel (300x7.5 mm ID) columns packed with PS-DVB gel, Pore Size: 102+103+104 A,

    Particle Size: 5 m, Tc= 40 oC

    Eluent: THF (HPLC grade from J.T. Baker), Flow rate: 1 mL/min

    Standards: PS1 MIX A, MIX B and PS 162

    Injection volume: 100 L

    Inj. C: ~1 mg/mL/component

    Detector: RI, TD = 40 oC

    Page 45

  • APC of 16 PS Standards

    APC Conditions:

    Column set: 3 APC columns (150x4.6 mm ID) packed with BEH TMS particles,

    Pore Size: 45+125+450Å, Particle Size: 2.5 µm, Tc= 40 oC

    Eluent: THF (Certified grade from Fisher), Flow rate: 1 mL/min

    Injection volume: 10 µL, Inj. C: ~0.07 mg/mL/component

    Detector: RI, TD = 40 oC

    ~200 – 1M

    Page 46

    Run time: 6 min

    1.8 5.6

    RI d

    ete

    cto

    r re

    sp

    on

    se

    , [m

    V]

    3 4 5

    1 1

    30K

    560K

    310K

    200K

    120K

    98K

    66K

    44K

    30K

    22K

    11.6

    K

    7000

    3250

    1700

    580

    220 (

    BH

    T)

  • Literature

    1. Mori, S & Barth, H (1999) Size Exclusion Chromatography,

    Springer Verlag, Berlin, Germany

    2. Wu, C-S (2003) Handbook of Size Exclusion Chromatography

    and Related Techniques. Marcel Dekker, New York, NY, U.S.A.

    3. Striegel, AM, Yau, WW, Kirkland, JJ & Bly, DD (2009) Modern

    Size-Exclusion Chromatography. John Wiley & Sons, Chichester,

    UK

    4. Polymer Handbook, 4th edition; Editors: Brandrup, E. H.

    Immergut, and E. A. Grulke, Willey-Interscience, 1999

    5. GPC/SEC/APC Vendor’s webpages:

    www.agilent.com

    www.jordilabs.com

    www.malvern.com

    www.phenomenex.com

    www.pss-polymer.com

    www.shodex.com

    www.tosohbioscience.com

    www.waters.com

    www.wyatt.com

    Page 47

    http://www.agilent.com/http://www.jordi.com/http://www.malvern.com/http://www.phenomenex.com/http://www.pss-polymer.com/http://www.shodex.com/http://www.tosohbioscience.com/http://www.waters.com/http://www.wyatt.com/

  • Safety Considerations

    Safety should be the highest priority

    while working in the lab

    Wear appropriate PPEs (lab coat,

    safety glasses, gloves, goggles, face

    shield, apron, etc.)

    Sample prep steps/handling should be

    performed in the hood or in a well

    ventilated enclosure to minimize

    exposure of operator and lab personnel

    to chemical vapors

    Spills – know the spill kit location,

    handle by institution policy

    All waste generated should be

    disposed off in the proper waste stream

    according to institution’s policy

    Know eye wash and safety shower

    locations

    Ergonomics

    Page 48

  • Summary

    Page 49

    Sample preparation is viewed as a routine task rather than as an integral

    component in the analytical process, and as a result, it has long been

    undervalued as a science and underdeveloped as a technology.

    There is no universal solvent nor GPC/SEC/APC method

    Solvent /Eluent selection is driven by

    Sample Solubility

    Column Packing Compatibility

    GPC/SEC/APC method development / improvement is driven by

    The analysis goal which defines the selection of:

    Column(s)

    Eluent

    Flow rate

    Injected mass (injection volume and concentration)

    Temperature

    Calibration Standards

    Detection Options

  • THANK YOU FOR ATTENDING!

    Page 50

    ANY QUESTIONS?