Ronald E, MajorsAgilent TechnologiesWilmington, DE USAron_majors@agilent.com

Continuing Innovations inReversed-Phase HPLC Column

Technology

Pittcon 2010March 4, 2010Orlando, FL

Outline of Talk*

• RPC Innovations in Three Areas:

• Selectivity

• Stability

• Efficiency

• Future Directions

• Focus on commercial products, not research products

• Approach innovations from an historical perspective

Reversed Phase Trivia

• First reported RPC experiment (1950- A.J.P. Martin and Howard,Biochem. J. 46, 532 (1950)- LL partition paraffin oil and n-octane on

diatomaceous earth

• First reported siloxane bonded phase used in RPC (1967-Aue andHastings, Dalhousie University, Nova Scotia)

• First reported commercial RPC packing, Permaphase (polymer) onZipax (1972-Kirkland, Dupont)

• First reported commercial RPC microparticulate packing, MicroPak-CH,5-10-µm silica with monomeric C18 (1972-Majors, Varian)

• Reversed phase chromatography named by Csaba Horvath (date-unknown but around 1973)

• Approx. 60-70% of all HPLC work performed by RPC [Horvath &Melander, J. Chrom. Sci. 15, 393 (1977)]

• 70-80% of all HPLC work performed by RPC (J. Chrom. Sci, Sept, 1980)

• LCGC 2009 survey showed 94% of all chromatographers use RPC

HPLC Analytical Column Pittcon Introductions by Phase(1985-2010)*

0

10

20

30

40

50

60

70

1985

1987

1989

1991

1993

1995

1997

1999

2001

2003

2005

2007

2009

RPC

Specialty

IEX

NP

SEC

* Extracted from my LC/GC Pittcon Articles

RPC

Specialty

SECNPC

IEX

Nu

mb

er

of

Co

lum

ns

2010

(for RPC columns, 915 cumulative total)

HPLC Mode Usage versus Year (1984-2009)*(Normalized to 100%)

0

10

20

30

40

50

60

70

1984 1985 1986 1987 1991 1994 1997 2007 2009

RPC

NPC

LSC

Ion Exch

SEC

HILIC

Chiral

OtherRe

lati

ve

%o

fU

sa

ge

LCGC Surveys

Innovations in RPC Selectivity

• Base packing contribution

• Silica type (acidity), surface area, pore size, pore volume, etc.

• Other particles: alumina, zirconia, titania, polymer

• Chemical moiety contribution

• Bonding chemistry

• Monomeric- and polymeric-bonding (coating)

• Carbon loading, endcapping

• Mobile phase contribution

• Will not be covered here

The Surface of Silica Supports

OH HO OH OH

AssociatedSilanols

Internal Metal(activated silanol)

(most acidic)

Surface Metal

decreasing acidity

GeminolSilanols

FreeSilanols

OH

OH

Si Si Si Si

SiMM+ +

Original ZORBAX, 1973 and other type A silicas

(basic compound can tail)

ZORBAX Rx-Sil, 1987 and other Type B silicas

(basic compounds have less tailing; lowereffective silanol pKa)

Conditions: Flow Rate: 2.0 mL / min.

Mobile Phase: 5% 2-Propanol in Heptane

Chromatographic ImprovementUsing Highly PurifiedType B Zorbax Rx-Sil

CH2 -OH.

CH2 -OH

9

0 5 10 15Time (min)

OCH CHCH NHCH(CH )2 32 2

OH

Zorbax StableBond with Rx-SILImproves Peak Shape

Silica Type A – More AcidicColumn: ODS, 4.6 x 250 mm, 5 mPlates: 92

USP Tf (5%): 2.90

PropranololpKa 9.5

Silica Type B– High Purity, Rx-SilColumn: SB-C18, 4.6 x 150 mm, 5 mPlates: 6371

USP Tf (5%): 1.09

Mobile Phase: 75% 50 mM KH2PO4, pH 4.4 : 25% ACN Flow Rate: 1.5 mL/min

0 5 10

Time (min)

Different C18 Bonded Phases for MaximumSelectivity

Eclipse Plus C18

Eclipse

XDB-C18

Extend-C18

StableBond

SB-C18

Mobile phase: (69:31) ACN: waterFlow 1.5 mL/min.Temp: 30 °CDetector: Single Quad ESIpositive mode scanColumns: RRHT4.6 x 50 mm 1.8 um

Sample:1. anandamide (AEA)2. Palmitoylethanolamide (PEA)3. 2-arachinoylglycerol (2-AG)4. Oleoylethanolamide (OEA)

1 2

3

4

1 2 3 4 5

1 2

3

4

1 2 3 4 5

1 2,3 4

1 2 3 4 5

1 234

min1 2 3 4 5

1st choiceBest Resolution& Peak Shape

2nd choiceGood alternate selectivity dueto non-endcapped

3rd choiceGood efficiency & peak shapeResolution could be achieved

4th choiceResolution not likely,Other choices better, for thisseparation.

Multiple bonded phasesfor most effective method

development.Match to one you are

currently using.

RPC Bonded Phase SelectivityDifferences in 30% ACN

1

mAU

0

100

1 2

mAU

0

100

1 2

mAU

0

100

1 2

mAU

0

100

1 2

mAU

0

100RRHT SB-C184.6 x 50 mm, 1.8 m

RRHT SB-Phenyl4.6 x 50 mm, 1.8 m

RRHT Eclipse Plus C184.6 x 50 mm, 1.8 m

RRHT SB-AQ4.6 x 50 mm, 1.8 m

RRHT SB-CN4.6 x 50 mm, 1.8 m

5 Different bonded phasescompared

Analysis time of each run isonly 2 minutes

Comparison done in optimum% organic

The fast runs mean acomparison can be done even ifyou have a good separation onthe C18

More chances to optimize!

0

100

200

300

400

500

600

700

800

C18 C

8

C4-

C6

Polym

er

Phenyl C

N

C1-

C3

Fluor

inat

ed

Polar

-em

bedded C

30

Oth

er

#o

fC

olu

mn

s

Reversed Phase Columns Introduced 1973-2010*

*My J. Chromatog. Sci, Anal. Chem. & LCGC Articles

Shape Recognition in RPCSeparation of EPA 16 Priority Pollutant PAHs

NIST Standard Reference Material 1647

Through the adjustment oftemperature the monomericphase can yield a shape selectiveseparation (decreasedtemperature) and the polymericphase can loose shape selectivity(increased temperature).

C.Rimmer, K.Lippa, & L. Sander, LCGC No. America, Oct. 2008

Rapid Separation of PAHs on Polymeric RPCColumn

4.6x50mm, 1.8µm

Conditions:Agilent 1200SLDAD 220,4nm No Ref. DADStop Time = 5.60minFlow 2.00 ml/minMobile Phase A = Water; B = AcetonitrileGradient: Time (Min) % B

0.00 453.5 1004.9 1005.2 45Stop Time = 5.6

Temp. = 25° C50 nanogm on Col for each Componentno Mixer & no Pulse Dampener

Rs = 2.0

min0.5 1 1.5 2 2.5 3 3.5 4 4.5 5

mAU

0

20

40

60

80

100

120

140

160

1

1

2

3

4

56

7

8

9

1011

12

13

14

1516

17

1 = Toluene2 = Naphthalene3 = Acenaphthylene4 = Acenaphthene5 = Fluorene6 = Phenanthrene7 = Anthracene8 = Fluoranthene9 = Pyrene

10 = Benzo(a)anthracene11 = Chrysene12 = Benzo(b)fluoranthene13 = Benzo(k)fluoeanthene14 = Benzo(a)pyrene15 = Dibenzo(a,h)anthracene16 = Benzo(g,h,i)perylene17 = indeno(1,2,3-c,d)pyrene

RPC Phases Beyond the “Regulars”

• Different alkyl chain lengths

• C12 (1997)

• C14 (1996)

• C20 (1994)

• C22 (1980)

• C27 (2005)

• C30 (1994)

• Mixed alkyl chains

• C18-Short alkyl (2002)

RPC Phases Beyond the “Regulars”(continued)

• Aryl phases

• Diphenyl (1991)

• Biphenyl (2008)

• Mixed alkyl-aryl

• C18-phenyl (1993)

• C6-phenyl (1998)

• Alkyl-polar

• Phenyl-CN (2008)

• C18-urea (2001)

• Phenyl-PEO (1994)

Biphenyl Diphenyl

Restek Cartoons

RPC Phases Beyond the “Regulars”(continued)

• Polar-embedded Phases

• C18-carbamate (1998)

• C8-carbamate (1997)

• C18-polar embedded (2003)

• Alkylamide (1997)

• C8-amide (1999)

• C14-amide (1999)

• C23-amide (2000)

• C16-sulfonamide (2004)

• Phenyl-ether linkage (2001)

Bonus-RP, pH 2-81. polar alkyl phase2. triple endcapped3. uses bulky silanes

O R

Si

Si

O

Si

O R

R1

CH3

CH3

CH3

CH3

R

R1

CH3

R1

R1

R1

CH3

R1

Si

Si

PG

PG

PG

PG = amideR = isopropyl

RPC Phases Beyond the “Regulars”(continued)

• Alkyl-Ion Exchange

• C18-SAX (2001)

• C18-WAX (2007)

• C18-SCX (2002)

• C18-WCX (2007)

• C18-Basic group (2007)

• Phenyl-SCX (2006)

• C18-SCX-SAX (2009) (trimodal)

RPC Phases Beyond the “Regulars”(continued)

• Fluorinated Phases

• Pentafluorophenyl (PFP)/pentafluorobenzene (PFB)(1989)

• Fluorocarbon (1984)

• Fluorophenyl (1985)

• Perfluoroalkyl (2002)

RPC Phases Beyond the “Regulars”(continued)

• Other

• Hypercarb (Graphitized carbon)

• Graphitized carbon on ZrO2

• Pyridine (2008)-SFC

• PEG (2002)

• Cholesterol (2003)

• Hydrid (Type C silica) (2008)

• AQ-type phases

Polymeric RPC Columns

• Wide pH range (1-14)

• No silanols present

• Lower efficiency than silica-based columns (~1/3)

• Polymer-coated

• PBD on silica and alumina (1980)

• PBD on zirconia

• Representative chemistries

• PS-DVB (1977)

• C18-PS-DVB (1980)

• Polymethacrylates (1985)

• Polyvinylalcohol (1989)

• DVB (1990)

• PS-DVB-methacrylate (1996)

• DVB-methacrylate (1996)

Specialty RPC Columns

• HPLC columns developed for specific separations that aredifficult to achieve on a standard column.

• Sometimes manufacturers will use a standard column but test itspecifically for a certain class or compounds and provide arecommended set of chromatographic conditions.

• In some cases, the specialty column comes as part of a “totalsolution” kit with reagents, standards and a method.

• Most specialty columns will be delivered with a testchromatogram from an analysis performed at the factory beforeshipment and some are guaranteed for a specific separation

Examples of Specialty RPC Columns

• Fatty acid (1977)

• PAHs (1985)

• PTH-amino acid (1990)

• Triglyceride (1990)

• Peptide/protein (early 1970’s)

• Fullerenes (Bucky Balls) (mid-90’s)

• e.g. pyrene, pentabromobenzene

• Polar-embedded

Innovations in RPC Column Stability

• Packing base material

• Chemical bonding

• Packed bed

Base Material for All Columns (Not Just RPC)Introduced at Pittcon in Last 10 Years

0

100

200

300

400

500

600

700

Silica

Polym

er

Hybrid

Zirconia

Monolit

hs

#of

Colu

mns

SILICA is THE Most Popular LCPacking

• Spherical Shape

• Narrow Particle SizeDistribution

• Uniform Porosity

• Narrow Pore SizeDistribution

• Porous Throughout

• Choice of Particle Size

• 1.8, 2, 2.5 3, 5, 10, 15, 20m

• Choice of Pore Sizes

• 60-120, 300, 1000Å

• Surface Easily Modified

• Choice of Bonded Phases

• Compatible with Water and AllOrganic Liquids

• Insoluble

• Unreactive

Unfavorable Physical Characteristics:

Favorable Physical Characteristics:

• Soluble at high pH (pH > 9)

• Surface is weakly acidic (-Si-OH), ionize at mid-pH

• Typical bonded phase pH range 2-8 (monomeric bonding)

Monomeric vs. Polymeric Bonding

figure courtesy of Grace Davison

Monomeric Bonding

Polymeric Bonding

Trifunctional silane

Highly reproducible

X = chloro- or alkoxy

Mechanism of Silica Bonded Phase ColumnFailure at Low pH

• Bonded phase cleavage

• Three Current Solutions:

• Hybrid phases

• Sterically-protectedbonded phases

• Bidentate bonded phases

StableBond Reaction to Make aSterically-Protected Surface (Kirkland,

1991)

CH 3HC

SiX R

H3C

CH 3H 3CCH

HC

X = Cl, OEt , etc .

R = CN, C8, etc .

Si

CH 3

H 3C

SiOH + Si

R

CH 3

H 3C

OCH

• Diisopropyl silanes or diisobutyl silanes (C18)

ZORBAX StableBond Bonding(Kirkland, 1991)

•Low pH stability – down to pH 1

•Non-endcapped for selectivity andlifetime

•Patented sterically protectingbonding

•6 different selectivities - C18, C8, CN,Phenyl, C3, Phenyl-hexyl

•For most sample types at low pH

O

R

R

Si

OH

Si

O

Si

R

R

R

O

OH

R

Hybrid Silica-Carbon Particle (Xterra, Waters 1999)

(courtesy of Waters)

Acquity 2004XBridge BEH 2005

Mechanism of Silica Bonded Column Failureat High pH

• Silica Dissolution!

• Current Solutions:

• Protect the Silica

• High coverage, exhaustive endcapping

• Long bonded phase chains

• Bidentate bonding

• Polymeric phase

• Coated polymeric phase on inorganic support

• Hybrid Particle

ZORBAX Extend-C18 (Kirkland, 2000)

• Superior high pH stability –up to pH 11.5 with silicaparticles

• Excellent reproducibility

• Patented bidentate, C18bonding

• Double endcapping

C18SiO

O Si C18

C18SiO

O Si C18

Am

ount

ofS

ilica

Dis

solv

ed,m

g

Volume of Eluent, Liters0 2 4 6 8 10 12 14 16

0

20

40

60

80

100

120

140

160

180

Eclipse XDB-C8Eclipse XDB-C18Extend-C18

Lifetime of ZORBAX Extend-C18at High pH (Kirkland, 2000)

Columns: 4.6 x 150 mm, 5 m

Purge: 50% ACN / 50% 0.02 MK2HPO4, pH 11

Flow Rate: 1.5 mL/min

Temperature: 25°C

Detection: Silicate concentration bysilicomolybdate color reaction

b) c)a)

Chromatograms to Illustrate “Phase Collapse” in Reversed PhaseChromatography with Highly Aqueous Mobile Phase

a) 40:60 H2O:MeCNb) 100% H2O (30 min)c) Recondition w/

40:60 H2O:MeCNd) & e) same

experiment(all w/ 1% HOAc)

d) e)

High DensityC18 phase

Polar-embeddedC14 phase withether linkage

SiO2

H2O

H2O

H2O

H2O

H2O

H2O

H2O

H2O

H2O

H2O

H2O

CH3OH

CH3OH

CH3OH

CH3OH

CH3OHCH3OH

CH3OH

CH3OH

CH3OH

“Phase Collapse” in Reversed Phase ChromatographyConfiguration of Long Chain Bonded Alkyl Phases in

Water-Methanol Mixtures

Bonded moieties are fully extended from silica gel surface, are solvated and ableto interact with analytes

(Before)

SiO2

H2O

H2O

H2O

H2O

H2O

H2O

H2O

H2O

H2O

H2OH2O

H2O

H2O

H2O

H2O

H2O

Bonded moieties are self-associated and in a “collapsed” structure

“Phase Collapse” in Reversed Phase ChromatographyConfiguration of Long Chain Bonded Alkyl Phases

in 100% Water

(After)

a) b)

Analytes Properly Retained

Analytes PartiallyRetained or Unretained

a) Pore Structure with pressure using a 100% aqueous mobile phase and alkyl chains in thepore properly solvated; analytes can partition into the pore and interact with nonpolarbonded phase

b) b) Pore Structure after stopping the flow to allow expulsion of water from the pores; withflow resumed the pores are still dewetted and analytes cannot enter pores and have littleor no retention

Possible Mechanism of Pore Dewetting (“Phase Collapse”)for Reversed-Phase Chromatography in a Highly Aqueous

Mobile Phase

Ref. J. E. O’Gara et al, LC/GC 19 (6)632-642 (2001).

Innovations in Efficiency of RPC Columns

• Particle size reduction

• Non-porous

• Porous

• Superficially porous*

• Monoliths

* also called pellicular, porous layer beads, fused core, solid core

Future Directions in Particle Size Development(1973)

Majors & MacDonald, J. Chromatogr. 83, 169 (1973)

Extrapolated line

1998* 1.5 µm*(non-porous) 30,000

History of Commercial HPLC Particle Development

2003 1.8 µm 32,500

2007/2008 2.7 µm (pellicular) 32,000***

Year(s) ofAcceptance

Particle Size Most PopularNominal Size

Plates / 15cm(Approximate)

1950’s 100µm 100

1967 50 µm(pellicular) 1,000

1972 10 µm 6,000

GlassBead

Irregularly-Shaped

12,000

1992 3-3.5 µm 22,000

1982 5 µm

2000 2.5 µm 25,000

1999 5.0 µm (pellicular) 8,000**

* non-porous silica or resins

** 300 A pore for protein MW 5,700*** 90-120 A pore

Particle Size Usage in Analytical RPCColumns versus Year*

0

10

20

30

40

50

60

70

1984 1985 1986 1987 1994 1997 2007 2009

<2

3-3.9

4-5

6-10

%o

fP

art

icle

Siz

es

ParticleSize, µm

* LCGC Magazine surveys

Average Particle Sizes for HPLC ColumnsIntroduced at Pittcon 2010*

Nu

mb

erof

Col

um

ns

Aver. Particle Size, Microns

0

2

4

6

8

10

12

14

<2 2.1-2.9 3.0-4.0 5 7 10 >10

Sub-two micron & superficially porous (2.7-µm) driven by highthroughput applications & analysis of complex samples

R. Majors, LCGC No. Amer. March, 2010

Solutes degradation,Limited number of stable

stationary phases

High efficiencymaintained at high

mobile phase velocity

Yes (above100°C)

High Temperature

Limited commercialphases

High plate counts atrelatively lower pressure

NoSuperficially

Porous Particle

Batch to batchreproducibility, Limitedcolumn dimensions &

phases

High columnpermeability

NoSilica Monolith

Extra-columnbroadening, frictional

heating

Very high plate countsin short analysis times

Yes/maybeSub-2 m Particle

Major LimitationsKey AdvantagesSpecializedInstrumentRequired

Approach

Recent Efficiency Improvements in HPLC

Column Scalability: Change in Column Configurationto Increase Speed While Maintaining Resolution

min1 2 3 4 5 6 7 8

4.6 x 50-mm, 1.8 µm

min1 2 3 4 5 6 7 8

mAU

0

10

20

30

40

50

60

70

4.6 x 250-mm, 5 µmR4,3=9.30R3,2=3.71R2,1=4.31

8 min.

min1 2 3 4 5 6 7 8

mAU

0

10

20

30

40

50

60

4.6 x 100-mm, 3.5 µm

3 min.

R4,3=8.53R3,2=3.37R2,1=3.69

mAU

0

10

20

30

40

50

60

1.5 min.Column: Zorbax SB-C18A= 0.2% FA, B=AcCN w 0.2%FA (98:2)F=1.5 mL/min Inj. Vol: 2-,4-, 6-ul, respectively;Detector: DAD, 254-nm; Flowcell: 3uL, 2 mm flow path

R4,3=9.30R3,2=3.61R2,1=3.87

1-methylxanthine; 2) 1,3-dimethyluric acid; 3) 3,7-dimethylxanthine; 4) 1,7-dimethylxanthineSolutes:

1

2

3

4

Commercial 2- and Sub-2-µm Totally Porous HPLC Columns*

2.0Rapid aSBAgela Technologies

1.9Pursuit UPSVarian

2.0PrestoImakt

1.8Epic Sub-2ES Industries

1.7Fortis 1.7Fortis Technologies

1.9Hypersil GoldThermo

2.0TSKgel SuperODSTosoh Haas

1.7Acquity BEHWaters

2.0Ultra-FastYMC

2.0ZirchromZirchrom

2.0Capcell PackShiseido

1.7Emerald, EpitomizeOrachem Technologies

2.0LunaPhenomenex

1.9Pinnacle DB/ Ultra IIRestek

1.7GP-8 and GP-18Sepax

1.5PathfinderShant Laboratories

1.8NucleodurMacherey-Nagel

1.8Cogent Diamond & Silica-CMicroSolv Technology

1.8MicrosilMicro-Tech Scientific

2.0LaChromUltraHitachi

1.8BlueOrchidKnauer

1.8ProntoPEARL TPP Ace-EPSBischoff

1.5VisionHTAlltech (Grace Davison)

1.8Zorbax Rapid Resolution HT/HDAgilent Technologies

Aver. dP, µmProduct NameManufacturer

* Non-porous & Superficially Porous Particles Not Included

Commercial 2- to 3-µm Totally Porous HPLC Columns*

2.5PathfinderShant Laboratories

2.5Kromasil EternityAkzo Nobel

2.5XBridge, SunFireWaters

2.2GP-8 and GP-18Sepax

2.2XRShimadzu

2.1, 2.5FortisFortis Technologies

2.4, 2.8Pursuit UPS and XRSVarian

2.5Luna HST and SynergiPhenomenex

2.3TSK-Gel ODS HTPTosoh Bioscience

Aver. dP, µmProduct NameManufacturer

* Non-porous & Superficially Porous Particles Not Included

Monolith Silica Support(Merck Chromolith, 2000)

Monolith Rod MacroporousStructure

(2-µm)

MesoporousStructure(13-nm)

Total Porosity 80%

Van Deemter Plot A: HETP vs. Linear VelocitySmall Porous Particle Columns and Silica Monolith (Chromolith)

0.0000

0.0005

0.0010

0.0015

0.0020

0.0025

0.0030

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4

HET

P(c

m/

plat

e)

Interstitial Linear Velocity (ue)

Dimensions: 4.6 x 50/30/20mmEluent: 85:15 ACN:WaterFlow Rates: 0.05 – 5.0 mL/minTemp: 20°CSample: 1.0L Octanophenone in Eluent

ZORBAX 5.0mZORBAX 3.5mVendor A 2.5mVendor B 2.0mZORBAX 1.8mMonolith

Equivalent to ~ 3.5-µm particle

Mobile Phase:A-ACNB-H2O

Gradient:t A B flow[min] [%] [%] [ml/min]

0,0 15 85 1,500,4 15 85 1,502,0 100 0 1,002,5 100 0 1,002,6 15 85 1,003,0 15 85 1,00

Injection: 0,5µlDetection: 240 nm UVTemperature: 40°C

Sample: 1.) Fluoxymesterone2.) Boldenone3.) Methandrostenolone4.) Testosterone5.) Methyltestosterone6.) Boldenone acetate7.) Testosterone acetate8.) Nandrolone propionate9.) Testosterone propionate

10.) Nandrolone phenylpropionate11.) Testosterone isocaproate

0 0,5 1 1,5 2 2,5t [min]

Chromolith® FastGradient (50-2mm)Fast Analysis of Steroids (Doping Substances)

(Courtesy of Merck KGaA)

5 µmTotally Porous Particle

Comparison of Diffusion DistancesTotally porous silica versus superficially porous silicas

2.5 µm

Required diffusion distancefor a molecule

0.25 µm

5 µmSuperficially Porous Particle

2.7 µmSuperficially Porous Particle

Poroshell 300 A Poroshell 120 A

0.50 µm

1.7µm4.5 µm

2000 2010

Poroshell 120 Columns for HPLC and UHPLC

• 80-90% efficiency of sub 2 µm• At ~40-50% lower pressure• 2X efficiency of 3.5 µm (totally

porous)• A 2.7 µm particle size• A 2um frit to reduce clogging• A 600 bar pressure limit

• The particle has a solid core (1.7µm) and porous outer layer with a0.5 µm diffusion path

March 8, 2010Confidentiality Label

53

Poroshell 120 columns have:

Poroshell 120 Pore and Particle SizeDistribution

Pore Width (Å)10 100 1,000

Po

reV

olu

me

(cm

³/g

)

0.00.0

0.5

1.0

BJH Adsorption dV/dlog(w) Pore Volume Particle Size Distribution Comparisonwith Totally Porous Particles

Poroshell 120 particles have an averagepore size of 120 Å.

Normalized Particle Size Distribution

Poroshell 120

1.8um totally porous

3.5um totally porous

5.0um totally porous

Backpressure Comparison of 2.7µmPoroshell 120 with 1.8µm Totally Porous

Particles

• Superficially porous particles have 40%-50% back pressure of 1.8 µm totallyporous particles.

Column Pressure (bar) vs Flowrate (mL/min)

(60:40 ACN:H2O, all columns 4.6mm x 50mm)

0

50

100

150

200

250

300

350

400

450

500

0 1 2 3 4 5 6

Flow Rate (mL/min)

Pre

ssu

re(b

ar)

Poroshell 120 SB-C18, 2.7µm

RRHT SB-C18 1.8µm

System Pressure1.8 µm totally porous particles

2.7 µm Poroshell 120

Van Deemter Curves – Sub-2 µm, 3.5 µm,Superficially Porous

Van Deemters, 60/40 CH3CN/H2O, with RRLC measuring heptanophenone

0

2

4

6

8

10

12

0 2 4 6 8 10 12

u (mm/s)

h

Agilent Poroshell 120 EC-C18, 3.0 mm x 100 mm, 2.7 um (USCFX01009)

Supelco Ascentis Express C18, 3.0 mm x 100 mm, 2.7 um (USKJ001754)

Phenomenex Kinetex C18, 4.6 mm x 100 mm, 2.6 um (501286-43)

Agilent ZORBAX Eclipse Plus C18, 3.0 mm x 100 mm, 1.8 um (USUYB01455)

Agilent ZORBAX Eclipse Plus C18, 3.0 mm x 100 mm, 3.5 um (USUXV01435)

min0 0.2 0.4 0.6 0.8

mAU

50

100

150

200

250

min0 0.2 0.4 0.6 0.8

mAU

0

50

100

150

200

Fast Analysis (5cm)

2.7µm Poroshell 120EC-C182.1x5 cmF = 1.94 mL/min, 40°C56% ACN, 44% Water549 bar

tr= 0.407 minN = 5,514Log(t0/N)=-3.20

t0= 0.058 min

t0= 0.060 mintr= 0.474 minN = 6,194Log(t0/N)=-3.23

1.8µm ZORBAX RRHDEclipse Plus C182.1x5 cmF = 1.82 mL/min, 40°C59% ACN, 41% Water1003 bar

min0 20 40 60

mAU

0

50

100

150

200

250

min0 20 40 60

mAU

0

50

100

150

200

Long Analysis (55cm)

1.8µm ZORBAX RRHDEclipse Plus C182.1x55cm (3x15cm, 10cm)F = 0.23 mL/min, 40°C59% ACN, 41% Water1001 bar

t0= 5.245 min

tr= 41.453 minN = 105189Log(t0/N)=-2.56

tr= 40.185 minN = 97,363Log(t0/N)=-2.49

2.7µm Poroshell 120EC-C182.1x55cm (3x15cm, 10cm)F = 0.202 mL/min, 40°C56% ACN, 44% Water547 bar

t0= 4.729 min

Conclusions/Future

• RPC will continue to dominate HPLC separations

• Silica monoliths will continue to improve but will they becommercialized?

• Polymeric monoliths will become more practical for smallmolecule separations (less IP involved)

• Designer RPC materials for orthogonal separations (LCXLC) andfor “walkup” RPC method development “scounting” systems

• Challenges:

• Packing small diameter columns efficiently with smallparticles

• Instrumental design keeping up with column developments(e.g. extra column effects, particle size, flow rate capability)

• LCGC Survey top reader dis-satisfiers: Column-to-columnreproducibility (21%); lifetime (17%); price (14%)

Acknowledgments

• Manufacturers who supply me data for my Pittcon articlesin LCGC Magazine

• Jason Link and other chemists at Agilent for providingslides from their presentations

• And you the audience for listening to my “marathon”presentation