making sub-2 micron particle technology easy - agilent sub-2... · making sub-2 micron particle...

Making Sub-2Micron Particle

Technology Easy

LC Columns and Consumables

John PalmerChromatography SpecialistOctober 12, 2007

Bonded phase

Bonded phase

• Differential partitioning of the components into the stationary and mobile phases.

• Separation controlled by chemical interaction of mobile phase/sample/bonded phase

Mobile phase

The Separation Process

Separation Mechanism

• As Bands Travel the Length of the Column the Respective Distance(ΔL ) Increases

• As Bands Move Through the Column Dispersion (while in the Bonded Phase and Mobile Phase) Causes the Band Width to Increase

• Choosing a High Efficiency Short Column Minimizes On-Column Dispersion and Improves Resolution

W1 W2ΔLL

Longitudinal diffusion

Smaller Particles Improve HETP by Minimizing Dispersion Effects

Plat

e H

eigh

t H

Linear Velocity u

Eddy Diffusion

Sum Curve: van-Deemter

Resistance to Mass Transfer

H = A + B/u + C uLarge Particle

SmallParticle

The smaller the plate height, the higher the plate number and the greater the chromatographic resolution

u opt

H min

Smaller Particles Maintain Efficiency Over Wider Flow Rate Ranges

0.0000

0.0005

0.0010

0.0015

0.0020

0.0025

0.0030

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0

Column: ZORBAX Eclipse XDB-C18 Dimensions: 4.6 x 50/30mmEluent: 85:15 ACN:WaterFlow Rates: 0.05 – 5.0 mL/minTemp: 20°CSample: 1.0μL Octanophenone in

Eluent

Volumetric Flow Rate (mL/min)

HET

P (c

m)

5.0μm3.5μm1.8μm

H = A + B/u + Cu

Smaller particle sizes yield flatter curves, minima shift to higher flow rates

ZORBAX RRHT Columns Provide the Efficiency of Longer Columns for More ProductivityColumnLength(mm)

Resolving Power

N(5 µm)

Resolving Power

N(3.5 µm)

ResolvingPower

N(1.8 µm)

Typical Pressure

Bar (1.8 µm)

150 12,500 21,000 32,500 >420

100 8,500 14,000 24,000 420

75 6000 10,500 17,000 320

50 4,200 7,000 12,000 210

30 N.A. 4,200 6,500 126

15 N.A. 2,100 2,500 55

AnalysisTime

PeakVolume

SolventUsage

Analysis Time*

-33%

-50%

-67%

-80%

-90%

* Reduction in analysis time compared to 150 mm column• pressure determined with 60:40 MeOH/water, 1ml/min, 4.6mm ID

Pressure Equation Defines Physical Causes�

ΔP = ηFLK0πr2dp

2

viscosity flow length

specificPermeability

columnradius

particlediameter

The particle size term is absolute, and does not consider the particle size distribution

Ultrafast HPLC (UFLC)First Publication High Resolution/Speed LC with standard Agilent 1100 LC and Zorbax 1.8um particle

•J. Chrom Sci., 38, Dec 2000, pp 535-544

• author J. J. Kirkland

•Review article with 38 references

•Binary 1100 pump, using 1.8 um 4.6 x 30 mm columns

•Appl’s demonstrating 15 and 24 sec separations

How Does ZORBAX RRHT EBT Manage Pressure for Universal Application?

1. Investigate the effect of Particle Size Distribution (PSD) on performance to develop new packing bed process

2. Take extra steps to minimize fines and therefore lower column backpressure.

3. INTENTIONALLY leave a small, controlled fraction of slightly larger particles in the commercial product because: Slightly broader PSD packings resulted in LITTLE loss of column efficiency compared to the narrow PSD packings.Narrower PSD packings resulted in columns with MUCH higher back pressures. The narrower PSD packing produced a backpressure that was 25% higher than the packing with an optimized PSD

Actual Example of Particle Size Distribution on ZORBAX 1.8um Particles

Number Average Plot of ZORBAX 1.8 um material

Differential Number

6543210

500

1000

1500

2000

2500

3000

3500

Particle Diameter (μm)

Num

ber

Number StatisticsMean: 1.740 umMedian: 1.727 umMode: 1.732 um95% Conf. Limits: 1.739 – 1.742 umSD.: 0.271 um

%> 10 90um 2.04 1.47

Small number of “off median” particles visible in number average plot

Van Deemter Curves for Narrow vs. Engineered Particle Size Distribution 1.8μm SB-C18

0

1

2

3

4

5

6

7

8

9

10

0 2 4 6 8 10 12 14 16 18 20Reduced Linear Velocity (ν)

Red

uced

Pla

te H

eigh

t (h)

Production 1.8μm – Engineered PSD

Narrow 1.8μm – Narrow PSD

7793 Plates @

1.6 mL/min(-3.4%)

8065 Plates @

2.0 mL/min

Columns: ZORBAX SB-C18Dimensions: 4.6 x 30mm, 1.8 μmEluent: 85:15 ACN:WaterFlow Rates: 0.05 – 5.0 mL/minTemp: 20°CSample: 1.0μL Octanophenone in

EluentDetection: UV 245nm

h vs. υ Fit CoefficientsProduction PSD Narrow

PSDVD A: 0.795 0.760

B: 3.97 4.26C: 0.085 0.077r2: 0.9994 0.9998

Particle Size Distribution – Agilent vs. WatersAgilent has a tighter, narrow PSD

Packing from a new Waters Acquity 2.1 x 100mm, 1.7um column

HETP vs. Volumetric Flow RateSmall Particle Columns including Monolith

0.0000

0.0005

0.0010

0.0015

0.0020

0.0025

0.0030

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0

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

Eluent

Volumetric Flow Rate (mL/min)

(cm

/pla

te)

HET

P

ZORBAX 5.0μmZORBAX 3.5μmVendor A 2.5μmVendor B 2.0μmZORBAX 1.8μmMonolith

Agilent RRHT Columns Show No Selectivity Differences with Changes in Particle Sizes Under Same Conditions

Column: 4.6 x 50mm Eclipse XDB-C18 Mobile Phase A= pH 4.5 Na Acetate, B=MeOH (60:40)Flow Rate =1 mL/min Inj. Vol: 2 ul Flowcell: 3uL, 2 mm flow path

1. Caffeine (I.S.)2. Allobarbital3. Phenobarbital4. Butabarbital5. Hexobarbital

min1 2 3 4 5 6

min1 2 3 4 5 6

min1 2 3 4 5 6

5 µm

1.8 µm

3.5 µm t0=0.44 minα 3,2=1.20

t0=0.44 minα 3,2=1.20

t0=0.46 minα 3,2=1.22

α 4,3=1.97

α 4,3=1.94

α 4,3=1.97

α 5,4=1.13

α 5,4=1.16

α 5,4=1.14

Why Worry About pH?pH, pKa and Weak Acids

At pH 4.2 – the sample exists as benzoic acid and the benzoate ion in a ratio of 1:1. Peak shape can be poorAt pH 5.2 – 91% of the sample exists as the benzoate ion. RP retention decreases.

At pH 3.2 – 91% of the sample exists as benzoic acid. RP retention increases.

RCOOH RCOO- + H+

Ka = 6.4 x 10-5

pKa = 4.2

Ka =[RCOO-][H+]

[RCOOH]

+ H+

COOH COO_

Why Worry About pH?pH, pKa and Weak Bases

At pH 9 – the sample exists as protonated and unprotonated diphenhydramine in a ratio of 1:1. Peak shape can be poor.At pH 10 – 91% of the sample exists as unprotonated diphenhydramine.At pH 8 – 91% of the sample exists as protonated diphenhydramine.

Ka =[R3N][H+][R3NH+]

Ka = 1 x 10-9

pKa = 9

R3NH+ R3N + H+

CHOCH CH N2 2

CH3

CH3

∅

∅

+H

CH3

CH3

∅

∅

CHOCH CH N2 2 + H+

pH vs. Selectivity for Acids and Bases

5

SCD

++

2 +

1

1.5

1.0

0.5

0.0

-0.5

log k«

3 4 5 6 7 8 pH

A B C

2

4

5

3

3 5 7 9ELUENT pH

40

30

20

10

10Re

tentio

n

+++

3

1 7,12 - OC

UDCSOC4

6

C12-OC

J.C. 268(1983) 1

J.C. 111(1975) 149

Column: Nucleosil-C18Mobile Phase: 45% ACN/55% phosphate bufferSample: Bile Acids

Column: μBondapak-C18Mobile Phase: 60% 25 mM phosphate buffer

40% Methanol1. Salicylic acid2. Phenobarbital3. Phenacetin4. Nicotine5. Methamphetamine

• Retention and selectivity can change dramatically when pH is changed.

0 5

1

2,3

4

5

Time (min)

7

6

0 5 10

1

2

3

4

Time (min)

6

5

7

High pH Improves Retention and Resolution of Antihistamines

Peak Shape and Retention of this sample of basic compounds improves at high pH. Why?

Column: ZORBAX Extend-C18, 4.6 x 150 mm, 5 μ m Mobile Phase: See Below Flow Rate: 1.0 mL/min Temperature: RT Detection: UV 254 nm Sample: 1. Maleate 2. Scopolamine 3. Pseudoephedrine 4. Doxylamine 5. Chlorpheniramine 6. Triprolidine 7. Diphenhydramine

pH 730% 20 mM Na2HPO470% MeOH

pH 1130% 20 mM TEA70% MeOH

tR = 8.5 tR = 11.4

Resolution Equation Which Parameter Has Most Effect on Resolution?

Rs =N

4

k'

k'+1

(α-1)

000990P2.PPT

Plates Selectivity Retention

••α

For the Answer Let’s Take Look at the Next Slide!

Resolution as a Function of Selectivity, Column Efficiency, or Retention

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

Res

olut

ion

Increase NIncrease AlphaIncrease k'

Plates: 5000 10000 15000 20000 2500Alpha: 1.10 1.35 1.60 1.85 2.1k’: 2.0 4.5 7.0 9.5 12.0

Selectivity Impacts Resolution Most• Change bonded phase • Change mobile phase

Rs = N½/4 • (α-1)/α • k’/(k’+1)

ZORBAX RRHT Bonded Phase Choices

ZORBAX Eclipse XDB – first choice for method development, general purpose bonded phase, for most applications, use up to 60°C

ZORBAX Eclipse PLUS– excellent choice for method development, optimized for basic compounds but applicable for neutrals and acids, use up to 60°C

ZORBAX StableBond – good for low pH and high temperature applications, for those who want to raise temperature for faster analysis

• SB-C18 – use up to 90°C and down to pH <1 (high temp only at low pH)• SB-C8 – use up to 80°C and down to pH 1• SB-CN – use up to 80°C at pH 2• Note: Acquity can only go up to 75°C

ZORBAX Extend-C18 – good at high pH – up to pH 11.5 at ambient temperature

Match Method pH and Column ChoiceChoose the Best Bonded-Phase for Each pH Range

StableBond, pH 1-61. Uses bulky silanes2. Non-endcapped

Eclipse Plus and XDB, pH 2-91. eXtra Densely Bondeddimethylalkylsilanes2. proprietary double-endcapping

*

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

Extend-C18, pH 2-11.51. unique bidentate structure2. double endcapped*

O

R

R

Si

OH

Si

O

Si

R

R

R

O

OH

R

R1

R1

R1

SiO

SiO

Silica Support

C18C18

CH3

CH3

CH3

CH3

CH3

CH3

O

SiO

O

O

O

Si

Si

Si

CH3

CH3

CH3

CH3CH3

CH3

Si

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

Eclipse Plus C18 – Best Peak Shape for Bases without Tailing – Standard Test Mix 1

Eclipse Plus C18

Ace 5 C18

Discovery HS C18

Gemini C18

Luna C18(2)

SunFire C18

XTerra MS C18

XBridge C18

min0 0.5 1 1.5 2 2.5 3 3.5

1. Pyridine (Base) 2. Phenol (Acid) Mobile Phase: 60/40 Water/ACN

12

1 2

1 2

12

12

1

1

2

2

1 2

Start Method Development with RRHT Columns: Different ZORBAX RRHT C18 Bonded Phases for Max Selectivity

Eclipse Plus C18

Eclipse XDB-C18

Extend-C18

StableBondSB-C18

Mobile phase: (69:31) ACN: waterFlow 1.5 mL/min.Temp: 30 °CDetector: Single Quad ESI positive mode scan Columns: RRHT 4.6 x 50 mm 1.8 um

1 23

4

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

1 2 3 4 5

1 23

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 due to non-endcapped

3rd choiceGood efficiency & peak shapeResolution could be achieved

4th choiceResolution not likely,Other choices better, for this separation.

Multiple bonded phases for most effective method development.

More RRHT Bonded Phase Choices Allow for Optimized Method Development

Conditions: Columns: RRHT 4.6 x 50mm, 1.8um Mobile Phase: Acetonitrile (40%):Buffer 60% pH=2.4 25mM NaH2PO4Flow Rate: 1.85 mL/min Detection: DAD, 254 nm LC: Agilent 1200 RR LC Sample: As listed, 1 uL injection

Change in Elution Order Peaks 3,4 & 5

Sample:1. Tolmetin2. Naproxen3. Diflunisal4. Ibuprofen5. DiclofenacFor SB-Phenyl

SB-Phenyl 1.8um

SB-C8 1.8um

SB-C18 1.8um

SB-CN 1.8um

min0 1 2 3 4 5

mAU

0

50

100

150

200

250

300

350

400

450

5 Choices in SB family can be used for method optimizationRRHT SB-Phenyl best choice - more resolution in half the time!

Best Choice!

2X the needed timeNot the best choice!

The Complete List of RRHT 600 bar Columns As of April 9, 2007

Dimensions Eclipse Plus C18 Eclipse Plus C8Eclipse

XDB-C18 Eclipse XDB-C8 Extend-C184.6 x 150 959994-9024.6 x 100 959964-902 959964-906 928975-902 928975-906 728975-9024.6 x 50 959941-902 959941-906 927975-902 927975-906 727975-9024.6 x 30 959931-902 959931-906 924975-902 924975-906 724975-9024.6 x 20 926975-902 926975-906 726975-9023.0 x 150 959994-3023.0 x 100 959964-302 959964-306 928975-302 928975-306 728975-3023.0 x 50 959941-302 959941-306 927975-302 927975-306 727975-3023.0 x 30 924975-302 924975-306 724975-3023.0 x 20 926975-302 926975-306 726975-3022.1 x 150 959794-9022.1 x 100 959764-902 959764-906 928700-902 928700-906 728700-9022.1 x 50 959741-902 959741-906 927700-902 927700-906 727700-9022.1 x 30 959731-902 959731-906 924700-902 924700-906 724700-9022.1 x 20 926700-902 926700-906 726700-902Dimensions SB-C18 SB-C8 SB-Phenyl SB-CN SB-AQ Rx-Sil4.6 x 150 829975-902 829975-906 829975-912 829975-905 829975-9144.6 x 100 828975-902 828975-906 828975-912 828975-905 828975-914 828975-9014.6 x 50 827975-902 827975-906 827975-912 827975-905 827975-914 827975-9014.6 x 30 824975-902 824975-906 824975-912 824975-905 824975-9144.6 x 20 826975-902 826975-9063.0 x 150 829975-302 829975-306 829975-312 829975-3053.0 x 100 828975-302 828975-306 828975-312 828975-305 828975-314 828975-3013.0 x 50 827975-302 827975-306 827975-312 827975-305 827975-314 827975-3013.0 x 30 824975-302 824975-306 824975-3053.0 x 20 826975-302 826975-3062.1 x 150 820700-902 820700-906 820700-912 820700-9052.1 x 100 828700-902 828700-906 828700-912 828700-905 828700-914 828700-9012.1 x 50 827700-902 827700-906 827700-912 827700-905 827700-914 827700-9012.1 x 30 824700-902 824700-906 824700-912 824700-905 824700-9142.1 x 20 826700-902 826700-906

http://www.chem.agilent.com/Scripts/PDS.asp?lPage=9818 (Main product page)http://www.chem.agilent.com/Scripts/Generic.ASP?lPage=11424&indcol=N&prodcol=N (P/N List)

2 Recommended Starting Choices:• 959941-902• 959741-902They are Eclipse Plus C18 Columns.

http://www.chem.agilent.com/Scripts/PDS.asp?lPage=9818

http://www.chem.agilent.com/Scripts/Generic.ASP?lPage=11424&indcol=N&prodcol=N

Equivalent Selectivity and Improved Efficiency ZORBAX Eclipse Plus Substituted for Original Column

min0 2 4 6 8 10 12

min0 2 4 6 8 10 12

Waters X-Bridge C18, 4.6 x 150 mm, 5 um

ZORBAX Eclipse Plus C18, 4.6 x 150 mm, 5 um

N=41000TF=1.13

N=14000TF=1.25

N=60000TF=1.00

N=23000TF=1.01

Gradient: 10 to 30% B/15 min. A: 0.1% Formic Acid, B: 0.1% FA in ACN Flow: 1 mL/min. Temperature 40°CElution order: sulfanilaminde, sulfadiazine, sulfathathiazole, sulfamerazine, sulfamethazine, sulfamethoxazole

Let Us Take A Break!

RRLC Instrument Considerations:Can the HPLC in your lab today handle RRLC ?

Gradient delay volume

Extra-column volume

Data rate

•Things to consider on your system:•Gradient delay volume – effects column re-equilibration and gradient profile

•Extra-column volume – effects peak dispersion and peak width for Isocratic separations

•Data Rate – match to expected peak widths-If rate is too slow sensitivity and peak detection suffer in Iso and Gradient separations

Binary Pump SL• Configurable delay volume (120µl and 600-800µl) for

uncompromised support of narrow and std bore columns (2.1 – 4.6mm ID)

– Standard delay volume (600 – 800 µl) for uncompromisedsupport of std bore RRLC and HPLC on one configuration

– Low delay volume (120 µl) for uncompromised support of narrow LC and LC/MS (both RRLC and HPLC)

• 0.05 – 5 ml/min flow rate range for highest speed on narrow and standard bore RRHT columns (full P range)

• 600 bar pressure for higher speed and resolution in RRLC• Highest RT precision and lowest mixing noise by new

piston control algorithms (EDC – Electronic Damping Control)*

*patents filed

1200 RRLC System Benefits of Key Modules Features

1200 Series Binary Pump SL – Configurable Delay Volume

Low delay volume

120 µl

Disconnect only here!

BA

Pressure Sensor

PurgeValve

600bar Damper

400µl Mixer

Pressure-Sensor

Purge Valve

Mixing-T

Pressure-Sensor

Purge Valve

Mixing-T

Damper

400µl Mixer

Std delay volume

600-800 µlBA

600bar Damper

400µl Mixer

Pressure Sensor

Purge Valve

Disconnect only here!

Flow Path

min0 0.2 0.4 0.6 0.8 1

mAU

0

100

200

300

400

500

600

700

0.20

9

0.37

0

0.50

5

0.62

00.

664

0.72

5

0.82

1

0.90

8

0.98

6

1200 Rapid Resolution

Suppler B

Column RRHT, SB-C18,

2.1 x 50mm, 1.8um

A BEH,

2.1 x 50mm,1.7umAnal. Time 0.949 min 0.914 min

Resolution 3.69 3.50

Precision 0.025 – 0.094 %RSD 0.18 – 0.30 %RSD

1200 Series Rapid Resolution System - efficient use of pressure

Pressure 440 bar 700 bar (+60%)

Sample: Phenones Test MixFlow Rate: 1 ml/minGradient: 35-95% ACN in 0.9

minTemperature: 50°Injection volume: 1 µlInjection Technique: ADVR,

OI, MCOWL: 245 nmData Rate: 80 Hz

Better performance at dramatically lower pressure than other U-HPLC designs

1200 RRLC and RRHT Column FlexibilityConventional to High Speed HPLC

Conditions:A=pH 4.5 Na Acetate, B= Methanol (60:40)1.0mL/minFlow cell: Micro/High Pressure 6mm, 1.7µLDetection: UV 254 nmInjection Volume: 2 uL

min0 1 2 3 4 5 6 7

Rapid Resolution HTEclipse Plus C18

4.6 x 50 mm, 1.8µm

Rapid ResolutionEclipse Plus C18

4.6 x 150 mm, 3.5 µm

Eclipse Plus C184.6 x 250 mm, 5 µm

min0 5 10 15 20 25 30

min2 4 6 8 10 12 14 16 18

Sample: Phenobarbital 1. Caffeine 2. Allobarbital 3. Phenobarbital 4. Butabarbital 5. Hexobarbital

main comp. • Zorbax SB C18 2.1mm x 150mm, 1.8µm• 20-95%ACN (0.1%TFA) in 14min, 2min hold,

5min 20%ACN, 55°C, 0.5ml/min• total runtime 21 min

1

2

3

45

6

78

Customer Sample 1

1200 Rapid Resolution System – PerformanceIncreased Resolution in Drug Development Impurity Testing

• Acquity BEH C18 2.1x100mm,• 5-80% ACN (50mM NaClO4/0.1%H3PO4) in

12min, in 0.5min to 5% ACN, 8.5min 5% ACN, 0.5ml/min, 40°C,

• total runtime 21 min

1 2 3

4

56

main comp.

• Two more baseline separated impurities • Higher sensitivity, lower noise• Same runtime

Stepwise Scale-up to Rapid Resolution LCFrom 1100 to 1200 RRLC in two steps – Example 1

Actual: 1100

Quat System

> 5 min> 6 min> 3 sec

5 - 12,0004.6 mm50 mm

0.2 - 10ml/min80 C

400bar

ALS

TCC

VWD/MWD/DAD

Quat Pump

Degasser

Step 1: 1100/1200

„Bin SL“ System

h-ALS SL

TCC

VWD/MWD/DAD

Bin Pump SL

µ-Degasser

> 1.5min> 2min

> 1.5 sec5 - 30,000

2.1 - 4.6 mm50 - 150 mm

0.05 - 5 ml/min80 C

600 bar

Analysis TimeCycle timesPeak Width

NColumn ID

Column Length Flow rates

TemperaturePressure

Step 2: 1200

Rapid Resolution System

h-ALS SL

TCC SL

DAD SL

Bin Pump SL

u-Degasser

> 0.2min> 0.4min> 0.2 sec

5 - 60,0002.1 - 4.6 mm20 - 150mm

0.05 – 5 ml/min100 C

600bar

+ Speed+ Resolution+ Sensitivity

+ MS-Robustness+ Data Security & Traceability

+ Qualification (Degasser, ACE)+ Compatible with conv. HPLC

+ Resolution+ Speed

+ MS-Compatibility+ Solvent Saving

+ Compatible with conv. HPLC

Preserve your existing 1100 LC module investment:

Agilent HPLC System Flexibility

• Agilent LC systems are designed for flexible use with a wide range of flow and pressure capabilities

– Many of the New 1200 Modules (i.e. 80 Hz DAD) are compatible with older 1100 systems

– 1200 RRLC systems can run current, legacy and new high performance methods

– Engineered Column Technology Allows Higher Performance Separations with:

– Existing 1100 System– Enhanced 1100 System – Higher Pressure, Enhanced Flow Path 1200SL System

Separation needs will determine your choice!

New RRHT-1100 Upgrade Kits Make Conversion of 1100 for use of RRHT Columns

5188-5323 4.6 mm RR HT 1100 Fast LC Kit – VWD- 0.17 mm capillaries, 80 ul mixer, 5 ul semi-micro VWD cell- two 4.6 x 50 mm Zorbax 1.8 um columns

5188- 5324 4.6 mm RR HT 1100 Fast LC Kit – DAD- 0.17 mm capillaries, 80 ul mixer, 5 ul semi-micro DAD cell- two 4.6 x 50 mm Zorbax 1.8 um columns

5188-5328 2.1 mm RR HT 1100 Fast LC Kit – MS/DAD- 0.12 mm capillaries, 80 ul mixer, 1.7 ul micro DAD cell- 0.12 mm std ALS and WP ALS needle seats- two 2.1 x 50 mm Zorbax 1.8 um columns

Possible Instrument Configurations for Cost Effective Fast and Ultra-Fast HPLC

* Pieces to upgrade, in kits

High pressureGradient pump

Std or WellPlate sampler

Diode Array Detector

Standard assembly without standard mixer

0.12 x 400 mm capillary

DAD equipped with a 1.7 μL flow cell

Mass Spectrometer

0.13 x XX mm PEEK Capillary

High pressure Gradient pump

Std or WellPlate sampler

Rapid ResolutionHT Column

Diode Arraydetector

Waste

Standard assembly without standard mixer and with 80 μL mixer




DAD equipped with a 5uL or 1.7 μL flow cell

3 μL heat exchanger

Thermostatted Column

compartment

4.6 mm ID columns 2.1 mm ID columns

Cell Inlet Capillary

Cell Outlet Capillary

Rapid ResolutionHT Column



3 μL heat exchanger

Thermostatted Column

compartment

DAD equipped with a 500 nL flow cell

Cell Inlet Capillary

Cell Outlet Capillary 0.125 x 80 mm capillary


The Agilent 1200 LC SystemsOne Comprehensive Series of Solutions for your Applications

Application 1200 RRTM LC 1100/1200 Binary

1100/1200 Quaternary

Ultra-fastcolumn length 15 – 50mm

YES NODelay volume too large

NODelay volume too large

RRLC – Narrow

(2.1)High-resolutioncolumn length 100 – 150mm

YES NODelay volume too large, pressure limit too low

NODelay volume too large, pressure limit too low

Ultra-fastcolumn length 15 – 50mm

YES YES YESModifications

recommended to reduce delay volume

RRLC – Std

(3.0, 4.6)

High-resolutioncolumn length 100 – 150mm

YES NOPressure limit too low

NOPressure limit too low

HPLC – Narrow bore (2.1) YES YESModifications

recommended to reduce delay volume

NODelay volume too large

HPLC – Standard bore (3.0, 4.6) YES YES YES

Transferring a Method- Binary to QuaternaryThe Proof that a binary to quaternary works!

min0.5 1 1.5 2 2.5

mAU

0

100

200

300

400

500

600

DAD1 A, Sig=210,4 Ref=off (FASTLC\02090510.D)

1.5

45 1

.606

1.8µm 4.6x50mm Binary Pump

1.8µm 4.6x50mmQuat Pump

AU

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

Minutes0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40

Com

poun

d "F

" - 1

.434

Com

poun

d "G

" - 1

.493

1.65

41.

709

Com

poun

d "D

" - 1

.736

Run time 2.3 minutes

Run time 1.8 minutes

Run time differs by 0.5 minutes due to larger delay volume in quaternary pump.But separation acceptable to the customer.Making use of available instruments with some limitations.

Higher Temperature as an Aid to Faster Operation

Higher Temperature:

Temperature should always be considered as a parameter during speed optimization

Provides more rapid mass transfer:• Improves Efficiency – enhances resolution• Decreases analysis time – faster separations with no loss in resolution

Decreases Mobile Phase Viscosity• Lowers backpressure – allows for higher flow rates, faster separations,

greater efficiency and use of sub 2-micron columns

Can change selectivity – optimize resolution

High Temperature for Improved Selectivity – What Column Compartment is Required?

20°C

60°C

40°C

30°C

90°C

Salicylic acid

min0 1 2

0 1 2 345

0 1 2 345

0 1 2 345

0 1 2 345

3 4

Salicylic acidCoelution

Salicylic acid

Salicylic acid

Salicylic acid

Column: RRHT SB-C184.6 x 50mm, 1.8um

Increased T Decreases Run Time and Sharpens Peaks Improving Resolution

1200 Series TCC SL Low volume Pre-column Heater and Post-column Cooler

• L-shaped pre-column heater • Volume: 1.6 µl• Mounted on carrier

• U-shaped Post-column cooler • Volume: 1.5 ul• Mounted on Carrier

Holesto attachcarrier

Choice of Valves

Plumb multiple columns – have it your way !

The New 1200 Thermostatted Column Compartment SLPost-column coolingNoise reduction at high flow rates and high temperatures

0.685 - Anthracene

min0.5 1 1.5 2 2.5

mAU

-0.05

0

0.05

0.1

0.15

0.2

0.25

• Column Temperature: 80°C • Detector-in Temperature: 50 °C• Noise = 90 uAU• S/N = 3.3

• Column Temperature: 80°C • Detector-in Temperature: 24 °C• Noise = 30 uAU• S/N = 10• No Peak Broadening

Column: 4.6 x 50mm, 1.8um

Flow Rate: 4ml/min

Detector celltemperature

Detector celltemperature

Data Rate

Peak Width

Resolution Peak Capacity

80 Hz 0.300 2.25 60

40 Hz 0.329 2.05 55

20 Hz 0.416 1.71 45

10 Hz 0.666 1.17 29

5 Hz 1.236 0.67 16

min0.1 0.2 0.3 0.4 0.50

80Hz

PW=0.30sec

40Hz

PW=0.33sec

20Hz

PW=0.42sec

10HzPW=0.67sec

5HzPW=1.24sec

Sample: Phenones Test MixColumn: Zorbax SB-C18, 4.6x30, 1.8umGradient:: 50-100%ACN in 0.3minFlow Rate: 5ml/min

80Hz versus 10Hz (20Hz) Data Rate• Peak Width: – 55% (– 30%)• Resolution: + 90% (+ 30%)• Peak Capacity: + 120% (+ 40%)• App. Column Eff.: + 260% (+ 70%)

High Speed LC with RRLC and RRHT ColumnsMatch Detector Speed to Peak Width for Optimum Detection

Chromatographic Results with Second Source Lamp at 214 nm Wavelength - Small Peaks

Lamp fromLamp from AgilentAgilentTechnologiesTechnologies

Lamp from Second SourceLamp from Second Source

Peak 1Peak 1 S/N = 150S/N = 150

Peak 2Peak 2 S/N = 400S/N = 400

Peak 3Peak 3 S/N = 300S/N = 300

Peak 1Peak 1 S/N = 15S/N = 15



Narrow Peaks Require Faster MS Scan Speeds

min0.1 0.2 0.3 0.4 0.5 0.6 0.7

50000

100000

150000

200000

MSD1 TIC, MS File (C:\CHEM32\1\DATA\FAST SCAN 7_05\SULFA1000034.D) MM-ES+APCI, Fast Scan, Frag: 150

0.10

2

0.16

7

0.43

2

0.48

1

PW = 0.8 sec

RRHT SB-C18 2.1 x 50 x 1.8 micron column, 5% MeOH w 1 mM ammonium acetate to 50% in 1 minute

Data acquired with 6140 MS – fast enough for RRHT columns.

Increased Isocratic HPLC Speed with 1.8u Particles

1. Reduce Column Length and Particle Size

2. Maintain Flow Rate

3. If Resolution and Pressure is Within Goal, Increase Flow Rate

4. Approaching Pressure Limit of Instrument ? Increase Temperature to Lower Viscosity and Lower Pressure

5. Increase Flow Rate

6. Approaching Flow Rate Limit of Instrument ? Decrease Diameter Column and Reduce Flow Rate Proportionately

7. Repeat Steps 3, 4 and 5 until Speed Goal Achieved ISOCRATIC ELUTION

Faster AnalysisShorter Column, Smaller Particle, Same Flow

Original methodZORBAX LC column Extend-C184.6 x 150 mm, 5 μm3 μL inj.

min0 2 4 6 8 10

mAU

0

50

100

150

200

250

min0 2 4 6 8 10

mAU

-20

0

20

40

60

80

100

120

Mobile phase: (70:30) MeOH: 50 mM pyrrolidine buffer Flow = 1.0 mL/min, Temp. : ambient

ZORBAX RRHT column Extend C184.6 x 50 mm, 1.8 μm1 μL inj.

2

2 3

5

4

516

614

3

3x faster

Simplified Method Transfer for Increased Gradient Speed

1. Shorten the Column Length2. Adjust Gradient Time by same Factor 3. Maintain Flow Rate4. If Resolution and Pressure is Within Goal,

Increase Flow Rate and Lower Gradient Time5. Approaching Pressure Limit of Instrument ?

Increase Temperature to Lower Viscosity and Lower Pressure

6. Increase Flow Rate and Lower Gradient Time7. Approaching Flow Rate Limit of Instrument?

Decrease Diameter Column and Reduce Flow Rate Proportionately

8. Repeat Steps 4, 5, 6 and 7 until Speed Goal Achieved

GRADIENT ELUTION

0 10 20 30 40

Relationship of k* and Key Gradient Parameters

Time (min)

100% B

100% B

100% B

100% B

tg= 40

tg= 20

tg= 10

tg= 5

1/k* = gradient steepness = b

tg F

S Δ%B Vm

k* ∝

ΔΦ = change in volume fraction of B solvent

S = constantF = flow rate (mL/min.)tg = gradient time (min.)Vm = column void volume (mL)

0% B

0% B

0% B

0% B

1

1

33

5

5

22

4

4

Time (min) Time (min) Time (min)0.0 2.5 5.0 7.5 10.0 12.5 15 0.0 2.5 5.0 7.5 0.0 2.5 5.0

1

3

5

2

4

Length = 150 mm, 5 μmtG = 18 minTotal Time ~ 30min

A. B. C.

Length = 75 mm, 3.5 μmtG = 9 minTotal Time ~ 15 min

Length = 50 mm, 3.5 μmtG = 6 minTotal Time ~ 8min

Column Diameter: 4.6 mm Mobile Phase: A: 25 mM Na2HPO4, pH 3 B: MeOH Gradient: 45 – 90% B in tG min Flow Rate: 1.0 mL/min Temperature: 35°C Sample: Cardiac Drugs 1. Diltiazem 2. Dipyridamole 3. Nifedipine 4.

Lidoflazine 5. Flunarizine

Reducing Column Length Reduces Run TimeGradient Time ∝ Column Length

Rapid Resolution LCUltra Fast, Resolution Maintained, Sensitivity Improved

Optimized Gradient

Time (min)

1

2

4

5

3

0.0 0.5 1.0 1.5 2.0 2.5 3.0

Column: Rapid Resolution Eclipse XDB-C8, 4.6 x 50 mm, 3.5 μm

Mobile Phase:A: 55% 25 mM Na2HPO4, pH 3B: 45% MeOH

Gradient: 42 – 90% B in 2 minFlow Rate: 3 mL/minTemperature:35°CSample: Cardiac Drugs

1. Diltiazem2. Dipyridamole3. Nifedipine4. Lidoflazine5. Flunarizine480 Samples/day

Run Time 1.8 minEquilibration 1 minTimeTotal Analysis 2.8 minTime

F= 1.20ml/minT = 40°CAnalysis Time = 11minSolvent Cons. = 13.2ml

High Resolution:4.6mm x 150mm 5.0µm

min0 2 4 6 8 10 12

F = 4.80ml/minT = 40°CAnalysis Time = 1.05minSolvent Cons. = 5.1ml

High Speed:4.6mm x 50mm 5.0µm

min0 0.2 0.4 0.6 0.8 1

F= 1.00ml/minT = 40°CAnalysis Time = 1.1minSolvent Cons. = 1.1ml

High Speed & Resolution:2.1mm x 50mm 1.8µm

min0.2 0.4 0.6 0.8 10

Agilent 1200 Rapid Resolution System – SpeedConventional LC → UFLC → RRLC

Max Speed at T = 95oC2.1mm x 50mm 1.8um

F= 2.40ml/minT = 95°CAnalysis Time: 0.4minSolvent Cons. = 1.0ml

PWHH = 197msec

min0.2 0.4 0.6 0.8 10

> 20x faster !

Rapid Resolution Compendium CD-ROM

(Please Ask For A Copy – Pub No 5989-5130EN)

Tools

• Application Examples

• Technology Overview

• Rapid Resolution in The Scientific and Trade Press and Conference Posters

• System Configurator (guide to reduce dead volume and extra column effects)

• Method Translator Program

Mini-Demo Method Translator

Agilent HPLC Method TranslatorA Simple to Use Tool to Move Methods to RRHT

Why Filter the Sample? Extreme Performance Requires Better Sample “Hygiene”

• Prevents blocking of capillaries, frits, and the column inlet

• Results in less wear and tear on the critical moving parts of injection valves

• Results in less downtime of the instrument for repairs

• Produces improved analytical results by removing potentially interfering contamination

Choice of Membranes

• Regenerated Cellulose (RC)• Universal hydrophilic membrane, compatible with most

solvents - aqueous and organic• High purity, extremely low extractables and lowest protein

binding• PTFE

• Good chemical compatibility with aqueous and organic solvents as well as for acids and bases

• Hydrophobic• Nylon

• Universal filter for aqueous and organic solutions• Hydrophilic

• Cellulose Nitrate• Compatible with many, but not all, aqueous or non-

aqueous solvents• Most commonly used as a pre-filter

• Cellulose Acetate (CA)• Only compatible with aqueous solvents• Used for proteins and protein-related samples

Regenerated Cellulose

Nylon

PTFE

Magnified views showing structural differences of some membrane types

In-Line Filters Provide Good Insurance Against System OverPressure

NEW RRLC In-line filter and fitting – max 600 bar

Protect RRHT columns with efficient in-line filter with 0.2 µm pore size frits

Description Part number PorosityFrit diameter

Flow rate

Part number Replacement Frits

RRLC In-line filter, 2.1 mm, max 600 bar

5067-1551 0.2 µm 2.1 mm <1 mL/min

5067-1555 (10/pk)

RRLC In-line filter, 3.0 & 4.6 mm, max 600 bar

5067-1553 0.2 µm 4.6 mm 1 - 5 mL/min

5067-1562 (10/pk)

Summary of Fundamental Aspects Concerns of Routine Use of 1.8µm Columns

• Base Your Column and Instrument Choice on Typical Sample Needs

• Optimize Chemistry for Best Separation

• 1200 SL Flexible Capability Allows Better Asset Management by Running Current, Legacy and Extreme Methods

• Engineered Column Bed Design Minimizes Pressure and Allows Better Utilization of All Assets

• Use Filtration to Extend Column Life Time and Increase Instrument Up Time

• Attend to Detection Data Rates

• Optimize Instrument Volume

• Make Sure Instrument “Parts” are Compatible with High Performance Needs

New Convenience for Column Installation in RRLC Instruments

Easy, hand tighten column connection with Polyketone fittings up to 600 bar PN: 5042-8957 (10/pk)

Wrap-up E-Seminar Questions

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