Rapid Resolution High Definition LC Columns · Rapid Resolution High Definition LC Columns ... What...

Group/Presentation Title

Agilent Restricted

Month ##, 200X

Rapid Resolution

High Definition

LC Columns

New Columns for Use with

the 1290 Infinity LC

Edward Kim

Application Engineer

August 11, 2010


Agilent Restricted

Month ##, 200X

What are Rapid Resolution High Definition (RRHD)

Columns?

RRHD columns are the columns designed to be compatiblewith the 1290 Infinity LC – and be backwards compatible to other Agilent instruments with lower pressure limits (though not the first choice) and fully UHPLC compatible

RRHD columns can be used up to 1000-1200 bar and represent a complete Agilent solution with the 1290 LC.

They are 1.8um ZORBAX columns packed to withstand the higher pressures of the 1290 LC.

Like other ZORBAX columns we will maximize compatibility across instruments and labs by maintaining scalabilityacross particle sizes.


Agilent Restricted

Month ##, 200X

What is Different Between RRHT 1.8um Columns

and RRHD 1.8um Columns

The pressure limit of the column

• RRHD columns are designed for 1000-1200 bar use, RRHT columns are designed for 600 bar use on the Rapid Resolution LC (1200 SL)

• RRHD columns use improved hardware and packing processes

– the packing pressure and process are proprietary

– improved hardware is being transferred to RRHT columns (for consistency)

This impacts the backpressure the column

1. These columns operate at a higher backpressure than a 600 bar column – higher pressure packing = higher packing density, more backpressure

2. A 50mm length RRHD can not be used on a 400 bar instrument but can be used on a 1200 RRLC (600 bar instrument)

3. The 100 and 150mm length columns are only recommended for use on the 1290 Infinity LC (or UHPLC instruments)


Agilent Restricted

Month ##, 200X

LC Beyond 600 bar Requires New RRHD Columns

Agilent ZORBAX RRHT Eclipse Plus C18, 2.1 x 100 mm, 1.8 um; PN 959764-902,

SN USUXU01664, LN B08101

y = 935.21x + 7.1903

R2 = 0.9993

y = 1025.3x + 13.279

R2 = 0.9973

y = 1817.7x + 21.259

R2 = 0.9946

0

100

200

300

400

500

600

700

0 0.1 0.2 0.3 0.4 0.5 0.6

Flow Rate (mL/min)

Pre

ssu

re (

bar

)

Acetonitrile/Water(60:40)

Acetonitrile/Water(50:50)

Methanol/Water(60:40)


Agilent Restricted

Month ##, 200X


Agilent Restricted

Month ##, 200XPage 5

Comparison of Pressure Generated on RRHT and

RRHD 1.8um Columns

RRHT 1.8um 600 bar RRHD 1.8um 1200 bar

2.1 x 50mm 292 bar 350 bar

2.1 x 100mm 452 bar 566 bar

2.1 x 150mm 660 bar 750 bar

The RRHD columns are ideal for use on the 1290 Infinity LC or on other

UHPLC instruments.

To withstand maximum pressure requires a denser packed bed that

generates a little more backpressure.

This is necessary for optimum lifetime and use up to 1200 bar.

Change in pressure may vary a little until optimized columns ship!

40:60 water:acetonitrile, Flow Rate: 0.5 mL/min, ambient temperature


Agilent Restricted

Month ##, 200X

RRHT, 600 bar and RRHD Column Comparison

Blue = RRHT, Red=RRHDColumns: 2.1 x 50mm, 1.8um Flow: 0.6 mL/min Sample: Alkylphenones

Expected Differences due to higher packing density:

RRHD – longer retention, same selectivity

Higher Back Pressure of RRHD = 434 bar

vs. Back Pressure of RRHT = 323 bar

N = 11,041N=11,168


Agilent Restricted

Month ##, 200X

Gain N and Rs w/ Densely Packed RRHD columns


Agilent Restricted


Rs3,2: 4.16

N6 : 9200

Rs3,2: 10.71

N6 : 33300

Rs3,2: 8.38

N6 : 18700

RRHT 1.8um 600 bar RRHD 1.8um 1200 bar

Mobile Phase: 40:60 water:acetonitrile, Flow Rate: 0.5 mL/min, Temp: ambient, Columns: RRHD/RRHT SB-C18, 2.1 mm ID,

Lengths as noted Peaks: 1: uracil, 2acetophenone, 3 propiophenone, 4 butyrophenone, 5 valerophenone, 6 hexanophenone

1

2

6

54

3

Rs3,2: 5.64

N6 : 10700

Rs3,2: 11.98

N6 : 34100

Rs3,2: 9.02

N6 : 21100

50 mm

100 mm

150 mm

min0 0.5 1 1.5 2 2.5 3 3.5 4 4.5

min0 0.5 1 1.5 2 2.5 3 3.5 4 4.5

min0 0.5 1 1.5 2 2.5 3 3.5 4 4.5

min0 1 2 3 4

min0 1 2 3 4

min0 1 2 3 4

0

The additional packing results in more retention, then Rs and efficiency.


Agilent Restricted

Month ##, 200X

Optimum Flow Rates and Efficiencies-Van Deemters

John W. Henderson JrGroup/Presentation

Title

Agilent Restricted

0

2

4

6

8

10

12

14

0 1 2 3 4 5 6 7 8

h

u (mm/s)

DAZ01002

LFS Column

performance

report results

N=20600

TF 1.02

P=324

F=0.3

RRHD Eclipse Plus C18, 2.1 x 100 mm, 1.8 um;

PN 959758-902, SN DAZ01002, LN B009013

Naphthalene k=4.0

RRHT Eclipse Plus C18, 2.1 x 100 mm, 1.8 um;

PN 959764-902, SN USUXU01664, LN B08101

Heptanophenone k=10.8

N=24900

TF 1.08

P=520

F=0.489

N=24900

TF 1.08

P=550

F=0.520

N=22300

TF 1.01

P=535

F=0.571

2.1 x 100 RRHD Optimum flow rate is about 0.5 mL/min. (60% MeCN, Themostatted 26°C, ~LC Performance Report

Conditions)

Note pressure of more than 500 bar on 100mm length!

With just acetonitrile and water – new 1290 Infinity LC ideal

Van Deemters are very similar, this is expected.


Agilent Restricted

Month ##, 200X

Why Is It Important to Know the Optimal Flow Rate?

Efficiency is Highest at Optimal Flow Rate

John W. Henderson JrGroup/Presentation

Title

Agilent RestrictedMonth ##, 200X

min0.5 1 1.5 2 2.5 3

3.5

min0.25 0.5 0.75 1 1.25 1.5 1.75

2

DAZ01002

LFS Column

performance

report results

N=20600

TF 1.02

P=324

F=0.3

DAZ01002

N=25100

TF 1.08

P=550

F=0.520

Sub-Optimal Optimal

0.3 mL/min 0.5 mL/min

N= 20,600 N= 25,100

P= 324 bar P = 550 bar

At sub optimal flow rates the efficiency is lower and customers may

not see the benefits of a 1.8um column or UHPLC technology.


Agilent Restricted

Month ##, 200X



The column hardware – on the inside

• We redesigned the hardware to improve available frit surface area to reduce the likelihood 2.1 mm ID columns will plug

• We increased the frit pore size to 0.5um to also reduce the likelihood a column will plug

The column label – on the outside

• We now use a colored label so customers can visually tell the difference between a 600 bar RRHT column and new RRHD column

Available column dimensions

1. Initially the columns will only be available in 2.1 mm ID

2. We will test 3.0 mm ID

3. 4.6 mm ID will not be available due to technical limitations in this configuration, other 4.6 mm ID with lower pressure limits can be used on the 1290 Infinity LC with appropriate pressure limits entered


Agilent Restricted

Month ##, 200X

Examples of the RRHT and RRHD Columns

RRHT columns show this groove in the column between the end of the

fitting and the column body. They also have a white label.

RRHD columns have a light blue label

RRHD columns have no visible groove on the column between

end of the fitting and the column body.


Agilent Restricted

Month ##, 200X



The part numbers!

• These new columns will have new part numbers (table below)

• The column shipping with the 1290 Infinity LC will be the ZORBAX Rapid

Resolution High Definition (RRHD) Eclipse Plus C18, 2.1 x 50mm, 1.8um

PN Description

959759-902 RRHD Eclipse Plus C18, 2.1 x 150mm, 1.8um



859700-902 RRHD SB-C18, 2.1 x 150mm, 1.8um

858700-902 RRHD SB-C18, 2.1 x 100mm, 1.8um

857700-902 RRHD SB-C18, 2.1 x 50mm, 1.8um

859700-906 RRHD SB-C8, 2.1 x 150mm, 1.8um

858700-906 RRHD SB-C8, 2.1 x 100mm, 1.8um

857700-906 RRHD SB-C8, 2.1 x 50mm, 1.8um


Agilent Restricted

Month ##, 200X

What’s Not Different Between the RRHT 1.8um and

RRHD 1.8um Columns

The ZORBAX packing materials

• These are still the same ZORBAX 1.8um packing materials with the same

bonded phases - Eclipse Plus, StableBond etc.

• The packing material still has the same particle size distribution – the

change in pressure is only related to change in packing density, not particle

size distribution

– We could have changed it, but there was no performance advantage

• We will start with our most popular C18 phases – Eclipse Plus C18 and

StableBond C18

– Eclipse Plus delivers better peak shape than the BEH Hybrid material

– StableBond C18 is still the best column for low pH lifetime

– ZORBAX columns can deliver excellent performance


Agilent Restricted

Month ##, 200X

Updated Peak Shape Comparisons – Why Compare

Chromatograms with Dextromethorphan?

Dextromethorphan is a strongly basic compound that generates

the best results in LC at low pH.

• Eclipse Plus claims superior peak shape so comparisons for peak shape

are most relevant

Peak shape is a critical issue and poor peak shape is common

with this compound.

• Peak shape continues to be a major driver in column choice for customers

Because of this, efficiency can also be low on columns so it is a

critical parameter to monitor.

• High efficiency and good peak shape provide the most quantitative

accuracy


Agilent Restricted

Month ##, 200X

Peak Shape of Comparison of sub 2 um columns

Eclipse Plus is Superior to Acquity BEH C18

min1 2 3 4 5 6

5.837

min1 2 3 4 5 6

5.503

ACQUITY BEH C18

2.1 x 50 mm, 1.7 um

P = 228 Bar N = 10107,

Peak Width = 0.137 min,

Tf= 1.39

RRHT Eclipse Plus C18

2.1 x 50 mm, 1.8 umP = 202 Bar N = 10881,

Peak Width = 0.124 min,

Tf= 1.23

Mobile Phase = 80% 25 mM phosphate (pH 3): 20% ACN, Flow Rate = 0.3 mL/min Detection: 205nm

Analyte: Dextromethorphan

Tf = 1.4

Tf = 1.2


Agilent Restricted

Month ##, 200X

Peak Shape Comparison of 2.5 & 1.8 um ColumnsEclipse Plus Delivers Peak Shape and Efficiency

min1 2 3 4 5 6 7 8 9

5.08

min1 2 3 4 5 6 7 8 9

6.73

min1 2 3 4 5 6 7 8 9

6.90

min1 2 3 4 5 6 7 8 9

5.50

XBRIDGE C18 2.1 x 50 mm, 2.5 umP = 134 Bar N = 6094, PW = 0.153 min, TF= 1.92

Luna HST C18 2.0 x 50 mm, 2.5 um P = 128 Bar N = 4953, PW = 0.225 min, TF= 1.09

Shim Pak XR C18 2.0 x 50 mm, 2.5 um P = 159 Bar N = 6940, PW = 0.195 min, TF= 1.42

Eclipse Plus C18 2.1 x 50 mm, 1.8 umP = 202 Bar N = 10881, PW = 0.124 min, Tf = 1.23

Mobile Phase = 80% 25 mM phosphate (pH 3): 20% ACN, Flow = 0.3 mL/min (2.1mm), 0.27 mL/min (2.0mm ID)

Detection: 205nm Sample: Dextromethorphan

Acquity and XBridge are the same packing material, yet we see worse performance on

the XBridge – maybe not as optimized packing process as for Acquity?


Agilent Restricted

Month ##, 200X

Comparing RRHD to Competition on 1290 Infinity


Agilent Restricted


min0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8

mAU

0

50

100

150

200

250

300

350

min0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8

mAU

0

50

100

150

200

250

300

350

min0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8

mAU

0

50

100

150

200

250

300

350

α3,2: 1.36

Rs3,2 5.64

N6 : 10700

TF6: 1.04

α3,2: 1.22

Rs3,2 3.69

N6 : 9400

TF6: 1.17

α3,2: 1.29

Rs3,2 4.58

N6 : 11000

TF6: 1.14

RRHD SB-C18 2.1 x 50mm, 1.8um USCES1057

Acquity HSS C18 SB

2.1 x 50 mm 1.8um

Acquity BEH C18

2.1 x 50 mm, 1.7um

1

2

6

54

3

Mobile Phase: 40:60 water:acetonitrile, 0.1% Formic Acid Flow Rate: 0.5 mL/min, Temp: ambient, Det: UV 254 nm Columns:

RRHD SB-C18, 2.1 x 50mm, 1.8um and competitive Peaks: 1: uracil, 2acetophenone, 3 propiophenone, 4 butyrophenone, 5

valerophenone, 6 hexanophenone

StableBond is still the best column for low pH lifetime on the market!


Agilent Restricted

Month ##, 200X

These Columns Are Now Available!

Customers receiving beta test or pre-release LC’s can receive beta test or pre-release columns

• These customers are a critical part of our beta testing program and we want feedback from all of them

• Feedback to date has been excellent – though mostly verbal

A RRHD column will ship with every 1290 Infinity LC

• Part number 959758-902 – RRHD Eclipse Plus C18, 2.1 x 50mm, 1.8um

• This allows quick evaluation of performance and uses our best performing bonded phase for peak shape – Eclipse Plus


Agilent Restricted

Month ##, 200X

The Technical Details….

Status Report

Currently we have made more than 100 columns available in a working hardware design

We are finalizing hardware design – these are small details on the internal part of the hardware and impact nothing visible to the customer

Every column configuration is tested multiple times to a minimum of 5000 injections to be considered optimized

We have good packing processes for some of the phases that meet these requirements (one shown earlier)

We will continue to optimize the packing process for the multiple bonded phase and configurations so that they are ready for introduction

We will continue to add part numbers and phases each month after initial intro – but this will be driven by requests/customer needs and sales


Agilent Restricted

Month ##, 200X

RRHD Columns Tested to Minimum Lifetime of

5000 Injections at 1000 Bar

RRHD 2.1 x 100mm, 1.8um

2157622200

2120521480

0

500

1000

1500

2000

0 1000 2000 3000 4000 5000 6000

Injections

Bar

0

5000

10000

15000

20000

25000

N

Pressure

QC Plates


Agilent Restricted

Month ##, 200X

Overlay of First and Last Injections of QC Test for

Lifetime of RRHD 2.1 x 100mm, 1.8um Column

min0.5 1 1.5 2 2.5 3 3.5 4 4.5

mAU

0

5

10

15

20

25

30

35

40

Inj #1 = N4= 21,480

Inj #5000 N4= 21,205

Column QC Test: Mobile Phase: ACN:water Flow Rate: 0.3 mL/min Sample: Uracil, Phenol, 4-

Chloronitrobenzene, Naphthalene

The QC chromatogram shows consistent performance over the 5000 injections.


Agilent Restricted

Month ##, 200X

Pressure of RRHT Column with New Hardware

200.0

225.0

250.0

275.0

300.0

325.0

350.0

375.0

400.0

425.0

450.0

475.0

500.0

525.0

550.0

575.0

600.0

0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 6000

Pressure

Linear(Pressure)

Column Test of RRHT Column with new RRHD

Type Hardware

Consistent pressure for over 5500 injections (test ended).

Trend line is straight!Conditions: Mobile Phase: 70% Methanol/30% water Flow Rate: 2mL/min flow Temp: RT

Detection: 254nm, VWD Injection volume: 2ul Sample: QC sample 1B - Uracil, phenol,

4-Cl nitrobenzene, toluene Run time 1.1 minutes

Column: RRHT Eclipse Plus C18, 1.8um


Agilent Restricted

Month ##, 200X

How Do They Compare On Performance….

Use and Lifetime at 1000 bar or more…

• Current feedback from beta testers is excellent

• Roche feedback indicated use at over 1000 bar with continued good

performance

• Column was taken above 1000 bar though focus is on 1000 bar for

routine use and higher pressure as the “room” left for pressure

changes, shifts due to gradients etc.

• We are expecting more data to be available very soon in this area

and we will update as it is available


Agilent Restricted

Month ##, 200X

Gain Efficiency and Resolution with Long Length

150 +100 mm = 250 mm 1.8um @ 1000 bar


Agilent Restricted


α3,2: 1.15

Rs3,2: 6.75

N6 : 49200Pw2: 0.017 min

Pw6: 0.029 min

α3,2: 1.15

Rs3,2: 4.92

N6 : 29600Pw2: 0.015 min

Pw6: 0.023 min

Mobile Phase 20:80 water:acetonitrile, F=0.5 mL/min, ambient temperature,

ZORBAX RRHD SB-C18, 1.8um columns

150 mm (P:560bar)

150 +100 mm (P: 960 bar)1. Uracil

2. Acetophenone

3. Propiophenone

4. Butyrophenone

5. Valerophenone

6. Hexanophenone

1

2

6

5

43

1

min0 0.5 1 1.5 2 2.5 3 3.5

mAU

0

50

100

150

200

250

300

min0 0.5 1 1.5 2 2.5 3 3.5

mAU

0

50

100

150

200

250

300

Highest Rs in less than 3 minutes, 250mm, 1.8um at 1000 bar


Agilent Restricted

Month ##, 200X

High Resolution Separation with Long 1.8um

Columns


Agilent Restricted


min0 2 4 6 8 10 12 14

mAU

0

50

100

150

200

DAD1 A, Sig=254,4 Ref=off (E:\090422JWH\PRESSURE000027.D) PMP1, PMP1B, Pressure

Mobile Phase A: water B:acetonitrile, F=0.25 mL/min, ambient temperature,

Columns: ZORBAX RRHD, SB-C18, 2.1 x 150+100 mm, 1.8um

Gradient 10-100% B/15 min.

Pressure 800- 900 bar

Low point of gradient about 480 bar

Long column or columns in series provide max Rs


Agilent Restricted

Month ##, 200X

Fast Analysis on ZORBAX RRHD Eclipse Plus C18

min0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8

mAU

0

100

200

300

400

500

600

DAD1 D, DAD1C, DAD: Signal C, 280 nm/Bw:10 nm Ref 400 nm/Bw:50 nm (G:\1290\AOX JASMIN\AOX-AM2-45C-101.D)

0.2

72

0.4

15

0.4

80

0.7

57

0.8

83

1.0

11

1.0

55

1.5

24

1.5

56

DAD1 E, DAD1D, DAD: Signal D, 255 nm/Bw:10 nm Ref 400 nm/Bw:50 nm (G:\1290\AOX JASMIN\AOX-AM2-45C-101.D)

1.6

84

Eclipse Plus C18, 1.8um,

Pmax 1070 bar

PG

THBP

TBHQNDGA

BHA

Ionox-100

OGDG

BHT AP

Data Courtesy of: Gerd Vanhoenacker

Research Institute for Chromatography

Kennedypark 26 8500 Kortrijk Belgium


Agilent Restricted

Month ##, 200X

Fast Analysis on Agilent Zorbax Rapid

Resolution High Definition Eclipse Plus C18

MethodColumn ZORBAX RRHD Eclipse Plus C18 2.1 x 50 mm, 1.8 µm

(SN USDAY01001)Mobile phase A=0.02% H3PO4, B=acetonitrile/methanol 3/1Flow rate 1.6 mL/minGradient 0-1.85 min 30-100% BInjection volume 1 µLDetector Sig=280/10 nm, Ref=400/50, 80 Hz

Sig=255/10 nm, Ref=400/50, 80 HzTemperature 45°CSample Antioxidant mixture, ca. 100 ppm

Data Courtesy of: Gerd Vanhoenacker Research Institute for Chromatography

Kennedypark 26 8500 Kortrijk Belgium


Agilent Restricted

Month ##, 200X

3 minute analysis, Zorbax Eclipse + C18 2.1x100mm, 1.8umSeiko TOF, 100pg on-column, 10 Hz Acquisition.162/220 Pesticides Identified using TOF,

Customer Example Generated on 1290 with RRHD

Eclipse Plus C18


Agilent Restricted

Month ##, 200X

What Applications Are Planned on the RRHD

Columns? – CSD Led Information

Note: A consolidated applications plan is available and covered

in another presentation

Applications Categories for CSD

1. Proof of Performance

1. Lifetime, Efficiency

2. Resolution/Peak Capacity Message

1. Resolution per time based on improving resolution with different

gradient times and column lengths

3. Comparisons to Acquity


Agilent Restricted

Month ##, 200X

CSD Initial Applications Plan for RRHD Columns

General App Samples Columns

Lifetime at 1000 and 1100 bar Formic acid mp, dirtier sample All

Efficiency -simple tests of each dimension QC and Waters acid base neutral mix

2.1 x 50mm,

2.1 x 100mm

Van Deemters, consideration of frictional

heating, limited to 2.1 mm and 3.0 mm ID? Phenones - RRLC checkout sample

2.1 x 50mm,

2.1 x 100mm

Efficiency - 2 columns in series Neutrals - and complex pharma mix 2.1 x 100mm

Comparisons to Acquity Widest range of sample types, show off selectivity

2.1 x 50mm,

2.1 x 100mm

Demonstrate use on an Acquity system -

1000 bar means any 1000 bar LC (Prove

fittings work appropriately)

Duplicate Acquity app - target big pharma

examples so that we can sell columns where they

already have experience

2.1 x 50mm,

2.1 x 100mm

Efficiency - 2.1 x 150mm in 10/20/30

minute increments - demonstrate a short

and long run with efficiency/Rs per time

Several samples of different complexity - peptide

map (most complex), pharma sample with

impurities, sample with at least 5 components with

at least one closely eluting 2.1 x 150mm


Agilent Restricted

Month ##, 200X

Summary

New ZORBAX RRHD – Rapid Resolution High Definition –

LC columns for the 1290 Infinity LC are designed for the

demands of this UHPLC and any other UHPLC.

RRHD columns are made from the same ZORBAX 1.8um

particles and bonded phases in the ZORBAX RRHT columns

for the RRLC.

RRHD columns deliver long lifetime and high resolution over

an expanded performance range compared to the RRHT

columns

Practically speaking they can go to the high pressure limits

with new hardware design and packing processes.

Rapid Resolution High Definition LC Columns · Rapid Resolution High Definition LC Columns ... What...

Documents

Transcript of Rapid Resolution High Definition LC Columns · Rapid Resolution High Definition LC Columns ... What...