Rapid Resolution - Practical Considerations - Series 6 ... · Rapid Resolution - Practical ConsiderationsPractical Considerations - Series 6 LC Columns and Consumables Ed Kim Application

Rapid Resolution -Practical Considerations -Practical Considerations -

Series 6

LC Columns and Consumables

Ed KimApplication EngineerMay 6, 2009

What are the Advantages of Sub 2-Micron Columns?Columns?1. Faster analyses – up to 10 – 20X faster

Small particles generate equivalent efficiency in a shorter column p g q ylengthThis allows equivalent resolution in less time – or faster analyses

2. Higher resolution analyses in equivalent or less time2. Higher resolution analyses in equivalent or less time

Improved resolution can be critical to some analysesLonger columns with small particles can provide extremely high resolution or peak capacityresolution or peak capacitySmaller particles generate more efficiency than larger particles in the same length column

3 Hi h iti it3. Higher sensitivity

4. Lower costs – time, solvent etc.

RR-Practical Considerations Agilent Restricted

5/6/2009

What Are the “Limitations” of Sub 2-micron Columns? or

“Are They Easy Enough to Use?”Are They Easy Enough to Use?

1. Backpressure and particle size distribution – is there a balance?

2. Are there enough choices to do the separation you want?

3. What extra preparation/care do you need to take to use the columns f ll ?successfully?

4. Are there sample limitations for these columns?

5 Can you use the same range of mobile phases and conditions5. Can you use the same range of mobile phases and conditions (temperature etc.) with sub 2-micron columns?

6. What LC instrumentation is required to achieve maximum benefit from sub 2-micron columns?


5/6/2009

Advantages – Faster Analyses

What makes the analysis faster?• Theory – van Deemter curves

Are faster analyses easily achieved?• Theory/Practice

What does it take to go 5X, 10X, or 20X faster?

Are there practical examples to go with this?Are there practical examples to go with this?


5/6/2009

van Deemter – The Theory BehindFor small molecules, smallerFor small molecules, smaller particles result in faster diffusionResult: faster chromatography

ate

heig

ht

l ti l ll ti l

The smaller the theoretical plate height H, the better the Resolution!

oret

ical

Pla

Resulting Van-Deemter curve

H = A + B/u + C x ularge particle small particle

Theo

H

curve

Resistance to Mass Transfer

H min

Multipath Term

Longitudinal Diffusion


5/6/2009

linear flow uu opt

van Deemter Curves1.8um Rapid Resolution HT Columns1.8um Rapid Resolution HT Columns

0.0045

1 8 RRHT

Higher Speed – Higher Resolution – Higher Sensitivity

0 0030

0.0035

0.0040 Columns: ZORBAX Eclipse XDB-C18Dimensions: 1.8 μm, 4.6 x 50mmEluent: 85:15 ACN:WaterFlow Rates: 0.05 – 5.0 mL/min

1.8um RRHT columns provide flow rate independent efficiency gain!

0.0020

0.0025

0.0030

(cm

/pla

te)

Temp: 20°CSample: 1.0μL Octanophenone in Eluent

5.0 μm

efficiency gain!

Therefore use faster flow rates for faster analyses!

0.0005

0.0010

0.0015

HET

P

3.5 μm

1 8 m

analyses!

-0.0005

0.0000

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6

1.8 μm 260,740 N/m @ 2mL/min 5.0 mL/min


5/6/2009

Interstitial Linear Velocity (ue- cm/sec)

All scaling calculations to transfer methods to RRHT, 1.8um particles are done using the Agilent MethodTranslator

For free –P/N 5989-5130EN


5/6/2009

Contributed Example Shows Minimum 4X Faster Method

Compounds A-I monitored4X shorter, with no loss in RsRs of all minor compounds

…maintained or improved

35 min.


5/6/2009

9 min

10X Shorter Analysis Time with a 4.6 x 50mm RRHT Column – Same Method, Shorter ColumnRRHT Column Same Method, Shorter Column

RRHT, SB-C184.6 x 50mm

15 min 1.5 min

QA/QC Analyses are sped up 10XNo change in resolution – system suitability analysis the sameSensitivity improvedSame mobile phase, other conditions used


5/6/2009

USP Methods with Rapid Resolution HT Columns –10X Faster with Shorter Columns10X Faster with Shorter Columns

2

USP Analysis of GuaifenesinSample: 1. Guaifenesin – 0.04 μg/μL, 2. Benzoic Acid – 0.10 μg/μL

USP L1 Column: ZORBAX Eclipse XDB-C184.6x250mm, 5μm

Eluent: 40% Methanol:60% Water:1.5% Glacial Acetic AcidFlow: 1.0 mL/min, Injection: 8μL, Temp: 25 °C

1 Peak Tr N Rs1 6.63 12,737 02 11.19 18,552 15.8

min0 2 4 6 8 10 12

12 Rapid Resolution HT Column (L1)

ZORBAX Eclipse XDB-C18, 4.6x50mm, 1.8μmEluent: 40% Methanol:60% Water:1 5% Glacial

Peak TR N Rs 1 1.40 11,421 0

Key Parameters-Changed Column Length

min0 2 31

Eluent: 40% Methanol:60% Water:1.5% Glacial Acetic AcidFlow: 1.0 mL/min, Injection: 2μL, Temp: 25 °C

,2 2.33 12,909 12.3

gParticle Size

Key Parameters-SameFlow RateEluent

12

Rapid Resolution HT Column (L1)ZORBAX Eclipse XDB-C18, 4.6x30mm, 1.8μmEluent: 40% Methanol:60% Water:1.5% Glacial Acetic AcidFlow: 1.0 mL/min, Injection: 2μL, Temp: 25 °C

Peak TR N Rs 1 0.85 5,855 02 1.43 7,300 8.6

Injection SizeTemperature


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min0 21

Flow: 1.0 mL/min, Injection: 2μL, Temp: 25 C

Rapid Resolution HT Columns – Up to 20X Faster at High Temperaturesat High Temperatures

Up to 20x faster than conventional HPLC • With same or better performance (resolution, precision, sensitivity, carry over)

HPLC, 40°CSB-C184.6 x 150mm, 5µm1 20 l/ i 40°C

• Compliant with strictest regulatory performance requirements

RRHT SB-C182 1 50 1 8 RRLC 40°C

1.20ml/min, 40°CAnalysis Time = 11min

min0 2 4 6 8 10

2.1mm x 50mm 1.8µm1.00ml/min, 40°CAnalysis Time= 1.1min

RRLC, 40 C10x faster

min0.2 0.4 0.6 0.8 10

RRHT SB-C182.1mm x 50mm 1.8µm2.40ml/min, 95°CAnalysis Time: 0.4min

RRLC, 95°C27x fasterPW = 197msec


5/6/2009

0.2 0.60 0.4 min

What are the Advantages and Limitations of Higher Resolution Analyses?Higher Resolution Analyses?Advantages• More certainty that everything is resolved.• High peak capacity for analyzing complex samples.

LimitationLimitation• Longer RRHT columns will generate more back pressure and require

the RRLC


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Longer Eclipse Plus RRHT Columns Improve Efficiency Achieve Baseline Resolution from 0Efficiency Achieve Baseline Resolution from 0

Rs5,4 = 0 4.6 x 50 mm 1.8um Mobile phase: (69:31) ACN: waterFlow 1.5 mL (/min.Temp: 30 C

S Q S

1200000 1 25

186

Rs 1 384 6 x 100 mm 1 8um

Detector: Single Quad ESI positive mode scan

min2 4400000

3186bar

Rs5,4= 1.38

Rs = 1 61

4.6 x 100 mm 1.8um1600000

326bar

Rs5,4= 1.61

4.6 x 150 mm 1.8um1800000

2 4 6 8200000

12

5

413

Sample: 1. Anandamide (AEA) 2. Palmitoylethanolamide (PEA) 3. 2-arachinoylglycerol (2-AG) Rs5,4 = Baseline

min2 4 6 8 10

400000

34

413bar


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4. 1(3)-arachidonylglycerol5. Oleoylethanolamide (OEA)Baseline

Greater Peak Capacity Means More Efficiency and More Certain SeparationsMore Certain Separations

7 ImpuritiesAll 7 Baseline S t d!

4 Impurities2 Not Baseline Separated!

Customer Example Isocratic Impurity Method

Separated!Zoom of critical time range @ 7min

4.6 x 150, 1.8um4.6 x 150, 5um93 bar

490 barN = 28669

93 barN = 7259

More Peak Capacity Means Greater Certainty Everything is Resolved!!


5/6/2009

More Peak Capacity Means Greater Certainty Everything is Resolved!!

Standards - Improve Resolution with the Same Column Selectivity - Smaller Particle Size (1.8um)Column Selectivity Smaller Particle Size (1.8um)

Column: SB-C18, 4.6 x 50 mm Mobile Phase A= 0.1% formic, B=0.1% formic in MeCN (92:8) Flow Rate =1.5mL/min Inj. Vol: 2 ul Detection: UV 254 nm Flowcell: 2uL, 3 mm flow path

1. 1-methylxanthine2. 1,3-dimethyluric acid3. 3,7-dimethylxanthine

(theobromine)4. 1,7-dimethylxanthine

mAU

30405060 5 µm α2,1= 1.36

α3,2= 1.27

min0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.80

1020 α4,3= 1.79

mAU

20

30405060 1.8 µm

α2,1= 1.36

α3,2= 1.27

α = 1 79

min0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.80

1020 α4,3= 1.79

The selectivity is the same but resolution of standards is improved.


5/6/2009

Will actual samples containing theobromine look good?

RRHT Columns are for Real Samples! Samples Prepared and Filtered onlyPrepared and Filtered only

theobromine Hot Cocoa MixTheobromine is dominant in chocolate and not in tea (as expected).

caffeine

min0.5 1 1.5 2 2.5 3

Chocolate syrup

Tea bagtheobromine

min0.5 1 1.5 2 2.5 3

min0.5 1 1.5 2 2.5 3

Column: SB-C18, 4.6 x 50 mm Mobile Phase A= 0.1% formic, B=0.1% formic in MeCN (92:8) Flow Rate =1.5mL/min


5/6/2009

, , ( )Inj. Vol: 2 ul Detection: UV 254 nm Flowcell: 2uL, 3 mm flow path

Comparison of Peak Capacity with Change in Particle SizeParticle Size

mAU

100

120RRHT Eclipse XDB-C18,

2.1 x 100mm, 1.8µ, 600 Bar

Uracil35% increase in peak capacity!!

20

40

60

802.1 x 100mm, 1.8µ, 600 Bar

C-14 AlkylphenonePeak Capacity = 461 (n=10)

min0 2 4 6 8 10 12 14 16 180

20

mAU100

Rapid Resolution Eclipse XDB-C18,

40

60

80 2.1 x 100mm, 3.5µ, 400 Bar

Peak Capacity = 343 (n=10)

min0 2 4 6 8 10 12 14 16 180

20

Conditions: Column: as shown Mobile Phase: A:water B: ACN Gradient: 50 – 100% B in 20 minutes Flow Rate: 0.5 mL/min


5/6/2009

Injection Volume: 2 uL Sample: alkylphenones C3-C10,12,14 Temperature: 40°C Detection: 254 nm (80 Hz)

Particle Size - More Peak Capacity with 1.8um RRHT Columns - Peptide Map of BSARRHT Columns Peptide Map of BSA

673Peak CapacityColumn used

SB C18 2 1 150 1 8

mAU

40

50

Conditions: Columns: as listed, Mobile Phase: A:0.1% TFA in Water B:0.08% TFA in ACN Gradient: 5% B to 60%B in 25 min. Temperature: 80°C Sample: BSA tryptic digest

StartingPressure380 bar673SB-C18, 2.1x150mm, 1.8µ

10

20

30

35% More peak capacity, more resolution

380 bar

min14.5 15 15.5 16 16.5 17 17.5 18 18.5 19

-10

0

mAU

502SB-C18, 2.1x150mm, 3.5µ40

60

Less peak capacity less resolution

StartingPressure105 barPeak Capacity

0

20

Less peak capacity, less resolution

S ll ti l i h k t k it


5/6/2009

min15 16 17 18 19

-20 Smaller particle size = sharper peaks = greater peak capacity

Time Savings, Scalability and Improvement in Sensitivity with RRHT Eclipse Plus – 4.6 mm IDSensitivity with RRHT Eclipse Plus 4.6 mm IDColumns: Eclipse Plus C18, as described below. Mobile Phase: A: water, B: MeOH, (15:85)Temperature: 25°C Flow: 1 mL/min. Detection: 310, 4 nm, 0.5 s response time, semi-micro flow cell, Sample: Sunscreens

1.8 um Rs3,2= 6.51 Rs same,

4.6 x 50 mm, 1.8 µm

4.6 x 150 mm, 5 µmP=82 bar 5um Rs3,2= 6.41mAU

150

scalability demonstrated

P=208 bar

23100

Time savings, improved productivity with

Pressure below 400 bar, try before buy

1. 2-hydroxy-4-methoxybenzophenone

2. Padimate-O3 2 ethylhexyl trans 4

0

50

productivity with RRHT

3. 2-ethylhexyl trans-4-methoxycinnamate

4. 2-ethylhexyl salicylate8 10 12 min2 4 6

This blow-up of a chromatogram shows the added sensitivity achieved with a 1.8um column.


5/6/2009

p g yThe chromatograms are on the same scale and we are looking at the same 2 peaks in each.

Questions on Uses and Limits of Sub 2-micron columns?columns?1. Particle size distribution and backpressure – can you lower

the backpressure and not compromise results?

2. Are there enough choices to do the separation you want to do?

3. What extra preparation/care do you need to take to use the columns successfully?

4 Are there sample limitations for these columns?4. Are there sample limitations for these columns?

5. Can you use the same range of mobile phases and conditions (temperature etc.) with sub 2-micron columns( p )

6. What LC instrumentation is required to achieve maximum benefit from sub 2-micron columns?


5/6/2009

Particle Size Distribution for LC Columns

Particle Size Distribution should have a ratio of smallest to largest of 1:2, 1:1.5 would be ideal based on 90:10, 10:90 ratios

This is because:• Smallest particles, or fines, produce a greater flow resistance – or higher

b kback pressure• Therefore reducing fines is critical to lower back pressure on any

particle size distribution • Largest particles determine maximum efficiency and increase band

broadening A particle size distribution occurs in the manufacturing process• Number average particle size • Volume average particle size – always larger than the number-average


5/6/2009

Actual Example of Particle Size Distribution on ZORBAX 1.8um ParticlesZORBAX 1.8um Particles

Number Average Plot of ZORBAX 1.8 um materialDifferential Number

2500

3000

3500 Number StatisticsMean: 1.740 umMedian: 1.727 umMode: 1.732 um

1500

2000

2500

Num

ber Mode: 1.732 um95% Conf. Limits: 1.739 – 1.742 umSD.: 0.271 um

%> 10 90 Ratio

500

1000um 2.04 1.47 1.39

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

6543210

Particle Diameter (μm)

g p

PSD is “ideal” in terms for largest:smallest particles for high efficiency


5/6/2009

PSD is ideal in terms for largest:smallest particles for high efficiencyLow fines means lowest back pressure.

Actual Example of Particle Size Distribution on ZORBAX 1.8um ParticlesZORBAX 1.8um Particles

Volume Average Plot of ZORBAX 1.8 um materialDifferential Volume

8000

10000

3 )

Volume StatisticsMean: 1.889 umMedian: 1.805 umM d 1 761

4000

6000

lum

e (u

m3 Mode: 1.761 um

95% Conf. Limits: 1.887 – 1.892 umSD.: 0.401 um

%> 10 90 Ratio

2000

4000

Vo

%> 10 90 Ratioum 2.23 1.57 1.42

Volume Average Plot shows “off median” particles in distribution due to particle

Particle Diameter (μm)1 2 3 4 5 6

0

particles in distribution, due to particle engineering


5/6/2009

(μ )

High Performance - Van Deemter Curves for Narrow vs. Production Particle Size Distribution 1.8μm SB-C18μ

9

10Columns: ZORBAX SB-C18Dimensions: 4 6 x 30mm 1 8 μm

h vs. υ Fit CoefficientsProduction PSD Narrow

7

8

ight

(h)

Dimensions: 4.6 x 30mm, 1.8 μmEluent: 85:15 ACN:WaterFlow Rates: 0.05 – 5.0 mL/minTemp: 20°CSample: 1.0μL Octanophenone in EluentD t ti UV 245

Production PSD Narrow PSDVD A: 0.795 0.760

B: 3.97 4.26C: 0 085 0 077

4

5

6

d Pl

ate

Hei

7793 Plates @1.6 mL/min

8065 Plates @

Detection: UV 245nmC: 0.085 0.077r2: 0.9994 0.9998

1

2

3

Red

uced Production 1.8μm – Optimum PSD

Narrow 1.8μm – Narrow PSD

(-3.4%)@

2.0 mL/min

R ld f 2 0 t 1 96!

0

1

0 2 4 6 8 10 12 14 16 18 20

Narrow 1.8μm Narrow PSDRs would go from 2.0 to 1.96!This is not noticeable in practice!


5/6/2009

Reduced Linear Velocity (ν)

Lower Back Pressure for Production vs. Narrow Particle Size Distribution 1.8μm SB-C18Particle Size Distribution 1.8μm SB C18

250

300

Narrow 1.8μm – Narrow PSDColumns: ZORBAX SB-C18Dimensions: 4.6 x 30mm, 1.8 μmEluent: 85:15 ACN:Water

200

250

ar)

Eluent: 85:15 ACN:WaterFlow Rates: 0.05 – 5.0 mL/minTemp: 20°CSample: 1.0μL Octanophenone in EluentDetection: UV 245nm

100

150

ress

ure

(Ba

25% HIGHER Pressure at Optimum Flow (2.0 mL/min)

Production 1.8μm – Optimum PSD

Lower Pressure

50

100Pr

System Pressure (No Column)

00.0 1.0 2.0 3.0 4.0 5.0

Flow Rate (mL/min)


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Flow Rate (mL/min)

1. The Complete List of RRHT 600 bar ColumnsEclipse

Dimensions Eclipse Plus C18 Eclipse Plus C8 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 302

2 Recommended Starting Choices:

3.0 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 728700-902

• 959941-902• 959741-902They are Eclipse Plus C18 Columns.2.1 x 50 959741-902 959741-906 927700-902 927700-906 727700-902

2.1 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 901

C18 Columns.

4.6 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)


5/6/2009

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)

Are all the C18’s Different Selectivity Choices?

Eclipse Plus C18

M bil h (69 31) ACN t

1 23

41st choiceBest Resolution& Peak Shape

StableBondSB C18

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

1 2 3 4 5

1 234

p

2nd choiceGood alternate selectivity due t d d

Eclipse

SB-C18 Columns: RRHT 4.6 x 50 mm 1.8 um

Sample:1. anandamide (AEA)2 P l it l th l id (PEA)1 4

min1 2 3 4 5

to non-endcapped

3rd choice EclipseXDB-C18

2. Palmitoylethanolamide (PEA)3. 2-arachinoylglycerol (2-AG)4. Oleoylethanolamide (OEA)

1 23

4

1 2 3 4 5

3 choiceGood efficiency & peak shapeResolution could be achieved

4th choice Each C18 offers something

Extend-C181 2,3 4

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

unique.While Eclipse Plus C18 might be the best general choice, other separations may be

better with others


5/6/2009

1 2 3 4 5

better with others.

More RRHT Bonded Phase Choices Allow for Optimized Method Development

mAU

400

450

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

2X the needed timeSB-C18 1.8um

300

350

Not the best choice!

Change in Elution Order Peaks 3,4 & 5

S lSB-Phenyl 1.8um

SB-C8 1.8um

200

250

Best Choice!Sample:1. Tolmetin2. Naproxen3. Diflunisal4. Ibuprofen5 Diclofenac

SB Phenyl 1.8um

50

100

150

Conditions: Columns: RRHT 4.6 x 50mm, 1.8um Mobile Phase: Acetonitrile (40%):Buffer 60% pH=2.4 25mM NaH2PO4

5. DiclofenacFor SB-PhenylSB-CN 1.8um

min0 1 2 3 4 5

0


5/6/2009

Flow Rate: 1.85 mL/min Detection: DAD, 254 nm LC: Agilent 1200 RR LC Sample: As listed, 1 uL injection

How To “Match” a Column to a ZORBAX RRHT Column

General Phase Type Starting ZORBAX Choice

Typical “endcapped” C18 or C8 bonded phases, newer columns Eclipse Plus C18 or C8

Endcapped C18 or C8 columns olderEndcapped C18 or C8 columns, older generation Eclipse XDB-C18 or C8

Non-endcapped columns StableBond C18

Older types of columns StableBond C18, C8 etc.

Aqueous “type” columns SB-AQ

CN or Phenyl SB-CN, SB-Phenyl


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2. What Extra Precautions Do You Need to Use RRHT Columns Successfully?RRHT Columns Successfully? 1. At column installation

Solvent changesgLC System cleanlinessColumn Equilibration

2 Column use2. Column useAre you operating at the pressure you anticipate?Check your gradient

3. Mobile phaseAre there limitations?

4 S l id ti4. Sample considerationsParticulate free samplesInjection solvent


5/6/2009

j

RRHT Column Installation Recommendations1 Purge the pumps (connections up to the column) of any buffered mobile phases1. Purge the pumps (connections up to the column) of any buffered mobile phases.

Flush at least 5 mL of solvent before attaching the column to instrument. Goal: Eliminate any dried out or precipitated buffer from the system so it doesn’t wash onto the column and plug the frit.

2. Flush the column with your mobile phase (compatible with the solvents the column was shipped in) starting slowly at 0.1 mL/min for a 2.1 mm ID column, 0.2 mL/min for a 3.0 mm ID column, and 0.4 mL/min for 4.6 mm ID. Goal: Avoid a pressure spike when the new mobile phase reaches the column. This p p poccurs when the different solvents mix. The low flow rate allows this to happen without causing an unanticipated pressure change.

3. Increase the flow rate to the desired flow over a couple of minutes. Goal: anticipate the final operating pressureanticipate the final operating pressure

4. Once the pressure has stabilized, attach the column to the detector.

5. Equilibrate the column and detector with 10 column volumes of the mobile phase i t G l d ibl h t h f th 1stprior to use. Goal: reproducible chromatography from the 1st run

6. If you are running a gradient, check that the pressure range of the gradient –which may be 100 – 130 bar or more, will not cause the system to overpressure, before starting any sequence Goal: no surprises good unattended operation


5/6/2009

before starting any sequence. Goal: no surprises, good unattended operation over 100’s or 1000’s of injections.

RRHT Column Installation Recommendations (cont.)(cont.)4. Once the pressure has stabilized, attach the column to the detector.

5. Equilibrate the column and detector with 10 column volumes of the5. Equilibrate the column and detector with 10 column volumes of the mobile phase prior to use. Goal: reproducible chromatography from the 1st run

6 If you are running a gradient check that the pressure range of the6. If you are running a gradient, check that the pressure range of the gradient – which may be 100 – 130 bar or more, will not cause the system to overpressure, before starting any sequence. Goal: no surprises good unattended operation over 100’s or 1000’s ofsurprises, good unattended operation over 100 s or 1000 s of injections.

7. Avoid overtightening fittings/replace fitting to column periodically – use polyketone fittings for quick changes Goal: good connections nopolyketone fittings for quick changes. Goal: good connections, no extra volume, no overtightening of fittings.


5/6/2009

Mobile Phase and Sample Recommendations to Avoid High PressureAvoid High PressureIf the system has been sitting with buffer in it, flush the injector as well as the column. This prevents any bacterial growth in the injector from transferring to the column and plugging the fritthe column and plugging the frit.

Replace bottles of mobile phase buffer every 24 – 48 hours. Do not top off the bottle with more mobile phase, replace the buffer with a fresh bottle

Do not use a high buffer salt mobile phase (>50mM) in combination with high ACN concentrations due to possible precipitation.

Filter all aqueous buffers prior to use through a 0.2 um filter.

Use solvents that are high quality chromatography grade solvents (HPLC or MS grade).

Filter all samples with particulates through an appropriate 0 2um filterFilter all samples with particulates through an appropriate 0.2um filter. Particulates can clog the inlet frit on the column and cause high pressure and short column lifetime.

Install an in line filter to catch particulates before they get to the column


5/6/2009

Install an in-line filter to catch particulates before they get to the column.

RRLC In-line Filter and Polyketone Fittings –Making RRLC and RRHT Columns Easy to UseMaking RRLC and RRHT Columns Easy to Use

Description Part number PorosityFrit diameter

Flow rate

Part number Replacement Frits

RRLC In lineRRLC 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 5067 1553 0 2 µm 4 6 mm 1 - 5 5067-1562

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

mm, max 600 bar

5067-1553 0.2 µm 4.6 mm mL/min (10/pk)

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

Si l i t t d t d th lif ti f th l dSimple accessories can protect and extend the lifetime of the column and finger tight fittings are easy to use making column and tubing changes fast and leak free.


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Sample and Injection Solvent Considerations – Are There Limits?There Limits?1. Injecting samples with particulates will plug the frit – filter

before use.

2. The injection solvent should be miscible with the mobile phase in the column at injection (gradient or isocratic).

3. Sample should be soluble with the initial mobile phase composition.

4 Injection solvent matched to initial mobile phase is ideal for4. Injection solvent matched to initial mobile phase is ideal for chromatographic efficiency and peak shape, but you can inject in DMSO

5. You can inject protein containing samples or other “dirty” samples, but you will need to clean up the column.


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You Can Inject Dirty Samples – Customer Samples/ExamplesSamples/ExamplesExample 1:• RRHT SB-C18, 2.1 x 50mm, 1.8um 600 bar columnsRRHT SB C18, 2.1 x 50mm, 1.8um 600 bar columns• Protein sample dissolved in DMSO• Gradient mobile phase• High pressure LC

ObservationAft l 100 i j ti l i• After several 100 injections pressure on column increases

Question• Is the column still usable?Is the column still usable?


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Example #1

Adsorbed material washing off – this column flushed with ACN:water

Pressure trace while column is equilibrating,the pressure increases are smalleras flow rate is increased because of materialthat has been washed off

Fl hi l d b d l t d lif ti

that has been washed off


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Flushing column removes adsorbed sample, extends lifetime.

Example 1: Excellent Performance Achieved After “Cleaning” ColumnCleaning Column

Adsorbed material washing

Pressure change as adsorbed material eluted

goff

QC chromatogram

Pressure, efficiency return to initial levels after cleaning.C ti d i ibl Fl hi i d d t t d lif


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Continued use is possible. Flushing is recommended to extend life.

Photo of Frits – No contamination of Frits

Frits are relatively clean indicating that a plugged frit with particulates was not the problem, but material from the sample adsorbing to the column was the issuecolumn was the issue. Few small black particles – typical of wear on any LC system


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Example #2: Protein Precipitated Samples “Dirty Sample” Pressure vs. Injection #Sample Pressure vs. Injection #

150

Column: ZORBAX RRHT SB-C18

130

140Column: ZORBAX RRHT SB C182.1 x 30mm, 1.8 μm

120 End Press

Pressure Increase of only 8 bar over 600+ injections!!

100

110

900 100 200 300 400 500 600 700

Inj #Protein precipitated filtered – only sample preparation


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Protein precipitated, filtered only sample preparationWith no particulates pressure increase is minimal.

Success #1: RRLC Gradients with the Agilent 1200 SL QA/QC Analysis: SB-C18 3.0 x 50 mm 1.8 um

GAT-XXX5708492-1

mAU3000

Customer Sample - drug intermediate QA-sample

1 Blanks1 T t l

2000

2500GAT-XXX5708492-1

mAU

2500

1 Test-sample (ref. of main comp. and imp.)

2 Std.-samples (reference of main comp.)

1 Sample2 Std.-samples

70 C, pc cooling 21 C flow = 2.8 ml/min A= (2mM Phos, pH=2.0), B=ACN50 90%B in 3 0min 90%B

1000

1500

XXX6087077

1500

2000S d sa p es

1 Sample1 Std. Sample

Original method was

50-90%B in 3.0min, 90%B 1.5minanalysis-time = 4.5 minpost-time = 1.0minDAD SL: 214 & 266nm

min0 5 10 15 20 25 30

0

500XXX6087077

XXX6087077500

1000Original method was 35 min run time= 5 hours 15 min = half a day!!!

30

min0.5 1 1.5 2 2.5 3 3.5 4.0

0

RRLC Method:5.5 min run time

49 i til b t h l !!!RR-Practical Considerations

Agilent Restricted5/6/2009

= 49 min until batch release!!!

1200 RRLC – Achieve Maximum Results with RRHT Columns for High Speed and High Resolution LC

Increased linear flow rates for higher analysis speedHigher Column and System Temperature: 80°C => 100°C

C & S

Columns for High Speed and High Resolution LC

Higher Allowed Column & System Pressure: 400bar => 600barMinimized column pressure: – 25%Minimized system pressure: – 30% at 1ml/minMaintained high flow rates: 0 05 5ml/minMaintained high flow rates: 0.05 – 5ml/min

High Method FlexibilitySupport of narrow (2.1mm) and analytical (3.0 and 4.6mm)

columnsSupport of RRLC and conventional HPLC on one unitBroad Range of >100 RRHT 1.8um ColumnsSame Chemistry for 1.8, 3.5, 5 and 7um particle columns

Fast, easy, secure method transferIncreased efficiency for higher resolution/peak capacity

Longer columns: 50mm => 150mm


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Conclusions

The key advantages or Rapid Resolution HT (1.8um) columns are improved speed and resolution.

Th “li it ti ” f RRHT l f d ithThe “limitations” of RRHT columns are very few and with a suitable focus on reducing particulates excellent results can be obtained.

All normal mobile phases and normal conditions can be used on RRHT columns.

RRLC instruments provide the ideal tool for achieving high resolution and high throughput results with RRHT columns.


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Agilent LC Columns and Agilent J&W GC Columns Scientific Technical SupportColumns Scientific Technical Support

800-227-9770 (phone: US & Canada)*

302-993-5304 (phone)

For LC columnsFor LC columns

Select option 4, then option 2

For GC Columns

* Select option 4, then option 1.

www.agilent.com/chem


5/6/2009

Documents

Rapid Resolution - Practical Considerations - Series 6 ... · Rapid Resolution - Practical ConsiderationsPractical Considerations - Series 6 LC Columns and Consumables Ed Kim Application