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Practical Applications of Method Translation Using g

the Agilent Method Translation Tool

eSeminar and Workshop

Thomas J. Waeghe, Ph.D.Inside Application EngineerAgilent TechnologiesLife Sciences and Chemical Analysis

Practical Applications of Method Translation Using the Agilent Method Translation Tool

Title4/16/2008

Objectives for Today’s e-Seminar and Workshop

• Demonstrate the practical use of the Agilent Method Translation tool for fast, easy, and successful method transfer to smaller volume columns

• Review and discuss the variables that are mostReview and discuss the variables that are most important for successful translation of isocratic and gradient methods

• Review several completed method transfer• Review several completed method transfer examples using the Agilent Method Translation Tool

• Work through several real examples submitted by customers


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Agenda for Today

Successful Method Translation– Separation Goals and Method Performance Criteria– Isocratic separations

• Which instrumentation must you have to get started• Which instrument and method parameters afford optimal resultsp p• Considerations for successful implementation• Agilent Method Translator for isocratic separations

Gradient separations– Gradient separations• Review of gradient retention parameters• Instrument considerations• Agilent Method Translator for gradient separations

Workshop with submitted examples


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Separation Goals and Method Performance Criteria

Separation Goals and System Suitability• Resolution (≥ 2)

Method Performance CriteriaAccuracy

• Peak shape (USP Tf close to 1 [< 2])

• Injection Repeatability (areas, Tf, etc., [RSD 0.1 - 0.25%])

Precision• Repeatability• Intermediate precision

aka Figures of

Merit

• Absolute retention ( 1 < k > 10)

• Relative Retention (α or k2/k1)

• Signal to Noise Ratio (> 10)

• Reproducibility• RobustnessSelectivity/Specificity

• Signal-to-Noise Ratio (> 10)

AVOID THESE for System Suit. Criteria

Linearity

Range

Quantitation Limit (LOQ 10x S/N)Column efficiency (theoretical plates)

Absolute retention

Quantitation Limit (LOQ, 10x S/N)

Detection Limit (LOD, 3x S/N)


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An Approach for Isocratic Method Translation

• Assess and document current method performance and parameters

• Assess current instrument configuration

• Set performance goals for method to be translated

• Determine which column geometry will provide necessary efficiency

Instrument needs vs method performance goals will depend on• Instrument needs vs. method performance goals will depend on requirements for column size and particle size to get desired Rs

• Instrument extracolumn volume, detector data rate• System pressure limitations

• Adjust injection volume for smaller column volume

• Assess injection repeatability and sample solvent composition• Assess injection repeatability and sample solvent composition robustness

• Adjust flow rate vs. system max. pressure relative to method


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performance goal for analysis time.

Isocratic Method: Document current method performance and parameters, and instrument configurationCurrent Method Performance• Limiting Resolution for Critical Pair(s)• Peak Shape(s) (USP Tf)

Method Parameters

• Column length, id and particle size• Peak Shape(s) (USP Tf)• Injection Repeatability

(pooled RSD duplicate injs)• Signal to Noise Ratio

• Flow Rate

• Mobile Phase Composition (viscosity)

• Column Temperature• Signal-to-Noise Ratio

Instrument Configuration• Extracolumn Volume

• Column Temperature

• Injection Volume

• Sample concentration and Sample • Extracolumn Volume• Tubing ID and length• Flow Cell Volume

Solvent Composition

• Nominal Backpressure

• Detector Data Rate• Flow Cell Pathlength• System Maximum Pressure


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Isocratic Method ExampleSituation:You have isocratic method for tocopherols developed for 4.6 mm i.d. columns in 150 mm length. Run time is ~14 min.

Pump: Agilent 1100 quaternary systemAutosampler: Standard autosamplerTCC: 1100 standardDetector: 1100 DAD, max. data rate 20 Hz

Typical setting, PW = 0.05 min.Flow Cell: 13 μL, 10 mm path lengthFlow Rate: 1.0 mL/min.Column temp. 23ºCGoals: Decrease run time and improve throughput (5X, if possible)

S l t d t (i li ll l id h tSave solvent usage and waste (implies smaller column id or shorter run at higher flow rate)

• Can anything be done to speed up these methods with existing equipment?

What modifications can be made and which are most important?


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• What modifications can be made and which are most important?

Isocratic method on Conventional ColumnTocopherols

Column: ZORBAX Eclipse XDB-C18Mobile Phase: 95% ACN: 5% WaterTemp: 23ºCRRHT

p

Conventional4.6 x 150 mm 5 μm

Flow Rate: 1 mL/minP = 37 bar

Temp: 23 CInjection volume: 1 uL

RRHT4.6 x 50 mm 1.8 μm

Flow Rate: 3 mL/minPressure = 229 bar

P = 37 barmAU

60

80

Rs ~ 4.4Rs ~ 5.2

20

40 1.7 min13.5 min

Sample: Vitamin E – α, β, γ-tocopherols in gel capE li XDB C18 i d fi t h i f th d

min0 2 4 6 8 10 12 140


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Eclipse XDB-C18 is a good first choice for many methods.

Assess Your Current Method

Assess your current method4.6 x 150 mm, 5 μm column

Questions to ask?What is the mobile phase composition?

1.0 mL/minRT last = 14 minutes

What is the current backpressure?Injection Volume?Data Rate/Peak Width?What is your limiting resolution with current method?What size column can deliver the

l ti d?resolution you need?Can your current instrument be used to apply the shorter column with smaller particle size?Which changes in method parameters are necessary and can you get the same or similar performance and results?


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Efficiency Ranking of Various Column Geometries and Typical Backpressuresand Typical Backpressures

This RRHT column Replaces These Longer Columns50 mm, 1.8 μm 150 mm, 5 μm, 100 mm, 3.5 μm

100 mm, 1.8 μm 250 mm, 5 μm


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Tocopherol method translation

Current Method4.6 x 150 mm, 5 µm XDB-C18Viscosity of 95:5 ACN/water at 23ºC is

Translated Method4.6 x 50 mm, 1.8 um RRHT XDB-C18Column length in shorter dimensions with 1.8

ti l i 4 6 50 RRHT~0.43 cp.Flow Rate is 1 mL/minBackpressure is 37 bar

µm particles is 4.6 x 50 mm RRHTAt 1 mL/min expected backpressure is 79 bar + ~10 bar (a/s and flow cell) or ~90 barExpected run time will be 1/3 of 14 minutes or

Standard flow cell (13 µL)Standard 0.17 mm tubing throughoutLimiting Resolution ~4.4

p4.67 minutesTry 3 mL/min for run time of 1/9 of 14 min. or 1.55 min.Predicted pressure is 238 barg

Peak Width required 0.1 minResponse Time = 2 sec or Data Rate = 2.5 Hz is adequate

Predicted pressure is 238 barLimiting resolution will be approximately the same (4.4) or 4.4 x SQRT(13043/12077) = 4.2, IF no band broadening due to extracolumn volume or data rate.Standard DAD or MWD at fastest setting (20 Hz) with 0.17 mm id tubing adequate but not optimumChoose 0.12 mm i.d. tubing and 5 µL flow cell


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g µfor better results

Agilent Method Translator


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Isocratic Method: Translation Tool 13 uL flow cell and 0.17 mm tubing

21.6 uL tubing vol + 13 uL

flow cell

Effective N hurt by EC vol.


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For isocratic runs the 2nd row must be set to same %B as row 1

Use 5 uL flow cell and 0.12 mm id tubing

Improvement in N effective


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Adjust % max. pressure until desired flow

Speed Optimized at 3 mL/min

rate

Adjust to 3 mL/min

Click radioClick radio button to

allow % max pressure

adjustment


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Comparison of Conventional Isocratic Method vs. Translated Method at 3 mL/min

Column: ZORBAX Eclipse XDB-C18Mobile Phase: 95% ACN: 5% WaterTemp: 23ºCRRHT

Translated Method at 3 mL/min

Solvent used 15 mL

Conventional4.6 x 150 mm 5 μm

Flow Rate: 1 mL/minP = 37 bar

Temp: 23 CInjection volume: 1 uL

RRHT4.6 x 50 mm 1.8 μm

Flow Rate: 3 mL/minPressure = 229 bar

P = 37 barmAU

60

80

Rs ~ 4.4Rs ~ 5.2Solvent used 5.1 mL

20

40 1.7 min13.5 min

min0 2 4 6 8 10 12 140

Sample: Vitamin E – α, β, γ-tocopherols in gel capE li XDB C18 i d fi t h i f th d


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Eclipse XDB-C18 is a good first choice for many methods.

Flow Cells for RRLC

13 µl Standard Flow Cell: For highest sensitivityHigh-demanding quantitative work, e.g. analytical method development QA/QCanalytical method development, QA/QC

2 µl Micro Flow Cell: For highest resolutionUltra fast semi quantitative workUltra-fast semi-quantitative work, e.g. Screening Experiments, HT LC/MS/UV

5 µl Semi-micro Flow Cell: Best compromise of sensitivity and resolutionBest compromise of sensitivity and resolutionFor good quantitative and qualitative results, e.g. Screening, HT LC/MS/UV, Early Formulation Studies

Dimension Sensitivity* Resolution*

13 µl / 10 mm +++ +

* D d l ti l diti d l di i

5 µl / 6 mm ++ ++2 µl / 3 mm + +++


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* Depends on analytical conditions and column dimension

Choosing the flow cell size


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Peak Width Setting – Response Time – Data Rate and Sensitivityy

Don‘t use for > 0.15 sec peak width!

> 0.15 sec

> 0.3 sec

> 0.6 sec

Recommended settings in ultra-fast LC with 50% peak width between 0.15 and 0.6 sec

For 50% peak width between 0.6 and 1.2 sec

> 1.2 sec

> 3 sec

> 6 sec

Notes: • Noise level changes ~ proportional to the

square root of the change in data rate.• For optimum selectivity and sensitivity the

Peak Width should not be chosen smaller> 12 sec

> 24 sec

> 51 sec

Peak Width should not be chosen smaller than necessary.

• For 50% peak width between 0.3 and 0.6 seconds Peak Width of > 0.005 min is recommended, which correspondes to 40Hz

Peak Width = Peak Width at 50% Peak Height

data rate.• Only for peaks narrower than 0.3sec at half

height, Peak Width of > 0.0025min (80Hz data rate) should be used.

• For highest sensitivity in ultra fast LC the• Set at fastest rate and then decrease data rate until

peak width increases and S/N is optimum


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• For highest sensitivity in ultra-fast LC the slit can be increased to 8 or 16nm.

peak width increases and S/N is optimum

Detector Data Acquisition Rates – Effects on Peak Width, Resolution and Peak Capacity in UFLC

Data Peak Resolution Peak 1.2

1.4

c

60

70

, p y

Rate Width Capacity

80 Hz 0.300 2.25 61

40 Hz 0.329 2.05 560.6

0.8

1

Peak

Wid

ths

/ se

c

20

30

40

50

Peak

Cap

acity

Peak Width [s]

Peak Capacity

20 Hz 0.416 1.71 44

10 Hz 0.666 1.17 28

5 Hz 1.236 0.67 16

0.2

0.4

0 20 40 60 80 100Data Rate [Hz]

0

10

80Hz versus 20Hz Data Rate:– 40% Peak Width => +40% Peak Capacity+ 30% Resolution => + 70% Apparent Column Efficiency08

1

1.2

1.4

dths

/ s

ec

1.5

2

2.5lu

tion

Peak Width [s] pp y

80Hz versus 10Hz Data Rate:– 120% Peak Width => +120% Peak Capacity+ 90% Resolution => +260% Apparent Column Efficiency0.2

0.4

0.6

0.8

0 20 40 60 80 100

Peak

Wid

0

0.5

1 Reso

l

Resolution (4,5)


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0 20 40 60 80 100Data Rate [Hz]

Data Rate and Slit Width Effect on S/N Ratio (DAD and MWD, VWD data rate)(DAD and MWD, VWD data rate)

S/N can be optimized with data rate Slit width can be increased to improve S/N (2 uL and 5 uL cells)


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to improve S/N (2 uL and 5 uL cells)

Break 1: Gradient Methods Next

Questions?


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Translating Gradient Methods


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Advantages of Gradient Elution

Complex samples are analyzed in a single HPLC runAnalysis time is reducedAnalysis time is reducedAll peaks elute with the same bandwidthMore peaks can be baseline resolved per unit timeMore peaks can be baseline resolved per unit time

– higher peak capacity than isocratic methodSignal-to-Noise ratios and LOD/LOQ are relatively the g ysame during a gradient run (barring ghost peaks, anomalies, etc.!)

peaks don’t broaden with increasing retention time as– peaks don t broaden with increasing retention time as they do in an isocratic separation)


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100% B Gradient Steepness AffectsRetention (k*) and Resolution

100% B

tg= 5

87tg Fk* =0% B

This equation

tg= 10 1/k* ∝ gradient steepness = b

ΔΦ Vm Sk

governs gradient retention and

selectivity

100% B

t =

ΔΦ = change in volume percent of B solvent (%)

S = property of sample compoundF fl t ( L/ i )

0% B

100% B

tg= 20

F = flow rate (mL/min.)tg = gradient time (min.)

Vm = column void volume (mL)0% B

0 10 20 30 40

tg= 400% B

• S ≈ 4–5 for small molecules• 10 < S < 1000 for peptides

and proteins


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0 10 20 30 40

Time (min)000995P1.PPT

To Increase Gradient Resolution by Changing Gradient Retention (k*) Use:( )

A longer gradient time tGA h t l VA shorter column Vm

A higher flow rate F

A shorter organic range %B

87 t F87 tg F

S (Δ%B) Vm

k* =


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Transferring a Gradient Method to a Small(er) Column

Examine the current method– Column length and i.d., particle size, N– Injection volume– Injection precision– Gradient program

• Initial Hold Time

It’s much easier to transfer a linear gradient than one with multiple

segments and hold times

• Linear gradient segments• Isocratic holds during gradient

– Delay Volume– Resolution of critical pair(s)– Backpressure

Can you trade excess resolution for time or can you get the same ffi i (N) ith h t l ?efficiency (N) with a shorter column? – Calculate critical pair resolution on shorter column(s) with smaller particle size(s)– Calculate expected pressure at one or more flow rates on shorter column


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Transferring Gradient Methods to Smaller Diameter Columns and to Different Instruments

To Transfer Gradient Separations,Average retention factor for k* must match, andg ,Effective delay times must match (or ratio of gradient volume/column volume must be same)

Also Important for Gradient Separations• Column re-equilibration time (post time)• System/Dwell volume—volume from point of mixing to y p g

column– How to measure and account for it– Correct for differences between instruments


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Gradient Separations: Considerations When Translating Existing Gradient methodsIsocratic SeparationsIsocratic Separations

Sample load (Vinj, [analyte])

Sample solvent strength

Gradient SeparationsSame as Isocratic Separations plus…

Extracolumn volume

– Flow cell volume – Injection volume

Delay Volume– Same instrument (different pressures)– Different instrument (for example,

Capillary 1100 vs Binary 1100)Injection volume– Tubing volume

Injector precisionC ith V

Capillary 1100 vs. Binary 1100)Gradient Time

– Adjust relative to equation for gradient retention

– Can vary with Vinj

– Data Rate– Too fast, too much noise

– Keep k* constant

Gradient Delay Time– Gradient delay time must be same as for

– Too slow, loss of N larger column separation– Ratio of gradient volume/column volume

must be same as for larger column

Column Equilibration Time (Post Time)87 tg F

S (D%B) Vk* =


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q ( )S (D%B) Vm

Gradient Separations – What is Delay Volume?

Also known as

Dwell Volume

DelayVolume

• Delay Volume = volume from formation of gradient to the column


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• Behaves as isocratic hold at the beginning of gradient.

Comparison of System Delay Volumes

Pump w/o mixerw/ mixer

1090 1050 1100 Quat. 1100 Bin.

300-5001050 1250

180-480600 900

800-1100n/a

800-1100n/aw/ mixer

Mixer

Autosampler Standard

1050-1250

750

V (loop)

600-900

420

300 + V (inj)

n/a

n/a

300 + V (inj)

n/a

n/a

327 + V (inj)Autosampler StandardBypass

Column compartment StandardBypass

V (loop)N/A

4.1 or 8.20

300 + V (inj)6.2

3 or 60

300 + V (inj)6.2

3 or 60

327 + V (inj)8

15 ul0Bypass 0 000

Min RangeMax Range

304-5041058-1258

189-489906-1206

1203-14061242-1442

Delay Volume Comparison: 1100/1200 Series Binary Pump vs. 1200 Series Binary Pump SLBinary Pump vs. 1200 Series Binary Pump SL

Binary pump SL (pressure range up to 600 bar):y p p (p g p )Standard delay volume configuration: 600-800μL (incl. damper and mixer)

Low delay volume configuration: 120μL (virtual damper)y g μ ( p )

Damper volume: 80-280μl

Binary pump (pressure range up to 400 bar):Standard delay volume configuration: 600-900μL (incl. damper and mixer)Standard delay volume configuration: 600 900μL (incl. damper and mixer)

Reduced delay volume configuration: ~200μL (damper needed)

Damper volume: 180μl + 1μl per bar


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


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Features of the Agilent Method TranslatorBasic mode with certain pre-set parameters:as c ode ce a p e se pa a e e s

1.3.

2. 6.

4

5.

Enter the parameters of your existing method and the parameters of the

4.


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p y g pdesired column you would like to convert to.

Features of the Agilent Method TranslatorAdvanced mode – all calculation parameters in your hands:d a ced ode a ca cu a o pa a e e s you a ds

1 2.1.

[mL] [mL]

3.

4.

More to enter but much more information returned

3


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More to enter but much more information returned

Analysis of impurities of an active pharmaceutical ingredient by Does it work? - Example

conventional HPLC (4.6mmID x 250mm, 5.0µm):

mAU HN

CH3CH3

30

40 OH

OCH3

Main Compound

20

30

OH

HN

CH3CH3

OCHN

CH3CH3CHCHN

33

H

NCH3CH3

H

10

OCH3Impurity A

O CH3

Br

Bromanisole

OCH3

Impurity B

OCH3

Impurity C

OH

OH

Impurity D

0


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min0 2.5 5 7.5 10 12.5 15 17.5 20

Does it work?Converting to a 4.6 x 100 mm, RRHT column:


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mAU Conventional HPLC mAU35

Does it work? YES

30

40

25

30

35mAU

20

20

30

15

20

10

15

10

5

10

5

4 6 mm ID x 250 mm 5 0µm Zorbax SB C18

min0 2.5 5 7.5 10 12.5 15 17.5 20

0

4 6 mm ID x 100 mm 1 8µm Zorbax SB C18

min0 1 2 3 4 5 6 7 8

0

min0 0.5 1 1.5 2 2.5 3 3.5 4 4.5

0

4 6 mm ID x 100 mm 1 8µm Zorbax SB C184.6 mm ID x 250 mm, 5.0µm Zorbax SB C18

0.00 min 5% B20.00 min 90% B23.00 min 90% B23 01 min 5% B

4.6 mm ID x 100 mm, 1.8µm Zorbax SB C18


Simple Conversion4.6 mm ID x 100 mm, 1.8µm Zorbax SB C18


Speed Optimized


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23.01 min 5% B30.00 min 5% B23.01 min 5% B9.20 min 5% B4.99 min 5% B6.5 min 5% B

Advanced Mode: Select worst case viscosity for ACN/water at 40ºCACN/water at 40 C

0.75 cp


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Advanced mode, Simple Conversion


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Advanced mode, Resolution Optimized


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How to Use Rapid Resolution HT and other Low Volume HPLC Columns Effectively on Agilent 1200Volume HPLC Columns Effectively on Agilent 1200 and 1100 HPLCs

• Use data acquisition rate of 0 1 secUse data acquisition rate of 0.1 sec• Use DAD SL for 80 Hz data acquisition• Short lengths of 0.12 mm i.d. tubing or smaller (watch pressure)• Thermostated column compartment plumbed through 3 μL side• For 2.1 mm id columns at elevated temps, use low vol. heat

exchangersg• For gradients - 80 μL (p/n 5022-2165) or no mixer and injector

bypass (not relevant for quaternary systems)• Recommend micro and well plate autosamplers (ADVR “on”)• Recommend micro and well plate autosamplers (ADVR on )• Otherwise, use injector program to reduce delay volume


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1100 System Configuration for Ultra-fast LC Recommendations for System Setup and Connecting Capillaries

Replace standard mixer of Binary Pump with 80 μL filter (p/n 5064-8273) to reduce delay volumneUse low volume, 3ul heat exchanger of TCC G1316A to th t t l t

1100 Binary Pump (G1312A) thermostate eluent

For 4.6 and 3mm columns use shortest possible 0.17mm ID connecting capillaries

Note: In ultra-fast applications the typical flow rate range using 4.6 and 3mm ID columns is 1-5 ml/min. At such higher flow

Pump (G1312A)

1100 WPS(G1367A)

4.6 and 3mm ID columns is 1 5 ml/min. At such higher flow rates the larger delay volume of 0.17mm ID capillaries doesn’t have a measurable negative impact on chromatographic performance.

For 2.1 and 1mm columns use shortest possible 0.12 or 0 1mm ID capillaries

3 μL heat exchanger

1100 TCC(G1316A)

0.1mm ID capillariesNote: In ultra-fast application the typical flow rate range using 2.1 and 1 mm ID columns is between 0.1-1 ml/min. At these lower flow rates smaller ID connecting capillaries should be used to minimize system delay volume and extra column peak dispersion/band broadening

4.6mm ID, 1.8umRRHT Column

dispersion/band broadening. Inlet tubing of the flow cell should be directly connected to the column.

Note: If this is not possible an appropriate low-volume connection should be used (capillary of small ID, i.e. 0.12 mm

0 17 d ZDV i )

Waste

1100 DAD SL(G1315C)


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or 0.17mm and ZDV-union).

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

150 mm or shorter cols with 2.1, 3.0, 4.6 mm

IDs

Actual: 1100

Quat System

Step 1: 1100/1200

„Bin SL“ System

Degasser

Step 2: 1200

Rapid Resolution System

Bin Pump SL

u-Degasser

50 mm or shorter cols with 3.0 or

4.6 mm IDs

Quat System

ALS (WPS?)

Quat Pump

Degasser

h-ALS SL

TCC

Bin Pump SL h-ALS SL

TCC SL

DAD SL80 Hz max

TCC

VWD/MWD/DAD

VWD/MWD/DAD+ Speed

+ Resolution+ Sensitivity

+ MS-Robustness+ Data Security & Traceability

+ Speed+ Resolution

+ MS-Compatibility

20 Hz max

20 Hz max

max

> 5 min> 6 min

> 1.5min> 2min

Analysis TimeCycle times

> 0.2min> 0.4min

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

+ MS Compatibility+ Solvent Saving

+ Compatible with conv. HPLC

> 3 sec5 - 12,000

4.6 mm50 mm

0.2 - 10ml/min80 C

> 1.5 sec5 - 30,000

2.1 - 4.6 mm50 - 150 mm

0.05 - 5 ml/min80 C

Peak WidthN

Column IDColumn Length

Flow ratesT t

> 0.2 sec5 - 60,000

2.1 - 4.6 mm20 - 150mm

0.05 – 5 ml/min


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80 C400bar

80 C600 bar

TemperaturePressure

100 C600bar

Stepwise Scale-up to Rapid Resolution LCFrom 1100 to 1200 RRLC in two steps – Example 2

150 mm or shorter cols with 2.1, 3.0, 4.6 mm

IDs

Actual: 1100

Bin System

Step 1: 1100/1200

„DAD SL“ System

Degasser

Step 2: 1200

Rapid Resolution System

binPumpSL

u-Degasser

50 mm or shorter cols with 3.0 or

4.6 mm IDs

Bin System

ALS

BinPump

Degasser

h-ALS SL

ColCom

binPump h-ALS SL

ColCom SL

DAD SL80 Hz max

ColCom

VWD/MWD/DAD

DAD SL + Speed+ Resolution+ Sensitivity

+ Solvent Saving+ MS-Robustness

+ Speed+ Data Security & Traceability

+ Compatible with conv. HPLC

max80 Hz max

> 1 min> 2 min

> 0.2min> 0.4min

Analysis TimeCycle times

> 0.2min> 0.4min

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

Compatible with conv. HPLC

> 1.5 sec8 - 12,000

3 - 4.6 mm50 mm

0.05 - 5 ml/min80 C

> 0.3 sec5 - 22,000

3 - 4.6 mm20 - 100 mm

0.05 - 5 ml/min80 C

Peak WidthN

Column IDColumn Length

Flow ratesT t

> 0.2 sec5 - 60,000

2.1 - 4.6 mm20 - 150mm

0.05-5ml/min100 C


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80 C400bar

80 C400 bar

TemperaturePressure

100 C600bar

Optimizing Gradient Separations With 1.8 um RRHT Columns: 10 X Faster Analysis

RRHT SB-C182.1 x 50mm, 1.8um

Conditions: Column: SB-C18, Dimensions listed below, Gradient: 10 – 90% ACN/25mM H3PO4, Gradient time: tG, as notedCPAH’s = Chlorphenoxyacid herbicides – environmental sample

i0 25 0 5 0 75 1 1 25 1 5 1 75 2

Temp: 50°CFlow: 1 mL/minGradient (tG): 2.4 min

C.

min0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.25Rapid Resolution SB-C183.0 x 150mm, 3.5umTemp: 25°CFlow: 1.0 mL/minGradient (tG) : 18 min

B. Key Parameters• Particle size

Fl R t( G)

SB-C184 6 x 250mm 5um

0 2 4 6 8 10 12

A

• Flow Rate• Gradient Time• Column Length• Column ID• Temperature

min5 10 15 20 25

4.6 x 250mm, 5umTemp: 25°CFlow: 1mL/minGradient (tG): 30 min

A.p

Rsoptimized


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min5 10 15 20 25Sample: CPAH= Chlorophenoxy herbicides : Picloram, Chloramben, Dicamba, Bentazon, 2,4-D, Dichlorprop, 2,4,5-TP, Acifluorfen.

Translation to 3 0 x 150 mm 3 5 um 18 min gradient


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Translation to 3.0 x 150 mm, 3.5 um 18 min gradient

3.0 x 150 mm, 3.5 um, Resolution Optimized


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Scaling Gradients from 4.6 mm I.D. Columns to Solvent Saver Plus Column-Organic Acids

mAU

40506070 4.6 x 250 mm SB-C18, 5-um

57 mL solvent used25 uL std injection

1.5-mL/min; tg= 38

1

25 7

60

0102030

0min5 10 15 20 25 30 35

mAU70

4.6 x 150 mm SB-C18, 3.5-um

gmin3 4

56 8

10

010

20

3040

504.6 x 150 mm SB C18, 3.5 um 33 mL solvent used

15 uL std injection

1.0-mL/min; tg= 33 min

20

40

60

30 35-10

mAUmin0 5 10 15 20 2580

3.0 x 100 mm SB-C18, 3.5-um10.5 mL solvent used

6 uL injection with INJ Program

min0 5 10 15 20 25 30 35-20

0

20

Analytes 1) gallic acid 3) protocatechuic acid 5) syringic acid 7) salicylic acid

0.5-mL/min; tg= 21min


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Analytes 1) gallic acid 3) protocatechuic acid2) hydrocaffeic acid 4) gentisic acid

5) syringic acid6) sinapinic acid

7) salicylic acid8) caffeic acid

Summary

• Method conversions are an opportunity to increase lab productivity significantly.

• The Agilent Method Translator is easy to use and can make your method translations to smaller columns much quicker and successful.

• Maintain resolution and avoid any change of selectivity y g y

• Proper choice of column size and efficiency,• Careful selection of method parameters.

• System optimization may be required to use smaller columns and/or smaller particle sizes (tubing, flow cell, delay volume, data rate)

• Increased operating pressure may result – ensure that system hasIncreased operating pressure may result ensure that system has adequate capacity for standard and increased pressure operation across the flow range of routine and optimized methods


4/16/2008

Workshop

Examples

Isocratic• Isocratic

• Gradient


4/16/2008

Practical Examples of Method Translation 041608 › cs › library › eseminars › ...• Which...

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