practical examples of method translation 041608 - agilent · rt last = 14 minutes questions to ask?...

Practical Applications of Method Translation Using the Agilent Method Translation Tool

Title10/28/2008

Practical Applications of Method Translation Using

the Agilent Method Translation Tool

eSeminar and Workshop

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


10/28/2008

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 most important for successful translation of isocratic and gradient methods

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

• Work through several real examples submitted by customers


10/28/2008

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 results• Considerations for successful implementation• Agilent Method Translator for isocratic separations

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

Workshop with submitted examples


10/28/2008

Separation Goals and Method Performance Criteria

Separation Goals and System Suitability• Resolution (≥ 2)

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

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

• Absolute retention ( 1 < k > 10)

• Relative Retention (α or k2/k1)

• Signal-to-Noise Ratio (> 10)

AVOID THESE for System Suit. CriteriaColumn efficiency (theoretical plates)

Absolute retention

Method Performance CriteriaAccuracy

Precision• Repeatability• Intermediate precision• Reproducibility• RobustnessSelectivity/Specificity

Linearity

Range

Quantitation Limit (LOQ, 10x S/N)

Detection Limit (LOD, 3x S/N)

aka Figures of

Merit


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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 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 robustness

• Adjust flow rate vs. system max. pressure relative to method performance goal for analysis time.


10/28/2008

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

(pooled RSD duplicate injs)• Signal-to-Noise Ratio

Instrument Configuration• Extracolumn Volume

• Tubing ID and length• Flow Cell Volume

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

Method Parameters

• Column length, id and particle size

• Flow Rate

• Mobile Phase Composition (viscosity)

• Column Temperature

• Injection Volume

• Sample concentration and Sample Solvent Composition

• Nominal Backpressure


10/28/2008

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)

Save 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?


10/28/2008

Conventional4.6 x 150 mm 5 μm

Flow Rate: 1 mL/minP = 37 bar

Column: ZORBAX Eclipse XDB-C18Mobile Phase: 95% ACN: 5% WaterTemp: 23ºCInjection volume: 1 uL

RRHT4.6 x 50 mm 1.8 μm

Flow Rate: 3 mL/minPressure = 229 bar

Sample: Vitamin E – α, β, γ-tocopherols in gel capEclipse XDB-C18 is a good first choice for many methods.

min0 2 4 6 8 10 12 14

mAU

0

20

40

60

80

1.7 min13.5 min

Isocratic method on Conventional ColumnTocopherols

Rs ~ 4.4Rs ~ 5.2


10/28/2008

Assess Your Current Method

Assess your current method4.6 x 150 mm, 5 μm column1.0 mL/minRT last = 14 minutes

Questions to ask?What is the mobile phase composition?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 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?


10/28/2008

Efficiency Ranking of Various Column Geometries and 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


10/28/2008

Tocopherol method translation

Current Method4.6 x 150 mm, 5 µm XDB-C18Viscosity of 95:5 ACN/water at 23ºC is ~0.43 cp.Flow Rate is 1 mL/minBackpressure is 37 barStandard flow cell (13 µL)Standard 0.17 mm tubing throughoutLimiting Resolution ~4.4Peak Width required 0.1 minResponse Time = 2 sec or Data Rate = 2.5 Hz is adequate

Translated Method4.6 x 50 mm, 1.8 um RRHT XDB-C18Column length in shorter dimensions with 1.8 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 4.67 minutesTry 3 mL/min for run time of 1/9 of 14 min. or 1.55 min.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 for better results


10/28/2008

Agilent Method Translator


10/28/2008

Isocratic Method: Translation Tool

21.6 uL tubing vol + 13 uL

flow cell

13 uL flow cell and 0.17 mm tubing

Effective N hurt by EC vol.

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


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Use 5 uL flow cell and 0.12 mm id tubing

Improvement in N effective


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Adjust to 3 mL/min

Click radio button to

allow % max pressure

adjustment

Adjust % max. pressure until desired flow

rate

Speed Optimized at 3 mL/min


10/28/2008

Conventional4.6 x 150 mm 5 μm

Flow Rate: 1 mL/minP = 37 bar

min0 2 4 6 8 10 12 14

mAU

0

20

40

60

80

1.7 min13.5 min

Column: ZORBAX Eclipse XDB-C18Mobile Phase: 95% ACN: 5% WaterTemp: 23ºCInjection volume: 1 uL

RRHT4.6 x 50 mm 1.8 μm

Flow Rate: 3 mL/minPressure = 229 bar

Sample: Vitamin E – α, β, γ-tocopherols in gel capEclipse XDB-C18 is a good first choice for many methods.

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

Rs ~ 4.4Rs ~ 5.2Solvent used 5.1 mL

Solvent used 15 mL


10/28/2008

Flow Cells for RRLC

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

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

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

* Depends on analytical conditions and column dimension

Dimension Sensitivity* Resolution*

13 µl / 10 mm +++ +5 µl / 6 mm ++ ++2 µl / 3 mm + +++


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Choosing the flow cell size


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Peak Width Setting – Response Time – Data Rate and Sensitivity

Don‘t use for > 0.15 sec peak width!

> 0.15 sec

> 0.3 sec

> 0.6 sec

> 1.2 sec

> 3 sec

> 6 sec

> 12 sec

> 24 sec

> 51 sec

Peak Width = Peak Width at 50% Peak Height

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 secNotes: • 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 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 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 slit can be increased to 8 or 16nm.• Set at fastest rate and then decrease data rate until

peak width increases and S/N is optimum


10/28/2008

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

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

Data Rate

Peak Width

Resolution Peak Capacity

80 Hz 0.300 2.25 61

40 Hz 0.329 2.05 56

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

0.6

0.8

1

1.2

1.4

0 20 40 60 80 100Data Rate [Hz]

Peak

Widt

hs /

sec

0

10

20

30

40

50

60

70

Peak

Cap

acity

Peak Width [s]

Peak Capacity

0.2

0.4

0.6

0.8

1

1.2

1.4

0 20 40 60 80 100Data Rate [Hz]

Peak

Widt

hs /

sec

0

0.5

1

1.5

2

2.5Re

solu

tion

Peak Width [s]

Resolution (4,5)

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


10/28/2008

Data Rate and Slit Width Effect on S/N Ratio (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)


10/28/2008

Break 1: Gradient Methods Next

Questions?


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Translating Gradient Methods


10/28/2008

Advantages of Gradient Elution

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

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

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


10/28/2008

0 10 20 30 40

Time (min)

100% B

100% B

100% B

100% B

tg= 40

tg= 20

tg= 10

tg= 5

000995P1.PPT

1/k* ∝ gradient steepness = b

87tg F

ΔΦ Vm Sk* =

ΔΦ = change in volume percent of B solvent (%)S = property of sample compoundF = flow rate (mL/min.)tg = gradient time (min.)Vm = column void volume (mL)

0% B

0% B

0% B

0% B

• S ≈ 4–5 for small molecules

• 10 < S < 1000 for peptides and proteins

This equation governs gradient

retention and selectivity

Gradient Steepness AffectsRetention (k*) and Resolution


10/28/2008

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

A longer gradient time tGA shorter column Vm

A higher flow rate F

A shorter organic range %B

87 tg F

S (Δ%B) Vm

k* =


10/28/2008

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• 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 sameefficiency (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

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

segments and hold times


10/28/2008

To Transfer Gradient Separations,Average retention factor for k* must match, andEffective 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

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

Transferring Gradient Methods to Smaller Diameter Columns and to Different Instruments


10/28/2008

Gradient Separations: Considerations When Translating Existing Gradient methodsIsocratic Separations

Sample load (Vinj, [analyte])

Sample solvent strength

Extracolumn volume

– Flow cell volume – Injection volume– Tubing volume

Injector precision– Can vary with Vinj

– Data Rate– Too fast, too much noise– Too slow, loss of N

Gradient SeparationsSame as Isocratic Separations plus…

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

Capillary 1100 vs. Binary 1100)Gradient Time

– Adjust relative to equation for gradient retention

– Keep k* constant

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

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) Vm

k* =


10/28/2008

DelayVolume

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

• Behaves as isocratic hold at the beginning of gradient.

Gradient Separations – What is Delay Volume?

Also known as

Dwell Volume

Pump w/o mixerw/ mixer

Mixer

Autosampler StandardBypass

Column compartment StandardBypass

1090 1050 1100 Quat. 1100 Bin.

300-5001050-1250

750

V (loop)N/A

4.1 or 8.20

180-480600-900

420

300 + V (inj)6.2

3 or 60

800-1100n/a

n/a

300 + V (inj)6.2

3 or 60

800-1100n/a

n/a

327 + V (inj)8

15 ul0

Min RangeMax Range

304-5041058-1258

189-489906-1206

1203-14061242-1442

Comparison of System Delay Volumes


10/28/2008

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

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

Low delay volume configuration: 120μL (virtual damper)

Damper volume: 80-280μl

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

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

Damper volume: 180μl + 1μl per bar


10/28/2008

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


10/28/2008

Features of the Agilent Method TranslatorBasic mode with certain pre-set parameters:

Enter the parameters of your existing method and the parameters of the desired column you would like to convert to.

1.

2.

3.

4.

5.

6.


10/28/2008

Features of the Agilent Method TranslatorAdvanced mode – all calculation parameters in your hands:

More to enter but much more information returned

3.

4.

1. 2.

[mL] [mL]


10/28/2008

Analysis of impurities of an active pharmaceutical ingredient byconventional HPLC (4.6mmID x 250mm, 5.0µm):

min0 2.5 5 7.5 10 12.5 15 17.5 20

mAU

0

10

20

30

40 OH

HN

CH3CH3

OCH3

Main Compound

OH

HN

CH3CH3

OCH3Impurity A

O CH3

Br

Bromanisole

NCH3CH3

OCH3

Impurity B

CHCHN

33

OCH3

H

Impurity C

NCH3CH3

OH

H

OH

Impurity D

Does it work? - Example


10/28/2008

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


10/28/2008

4.6 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% B30.00 min 5% B

min0 2.5 5 7.5 10 12.5 15 17.5 20

mAU

0

10

20

30

40

Conventional HPLC



min0 1 2 3 4 5 6 7 8

mAU

0

5

10

15

20

25

30

35

Simple Conversion

min0 0.5 1 1.5 2 2.5 3 3.5 4 4.5

mAU

0

5

10

15

20



Speed Optimized

Does it work? YES


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

0.75 cp


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Advanced mode, Simple Conversion


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Advanced mode, Resolution Optimized


10/28/2008

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

• Use 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

exchangers• 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”)• Otherwise, use injector program to reduce delay volume


10/28/2008

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 thermostate eluentFor 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 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

Note: 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.

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 or 0.17mm and ZDV-union).

1100 Binary Pump (G1312A)

1100 WPS(G1367A)

4.6mm ID, 1.8um

Waste

3 μL heat exchanger

1100 TCC(G1316A)

RRHT Column

1100 DAD SL(G1315C)


10/28/2008

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 (WPS?)

TCC

VWD/MWD/DAD

Quat Pump

Degasser

Step 1: 1100/1200

„Bin SL“ System

h-ALS SL

TCC

VWD/MWD/DAD

Bin Pump SL

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

+ Speed+ Resolution

+ MS-Compatibility+ Solvent Saving

+ Compatible with conv. HPLC

50 mm or shorter cols with 3.0 or

4.6 mm IDs

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

IDs

20 Hz max

20 Hz max

80 Hz max


10/28/2008

Actual: 1100

Bin System

> 1 min> 2 min

> 1.5 sec8 - 12,000

3 - 4.6 mm50 mm

0.05 - 5 ml/min80 C

400bar

ALS

ColCom

VWD/MWD/DAD

BinPump

Degasser

Step 1: 1100/1200

„DAD SL“ System

h-ALS SL

ColCom

DAD SL

binPump

Degasser

> 0.2min> 0.4min> 0.3 sec

5 - 22,0003 - 4.6 mm

20 - 100 mm0.05 - 5 ml/min

80 C400 bar

Analysis TimeCycle timesPeak Width

NColumn ID

Column Length Flow rates

TemperaturePressure

Step 2: 1200

Rapid Resolution System

h-ALS SL

ColCom SL

DAD SL

binPumpSL

u-Degasser

> 0.2min> 0.4min> 0.2 sec

5 - 60,0002.1 - 4.6 mm20 - 150mm

0.05-5ml/min100 C

600bar

+ Speed+ Resolution+ Sensitivity

+ Solvent Saving+ MS-Robustness

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

+ Speed+ Data Security & Traceability

+ Compatible with conv. HPLC

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

50 mm or shorter cols with 3.0 or

4.6 mm IDs

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

IDs

80 Hz max

80 Hz max


10/28/2008

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

min0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.25

min5 10 15 20 25

RRHT SB-C182.1 x 50mm, 1.8umTemp: 50°CFlow: 1 mL/minGradient (tG): 2.4 min

Rapid Resolution SB-C183.0 x 150mm, 3.5umTemp: 25°CFlow: 1.0 mL/minGradient (tG) : 18 min

SB-C184.6 x 250mm, 5umTemp: 25°CFlow: 1mL/minGradient (tG): 30 min

0 2 4 6 8 10 12

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

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

A.

B.

C.

Key Parameters• Particle size• Flow Rate• Gradient Time• Column Length• Column ID• Temperature

Rsoptimized


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


10/28/2008

3.0 x 150 mm, 3.5 um, Resolution Optimized


10/28/2008

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

min0 5 10 15 20 25 30 35-20

0

20

40

60

30 35-10

010

20

3040

50

60

mAU

010203040506070

0min5 10 15 20 25 30 35

mAU70

mAUmin0 5 10 15 20 2580

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

5) syringic acid6) sinapinic acid

7) salicylic acid8) caffeic acid

4.6 x 250 mm SB-C18, 5-um

4.6 x 150 mm SB-C18, 3.5-um

3.0 x 100 mm SB-C18, 3.5-um

33 mL solvent used

10.5 mL solvent used

57 mL solvent used25 uL std injection

1.5-mL/min; tg= 38 min

15 uL std injection

1.0-mL/min; tg= 33 min

6 uL injection with INJ Program

0.5-mL/min; tg= 21min

1

23 4

56

78


10/28/2008

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

• 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 has adequate capacity for standard and increased pressure operation across the flow range of routine and optimized methods


10/28/2008

Workshop

Examples

• Isocratic

• Gradient

practical examples of method translation 041608 - agilent · rt last = 14 minutes questions to ask?...

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