Gilson, IncMiddleton, WIwww.gilson.com

GI2

Systematic Look at the Affects of Various Solvents,

Injection Techniques and Sample Amounts on

Preparative HPLC ColumnsGary Scharrer and Joan Stevens, Ph.D.

Slide 1

GI2 Gilson Inc., 2/21/2005

• Preparative Instrumentation• Performance Factors

• Solvent Selection• Mobile Phases• Sample Solubility• Sample Size

• Summary• Questions?

Outline

• Column Loading• Particle Size• Sample Carryover• Injection Techniques

Prep System Configurations

System A

• Gilson 215 Liquid Handler/Fraction Collector, equipped w/175-mm Z-arm, 819 Injection Module with 5.0-mL SS loop and bevel-tip probe (269x1.3x0.8 mm ID)

• Gilson 333/34 HPLC Pump, equipped with H3 pump heads (flow rates up to 200 mL/min.)

• Gilson 155 UV/Vis Dual-wavelength Detector (0.2-mm pathlength, 1.0 AUFS)

819 Injector

Figure 1.

Prep System Configuration

System B

• Gilson 215 Liquid Handler/Fraction Collector, equipped w/175-mm Z-arm, 845Z Injection Module w/5.0-mL SS loop, and bevel-tip probe (269x1.3x0.8mm ID)

845Z Injector

Figure 2.

• Gilson 322 HPLC Pump, equipped with H2 heads (flow rate up to 30 mL/min.)• Gilson 155 UV/Vis Dual-wavelength Detector (0.2-mm pathlength, 1.0 AUFS)

System/Injectors Used In Study

• 819 Injector – System A– Dispenses sample through an injection port to the

sample loop• 845Z Injector – System B

– Aspirates sample directly into the sample loop(Z-Mounted Valve)

• Above systems/injectors were both used interchangeablely in this study.

Solvent Selection• All compounds in mixture must be soluble in the

solvent selected at the desired concentration.– For polar compounds use acetonitrile, methanol or water

individually or in combination.– For more non-polar compounds use DMSO or DMF.

• Select container material which will not attract or adhere material (i.e. glass or polypropylene).– Losses due to this situation are difficult to quantify since the

loss occurs before sample injection.

• Select a solvent which matches the initial mobile phase as closely as possible.

Solvent Effects on Chromatography

2.0 3.0

ACN

ACN/H2O(75:25)

ACN/MeOH/H2O50:25:25

ACN/MeOH(50:50)

DMSODMF

3.0

Graph 1. Choosing a solvent and how it effects the chromatography is important for system design.

2.0 Min. Min. 2.0 3.0Min. 2.0 3.0Min. 2.0 3.0Min. 2.0 3.0Min.

Solvent Selection

Graph 2. The sample should - whenever possible -be dissolved in the mobile phase or a weaker solvent.

If the sample is dissolved in a too strong solvent, significant disturbances can occur in the chromatogram.

H2O / ACN100 : 0

H2O / ACN70 : 30

H2O / ACN50 : 50

H2O / ACN30 : 70

0 4 8 min 0 4 8 min 0 4 8 min 0 4 8 min

Area Decreases Due to Solvent Selection

0

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DMSO DMF ACN ACN/MeOH ACN/H2O

Are

a

38% Difference fromDMF Injection

17%6%

41%

DMSO DMF ACN ACN/H2OACN/MeOH

Are

a

12.5mg Injection

Biphenyl

Graph 3. This chart illustrates the response differences that occur with different solvents or solvent combinations. Assuming the DMF area as 100%, differences from 6% with ACN/MeOH to 41% with ACN/H2O are found.

Response Differences Due to Solvent Selection

0

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DMSO DMF ACN ACN/MeOH ACN/H2O

Ethyl Paraben

DMSO

Are

a

25 mg InjectionDMF ACN ACN/MeOH ACN/H2O

Graph 4. Another example of response differences due to solvent selection, but in this case the acetonitrile was the optimum solvent.

13%2% 1%

13%

Sample Loss Due to Container Materials

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5 10

Amount Injected (mg)

Caffeine Container Study

20

Standard Injection

ACN/MeOH - Glass

ACN/MeOH - Poly

DMSO - Glass

DMSO - Poly

Graph 5. The container material and solvent can affect sampsystem. In this case, caffeine transfer decreases of up to 29% are possi

to 18% are found.

le transfer into the analysis/purification ble. Even with the same solvent,

decreases of up

18 % Difference29 % Difference

Mobile Phases

Weak Mobile PhaseStrong polar or slightly soluble compounds may not elute

Strong Mobile PhaseSingle peaks may co-elute or are poorly resolved

Solvent MixturesOnly specific compound elutions are optimized

Gradient ElutionOptimized chromatogram with enhancedresolution and reduced retention time

retention time

Graph 6.

Sample Solubility• Determine sample polarity.

– Use polarity of sample as a guide for solubility.• Mobile phase compatibility

– Compounds could precipitate in the mobile phase if concentrations are high.

• Container material absorption– The possibility of the sample adhering to the

container surface must be considered.

Reverse Phase Solvent Polarity Chart

Non- Polar n-Propanol 4.0 210Tetrahydrofuran 4.2 215Isopropanol 4.3 210Ethanol 5.2 210Acetone 5.4 330Acetonitrile 6.2 190Acetic Acid 6.2 230Dimethylformamide 6.4 268Methanol 6.6 205Methyl sulfoxide 7.2 268

Polar Water 9.0 200

*Polarity index (P) = Measure of ability of solvent to interact as a proton donor, proton acceptor, or dipole.

Polarity Solvent Polarity Index UV Cutoff(P)* (nm)

Sample LoadingThis parameter is important for obtaining the purest and most concentrated fraction cuts.

To attain optimum results: – Use high concentration of sample – Keep injection size small

Better to have multiple injections into a more concentrated fraction than to manipulate more fractions.

Injection Volume Effects on Peak Area

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250 500 1000 2000 4000

Volume Injected (µL)

Are

a

Caffeine - 5 mg

Ethyl Paraben - 7.25 mg

Biphenyl - 3.25 mg

Graph 7. When the column is flooded with the injection solvent, regardless of load, peak area is suppressed until optimum injection volumes are obtained.

ACN/MeOH used as solvent

Chromatography Effects with Various Volumes

CaffeineEach injection = 5 mg

250 µL Injection

4000 µL Injection

500 µL Injection1000 µL Injection

2000 µL Injection

Graph 8. The effects of keeping the column load constant but varying the injection volumes can be seen as the chromatography degrades as the injection volume increases.

Injection Volume and Sample Loading

Caffeine Ethyl Paraben Biphenyl

Graph 9. As the injection volume and column load increase at the same rate the quality of chromatography degradation is amplified. Samples can still be fractionated, but the faction cuts will be more diluted and purity could also be reduced due to closely eluting impurities.

Injection of Caf/EP/Biphenyl

5 mL = 80/100/60 mg 2 mL = 40/50/30 mg1 mL = 20/25/15 mg

0.5 mL = 10/12.5/7.5 mg0.25 mL = 5/7.25/3.25 mg

5 mL Fraction start5 mL Fraction End

0.25 mL Fraction Start 0.25 mL Fraction End

GI1

Slide 18

GI1 Gilson Inc., 2/21/2005

Particle SizeReducing column packing particle size• Gains resolution• Saves solvent usage• Reduces solvent waste.• Reduces fraction volume.

Particle Size Reduction

5 micron @ 25 mL/min flow

3 micron @ 18 mL/min flowResolved Peaks

Chamomile Oil

Graph 10. Reducing from a 5 micron to 3 micron column particle size gives resolution to closely eluting compounds and also decreases solvent consumption by 28% due to slower flow rates, without loosing samp

Minutes

le turnaround efficiency.

Sample Carryover• Chemical binding • Connections

– Tubing– Injection Loop

• Physical Wear– Rotor seals

• Rinse station contamination– Probe– Injection Port

Chemical Binding

SolventRinse

Waste

Inside ProbeBinding

Flow

SamplingProbe

Outside ProbeBinding

Flow Thru Rinse StationFigure 3.

Sample Carryover

0

5000

10000

15000

25000

30000

Are

20000

250 1000 1500 2000 2500 3000 3500 4000

Rinse Volume (µL)

a

Graph 11. Sample carryover is a direct result of the components correctly between injections. Diff

ability/inability of the system to rinse the sampling erent rinse routines can be configured to meet the

ple shows the difference in changing the rinse sample situation, in this case biphenyl. The above examvolume for rinsing the sample probe.

<0.03

*= Used 845Z injection

%

<0.02%

Biphenyl

system<0.01%

Solvent blank after a 1000 µL injectionof a 15 mg/mL biphenyl solution*

<0.07%

Carryover Elimination Techniques

Waste

Probe

Injection PortSeal

Below seal rinse orStationary Injection Port Rinse

Figure 3.

Injection Valve

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

200 400 1000 2000

% C

arry

ove

Total Rinse Volume (µL)

r

Flow

Biphenyl

Probe movedbelow seal

Graph 13. Percent carryover versus total rinse volumeThis procedure rinses below the seal. Thewaste is pushed through the injection valveto waste.

0.00

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

200 400 1000 2000Total Rinse Volume (µL)

% C

arry

over

Carryover Elimination Techniques

Probe

Injection PortSeal

Above seal rinse

Waste

This procedure rinses above the seal. Thewaste is pushed out the top of the injectionport to the waste channel of the injection port.This procedure is combined with the standardbelow seal rinse.

Biphenyl

Flow

OverflowProbe moved above the seal

Graph 12. Percent carryover versus total rinse volume which Includes above and below rinses.

Figure 4.

Rinse Techniques

Graph 14. Carryover can be decreased substantially if the below seal rinse is combined with anabove seal rinse. The above seal rinse is a combination of one below seal rinse and three aboveseal rinses for the same total rinse volume as the single below seal rinse.

0.16

0.04

0.06

0.08

0.1

0.12

0.14

% C

arry

over

Below Seal Rinse CarryoverAbove Seal Rinse Carryover

0

0.02

200 400Tota

1000 2000l Rinse Volume (µL)

Injection Techniques• Standard Injection

– Sample is aspirated into tubing, followed by dispensing into the injector sample loop (819 Injector).

• Direct Loop Injection– Sample is aspirated directly through the sample probe into

the injection valve (845Z Injector).• Sandwich Injection

– Sample is aspirated into the injection path between plugs of strong solvent, typically DMSO, to protect the sample from precipitation or broadening (Either 819 or 845Z Injectors).

Sandwich Injection using Various Solvents and Varying DMSO Plug Size

0

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0 50 100 150

ACN/MeOH

ACN/MeOH/H2O

DMSO

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0 50 100 150

ACN/ MeOH

ACN/ MeOH/ H2O

DMSO

Injection of various solvents and plug sizes illustrate the performance differences in solvent selection. The response increases with increasing plug size but as the plug size increase the chromatography gets poorer.

0

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0 50 100 150

ACN/MeOHACN/MeOH/H2ODMSO

Are

a

DMSO Plug Size (µL)

Are

a

DMSO Plug Size (µL) DMSO Plug Size (µL)

Are

a

Caffeine

Biphenyl

Ethyl Paraben

Graph 15

Graph 16

Graph 17

Summary• Preparative HPLC Goals:

– Good peak shape– Concentrated fractions– Pure fractions

• Experimental Results Conclusion– Using the best suited solvent, inject the lowest amount of the

most concentrated sample solution possible.• Preparative HPLC Reminders

– Use injection techniques that will allow for the sample to be injected onto the system without interference with the mobile phase prior to reaching the column

– Optimize the solvent for solubility and chromatography.– Select a container material that it compatible with your

solvent/sample system.

Questions?

69 of 80 human therapeutic proteins on the market in 2003 sold at prices over $10,000/gram.

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