GI2 Systematic Look at the Affects of Various Solvents, Injection Techniques and ...€¦ · ·...
Transcript of GI2 Systematic Look at the Affects of Various Solvents, Injection Techniques and ...€¦ · ·...
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.
• 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
5000000
10000000
15000000
20000000
25000000
30000000
35000000
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
5000000
10000000
15000000
20000000
25000000
30000000
35000000
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
0
5000000
10000000
15000000
20000000
25000000
30000000
35000000
40000000
45000000
Are
a
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
0
2000000
4000000
60000008000000
10000000
12000000
14000000
16000000
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
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
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
0 50 100 150
ACN/MeOH
ACN/MeOH/H2O
DMSO
0
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
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
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
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.