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©2013 Waters Corporation 1
Troubleshooting and Tips &
Tricks (G1) for the Acquity Family
Tony Wiklund Nordic Application Chemist
©2013 Waters Corporation 2
Outline
General Recommendations — Care and Use
— Wash Solvent Selection and Optimization
— Using 1.0 mm columns
Preventative and Diagnostic Tools — Understanding Pressure Traces
— System and Column Equilibrations
Troubleshooting — Contamination
— Split Peaks
— Distorted Peaks
©2013 Waters Corporation 3
Recommended Guidelines of Use for the Solvent Manager
Use quality solvents, buffers and additives.
– J.T. Baker® LC/MS Grade, Burdick & Jackson: B&J Brand®, Fisher Optima® LC/MS Grade
Use Milli-Q water or equivalent
Filter Buffers
– 0.2 m filter membrane
– Keep concentrated stock solutions to prepare from
– Do not top off buffers. (Can promote microbial growth)
Do not block the degasser vent line
– trim if necessary (Do not put the line in the waste bottle)
Keep all 4 solvent lines primed
– Flush buffers out of system with water after use
– use 10-20% organic in water for storage
©2013 Waters Corporation 4
Recommended Guidelines of Use for the Solvent Manager
Keep seal wash primed.
– 90-95% water
Re-prime solvent lines before starting
– Sys Prep or Start-up
Use 100 µL mixer for TFA/ACN gradients at low wavelengths (for BSM only)
Run gradients starting with at least some organic 0.1%
– Provides more consistent and predictable gradient formation than from starting at 0%
Change aqueous mobile phases often – every 24 – 48 hr
– Remember: UPLC® flow rates are much lower, thus mobile phases are consumed at a lower rate and last longer
– Do not top of mobile phase but prepare new
©2013 Waters Corporation 5
Controlling Bacterial Growth in Aqueous Mobile Phases
ACQUITY UPLC® columns are packed with 1.7 µm particles and thus require 0.2 µm end frits
ACQUITY UPLC® columns are very fine and efficient filters
UPLC® column frits can plug due to bacterial growth in highly aqueous mobile phases
Bacteria is the most common cause of UPLC® column plugging and can easily be avoided by filtering through a 0.22 µm filter
pH 7 phosphate buffer is VERY susceptible to bacterial growth
Electron micrograph of Rod-shaped bacteria on
the bed side of the inlet frit removed from column.
©2013 Waters Corporation 6
Controlling Bacterial Growth in Aqueous Mobile Phases
Best way to discourage bacterial growth is to AVOID 100%
aqueous mobile phases where possible
Add small amount of organic modifier (e.g., ACN, MeOH) to
Mobile Phase A
– Adjust gradient profile accordingly
– Benefits include
o Reduction in bacterial growth
o Improved mobile phase mixing
Straightforward gradient profile adjustments (see Example)
©2013 Waters Corporation 7
Controlling Bacterial Growth in Aqueous Mobile Phases
Time %A %B %ACN
0.00 94.7% 5.3% 10%
2.00 94.7% 5.3% 10%
15.00 21.1% 78.9% 80%
20.00 5.3% 94.7% 95%
20.01 94.7% 5.3% 10%
30.00 94.7% 5.3% 10%
A = 95% Aqueous, 5% Organic
A = 95% 0.1% HCOOH, 5% Organic
B = Organic Modifier (e.g., ACN, MeOH, etc)
Time %A %B %ACN
0.00 90% 10% 10%
2.00 90% 10% 10%
15.00 20% 80% 80%
20.00 5% 95% 95%
20.01 90% 10% 10%
30.00 90% 10% 10%
A = 100% Aqueous
A = 0.1% HCOOH
B = Organic Modifier (e.g., ACN, MeOH, etc)
Original Gradient Method 100% Aqueous Mobile Phase A
New Gradient Method 95% Aqueous Mobile Phase A
100*A Phase Mobile in Aqueous %
Step inOrganic % Desired100%A
Calculations for each gradient step:
©2013 Waters Corporation 8
General Recommendations for Sample Manager (not applicable for FTN Sample Manager)
Use Default Conditions – 10 µL loop and PLUNO (partial loop uses needle overfill) injection will give good peak shape, good recovery, good linearity for range of injection volumes appropriate to 2.1 x 50 and 2.1 x 100 mm ACQUITY columns
Stay within the recommended limits for each injection mode and loop size
Use Load Ahead when cycle time is an issue
Use Full Loop when best precision and accuracy is critical
Use Partial Loop (pressure assisted) when sample consumption is critical
Characterize needle and loop volumes when changing weak wash solvents as well as loops and needles
Use steel tip needles for capmat covers on well plates
Always use the column stabilizer tube even if temperature is not used
©2013 Waters Corporation 9
Outline
General Recommendations — Care and Use
— Wash Solvent Selection and Optimization
— Using 1.0 mm columns
Preventative and Diagnostic Tools — Understanding Pressure Traces
— System and Column Equilibrations
Troubleshooting — Contamination
— Split Peaks
— Distorted Peaks
©2013 Waters Corporation 10
Washing Process
FTN – one Step
– Strong solvent (external only)
FL - Two steps
– Strong solvent
o Sample dilution and removal
– Weak solvent
o Strong solvent removal
o Avoid any risk of diffusion • Maintain chromatography profile
• Maintain good peak shape
©2013 Waters Corporation 11
Strong Wash Solvent
Function performed in the wash station
Flushes internal and external portion of the needle to prevent carryover
Typically stronger than sample and mobile phase to dissolve sample residue
Strong solvent should be no stronger than the concentration needed to reduce carryover to an acceptable level
Prime using the prime syringe function
Choose based on the chemistry application
100% organic solvent is acceptable
– Except THF
Default value is 200 µL (FL)
©2013 Waters Corporation 12
Weak Wash Solvent
Purges needle and syringe fluid path
Must be compatible with sample solvent
For best results, weak wash solvent should be equivalent
to the following (excluding buffers):
– mobile phase composition (for isocratic separations)
– initial gradient condition (for gradient separations)
– If you dilute the samples, match the weak wash solvent to
the sample diluent
Degassed for good hydraulic properties
Default value is 600 µL
©2013 Waters Corporation 13
Wash Solvent Considerations
As a general principle, strong and weak solvents should include the same organic species
– This may not always be practicable, especially in the case of “sticky” samples. You may, however use a 100% organic strong wash solvent
Do not use salt buffers in wash solvents
Wash volume ratio (weak to strong)
– Should be about 3:1, weak wash to strong
– Sufficient to ensure the weak wash flushes the strong from the needle and sample loop
For more details on solvents, see the section titled “Selecting weak wash and strong wash solvents” in the ACQUITY Operators Guide
©2013 Waters Corporation 14
Priming Recommendations
To change solvents on the BSM prime each line for 5 minutes.
To refresh solvents on the BSM prime the lines to be used for 1
minute.
To change solvents on the SM prime for 7 cycles (wash syringes
and sample syringe).
To refresh solvents on SM prime for 1 cycle (wash syringes and
sample syringe).
Always flush new columns with the detector flow cell
disconnected.
©2013 Waters Corporation 17
Full Loop Injection- Aqueous vs Organic Sample Diluent
AU
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
2.00
2.20
Minutes
0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60 0.65 0.70 0.75 0.80 0.85 0.90 0.95 1.00
Organic
Aqueous
©2013 Waters Corporation 18
Sample Pretreatment
Should you filter your
sample?
YES if…
– Particulates are present
– Doing it today
– Temperature affects
precipitation of sample
– Formulation present
Otherwise, NO.
Pre-column or Pre-filter?
Use a pre-filter when pressure
increases over time
— Filters can be replaced
When to use a pre-column guard?
— Definitely if you currently use
guards
— Sample matrices that might
deposit on head of column o Natural products
o Biological samples
©2013 Waters Corporation 19
Filtration and Buffers
Using Salt Based Buffers – Filter (0.2 µm filter membrane)
– Keep concentrated stock solutions to prepare from
– Do not top off buffers. (Can promote microbial growth)
– 48 hour changing
Filters – We generally recommend
o hydrophilic Teflon filters (PTFE)
o GHP filters for aqueous solutions
– Filtering organic and aqueous solutions through Nylon filters has exhibited leaching contaminants producing extra peaks
Filtering with SPE (Oasis®) + membrane filter works well with situations of cleaning poor water quality and questionable reagent quality
©2013 Waters Corporation 20
Outline
General Recommendations — Care and Use
— Wash Solvent Selection and Optimization
— Using 1.0 mm columns
Preventative and Diagnostic Tools — Understanding Pressure Traces
— System and Column Equilibrations
Troubleshooting — Contamination
— Split Peaks
— Distorted Peaks
©2013 Waters Corporation 21
1.0 mm ID Column Applications
Points to Consider
System Volume
– Lower Flow Rates = Longer Analysis Time
Proper Scaling
– Flow Rate
– Injection Volumes
o Proper loop size
– Injection Mass
Data Rates and Filtering Constants
– MS and UV
Gradient Elution
– Focusing Effect
Post Column Volumes
– Decrease in column ID + improper adjustment in post column
volumes = Broad Peaks
©2013 Waters Corporation 22
Outline
General Recommendations — Care and Use
— Wash Solvent Selection and Optimization
— Using 1.0 mm columns
Preventative and Diagnostic Tools — Understanding Pressure Traces
— System and Column Equilibrations
Troubleshooting — Contamination
— Split Peaks
— Distorted Peaks
©2013 Waters Corporation 23
System Equilibrated
Typical pressure trace indicating that the system is equilibrated and ready for injections
Note the pressure ripple in psi for the past minute = 18. This should normally be < 30 psi
indicating that the solvent manager is operating well.
Same distance between pump strokes
Same height of pump stroke
©2013 Waters Corporation 24
Pump Pressure plot Check Valve Problem
Primary A
Accumulator A
Accumulator B
Primary B
Inconsistent height of pressure trace
©2013 Waters Corporation 25
Pump Pressure plot Check Valve Problem
Inconsistent pressure in the accumulator heads
©2013 Waters Corporation 26
Outline
General Recommendations — Care and Use
— Wash Solvent Selection and Optimization
— Using 1.0 mm columns
Preventative and Diagnostic Tools — Understanding Pressure Traces
— System and Column Equilibrations
Troubleshooting — Carryover/Contamination
— Split Peaks
— Distorted Peaks
©2013 Waters Corporation 27
Carryover
Reasons for carryover:
– Sample is not totally solubilized in sample diluent
– Sample sticks to one part of the system
How to avoid Carryover
– Use the least concentrated sample possible
– Check solubility in the sample diluent
– Use other needles – Steel and PEEKSil,
Determine whether the issue is carryover or contamination?
– The system if contaminated it must be cleaned:
o Replace wash block needle seal o-ring
o Replacing bubble detector
o Replace loop and in BAD cases entire injection valve
©2013 Waters Corporation 28
Troubleshooting carryover
Run blanks
– Often an acid blank wash (Formic acid) can assist for stubborn
compounds
Does the injection mode affect the carryover?
– Generally Full Loop>Pressure Assist>PLUNO when conditions are
not optimized
Once you have the data call Waters to discuss
The following slides have some general guidelines that can be
effective
©2013 Waters Corporation 29
Carryover – new application
Run a simple test with a well behaved or well known sample
– For Waters this is usually caffeine
– Do not rule out matrix effects – do the samples and standards
behave in the same way?
If the well known assay is O.K. – “Wash Issue”
– Adjust wash solvents, composition and time
– For MS application, strong wash with as high as 10% Formic Acid
can be tried
If the washes do not help, change the material of the sample
needle.
– Generally, the PEEK needle absorbs hydrophobic, the steel absorbs
hydrophilic and the PEEK/sil is in the middle.
©2013 Waters Corporation 30
SM Performance – Carryover
10 mg/mL Human Insulin
Carryover – 0.0036%
Wash Conditions: 200 µL Strong (6:3:1 / 0.1% Phosphoric in Water:ACN:IPA) 600 µL Weak (0.01M HCl)
©2013 Waters Corporation 31
SM Performance – Carryover
Carryover – 0.0036%
Wash Conditions: 200 µL Strong (6:3:1 / 0.1% phosphoric in Water:ACN:IPA) 600 µL Weak (0.01M HCl)
©2013 Waters Corporation 32
Loop Contamination
Possible Loop Carryover Suspicion
If a loop was removed and then placed back on the valve
in the reverse position that it was installed,
chromatography performance might not be different,
however there might be a small void for contamination to
hide.
Make full loop injection and a full loop injection blank
1. OBSERVE for carryover
2. If no carryover, then its not the loop and refer back to the
NEEDLE SEAL
3. If carryover, then INSTALL NEW LOOP
©2013 Waters Corporation 33
Loop Contamination
Picture of valve and fitting
Only thing to do, is replace the loop.
Injection Valve
flow
Loop
Fitting
Hiding sample residue
©2013 Waters Corporation 34
If Changing Loops…
Modular design (removable “Pod”)
Use the design to your advantage. Remove the pod
anytime you need to install a new sample loop that is not
already pre-set.
©2013 Waters Corporation 35
Split Peaks
Why does my column yield split/distorted peaks?
– As in traditional liquid chromatography, several possible factors could be contributing to a split or distorted peak:
Poor tubing connections
– can result in voids forming, giving distorted peaks.
Are you using a column other than an ACQUITY UPLC® column?
– Inlet depths are different causing voids
What are you using as your weak needle wash?
– when choosing the weak needle wash use equivalent composition as the initial mobile phase composition
Blocked in-line filter
©2013 Waters Corporation 36
Split Peaks
What are the specific wash volumes, both strong and weak, set in the instrument method?
– If any strong wash is leftover, peak distortion can occur
What is the sample diluent?
– It might need to be similar to the mobile phase
What is the injection volume?
Is the sample overloaded?
Are you using a mobile phase pre-heater?
– Thermal mismatch of mobile phase and column
Have you allowed for proper column equilibration?
©2013 Waters Corporation 37
Effect of Contaminated/blocked In-line Filter on Peak Shape/Efficiency
Contaminated Frit P = 6400 psi* N = 7575
Debris from seal shedding, particulates from buffer, particulates from sample
Sample “Band”
Column
New Frit
P = 5100 psi* N = 9349
©2013 Waters Corporation 38
Original run showed only the two main peaks.
Not necessarily split peaks nor peaks with shoulders. Almost exhibits more of a co-elution of peaks??
Effect of a Poor Fitting/Poorly Cut Peak Tubing
©2013 Waters Corporation 39
Suspect Issue: Observed kink in needle or poor fittings
Remedy: Check all fittings and replace kinked needle with new needle
Note: Never try to bend needle to original state.
1. Problem not fixed with new needle
2. Fittings checked and tested on column position 1
3. Switched column to position 2 thru 4
4. Problem still observed during each change!!!
Effect of a Poor Fitting/Poorly Cut Peak Tubing
©2013 Waters Corporation 40
Effect of a Poor Fitting/Poorly Cut Peak Tubing
Injection: Column manager left valve in-line
Remedy: Since earlier tests on each column position resulted in the split peaks, proceeded to check fitting and tubing on outlet of valve to the detector
Problem resolved.
©2013 Waters Corporation 41
Over tightened Finger tight Fittings
Deformed Ferrule
Normal Ferrule
Reusable Fitting Installed Correctly
au
0.000
0.010
0.020
0.030
0.040
Overtightened Reusable Fitting
au
0.000
0.005
0.010
0.015
0.020
0.025
Minutes
1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40
©2013 Waters Corporation 42
In-line filter installation problem
N 5sigma = 5025 Tailing factor = 1.31
N 5sigma = 6959 Tailing factor = 1.17
Wrong position of ferrule
Correct position of ferrule
©2013 Waters Corporation 43
Additional Resources for Successful UPLC
www.waters.com/myuplc
Register with short name and serial number of your binary solvent manager
©2013 Waters Corporation 46
Conclusions
Good preparation and situational awareness of general lab
practices and LC pre-work increases efficiency.
Troubleshooting
– Adhere to the proper column volumes for column and system
equilibration.
– Use the Console to your advantage. It’s whole purpose is to be
a diagnostic tool. Observe for more trends through your
experiences
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