Troubleshooting and Tips & Tricks (G1) - Waters · PDF fileTroubleshooting and Tips & Tricks...

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Troubleshooting and Tips &

Tricks (G1) for the Acquity Family

Tony Wiklund Nordic Application Chemist


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


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


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


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.



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)



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:


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


Outline







— Split Peaks

— Distorted Peaks


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


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)


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


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


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.


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


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


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


Outline







— Split Peaks

— Distorted Peaks


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


Outline







— Split Peaks

— Distorted Peaks


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


Pump Pressure plot Check Valve Problem

Primary A

Accumulator A

Accumulator B

Primary B

Inconsistent height of pressure trace


Pump Pressure plot Check Valve Problem

Inconsistent pressure in the accumulator heads


Outline






Troubleshooting — Carryover/Contamination

— Split Peaks

— Distorted Peaks


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


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


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.


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)


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)


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


Loop Contamination

Picture of valve and fitting

Only thing to do, is replace the loop.

Injection Valve

flow

Loop

Fitting

Hiding sample residue


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.


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


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?


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


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


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!!!




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.


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


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


Additional Resources for Successful UPLC

www.waters.com/myuplc

Register with short name and serial number of your binary solvent manager


Additional Resources for Successful UPLC


Community Discussion section


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


Date post:	25-Mar-2018
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