ACQUITY UPLC Columns - Waters Corporation · PDF fileACQUITY UPLC® Columns Techniques for...

©2013 Waters Corporation 1

ACQUITY UPLC® Columns

Techniques for Maximizing Performance and Productivity

Esa Lehtorinne

[email protected]

Tel: +358-9-5659 6288 Fax: +358-9-5659 6282

Waters Finland Kutomotie 16 00380 Helsinki


Agenda

Introduction

Techniques for Maximizing UPLC® Column Lifetimes

Conclusion

Appendix


Introduction

Extremely LONG UPLC column lifetimes are being

achieved WW

Extremely SHORT UPLC column lifetimes can also

occur

How and why is there a difference?

– Method & sample dependent

How can we MAXIMIZE UPLC column lifetimes?


Introduction

UPLC® Technology MAY require small methodology

changes (but the benefits of UPLC Technology are

HUGE)

HPLC IS more forgiving than UPLC® technology

Previous HPLC method success does NOT guarantee

future UPLC method success


Introduction

Good news: – Most customers (~ 99 %) report NO issues with UPLC®

column technology in their applications

– Waters goal: 0 % UPLC® column problems

UPLC® columns will tell you quite quickly that you are doing something that needs improvement

It’s usually only ONE thing that requires attention BUT……

……The challenge: Determine that ONE thing


Maximizing Performance Overview

UPLC® columns act as very fine filters

A small procedural change may need to be made to

realize the full potential of UPLC® technology

–What this change is needs to be methodically

determined

OBJECTIVE: Avoid “guessing”

MOST COMMON REPORTED PROBLEM???

PLUGGING

How do we determine what is plugging the UPLC® columns?


Agenda

Introduction

Techniques for Maximizing UPLC® Column

Lifetimes

Conclusion

Appendix


Maximizing Performance Overview – Questions

Has someone from Waters been contacted?

Is an In-Line filter being used? VanGuard™ Pre-Column?

Do you filter your mobile phases? Samples?

–Membrane type? Porosity?

Did the method ever work on an HPLC column?

Have you changed the Inlet frit? Outlet frit?

– If so, where was the LOCATION of the plugging?

Plugging does not usually occur by itself – LOSS of peak shape, efficiency and/or resolution often occur simultaneously


Maximizing Performance Procedure Overview

Objective: Determine Method Compatibility

Objective: Check System & Solvents

Objective: Check Sample Preparation Procedure

Objective: Check Samples (Matrix)


Maximizing Performance Method Compatibility

1. Did the separation ever work on an HPLC system with HPLC columns? This will confirm sample/mobile phase/solvent compatibilities (e.g., PO4

3- & ACN).

• If this method and/or samples have never been run on an HPLC system, is it possible to run the method on an HPLC system with an HPLC column? Or UPLC® system with similar HPLC column dimensions?

• Check mobile phase miscibility by mixing mobile phase A with mobile phase B at the maximum %B conditions (or 95 % B, whichever is lower) {precipitation of buffer in high organic}

Objective: Determine Method Compatibility


Maximizing Performance

System & Solvents Compatibility

2. If you simply inject standards or blanks prepared in

mobile phase on a NEW COLUMN (or a column

with replaced frit(s)), what is the column

lifetime?

Don't skip this step!

• This tests the system, lab practices, solvent

quality and/or bacteria as the source of plugging





• If injecting standards or blanks results in column

plugging, check:

• Mobile phase quality for

1. Bacterial growth (most common)

2. Particulates

3. Precipitation

4. Leaching

• Condition of UPLC® system – contact Waters

Service Engineer to ensure system is

up-to-date (e.g., sinkers, bottles etc.)





Scanning Electron

Micrograph (SEM)

Inlet Side of Inlet Frit

Energy Dispersive Spectrometry (EDS)

Spectra

100 % aqueous mobile phases and water are susceptible

15 minutes is all it takes

when conditions are right

Fix: Prepare aqueous mobile phase daily (or add organic modifier)

Fix: During shutdown or

over a long period of time (such as a weekend), flush the system completely with water, followed by ≥ 10 % of an appropriate organic solvent (such as ACN or MeOH)

Primarily Carbon

Bacterial Growth


Bacterial Contamination Is NOT Unique to UPLC® Technology

Photo of Agilent 1200 sticker that is affixed to the HPLC system.




Bacterial growth is a very common source of UPLC® column plugging

Are you using a 100 % aqueous mobile phase? Using the system to mix isocratic mobile phase solvents?

– Prepare 100 % aqueous mobile phase daily

o Note: In-Line Filter may not get catch ALL bacteria

– Better solution:

o Gradient: add 5 % to 10 % organic modifier to aqueous mobile phase container and adjust gradient profile accordingly

o Isocratic: Pre-mix mobile phase solvents




Severe bacterial contamination?

–Remove and replace sinkers

o Bacteria are difficult to flush from sinkers

(and “Mission Impossible” from columns)

–Remove column, direct flow to waste

and flush system

–Flushing procedures described in

“Controlling Contamination in

UltraPerformance LC®/MS and

HPLC/MS Systems” (PN 715001307)

o e.g., ACN:MeOH:IPA:0.2 %

HCOOH in H2O (1:1:1:1)




Not Bacteria?

Important questions to ask at this point:

– Did these problems SUDDENLY occur? If so, what is different

now?

o Different UPLC method? Column? System change?

o Water system maintenance?

o Sample quality changed (e.g., NEW project/customer)

o Organic modifier batch/source?

– If always been this way, can method be run on different

system?




In rare cases, plugging can come from dissolved Molybdenum (Mo) leached from Hastelloy sinkers (marked: HX or XH)

Plugging due to Mo is VERY rare, usually associated with OUTLET frits being plugged

– Acidic mobile phases (or passivating with HX filter/sinker in place) prior to using neutral/basic mobile phases is the usual pattern

Solution: Remove HX filter/sinkers, flush system and replace with Critical Clean Stainless Steel filter/sinker (or no sinkers)

HX sinkers have been discontinued but some may still be in use in older systems

Filter, Solvent Bottle, Hastelloy, Pkg 7, 700002824




Root Cause: Hastelloy sinkers How:

• 16 % Mo in Hastelloy C-276 • In acidic and oxidizing solutions, Mo is leached from the HX sinkers • When switching mobile phase from acidic to neutral (or basic), dissolved molybdate may precipitate at a nucleation site such as column outlet frit

Interim Fix: Titanium sinkers Fix: “Critical Clean” SS sinkers

Energy Dispersive

Spectrometry (EDS)

Spectra

Filter, Solvent Bottle, Hastelloy, Pkg 7, 700002824

Solvent Filter, Titanium, Pkg 7, 700003530

Primarily Iron Chromium

Molybdenum

Scanning Electron

Micrograph (SEM)

BED Side of OUTLET Frit




Other sources of plugging

– Mobile phase filtration membrane dissolving

o Are you filtering your organic modifier prior to use? If so,

are you CERTAIN that it is compatible with the organic

modifier (e.g., polysulfone membranes are NOT compatible

with ACN)?

o SOLUTION: Avoid organic modifier filtration;

purchase pre-filtered (0.2 µm) solvents

• Recommended solvent brands (or their equivalents):

• J.T.Baker®: LC/MS Grade

• Burdick & Jackson: B&J Brand®

• Fisher: Optima® LC/MS Grade




Solvent Compatibility Chart, Pall Life Sciences




Customer was filtering ACN through POLYSULFONE membrane

and complained of EXTREMELY short UPLC column lifetimes

Inlet Frit Plugged With

Dissolved Polysulfone Membrane Material



System & Solvents Compatibility – Filter Membrane Pore Size

Cavity or space between closely packed particles

Cavity is ca. 1/8 x particle size:

5 µm => 0,6 µm 2,5 µm => 0,3 µm 1,7 µm => 0,2 µm

Remember also pore size of

in-line filters and other frits in LC

system

0,45 µm is sufficient for

conventional HPLC but

0,2 µm is MUST for UPLC




Other sources of plugging

– Mobile phase container leaching silicates at high pH

o Are you working at high pH (i.e., pH 10)?

o Are your columns plugging (OUTLET frit) after 100 – 200

injections?

o PROBLEM: High pH aqueous mobile phase is DISSOLVING

the glass of the mobile phase bottle

o SOLUTION: Use Borosilicate 3.3 glass bottles only

• Check with container manufacturer to confirm

high pH compatibility




Column 1: BEH UPLC column ALONE last only 100 to 200

injections under high pH (pH 10) conditions

Column 2: VanGuard™ Pre-Column connected to second UPLC

column & it protected the UPLC column but plugged instead of

column (indicating upstream source)

Bed Side of Outlet Frit

Plugged With Dissolved

Borosilicates from Mobile

Phase Container

Changing Bottle Brand

Eliminated the Problem




2. If you simply inject standards or blanks prepared in

mobile phase, on a NEW COLUMN, what is the

column lifetime?

Don't skip this step!

• This tests the system; lab practices;

solvent quality; and/or bacteria as

the source of plugging

• If no pressure increase is observed, go

to next step




Sample Preparation Compatibility

3. If long column lifetimes are observed in study #2, prepare

standards in water (or suitable solvent) and subject these

standards to the sample prep procedure. Inject these

prepared standards. What is the column lifetime?

• Now we introduce the sample prep procedure as a

variable since we have previously ruled out the system,

solvents and lab practices.

• If THIS results in column plugging, consider a different

sample preparation procedure and/or device

[filter rating/membrane type (brand), SPE (brand)]

• If no pressure increase is observed, go

to next step

Objective: Check Sample Preparation Procedure


Maximizing Performance Sample Matrix

4. All that is left now is the sample matrix. The

system, solvents, lab practices and sample

preparation device/process have been eliminated. If

you inject samples onto a clean, bacteria-free

UPLC® system/column and plugging or short

column lifetimes are now encountered, the sample

quality & preparation need to be investigated.



Maximizing Performance Sample Matrix

• By process of elimination, the sample matrix MUST be source of UPLC® column failure • What are the samples & matrix? Have the

samples/quality changed recently? Preparation procedure? Filtration details? Centrifugation? How long? RPMs?

• Are VanGuard™ Pre-Columns being used? In-Line Filter unit? Do THEY plug instead of UPLC column?

• Consult with your Waters Chemistry Business Unit (CBU) Specialist to determine sample preparation options and solutions (e.g., Oasis®, Ostro™, Sirocco™ etc.) which will prolong UPLC® column lifetime and provide more reliable results



Maximizing Performance Sample Matrix: VanGuard™ Pre-columns

VanGuard™ Pre-Columns

– Culmination of over two

years of research &

development

– 2.1 mm ID x 5 mm Length

configuration

– Direct-connect design

– Entire unit is disposable –

no cartridge or holder

– Same frit porosity, particle

size and chemistries as

ACQUITY UPLC® columns


Agenda

Introduction


Conclusion

Appendix


Maximizing Performance Conclusion

> 99 % of UPLC® columns are used without issue

To realize HUGE potential of UPLC® Technology,

small procedural changes may be required

–Analogous to past technology changes required to

utilize 3 µm particle or microbore LC columns

–Previous HPLC method success does NOT guarantee

future UPLC method success

Most customers realize thousands of injections per

UPLC® column right out of the box

– Maximizing UPLC® column lifetimes can be realized in an

efficient, systematic and logical way – Waters can help in this!


Agenda

Introduction


Conclusion

Appendix


Prequel to Maximizing Performance Procedure

1. Determine of the location of the plugging

(then determine the source of the plugging via

Maximizing Performance Procedure).

Thought Process First - Location: Where is the column plugged? Second - Source: What is plugging the column?



1. Determine of the location of the plugging

(then determine the source of the plugging via

Maximizing Performance Procedure).

Thought Process First - Location: Where is the column plugged? Second - Source: What is plugging the column?

FIRST: INLET OUTLET

SECOND: WHAT?



2. Change the Inlet frit in order to determine the

location of the plugging*.

• Columns are acting like fine filters. Change

the Inlet frit.

• If this does not eliminate column plugging,

change the Outlet frit via the same procedure.

The remaining steps are (fairly) easy.

(*) – ACQUITY UPLC® Columns Inlet Frit

Replacement Procedure


Inlet Frit Replacement Introduction

ACQUITY UPLC® Columns Frit Replacement Procedure

– Simple, yet valuable procedure designed to find LOCATION

(not source) of column plugging

– Demonstrates that the PACKED bed is not the problem

– Simple and takes only a couple of minutes

– No special items/tools needed

– CUSTOMER & Waters representative should perform this

operation together in CUSTOMER laboratory


Inlet Frit Replacement Definition

ACQUITY UPLC® Columns Frit Replacement Procedure

–An investigative procedure: NOT a means to

prolong column lifetime

–An investigative procedure: SHOULD NOT be

required on a regular basis (something is wrong if

this NEEDS to be done often in order to alleviate

column pressure)


Inlet Frit Replacement Before You Start

Safety First

–Safety glasses, gloves and lab coat are recommended prior to beginning this procedure

–Consider performing procedure in laboratory fume hood

–Confirm with UPLC® column user the types and quality of samples injected onto column

–Do not open the column if you feel uncomfortable doing so because of type of samples injected (e.g., radioactivity, infectious diseases etc.)


Inlet Frit Replacement Items Needed

3/16” wrench PN 600000142

5/16” Wrench

PN 605000115

UPLC® Column

Replacement frits and/or endnuts*

*Note: ACQUITY UPLC® In-Line Filters are

ACTUAL column frits

(*) - See next slide


ACQUITY UPLC® Column Replacement Parts


Inlet Frit Replacement Overview

1. Remove Inlet nut (end fitting). The frit will be inside nut.

2. Remove frit from nut (end fitting) {if necessary, turn nut upside down and tap on flat surface to dislodge frit from inside the inlet nut}.

3. Inspect the column sealing surface for excess material that may have escaped as the nut is removed.

4. Wipe the sealing surface of the column, do not disturb the exposed packed bed (and observe if bed is discolored or voided).


Inlet Frit Replacement Overview (cont.)

5) Place new frit inside nut.

6) Hand-tighten the assembled frit/nut onto the

column.

7) Torque inlet nut to column at approximately

1/2 turn past hand tightened.


Inlet Frit Replacement Remove Inlet Nut

1. Lay column flat on work surface

2. Place 5/16” wrench on column end nut flats

3. Place 3/16” wrench on column tube flats

4. Turn ¼ turn counter clockwise until nut is loose

5. Do not remove inlet nut from column yet

5/16” wrench 3/16”

wrench


Inlet Frit Replacement Remove Inlet Nut

Loosened Inlet Nut, Column in Vertical

position

1. Remove wrenches

2. Pick-up column so that loosened inlet frit is now on top and orient column so that it is perpendicular to work surface

3. Unscrew inlet frit, taking care that no packing material falls out of column


Inlet Frit Replacement Remove Frit From Inlet Nut

1. Remove inlet frit

2. If inlet frit is stuck inside nut, tap inlet nut on work surface until frit becomes dislodged

Inlet Frit Inside Inlet

Nut

Note: It is not unusual

that a small amount of packing material adheres to

the inlet frit


Inlet Frit Replacement Inspect Sealing Surface

Used Inlet Frit

Inlet Nut

Column with Inlet Nut Removed 1. With inlet nut removed, inspect

the sealing surface

2. Wipe the steel sealing surface of the column

3. Do not disturb the exposed packed bed

4. Observe if bed is discolored or voided


Inlet Frit Replacement New Inlet Frit Installation

1. Place new inlet frit inside inlet nut

Inlet Nut with New Inlet Frit

Used Inlet Frit


Inlet Frit Replacement Hand Tighten Inlet Nut/Frit & Column

1) Pick up column

(keep vertical

until nut/frit

assembly is

hand tight)

2) Place assembled

inlet nut/frit onto

UPLC® column

3) Hand tighten

Assembled Inlet Frit/Nut


Inlet Frit Replacement Tighten Inlet Nut/Frit & Column

1. Place 5/16” wrench on column end nut flats

2. Place 3/16” wrench on column tube flats

3. Tighten assembled inlet nut/frit and column approximately ½ turn past hand tightened

5/16” wrench

3/16” wrench


Inlet Frit Replacement Tighten Inlet Nut/Frit & Column

Assembled UPLC® Column


Inlet Frit Replacement Overview (cont.)

5) Place new frit (from PN 205000343 or PN 700002775) inside nut.

6) Hand-tighten the assembled frit/nut onto the column.

7) Torque inlet nut to column at approximately

1/2 turn past hand tightened.

8) Re-Check column for lower back pressure.


Inlet Frit Replacement Procedure Conclusions

Procedure designed to determine the LOCATION of UPLC®

column plugging

– It is not designed to prolong UPLC® column lifetimes

(although it CAN)

– It should not be necessary to perform this procedure on a

regular basis

If INLET frit is not the LOCATION of the plugging,

change OUTLET frit using the same procedure

Use THIS column for UPLC Maximizing

Performance Procedure


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ACQUITY UPLC Columns - Waters Corporation · PDF fileACQUITY UPLC® Columns Techniques for...

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