Life Sciences

Pall Laboratory for the Quality Control

Brice MONTEIL MENA Area Manager
Filtration. Separation. Solution. SM

Agenda
Pallchek presentation HPLC Analytical Sample Prep Mobile phase Microbiology

Analytical Filtration
1. What is Filtration 2. Types of Media 3. Filter Media Properties 4. How do we make membranes? 5. Chromatography and HPLC 6. Filtration and Sample Preparation
Choosing the best device

What is Filtration?
The process of removing particles from a
liquid or gaseous stream as it passes through a porous medium.

Why Use Filters?

Collect and/or Concentrate a Sample for
Analysis Clarify a Solution or Gaseous Stream Sterilize a Solution or Gaseous Stream Protect Critical Instrumentation from Particles or Liquids

Types of Media
Depth Media
Membranes Combination Filters

What is Depth Media

A Depth Filter is a filter consisting of
either multiple layers or a single layer of a medium having depth, which captures contaminants within its structure, as opposed to on the surface.

Membrane Filtration
A Membrane Filter typically traps
contaminants larger than the pore size on the addressed surface of the membrane.

A Combination Filter
A Combination Filter combines
different membrane pore sizes, or combines depth media and a membrane filter to create selfcontained serial filter units. They can offer an economical alternative to using individual prefilters and final filters.

Important Filter Media Properties

Pore Size Rating Hydrophilic or Hydrophobic Porosity (Flow rate) Chemical Compatibility Extractables Surface Area

Pore Size Ratings

Absolute Rated
Specifies the pore size at which a
challenge organism of a particular size will be retained with 100% efficiency under specific test conditions
Example: 0.2m rated membranes must retain
100% of the microorganism Brevundimonas diminuta according to ASTM F838-83 methodology

Pore Size Ratings

Nominal Rated

Specifies the pore size at which a challenge particle of a particular size will be retained with an efficiency of 60-98%. Nominally rated filters vary significantly in the filtration industry

Hydrophilic or Hydrophobic
Hydrophilic - Possessing an Affinity
for Water (likes water) Hydrophobic - Lacking an Affinity for or Repels Water (dislikes water)

Will Not wet in water but will wet in low surface tension liquids, such as organic solvents

Porosity
The measure of quantity of all the open
spaces (pores) in the membrane Membranes are generally 50% to 90% open space Directly proportional to the flow rate of the membrane (more pores, higher flow) Not pore size, but more a reflection of number of pores

Chemical Compatibility
The filter medias resistance to
select chemicals

Pore structure should not be adversely effected by chemical exposure

Least Resistant FP Vericel Supor HT-Tuffryn Nylon GN Metricel

Most Resistant PTFE GHP Metricel PP

Extractables
Contaminants eluting from the filter
media or device which may adversely effect effluent quality

HPLC Analysis - Add extraneous peaks Cell Culture - Kills cells Micro Analysis - Inhibits growth / effects recovery Environmental Analysis - Metals contaminants

Extractables
Contaminants may be:

Wetting agents in filter media Sterilization residuals (EtO) Additives in polymer or housing components (colorants, mold release agents, etc..)
Remember its not just about the filter, consider the housing

Surface Area
Doubling surface area will yield at least two times more service life and as much as four times more.

How do we make membranes?

Membranes can be made in the 4 following ways

Phase-Inversion Stretching Track-Etching Sintering

Phase-Inversion
Used to produce most of BioSciences
membranes Most versatile method for producing membrane Three major components are combined

Polymer Solvent Pore former

Phase-Inversion
Polymer

Makes up the body of the membrane

Solvent

Dissolves the polymer into a liquid state

Inability to dissolve the polymer Polymer forms around the pore former droplets

Pore Former

Stretching
Commonly used to produce PTFE
membrane A dense plastic film is carefully stretched in all directions Pores are formed under the conditions of the stretching

Some Pall BioScience Membranes

Supor(polyethersulfone) Biodyne (nylon 6,6 with +,-, or neutral
charge) HT Tuffryn (polysulfone) FP Vericel (polyvinylidene fluoride-PVDF) GN-4 & GN-6 Metricel (mixed cellulose esters) GHP (hydrophilic polypropylene) MetricelPolypropylene Zylon PTFE or Teflon (polyterafluoroethylene)

The basic HPLC

Mobile phase. Usually organic solvent mixture. Needs to be of high grade and filtered use the SolVac. Detector. Can be ultraviolet, refractive index or fluorescence. The detector contains a flow cell

mobile phase carries sample though HPLC

Eluent Reservoir

Pump

Sample Injector

Column

Detector

Fraction Collector

Pump is used to force eluent (mobile phase) through the column and detector. The pump should provide constant Flow.

Recorder
The Column, usually packed with small particles of porous silica, alumina, or organic resin. This is where the multistage separation Process occurs.

Sample Prep
Both samples and mobile phase
should be filtered. This will reduce system downtime and maintenance. Reduce incorrect results, noisy baselines caused by contamination and noise in the detector due to release of dissolved gases in the mobile phase. Protect columns.

Selecting membrane pore size

The filter pore size should be determined
based on the column packing size. As you can see the column packing particles touch each other. Ideally, you would not want contamination to fit into the space between the particles of packing. This space is labelled the Flow Path.

 We can use basic mathematics to

determine the size of the flow path If the column packing is 3 m in diameter, the flow path is 0.43 m.

When an HPLC column has a

packing size of 3 m or smaller, you should use a 0.2 m filter because a 0.45 m filter may let particles through that will plug the column.

 When filtering solvents and chemicals selecting

a chemically compatible membrane is important. Aqueous Samples Choose a hydrophilic membrane, for example, Pall
GHP, PES, Nylon, or PVDF membranes.

Selecting the correct membrane

Aggressive Organic Solvents

Choose a chemically resistant membrane, for
example PTFE.

Aqueous and Organic-solvent Solutions

Pall Life Sciences patented hydrophilic
polypropylene (GHP) membrane is a universal membrane for both aqueous and organic applications.

Extractables
It is important to use a clean, HPLC certified device

Contaminants eluting from the filter media or

device which may effect effluent quality

 Filters come in a variety of sizes ranging from

the area within a single well of a 96-well plate, to spin filters and syringe filters Devices are also available with a single layer or combination filter (with a prefilter) By increasing surface area and using a prefilter we will increase throughput If sample size is small we must consider holdup volume (the amount of sample that will be left in the device)

Selecting the best device

 Acrodisc

PSF Syringe Filter

Three Powerful Benefits in One

GxF multi-layered prefilter provides maximum throughput

The Acrodisc PSF syringe filter has a
serial prefilter to allow for two to four times the throughput and faster flow rates than standard devices. The multi-layered prefilter traps particulate rated from > 40 to 1 m.

Automation certification
Caliper (Zymark) certifies Pall syringe
filters for worry-free performance in their automated workstations.

Best protection for HPLC columns Extending column life up to 46 times

Plugging is the most frequently encountered
cause of High Performance Liquid Chromatography (HPLC) column failure. Injection of samples containing particulates will eventually block the column inlet, cause high column back-pressure, and shorten the normal lifetime of the column. Pall Acrodisc PSF syringe filters with GHP (hydrophilic polypropylene) membrane offer the most efficient removal of particulate and prolong the life of HPLC system components.

Retention Efficiency / Column Life Study

Examine how much longer an HPLC column might last when samples are filtered with Pall Acrodisc premium syringe filters
Inject UNFILTERED mixture of latex spheres Repeat injections until column plugs Inject FILTERED mixture into new column Repeat injections until column plugs Plot pressure vs. injection number

Results

Your experience
Have you ever had a column plug
unexpectedly? Have you ever had unexpected peaks in your chromatograms? Has your analyte concentration change after filtration? Do you consider analyte adsorption in your filter equivalency testing?

Other Pall Sample Prep Products

AcroPrep 24 24 x 1.9 mL well HPLC certified
for low extractables. GHP Nanosep Various pore in a centrifugal size and device. membrane HPLC certified configurations. for low extractables. 500 L sample volume.

AcroPrep
96 well filter plates 1ml size wells

Filtering Mobile Phase

Traditionally labs will
use a 47mm glass filter funnel to filter Glass filter funnels are easy to break They are open systems, therefore it is easy to spill aggressive solvents

SolVac Filter Holder

Draws directly from
HPLC solvent bottle. Less likely to spill aggressive solvents than glass funnels or disposable cups. Durable plastic construction. Less likely to break than glass funnels or assemblies.

Using the Solvac its safe and easy

Which membrane should I use?

Pall supplies a whole range of 47mm
membranes which will fit into the SolVac filter holder We would recommend our GHP PP membrane for the majority of requirements.

Summary
Filtration should be used in chromatography Samples and mobile phase should be
filtered Choose membrane based on compatibility Choose EFA based on volume to be filtered Choose pore size based on column bead size Not all 0.45m filters are equal Filtration does extend column life

Microbiology

Microbiology
Micro-organisms can halt the production
of pharmaceuticals and cause disease. The accurate and reliable detection and identification of micro-organisms is critical.

Pall Corporation began providing

membranes for microbial analysis over 50 years ago.

The MF Technique

Advantages of MF technique
Permits usage of large sample volumes. Reduced preparation time. Provides presence or absence
information within few days. Effective and accepted technique. Used in water, pharmaceutical, cosmetics, food and beverage industries.

What do we need to do the MF technique?

Vacuum Pressure Pump with VacuShield Filtering Flask Stainless Steel Forceps-smooth tip Petri Dishes-50mm Media Tubing Bunsen Burner Catch bottle

Microbiology
For microbial analysis, where culturing of
sensitive organisms can be difficult and identification is critical for process control and public safety, the mixed cellulose ester GN Metricel membranes set the standard worldwide. These membranes provide uniform and consistent growth of organisms to ensure quality in pharmaceutical products.

Why use a Pall membrane?

The GN6 mixed cellulose esters
membrane is most accepted filter media for microbiological analysis. The unique dot grid pattern provides easy quantification of bacterial colonies without growth inhibition or enhancement.

GN-6 Metricel Membrane

Mixed Cellulose

Ester > 90% Recoveries 0.45m Pore Size White Membrane Black Gridlines E. coli, Total Coliform, Total Bacteria

Supor 200 Membrane

Polyethersulfone > 90% Recoveries 0.2m White Membrane Black Gridlines Pseudomonas sp. analysis

Metricel Black Membrane

Polyethersulfone > 85% Recoveries 0.45m and 0.8m Black Membrane White Gridline Yeast, Mold, Light Colored Bacteria

MicroFunnel Filter Funnels

MicroFunnel Filter Funnels

Increase laboratory efficiency with
convenient, ready-to-use disposable filter funnels Test any aqueous solution for microbial contamination using the principles of the Membrane Filter (MF) Technique. Ideal for quality control analysis of aqueous fluids used in pharmaceutical production. Individually labelled for lot traceability.

MicroFunnel Filter Funnels

47 mm, 100 mL and 300 mL size GN-6, Supor, or Metricel Black membrane 0.2 gridded or plain Supor 0.45 m gridded Removable membrane - Culture in place or remove filter for culturing on agar plate. Gamma irradiated

MicroFunnel Filter Funnels Easy snap apart design

Fits directly on Manifold Individually Bagged LP Version has additional Petri Dish
with Dry Absorbent Pad Extensive Literature

What do I need to use with a funnel?

Pump, tubing, vacushield, catch vessel

Forceps Manifold (this is a single place)

Petri dishes (depending on the funnel) Media

MicroFunnel Plus Filter Funnel

Combines sample
cup and filter funnel into one product to prevent contamination of the sample. Optimized for use in hot loop water applications.
The Supor MF Plus has been validated with samples hotter than 90C.

MicroFunnel Plus Filter Funnel

Innovative design No other product like it
on the market. Patented design Current method required collection of sample in separate cup. MicroFunnel Plus filter funnel eliminates the need for a separate sample cup.
The filter has been validated with samples hotter than 90C.

Hot water loop

Water that is constantly circulating at elevated temperature of 70 - 90C or higher. The high temperature and constant circulation helps control any growth of microorganisms. Water is used to produce Sterile WFI or simply purified water.

Design Features

How to use it ?

MicroFunnel Benefits
Less material to order inventory dispose of Less potential for
contamination no sample transfer filtered air drawn in

 Fluid is sampled

MicroFunnel Plus AP filter funnel

into the port on the lid so it never comes in contact with the environment. Provides added contamination control for sampling fluids for microbial testing.

MicroFunnel Plus AP filter funnel

Vented Lid Supor Membrane Seal at Base Vent Tab

Benefits :

Contamination Control
Aseptic sampling port No sample transfer Filtered air drawn in

Time Saving
Less handling