AgendaPallchek presentationHPLCAnalytical Sample PrepMobile phaseMicrobiology

Analytical FiltrationWhat is FiltrationTypes of MediaFilter Media PropertiesHow do we make membranes?Chromatography and HPLCFiltration and Sample PreparationChoosing 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 AnalysisClarify a Solution or Gaseous StreamSterilize a Solution or Gaseous StreamProtect Critical Instrumentation from Particles or Liquids

Types of MediaDepth Media

Membranes

Combination Filters

What is Depth MediaA 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 FiltrationA Membrane Filter typically traps contaminants larger than the pore size on the addressed surface of the membrane.

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

Important Filter Media Properties

Pore Size RatingHydrophilic or Hydrophobic Porosity (Flow rate)Chemical CompatibilityExtractablesSurface Area

Pore Size RatingsAbsolute RatedSpecifies the pore size at which a challenge organism of a particular size will be retained with 100% efficiency under specific test conditionsExample: 0.2m rated membranes must retain 100% of the microorganism Brevundimonas diminuta according to ASTM F838-83 methodology

Pore Size RatingsNominal RatedSpecifies 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 HydrophobicHydrophilic - 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

PorosityThe measure of quantity of all the open spaces (pores) in the membraneMembranes are generally 50% to 90% open spaceDirectly proportional to the flow rate of the membrane (more pores, higher flow)Not pore size, but more a reflection of number of pores

Chemical CompatibilityThe filter medias resistance to select chemicalsPore structure should not be adversely effected by chemical exposurePTFE GHPMetricel PPFP VericelNylonHT-TuffrynSuporGN MetricelMost ResistantLeast Resistant

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

HPLC Analysis - Add extraneous peaksCell Culture - Kills cellsMicro Analysis - Inhibits growth / effects recoveryEnvironmental Analysis - Metals contaminants

ExtractablesContaminants may be:

Wetting agents in filter mediaSterilization residuals (EtO)Additives in polymer or housing components (colorants, mold release agents, etc..)

Remember its not just about the filter, consider the housing

Surface AreaDoubling 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-InversionStretchingTrack-EtchingSintering

Phase-InversionUsed to produce most of BioSciences membranesMost versatile method for producing membraneThree major components are combinedPolymerSolventPore former

Phase-InversionPolymerMakes up the body of the membraneSolventDissolves the polymer into a liquid statePore FormerInability to dissolve the polymerPolymer forms around the pore former droplets

StretchingCommonly used to produce PTFE membraneA dense plastic film is carefully stretched in all directionsPores are formed under the conditions of the stretching

Some Pall BioScience MembranesSupor(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)MetricelPolypropyleneZylon PTFE or Teflon (polyterafluoroethylene)

EluentReservoirPumpSampleInjectorColumnDetectorFraction CollectorRecordermobile phase carries sample though HPLCPump is used toforce eluent (mobile phase)through the column and detector.The pump should provide constantFlow.The Column, usually packed withsmall particles of porous silica, alumina,or organic resin.This is where the multistage separation Process occurs.Detector.Can be ultraviolet, refractive indexor fluorescence.The detector contains a flow cellMobile phase.Usually organic solvent mixture.Needs to be of high grade and filtered use the SolVac.The basic HPLC

Sample PrepBoth 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.

Selecting the correct membraneWhen filtering solvents and chemicals selecting a chemically compatible membrane is important.Aqueous SamplesChoose a hydrophilic membrane, for example, Pall GHP, PES, Nylon, or PVDF membranes.Aggressive Organic SolventsChoose a chemically resistant membrane, for example PTFE.Aqueous and Organic-solvent SolutionsPall 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 deviceContaminants eluting from the filter media or device which may effect effluent quality

Selecting the best deviceFilters come in a variety of sizes ranging from the area within a single well of a 96-well plate, to spin filters and syringe filtersDevices are also available with a single layer or combination filter (with a prefilter)By increasing surface area and using a prefilter we will increase throughputIf sample size is small we must consider hold-up volume (the amount of sample that will be left in the device)

Acrodisc PSF Syringe Filter

Three Powerful Benefits in One

GxF multi-layered prefilter provides maximum throughputThe 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.1

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

2

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.3

Retention Efficiency / Column Life StudyExamine how much longer an HPLC column might last when samples are filtered with Pall Acrodisc premium syringe filters

Inject UNFILTERED mixture of latex spheresRepeat injections until column plugsInject FILTERED mixture into new columnRepeat injections until column plugsPlot pressure vs. injection number

Results

Your experienceHave 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?

ReviewThe Value of Pall Acrodisc PSF syringe filters comes from:PSF extends HPLC column life better than competitorsPSF has lower extractables than competitorsAnalyte adsorption is negligible with PSF

No competitor has this data!

Other Pall Sample Prep ProductsGHP Nanosep in a centrifugal device.HPLC certified for low extractables.500 L sample volume.

AcroPrep 2424 x 1.9 mL wellHPLC certified for low extractables.Various pore size and membrane configurations.AcroPrep 96 well filter plates1ml size wells

Filtering Mobile PhaseTraditionally labs will use a 47mm glass filter funnel to filterGlass filter funnels are easy to breakThey are open systems, therefore it is easy to spill aggressive solvents

SolVac Filter HolderDraws 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 holderWe would recommend our GHP PP membrane for the majority of requirements.

SummaryFiltration should be used in chromatographySamples and mobile phase should be filteredChoose membrane based on compatibilityChoose EFA based on volume to be filteredChoose pore size based on column bead sizeNot all 0.45m filters are equalFiltration does extend column life

Microbiology

MicrobiologyMicro-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 techniquePermits 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 VacuShieldFiltering FlaskStainless Steel Forceps-smooth tipPetri Dishes-50mmMediaTubingBunsen BurnerCatch bottle

MicrobiologyFor 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 MembraneMixed Cellulose Ester> 90% Recoveries0.45m Pore SizeWhite MembraneBlack GridlinesE. coli, Total Coliform, Total Bacteria

Supor 200 MembranePolyethersulfone> 90% Recoveries0.2mWhite MembraneBlack GridlinesPseudomonas sp. analysis

Metricel Black MembranePolyethersulfone> 85% Recoveries0.45m and 0.8mBlack MembraneWhite GridlineYeast, Mold, Light Colored Bacteria

MicroFunnel Filter Funnels

MicroFunnel Filter Funnels

Increase laboratory efficiency with convenient, ready-to-use disposable filter funnelsTest 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 sizeGN-6, Supor, or Metricel Black membrane0.2 gridded or plain Supor 0.45 m griddedRemovable membrane - Culture in place or remove filter for culturing on agar plate.Gamma irradiated

MicroFunnel Filter Funnels

Easy snap apart designFits directly on ManifoldIndividually Bagged LP Version has additional Petri Dish with Dry Absorbent PadExtensive Literature

What do I need to use with a funnel?Manifold (this is a single place)ForcepsPetri dishes(depending on the funnel)MediaPump, tubing, vacushield, catch vessel

MicroFunnel Plus Filter FunnelCombines 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 FunnelInnovative designNo other product like it on the market.Patented designCurrent 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 loopWater 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 BenefitsLess material toorderinventorydispose ofLess potential for contaminationno sample transferfiltered air drawn in

MicroFunnel Plus AP filter funnelFluid is sampled 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 funnelVent TabSupor MembraneSeal at BaseVented LidBenefits : Contamination Control Aseptic sampling port No sample transfer Filtered air drawn in Time Saving Less handling

Remember we usually require a force to push the liquid/gas through the filtration media this may be a positive pressure or a vacuum.Absolute rating is something that is very important in sample prep. Because sample prep devices are not sterilised a lot of plastic injection molding companies will buy large roles of membrane and produce very cheap syringe filters. Often these companies will not state if these syringe filters are norminal or absolute.

HPLC High performance liquid chromatography is a widely used type of chromatography used in analytical chemistry and biochemistry labs.

The sample is injected into the system and is carried through a chromatography column at high pressure by a mobile phase. The column is where the separation takes place.

The mobile phase is usually an organic solvent mixture, or sometimes scientists will use buffers as mobile phases.

Some customers will say that they do not need to filter their mobile phase as they use a HPLC solvent There are various arguments for and against filtering. To see these arguments in full then read the various forums and message boards on the web!

HPLC solvents can still contain contaminants, these may include dust particles and particulates from bottle tops. Remember that filtering under vacuum will also remove dissolved gas. Most customers mix their own mobile phases which they should filter.

Some customers will say they do not need to filter samples as they use a guard column. Some users believe a guard column alone will protect their instrument. Guard columns provide additional protection, but not complete protection as they will not retain microscopic particulate. They are more of a chemical protaction they are not rated to remove particulate.

Sample filtration is still recommended by instrument manufacturers to avoid downtime and sample loss.

Extractables are a major issue in sample prep filtration. It is important to use clean, HPLC certified devices.

Here is a chromatograph comparing a blank sample of ACN, filtered sample using competitor PTFE hydrophilic syringe filter, and a filtered sample using a Pall GHP acrodisc.

Note the extra peaks after filtering with the competitor device. These peaks are due to extractables. These extrables are likely to be wetting agents chemicals added to make hydrophobic membranes hydrophilic, but that could also be colorants, mould release agents or additives added to the plastics.

Pall devices are very clean it should be noted that no colorants are used in the plastics, all of the printing is done on the top of the device, so no colorant may come into contact with solution and leach out.

Lets now look at some of the devices available starting with the Acrodisc PSF.For difficult-to-filter samples, it is best to use a syringe filter with a glass fiber prefilter over the membrane. The Acrodisc PSF syringe filter with GxF multi-layered glass fiber prefilter is the best option for extremely particulate-laden samples.

The Acrodisc PSF syringe filter has a serial glass fiber (GxF) prefilter to allow for maximum throughput and faster flowrates than standard glass fiber prefilter devices. The multi-layered prefilter, rated from > 40 to 1 m, trapsparticulate, thereby extending filter life.

A number of customers, especially in the larger pharmaceutical accounts, will use automated (robotic) systems to do their sample preparation work.

These customers will require filters that are reliable and will move consistently within the automated sample handling apparatus.

Acrodisc PSF syringe filters from Pall Life Sciences are the only syringe filters to receive Caliper Life Sciences Automation Certified guarantee. This certification is granted to syringe filters that meet the stringent requirements for automated dispensing, robotic handling, and fit. Our certified filters operate in the Benchmate, Prelude, TPWII, and Multi-Dose workstations.

Injection of samples containing particulates will eventually block the column inlet, cause high column back-pressure,and shorten the normal lifetime of the column.

Our experiment is a study that involved examining column life without filtration compared to column life when samples werefiltered.

DEMO Now is a good time to run the latex sphere demo.

Conclusion - Latex sphere retention was tested for filters from three manufacturers. Compared to filters from two other vendors, Pall Acrodisc PSF syringe filters significantly prolonged HPLC column life with virtually no back-pressure increase after 972 injections.

Without filtration, the column failure due to plugging occurred after only 21 injections. After the 0.05% latex-sphere solution was filtered with competitive filters, the columns were plugged after 37 and 487 injections,respectively.

Pall Acrodisc PSF syringe filters with GHP membranes were able to retain an average of 94.9% of the 0.45 m averagediameter latex spheres. By comparison, competitors syringe filters only removed averages of 90.5% or 33.0% of the0.45 m average diameter latex spheres, Pall syringe filters showed greater lot-to-lot consistency with relative standard deviation (RSD) of 3.5%.

Among 0.45 m rated syringe filters from three manufacturers, the Pall Acrodisc PSF syringe filters with GHP membranehave the highest average retention efficiency (94.9%) of latex spheres, as well as greater lot-to-lot consistency.

It has been shown that it is imperative that samples be filtered prior to their introduction into an HPLC system. The data demonstrates equivalent filters from various manufacturers with the same removal rating differ in capabilities.

Using Pall Acrodisc PSF syringe filters with GHP membrane extended the column life 46 times with no significant increase in column back-pressure.

At this point it is best to show the glass filter funnel system.

Points to note;1. Its glass, and glass breaks.2. Its hard to align the membrane and clamp the funnel on top. 3. The clip is quite stiff to use.4. Once assembled the solvent needs to be poured into the top of the unit, spills could occur.5. The assembly is open, therefore if it was to be knocked over solvent would spill everywhere.The SolVac is a durable plastic filter holder, it works under vacuum, so it both filters and degases.

Some points to note:-

Polypropylene construction - Reusable chemically resistant to common HPLC mobile phases. Durable plastic less likely to break when tipped or dropped.

Innovative design - Versatile, fits most HPLC bottles.

Draws directly form solvent bottle - Eliminates need to repeatedly refill the filter funnel. Reduces likely hood of spilling aggressive solvents.

Reliable and leak proof patented Magnetic Seal -Allows easy placement of the membrane and eliminates the potential for tearing when metal clamps or threaded holders are used.

Membrane placement guides / Forceps Access Point - The circular rim of the membrane support has forceps access points to make 47mm membrane disc filter placement easy every time.

More EFA (effective filtration area) - The 10.2 cm sq EFA of the SolVac provides a bit more than the standard 47mm filter holder which allows for faster filtration and longer life. (~6%)

Remember - Always use a safety-coated receiving vessel that is less than 4 L and rated for vacuum applications. Failure to do so mayresult in implosion of the receiving vessel and potential injury to the user.

The same rules apply for selecting the correct pore size of the membrane:

0.2 m - columns < 3 m or smaller packings, microbore columns or microbial growth removal0.45 m - columns > 3 m or larger packingsNotes - Notes this is an example of the MF technique using reusable magnetic filter funnels.Obviously the important thing is to do the technique in an aseptic way.

It is good to point out that everything needs to be sterile, and that a separate petri-dish needs to be used.

The MF technique;

Permits usage of large sample volumes.Reduced preparation time.Provides presence or absence information within 24 hours.Effective and accepted technique.

Used in water, pharmaceutical, cosmetics, food and beverage industries.

Notes Pall provide 3 main membranes for microbiology. GN6 0.45um for total coliforms, Supor 0.2um Pseudomonas sp. Analysis and the Metricel Black for light coloured organisms, ie yeasts.

Our membranes are used as part of the Membrane Filtration Technique which permits usage of large sample volumes and provides presence or absence information within 24 hours.

The MF Technique is an effective and accepted technique, and is used in water, pharmaceutical, cosmetics, food and beverage industries.

Our membranes have a unique dot grid pattern which provides easy quantification of bacterial colonies without growth inhibition or enhancement. (see our catalogue for further information)

Notes The unique dot grid pattern prevents any growth inhibition which can be cause by membranes with solid grid lines.

Our dotted membrane is also prefered by microbiologists as it a lot more easier on the eye, especially when counting lots of membranes per day.Vacuum Pressure Pump with VacuShieldHand Operated Vacuum Pump and Filtering FlaskMicrobiology magnetic funnels (autoclavable must wrap in autoclave paper)Stainless Steel Forceps-smooth tipPetri Dishes-50mm + PadsMedia

Notes The MicroFunnel family is a group of ready to use disposable filter funnels.They are available in a number of different membrane configurations, sizes and pack sizes to suit your customers needs.Notes - Certified. Each lot is certified for microbiological analysis to provide added assurance of reliable results. This makes the funnels suitable for Pharmaceutical use.

Easy to use. Unique squeeze separation of cylinder from base allows easy access to membrane. breaks down and reusable hardware cannot be sterilized.One of the major feature of this product range is the unique squeeze separation of the filter funnel from the base. The products fit directly onto a Pall manifold, or are available with adapters for other vacuum manifolds/configurations

Notes - The 300 mL funnel is ideal for processing larger samples without the "pour and wait" of smaller funnels. Easy and accurate sample measurement. The 100 mL funnels are marked in 10 mL increments.The 100 mL increment is marked completely around the funnel for higher visibility. The 300 mL funnel is marked in 50 mL increments.

The funnels are available with GN6 0.45um for total coliforms, Supor 0.2um Pseudomonas sp. Analysis and the Metricel Black for light coloured organisms, ie yeasts.

Remember - Our membranes have a unique dot grid pattern which provides easy quantification of bacterial colonies without growth inhibition or enhancement.

An important product feature is that the membrane can be removed from the funnel or can be cultured in place so the membrane is left in place, the lid of the funnel is used as a lid for the base, and media can be injected into the bottom of the base unit and sealed using a red stopper.

Our products are gamma irradiated sterilised and not sterilised using cyto toxic reagents like EtO which can inhibit recovery.

Notes refer to our catalogue to veiw the full range of products available.

Easy to use. Unique squeeze separation of cylinder from base allows easy access to membrane. breaks down and reusable hardware cannot be sterilized.One of the major feature of this product range is the unique squeeze separation of the filter funnel from the base. The products fit directly onto a Pall manifold, or are available with adapters for other vacuum manifolds/configurations

NOTE even if a customer is not using a disposable funnel system in their micro lab still sell them a box! They can use them if their autoclave ever breaks down.

Notes This is a unique product. It is available in a 100ml and 300ml format. With either GN6 membrane or Supor membrane.The Supor membrane version of the funnel is validated for hot samples to help with the sampling two ceramic funnel holders are available (see the picture).

The main benefits for this product are 1. Remove the need for a separate sample cup and 2. Remove any risk of cross contamination.Notes The hydrophobic vented lid allows for filtration without removing the lid and ensures that there is no aerosol contamination.On the Supor model of the funnel there is a tear-away vent on the bottom of the funnel to allow for air expansion remember you may use this funnel with hot samples. On the GN6 version of the funnel there is a red plug you would not use the GN6 for hot samples, therefore expansion is not an issue.Note its good to point out here the picture of the ceramic holder which can be used to hold the unit if hot samples are being added to it.