The increasing use of affinity membranes with molecular recognition technology

The Increasing Use of Affinity Membranes with Molecular Recognition Technology

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urface modification of membranes has been developed for a number of applications for biotechnology and biomedical uses, including protein purification and isolating other biomolecules from complex biologically derived fluids. These types of separations are already being performed and they will become increasingly important over the next 10 years. There is a need, however, for a broad family of highly selective membrane materials that can address a number of analytical and process applications in the environmental and chemical process industries. Introduction of affinity

into these membranes applications has been slow due to the lack of: selective chemistries on a commercial basis for binding of inorganic cations and anions; membrane stability in the environments that it is exposed to; low cost methods for putting highly selective into the chemistries membrane format. In this article, Neil Izatt, IBC Advanced Technologies, Inc. (IBC), USA describes how they are commercializing a new family of affinity membranes that overcomes the above issues and that can be used in a number of analytical

and process scale applications previously una.vailable to membrane technology. IBC has developed a family of selective chemistries based on molecular recognition technology (MRT) that can be incorporated into stable and rugged membrane formats’. MOLECULAH RECOGNITION TECHNOLOGY (MRT) Molecular Recognition Technology (MRT) is based on research that won the Nobel Prize for chemistry in 1987 for three scientists (Donald Cram of UCLA, Charles Pedersen of DuPont and Jean-Marie

1

Figure I. Three IBC Core Technologies

1 Synthesis of Selective Ligand

2 Attachment of Ligand to Solid Support

3 Selective Washing and Eluting i Figure 1: Three IBC Core Technologies involving Molecular Recognition Technology

Pasteur Lehn of the Institute). In MRT, organic ligands are created that selectively bind targeted molecules in solution. The binding is based on charge, size and shape and is specific to the target of interest. IBC Advanced Technologies was founded in 1988 to commercialize molecular recognition technology to produce separation techniques that are rapid, specific and efficient. The company’s mission is to develop and market unique materials based on MRT. IBC is applying its core technologies to the development of innovative affinity membranes. These technologies include: the use of selective complexing molecules such as macrocycles; the use of these molecules with solid phases to form solid phase extraction particles and membranes; and the use of washing and eluting chemicals that allow extracted molecules to be recovered in pure form. Figure 1 illustrates the attachment of a macrocycle to a solid support. The first of the core technologies is the high selectivity IBC programs into its molecular recognition ligands. Molecular recognition uses one chemical structure, called the host, to recognize specific electronic and spatial features of another chemical, called the guest, to form a “host-guest” complex. A guest, such as a dissolved ionic species, can be selectively removed from solution by being complexed with the host chemical and thus be isolated for later recovery and/or measurement. IBC has developed a number of selective complexation ligands for cations, anions and neutral molecules. In contrast to many of today’s membrane and non-

r Species

lhterferenca Level (mgfi)

Agf2 Al+3 AUs+ CW Co+2 Crs6


OJ+2

F-

Fec2 Mo”~ NY.2 NO,-N Pb”+ SiO, Zn+2

membrane separation techniques, IBC’s chemistries, which incorporate the principles of molecular recognition, essentially ignore all non-target species, even those with similar electric charges, molecular shapes, or other target-like attributes. Complexes can be formed with a single molecular species, even when chemically similar molecules are present in higher concentration ratios of IO6 or more. This is significant in applications such as stored nuclear waste where removal of very low concentrations of radioactive ions must occur even in the presence of extremely high concentrations of nonradioactive ions. The reaction of a complexing agent with a metallic ion is u s e d i n s o m e liquid/liquid extraction and precipitation type separations. However, these techniques are not always appropriate for the application as they generate secondary extraction fluids, usually organic solvents, or finely divided precipitate. In many applications, solid phase extraction (SPE) media provide cleaner and more efficient separations. Highly selective affinity membranes are prepared by using the second core technology, covalent attachment at high density of MRT ligands to solid phases. These phases include polyacrylate, polyethylene or teflon membranes, silica gels and polymeric beads. Ligands directly attached to membranes are used as

IO 1.0

100 10 IO 50 IO 100 10

affinity membranes while the SPE particles are incorporated into membranes, s u c h a s 3M’s particleloaded membranes, traden a m e d EmporeTM. T h e membrane materials that this article describe are suitable for the harsh environments of many applications. The third core technology is the chemistry for selective washing, eluting and recovering of the target molecule. The target species can be recovered in pure form and quantitative amounts. Many applications involve the removal of trace levels of the target and thus use only a small fraction of the available reactive sites. Non-target species can become loosely associated with the remaining active sites and remain after the final sample liquid passes. By applying a variety of conditions such as pH change, water wash, or additional complexation, these loosely associated species can be extracted leaving behind the complexed target on the membrane. This can then be eluted in a liquid concentrate, or used in its pure form as retained on the membrane.

beads, such as produced by IBC, can be enmeshed into Empore without the use of binders or adh,esives. Because no coating ;is used, the affinity particle is fully exposed for contact with any fluid passing through the membrane and the chemistry and reaction kinetics of the particle are preserved. SELECTIVE SEPARATION MEMBRANES The joint IBC-3M m e m brane combines membrane and molecular recognition technologies. It is compatible with nearly all organic and inorganic solvents. The membrane is composed of polymer or chemically modified silica particles tightly bound in a web of PTFE micro-teflon and other fibrils. The particles are individually suspended so that the surface of each is free for maximum interaction with the sample during the separation. Since the particles are enmeshed and not coated, the mernbrane maintains all of the chemical and available surface area characteristics of the selective particle and demonstrates all of the physical performance properties of a filter-like membrane. The particles can comprise up to 95% of the membrane’s weight and are very small compared to particles in ion typical used exchange or SPE columns. Because they are small and closely packed, flow rates can be 10 to 100 times faster than columns yet achieve equal extraction efficiencies.This is the combined result of the particle size and the fibrils creating a very torturous, but uniform, path through the membrane. No charineling

or wall effects typical of columns are observed with these products. Since the reactive sites are reserved almost exclusively for the target species, particle capacity is used very efficiently. This highly efficient use of particle capacity is significant particularly in separating extremely low concentrations of the target species. The affinity membrane based on molecular recognition has been employed in a number of unique commercial process and analytical applications, and won the prestigious R&D 100 award in 1996 for innovation’. The benefits of this new technology compared to chromatography, SPE or conventional membranes include: increased selectivity for the isolation and/or purification of chemical species that have only small differences in size, functional group or structure; increased productivity available over a wide range of production scales and stream concentrations; robustness at extremes of pH, temperature, pressure; resistance to hydrolysis, organic solvents, impurity poisoning or reactive mixtures; attractive economics in use. APPLICATIONS Selective membranes based on MRT can be used in both process scale and analytical applications. Process applications that are particularly suitable to this approach display the following characteristics: ?? low levels of ions (< IO -5 g/l)

EMPORETM 3M has developed the ability to produce particle loaded membranes using their EmporeTM membrane technology. Empore membranes are thin, about 0.5mm, and are composed of an inert fibrous matrix such as polytetrafluoroethylene (PTFE). Affinity

ton Magnesium Calcium Strontium Lead Sodium Potassium Barium

Coacenttattoa (mg/k),

Gancentrqtian (mg/L)

10,000 300 3

t 0,UOO 10,000 2

1

iaao IO 0.1

IO

Iam 100 1

Figure 2. Number of ManipulstJons Required for Sr and Ra Analysis

EPA%

IBC13M

IEW3M

EPARa

(57)

Sr (61

Ra WI

(56)

EPA Sr = Method SOB, EPA RP = Methods 803.1, SO4

Figure 2: Number of Manipulations Required forsr and Ra Analysis

high stream flowrates (> 500 liter/minute) ?? low filtration requirements (no solids above 1 OCL) Analytical applications that may be particularly suitable to this approach have the following characteristics: ?? low levels of ions (< IO -5 g/l) ?? high levels of interfering species present ?? otherwise involve multiple sample prep steps Some of the applications where the technology is being commercialized are described below, and include PbExB and HgExB for heavy metal test kits, EmporeTM Rad Disks for radionuclide sample preparation and analysis; and SuperLig@ for waste characterization and process polishing.

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PBEXTM AND HGEXTM ANALYTICAL TEST KITS Process Description IBC and 3M have used the affinity membrane approach in a commercialization effort with Hach Company (Loveland, Colorado, USA), a leading supplier of wet chemistry test kits for analysis.3 Initial metal ion targets were Hg and Pb. For Hg, a membrane is used which incorporates AnaLi@ HgOl particles and for Pb, AnaLigB PbOl . A 25 mm diameter membrane disk is sealed in a syringe barrel type Hg or Pb extractor that can be fitted to a hand-operated vacuum pump. A measured volume of acidified or digested sample is drawn

through the appropriate Extractor. The absorbed target ion is washed with deionized water and then eluted off the disk. A colorproducing porphyrin is added to the eluate and the pH adjusted to above pH 13. Color measurement is performed with a Hach DW4000, or equivalent, UVVisible Spectrophotometer. For Hg, color adsorption at 412 nm is measured and 477 nm for Pb. A complexing agent is added to the eluate to produce a zero blank and color absorption is again measured. The difference represents the target ion present. Comparison with Existing Methods The new affinity membrane method allows the use of trained assistants rather than senior personnel. For these new Hach test kits, the need for advanced instrument techniques is eliminated such as cold vapor atomic absorption for Hg and graphite furnace operation for Pb. A relatively simple spectrophotometer can be used. The new test kits can also operate in the presence of a number of interferents, and still give excellent results. In addition to time, labor and material savings, the Hach test kits eliminate the use of several chemicals associated with health risks. Results Capture efficiency of Pb is 90-95% from several standard solutions. Estimated detection limit at the 99% confidence level was 10 pgram/liter for a 10 ml

sample. Precision for a 250 pgram/liter standard at the 95% confidence interval was 250 pgram/liter Pb2+ + 5 pgram/liter Pb2+. The high selectivity of the particles usecl make these applications work even in the presenlse of a number of potential interferents. Table I summarized the results of an intetference study for the Hg test kit. Pb results are similar. Note that the Hg level in the sample is at the pgram per liter level while the interferences are at the milligram level, a factor of 1000 greater than the Hg units. SR RAD DISKS8 Process Description IBC has pioneered .Ihe use of molecular recognition technology for radionuclide analysis4, Products for strontium cesium and analysis have been introduced. The process for the Sr Rad Disk@, which contains SuperLigB 620, involves passing a sample acidified to 2M witlh nitric acid through a 47 mm disk positioned on a vacuum filter apparatus at about 50 ml per minute. For direct counting, the disk is simply dried with a 20 ml acetone wash and placed in a planchet for counting. Counting is performed with a low background, gas proportional counter. For liquid scintillation, the disk is placed directly in a scintillation vial containing a cocktail such as Ultima Gold (Packard Division of Canberra of Meridan, Connecticut). The vial is then placed in a liquid scintillation counter. Alternate methods include the use of added tracers. Considerable work has been done to validate results using tracers with the Sr Rad Disk@. Typical is the addition of 85S~: to a sample being tested for g”Sr then passing through an Sr Rad [email protected] disk is counted on a liquid scintillation counter capable of evaluating dual labeled samples. A O-12 KeV window is used for 85Sr and a 12-500 KeV window for gOSr measurement. The reported tracer r e s u l t s w e r e a l l >95% recoveries.

gamma For spectroscopy, the original sample is processed through a disk and a second sample, is 85Sr with spiked processed through a second disk. The original sample is analyzed by either low background or liquid scintillation counting. The disk with the 85Sr is analyzed by gamma spectroscopy. The reported corrected tracer results are >95% recoveries. Interferences are reported in Table II. Comparison with Existing Methods The current US EPA Method 905 for radioactive determination strontium lists 56 steps. This new method using the IBW3M affinity membranes requires only 6 steps (Figure 2). Significant savings in analysis time and costs result by switching to the newer method. Data The data in Table II demonstrates the effectiveness of Sr Rad Disk in separating Sr2+ from the indicated ions. Typical environmental samples may contain an assortm e n t o f t h e s e cationic species. It is of interest that separations can be achieved at the high Mg2+, Caz+ and Na+ levels commonly found in these samples. Solutions containing Ca”+ at levels as high as 500 mg per liter have been tested and Sr2+ has been recovered at quantitative levels from one liter solutions containing one mg of Sr. Typical potable water levels of K+ are less than that shown in Table II. RA RAD DISKS Process Description The process for the Ra Rad Disk, which contains AnaLigB RaOl, is equally simple; a sample is acidified to 2 M with nitric acid and passed through a 47mm disk positioned on a filter apparatus at about 50 ml per minute. Once isolated on the disk, the investigator has a number of quantification options. Radiation can be measured directly from the disk but interpretation is difficult because of the multiple ingrowth paths occurring.

Where EPA Methods are required **‘%a may be determined by eluting the radium with 15 to 20 mL of basic 0.25 M EDTA solution then transferring this solution to a radon bubbler for determination by the radon emanation technique, EPA Method 903.1, 228Ra may be determined by allowing the ingrowth of 228A~ and the decay of 224Ra to occur, then eluting actinium with 15 to 20 mL of 0.5 M nitric acid. The eluate solution is processed for gas flow proportional counting by evaporation on a planchet, or by co-precipitation on yttrium oxalate as described in EPA Method 904.0. **%a and ***Ra can be measured simultaneously by heat sealing the dried disk in a 3.5 mil Mylar envelope and setting aside for 21 days to allow for equilibrium to occur between radium and their pr@@ny. The envelope is then placed directly on a gamma spectrdmeter detector endcap for counting. A multichannel analyzer is u s e d t o i d e n t i f y *14Pb

peaks at 325 KeV and 295 KeV for the quantification of 226Ra and 228A~ peaks at 338KeV, 911 KeV, and 969 KeV for quantifying 228Ra.

Comparison with Existing Methods The current methods for radioactive radium are tedious and time consuming. These new methods using the IBC/3M affinity membrane are making inroads into those laboratories faced with that problem. Again, significant savings in analysis time and costs are being demonstrated.

Data In Table II it is seen that the Ra Rad Disk effectively removes the Ra isotopes present in a one liter sample containing cations that might be present in an environmental sample, Mg2+, Ca*+ Na+ and K+ for example. ihe ibnic radii of Sr2+, Ba*+, and Pb2+ are most similar to Ra*+ therefore those separations are the most difficult.

FUTURE DIRECTIONS IBC has developed a number of technology platforms for use in affinity membranes. These include a library of selective chemistries that are selective for a large number of cations and anions, general selective chemistries that select a group of cations or anions, and chemistries for chiral organic molecules. Alliances with membrane manufacturers that IBC has formed in industry allow access to a number of membrane platforms including the Empore materials described in this article. In the future, the marriage of unique, selective chemistries to robust membrane formats will allow for the technology to be used in a number of process and analytical applications. These new membrane technologies can be expected to allow significant reductions in operating costs, process steps, and separations time in widespread applications in the chemical process and analytical industries.

REFERENCES 1. Goken, G.L., Bruening, R.L., Krakowiak, K.E., and Izatt, R.M., “Metal-Ion Separations Using SuperLigB Materials or AnaLigB Materials Encased in EmporeTM Cartridges and Disks”, presented at American Chemical

Society National Meeting, September, 1997, Las Vegas, NV. 2. R&D Magazine, September, 1996. 3. Heinzig, M.W., “Lead Determination with the New AnaLigB/Pb Ex Method”; Hach, Loveland, CO, 1996. 4. Seely, DC., Osterheim, J.A., “Radiochemical Analyses Using EmporeTM Disk Technology”, Fourth

Conference on methods and Applications of RadioAnalytical Chemistry, Kona, HI, April 6-I 1, 1997.

IBC Advanced Technologies, Inc., 856 East Utah Valley Drive, PO Box 98, American Fork, UT 84003, USA. Tel: + 180 I 763 8400. Fax:+1 801 763 8491.

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