Solid Phase Extraction Products PAGE: 1. Introduction of Solid Phase Extraction (SPE) Why Choose Nano-Micro Tech SPE

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1 Solid Phase Extraction Products PAGE: 1 Introduction of Solid Phase Extraction (SPE) SPE has been used increasingly in chemical analysis and purification broadly, and become the most popular technology of sample preparation. It is employed primarily in purification or concentration of samples before analysis in gas chromatography and liquid chromatography. SPE initially used bonded silica gel as the base sorbents. As technology evolves, polymer microspheres for SPE sorbents are gaining popularity and acceptance because of the shortcomings of bonded silica such as poor ph stability and low absorption capacity. SPE is a column chromatographic analysis process bearing much similarity to high performance liquid chromatography (HPLC) in the mechanism of separation and choice of reagents and solvents. When a sample passes through the extraction column filled with a sorbent, a part or all of the constituents interact with the sorbent and are retained in the column. A solvent or mixture of solvents is then used to wash out interferences. The target analyte is then quickly eluted out of the column by a desired solution of reduced volume. The goal of fast separation, purification, and concentration is therefore achieved. SPE can be carried out with various types of sorbents to perform extraction and separation of different analytes. Depending on the nature of the sorbents, SPE extraction materials can be divided into normal phase sorbents, reverse phase sorbents and ion exchange sorbents. Why Choose Nano-Micro Tech SPE Nano-Micro Tech is taking the lead in advancing the SPE technology. Using the latest techniques in microparticle preparation and surface modification, a series of highly efficient Nano-Micro Uni SPE sorbents have been developed. They have accurate particle sizes, narrow size distribution, large surface area, very high chemical stability, broad application ph range, optimized pore size, and uniform surface functional groups. Uni SPE sorbents therefore readily satisfy the need for high sensitivity in analyzing different materials. Nano-Micro Tech s unique particle technology not only ensures the consistency of the sorbents size and internal pore structure, but also brings about high recovery and good reproducibility for specific adsorption. The monodisperse particles provide benefits of even flow characteristics of the solvents and samples moving through the column. They also prevent the sorbent and analyte from breakthrough and minimize solvent usage. Figure1. Impact of broad particle size distribution on flow. Nano-Micro Tech is the world s first and only company that supplies monodisperse SPE sorbents, which has already impacted the current SPE market traditionally relied on sorbent particles of broad size distribution or irregular shape. In the past, monodisperse sorbents are only used in high-end chromatography and analysis columns. Under optical microscope (Figure 2), the first generation silica sorbents are irregular in shape and the second generation spherical polymer particles are broad in size distribution. The new generation Uni series SPE sorbents by Nano-Micro Tech is spherical with a uniform particle size. They also have a high surface area and high density of functional groups on the particle surface. Figure 2. Optical microscope of various SPE sorbents. Silica-Bonded SPE Second Generation SPE Nano-Micro Uni SPE

2 Solid Phase Extraction Products PAGE: 2 Particle size analysis shows that Nano-Micro Uni particles are monodisperse with Coefficient of Variation (CV) less than 3%, whereas those of the competitors have a broad particle size distribution from 10 to 50 µm with a CV larger than 10%. The monodisperse Nano-Micro Uni particles provide outstandingly high recovery, loading, selectivity, reproducibility, and elution concentration plus a reduction in solvent usage. Figure 3. Particle size distribution of Nano-Micro Uni (Green) and competitor (Red) SPE sorbents. As demonstrated and proven by customers, Nano-Micro Uni series columns have advantages of higher loading and recovery, lower solvent usage, faster flow rate, more concentrated elution and reproducibility than those of the competitors. Nano-Micro Tech s advanced manufacturing facilities and instruments as well as reliable procedures of quality control ensure the quality and reproducibility of the products. World s only supplier of monodisperse SPE sorbents. Recovery equal to or higher than globally known brand name SPE columns. High quality SPE sorbents similar to those employed in chromatography. Highest performance and consistent quality in SPE products Table1. Guide to the selection of Nano-Micro Tech SPE columns Samples soluble in aqueous or highly polar solvents Samples with positive charge or acidic Samples with negative charge or basic Samples neutral without charge UniPS-SAX UniPS-SCX UniBPC-SCX Use UniPS-SCX and UniPS-SAX jointly to remove charged impurities Samples soluble in organic solvents, mild or no polarity Samples with positive charge or acidic Samples with negative charge or basic Samples neutral without charge UniPS-SAX or UniBPC UniPS-SCX or UniBPC Uni BPC

3 Solid Phase Extraction Products PAGE: 3 It is important to select suitable Nano-Micro Tech Uni SPE products based on the need and sample properties. A proper selection of SPE columns will substantially facilitate the extraction process leading to satisfactory results. Listed in Table 1 are different types of sorbents for selection; Table 2 are different sizes of SPE columns and amounts of sorbents. For questions, please contact Nano-Micro Tech service department. Table2. Sample Volume vs. SPE Column Specifications Sample Volume Column Spec., cc/mg 1-10 ml 1 / ml 3 / ml 6 / 200 * For normal phase and reverse phase SPE, the amount of analyte to be extracted usually does not exceed 5% of SPE sorbent. For ion exchange SPE, the capacity of ion exchange resin also needs to be considered. The above table is for reference only. 500 ml ml 6 / 500 UniBPC Series, Water-Wettable Reverse Phase Sorbents Formulated specifically by Nano-Micro Tech, UniBPC SPE sorbents are produced with a special polymerization technique. They are reverse phase sorbents wettable by water with balanced hydrophilicity and hydrophobicity capable of satisfying most SPE needs. Monodisperse microspheres in UniBPC SPE sorbents are made with outstanding ability to retain polar compounds. They are quite suitable for the isolation of compounds of no or medium polarity. Because of a large surface area, UniBPC has a very high adsorption capacity which not only effectively reduces breakthrough, but also substantially decreases the volume of eluent passing through a compact column thereby raising the elution stability and speed. UniBPC is suitable for separation and isolation of a very wide range of organic compounds such as barbiturates, antibiotics, phthalazines, caffeine, drugs, dyes, aromatic oil, lipid soluble vitamins, fungicides, herbicides, agriculture chemicals, hydrocarbon compounds, phenols, phthalates, steroids, surfactants, water-soluble vitamins, etc. UniPS-SCX Series, Mixed-mode Cation Exchange and Reverse Phase Sorbents UniPS-SCX SPE sorbents are polymer microspheres with sulfate groups. Based on a mixed mode of strong ion exchange and hydrophobicity interaction, their function is to separate and analyze basic compounds such as amphetamines, melamine, chloropheniramine, phencyclidine, etc. Because of the high surface area of the microspheres and ion exchange capacity of sulfate groups, UniPS-SCX can effectively and steadily adsorb basic compounds, and retain them well in comparison with known products from the current leading companies. UniPS-SCX sorbents hold the stability of the bonded phase across the entire ph range of 1 to 14 and greatly widen the conditions for separation exploration. Furthermore, UniPS-SCX SPE columns have the unique monodisperse sorbents which enhance flow rate stability and improve recovery thus providing good product reproducibility on large scale manufacturing. UniPS-SAX Series, Mixed-mode Strong Anion Exchange and Reverse Phase Sorbents UniPS-SAX SPE columns are used for the isolation and purification of acidic compounds in basic or neutral medium. Based on polymer microspheres with quaternary ammonium functional groups, UniPS-SAX has a mixed mode of operation combining strong anion exchange and hydrophobicity. UniPS-SAX can quickly and steadily recover acidic compounds from the samples for purification, for example, estrone, adenine, nucleosides, etc.

4 Solid Phase Extraction Products PAGE: 4 Nano-Micro Tech provides a complete line of SPE sorbents as well as packing columns. Uni SPE sorbents Uni SPE columns SPE Operating Procedures Sorbent Conditioning/Activation: Condition SPE column is to completely wet the surface of the sorbent and prepare the sorbent to work with the sample. A properly conditioned sorbent ensures that all surface area is available for interaction with analyte to enhance analysis reproducibility. A typical step begins with rinsing the column bed with a certain amount of organic solvent, followed by a solvent similar in nature to the solvent used to dissolve the sample for analysis. After conditioning the sorbent, keep the SPE column from drying out as it may lower recoveries. Sample Loading/Application: Add the sample in the form of liquid and pass it through conditioned SPE column. Because of one or more types of interaction, for example, non-polar interaction with reverse phase column sorbent or charge interaction with ion exchange sorbent, the target analyte and some interference will be absorbed by the SPE sorbents. The ph and salt concentration of the sample loading liquid should be adjusted to increase the retention of the target analyte and reduce the absorption of interferences in the sorbent. Before loading the sample liquid into the extraction column, it should be filtered or centrifuged to avoid plugging the frit. It is important to control the flow rate when loading the sample. If the flow rate is too high, the pulling force from the mobile liquid could be stronger than the retention exerted by the sorbent especially in ion exchange interaction resulting in breakthrough. Nano-Micro Tech sorbent is composed of monodisperse microspheres with very high retention ability. Even under high flow rate, it still maintains a strong interaction between the sorbent and the retained analyte without incurring any recovery loss. Washing: Use a solvent with proper strength for washing the extraction column. The purpose is to rinse interferences through the column while leaving the target analyte in the column. To reach that goal the strength of the solvent should be strong to remove the absorbed interferences, but also weak enough to leave behind the retained analyte. A commonly used solvent for washing is water or a mixture of water and organic solvent. Elution: Use a solvent or mixture of solvents capable of eluting the target analyte off the sorbents. This step has a strong effect on the recovery. It requires an accurate control of the amount and flow rate of the eluent to ensure the reproducibility of the separation. The strength of the eluent is also important. A strong eluent may remove some intentionally retained interference, whereas a weak eluent may not break up the retention of the target analyte on the sorbent. To increase the analyte concentration or change solvents, the collected eluent containing the target analyte can be dried by blowing nitrogen over it and then re-dissolve the analyte in an appropriate solvent. * For purification of samples, only the first three steps are needed. In the third step, the analyte flows out the column with sample solvent while leaving the interferences on the sorbent.

5 Solid Phase Extraction Products PAGE: 5 Table 3. High Performance Solid Phase Extraction Sorbents Name Characteristics Application Uni BPC UniPS RPC-100 UniPS RPC-300 UniMC-RPC Uni BPC-SCX UniPS-SCX UniPS-SAX Matrix: PolyTATO/St Particle Size: 10, 20, 30, 40, 50, 60µm Surface Area: 850 m 2 /g Matrix: PS/DVB Particle Size: 3, 5, 10, 20, 30, 40, 50µm Surface Area: 800 m 2 /g Average Pore Size:100 Å Matrix: PS/DVB Particle Size: 3, 5, 10, 20, 30, 40, 50µm Surface Area: 700 m 2 /g Average Pore Size:300 Å Matrix: Polyacrylic Particle Size: 10, 20, 30, 40, 50, 60µm Surface Area: 500 m 2 /g Matrix: PolyTATO/St-SO 3 H Particle Size: 5, 10, 20, 30, 40, 50, 60µm Surface Area: 850 m 2 /g Ion Capacity: 1 meg/g Matrix: PS/DVB-SO 3 H Surface Area:700 m 2 /g Ion Capacity: 1 meg/g Matrix: PS/DVB-NR + 3 Cl Surface Area:700 m 2 /g Ion Capacity:0.3 meg/g Monodisperse reverse phase sorbent with balanced hydrophilic/hydrophobic surface, extracting polar and non-polar compounds such as Naproxen, Ibuprofen, Fenoprofen, Indomethacin, Caffeine, Theobromine, Doxepin and many more; Corresponding to Waters Oasis HLP or Phenomenex Strata X. Monodisperse polyaromatic sorbent for rapid adsorption of hydrophobic compounds such as phenols, surfactants, ethidium bromide, antibiotics, amino acids, and peptides, etc.; Corresponding to Varian Bont Elut LMS, Bond Elut PPL or PLRP. Monodisperse polyaromatic sorbent for rapid adsorption of hydrophobic compounds such as phenols, surfactants, ethidium bromide, antibiotics, amino acids, and peptides, etc.; Corresponding to Varian Bont Elut LMS, Bond Elut PPL or PLRP. Monodisperse aliphatic sorbent used for absorption and purification of insulin, fulvic and humic compounds, and antibiotics. Ionic species can be retained through hydrophobic and polar interactions used under isocratic conditions. reversed and strong cationic exchanger function, commonly used for clean up of basic compounds such as Amphetamine, Melamine, Chlorpheniramine, Methadone, Phencyclidine etc.; Corresponding to Waters Oasis MCS, Phenomenex Strata-X-C. reversed and strong cation exchanger function, commonly used for clean up of basic compounds such as Amphetamine, Melamine, Chlorpheniramine, Methadone, Phencyclidine etc.; Corresponding to Waters Oasis MCS, Phenomenex Strata-X-C. reversed interaction and strong anion function used for fractionation of acidic compounds such as Ortho-phosphotyrosine, Estrone Sulfate, Adenine Nucleotides from basic or neutral impurities.

6 Solid Phase Extraction Products PAGE: 6 Table 3. High Performance Solid Phase Extraction Sorbents (continued) Name Characteristics Application Uni BPC-SAX UniMC-SAX UniPS-WAX UniMC-WAX UniPS-WCX UniMC-WCX Matrix: PolyTATO/St- NR + 3 Cl Surface Area:800 m 2 /g Ion Capacity:0.3 meg/g Matrix: Polyacrylic-Quaternary Amine Particle Size: 10, 20, 30, 40, 50µm Surface Area: 300 m 2 /g Ion Capacity: 2.0 meg/g Matrix: PS/DVB-NH 2 Surface Area: 700 m 2 /g Ion Capacity: 0.3 meg/g Matrix: Polyacrylic-NH 2 Particle Size: 10, 20, 30, 40, 50µm Surface Area: 300 m 2 /g Ion Capacity: 2.0 meg/g Matrix: PS/DVB-COOH Surface Area:700 m 2 /g Ion Capacity: 0.3 meg/g Matrix: Polyacrylic-COOH Particle Size: 10, 20, 30, 40, 50, 60µm Surface Area: 300 m 2 /g Ion Capacity: 2 meg/g reversed interaction and strong anion function used for fractionation of acidic compounds such as Ortho-phosphotyrosine, Estrone Sulfate, Adenine Nucleotides from basic or neutral impurities. Monodisperse strong anion sorbent used for purification of strong acid compounds. reversed interaction and weak anion function used for fractionation of acidic compounds such as Ortho-phosphotyrosine, Estrone Sulfate, Adenine Nucleotides from basic or neutral impurities. Weak anion sorbent used for purification of strong acid compounds. reversed interaction and weak cationic exchanger with carboxylic acid group used for isolation and purification of cytochrome C, amines, drugs, metal ions, thrombin etc. Monodispersed polyacrylic weak cationic exchanger with carboxylic acid group used for isolation and purification of cytochrome C, amines, drugs, metal ions, thrombin etc.

7 Solid Phase Extraction Products PAGE: 7 Table 4. SPE chromatographic column Product Name Matrix Particle Size Pore Size Functional μm Å Group BPC60 PolyTATO/St Uni BPC60 PolyTATO/St Uni BPC40 PolyTATO/St Uni BPC30 PolyTATO/St Uni PS40 PS/DVB BPC60-SCX PolyTATO/St SO 3 H Uni BPC60-SCX PolyTATO/St SO 3 H Uni BPC40-SCX PolyTATO/St SO 3 H Column Specification Uni BPC30-SCX PolyTATO/St SO 3 H Uni PS40-SCX PS/DVB SO 3 H BPC60-SAX PolyTATO/St NR + 3 Cl - 20mg/1cc 60mg/3cc Uni BPC60-SAX PolyTATO/St NR + 3 Cl - 20mg/1cc 60mg/3cc Uni BPC40-SAX PolyTATO/St NR + 3 Cl - Uni BPC30-SAX PolyTATO/St NR + 3 Cl - Uni PS40-SAX PS/DVB NR + 3 Cl - BPC60-WCX PolyTATO/St COOH Uni BPC60-WCX PolyTATO/St COOH Uni BPC40-WCX PolyTATO/St COOH Uni BPC30-WCX PolyTATO/St COOH

8 Solid Phase Extraction Products PAGE: 8 Uni PS40-WCX PS/DVB COOH BPC60-WAX PolyTATO/St NR 2 Uni BPC60-WAX PolyTATO/St NR 2 Uni BPC40-WAX PolyTATO/St NR 2 Uni BPC30-WAX PolyTATO/St NR 2 Uni PS40-WAX PS/DVB NR 2