Bio-Rad MP-1M User Manual

AG®1, AG MP-1

and AG 2

Strong Anion Exchange Resin

Instruction

Manual

Table of Contents

Page

Section 1 Introduction ............................................ 1

Section 2 Technical Description............................. 1

Section 3 Mechanism .............................................. 5

Section 4 Resin Conversion.................................... 10

Section 5 Instructions for Use................................ 14

5.1 Batch Method.................................................... 14

5.2 Column Method................................................ 15

Section 6 Sample Protocols

Section 7 Applications............................................. 23

Section 8 Product Information............................... 32

Section 9 Technical Information............................ 39

for Anion Exchange Resins.................... 18

6.1 Separation of Metal Ions................................... 18

6.2 Glucose Binding on AG 1-X8 Resin ................ 21

Section 1 Introduction

AG 1, AG MP-1 and AG 2 resins are strongly basic anion exchangers. They are capable of exchanging anions of acidic, basic, and neutral salts, and ampholytes on the basic side of their pI. Strong anion exchange resins are used for sample preparation, enzyme assays, metal separations, and peptide, protein, and nucleic acid separations.

Section 2 Technical Description

Strongly basic anion exchange resins are available as Analytical Grade AG 1 and AG 2 resins, AG MP-1 macroporous resin, and Biotechnology Grade AG 1 resin. The Analytical Grade AG 1, AG MP-1 and AG 2 resins have been exhaustively sized, purified, and converted to make them suitable for accurate, reproducible analytical techniques. Biotechnology Grade AG 1 resin is analytical grade resin which is certified to contain less than 100 microorganisms per gram of resin.

AG 1 and AG 2 resins are strongly basic anion exchangers with quaternary ammonium functional groups attached to the styrene divinylbenzene copolymer lattice. The amount of resin crosslinkage determines the bead pore size. A resin with a lower percentage of crosslinkage has a more open structure permeable to higher molecular weight substances than a highly crosslinked resin. It also has a lower physical resistance to shrinking and swelling, so that it absorbs more water and swells to a larger wet diameter than a highly crosslinked resin of equivalent dry diameter. For example, the lower crosslinked resins, particularly AG 1-X2 2% crosslinked resin, are useful for the sorption and fractionation of relatively high molecular weight substances such as peptides, ribo- and deoxyribonucleotides, and uranium. The higher crosslinked resins, particularly AG 1-X8 8% crosslinked resin, are used for sorption, exchange, and separation of low molecular weight inorganic anions, and in applications such as cyclic nucleotide assays and fractionation of organic acids. Table 1 shows the approximate molecular weight

exclusion limits in water for resins of various crosslinkages.

Table 1. Approximate Molecular Weight Exclusion Limits for Ion Exchange Resins in Water

Percent Approximate MW Exclusion Limit

Crosslinking for Globular Molecules

2% 2,700 4% 1,400

8% 1,000 10% 800 12% 400

AG 2 resin is similar to AG 1 resin, but is slightly less basic and slightly less resistant to oxidation due to differences in the structure of the quaternary functional group. It offers advantages in certain applications. For example, it is capable of separating sugars, sugar alcohols, and glycosides using a step gradient and borate buffers without isomerizing some sugars, as AG 1 resin tends to do.

Each AG 1 resin is supplied in the chloride form. Selected resins are available in the acetate, formate, and hydroxide form. These ionic forms may be considered more activated forms than the chloride form, as may be deduced from the order of selectivity information given in Tables 2 and 3. AG 1 resins purchased in the more active forms may be converted to any other form. The chloride ion, because of its higher selectivity for the resin, is relatively difficult to replace with formate, acetate, hydroxide, or fluoride. Thus, if various ionic forms are to be used, the formate or acetate forms provide flexibility and convenience (see Table 3). Formate and acetate forms may be used to separate most low molecular weight biological compounds, such as nucleotides, hormones, peptides, and carboxylic acids. AG MP-1 resin is the macroporous equivalent of AG 1 resin. Its effective surface area approximates 23 square meters per dry gram, 20% porosity.

The physical properties of the resins are listed in Table 2. The anion exchange resins are thermally stable and resistant to solvents (alcohols, hydrocarbons, etc.), reducing agents, and oxidizing agents.

Table 2. Guide to Analytical Grade Anion Exchange Resins

Resin Active Order of Thermal Solvent to Oxidizing Type Group Selectivity Stability Stability Agents

AG 1

R-CH2N+>phenolate OH-form, Very good Slow

and (CH3)3>HSO4>ClO3fair to 50 °C; solution AG MP-1 >NO3>Br> Cl-and other in hot 15% Resins CN>HSO3> forms, good HNO3or

AG 2 R-CH Resin (CH3)2>HSO4>ClO3to 30 °C; Cl

NO2>Cl> to 150 °C conc. H2O HCO3>IO3> H2COO>Ac> OH>F

N+phenolate>I OH-form, Very good Slow

C2H4OH >NO3>Br> forms, good in hot 15%

CN>HSO3> to 150 °C HNO3 or NO2>Cl>OH conc. H2O >IO3>H2COO >Ac>F

Resistance

solution

Section 3 Mechanism

In an ion exchange procedure, the counterions on the resin are replaced by sample ions that have the same charge. With anion exchange resins such as AG 1 and

AG MP-1, neutral species and cations do not interact with the resin. In the chloride form of AG 1, AG MP-1, and AG 2 resin, the counterion on the resin is Cl

. A resin can be converted from one ionic form to another. Usually the resin is used in an ionic form with a lower selectivity for the functional group than the sample ions to be exchanged. The sample ions are then exchanged onto the resin when introduced, and can be eluted by introducing an ion with higher affinity for the resin or a high concentration of an ion with equivalent or lower affinity. Table 3 shows the relative selectivity of various counterions. In general, the lower the selectivity of the counterion, the more readily it exchanges for another ion of like charge. The order of selectivity can also be used to estimate the effectiveness for different ions as eluants, with the most highly selective being the most efficient. Finally, the order of selectivity can be used to estimate the difficulty of converting the resin from one form to another. Conversion from a highly selected to a less highly selected form requires an excess of the new ion.

Table 3. Relative Selectivity of Various Counterions

Relative Selectivity Relative Selectivity

Counterion for AG 1 and for AG 2 Resin

AG MP-1 Resins

1.0 1.0 Benzene sulfonate 500 75 Salicylate 450 65 Citrate 220 23

I Phenate 110 27

HSO

ClO

HSO

BrO

HCO

HPO

Formate 4.6 0.5

175 17

85 15 74 12 65 8 50 6 28 3 27 3 27 3 24 3 22 2.3

6.0 1.2

5.5 0.5

5.0 0.5 Acetate 3.2 0.5

Propionate 2.6 0.3

1.6 0.3

The AG 1 and AG MP-1 resins are available in several particle size ranges. The flow rate in a chromatographic column increases with increasing particle size. However, the attainable resolution increases with decreasing particle size and narrower size distribution ranges. Particle size is given either in mesh size or micron size. The larger the mesh size number, the smaller the particle size. Table 4 shows wet mesh and equivalent micron diameters.

Table 4. Wet Mesh and Equivalent Micron Diameters

Wet Mesh

(U.S. Standard)

16 20 40 50 80 100 140 200 270 325 400

Micron Diameter

(1 µm = 0.001 mm) 1,180 850 425 300 180 150 106 75 53 45 38

Large mesh material (20-50 and 50-100 mesh) is used primarily for preparative applications and batch operations where the resin and sample are slurried together. Medium mesh resin (100-200) may be used in

batch as well as column applications. Medium mesh is an ideal, general purpose particle size for use in analytical and preparative scale column chromatography. Fine mesh material (200-400 and minus 400 mesh) is used for high resolution analytical separations.

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