《食品分析》课程实验室操作参考手册（Amersham Biosciences）03 离子交换层析手册 Ion Exchange Chromatography Principles and Methods

The resolution(R)is determined from the chromatogram as shown in Figure 3. Absorption R.=2CYms-Ym) Yolume Fig.3.Determination of the resoution (R)between two peaks The resolution is defined as the dis ance between peak maxima comp If Rs=1.0(Fig.)then%purity has been achieved at 9%of peak recovery the separation parameter. The resolut on achievable in a sy vity,the efficiency and the cap city of o the product of the select tem,the three most important para- meters to control in column chromatography.The analytical expression for Rs is: R=1/4 (N) selectivity capacity efficiency 14

The resolution (Rs) is determined from the chromatogram as shown in Figure 3. Fig. 3. Determination of the resolution (Rs) between two peaks. The resolution is defined as the distance between peak maxima compared with the average base width of the two peaks. Elution volumes and peak widths should be measured with the same units to give a dimensionless value to the resolution. Rs is a measure of the relative separation between two peaks and can be used to determine if further optimization of the chromatographic procedure is necessary. If Rs = 1.0 (Fig. 4) then 98% purity has been achieved at 98% of peak recovery, provided the peaks are Gaussian and approximately equal in size. Baseline resolu￾tion requires that Rs ³1.5. At this value purity of the peak is 100%. Note: A completely resolved peak is not equivalent to a pure substance. This peak may represent a series of components which are not resolvable using the selected separation parameter. 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I@M? selectivity capacity efficiency

9gB如m o 100%A+ 100%E Fig.4.Separation results with different resolutions. Capacity factor k is a measure of the r rom the equation: VRI-Ve capacity factor k= V In the equation for Rk is the average ofk and k2 Adsorption techniques such as ion exchange chrom

Fig. 4. Separation results with different resolutions. Capacity factor The capacity or retention factor k is a measure of the retention of a component and should not be confused with loading capacity (mg sample/ml) or ionic capaci￾ty (mmol/ml). The capacity factor is calculated for each individual peak. For example k for peak 1 in Figure 5 is derived from the equation: In the equation for Rs, k is the average of k1 and k2. Adsorption techniques such as ion exchange chromatography can have high capacity factors since experimental conditions can be chosen which lead to peak retention volumes greatly in excess of Vt (Vt is also often denoted Vm). 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J5 @? ?@ @? @? 7H 3L ?@ 3L @? ?J5? N1 J5 N1 @? ?7H? ?@ 7H ?@ @? J5 ?@ @? ?@ @? ?W.Y ?3L? @? ?3L? @? W.Y? ?N1? ?J5? ?N1? @? ?W.Y @? ?7H? 3L @? W.Y? @? ?@ N1 @? 7H 3L ?@ ?@ @? ?J5? N1 J5 ?3L? @? W.Y? ?@ 7H ?N1? @? ?W.Y ?3L? @? 3L @? O.Y? ?N1? ?J5? N1 @? O20Y @? ?7H? ?3L? @? O20M @?g@?g?@ ?N1? @? O20M 3Lg@?gJ5 3L @? O20M N1g@?g7H V/X? @? O20M ?3L?f@?f?J5? ?N1? @? O20M ?V/Xf@?fW.Y? 3L @?@?@? ?O2@Y? ?O2@0MO20M V/K?e@?e?O.Y ?V4@6X@?O2@0Y? V@@@@Y V/X?f@Khg ?V/Kf@@6Khf V@6Ke@@@@6Xhe @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@)he @?@? @?e@Ke@? ?I4@0Mhf @? 3L?J@@6X@?@?@??@@@@@?W26X?he @? N1?7@?B@@?@?@??@?@?@?7YV1?he @? ?@?@@??@@?@?@??@?@?@?@@@@?he @?@?@? ?3T@@=C@@?@?@??@?@?@?3X?hf ?V+MI40R'?@@@??@?@?@?V4@hf @6K?e@?e?O@? @@@@6X@?O2@@@? @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ @@@0M?@?I4@@@? @0M?e@?e?I@? W&?W26X?W26Xhe?W-Xhf@? W&?W26X?W26Xh?@@6X? *@?7<B1?7<B)T-X?g?7R1hf@? *@?7<B1?7<B)T-X?f?@?B1? N@?@e@?@??@@?)T-XfJ@?@L?he@? N@?@e@?@??@@?)T-Xe?@?C5? @@6T.??@?@e@?@??@V+R@>)X?e7@@@1? @@6T.??@?@e@?@??@V+R@>)X??@@@U? @?40Y??@?3=C5?3=C5eJ@@?,?e@?e@? @?40Y??@?3=C5?3=C5eJ@@?,??@?V1? ?@?V40Y?V40Ye.MI+Y?e@?e@? ?@?V40Y?V40Ye.MI+Y??@@@@? 15 Vt capacity factor k = VR1 - Vt

Adsorption Yo Efficiency cacahawaeihnm where wh is the peak width N=5.54 at half peak height and is expressed as the number of theoretical plates(N)for the column under spe- hae,HE).hcte bed ngh( H=UN Na e.g.acetone. 16

be seen in contrast with the technique of gel filtration where capacity is limited since all peaks must elute within the volume (Vt - V0). Fig. 5. Hypothetical chromatogram. V0 = void volume, VR1 = elution volume for peak 1, VR2 = elution volume for peak 2, Vt = total volume, wb1 = peak width for peak 1, wb2 = peak width for peak 2. Efficiency The column efficiency is related to the zone broadening which occurs on the column and can be calculated from the expression: VR1 where wh is the peak width wh at half peak height and is expressed as the number of theoretical plates (N) for the column under spe￾cified experimental conditions. Efficiency is frequently stated as the number of theoretical plates per metre chromatographic bed, or expressed as H (height equi￾valent to a theoretical plate, HETP), which is the bed length (L) divided by the plate number. H = L/N Since the observed value for N depends on experimental factors such as flow rate and sample loading, it is important that comparisons are done under identical con￾ditions. In the case of ion exchange chromatography, efficiency is measured under isocratic conditions, using a substance which does not interact with the matrix, e.g. acetone. ?@g@? @? ?@g@? @K J@L??W2@@?W2@@?W26X?)T2@@6X?@@@?W26X?)T-X? 7R1??7<?@?*Ue?7<B1?@(Y@?B1?@?@?7<B1?@(R1? ?J@@@L?@e@?V46X?@e@?@H?@e@?@?@?@??@?@H?@? ?7<?B1?3=?@?eS,?3=C5?@??@?C5?3X@?3=C5?@e@? ?@e?@?V4@@?@@0Y?V40Y?@??@@0Y?V4@?V40Y?@e@? ?@?@ @@@@ ?J@@L? ?7@@1? J@@@@? @@@@5? ?@H? ?@?@?@?@?@?@?@?@?@?@?@?@ W& W26X ?W26X? ?@ *@ .MB1 ?.MB1? ?@ N@ J5 ?J5? ?@ ?@ ?W.Y ?*U? ?@ ?@ W.Y? ?N1? ?@ ?@ 7Y ?/KC5? ?@ ?@ @@@@ ?V40Y? ?@?@?@?@?@?@ ?O-K O-K? W-K? ?@ ?W20R46X W20R46X? ?W.R46K? ?@ ?7<?eI/X? 7<e?I/X W.Y??I'@ ?@ J5f?N1? ?J5?fN1 7HfV'L? ?@ 7Hg3L ?7H?f?3L? ?J5?f?N1? ?@ ?J5?gN1 J5g?N1? W.Y?g@? ?@ ?7H?g?@ 7Hh@? 7Hh@? ?@ ?@h?@ @?h@? @?h@? ?@ J5h?@ @?h3L ?@ 7Hh?3L? ?J5?h3L ?J5?hN1 ?@ @?h?N1? ?7H?hN1 ?7H?h?@ ?@ @?he@? ?@he?@ ?@he?@ ?@?@ @?he@? ?@he?@ ?@he?@ ?@ ?J5?he@? ?@he?@ ?@he?3L? ?@ ?7H?he@? J5he?@ J5he?N1? ?@ ?@hf@? 7Hhe?@ 7Hhf@? ?@ ?@hf3L @?he?3L? @?hf@? ?@ ?@hfN1 @?he?N1? ?@ J5hf?@ @?hf@? @?hf@? ?@ 7Hhf?@ @?hf@? @?hf3L ?@ @?hf?@ ?J5?hf@? ?J5?hfN1 ?@ @?hf?@ ?7H?hf@? ?7H?hf?@ ?@ @?hf?@ ?@ @? ?@ ?@ ?@ ?J5?hf?@ ?@ @? ?@ ?@ ?@ ?7H?hf?3L? ?@ 3L ?@ ?@ ?@ ?@ ?N1? ?@ N1 ?@ ?3L? ?@ ?@ @? J5 ?@ J5 ?N1? ?@ ?@ @? 7H ?@ 7H @? ?@ ?@ @? @? ?@ @? @? ?@ J5 @? @? ?@ @? @? ?@ 7H @? @? ?@ @? @? ?@ @? 3L @? ?3L? @? 3L ?@ @? N1 ?J5? ?N1? ?J5? N1 ?@ @? ?@ ?7H? @? ?7H? ?@ ?@ ?J5? ?@ ?@ @? ?@ ?@ ?@ ?7H? ?@ ?@ @? ?@ ?@ ?@ ?@ ?3L? ?@ 3L ?@ ?3L? ?@ ?@ ?N1? ?@ N1 ?@ ?N1? ?@ ?@ @? J5 ?@ J5 @? ?@ J5 @? 7H ?@ 7H @? ?@ 7H 3L @? ?3L? @? 3L ?@ @? N1 @? ?N1? @? N1 ?@ @? ?@ @? @? @? ?@ ?@ @? ?@ @? @? @? ?@ ?@ @? ?@ ?J5? @? ?J5? ?@ ?@ ?J5? ?3L? ?7H? 3L ?7H? ?3L? ?@ ?7H? ?N1? ?@ N1 ?@ ?N1? ?@ @? ?@ @? @? ?@ ?@ ?@ @? ?@ @? ?@ @? @? ?@ ?@ ?@ @? ?@ @? ?@ @? @? ?@ ?@ ?@ @? ?@ @? J5 3L @? J5 ?3L? J5 3L ?@ @? 7H N1 @? 7H ?N1? 7H N1 ?@ @? @? ?@ @? @? @? @? ?@ ?@ @? @? ?3L? @?hf?J5? 3L ?J5? ?3L? ?@ @? ?J5? ?V/X @?hf?7H? V/X? ?7H? ?V/X ?@ @@@@@?hf?7H? N1 @@@@@?heJ5 ?N1? J5 N1 ?@ N@@@H?hfJ5 ?3L? N@@@H?he7H 3L 7H ?3L? ?@ ?@@@ 7H ?V/X ?@@@he?J5? V/X? ?J5? ?V/X ?@ ?3@5hf?J5? V/X?hf?3@5he?7H? ?V/X ?7H? V/X? ?@ ?N@Hhf?7H? ?N1?hf?N@HheJ5 N1 J5 ?N1? ?@ @?hfJ5 3L @?he7H ?3L? 7H 3L ?@ ?O&Y V)K? @? ?V)K @? V)K? ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ ?@ @? ?@ ?@ ?@ @? ?@ @?@?@? @? ?@hf?@?@?@ ?@ ?@ ?@?@?@ @? @?@?@? ?@?@?@L? ?@?@?@ @?@?@?@? ?@?@?@)X ?@?@?@W-X? 3T@T5?@? V+R+Y?@?@? ?3T@T(R/ ?V+R'UN)K? ?3T@T@@?,? ?V+R+MI'U? ?S,? ?@e?@g@?g ?3L?J5g@?g ?N1?7H?W26X?@?@??@?@ @?e@? @? ?@@@ ?.Y? 3T5??7<B1?@?@??@?@ 3L?J5? N@H??@e@?@?@?J@?@ N1?7H? ?@e?3=C5?@?3T&@?@ ?3T5 ?@e?V40Y?@?V+R'?@ ?@6Kh?N@H ?@@@@6K?g@?eW-X? ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@g@?e7R1? ?@@@@0M?he@?@? ?@0M 3T5? V+Y? ?@e?@ ?3L?J5 ?N1?7H 3T5? ?@6KhN@H? ?@@@@@g?@e?@@@@?@? @@@@@@@@@@@@e @Kg@@6Kf@@@@@@e ?I4@0Mf /T26X?W26Xf N@<B1?7YV1f ?@e@?@@@@f ?@e@?3Xg ?@e@?V4@@f ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?f?@e?@KO.?@? ?@@@0Mhe?@@@U?@? ?@0Mhf?@?B1?@? ?@e@?@? ?@e?@ ?3L?J5 ?N1?7H 3T5? ?@K?gN@H? ?@@6K?f?@ ?@@@@6X?e?@e?@ ?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@)?g?@ I4@0M?h?@?@ ?@e?@ ?3L?J5 ?N1?7H 3T5? ?@6KgN@H? ?@@@@6X?e?@e?@@@6T-X @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@)?e?@e?@?W@0R/ ?@@@0Mh?@@@U? ?@0Mhe?@?B@6K? ?@e@@@@ N = 5.54 16 ( ) 2

s and important deve h designed for analytical and micropreparative After bead sie,thes Co/ and the importance of column packing increases in a pro Selectivity ty (a)defines the ability of the s gram(Fig.5)using the expression VR2-Vo VR? as VRI-Vo VRI 8 Good selectivity Bad selectivity low efficiency Fig.6.The effect of selectivity and efficiency on resolution

One of the main causes of zone broadening in a chromatography bed is longitudi￾nal diffusion of the solute molecules. The effect is minimized if the distances avai￾lable for diffusion, in both the mobile phase and stationary phase, are minimized. In practice this is achieved by using small uniform bead sizes and important deve￾lopments in ion exchange chromatography have been the introduction of 10 µm and 15 µm diameter particles such as MonoBeads and SOURCE, to give high effi￾ciency preparative media. The highest efficiency is achieved with the non-porous, 3 µm diameter MiniBeads, designed for analytical and micropreparative applications. After bead size, the second major contributory factor to efficiency is good experi￾mental technique. Badly, unevenly packed chromatography beds and air bubbles will lead to channelling, zone broadening and loss of resolution. Good separations require well packed columns and the importance of column packing increases in direct proportion to the performance required. Selectivity The selectivity (a) defines the ability of the system to separate peaks i.e. the distan￾ce between two peaks. The selectivity factor can be calculated from the chromato￾gram (Fig. 5) using the expression k2 VR2 - V0 VR2 a = = Å k1 VR1 - V0 VR1 Good selectivity is a more important factor than high efficiency in determining resolution (Fig. 6) since Rs is linearly related to selectivity but quadratically related to efficiency. This means that a four fold increase in efficiency is required to dou￾ble the resolution under isocratic conditions. Fig. 6. The effect of selectivity and efficiency on resolution. 17 ?W2@6X @?hf@?hf@?f?@ @@@6X?h@?hf@?hf@? 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experimental conditions,and thus the selectivity,can be manipulated which gives ion exchange chromatography the potential of extremely high resolution Capacity may be expressed as total ionic capacity,available capacity or dynamic capacity. The total ionic capacity is the number of charged substituent s ram dry actual amount of protein which can be bound to an ion exchanger,under dc lable capacity for is refer The properties of the protein. The properties of the protein whi h determine the available tionship.The capacity of an ion exchanger is thus different for different proteins. On oecules which ar small enough to enter the pores will exhibit a highe r available capacity than those molecules which are restricted to the charged substituents on the surface of the gel Similarly,since the interaction is ionic,the protein's charge/pH relationship must be such that the p t net cha rge,at a su number of the charged substituens.High availabl capacity is obtained by having a matrix w croporous and high 18

Selectivity in ion exchange chromatography depends not only on the nature and number of the ionic groups on the matrix but also on the experimental conditions, such as pH and ionic strength. It is the ease and predictability with which these experimental conditions, and thus the selectivity, can be manipulated which gives ion exchange chromatography the potential of extremely high resolution. Capacity The capacity of an ion exchanger is a quantitative measure of its ability to take up exchangeable counter-ions and is therefore of major importance. The capacity may be expressed as total ionic capacity, available capacity or dynamic capacity. The total ionic capacity is the number of charged substituent groups per gram dry ion exchanger or per ml swollen gel. Total capacity can be measured by titration with a strong acid or base. The actual amount of protein which can be bound to an ion exchanger, under defi￾ned experimental conditions, is referred to as the available capacity for the gel. If the defined conditions include the flow rate at which the gel was operated, the amount bound is referred to as the dynamic capacity for the ion exchanger. Availa￾ble and dynamic capacities depend upon: The properties of the protein. The properties of the ion exchanger. The chosen experimental conditions. The properties of the protein which determine the available or dynamic capacity on a particular ion exchange matrix are its molecular size and its charge/pH rela￾tionship. The capacity of an ion exchanger is thus different for different proteins. On a porous matrix used for ion exchange chromatography, molecules which are small enough to enter the pores will exhibit a higher available capacity than those molecules which are restricted to the charged substituents on the surface of the gel. Similarly, since the interaction is ionic, the protein’s charge/pH relationship must be such that the protein carries the correct net charge, at a sufficiently high surface charge density, to be bound to a particular ion exchanger under the chosen buffer conditions. The properties of the ion exchange matrix which determine its available capacity for a particular protein are the exclusion limit of the matrix, and the type and number of the charged substituents. High available capacity is obtained by having a matrix which is macroporous and highly substituted with ionic groups which maintain their charge over a wide range of experimental conditions. Non-porous 18