An initial estimate of the weight of injected sample is possible, based on 1 Equation 15.5, 2 a value ofα, 3 the column capacity w s, and 4 a rough estimate of sample purity %-product in
Trang 1746 PREPARATIVE SEPARATIONS
0.005 g A 0.05 g B
0.05 g A 0.005 g B
A
B
0.5 g A
5 g B
5 g A 0.5 g B T-P
2 g A
20 g B
20 g A
2 g B
(a)
(b)
(c)
Figure15.9 Isocratic separation of a two-component sample as a function of sample size Computer simulations based on the Langmuir isotherm; Conditions: 250× 50-mm column (7-μm), 210 mL/min flow rate, N = 800; k = 1 and 1.5, respectively Sample weights
indi-cated in figure Adapted from [9]
(Section 9.3.1) If there is any doubt as to the identity of the product peak in these initial separations, this can be confirmed by a separate injection of pure product
Step 2 of Figure 15.8 Following the adjustment of %B in step 1, separation
conditions are varied for the best possible separation of the product peak from adjacent impurity peaks Usually the product peak should be placed midway between the adjacent impurities on each side Previous chapters provide a detailed discussion
of how selectivityα can be optimized, depending on the kind of sample and whether
NPC or RPC is used (see Table 2.2 for conditions that affect α) Because of the
importance of maximizingα in prep-LC (Eq 15.5), more work on step 2 may be
warranted than for analogous analytical separations Unlike the case of analytical separation, in prep-LC it is important—if possible—to avoid separation conditions that result in>50% ionization of the product molecule (see Section 15.3.2.1 and
the discussion of Fig 15.6) Large changes inα (without ionizing the product) are
most likely to be achieved by a change in B-solvent or the column
Trang 2The same conditions used for this optimized separation can be used to assay fractions collected during prep-LC (but with the initial small-scale column) If
the resolution of the product peak is R s 2 (desirable for prep-LC), the assay separations can be carried out with a shorter column and increased flow rate to speed up fraction analysis
Step 3 of Figure 15.8 An initial estimate of the weight of injected sample is
possible, based on (1) Equation (15.5), (2) a value ofα, (3) the column capacity
w s, and (4) a rough estimate of sample purity (%-product in the sample) For a
150× 4.6-mm column with 10-nm pores, and a product that does not ionize in the mobile phase, the column capacity can be estimated as w s≈ 150 mg (≈100 mg/g of column packing), from which the weight of sample for T-P separation
is w s ≈ (1/6) × (150) × ([%-product]/100) × ([α − 1]/α)2; if the product is partly
or completely ionized, the allowed sample weight can be much lower than the latter estimate Following a separation with this estimated sample weight, sample weight can be increased or decreased by trial and error to achieve T-P separation Alternatively, the use of fully automated equipment allows a number of trial separations where sample size is varied; from such experiments the correct sample weight can be quickly determined Once a promising separation is identified in this way, the product peak should be collected and assayed, in order to confirm≈100% recovery and purity It may also be worthwhile at this point to see if an increase in flow rate can maintain the latter separation, but with a reduced run time The object
of prep-LC is usually maximum production of purified product in minimum time, which favors short run times
Step 4 of Figure 15.8 The final step in Figure 15.8 (scale-up) completes method
development The desired scale-up factor can be calculated from the results of step 3 (see Section 15.1.2.1), taking into account the availability of (1) columns of different i.d and (2) equipment that can provide the required flow rate A final separation with this larger column can then be carried out, allowing verification of the product recovery and purity obtained with the previous (smaller) column Scale-up should result in essentially the same purity and recovery of product as found for the small-scale separation
15.3.2.5 Fraction Collection
The usual goal of fraction collection—whether carried out manually or with an automated system—is to obtain a maximum yield of adequately pure product, with
as little effort as possible The initial step is to collect a number of fractions across the product peak, followed by their analysis for content and purity These results can be used to determine the time during which the product peak should be collected (best ‘‘cut points’’) in the final separation(s), so as to achieve the purification goals (Section 15.4.1) Prior to finalizing the prep-LC procedure, a trial separation can be carried out to confirm the latter cut points A few small fractions around each cut point can be collected for this purpose Once the separation procedure and cut points are finalized, only a single product fraction need be collected However, additional fractions can provide insurance against unanticipated changes in the separation
Trang 3748 PREPARATIVE SEPARATIONS
A detailed study of severe column overload (i.e., sample sizes larger than those that correspond to T-P separation) is beyond the scope of the present book; however, it
is useful to consider certain aspects of such separations Such severely overloaded separations can result in a greater production of purified product per hour with reduced consumption of the mobile phase, as well as requiring smaller columns and smaller scale equipment—all of which can be of great practical importance The disadvantage of such separations is that more effort is required for method development, and individual separations usually require the collection and analysis
of several product fractions so that only adequately pure material is obtained It may also be necessary to re-process product fractions that are insufficiently pure The interested reader is referred to several texts [4, 9, 10] for further study
15.4.1 Recovery versus Purity
As sample weight increases to the point of severe overload, the prediction of individual peak shapes becomes more uncertain This is illustrated in Figure 15.9
for small-sample (Fig 15.9a), T-P (Fig 15.9b), and severely-overloaded (Fig 15.9c)
separations of a sample where the relative concentrations of the two components
A and B vary from 1:10 to 10:1 Thus we can see what happens to a minor peak that elutes either before or after the (larger) product peak For severe overload
(Fig 15.9c), when the impurity peak precedes the product peak, it is displaced
and compressed so that peak height increases There is also some overlap of the two peaks When the impurity peak follows the product peak, it is dragged into the product peak (so-called tag-along effect) The relative importance of these two effects can be difficult to predict, so the optimum sample weight must be determined experimentally This optimum weight will also vary with the relative concentrations
of product and impurities
0
10
20
30
40
50
60
70
80
90
100
Purity (%)
bovine insulin porcine insulin
Figure15.10 Plot of recovery against product purity for a given sample load (5 mg) for a 1:1 mixture of bovine and porcine insulins Bovine insulin (solid line); porcine insulin (dashed line) Conditions: 250× 4.6-mm (20-μm) C8 column; 10–29% acetonitrile–0.1% aqueous
TFA in 10 minutes Adapted from [13]
Trang 4(c) (b)
Isocratic elution
Gradient elution
A,
A’
B, B’
A A’
B, B’
A A’
B, B’
3
10 11 12 13 (min) 10 11 12 13 (min) 10 11 12 13 (min)
3 4 5 6 (min) 3 4 5 6 (min) (min)
6 5 4
A,
A’
B, B’
A A’
B, B’
B, B’
Figure15.11 Similar effects of column overload in corresponding separations: (a) isocratic and (b) gradient elution Separation of two xanthines (β-hydroxyethyltheophylline [A] and
7β-hydroxypropyltheophylline [B]) with k (isocratic) equal k∗(gradient) Sample weights
shown in figure Peaks labeled Aand Bare for the injection of samples of pure of A or B; peaks labeled A and B are for the separation of mixtures of A and B Adapted from [2]
15.4.2 Method Development
The selection of an optimum sample size for severely overloaded prep-LC can start
with an optimized T-P separation (as in Fig 15.2b), followed by injecting successively
larger sample weights For each separation (or sample weight), a number of fractions that surround and include the product peak are collected and assayed, and the results are collected within a spreadsheet Based on pooling the purest fractions, product recovery (or yield) can then be plotted against product purity, as in the examples of Figure 15.10 for two different products (bovine and porcine insulin) Similar plots will result for different sample weights, allowing selection of the most attractive sample weight To a first approximation, the maximum weight of purified product with some predetermined purity (e.g., 98%) can be established in this way For the
Trang 5750 PREPARATIVE SEPARATIONS
examples of Figure 15.10 the recovery of 98%-pure material would be 92 and 42%, respectively, for bovine and porcine insulin Similar plots for different sample sizes might result in a better compromise between the weight of purified product and its recovery
The separation of Figure 15.10 was carried out with RPC using gradient elution However, the same approach would be used for NPC or isocratic elu-tion The principle of estimating sample size is the same regardless of whether isocratic or gradient elution chromatography is being used Optimum separation conditions— other than sample weight—may not be the same for T-P as compared with severely overloaded separation In both cases a very considerable experimental effort can be required in order to simultaneously optimize both sample size and separation conditions
15.4.2.1 Column Efficiency
As the (small-sample) separation factor α0 increases for T-P separation, and a larger sample weight becomes possible, the effect of the small-sample column plate
number N0 on product resolution decreases (Eq 15.4a) Smaller values of N0 are therefore required, with little effect on the recovery or purity of the product This
is no longer the case for severely overloaded separations Displacement effects as in
Figure 15.9c, for a small peak that precedes a large peak, can improve separation This is better shown in the isocratic separations of Figure 15.11c, where in the
absence of sample displacement peak B would completely overlap peak A Because
of displacement, there is some separation of the two peaks (compare the similar
situation of Fig 15.9c) Sample displacement is highly advantageous in severely
overloaded separation, but unlike the case of T-P separation, it appears to be
favored by higher values of N0[11] As a result large-scale separations are generally carried out with moderately efficient columns that use particle diameters of 10 to 15 μm
15.4.2.2 ‘‘Crossing Isotherms’’
This unusual behavior can arise for solutes with different saturation capacities An example is seen in the separation of alcohols from phenols [13], where alcohols can
have significantly higher saturation capacities than phenols Figure 15.12a shows
the RPC separation of benzyl alcohol and phenol for a small sample (10 μg),
where benzyl alcohol elutes last Figure 15.12c shows the same separation for
a larger sample (1-mg phenol, 3-mg benzyl alcohol); the two peaks are almost completely separated When the order of elution of a phenol and alcohol are reversed, while the weights of early- and late-eluting compound are held the same
(as in Fig 15.12a,c), a very different result is obtained; see Figure 15.12b,d for the separation of phenethyl alcohol and p-cresol In the overloaded separation of Figure 15.12d, peak overlap is almost complete—contrasting strongly with the analogous separation of Figure 15.12c.
The reason for the contrasting separations of Figure 15.12 is somewhat complicated, but can be pictured in terms of ‘‘crossing isotherms’’—as illustrated in
Figures 15.12e (phenol and benzyl alcohol) and Figure 15.12f (phenethyl alcohol and p-cresol) For the separation of phenol and benzyl alcohol, the isotherms do not cross (Fig 15.12e) because phenol is always more retained than benzyl alcohol, and
Trang 6(min) 10 8 6
10 8 (min) 10 12 (min)
8 10 12 (min)
OH CH2OH
CH2OH
CH2OH
CH3
CH3
CH3
CH2CH2OH
CH2CH2OH
CH2CH2OH
OH
OH
OH
OH OH
Figure15.12 Example of crossing-isotherm behavior, with decrease in allowed sample weight for touching-peak separation Conditions: 150× 4.6-mm (5-μm) C18column;
methanol-water mobile phases; 1.0 mL/min (a) 3μg phenol and 7 μg benzyl alcohol (BA);
(b) 3 μg 2-phenylethanol (PE) and 7 g p-cresol; (c) same as (a), except 4-mg sample weight; (d) same as (b), except 4-mg sample weight; (e, f ) hypothetical isotherms corresponding to separa-tions of phenol-benzyl alcohol and 2-phenylethanol/p-cresol, respectively Adapted from [14].
the two compounds are well separated For the separation of phenethyl alcohol and
p-cresol (Fig 15.12f ), the greater retention of p-cresol for a small sample, combined
with its smaller column capacity, leads to crossing of the isotherms for a sufficiently large sample For the latter sample weight, the two compounds are equally retained,
with no separation—as observed in the separation of Figure 15.12d The latter
explanation is intentionally oversimplified
While gradient elution is often used for analytical separations and small-scale prep-LC, its use for large-scale separations can be less convenient and more costly
Trang 7752 PREPARATIVE SEPARATIONS
An exception to this generalization occurs for the separation of large biomolecules because their isocratic retention can vary greatly for small changes in %B (Section 13.4.1.4), making isocratic elution impractical or impossible An example of the industrial-scale purification of biosynthetic human insulin by gradient elution is discussed in Section 13.9.2 Method development for gradient separations closely parallels that for isocratic separation, as discussed in Chapter 9 Thus, when the
gradient retention factor k∗is the same as k for isocratic elution, and other conditions
are the same (‘‘corresponding’’ separation; Section 9.13), the separation of a product peak from its impurities will be the same for both isocratic and gradient elution Similarly any change in conditions that can improve isocratic selectivity can be used
in the same way to improve gradient separation Consequently virtually everything that applies for isocratic prep-LC in Section 15.3 applies equally for gradient elution This will simplify our remaining discussion of gradient prep-LC in this section For additional information about gradient prep-LC, see [2]
15.5.1 Isocratic and Gradient Prep-LC Compared
Figure 15.11a– c was used previously to compare the effect of sample size on an
isocratic separation, where only the weights of two compounds in the sample are
varied A similar series of separations is shown in Figure 15.11d –f for the gradient
separation of the same sample (compounds A and B) with the same conditions (except that gradient steepness replaces %B) In each case chromatograms are overlaid for (1) the separate injection of each compound (A and B), and (2) the injection of the mixture (A plus B); see the related discussion of Section 2.6.2 and Fig 2.24 The isocratic and gradient chromatograms for separations of equal sample
weights (e.g., Fig 15.11a vs d, b vs e, c vs f ) are seen to be virtually identical,
with the exception of the more rounded (‘‘shark-fin’’ shaped) peaks for overloaded
gradient elution in Figure 15.11d –f
This similarity of isocratic and gradient separations under comparable con-ditions was discussed in Section 9.1.3 For equivalent results as in Figure 15.11 for ‘‘corresponding’’ isocratic and gradient separations, the retention factor for
each peak in the isocratic (k) and gradient (k∗) separations must be approximately equal, and all other separation conditions (column, A- and B-solvents, flow rate,
temperature) must be the same In the gradient separations of Figure 15.11d –f , separation conditions were adjusted so that (small-sample) values of k∗ were equal
to isocratic values of k in Figure 15.11a–c As discussed in Section 9.2, values of k∗
are determined by gradient conditions:
k∗= 0.87t G F
Here t G is the gradient time, F is flow rate, Δφ is the change in φ ≡ 0.01 × (%B)
during the gradient, S is related to the change in k for a given change in φ or %B
(equal to d[log k] /dφ), and V m is the column dead-volume (mL)—which can be
determined from an experimental value of t0 and the flow rate F (Section 2.3.1;
V m = t0F) Changes in isocratic separation as a result of a change in %B can be
replicated in gradient elution, by a change in gradient time t G (Eq 9.5), so that the
new values of both k and k∗are the same
Trang 8B
f*
t G= 30
SA= S B
(Parallel)
SA< S B
(Divergent)
SA> S B
(Convergent)
A
B
A
B
(c)
Figure15.13 Effect of unequal values of S on the overload separation of two peaks by gradient
elution Adapted from [2]
15.5.2 Method Development for Gradient Prep-LC
The general plan of Figure 15.8 for isocratic method development can be followed for gradient elution also, with a few changes The selection of initial conditions will be virtually the same, except than the initial separation will be carried out with a 0–100%B gradient, followed by narrowing the gradient range in most cases
so as to shorten run time (Section 9.3.4) The optimization of the initial gradient separation for improved selectivity (step 2) can be carried out in the same way as for isocratic elution (Section 9.3.3), except that the goal is a maximum resolution for the product peak, rather than acceptable resolution of all peaks in the chromatogram Aside from the choice of conditions for maximumα, the gradient program can be
further modified so as to minimize separation time, while maintaining the resolution
of the product peak from its adjacent impurity peaks (see Sections 9.3.4, 9.3.5) Maximizing sample weight (step 3) and scale-up (step 4) then proceed in exactly the same way as for isocratic prep-LC For further details, see [2]
A complication not found for isocratic prep-LC is observed occasionally in
gradient prep-LC When two adjacent peaks have different values of S, this can affect
the sample weight for T-P separation, as illustrated conceptually in Figure 15.13
Figure 15.13a gives the T-P gradient separation for the case of equal S-values for
two compounds (‘‘parallel’’ case), which is often a close approximation for most
samples At the top of Figure 15.13a is a plot of log k∗ versusφ* for each peak,
whereφ* is the value of φ (≡0.01 × %B) when the peak is at the column mid-point
Trang 9754 PREPARATIVE SEPARATIONS
(see Eq 9.5a); values of φ* in Figure 15.13 track the time during the separations
shown at the bottom of Figure 15.13a– c The dotted lines connect the log k∗–φ∗ plot for compounds A and B to (small-sample) peaks in the chromatogram below
The values of k∗andφ* for each peak are determined by gradient conditions, with
an assumed gradient time of 30 minutes for each separation in Figure 15.13 Now assume that a large enough sample weight has been injected to allow
peak B to cover the space between the two small-sample peaks (T-P separation), giving the wide cross-hatched peaks in the chromatogram of Figure 15.13a We see that the vertical separation of the two log k∗ versusφ plots is constant and equal
to logα for each value of φ Thus, at the beginning of elution of overloaded peak
B (at a lower value of φ*, corresponding to the elution of a small sample of A),
α is the same (= α o) as at the end of elution; that is, the separation factor is not
a function of sample weight (Note that Eq 15.5, which relates sample weight for T-P separation to values ofα o , assumes approximately equal values of S for the two
adjacent peaks.)
Figure 15.13b is similar to that of 15 13a (same weight of injected sample for
T-P separation in Fig 15.13a), except that now the plots of log k∗ versus φ* are
no longer parallel but diverge for lower values ofφ* (‘‘divergent’’ case); that is, the
value of S for compound B is greater than for compound A For higher loading of
the column (at lower values of φ*), the vertical separation of the two log k∗–φ*
curves increases, corresponding to an increase inα with increasing sample weight A
larger value ofα means a larger sample weight for T-P separation (Eq 15.5), so the
same injected weight of sample as in Figure 15.13a is no longer sufficient to cause
the peaks to touch That is, the divergent case allows a larger weight of injected
sample (other factors equal), compared to the equal-S case of Equation (15.5) and Figure 15.13a.
Figure 15.13c illustrates the third possibility: log k∗–φ* plots that converge
for smaller φ* (‘‘convergent’’ case); namely S for compound B is less than for
compound A Now α decreases with increasing sample weight, and injection of
the same weight of sample as in Figure 15.13a for T-P separation leads to a more
rapid column overload with overlap of the two peaks When convergent behavior
is suspected (because of lower than expected sample weights for T-P separation), further changes in separation conditions should be considered—with the goal of reversing the elution order of the two peaks (product and nearest impurity bands)
A similar approach can also be used to minimize the problem of crossing isotherms
(Section 15.4.2.2) For a further discussion of the consequences of unequal S-values
in gradient prep-LC, see [2]
Production-scale separations are well beyond the scope of this book, but the simple theory and practice outlined here is still pertinent Separations of this kind are usually highly optimized, so as to result in the highest possible production rate for the desired product, with the required purity and recovery At this scale, process economics are of primary importance; the goal is a combination of purity, recovery, and production rate that yield the lowest cost per kg of the desired product, including the cost of removing mobile phase from the purified product Separation conditions
Trang 10are usually developed empirically, using the approach of Figure 15.10, for samples much larger than correspond to T-P separation For an example, see Section 13.9 for the production-scale separation of rh-insulin
For production-scale separations, the use of simulated moving bed (SMB) techniques are increasingly important This is a binary separation technique that relies on the simulation of a countercurrent separation system by the use of multiple columns and switching valves The reader is referred to specialized texts on this topic [14] Although this approach has been used for many decades in the petroleum
industry (the Molex process for isolation of p-xylene) and the food industry (the
Sorbex process for fructose-rich syrups), it has only been used by the pharmaceutical industry since the 1990s The use of countercurrent separation with a continuous sample input and product output gives a more effective use of the chromatographic bed than the traditional procedures discussed in this chapter SMB thus reduces both the size of the columns and the amount of solvent used (and therefore costs);
it is widely used The column efficiency required for countercurrent separations
is relatively low, which allows the use of very short ‘‘pancake’’ columns For example, successful enantiomer separations are carried out under SMB conditions with columns of 800- or 1000-mm i.d., but only 100 mm in length (e.g., packed with 20-μm particles)
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NJ, 2007, ch 7
3 Azeotropes: http://en.wikipedia.org/wiki/Azeotrope (data)
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and Non-linear Chromatography, 2nd ed., Academic Press, Boston, 2006.
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Agents, Wiley-VCH, New York, 2005.
11 G Guiochon and S Ghodbane, J Phys Chem., 92 (1988) 3682.
12 G B Cox and L R Snyder, J Chromatogr., 590 (1992) 17.
13 G B Cox and L R Snyder, J Chromatogr., 483 (1989) 95.
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Wiley-Blackwell, New York, 2005