CHAPTER 9

Fraction Collection
Kevin D. Altria
1. I n t r o d u c t i o n Fraction collection by CE is a potentially important area in which there are several approaches necessary (18) to yield sufficient material following fraction collection. Even using optimized conditions, the amounts collected employing preparative CE are extremely small and are on the order of nanogram quantities. However, these levels can be useful to perform operations such as: (1) peak purity verification by reinjection on CE or onto another analytical method, such as HPLC, and (2) sample identity confirmation by characterization of the material by another technique, such as amino acid sequencing or off-line mass spectrometry. The key advantage to preparative CE compared to, for example, preparative HPLC, is that the fraction collection can be readily performed from tiny sample volumes, since typical CE injection volumes are only 150 nL/injection. The technicalities of performing preparative CE are demanding and require use of a programmable autosampler. In principle, however, the operation of preparative CE is identical to normal CE, and standard equipment is employed. Sample solutions are injected, separated, and detected in a conventional fashion. The separated components are then collected as they emerge from the detector end of the capillary. To allow this collection, it is necessary to stop the separation voltage and position the autosampler tray such that the capillary then dips into a collection vial, containing a few microliters of water or dilute buffer. The voltage is then restarted, and the required sample components migrate out of the capillary into the collection vial. When all the sample zone has emerged from the capillary, the voltage is stopped.
From Methods in Molecular Biology, Vol 52 Capillary Electrophoresis Edited by K Altria Copyright Humana Press Inc , Totowa, NJ

99

100

Altria

The times required for calculating the exact time for the switching between vials can be calculated from the total capillary length, the distance to the detector, and the migration time of the peak of interest (1). Therefore, the switching time to collect a peak detected at 7.8 min on a 57-cm long capillary (50 cm to the detector) is: 57/50 X 7.8 = 8.9 mm (1)

The exact time of the switch should, however, be programmed to be at least 0.1 min before the expected time to ensure the entire peak is collected. The length of peak collection should also be sufficient to cover collection of the required peak only. Typically, if the required peak is well resolved from interfering peaks, then the collection step may be for 0.5 min. Using this example of a peak that was detected at 7.8 min, the following method may be appropriate: Step 1: Rinse with generation solution (0. \M NaOH or similar). Step 2: Rmse with electrolyte solution. Step 3: Set temperature. Step 4: Inject sample. Step 5: Apply voltage for 8.8 mm (capillary dipping into electrolyte vial at detector end). Step 6. Apply voltage for 0.5 mm (capillary dipping into fraction collection vial at detector end). There are three main approaches to optimizing the amount collected by preparative CE. These are to use concentrated sample solutions, perform several sequential runs, or employ relatively large-bore capillaries. 2. U s e of C o n c e n t r a t e d S a m p l e S o l u t i o n s The limitation is that resolution may be lost with increased sample loading. For example, 25 mg/mL fluparoxan solutions (1) and 5 mg/mL calcitonin solutions (2) have been used in micropreparative work. 3. U s e of Wide-Bore Capillaries Increasing the bore dramatically increases the volume of sample injected into the capillary. However, the required resolution may be lost at these higher injection volumes, and considerations must be given to joule heating effects. A 100 p, x 57 cm polyacrylamide-coated capillary has been used (S) to collect peptide components from a four-component

Fraction

Collection

101

^v.

Fig. 1. The preparative separation of sample containing four peptides (reproduced from ref. 3). text mix of peptides. The fractions were collected into a 10-^L microvial of separation buffer. The separation was operated under stacking conditions (see Chapter 16), which allowed a 20-s injection on this relatively large-bore capillary, which is equivalent to a 500-nL injection volume. Figure 1 shows the preparative separation of the sample. The collected fractions were reexamined by CE to confirm purity levels. Fifty nanograms of the individual peptides were collected (3). This was sufficient material to conduct further analyses, such as amino acid sequencing. 4. R e p e a t e d I n j e c t i o n s Repeated injections in an automated sequence would also increase the total amount collected. This facility is possible with well-controlled methods where very consistent migration times are obtained. Therefore, particular attention should be paid to the use of appropriate rinse cycles and to operation using a constant temperature. It may be advisable to perform two test injections prior to starting a preparative sequence to

102

Altria

allow the system to equilibrate and to reach a constant temperature {see Chapter 3, Section 10. on good working practices). For example, a fraction sample of a fluparoxan impurity was obtained following 27 injections of a drug substance solution. Injection sequences of up to 30 injections were shown (4) to give collection yields of 70-100% for injected amounts of various peptides and proteins. The expected amount collected can be calculated for a given number of injections using a particular set of conditions (1). For example, 27 replicate injections of a 25 mg/mL solution containing a 2.5% impurity, using injection settings that give a 11.8-nL injection volume with collection of the fraction into 20 \ih of water, yielded a 10-ppm (mg/L) solution. Each injection is equivalent to a loading of 295 ng. However, the impurity is only present at 2.5%. Therefore, each injection gives a loading of 7.4 ng of the impurity. Twenty-seven replicate injections give a total amount of impurity collected as 198 ng into 20 ^L of water. This gives a final solution concentration of 10 \iglmh (ppm), which is suitable for further investigations.

5. Notes
1. The configuration on the ABI instrument requires that the detector end reservoir is fixed and cannot be used for fraction collection purposes. Therefore, a scheme has been devised (4) m which sample solution is vacuum-injected and a rinse cycle is used to suck the sample slug to the detector end of the capillary. A negative vohage is then applied, causing the sample components to migrate back along the capillary, pass through the detector, and emerge from the capillary at the sample introduction end of the capillary. Again the voltage is stopped immediately prior to emergence of the sample zone from the capillary, and the capillary is dipped into a collection vial and the voltage restarted. A user bulletin (user bulletin number 4) is available from ABI that gives precise details of this operation. 2. Bundles of capillaries in an array format (5) have been shown to be of use m particular applications. Beckman Instruments are expected soon to launch a clinical CE instrument (Tradename, Paragon) in which several capillaries are simultaneously employed. This instrument would have obvious application to preparative operations.

References
I Altna, K. D. and Dave, Y. K. (1993) Peak homogeneity determination and micropreparative fraction collection by capillary electrophoresis in pharmaceutical analysis. J. Chromatogr 633,221-225.

Fraction Collection

103

2. Camilleri, P , Okafo, G. N , Southan, C , and Brown, R (1991) Analytical and micropreparative capillary electrophoresis of the peptides from calcitonin Anal Biochem 198,36-42 3. Schwer, C and Lottspeich, F. (1992) Analytical and micropreparative separation of peptides by capillary zone electrophoresis using discontinuous buffer systems J Chromatogr. 623, 345-355. 4. Albin, M., Chen, S -M., Louie, A , Pairaud, C , Colbum, J., and Wiktorowicz, J. (1992) The use of capillary electrophoresis in a micro-preparative mode methods and applications. y4na/ Biochem 206,382-388. 5. Huang, X. C , Quesada, M. A , and Mathies, R A (1992) DNA sequencing using capillary array electrophoresis, ^na/ Chem 64,2149-2154 6. Herold, M. and Wu, S -L (1994) Automated peptide fraction collection in CE,LCGC Int 7, 554-558. 7. Lee, H. G. and Desiderio, D. M. (1994) Preparative capillary zone electrophoresis of synthetic peptides. Conversion of an autosampler into a fraction collector J Chromatogr 686,309-317. 8 Lee, H. G. and Desiderio, D. M (1994) Optimisation of the loading limit for capillary zone electrophoresis of synthetic opiod and tachykinin peptides, a study of the interactions among the amount of peptide, resolution, saturation, injection volume and the capillary diameter. / Chromatogr 662, 35-45.