Simultaneous Preparative HPLC Fraction Collection and Column Regeneration Application Note 234 Gary Scharrer and Joan Stevens, Ph.D. (Gilson, Inc.) Introduction Improving separation efficiency and accelerating elution can enhance sample throughput in many ways (e.g., use of shorter columns, smaller silica particle size and temperature elevation). However, these so called enhancements result in fundamental changes in methodology. Developing and validating new methods, along with documentation revisions, often outweighs any benefits from increased efficiency. This application employs a dual-gradient pump configuration in combination with dual-column switching and automated column reporting. The system used a standard gradient and a gradient reequilibrate method in parallel. The alternation pattern (switching between two columns) produced higher throughput. Materials & Methods Instrumentation and Software Gilson 215 Liquid Handler: equipped with 175-mm Z-arm, 819 Injection Module with 7010 Rheodyne valve and 5-mL sample loop, and beveled-tip probe (269 x 1.5 x 0.8 mm ID) Gilson 321 HPLC Pump Gilson 322 HPLC Pump Gilson 155 UV/VIS Dual-wavelength Detector: equipped with semiprep flow cell, 0.2-mm path length VALVEMATE Valve Actuator: equipped with 10-port, 2-position switch valve Accessories and Reagents Phenomenex Luna Combi-HTS C18 Column (5µ, 50 mm x 21.2 mm) Sample mixture: caffeine, ethyl paraben and biphenyl (compounds were dissolved in 50/50 acetonitrile/methanol at a concentration of 20 mg/ml, 40 mg/ml and 20 mg/ml, respectively) Mobile phase: solvent A, water/0.1% TFA; solvent B, acetonitrile/0.1% TFA June 2005 Page 1 of 6 319334-01
Procedure The following 10-port, 2-position valve configuration was used: 10-port, 2-position Switch Valve Position = 0 Waste Figure 1. Position 0 (shown) was used to regenerate column A, and used column B for analysis. The Gilson HPLC system was programmed to run the Analysis Run gradient as shown in Table 1 (total run time was 5.45 minutes). The Regeneration Gradient switches the column mobile phase and flow conditions, after eluting the compounds of interest, to regeneration conditions. At time 5.45 minutes within the analysis run, the column is switched to the regeneration system and then undergoes a regeneration (cleaning) run while the second column is switched in-line for analysis. This switching process continues until all samples are analyzed. It should be noted that the gradients shown in Tables 1 and 2 allowed for pumps with different flow capabilities. Also, it is imperative to first activate the control method. Time Table 1: Analysis Run Gradient Flow Rate (ml/min) Mobile Phase Mixture* 0.02 10 95% A:5% B 0.28 10 95% A:5% B 0.50 25 95% A:5% B 3.00 25 5% A:95% B 5.00 25 5% A:95% B 5.45 10 5% A:95% B *Solvent A: water/0.01% TFA, Solvent B: acetonitrile/0.01% TFA Time Table 2: Regeneration Gradient Flow Rate (ml/min) Mobile Phase Mixture* 0.02 15 5% A:95% B 2.00 15 5% A:95% B 4.00 15 95% A:5% B 4.50 10 95% A:5% B *Solvent A: water/0.01% TFA, Solvent B: acetonitrile/0.01% TFA. June 2005 Page 2 of 6 319334-01
The following diagram represents the timeline of the switching process. Figure 2. Gradient illustration of alternating columns. The following control methods were used for the two-column regeneration application: Table 3: UniPoint Instructions for the Analysis Control Method Time Device(s) Command Comment 1 0.02 water/acn FL3(mL/min): 100% water, INIT_ flow and conditions to equal REGEN system 2 0.04 injector wait to display BUSY REGEN system waiting for READY 3 0.28 water/acn FLOW1(mL/min): 100% water, INIT_ 4 0.30 prep FC injection <start>0.1, 30.0, 50.0, SAMPLE_ LOCATION, SAMPLE VOLUME, 450 flow and condition ready for injection inject sample 5 0.32 system controller synchronize wait for injection 6 0.36 data channels start chromatogram channels start data acquisition 7 0.38 UV/VIS detector autozero channels autozero detector 8 0.50 water/acn FLOW1(mLmin): 100% water, INIT_ start gradient 9 0.80 system controller synchronize set to fraction collection state 10 1.02 fraction collector set collection and travel depths 3,3 fraction collection parameter 11 1.04 fraction collector set fraction by time inside a peak 1.0 fraction collection parameter 12 1.06 fraction collector set peak level PEAK_LEVEL fraction collection parameter 13 1.08 fraction collector set fraction site FC_SITE fraction collection location 14 1.10 fraction collector start collection start fraction collection 15 3.00 water/acn FLOW1(mL/min): 100% water, END_ end gradient 16 5.00 water/acn FLOW1(mL/min): 100% water, END_ end gradient until sample elution 17 5.02 data channels stop chromatogram channels stop data acquisition 18 5.04 fraction collector stop collection stop fraction collection 19 5.45 water/acn FL3(mL/min): 100% water, END_ reduce flow to REGEN system 20 5.55 injector write to display READY switch column June 2005 Page 3 of 6 319334-01
Time 1 0.01 VALVEMATE 36 2 0.12 water/acn 3 0.14 water/acn 4 2.00 water/acn 5 4.00 water/acn 6 4.50 water/acn Table 4: UniPoint Instructions for the Regeneration Control Method Device(s) Command Comment set valve position variable VALVE_ POSITION FL3(mL/min): 100% water, INIT_ FL3(mL/min): 100% water, END_ FL2(mL/min): 100% water, END_ FL2(mL/min): 100% water, INIT_ FL3(mL/min): 100% water, INIT_ Switch column to ANALYSIS system REGEN system switching conditions REGEN starting conditions, ANALYSIS system end conditions Rinse column with organic Return gradient to ANALYSIS initial conditions REGEN column waiting conditions Using multiple flow and mobile phase variables in the above methods allows the flexibility to adjust independent gradient profiles on both systems. Results The injected sample consisted of three components: caffeine, ethyl paraben and biphenyl. A partialloop injection was used for this application. Chromatographic conditions and software are described in the Materials & Methods section. Figure 3. Fraction collection chromatogram of caffeine, ethyl paraben and biphenyl. June 2005 Page 4 of 6 319334-01
The elimination of the column switching step results in a time savings of 4 minutes. Results are presented in Table 5. Identical column regeneration times were used (see Figure 2). Table 5: Time Comparison of With Column Switching vs Without Column Switching Without Column Switching With Column Switching Sample Analysis 5.45 5.45 Organic Rinse 2.00 0.00 Return to Initial 2.00 0.00 Total Time 9.45 5.45 With a time savings of 4 min/sample for this gradient, the following table illustrates the impact of this application. Table 6: Cumulative Time Savings Sample Runs* Time Savings (minutes) 25 100 50 200 75 300 100 400 *Samples ran consecutively. June 2005 Page 5 of 6 319334-01
By column switching, sample throughput can be almost doubled. The precision and reproducibility associated with injecting each sample six times (or 12 switching events with the three compounds) are presented in Table 7. Table 7: Precision and Reproducibility of Sample Mixture Compound Column A Avg. Retention Time Column A Retention Time CV (%) Column B Avg. Retention Time Column B Retention Time CV (%) Caffeine 1.79 0.58 1.79 1.00 Ethyl Paraben 2.68 0.62 2.68 0.56 Biphenyl 3.66 0.41 3.66 0.54 Conclusion The Gilson HPLC preparative system shows that by employing a single 10-port, 2-position VALVEMATE, two columns can be used to speed analysis time. The ability to switch between two columns and maintain precision and reproducibility is a valuable tool. Also by using UniPoint System Software s unique ability to use variables, a number of different pumping systems with different capabilities can be adjusted for various flow rates and pumps. Gilson, Inc. World Headquarters P.O. Box 620027, Middleton, WI 53562-0027 USA Telephone: (1) 800-445-7661 or (1) 608-836-1551 Fax: (1) 608-831-4451 Gilson S.A.S. 19, avenue des Entrepreneurs, F-95400 VILLIERS LE BEL France Telephone: (33-1) 34 29 50 00 Fax: (33-1) 34 29 50 20 www.gilson.com sales@gilson.com, service@gilson.com, training@gilson.com June 2005 Page 6 of 6 319334-01