Varian Galaxie Chromatography Data System for Preparative HPLC

Varian Galaxie Chromatography Data System for Preparative HPLC By Gary Burce Varian, Inc. 2700 Mitchell Drive, Walnut Creek, CA 95498 USA Abstract Galaxie is an ideal chromatography data system for the chemist doing preparative and semi preparative HPLC. It controls the Varian ProStar and PrepStar HPLC modules, which are ideal for prep chromatography. These modules have high flow rate and extended detection range, making it easy to scale up from analytical to prep separations. Galaxie has real time display for estimated peak purity when using the Varian ProStar 325 UV-Vis Detector or the Varian ProStar 335 Diode Array Detector. Based on this purity information, or other peak characteristics, the chromatographer can make real time changes in sample collection. After the run is done, Galaxie clearly displays both graphically and in tabular form, what parts of eluant have gone into which collection vessel or waste. If a Diode Array Detector is being used, Galaxie can quickly and easily estimate the purity of each peak and fraction collected. Introduction...3 Control of Preparative HPLC Instrumentation...3 Galaxie Pump Control...3 Galaxie Detector Control...3 Galaxie Autosampler Control...4 Galaxie Fraction Collector Control...4 Galaxie CVM Control...6 Real Time Purity Display...7 Real Time Control of Instrument Functions Through Galaxie...9 Fraction Marks...9 Post Run Purity Determination...10 1 of 15

Information Display...11 Conclusions...14 2 of 15

Introduction Data system requirements for preparative HPLC are different from those for analytical HPLC. Control of pumps that provide wider flow rate ranges, detectors with larger linear dynamic ranges, and fraction collectors that collect from the smallest to largest fractions, are necessary for preparative chromatography. Preparative chromatography also requires more real time instrument interaction so that precious samples can be preserved when impurities are detected. The benefits of preparative chromatography include purity determination for the entire peak, or fractions into which the peak has been divided. Lastly, preparative chromatography needs good post run display of information so the content and purity of the material in the fractions can be clearly determined. Galaxie Chromatography Data System (CDS) software meets all of the requirements for a good preparative chromatography data system. Galaxie controls the Varian ProStar and PrepStar HPLC modules, which are ideal for prep HPLC. Galaxie displays real time peak purity information so the chromatographer can easily take manual control of peak collection if needed. Galaxie displays the part of the fraction that has been deposited in each vial, and the purity of the material in the vial, in both graphical and tabular formats. Control of Preparative HPLC Instrumentation Galaxie Pump Control Galaxie controls all of the ProStar and PrepStar HPLC modules. The ProStar 210/218 family of pumps can pump from 10 μ/per minute at 8700 psi for the 5 ml/min pump head to 200 ml/min at 3500 psi for the 200 ml/min pump head. The SD-1 pump can pump up to 3.2 L/min. These flow rate ranges are suitable for columns up to 6 inches in diameter. Galaxie Detector Control Galaxie controls the ProStar 325 UV-Vis Detector and the ProStar 335 Diode Array Detector. These detectors have dual path flow cells (Figure 1), which automatically switch from the long path (for best sensitivity) to the short path for an extended linear dynamic range. Depending on the flow cell used, the linear dynamic detection range can be more than 70 AU, which allows Galaxie to be able to detect small impurities while simultaneously keeping any large peaks on scale. 3 of 15

Figure 1 Dual path flow cell for the ProStar 325 UV-Vis and ProStar 335 DAD detector Galaxie Autosampler Control Galaxie controls the ProStar 410 AutoSampler. This autosampler can use 10 ml sample vials and inject up to 10 ml per injection. If larger injection volumes are needed, one of the ProStar 210/218 pumps controlled by Galaxie can be used as an injection pump to load any amount of sample onto the column. Galaxie Fraction Collector Control Galaxie controls the ProStar 701 Fraction Collector. The ProStar 701collects fractions in vessels ranging from small tubes to funnels that attach to reservoirs of unlimited size. Galaxie s control of the ProStar 701 provides a complete choice of how the collection of samples is determined. Galaxie controls the basic modes of collection, by drop or by time, whether the fraction collection pattern is directly across the tray from left to right, or back and forth in a serpentine fashion. Non-peak material can be collected or diverted to waste. (Figure 2) 4 of 15

Figure 2 Galaxie ProStar 701 Fraction Collector control of basic fraction collector operation. Galaxie has a complete set of parameters for peak sensing. Time, detector signal slope, or combinations of these parameters can be used to control peak sensing. Peak sensing can be started or stopped at any time during the run and turned on or off so that regions of the chromatogram can be excluded from collection. This provides better selectivity for collecting samples. (Figure 3) Figure 3 Galaxie control of the fraction collection peak sensing modes. Galaxie uses a wide range of collection vessels from regular tubes to bottles and beakers. (Figure 4) Figure 4 Galaxie configuration of the collection vessels Galaxie controls the chain of collection. It is possible to overlay the replicate injections over the same set of collection vessels, or choose to collect replicates independently and pool fractions later. It is also possible to prevent a run from starting if there are an insufficient number of collection tubes remaining in the rack. (Figure 5) 5 of 15

Figure 5 Further Galaxie control of the ProStar 701 Fraction Collector. Galaxie CVM Control Galaxie controls the ProStar 500 CVM (Column Valve Module). The ProStar 500 CVM can have several different types of automated valves installed. These include column switching, solvent selection, and fraction collection valves. A column switching valve can be used to automatically switch between a 4.6 mm analytical column and a 1in. or 2in. prep column (Figure 6). This feature gives prep chromatographers the ability to develop a method for the analytical column and automatically scale up the method to the prep column with no changes to the hardware. Figure 6 ProStar 500 CVM with analytical and a prep column installed The ProStar CVM also controls both a diverter valve and fraction collector valve. The diverter valve is used to direct the output of the HPLC system either to waste or to the fraction collector valve. The fraction collector valve can direct the output of the HPLC system to any of 10 or 12 different collection 6 of 15

vessels (Figure 7). This is all done on a time-programmed basis, controlled from Galaxie. It is a simple and economical way to collect fractions when injections are done infrequently. Figure 7 ProStar 500 CVM diverter and fraction collector valve interconnection Real Time Purity Display The ProStar 325 UV-Vis and the ProStar 335 PDA detectors can generate peak purity information as peaks are being eluted. The ProStar 325, when configured in the dual wavelength mode, provides a real time wavelength ratio plot output. This output is determined by the ratio of the two wavelengths specified in the method. This ratio also has a threshold value which can be set so the ratio is plotted only when a peak is detected; not when the signal is recording baseline. The ratio will be constant (square waveform) during detection of an entire pure peak. If the shape of the plot deviates from the square wave form during the detection of a peak, then the peak can be considered as no longer spectrally pure, and the chromatographer can interactively move the collection of the impure eluting fraction to a different vial. The ProStar 335 Diode Array Detector also displays real time purity information. This detector uses wavelength ranges designated by the chromatographer and generates a weighted average wavelength. This is called the purity parameter and is described in the equation below. The purity parameter is less sensitive to absorbance near the noise level because it uses the square of the absorbance to weight the wavelengths. This is better than a simple wavelength ratio because it 7 of 15

uses spectral comparisons at many wavelengths to identify impurities. This type of purity determination is ideal for real time situations because it is easy to calculate and does not require any spectral information about the apex of the peak when a peak is first detected. Galaxie controls the purity parameter by using both a user definable wavelength range and a threshold parameter for this calculation as shown in Figure 9. Figure 8 Galaxie control parameters for the ProStar 335 real time purity parameter. The threshold parameter causes the calculation to begin only when the absorbance has increased above the preset threshold level. In this way, the purity calculation is done only while a peak is eluting, making it much easier to identify the pure/impure regions of each peak. The purity parameter will remain constant during the elution of a pure peak as shown in Figure 9. Figure 9 Purity parameter and chromatogram of a series of peaks in Galaxie 8 of 15

In Figure 9 the fourth and fifth peaks are not baseline resolved. The purity parameter plot changes during this part of the chromatogram indicating that this portion of peak 4 is not pure. At the point where the purity parameter begins to change, the chromatographer can use Galaxie to interactively move the fraction collector to the next collection vessel or even the next rack, and collect the unresolved peaks portion of the chromatogram in a separate vial. In this manner, the purity of the material in the vessel, which contains peak 4, will not be compromised. Note that the purity parameter of peak 4 is indicating that it is pure even at an absorbance of over 2.5 AU. Real Time Control of Instrument Functions Through Galaxie As the chromatogram is displayed in real time in the chromatogram view above the status window in Galaxie, it is possible to take control of the fraction collector through a series of simple buttons as shown in Figure 10 below. Figure 10 Interactive control of the ProStar 701 Fraction Collector through the Galaxie status screen The chromatographer can Move To a particular collection position or just go to the Next Tube or even Change the Rack in which the sample is collected. When the button is pushed, the change occurs immediately, preserving the purity of the material collected while continuing to collect material that might prove to be a mixture. Alternatively, the chromatographic eluant can be directed to the drain. Fraction Marks As the fraction collector moves from one collection vessel to the next, or when the flow is redirected to waste, Galaxie records the time when the vessel or diverter valve position was changed. Galaxie then displays these as fraction marks on the screen in real time as well as on the post run chromatogram display as shown in Figure 11. Figure 11 Galaxie display of chromatogram and fraction marks 9 of 15

In Figure 11, fraction marks are clearly shown. These indicate when collection started and stopped, and when the eluant was directed to waste. They are color coded to match the fraction collector vessel display so it is easy to see what part of the chromatogram was deposited into each tube. Post Run Purity Determination With the Varian ProStar 335 Diode Array Detector, Galaxie not only determines peak purity in real time but can also determine peak purity post run. Any other vendor s Diode Array Detector controlled by Galaxie can only have peak purity determined after each run is completed. This purity calculation is controlled by peak detection and can be automatically reported at the end of the run. Control of the peak purity calculation parameters is an integral part of the Galaxie method as shown in Figure 12. Figure 12 Galaxie control of peak purity determination Peak purity is determined by comparing the spectra at the peak apex (or reference spectra selected from a library) with all the other spectra in the peak using a correlation algorithm once a background noise spectrum has been subtracted from each of the spectra in the peak. There are three types of purity calculation algorithms that can be used. The Dissimilarity algorithm is the most sensitive to impurities and is therefore best suited for preparative chromatography. The user can also determine what wavelength range will be used for a purity determination. Lastly, the user can determines the purity threshold limit values that will be used to determined which regions of a peak can be regarded as pure, medium purity, or impure. 10 of 15

Information Display Galaxie can report preparative chromatographic results in several ways. The fraction log and a graphical display of the chromatogram can be printed as shown in Figure 13. Figure 13 Galaxie display of a chromatogram with fraction marks and fraction log In the report shown in Figure 13, the fractions collected are graphically displayed on the chromatogram as time delimited, colored fraction marks. When the eluant has been directed to waste, these sections are labeled as Waste both on the chromatogram and in the report. Details about each fraction are listed in the report along with a colored bar that indicates where the collection of a particular fraction was started and stopped and where it was deposited in the fraction collector tray. If the ProStar 335 or another Diode Array Detector is being used, Galaxie can display the purity of each detected and identified peak in the chromatogram. This display can be either graphical as shown in Figures 14 and 15 or numerical as shown in Figure 16. 11 of 15

Figure 14 Galaxie graphical display of peak purity for the peak at 6.9 minutes in Figure 13 Figure 15 Galaxie graphical display of peak purity for the peak at 9 minutes in Figure 13 In the above figures, green shows the pure region of a peak and yellow represents the region of medium purity. A red region would indicate an impure peak. In Figure 14, the peak eluting between 5.8 and 7 minutes shows that most of the peak was pure but that there was some impurity at the beginning of the peak. In Figure 15, the peak eluting between 7.2 and 9 minutes was shown to be completely pure. The purity results are calculated for each detected peak in the chromatogram whether or not these peaks are identified in the peak identification table. Figure 16 shows a numerical report of peak purity for each detected peak in the chromatogram. 12 of 15

Figure 16 Galaxie numerical report of peak purity In Figure 16, the peak labeled Peak 3 shows the numerical purity determination corresponding to the plot in Figure 14. About 5% of the peak area is considered of medium impurity. The peak labeled Peak 4 corresponds to the plot in Figure 15. The entire peak is considered pure. Galaxie offers chromatographers several different ways to display peak information for different peaks in the chromatogram. Fractions are often collected by peak with each chromatographic peak being collected in a different vessel. However, a large peak might be fractionated across several collection vessels. In this situation, it would be desirable to determine the purity of the material in each individual vessel. Although peak related parameters (threshold/slope) are usually used to collect fractions, in the chromatogram in Figure 16, only time was used to collect the fractions shown. These fractions do not correspond to any of the concomitant peaks so the initial peak processing will not provide information about the purity of peak fraction collected. With Galaxie, you can change the peak processing by shifting the valley between the two peaks in fraction 2 so that the peak start and peak ends correspond with the start and end of the fraction. This is shown in Figure 17. 13 of 15

Figure 17 Shifting the peak start and end to correspond with Fraction 2. The peak start and end, represented by the small triangle topped dotted lines have been adjusted to correspond to the fraction that was collected. The peak end was moved a little beyond the end of the fraction so that any of the material that might have gotten into the collection vessel would be considered as part of the peak. Once the peak was identified, the peak purity was automatically calculated and is displayed in Figure 18. The material in Fraction 2 is pure. Figure 18 Purity of the Fraction 2 Conclusions Galaxie is an outstanding chromatographic data system for preparative chromatography. Galaxie has full control of the modular Varian ProStar HPLC systems who s high pump flow rates, extended detector linear dynamic ranges and flexible fraction collector control make them ideal for preparative chromatography. When used with the ProStar 325 UV-Vis Detector or the ProStar 335 Diode Array Detector, Galaxie provides a real time estimate of compound purity on the instrument status screen. The chromatographer can easily identify whether a peak is pure or impure, and act accordingly in order to preserve the purity of the collected fractions. 14 of 15

Galaxie has real time control of the fraction collection functions of both the ProStar 701 Fraction Collector and the ProStar 500 CVM. When used with the chromatogram and purity output of the ProStar 325 or 335, the chromatographer can immediately switch to any collection vessel or waste when a peak begins to contain impurities. This will preserve the purity of the collected samples. When used with a Diode Array Detector, Galaxie has highly sophisticated and flexible peak purity algorithms, which can easily and quickly identify whether the material in one peak or one fraction is pure. Galaxie displays peak purity and fraction collection information in both graphical and tubular form to make is easy to assess purity of the material that has been collected. 15 of 15