Preparative HPLC: Factors and Parameters that Directly Affect Recovery of Collected Fractions

Transcription

1 Preparative HPLC: Factors and Parameters that Directly Affect Recovery of Collected Fractions Gary Scharrer and Joan Stevens, Ph.D. Gilson, Inc Middleton, WI

2 Abstract Purification of synthetic, natural and biological compounds in any quantity usually requires the use of preparative HPLC. Collecting fractions from the preparative HPLC is the common approach to achieving the purified compound(s) of interest. Collection of fractions however is not trivial and is affected by many conditions and parameters within preparative HPLC. In the perfect world chromatographers would be able to optimize for each compound needing to be purified, however this not the case. The present study will examine a multitude of factors and parameters that directly affect fraction collection recovery. Some of the parameters to be discussed are column performance, mobile phase constituents, detector setting, and collection parameters such as collection via level versus slope and delay volumes. The data presented will offer guidelines for all chromatographers purifying compounds by preparative HPLC and can be implemented into existing systems.

3 Outline Preparative Systems Fraction Collection Factors Column Performance Mobile Phase Constituents Detector Settings Peak Level Collection Slope Collection Delay Volume Summary

4 Prep System Configurations 819 Injector System A Figure 1 Gilson 215 Liquid Handler/Fraction Collector, equipped w/175-mm Z-arm, 819 Injection Module with 5.0-mL SS loop and bevel-tip probe (269x1.3x0.8 mm ID) Gilson 333/34 HPLC Pump, equipped with H3 pump heads (flow rates up to 200 ml/min.) Gilson 155 UV/Vis Dual-wavelength Detector (0.2-mm pathlength, 1.0 AUFS)

5 Prep System Configuration 845Z Injector System B Figure 2 Gilson 215 Liquid Handler/Fraction Collector, equipped w/175-mm Z-arm, 845Z Injection Module w/5.0-ml SS loop, and bevel-tip probe (269x1.3x0.8mm ID) Gilson 322 HPLC Pump, equipped with H2 heads (flow rate up to 30 ml/min.) Gilson 155 UV/Vis Dual-wavelength Detector (0.2-mm pathlength, 1.0 AUFS)

6 System/Injectors Used In Study 819 Injector System A Dispenses sample through an injection port to the sample loop 845Z Injector System B Aspirates sample directly into the sample loop (Z-Mounted Valve) Above systems/injectors were both used in this study.

7 Factors that Affect FC Recovery Column Performance Compound Polarity Peak shape due to polarity can directly influence fraction collection parameters. The following data includes compounds of high to low polarity (caffeine, ethyl paraben, and biphenyl). Gradient versus Isocratic Elution Peak broadening due to elution type can affect time and efficiency of fractionation. Typically gradients are used to shorten purification time and give better peak shapes.

8 FC Factors (cont.) Mobile Phase Constituents Compound polarity in gradient program selection Injection Technique Standard Sample is dispensed into sample loop and is injected into the mobile phase via a switching valve. Sandwich Sample is placed between plugs of solvent, to protect from pre-column precipitation, prior to injection. The protected sample is then dispensed and injected as in the standard technique.

9 FC Factors (Cont.) Detector Settings Peak Width Set at 0 to acquire raw data with no smoothing effects. Sensitivity Level of detection desired. This parameter should not be confused with fraction collection sensitivity. Amount of fraction collected with be affected by increasing or decreasing this parameter when using peak level for collection.

10 Collection Parameters Peak Level Start Collection Stop Collection Level Disadvantages Loss of Compound No separation of multiple peaks Figure 3. Peak Level is the fraction collection parameter which uses a specified input set at a percentage of full-scale to start and end collection.

11 Affects of Peak Level Changes All peaks above level are collected. Peaks below level not collected. Recovery reduced Level Level Figure 4. As peak level decreases the recovery increases, any peaks that are at or above peak level setting are collected.

12 Peak Level vs. Recovery Recovery (%) Caffeine Ethyl Paraben Peak Level = 100 Peak Level = 50 Peak Level =25 Peak Level = 10 Peak Level = 5 20 Biphenyl Peak Level Graph 1. As peak level increases, recovery starts to drop. If the peak level is set too high, a large percentage could be lost to waste, and if set too low, peak purity could be affected, by collecting closely eluting compounds.

13 Collection Parameters Peak Slope Peak Slope is the parameter which uses peak sensitivity and peak width to determine peak collection Slope Minutes peak sensitivity -% height of smallest peak Peak sensitivity is the height of the shortest peak, measured In % of full scale. The peak sensitivity should be slightly greater Than the highest baseline noise. Peak Width is the duration in minutes, of a typical Gaussian peak at half-height. The peak width should be slightly longer than the width of the narrowest peak. peak width peak width time at ½height Figure 5

14 Slope vs. Recovery Sensitivity vs. Recovery 0.08 Peak Width Peak Width vs Recovery Peak 5% Recovery (% ) Caffeine Ethyl Paraben Biphenyl Recovery (%) Caffeine Ethyl Paraben Biphenyl Peak Sensitivity (%) Peak half height Graph 2 & 3. Slope is the combination of peak width and sensitivity. Above shows the impact of each factor. As the sensitivity increases, recovery decreases and as the peak width measured at ½ height increases recovery increases.

15 Collection Parameters (Cont.) Delay Volume (The Parameter used to compensate for the delay of the compound to reach the fraction collector from the detector). Factors which affect delay volume are: Flow rate System Plumbing Distance from detector cell to fraction collection valve Tubing Diameter

16 Delay Volume The volume it takes the sample to travel from the detector to the fraction collector dispense head. Tube Volume Equation V total =Tubing Length (in.) x (Tubing ID(in.) 2 )/2 x 3.14 Fraction Collector Detector Figure 6 Column To determine the delay volume accurately the volume of all of the flow components between the detector and fraction collection vessel must be added to get the total delay volume.

17 Affects of Delay in Fraction Collection Actual Peak Collection Start Fraction Collection End Fraction Collection Start Actual Peak Collection End Fraction Collected Fraction Lost Compound Recovered Delay Volume (Actual Peak Collection Start Fraction Collection Start) Figure 7. This figure illustrates the large amount of compound which could be lost if the delay volume is not considered. This also illustrates the illusion of collecting a pure fraction while obtaining possible impurity peaks.

18 Affects of Tubing Dimensions on Delay Volume 2000 Detector 0.26 min Collection Valve 0.26 min 0.26 min mvolts Minutes Graph 4. Delay retention between the detector and collection valve using 20ft x 0.04 in. ID tubing, flow 25 ml/min. Compounds used where caffeine, ethyl paraben, and biphenyl. Inner Diameter [inch] Inner Diameter [mm] Volume [per cm in µl] Volume [per inch in µl] 0.020" 0.50 mm " 0.75 mm " 1.00 mm

19 Calculation of Delay Volume Gilson Detector Prep Outlet Tubing (11.58 µl/inch x 24 inches) µl 3-way valve preparative for 215, FC µl Determine items to be included in the delay volume calculation Outlet detector flow cell tubing Inlet tubing from coupler to 3-way collection valve Collection valve The low mount collection valve is immediately before the collection vessel no additional volume components are necessary. Add all of the above volumes together to obtain a total delay volume. It is important to verify your delay volume calculation prior to injecting and collecting a valuable sample. A 2.5 mg/ml solution of Methylene Blue dye in water could be used a visual test solution.

20 Affects of Tubing Size on Delay Volume Delay Volume (ml) in ID 0.03 in ID 0.02 in ID Tubing Length (ft) Graph 5. Delay volume is dependent on tubing length and inner diameter or the volume of mobile phase between the detector and the collection vessel. This graph illustrates the effect of these factors on the delay volume.

21 Delay Volume vs. Recovery 5ft x 0.03in. ID % Recovery Biphenyl Caffeine Ethyl paraben Delay Volume (ml) Graph 6. If the delay volume is set incorrectly, the recoveries could be drastically affected.

22 Summary Fraction collection goals: High recovery Pure sample Consider all parameters as critical for optimum recovery Chromatography Detector settings including wavelength Collection Parameters Delay volume or time Incorrect or ignored parameters can cause recovery losses of over 50%. With 69 of 80 human therapeutic proteins on the market in 2003 sold at prices over $10,000/gram, losses of 50% would be $5000/gram. Can you afford to ignore these factors?