UPC2 Method Development Customer Training doc

ACQUITY Ultra Performance Convergence Technology UPC2Method DevelopmentGuidelinesUPC2 General Theory and Discussion1.This section will look at parameters that will affect a customer’s UPC2separation. Developing an UPC2 separation, requires understanding the sample chemistry as well as understanding what parameters in the UPC2 effect aseparation.2.Can you explain Density?a.Density is a physical property where Density(p) = Mass(m) ÷ Volume(v),Example:b.The density of CO2 at supercritical state is 1100 psi & 31° =467.6 kg/m3.c.The density of CO2 changes with temperature.d.The density of CO2 changes with pressure.e.The relationship of CO2 density (compressibility), pressure, andtemperature all affect a chromatographic separation and can be used inUPC2 method development. This is discussed in the next several pages.3.Why are “ACQUITY UPC2 BSM” temperatures set at 2 degrees for thePrimary A Pump head and 13 degrees for the Accumulator Pump head?a.Concept A: The density of liquid CO2 varies with temperature. The densityof the co-solvent (such as Methanol) will not vary much with temperature.b.Concept B: The chart below shows the density for methanol vs. liquid CO2at different temperatures and pressures. Liquid CO2 density changes withtemperature while methano l’s density is only a weak function of pressure.c.Concept B: Mass flow will vary with temperature for a given volumetricallymetered flow rate. Mass flow is an intrinsic attribute of the SFC separationmechanism.Note: The UPC2 BSM pump is not mass flow, or density based.d.Concept C: The greater the mass flow of CO2 (higher the density), thegreater the solvating power (or solvent strength) of liquid CO2.e.Concept D: By controlling the UPC2 BSM temperature we can control themass flow and density, thus controlling the separation based upon density.f.Concept E: The chart shows that a colder metering pump delivers densercarbon dioxide and presents smaller elution volumes for equivalentvolumetric compositions, ABPR settings and column temperatures.g.Answer: By controlling the UPC2 BSM pump temperatures to 2 degreesfor the primary and 13 degrees for the accumulator, we can speed the elution time because of CO2 density.Note: This was the same gradient and samples run on both systems.UPC2 Method Development Considerations1.UPC2 method development requires knowledge of many different variables aslisted below.2.These properties are ranked in priority of method development and factors thatcan be changed to affect a separation.3.This section will look at some of these parameters that will affect a customer’sseparation. Developing an UPC2 separation, requires understanding the sample chemistry as well as understanding what parameters in the UPCNote: This section is a start point for you and your customers looking to develop a separation.Sample Solute Properties1.Question 1: Is the sample soluble in Methanol, or less polar solvent?What is the Log P for the sample?h.Log P is used as a measure for water solubility.∙If the answer is yes, then it is a good candidate for UPC2 separation.∙If the answer is no, (only soluble in water), then it is a better candidate for UPLC reverse phase separations.a.Below is a list of recommended starting diluents to test sample solubility.These solvents are listed below based upon polarity (most common are in Bold). The most common diluent is 90/10 Heptane/IPA or IPA (less polarthe better).2. Question 2: What are some sample diluents and how does the samplediluent affect Peak distortion with UPC2?a.Answer A: In traditional SFC, Methanol is the most common solvent. Thisis not advised with UPC2 because peak distortion can occur due to the thestrong solvent effect of the sample diluent compared to the mobile phase.Because of these peak distortion issues, it is advised to inject a sampleusing a non polar diluent such as:∙80%/20% Heptane/2-Propanol∙Heptane/THF∙THF, Hexane∙Chlorobutane,∙Dichloromethane, Chloroformb.The chromatograms below illustrate sample diluent effects of a UPC2system with a small column ID and differing injection volumes.c.Notice that Methanol (most non-polar) delivers the most peak distortion,especially as ID of column decreases and volume injected on columnincreases (this is amplified with larger injection volumes).Column Selection1.Question 1: What columns are available for UPC2?a.Waters has wide array of chemistries that will work with UPC2 for achiralseparations.The chemistries are available in 1.7 and 3.5um size. Columns are available in 2.1 or 3.0 size ID’s.pound selectivity varies with the different column chemistries. Formethod development, it is suggested to start with the BEH and BEH-2EP columns, as these have a wide difference in sample selectivity. The HSS SB C18 is most appropriate for non-polar compounds.ing2.Question 2: What are recommended UPC2 column configurations?a.3.0 mm ID columns are a good compromise between sampledispersion, and achieving fast linear velocities.b.The chart shows predicted backpressures using different ID’s with samecolumns lengths (50 cm). A good start point for UPC2 column dimensions are the 1.7 um 3.0 mmx100 mm ID columns, which covers a completerange of flow rates and pressures.Note: 1.7um - 2.1mm ID columns for UPC2 configuration 2 and 3 systems will greatly reduce predicted pressures. 2.1 ID columns should be used only when the need for chromatographic resolution requires it.3.Question 3: How do use a new UPC2 column at system startup?a.Answer: ACQUITY UPC2 column are shipped dry. They should be flushed withat least 10 column volumes of the CO2 and co-solvent (95:5 CO2/Methanol).The chart below shows the column volumes for each UPC2 column dimension.4.Question 4: How should I care and store a UPC2 column?a.Answer A: For storage, it is recommended to flush an ACQUITY UPC2 columnwith 3-5 column volumes of 100% CO2 only. Do not store the column inMethanol or other co-solvent.5.Question 5: Can I clean or regenerate an UPC2 column?a.Answer A: Flush the column with 100% C02 for 5 minutes at 40C and ABPRset at 2000psi.b.Answer B: Full regeneration requires the use of Waters UPLC with the stepsbelow (See the UPC2 column Care and Use for full details).Gradient and Co-Solvent Selection1.Question 1: What are typical starting co-solvent and gradientconditions?a.Answer 1: Co-Solvent added to CO2 generally decreases a component'sretention time. As you change the amount of co-solvent, the polarity and density of the mobile phase is changed. Both factors contribute to adecrease in retention time.b.Answer 2: Mixtures of CO2 and co-solvents are supercritical in the columnwhen co-solvent percentages are approximately less or equal to 20%. Co-solvents percentages above 20% will create mixtures that are mostly liquid phase and will affect retention times, but may not be in a linear fashion.c.Co-solvents selection can affect retentively and selectivity. This showsMethanol reduces retention time and peak tailing (for bases) versusIsopropanol or Acetonitrile.d.The most common co-solvent is Methanol, though a variety of other co-solvents can be selected. In addition, solvent blends can be used for different selectivity and retention time changes. Below is a list of some of the most popular solvents, with the most widely used is highlighted in black.e.General recommendation of gradient %:∙Gradient for UPC2 typically are shallower than reverse phaseseparations.∙Suggested start conditions for UPC2 are from 2% co-solvent to 20-35% co-solvent, application and system pressure dependent.∙The stronger the polarity of the co-solvent the lower the retentiontime.∙Gradients strengths are rarely used above 50% modifier strength(due to pressure limitations).Co-Solvent Additives1.Question 1: What are co-solvent additives and when do I use them?a.Additives are buffer salts, acids and bases that are added to the modifier orco-solvent to change the separation of polar analytes. The additives candecrease the secondary or undesired interaction between the column(stationary phase) and the anayte. The addition of additives can improve peak shape and change retentivity of polar compounds by reducing ionic interactions between the stationary phase and the compounds.Note: Neutrals will usually not be affected by these additives though acid and basic molecules will: Neutrals may change retention time because additives sometime affect the stationary phase.2.Question 2: What are the recommended co-solvent (or modifier)additives for strong basic polar compounds?3.Basic additives added to the co-solvent can improve peak shape and/orelution of basic analytes, as highly basic analytes are relatively insoluble in pure CO2.a.Below is an example of how the following additives change peak shape forthese highly basic compounds.b.Acid additives are not recommended for basic compounds (except for ion-pairing reagents).c.Some common additives are:Diethyamine, Triethylamine, Ammonium Acetate, Ammonium Hydroxide4.Question 3: What are the recommended co-solvent additives foracidic polar compounds? What are the affects?a.Acids added to the co-solvent can improve peak shape and/or elution ofacidic analytes.b.Correct selection of the acid can affect peak shape for acids ore bases.This example shows that TFA gives better peak shape than Formic Acid(TFA is generally not recommended for MS).ABPR P RESSURE S ETTINGS1.Question 1: What does the ABPR settings do and what affectshappen on chromatography?a.UPC2Separations are affected by density. ABPR back pressure affectsretention time by changing the density of the CO2 mobile phase before the ABPR (or the release of pressure).b.As ABPR pressure increases, the overall system pressure increases.This also increases the density and thereby solvating power of the CO2, which leads to decreased retention times.2.Question 2: What are the appropriate ABPR settings for UPC2?a.Answer: The Convergence Manager range for ABPR is from 1500-6000psi. The next page will show suggestions for ABPR settings.b.Pressures below ~1800psi may affect the separation in a non-reproducible way as the condition changes from a supercritical phase toa more gaseous phase – which then affects the polarity and density.c. Higher pressures than ~3500psi may prohibit the pressure drop for thecolumn, causing system high pressure.d.General suggested ranges are from 1800-3500 psi (2.1mm ID columnsmay reduce the effective operation range).3.Question 3: Can I create an ABPR gradient by time?a.Answer A: You can set ABPR pressure gradients using a timegradient. Pressure gradients are linear steps. The example belowwould change the ABPR pressure linearly over 3 minutes from 2000to 3000psi.C OLUMN T EMPERATURE S ETTINGS1.Question 1: What chromatography effects happen with changes ofcolumn temperature?a.General UPC2 Column Temperatures change the CO2 density in thecolumn. Higher Column temperatures decrease density and increaseretention time.b.Lower column temperatures increase density and decrease retention time.c.The chromatogram on the next page shows the effects of temperature forUCP2 separations.2.Question 2: What are recommended column temperature ranges forUPC2?a.Recommended column temperatures ranges for UPC2 are 35-650C.b.A column temperature below 350C takes the CO2 out of the supercriticalphase in the column, and may produce retention time and selectivityreproducibility issues. This may also increase static and ABPRmaintenance.c.It is not recommended to leave a column connected outside the columnheater module. This is because CO2 needs to be in a supercritical phase inside the column (above 310C and pressures above 1074 psi).G ENERAL ACHIRIAL UPC2M ETHOD D EVELOPMENT S TRATEGY1.Step 1: Check for sample solubility, diluents, and conditions.2.If the sample is soluble as above, go to step 2 (next page).3.If the sample is not soluble, it is recommended to try Reverse Phase UPLCanalysis or other analytical technique.4.Step 2 – Develop a screening assay using 2 different UPC2 columns withfour different co-solvents generic conditions (as suggested below)..5.Did the injections work with any of the eight different method conditions?If so, go to the next page for sample and method optimization.6.Step 3 – Optimize Peak Shape or No Peak? Are the compounds acidic orbasic? Some method optimizations are detailed below:7.Step 4: Optimize Retention. If this is an issue, try using some of thesuggested additives as shown below. If not go to Selectivity.8.Step 5 – Need changes in sample selectivity? Try the changes as listedbelow:G ENERAL CHIRAL UPC2M ETHOD D EVELOPMENT S TRATEGY1.What is Chiral Chemistry?a.Answer: First the definition of Chirality / Enantiomers∙Contains 1 or more asymetric carbon (1 carbon with 4 different functional groups)∙Exists as a combination of enantiomers (different spatial disposition) ∙Mirror images of each others (like your hands)∙Enantiomers can have different bioavailability or biological activity∙This means only two peaks (right side and left side – with one chiral center)∙PDA-UV only as MS will still show the same molecular weight and structure.2.Question 2: Does Waters make Chiral columns for UPC2?a.Answer 1: Waters does not currently make or sell Chiral columns, thoughone of the major vendors is Chiral technologies. Its website address is: http://www.chiral.fr/3.Question 3: Are Chiral columns compatible with UPC2?a.Answer 1: Yes, though Chiral separations typically do take some methoddevelopment time for proper column selection.b.Answer 2: Typical column ID’s are 4.6mm x100mm for column ID’s, with3um-5um packings. Chiral columns are expensive, so strict adherence to the vendors care and use is required for proper operation and columnlifetime.c.Answer 2: Some column ID’s are larger and may require the use of theUPC2 30cm Column heater.4.Question 4: What is a good starting Chiral Method DevelopmentStrategy?1.Answer: Below is a suggested start point. Follow the achiral methoddevelopment suggestions (pages 16-17) for optimizing or refining theseparation.。