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Ed Kim Application Engineer Agilent Technologies, Inc January 14, 2009
Separation fundamentals Agilent Restricted December 11, 2007
Major HPLC modes Key Equations
• Resolution • van Deemter
Common terms & definitions Key parameters & conditions that affect them
• efficiency, selectivity, and retention
Role of pressure
Separation Fundamentals Agilent Restricted December 11, 2007
Volatile Carboxylic acids Sulfonamides Nitriles Amino acids Synthetic Glyphosate food dyes Aldehydes Ketones Glycols PG, OG, DG phenols Enzymes Aflatoxins Antibiotics Flavonoids Anabolica Natural food dyes Inorganic ions Sugars alcohols Sugars
BHT, BHA, THBQ antioxidents Alcohol
TMS derivatives of sugar Epoxides
Fat soluble vitamins Triglycerides Phospholipids
PCBs Essential Oils Polymer monomers
Fatty acid methylesters
Volatile – Gas Phase Volatile Volatility
Nonvolatile - Liquid Phase Nonvolatile
Separation Fundamentals Agilent Restricted December 11, 2007
Major Separation Modes of HPLC A Review
There are four major separation modes that are used to separate most compounds:
Reversed-phase chromatography (most popular) Normal-phase and adsorption chromatography Ion exchange chromatography Size exclusion chromatography
….Let’s look briefly at each mode
Separation Fundamentals Agilent Restricted December 11, 2007
Isomer separation. ethyl acetate. 2007 . Sample injection solvent is non-polar.Normal Phase or Adsorption Chromatography Key Points Column packing is polar ⌫ silica (strongest)>amino>diol>cyano (weakest) Mobile phase is non-polar • hexane. etc. iso-octane. methylene chloride. Retention decreases as polarity of mobile phase increases Reasons to choose normal phase Resolve strongly retained hydrophobic samples. Recovery in non-polar solvents is desirable. Separation of Nitroanilines on HPLC Column packed with silica gel using hexane (mobile phase component A) mixed with methylene chloride (mobile phase component B) Separation Fundamentals Agilent Restricted December 11.
RNA polymerase 2. etc.g. Chymotrypsinogen 3. Basic proteins on strong cation exchanger (-SO3-) 1. Lysozyme Separation Fundamentals Agilent Restricted December 11. formate.tetraalkylammonium Mobile phase is an aqueous buffer (e. phosphate.) Used infrequently Similarities to Ion-pair chromatography Well suited to the separation of inorganic and organic anions and …cations in aqueous solution. …. 2007 . sulfonic. e.Ion Exchange Chromatography In ion exchange: Column packing contains ionic groups.g.
The monomers elute after the polymer. Gel Permeation Chromatogram of Polybutadiene polymer on nonaqueous SEC (GPC) column. 2007 .Size Exclusion Chromatography (SEC) There are two modes: • non-aqueous SEC [sometimes termed Gel Permeation Chromatography (GPC)] • aqueous SEC [sometimes referred to as Gel Filtration Chromatography (GFC)] No interaction between the sample compounds and packing material • Molecules diffuse into pores of a porous medium • Molecules are separated depending on their size relative to the pore size molecules larger than the pore opening do not diffuse into the particles while molecules smaller than the pore opening enter the particle and are separated large molecule elute first. Column: PLgel mixed-D gel Mobile phase: Tetrahydrofuran (THF) Separation Fundamentals Agilent Restricted December 11. smaller molecules elute later The mobile phase is chosen mainly to dissolve the analyte Used mainly for polymer characterization and for proteins.
2007 .B) Enter Pores A Full entry Partial entry B Packing Pore C Molecule (C) Will be Excluded from Pores B C A Signal Molecules must freely enter and exit pores to be separated. followed by intermediate size molecules and finally the smallest molecules elute last.Mechanism of SEC Molecules (A. Time Separation Fundamentals Agilent Restricted December 11. Largest molecules elute first.
g Can be used for non-polar. water (buffer) + water-miscible organic solvent. Phenyl. e. C8. C3. Different sorption affinities between analytes results in their separation – More polar analytes retained less – Analytes with larger hydrophobic part are retained longer Mobile phase.Reversed-Phase Chromatography (RPC) Principle: Partition of analytes between mobile phase and stagnant phase inside the pore space + adsorption on the surface of bonded phase Nonpolar (nonspecific) interactions of analyte with hydrophobic adsorbent …surface – C18. …ACN* – water (buffer) + water-miscible organic solvent. e. MeOH.g. 2007 . polar. etc. ionizable and ionic molecules Gradient elution is often used *Non-aqueous reverse phase Separation Fundamentals Agilent Restricted December 11.
8μ RRLC y lectivit Se Gradient Retention Resolutio n Retention Factor UPLC Factor Tailing Rapid Resolution Rapid Resol ution HT RR H T Separation Fundamentals Agilent Restricted December 11.5μm Peak Shape m 1. 2007 .Chromatography Terms are All Around Us But what do they mean….. Gradient Steepness Plates e e Siz l artic P Efficiency Peak Capacity 3.
Chromatographic Profile Equations Describing Factors Controlling RS Retention Factor (tR-t0) t0 Selectivity k= α = k2/k1 Theoretical Plates-Efficiency N = 5.54 (tR / W1/2)2 Separation Fundamentals Agilent Restricted December 11. 2007 .
Chromatographic Terms Retention Factor Two compounds can be separated if they have sufficiently different retention factors – k’ values t’R t0 tR – t0 = t0 k’ = tR2 tR3 t R1 0 Separation Fundamentals Agilent Restricted December 11. 2007 .
2007 . relative retention. capacity factor) – this is used to measure how far apart the k’ values of two peaks are and if the separation can be achieved k’ α = 2 k’1 k’2 > k’1 α > 1 for a separation to take place Big α Separation Fundamentals Agilent Restricted December 11.Chromatographic Terms Selectivity factor Alpha. α (separation factor.
` 000995P1.) column void volume (mL) • S ≈ 4–5 for small molecules • 10 < S < 1000 for peptides and proteins In gradient separation the effective value of k (k*) for different bands will be about the same same. 2007 .Gradient Retention (k*) Selectivity in gradient elution is determined by the gradient retention factor k* = ΔΦ = S = F = tg = Vm = tg F S ΔΦ Vm change in volume fraction of B solvent constant flow rate (mL/min.) gradient time (min.PPT Separation Fundamentals Agilent Restricted December 11.
2007 .d.This Relationship Says that to Keep Relative Peak Position in the Chromatogram Unchanged Any Decrease in Column length Can be Offset by a Proportional Decrease in tG or F Increase in ΔΦ Column volume (i.) Decrease in tG or F Increase in ΔΦ ΔΦ (same column) tG • F Decrease in tG or F k* = S • ΔΦ • Vm Separation Fundamentals Agilent Restricted December 11.
More Chromatographic Terms Efficiency N . it is a measure of the relative peak broadening for an analyte during a separation – w (from our chromatogram). Column length tR N = 16 w 2 or L N= H HETP A Number of Theoretical Plates A Single Real Plate Separation Fundamentals Agilent Restricted December 11. For LC.Number of theoretical plates – This is one case where more is better! “Plates” is a term inherited from distillation theory. 2007 .
5 is baseline resolution R = 2 is highly desirable during method development R = 2 (tR2 – tR1) (w1 + w2) t0 Resolution is reduced based on how fat or wide the peaks are. 2007 . so thin is better! > N value Original < k’ values > k’ values >α value Separation Fundamentals Agilent Restricted December 11.More Chromatographic Terms Resolution The distance between two neighboring peaks R = 1.
gradient slope and dwell volume (gradients) Separation Fundamentals Agilent Restricted December 11.Resolution … Determined by 3 Key Parameters – Efficiency. Selectivity and Retention The Fundamental Resolution Equation Resolution can be expressed in terms of the components we have discussed thus far. 2007 . α = Selectivity – influenced by mobile and stationary phase N = Column Efficiency – influenced by length and particle size k = Capacity Factor (retention) – influenced by stationary and mobile phase.
0 Separation Fundamentals Agilent Restricted December 11. 2007 .00 2.00 6.35 4.1 12.0 10000 1.10 2.5 25000 2.Resolution—Effects of Selectivity.5 15000 1.00 Selectivity Impacts Resolution Most – Change bonded phase – Change mobile phase Plates are easiest to increase Increase N Increase Alpha Increase k' Resolution 4.0 20000 1.00 3.60 7.00 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Plates: Alpha: k’: 5000 1.00 Rs = N½/4 • (α-1)/α • k’/(k’+1) 0. and Retention 7.00 1.85 9.00 5. Efficiency.
Start Method Development with RRHT Columns: Different ZORBAX C18 Bonded Phases for Max Selectivity 1st choice Best Resolution & Peak Shape 1 2 3 1 2 3 4 5 4 Eclipse Plus C18 Mobile phase: (69:31) ACN: water Flow 1. 2007 . 1 2.6 x 50 mm 1. Separation Fundamentals Agilent Restricted December 11. anandamide (AEA) 2. Palmitoylethanolamide (PEA) 3. for this separation.3 4 Extend-C18 1 2 3 4 5 Multiple bonded phases for most effective method development.8 um Sample: 1. Other choices better. Temp: 30 °C Detector: Single Quad ESI positive mode scan Columns: RRHT 4. Match to one you’re currently using. 2-arachinoylglycerol (2-AG) 4. Oleoylethanolamide (OEA) 2nd choice Good alternate selectivity due to non-endcapped 1 3 2 4 StableBond SB-C18 5 min 1 2 3 4 3rd choice Good efficiency & peak shape Resolution could be achieved 1 2 3 4 Eclipse XDB-C18 4 5 4th choice 1 2 3 Resolution not likely.5 mL/min.
07 Peak 2: 1.55 3.: 40% MeOH.3 α 1.3 α 1.03 Peak 2: 1.55 Tailing factors: Peak 1: 1.01 Peak 3: 1.82 2 3 Tailing factors: Peak 1: 1.3 0 α 1. 60% water 0 1 Resolution 4.2 peaks 2.04 0 Eclipse XDB-C8 peaks 1.68 1.33 1.4 Resolution 6.43 1.01 2 3 4 min Separation Fundamentals Agilent Restricted December 11. 5 um m.96 Peak 3: 1.2 peaks 2.00 caffeine Eclipse Plus C18 peaks 1.6 x 150.2 peaks 2.29 1 4 min Cols: 4.03 Peak 3: 1.Small Differences in α Can Provide Large Differences in Resolution barbital sulfamethoxazole Eclipse Plus C8 Greatest Resolution for this sample peaks 1.15 3.p.12 4.31 1 4 min Resolution 3. 2007 .07 Peak 2: 0.97 2 3 Tailing factors: Peak 1: 1.
pressure Selectivity (α) helps best but… Is difficult to predict. so method development is slower (experience helps. 2007 .If α Has the Most Impact. DryLab) Separation Fundamentals Agilent Restricted December 11. model retention) Note: Software supported. . • Plate number increase is limited by experimental conditions (analysis time.. optimization for separation of multi-component mixtures can reduce method development time (ChromSword. Why Focus on N? It’s Easy High plate number (N) provides: • Sharp and narrow peaks • Better detection • Peak capacity to resolve complex samples But… • Resolution increases only with the square root of the plate number.
2007 .5um N = 10.000 theoretical plates 15 Separation Fundamentals Agilent Restricted December 11.000 theoretical plates 1.8um 1 3 5 7 9 Time (min) 11 13 N = 20.Peak Width Decreases with Increasing N – Column Efficiency 5um N = 5000 theoretical plates 3.
particle size) in a given amount of time. 2007 .Peak Capacity Better Peak Capacity – More Peaks Resolved Peak capacity is the number of peaks that can be separated (at a specified resolution.8μm Rs = +50% 5 2 peaks fit 3 peaks fit 1/3 More!! Separation Fundamentals Agilent Restricted December 11. 4 5μm 1. example R=1) by a given system (think column length. It is another measure of efficiency.
Putting it Together The van Deemter Equation Plate Height H H = A + B/u + C u Large Particle Small Particle Sum Curve: van-Deemter Resistance to Mass Transfer H min Longitudinal diffusion Eddy Diffusion u opt Linear Velocity u The smaller the plate height. 2007 . the higher the plate number and the greater the chromatographic resolution Separation Fundamentals Agilent Restricted December 11.
0μm 3.05 – 5.0020 0.0 2. Volumetric Flow Rate 0.5 3.5 1.0 mL/min Temp: 20°C Sample: 1.0μL Octanophenone in Eluent H = A + B/u + Cu 0.5μm 1. 2007 .0 0.0010 0.5 4.5 2.0005 5.0025 HETP (cm) Column: ZORBAX Eclipse XDB-C18 Dimensions: 4.0 1.0 Volumetric Flow Rate (mL/min) Smaller particle sizes yield flatter curves.6 x 50/30mm Eluent: 85:15 ACN:Water Flow Rates: 0.0030 0.5 5.0 3. minima shift to higher flow rates Separation Fundamentals Agilent Restricted December 11.0000 0.0015 0.Van Deemter Curve : HETP vs.8μm 0.0 4.
2007 .Key Chromatographic Parameters Impacted by Particle Size Optimum Particle Diameter Efficiency Pressure Peak Volume Analysis Time Separation Fundamentals Agilent Restricted December 11.
What About Pressure? Pressure Increases Exponentially with Decreasing Particle Size Equation For Pressure Drop Across an HPLC Column ΔP ΔP η η = θ L v dp2 = Pressure Drop = Fluid Viscosity = Column Length = Flow Velocity = Particle Diameter L v dp θ Many parameters influence column pressure Particle size and column length are most critical Long length and smaller particle size mean more resolution and pressure We can now handle the pressure = Dimensionless Structural Constant of Order 600 For Packed Beds in LC Separation Fundamentals Agilent Restricted December 11. 2007 .
flow rate must be reduced to 0.0 P psi 2500 1850 996 116 P bar 172 128 69 8 tR Min 3.2 mL/min Separation Fundamentals Agilent Restricted December 11. 30 cm column. 2007 . 6 mm mobile phase = 100% water *To achieve N= 13. = 4.0 1.0 1.000 on a 10 μm.0 1.0 9.0 0.0 1.6 1.d.6 6.2* k (last peak) = 5 column i.0 1.Calculated Effect of Particle Size on Key Chromatographic Parameters dp Flow μm mL/min 2 3 5 10 N = 13.5 3.000 L cm 6 10 15 30 Vm mL 0.6 1.5 3.0 90 t0 Min 0.
15 S/N = 42 4.6 x 150.5um 165 bar N = 14862 Rs = 1.37 S/N = 50 4.6 x 150. 1.6 x 150.Smaller Particle Size Columns Improve Resolution – But Pressure Increases Up to 60% higher resolution than in conventional HPLC Customer Example Isocr.8um 490 bar N = 28669 Rs = 1. 5um 93 bar N = 7259 Rs = 1.80 (+57%) S/N = 44 Separation Fundamentals Agilent Restricted December 11. Impurity Method Zoom of critical time range @ 7min 4 Impurities 2 Not Baseline Separated! 7 Impurities 6 Not Baseline Separated! 7 Impurities All 7 Baseline Separated! 4. 3. 2007 .
1 x 100mm column can be used with ACN below 400 bar. 2007 . especially with slightly elevated temperature. Make sure the buffer is soluble in the organic at all points in the run (gradient). the organic selected is more critical. But with MeOH you will need the 600 bar RRLC systems for almost all MeOH water mobile phases. % of organic solvent – there is a pressure maximum and minimum for organic:aqueous mobile phases and it differs depending on the organic • • A 2.Mobile Phase How Does it Impact Pressure 1. 2. Water < Buffer While buffers increase viscosity. Solvent viscosity – lower viscosity results in lower pressure • • Acetonitrile < Methanol The difference between MeOH and ACN can be dramatic and is the first thing to change if lower pressure is needed. Separation Fundamentals Agilent Restricted December 11.
Temperature = 40C 900 857 800 756 700 619 581 635 626 614 563 509 441 400 383 320 288 439 AcCN actual reading MeOH actual reading AcCN predicted 150mm MeOH predicted 150mm pressure (bar) at 40C 600 601 500 300 200 100 0 0 % Organic 20 40 60 80 100 (G1312A Binary Pump .5 ml/min.1% TFA) 120 Separation Fundamentals Agilent Restricted December 11.1 x 100 mm ACN vs.8 um Column Installed F = 0. Aqueous Fraction is 0. MeOH Back Pressure of 600 Bar 1100 with a SB-C18. 2007 .1 x 100 mm.Pressure on 2.Undamped with 600 Bar Pressure Transducer. 2. 1.
Flow Rate 50 mm 85% MeOH: 15% H2O 30mm 50mm 50mm 150mm 5% ACN: 95% H2O w/ 0. Acetonitrile runs at lower pressure as expected.1% FA 208 bar Ambient 1 mL/min 245 bar Ambient 1 mL/min 384 bar 30°C 200 bar 25°C 418 bar 70°C 2 mL/min 2. 30% ACN 40% H2O 0. 2007 . Separation Fundamentals Agilent Restricted December 11.1%TFA 15% ACN. and organic choice is critical. 70% NaAc: 60% MeOH: 0.1% FA:85%.Some Examples of the Pressure On RRHT Columns (4. longer columns can not.6 mm ID) Length Mobile Phase Pressure Temp.5 mL/min Short columns can run below 400 bar.5 mL/min 1.
2007 . mobile phase: 15% acetonitrile.6 x 50 mm.Increasing Temperature Lowers Operating Pressure Pressure.) Separation Fundamentals Agilent Restricted December 11.5 3 3.8 um 400 350 300 System back pressure (bar) 250 30C 200 60C 90C 150 100 50 0 0 0.5 4 4.5 1 1. 1.5 Flow rate (mL/min. Analgesics Analysis.5 2 2. 4.
2007 . faster separations. greater efficiency and use of sub 2-micron columns Provides more rapid mass transfer: Improves Efficiency – enhances resolution Decreases analysis time – faster separations with no loss in resolution •Can change selectivity – optimize resolution Separation Fundamentals Agilent Restricted December 11.Higher Temperature as an Aid to Method Development and Faster Operation Higher Temperature: Temperature should always be considered as a parameter during method development Decreases Mobile Phase Viscosity Lowers backpressure – allows for higher flow rates.
5 1 1.29 0.5 min 60°C Rs=2. 2007 .5 1 1.5 4 4.5 3 3.5 3 3.5 2 2. Selectivity Gradient of Ten Cardiac Drugs on SB-C18 RRHT 50°C Rs=1.27 0.5 3 3.5 min 70°C Rs=3.5 4 4.37 0.5 2 2.5 min Separation Fundamentals Agilent Restricted December 11.5 4 4.5 1 1.5 2 2.Use Elevated Temperature to Optimize Resolution.
Conclusion HPLC is a powerful analytical tool There are a variety of separation modes Most applications are RP It’s important to know what the terms mean We have introduced a number of chromatographic terms here Many build up from simple measurements taken on a chromatogram Key Equations Resolution Many parameters can affect resolution van Deemter Other things like pressure can affect our separation as well Does not have to be a limitation Separation Fundamentals Agilent Restricted December 11. 2007 .
Appendix Separation Fundamentals Agilent Restricted December 11. 2007 .
2007 . At column installation Solvent changes LC System cleanliness Column Equilibration 2. Column use Are you operating at the pressure you anticipate? Check your gradient 3. Mobile phase Are there limitations? 4.What Extra Precautions Do You Need to Use RRHT Columns Successfully? 1. Sample considerations Particulate free samples Injection solvent Separation Fundamentals Agilent Restricted December 11.
RRHT Column Installation Recommendations 1.2 mL/min for a 3. and 0. 5. This occurs when the different solvents mix. Flush at least 5 mL of solvent before attaching the column to instrument. Goal: Eliminate any dried out or precipitated buffer from the system so it doesn’t wash onto the column and plug the frit.4 mL/min for 4.1 mL/min for a 2. 0. 4. Goal: anticipate the final operating pressure Once the pressure has stabilized. Equilibrate the column and detector with 10 column volumes of the mobile phase prior to use. Flush the column with your mobile phase (compatible with the solvents the column was shipped in) starting slowly at 0.6 mm ID. 2007 . 2. Goal: reproducible chromatography from the 1st run Separation Fundamentals Agilent Restricted December 11. attach the column to the detector. Goal: Avoid a pressure spike when the new mobile phase reaches the column. The low flow rate allows this to happen without causing an unanticipated pressure change. Purge the pumps (connections up to the column) of any buffered mobile phases. Increase the flow rate to the desired flow over a couple of minutes.0 mm ID column. 3.1 mm ID column.
check that the pressure range of the gradient – which may be 100 – 130 bar or more. 8. Once the pressure has stabilized. Avoid overtightening fittings/replace fitting to column periodically – use polyketone fittings for quick changes. will not cause the system to overpressure.6. Separation Fundamentals Agilent Restricted December 11. Goal: good connections. good unattended operation over 100’s or 1000’s of injections. 9. 10. 2007 . RRHT Column Installation Recommendations (cont. Equilibrate the column and detector with 10 column volumes of the mobile phase prior to use. If you are running a gradient. will not cause the system to overpressure. before starting any sequence. Goal: reproducible chromatography from the 1st run If you are running a gradient. Goal: no surprises. no extra volume.) 7. Goal: no surprises. before starting any sequence. good unattended operation over 100’s or 1000’s of injections. no overtightening of fittings. attach the column to the detector. check that the pressure range of the gradient – which may be 100 – 130 bar or more.
2 um filter. This prevents any bacterial growth in the injector from transferring to the column and plugging the frit. Replace bottles of mobile phase buffer every 24 – 48 hours. Separation Fundamentals Agilent Restricted December 11. Filter all samples with particulates through an appropriate 0.Mobile Phase and Sample Recommendations to Avoid High Pressure If the system has been sitting with buffer in it. Particulates can clog the inlet frit on the column and cause high pressure and short column lifetime. Filter all aqueous buffers prior to use through a 0. Use solvents that are high quality chromatography grade solvents (HPLC or MS grade). 2007 . Install an in-line filter to catch particulates before they get to the column.2um filter. flush the injector as well as the column. Do not top off the bottle with more mobile phase. replace the buffer with a fresh bottle Do not use a high buffer salt mobile phase (>50mM) in combination with high ACN concentrations due to possible precipitation.
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