Scaling Up to Preparative Chromatography

Size: px
Start display at page:

Download "Scaling Up to Preparative Chromatography"

Transcription

1 Scaling Up to Preparative Chromatography Dr. Shulamit Levin home page: Strategy for Preparative Separation Selection of the appropriate mode of chromatography ptimization of the Separation (Stationary phase, Mobile Phase, Temperature, Additives) ptimization of the throughput (Sample amount: Column verloading) Adsorption Isotherm & Competition 1

2 Seven Basic Considerations in Choosing HPLC perating Parameters 1) Solubility - Hexane, Chloroform, Methanol, Water (buffer ph), other? ) Molecular Weight - Would GPC be useful in either the analysis or sample prep? 3) Functional Groups - Any ionizable groups? Acidic, Basic, or Neutral? 4) Sample Matrix - What amounts are expected in matrix for either analytical or preparative isolation? 5) Levels in Matrix - What amounts are expected in matrix for either analytical or preparative isolation? 6) Detectability - Any chromophores or fluorophores? Consider Redox or derivatization. Together with point #5, an appropriate detector is chosen. 7) How Do Species Differ - An important clue to manipulate selectivity in the separation, especially if compounds are similar in their structure. High Performance Liquid Chromatography Modes PRINCIPLE F SEPARATIN: ADSRPTIN NRMAL PHASE PARTITIN REVERSED PHASE SLUTES: LIPPHYLIC: ILS, FATS, LIPIDS MST F THE BIMEDICAL SUBSTANCES MBILE PHASE: RGANIC SLVENTS: n-hexane, HEPTANE, CHLRFRM, ALCHLS AQUEUS MIXTURES WITH METHANL, ACETNITRILE AND ADDITIVES (BUFFERS, IN -PAIRS)

3 High Performance Liquid Chromatography Modes PRINCIPLE F SEPARATIN: IN-EXCHANGE SIZE-EXCLUSIN BI-AFFINITY CHIRALITY SLUTES: INRGANIC INS, ACIDS, BASES PLYMERS, PRTEINS, NUCLEIC ACIDS PRTEINS & ENZYMES ENANTIMERS MBILE PHASE: AQUEUS BUFFERS, INIC SLUTINS AQUEUS BUFFERS R RGANIC SLVENTS AQUEUS BUFFERS AND SPECIAL ADDITIVES AQUEUS R RGANIC SLVENTS Strategy for Preparative Separation: Selection of the appropriate mode of chromatography ptimization of the Separation (Stationary phase, Mobile Phase, Temperature, Additives) ptimization of the throughput (Sample amount: Column verloading) Adsorption Isotherm & Competition 3

4 PERFRMANCE CRITERIA BY NE PEAK Application RETENTIN FACTR or CAPACITY RATI k' = t R - t 0 t 0 k = C s φ Cm ASYMMETRY FACTR B(10 h) A f = A (10 h) AU height Width (50) Minutes Area: 8501 USP Resolution: 4.48 USP Tailing: 1.11 K Prime: 4.0 Retention Time: 0.74 A B 10h Width(base) TAILING FACTR T f = A + B (5 h) A NUMBER F THERETICAL PLATES t R N = 16 ( ) w PERFRMANCE BY TW PEAKS SELECTIVITY FACTR α = k' ( ) k' (1) t R(1) t R() EXPERIMENTAL RESLUTIN Rs = t R () - t R(1 ) 1/ (w 1 + w ) t 0 w 1 w 4

5 R E S P N S E 0 0 VID VID ISCRATIC vs GRADIENT TIME (MIN.) Gradient enables wide scope of chemical entities General Introduction to Preparative Chromatography 5

6 HPLC System-Prep Solvent (Mobile Phase) HPLC Column (Packing Material/Stationary Phase) Injector AutoSampler Data Pump Detector Fraction Collector (Purified Analyte(s)) Modern Setup of Preparative Chromatography: Mass and UV directed Separations Makeup Pump MS Autosampler Column Splitters Gradient Pump Purified Fractions PDA Waste Trigger of Fraction Collector by Detector on MS ELSD 6

7 Purification of 0.1 µmole of 5 HEX labeled 5mer Non-labeled failure sequences Target Product Labeled failure sequences UV 60nm UV 539nm 6 10 Minutes Fountain et al., J. Chromatogr. B, 003, vol. 783, 61-7 Various Dimensions of Preparative Columns

8 Problem Definition: Purifications What quantity of material needs to be isolated? Is the material a major or minor component? Do you need to maintain biological activity? What degree of purity (or specific activity) is required? How will purity or activity be verified? If analyzed sample is to be collected; the above questions must be answered Preparative Chromatography Terminology: Different from Analytical Chromatography Sample Solubility Load - verload Throughput Purity Recovery/Yield from Column Recovery from Fractions Cost of Purification 8

9 Understanding the Chromatographic Process is Essential for Rational Scaling-up B+A Stationary Phase Mobile phase A B Distribution: K = Cs/Cm B A k' = Elution through the Column t R - t 0 t 0 k = C s φ Cm Chromatogram RETENTIN FACTR = Capacity Factor We know the measurement of k from the analytical work: Retention Factor: k' = t R - t 0 t 0 We use its thermodynamic expression to rationally scale up the separation: CAPACITY RATI k = C s C s V s = = φ K C m C m V m 9

10 ADSRPTIN ISTHERMS: THE KEY T RATINAL SCALE-UP k Langmuir type C s = C m linear Adsorption Isotherm φ Linear C [ s k = φ ] C m C S Non-Linear practical working limit in the analytical mode C s = a C m 1 + b C m Non-Linear 0 C m, mole / L C s = k φ C m Mechanism of Tailing C s = a C m 1 + b C m Linear C S Non-Linear 0 practical working limit in the analytical mode C m, mole / L 10

11 Mechanism of Diffuse Front Non-Linear Linear C s = ac α C S Non-Linear practical working limit in the analytical mode 0 C m, mole / L C s = k φ C m Example of the Lab Scale Purification of Single Stranded RNA 11

12 Mass verload Absorbance 6.5 µg injected Analytical load of 6 µg yields efficient peak shape Minutes 4 6 Preparative load of 5 mg generates mass overload peak shape Absorbance 5 mg injected Note that the back of the peaks of the analytical and prep loads are at the same retention ( ) 4 Minutes 6 Volume verload Absorbance 6.5µg injected in 5 µl of column volume: 0.65 Column Volume: 0.8 ml (800 µl) Wider peaks first observed at low retention Absorbance 0.65 mg injected in 500 µl of column volume: Minutes 6 Peak position shifts to higher retention in proportion to the injection volume Start of peak remains in the same position unless injected in a weaker solvent Minutes 4 6 1

13 Scaling Comparison verlaid 8.0e- 7.5e- 7.0e- 6.5e- 6.0e- 5.5e- 5.0e- AU 4.5e- 4.0e- 3.5e- 3.0e-.5e-.0e- 1.5e- 1.0e- 5.0e-3 Time Strategy for Preparative Separation Selection of the appropriate mode of chromatography ptimization of the Separation (Stationary phase, Mobile Phase, Temperature, Additives) ptimization of the throughput (Sample amount: Column verloading) Adsorption Isotherm & Competition 13

14 From Small Scale To Purification Parameters of Scaling Up and ptimization of the verloaded Chromatography Mass Load Injection Volume Flow Rate Gradient/Run-Time Duration Column Dimensions Particle Size Chemistry Chemistry of the Mobile phase 14

15 Scaling Mass Load and Injection Volume Mass Load Proportional to column volume Limited by solubility in mobile phase Injection volume Approximately proportional to column volume Approximately proportional to both length and crosssectional area Most strongly dependent on sample solvent Scale Up Equations - Mass V M prep = M anal V prep anal M prep = mass injected on prep column M anal = mass injected on analytical column V prep = volume of prep column M anal = volume of analytical column 15

16 M M M L L d d L = M 1 L is Mass is is is is is Mass Length Length 1 on Diameter of Diameter d where : d 1 on Column 1 of Scaling Equations Mass Column of of Column 1 Column Column 1 Column (Analytical) (Prep) Mass Scalability - XTerra Prep MS C 18 Sample: 3 mg load XTerra MS C18 5 µ particle, 4.6x50mm 1.8 ml /min 70 nm 13B/min over 4 min 3mg Components: xicillin Cloxicillin Dicloxicillin Mobile phase: A: Water with 0.1 Formic Acid B: ACN with 0.1 Formic Acid Sample: 51 mg load XTerra Prep MS C18 5 µ particle, 19x50mm 31mL /min 70 nm 13B/min over 4 min 51mg 17x Sample: 18 mg load XTerra Prep MS C18 5 µ particle, 30x50 mm 77mL /min 70 nm 13B/min over 4 min 18mg 43x Time 16

17 Mass Scalability - XTerra Prep MS C x50mm 3mg 19x50mm 51mg 17x 30x50 mm 18mg 43x Time R (cm) R L (cm) V (ml) Scaling Factor Flow Mass Load Buspirone Impurities II AU 4.8 mg Structure of Buspirone N (CH ) 4 N N N AU Minutes 9.6 mg Minutes 19. mg Column: SymmetryPrep C18, 7µm (3.9 x 150) mm Mobile Phase: 8 acetonitrile / TETA-MeCH ph 7.0 Flow Rate: 1.0 ml/min Detection: UV at 360 nm Sample: 1 mg/ml of Buspirone Injection: from 0.4 to 1.6 ml N 0.10 AU 0.00 El Fallah Minutes 17

18 Approximate Mass Loading Capacity Many factors affect the mass capacity of preparative columns. The listed capacities represent an average estimate Mass Capacity (mg) Diameter (mm) Length (mm) Reasonable Flow Rate(ml/min) Reasonable Injection Volume( µ l) Parameters of Scaling Up and ptimization of the verloaded Chromatography Mass Load Injection Volume Flow Rate Gradient/Run-Time Duration Column Dimensions Particle Size Chemistry Chemistry of the Mobile phase 18

19 Scale Up of Flow Rate * Same Linear Velocity to maintain N and Rs and Retention Time - Van Deemter Scaling Equations Flow Rate F F F d d 1 1 d = F 1 d is Flow 1 where : Rate for Column 1 is Flow Rate Column is Diameter of Column 1 is Diameter of Column (Prep) (Analytical) 19

20 Valerophenone Impurities a 5 µm, 3.9 x 150 a: 0.70 ml/min Structure of Valerophenone AU CH 3 (CH ) 3 C Minutes AU b 7 µm, 7.8 x 300 b: 5.60 ml/min Column: a: Symmetry C18 5 µm (3.9x150) mm b: Symmetry Prep C18 7 µm (7.8x300) mm Mobile Phase: 60 acetonitrile / 40 water Sample: 4 mg/ml Valerophenone solution a: µl injection b: 800 µl injection Detection: UV at 340 nm El Fallah Minutes Example: Calculations AU AU a b 5 µm, 3.9 x 150 a: 0.70 ml/min 5.00 Minutes µm, 7.8 x 300 b: 5.60 ml/min El Fallah 5.00 Minutes R (cm) R^ L (cm) V (ml) Flow Scaling (ml/min) Factor Column Volume = π x R x L 0

21 Parameters of Scaling Up and ptimization of the verloaded Chromatography Mass Load Injection Volume Flow Rate Gradient/Run-Time Duration Column Dimensions Particle Size Chemistry Chemistry of the Mobile phase Understanding the Concept of Column Volumes Column Volume = π x R x L 1

22 Column Volume to Run-Time Volume Relationship 5 x50 mm 5 x50 mm Column Volume: (πr L~ ) 5 ml Column Volume: (πr L~ ) 15 ml 5 min Run-Time at Flow = ml/min 5 min Run Time at Flow = 50 ml/min Run-Time Volume: Analytical 5 min x 1 ml/min = 50 ml No. of Column Volumes: Run-Time Vol/Column Vol 50/ 5 = 10 Run-Time Volume: Preparative 5 min x 5 ml/min = 150 ml No. of Column Volumes: Run-Time Vol/Column Vol 150 / = 10 Valerophenone Impurities a 5 µm, 3.9 x 150 a: 0.70 ml/min Structure of Valerophenone AU CH 3 (CH ) 3 C Minutes AU b 7 µm, 7.8 x 300 b: 5.60 ml/min Column: a: Symmetry C18 5 µm (3.9x150) mm b: Symmetry Prep C18 7 µm (7.8x300) mm Mobile Phase: 60 acetonitrile / 40 water Sample: 4 mg/ml Valerophenone solution a: µl injection b: 800 µl injection Detection: UV at 340 nm El Fallah Minutes

23 Example: Calculations AU AU a b 5 µm, 3.9 x 150 a: 0.70 ml/min 5.00 Minutes µm, 7.8 x 300 b: 5.60 ml/min El Fallah 5.00 Minutes R (cm) R^ L (cm) V (ml) Scaling Flow Run-Time (Min) Column Volumes Factor Column Volume to Gradient Volume Relationship 5 x50 mm 5 x50 mm Column Volume: (πr L~ ) 5 ml Column Volume: (πr L~ ) 15 ml 5 min Gradient at Flow = ml/min 5 min Gradient at Flow = 50 ml/min Gradient Volume: Analytical 5 min x ml/min = 50 ml No. of Column Volumes: Gradient Vol/Column Vol 50/ 5 = 10 Gradient Volume: Preparative 5 min x 50 ml/min = 150 ml No. of Column Volumes: Gradient Vol/Column Vol 150 / = 10 3

24 Scale Up Equations - Gradient V prep t prep = tanal V anal F F anal prep t prep = run time with prep column t anal = run time with analytical column V prep = volume of prep column V anal = volume of analytical column F prep = prep flow rate F anal = analytical flow rate Example: Scale-up Gradient SunFire TM C x50 mm Total Mass Load: 5 mg 1 3 Flow Rate: 1.4 ml/min Gradient: Time Profile (min) A B Injection Volume: 0 µl - Time SunFire TM C 18 19x50mm Total Mass Load: 85mg 1 3 Flow Rate: 3.9 ml/min Gradient: Time Profile (min) A B Injection Volume: 340 µl - Time Compounds: Metoprolol (50 mg/ml), xprenolol (5 mg/ml), Propranolol (5 mg/ml) 4

25 - - 1 Scale-up Gradient SunFire TM C x50 mm Total Mass Load: 5 mg Time SunFire TM C 18 19x50mm Total Mass Load: 85mg Time Compounds: Metoprolol mg/ml), xprenolol mg/ml), Propranolol mg/ml) (50 (5 (5 (Addition of 0.8 min to compensate for instrument s delay volume) Flow Rate: 1.4 ml/min Gradient: Time Profile (min) A B Injection Volume: 0 µl Flow Rate: 3.9 ml/min Gradient: Time Profile (min) A B Injection Volume: 340 µl R (cm) R L (cm) V (ml) Scaling Factor Flow Prep Calculator 5

26 Parameters of Scaling Up and ptimization of the verloaded Chromatography Mass Load Injection Volume Flow Rate Gradient/Run-Time Duration Column Dimensions Particle Size Chemistry Chemistry of the Mobile phase Chromatography HPLC Columns Internal Diameter 1mm -- 50mm Length 3cm -- 50cm 30mm 500mm 6

27 Scale Up Column s Dimensions Dimensions Mass capacity is proportional to column volume Elution volume is proportional to column volume Shorter columns preferred for speed and pressure Longer columns preferred for resolution Effect of Column Length Efficiency increases with length Backpressure increases with length Run time increases with length Cost increases with length 7

28 Effect of Column Length on Capacity Absorbance Length = 50 mm Load = 1 mg Length = 150 mm Load = 36 mg As can be seen in this example, there is a linear relationship between column length and loading capacity - 3X increase in column length generates a >3X increase in loading capacity Disadvantage of long columns: - higher pressure for equal run time (9x at 3x increase in length) - higher pressure at equal velocity and longer run time 0 Minutes 0 Effect of Column Length XTerra MS C 18, BD, 5 µm 19 X mm -6 Time X 50 mm Time As column length decreases, resolution between the minor impurity and main peak decreases. Loading capacity will also decrease. 8

29 XTerra Prep Columns for Speed 40 Reduction in Retention and Peak Volume Peak # 1 Peak # Peak # 3 13 ml 9 ml 13 ml Peak # 1 Peak # Peak # 3 8 ml 6 ml 7 ml Scaling to Smaller Columns Allows: Faster chromatography Peak volume reduction Less expensive column Depending on the application, how far is it possible to downsize? Plate-to-plate mapping, injecting from a 96 well plate and collecting fractions in another 96 well plate, analytical size columns are suitable for this work 9

30 Load Considerations Mass Capacity Proportional to column volume Injection Volume Scale in proportion to the column volume Usually larger for gradient separations Cannot be considered separately from sample solvent Sample Solvent Good solvent needed to load high sample concentrations Good sample solvents degrade chromatography Good chromatographic diluents give low sample concentrations Parameters of Scaling Up and ptimization of the verloaded Chromatography Mass Load Injection Volume Flow Rate Gradient/Run-Time Duration Column Dimensions Particle Size Chemistry Chemistry of the Mobile phase 30

31 Effect of Particle Size on Capacity Column: 4.6 x 50 mm DS-A 10Å 15 µm 0 mg Load As particle size increases, resolution of the minor impurities decreases ( ) Absorbance 5 µm 0 mg Load Well resolved components not affected by change in particle size 50 µm 0 mg Load Minutes Effect of Particle Size XTerra MS C 18, BD, 19 X mm 5 µm 15 mg of buspirone -6 Time µm 15 mg of buspirone -6 Time As particle size increases, resolution between the minor impurity and the main peak decreases. 31

32 Parameters of Scaling Up and ptimization of the verloaded Chromatography Mass Load Injection Volume Flow Rate Gradient/Run-Time Duration Column Dimensions Particle Size Chemistry Chemistry of the Mobile phase Prochlorperazine: Effect of Loading Capacity on the Separation of Impurities Column: a: Kromasil a: SymmetryPrep C18 7µm (4.6x150) mm b: Kromasil C18 7µm (4.6x150) mm AU Minutes Mobile Phase: 75 methanol / 5 0 mm phosphate buffer ph 7.0 Flow Rate: 1 ml/min Detection: UV at 54 nm Sample: 10 µl of 0.97 mg/ml solution AU b: SymmetryPrep CH N 3 N CHCH CH N CH S H CH S H Cl Minutes S 3

33 Impact of Column Chemistry Conditions: Columns: 4.6 x 150 mm, 5 µm Mobile Phase A: 0.1 HCH in H Mobile Phase B: 0.1 HCH in ACN Flow Rate: 1.4 ml/min Gradient: Time Profile (min) A B Injection Volume: 10.0 µl Sample Diluent: DMS Temperature: 30 o C Detection: 54 nm Instrument: Alliance 695 with 996 PDA N NH CH 3 H H N xacillin H S CH 3 CH 3 Cl H N NH CH 3 Cloxacillin H S N H CH 3 CH 3 Cl Cl H H NH S N N CH 3 H Dicloxacillin CH 3 CH 3 All C 18 Columns are NT the Same SunFire C 18 Rs:.1 W 1/ : 0.4 Peak splitting Luna () C 18 Rs: 1.31 W 1/ : 0.39 Kromasil C 18 Hypersil Gold Rs: N/A W 1/ : N/A Rs: 0.71 W 1/ : mg Total Load Zorbax C 18 Rs: N/A W 1/ : N/A Time Time 33

34 Parameters of Scaling Up and ptimization of the verloaded Chromatography Mass Load Injection Volume Flow Rate Gradient/Run-Time Duration Column Dimensions Particle Size Chemistry Chemistry of the Mobile phase Chemistry of the Sample Solution: Maximizing Sample Load Mobile phase solvent choice Buffer the mobile phase Control of chromatographic ph conditions Reduces breakthrough of sample Use volatile buffers Reduces sample handling and energy required for evaporation Minimizes contamination of purified fraction Minimizes risk of target decomposition Mass spectrometry compatible Mobile phase ph impacts load Low ph maximizes acid load High ph maximizes base load 34

35 Sample Solvent Affects Resolution and Peak Shape Chromatography run at ph 3.8 Sample solvent ACN Sample solvent DMS Sample solvent IPA Sample solvent MeH Sample solvent THF Poor resolution DMS in high aqueous mobile phases generates a high viscosity pressure spike (observed post injection) resulting in poor resolution of the chromatographic peaks. All weaknesses in HPLC system will become evident. Increasing Load in DMS: Mass verload Column: XTerra MS C 18, mm, 5 µm.mobile phase A: water + 50 mm formic acid; mobile phase B: acetonitrile + 50 mm formic acid. Flow rate: 1.8 ml/min. Gradient: B in 5 min. UV: 80 nm. 5.4 mg Load Peak begins to front 5.7 mg Load Very bad fronting 7.5 mg Load Breakthrough 1.5 mg Load When DMS is used as the diluent, mass loading is 5.4 mg. 35

36 Non-Ionic Loading: Acids XTerra MS C 18 Breakthrough Cl Cl N NH 50 mg/ml ph = H H S CH3 CH3 N CH3 H DMS 1.5 mg Load -1.3X higher loading Nice peak shape DMS + 50 mm FA Formic acid was added to DMS directly, then use this acidified DMS to dissolve sample 1.5 mg Load Time When acidic sample is loaded in its non-ionic state, mass loading is up to 1.5 mg on XTerra MS C 18 column. Non-Ionic Loading: Bases XTerra MS C 8 00 mg/ml ph = 11-0 Shoulder appears USP Tailing: 1.60 DMS 6 mg Load Nice peak shape USP Tailing: 0.78 DMS + 50 mm NH 4 H 6 mg Load Time When a basic sample is loaded in its non-ionic state, peak tailing improves dramatically. 36

37 Loading a Base under Acidic Mobile Phase Conditions Column: XTerra MS C 18, mm, 5 µm.mobile phase A: water + 50 mm formic acid; mobile phase B: acetonitrile + 50 mm formic acid. Flow rate: 1.8 ml/min. Gradient: 5-95 B in 5 min. UV: 80 nm. Breakthrough DMS 13.5 mg DMS+ NH 4 H 13.5 mg DMS 10.5 mg Time 6.00 Amitriptyline HCl Time 6.00 When NH 4 H is added to the DMS, no breakthrough was observed. 1.8X high mass load by adding base to the diluent. - DMS+ NH 4 H 18 mg Choice of Strong Solvent (Acetonitrile vs. Methanol) Column: XTerra MS C 18, mm, 5 µm.mobile phase A: water + 50 mm formic acid; mobile phase B: acetonitrile/methanol + 50 mm formic acid. Flow rate: 1.8 ml/min. Gradient: B in 5 min. UV: 54 nm. Acetonitrile, ph Methanol, ph Cl Cl H H NH S N CH3 N H Dicloxacillin CH3 CH3 5.4 mg 6 mg Applying the same gradient with both organic solvents, sample elutes later with methanol than with acetonitrile. Changing the organic solvent may improve peak shape due to the additional interaction with the sample. 37

38 Use of Buffers Sample: Propranolol 5 mm Buffer, ph 7 DI water, ph 7 DI water, ph t r = t t r = ACN 3 mg 0 ACN 3 mg 7 ACN 0.5 mg Buffered mobile phases enhance retention and mass loading. There is a high risk of breakthrough and retention loss leading to recovery problems when buffers are left out! Modifier Impact on Load AU Formic Acid 0.3 mg Total Mass Analytes: 1. xacillin. Cloxacillin 3. Dicloxacillin Minutes AU TFA 0.96 mg Total Mass Minutes As sample mass / volume loading increases, strong ph shifts can occur, affecting chromatographic peak shapes. 38

39 Mobile Phase ph Effect: Loading of Bases XBridge C 18 1, 3 Adjusted gradient to keep the same k values. X5 in y axis Bases 1. Nordoxepin. Doxepin 3. Amitriptyline 0.1 mg ph.3-1 Time X0 in y axis -8 Time Time mg 0.5 mg ph 7 ph 10 High ph for basic analytes results in the best loadability, as well as the best retention and separation.* *: Uwe D. Neue et al. J. Chromatography A. Vol. 1030, , 004. Adjusted gradient to keep the same k values. XBridge TM C Mobile Phase ph Effect: Loading of Acids Acids 1. xacillin. Cloxacillin 3. Dicloxacillin 0.8 mg ph.3-0 Time mg ph 7 0 Time mg ph 10-0 Time Low ph for acidic analytes results in the highest loadability, as well as the best retention and separation.* *: Uwe D. Neue et al. J. Chromatography A. Vol. 1030, ,

40 Loadability with Varied ph Flurbiprofen Amitriptyline ph 4.80 mg 10.5 mg ph 11.4 mg 13.5 mg Increased retention for targets in their non-ionic state. Utilizing ph to Manipulate Target Retention ph is the most powerful tool to manipulate retention of ionizable compounds Elution rder (Standard Gradient Mode) early eluting peaks can be isolated more quickly later eluting peaks can be purified at higher loadings and elute in higher concentration organic solvent Basic compounds chromatographed in their unionized state (ph units above pka) are retained longer and exhibit excellent peak shape A popular high ph volatile buffer is 0.1 NH4H 40

41 Example: Basic Test Compounds CH 3 N CH 3 H N CH 3 CH 3 Diphenhydramine xybutynin C(CH 3 ) 3 N H H Terfenadine Peak shape and retention comparison: Basic Compounds at Low and High ph AU AU Minutes XTerra Prep MSC x 50 mm, 5 µm ph 3.8 ph Minutes Analytes: 1. Diphenhydramine (.5 mg/ml) 3 mg Load. Terfenadine (0.15 mg/ml) 0.18 mg Load 41

42 Scale-up Strategy - Summary 1. Define the problem Find the chromatographic mode.. Develop and optimize the separation Increas selectivity > Maximize throughput Measure adsorption isotherm. 4. Increase sample mass and volume to the maximum while meeting purity objectives. Examine the interferences 5. Determine recovery Examine residuals on the column 6. Scale up to desired column size to meet throughput/load objectives. Keep the flow rate and sample load ratio 7. Pool fractions of comparable purity and rerun if necessary. 8. Check fraction purity using analytical column. 4

Dilution(*) Chromatography

Dilution(*) Chromatography WA20264 Poster # 184, HPLC 2002, Montreal, 4-5 June 2002 At-Column Column-Dilution for Preparative Dilution(*) Chromatography Cecilia Mazza, Jie Cavanaugh, Ziling Lu,Tom Sirard,Tom Wheat and Uwe Neue Waters

More information

Preparative Chromatography with Improved Loadability

Preparative Chromatography with Improved Loadability 05 LCGC RTH AMERICA VLUME 0 UMBER VEMBER 00 www.chromatographyonline.com Preparative Chromatography with Improved Loadability In combinatorial chemistry and drug development, as well as in the full-scale

More information

LC Technical Information

LC Technical Information LC Technical Information Method Transfer to Accucore.6 μm Columns Containing solid core particles, which are engineered to a diameter of.6μm and a very narrow particle size distribution; Accucore HPLC

More information

STUDY OF LOADABILITY AND SELECTIVITY OF PHARMACEUTICAL COMPOUNDS ON RPLC COLUMNS

STUDY OF LOADABILITY AND SELECTIVITY OF PHARMACEUTICAL COMPOUNDS ON RPLC COLUMNS STUDY OF LOADABILITY AND SELECTIVITY OF PHARMACEUTICAL COMPOUNDS ON RPLC COLUMNS Fang Xia, Jie Y. Cavanaugh, Uwe Neue, Jeffrey R. Mazzeo, Diane M. Diehl Waters Corporation, Milford, MA USA OVERVIEW- INTRODUCTION-

More information

Reversed Phase Solvents

Reversed Phase Solvents Part 1. General Chromatographic Theory Part 2. verview of HPLC Media Part 3. The Role of the Mobile Phase in Selectivity Part 4. Column Care and Use Reversed Phase Solvents 2 Solvents for RP Chromatography

More information

HPLC Preparative Scaleup of Calcium Channel Blocker Pharmaceuticals Application

HPLC Preparative Scaleup of Calcium Channel Blocker Pharmaceuticals Application HPLC Preparative Scaleup of Calcium Channel Blocker Pharmaceuticals Application Pharmaceuticals Author Cliff Woodward and Ronald Majors Agilent Technologies, Inc. 2850 Centerville Road Wilmington, DE 19808

More information

Reversed Phase Chromatography: Mobile phase Considerations

Reversed Phase Chromatography: Mobile phase Considerations Reversed Phase: Mobile Phase Considerations Dr. www.forumsci.co.il/hplc Example of a Reversed Phase Method with UV Detection 1 Chromatographic Process B+A Mobile phase Elution through the Column-movie

More information

Prep 150 LC System: Considerations for Analytical to Preparative Scaling

Prep 150 LC System: Considerations for Analytical to Preparative Scaling Andrew Aubin and Jo-Ann Jablonski Waters Corporation, Milford, MA, USA APPLICATION BENEFITS The Prep 150 LC System is an affordable, highly reliable system for preparative chromatography and is suitable

More information

High Performance Liquid Chromatography

High Performance Liquid Chromatography Updated: 3 November 2014 Print version High Performance Liquid Chromatography David Reckhow CEE 772 #18 1 HPLC System David Reckhow CEE 772 #18 2 Instrument Basics PUMP INJECTION POINT DETECTOR COLUMN

More information

High Performance Liquid Chromatography

High Performance Liquid Chromatography Updated: 3 November 2014 Print version High Performance Liquid Chromatography David Reckhow CEE 772 #18 1 HPLC System David Reckhow CEE 772 #18 2 1 Instrument Basics PUMP INJECTION POINT DETECTOR COLUMN

More information

Basic Principles for Purification Using Supercritical Fluid Chromatography

Basic Principles for Purification Using Supercritical Fluid Chromatography Basic Principles for Purification Using Supercritical Fluid Chromatography Jo-Ann M. Jablonski, Christopher J. Hudalla, Kenneth J. Fountain, Steven M. Collier, and Damian Morrison Waters Corporation, Milford,

More information

High Performance Liquid Chromatography

High Performance Liquid Chromatography High Performance Liquid Chromatography What is HPLC? It is a separation technique that involves: Injection of small volume of liquid sample Into a tube packed with a tiny particles (stationary phase).

More information

Chiral separations efficient, fast and productive

Chiral separations efficient, fast and productive Chiral separations efficient, fast and productive By Per Jageland*, Mattias Bengtsson and Kristina Hallman AkzoNobel Eka Chemicals AB, Separation Products SE 445 80 Bohus Sweden Introduction Chromatographic

More information

Minimizing Solvent Impact on Purification of Nitrogencontaining

Minimizing Solvent Impact on Purification of Nitrogencontaining Minimizing Solvent Impact on Purification of Nitrogencontaining Compounds J. Liu and P. C. Rahn Biotage Discovery Chemistry Group US 1725 Discovery Drive Charlottesville, VA 22911 USA 1 Abstract This paper

More information

Chromatography. Gas Chromatography

Chromatography. Gas Chromatography Chromatography Chromatography is essentially the separation of a mixture into its component parts for qualitative and quantitative analysis. The basis of separation is the partitioning of the analyte mixture

More information

Product Bulletin. ACE LC/MS and Rapid Analysis HPLC Columns. HPLC Columns

Product Bulletin. ACE LC/MS and Rapid Analysis HPLC Columns. HPLC Columns Product Bulletin HPLC Columns ACE LC/MS and Rapid Analysis HPLC Columns 0 mm, 30 mm, 35 mm and 50 mm column lengths.0,., 3.0, 4.0 and 4.6 mm column diameters Configured for High Sample Throughput Specially

More information

Luna 2.5 µm C18(2)-HST. Advantages of 2.5 µm for increasing the speed of analysis while maintaining high efficiency

Luna 2.5 µm C18(2)-HST. Advantages of 2.5 µm for increasing the speed of analysis while maintaining high efficiency Luna 2.5 µm C18(2)-HST Advantages of 2.5 µm for increasing the speed of analysis while maintaining high efficiency Table of Contents Part 1 Theory 1.1 Abstract...3 1.2 Introduction...3 Part 2 Set Up 2.1

More information

Shodex TM ODP2 HP series columns

Shodex TM ODP2 HP series columns HPLC Columns Shodex TM ODP2 HP series columns Better retention of highly polar substances Technical notebook No. 6 Contents 1. Introduction 1-1. Specifications 1-2. Eluent Compatibility of ODP2 HP Series

More information

LC and LC/MS Column Selection Flow Chart

LC and LC/MS Column Selection Flow Chart LC and LC/MS Column Selection Flow Chart To use the column selection diagram below, simply follow the path for your analyte and mobile phase. At the far right, follow your final column selection to the

More information

Successfully Scaling and Transferring HPLC and UPLC Methods

Successfully Scaling and Transferring HPLC and UPLC Methods Successfully Scaling and Transferring HPLC and UPLC Methods Esa Lehtorinne Esa_Lehtorinne@waters.com Tel: +358-9-5659 6288 Fax: +358-9-5659 6282 Waters Finland Kutomotie 16 00380 Helsinki 2013 Waters Corporation

More information

Hypersil BDS Columns TG 01-05

Hypersil BDS Columns TG 01-05 TG 0-0 Hypersil BDS Columns Introduction Hypersil BDS columns have gained a reputation over the years as one of the most robust, reproducible and reliable HPLC column brands available. This Technical Guide

More information

Chem 230, Fall, 2014 Homework Set # 3 Short Answer SOLUTIONS

Chem 230, Fall, 2014 Homework Set # 3 Short Answer SOLUTIONS Chem 230, Fall, 2014 Homework Set # 3 Short Answer SOLUTIONS 1. List two advantages of temperature programming in GC. a) Allows separation of solutes with widely varying retention factors in a reasonable

More information

Transferring Yesterdays Methods to Tomorrows Technology: The Evolution from HPLC to UPLC TM

Transferring Yesterdays Methods to Tomorrows Technology: The Evolution from HPLC to UPLC TM Transferring Yesterdays Methods to Tomorrows Technology: The Evolution from HPLC to UPLC TM Eric S. Grumbach Thomas E. Wheat Chuck Phoebe Joe Arsenault Jeffrey R. Mazzeo Diane M. Diehl UPLC TECHNOLOGY

More information

penta-hilic UHPLC COLUMNS

penta-hilic UHPLC COLUMNS penta-hilic UHPLC COLUMNS penta-hilic Highly retentive, proprietary penta-hydroxy-ligand Excellent peak shape for polar compounds with a variety of functional groups: acids, bases, zwitterions strong and

More information

UPLC Method Development and Validation

UPLC Method Development and Validation UPLC Method Development and Validation Rev. 2 2008 Waters Corporation Challenges of Method Development Methods are developed throughout the drug development process Samples vary in complexity Redundancy

More information

DETERMINATION OF DRUG RELEASE DURING DISSOLUTION OF NICORANDIL IN TABLET DOSAGE FORM BY USING REVERSE PHASE HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

DETERMINATION OF DRUG RELEASE DURING DISSOLUTION OF NICORANDIL IN TABLET DOSAGE FORM BY USING REVERSE PHASE HIGH PERFORMANCE LIQUID CHROMATOGRAPHY CHAPTER 9 DETERMINATION OF DRUG RELEASE DURING DISSOLUTION OF NICORANDIL IN TABLET DOSAGE FORM BY USING REVERSE PHASE HIGH PERFORMANCE LIQUID CHROMATOGRAPHY CHAPTER 9 Determination of drug release during

More information

LECTURE 2. Advanced Separation Science Techniques Present and Future Separation Tools

LECTURE 2. Advanced Separation Science Techniques Present and Future Separation Tools LECTURE 2 Advanced Separation Science Techniques Present and Future Separation Tools Jack Henion, Ph.D. Emeritus Professor, Analytical Toxicology Cornell University Ithaca, NY 14850 Lecture 2, Page 1 Contents

More information

High Performance Liquid Chromatography. Table 1: Allowed HPLC Adjustment of USP <621> and EP <2.2.46>

High Performance Liquid Chromatography. Table 1: Allowed HPLC Adjustment of USP <621> and EP <2.2.46> High Performance Liquid Chromatography HPLC-9 Ultra-high Speed Analysis of Ibuprofen within USP Allowed Limits by Nexera Method Scouting In recent years, high-throughput analytical techniques have

More information

Chromatographic Analysis

Chromatographic Analysis Chromatographic Analysis Distribution of Analytes between Phases An analyte is in equilibrium between the two phases [S 1 ] [S 2 ] (in phase 1) (in phase 2) AS [S2 ] K 2 A S [S1 ] 1 AS, A 1 S Activity

More information

Chapter content. Reference

Chapter content. Reference Chapter 7 HPLC Instrumental Analysis Rezaul Karim Environmental Science and Technology Jessore University of Science and Technology Chapter content Liquid Chromatography (LC); Scope; Principles Instrumentation;

More information

Open Column Chromatography, GC, TLC, and HPLC

Open Column Chromatography, GC, TLC, and HPLC Open Column Chromatography, GC, TLC, and HPLC Murphy, B. (2017). Introduction to Chromatography: Lecture 1. Lecture presented at PHAR 423 Lecture in UIC College of Pharmacy, Chicago. USES OF CHROMATOGRAPHY

More information

HPLC Background Chem 250 F 2008 Page 1 of 24

HPLC Background Chem 250 F 2008 Page 1 of 24 HPLC Background Chem 250 F 2008 Page 1 of 24 Outline: General and descriptive aspects of chromatographic retention and separation: phenomenological k, efficiency, selectivity. Quantitative description

More information

Liquid Chromatography

Liquid Chromatography Liquid Chromatography 1. Introduction and Column Packing Material 2. Retention Mechanisms in Liquid Chromatography 3. Method Development 4. Column Preparation 5. General Instrumental aspects 6. Detectors

More information

ANALYSIS OF BASIC COMPOUNDS

ANALYSIS OF BASIC COMPOUNDS COMPARISON OF Fully AND Superficially POROUS PARTICLE COLUMNS FOR THE ANALYSIS OF BASIC COMPOUNDS Kenneth J. Fountain, Jane Xu, Zhe Yin, Pamela C. Iraneta, Diane M. Diehl Waters Corporation GOAL Compare

More information

Welcome to our E-Seminar: Choosing HPLC Columns for Faster Analysis Smaller and Faster

Welcome to our E-Seminar: Choosing HPLC Columns for Faster Analysis Smaller and Faster Welcome to our E-Sear: Choosing HPLC Columns for Faster Analysis Smaller and Faster High Throughput/Fast LC Requires. Short columns 0 mm or shorter Small particle sizes. µm Rapid Resolution or new.8 µm

More information

Principles of Gas- Chromatography (GC)

Principles of Gas- Chromatography (GC) Principles of Gas- Chromatography (GC) Mohammed N. Sabir January 2017 10-Jan-17 1 GC is a chromatographic technique utilizes gas as the mobile phase which is usually an inert gas (Hydrogen, Helium, Nitrogen

More information

Comparison of different aqueous mobile phase HPLC techniques

Comparison of different aqueous mobile phase HPLC techniques June 009 ewsletter Pharmaceutical Analysis: What is Your Problem? SIELC Technologies, Inc., Prospect Heights, IL 0070 Pharmaceutical analysis involves liquid chromatography of various compounds, from active

More information

UPLC for Synthetic Peptides: A User`s Perspective

UPLC for Synthetic Peptides: A User`s Perspective Tides 2007 Conference Las Vegas, May 23, 2007 UPLC for Synthetic Peptides: A User`s Perspective Holger Hermann, Jens Donath* Lonza AG, Switzerland Welcome to Lonza! One of the leading CMO Production facilities

More information

Instrumental Chemical Analysis

Instrumental Chemical Analysis L2 Page1 Instrumental Chemical Analysis Chromatography (General aspects of chromatography) Dr. Ahmad Najjar Philadelphia University Faculty of Pharmacy Department of Pharmaceutical Sciences 2 nd semester,

More information

Barry E. Boyes, Ph.D. Consumables and Accessories Business Unit May 10, 2000

Barry E. Boyes, Ph.D. Consumables and Accessories Business Unit May 10, 2000 Barry E. Boyes, Ph.D. Consumables and Accessories Business Unit May 0, 000 HPLC Column Troubleshooting What Every HPLC User Should Know :00 a.m. EST Telephone Number: 86-650-06 Chair Person: Tim Spaeder

More information

Choosing Columns and Conditions for the Best Peak Shape

Choosing Columns and Conditions for the Best Peak Shape Choosing Columns and Conditions for the Best Peak Shape What is Good Peak Shape and Why is it Important? Good peak shape can be defined as a symmetrical or gaussian peak and poor peak shape can include

More information

BÜCHI Labortechnik AG. Loadability _Company_Presentation BUCHI. For internal use only

BÜCHI Labortechnik AG. Loadability _Company_Presentation BUCHI. For internal use only BÜCHI Labortechnik AG Loadability page 1 Load ability Loading Capacity Resolution Sample Solubility (injection) Conclusion 2 page 2 Loading Capacity(active sites/sample affinity) Competitor B Competitor

More information

for Acclaim Mixed-Mode HILIC-1 Column

for Acclaim Mixed-Mode HILIC-1 Column for Acclaim Mixed-Mode HILIC-1 Column Product Manual for ACCLAIM Mixed-Mode HILIC-1 Page 1 of 17 Product Manual for ACCLAIM Mixed-Mode HILIC-1 Column 5µm, 4.6 x 250mm, P/N 066844 5µm, 4.6 x 150mm, P/N

More information

Liquid storage: Holds the solvent which is going to act as the mobile phase. Pump: Pushes the solvent through to the column at high pressure.

Liquid storage: Holds the solvent which is going to act as the mobile phase. Pump: Pushes the solvent through to the column at high pressure. High performance liquid chromatography (HPLC) is a much more sensitive and useful technique than paper and thin layer chromatography. The instrument used for HPLC is called a high performance liquid chromatograph.

More information

HPLC. GRATE Chromatography Lab Course. Dr. Johannes Ranke. September 2003

HPLC. GRATE Chromatography Lab Course. Dr. Johannes Ranke. September 2003 HPLC GRATE Chromatography Lab Course Dr. Johannes Ranke Organisation The groups Start at 9:00 am End at 18:00 pm at the latest Friday, 19th we will finish at 2:00 pm Thursday, 11th: Lecture at 08:15 am

More information

Assay Transfer from HPLC to UPLC for Higher Analysis Throughput

Assay Transfer from HPLC to UPLC for Higher Analysis Throughput MAY 2005 SEPARATION SCIENCE REDEFINED 31 Assay Transfer from HPLC to UPLC for Higher Analysis Throughput A typical HPLC assay was transferred and optimized for a Waters ACQUITY UPLC system to achieve both

More information

William E. Barber, Ph.D. Applications Chemist October

William E. Barber, Ph.D. Applications Chemist October William E. Barber, Ph.D. Applications Chemist ctober 0 00 Secrets of Good Peak Shape in HPLC Time: :00 p.m. CET Telephone Number: + 44 0 76 088 Chairperson: John Vis The Secrets of Good Peak Shape in HPLC

More information

Utilizing ELSD and MS as Secondary Detectors for Prep HPLC and Flash Chromatography. Tips and Techniques to Optimize ELSD and MS based Purification

Utilizing ELSD and MS as Secondary Detectors for Prep HPLC and Flash Chromatography. Tips and Techniques to Optimize ELSD and MS based Purification Utilizing ELSD and MS as Secondary Detectors for Prep HPLC and Flash Chromatography Tips and Techniques to Optimize ELSD and MS based Purification Utilizing ELSD and MS as Secondary Detectors for Prep

More information

Method Development in Solid Phase Extraction using Non-Polar ISOLUTE SPE Columns for the Extraction of Aqueous Samples

Method Development in Solid Phase Extraction using Non-Polar ISOLUTE SPE Columns for the Extraction of Aqueous Samples Technical Note 101 Method Development in Solid Phase Extraction using Non-Polar ISOLUTE SPE Columns for the Extraction of Aqueous Samples This technical note includes by specific information on the extraction

More information

penta-hilic UHPLC COLUMNS

penta-hilic UHPLC COLUMNS penta-hilic UHPLC COLUMNS Highly retentive, proprietary penta-hydroxy-ligand penta-hilic Excellent peak shape for polar compounds with a variety of functional groups: acids, bases, zwitterions strong and

More information

2.1 2,3 Dichloro Benzoyl Cyanide (2,3 DCBC) and survey of. manufactured commonly for the bulk drug industry, few references were

2.1 2,3 Dichloro Benzoyl Cyanide (2,3 DCBC) and survey of. manufactured commonly for the bulk drug industry, few references were . Introduction.,3 Dichloro Benzoyl Cyanide (,3 DCBC) and survey of analytical methods,3-dcbc substance, is the although advanced intermediate,3-dcbc is a of lamotrigine well-known bulk drug chemical manufactured

More information

ProntoSIL C18-EPS Reversed-Phase HPLC Columns

ProntoSIL C18-EPS Reversed-Phase HPLC Columns ProntoSIL C8-EPS Reversed-Phase HPLC Columns Provides excellent separation of polar compounds Better peak shape for acids and bases Stabilized bonded phase for rugged, robust HPLC methods More retentive

More information

Nexera UC Unified Chromatography

Nexera UC Unified Chromatography Nexera UC Unified Chromatography The latest addition to the chromatography toolbox Dr. Gesa J. Schad Shimadzu Europa GmbH A brief history of SFC ϒ Late 1800 s: it was found that heavy, non-volatile organic

More information

7. Stability indicating analytical method development and validation of Ramipril and Amlodipine in capsule dosage form by HPLC.

7. Stability indicating analytical method development and validation of Ramipril and Amlodipine in capsule dosage form by HPLC. 7. Stability indicating analytical method development and validation of and in capsule dosage form by HPLC. 7.1 INSTRUMENTS AND MATERIALS USED 7.1.1 INSTRUMENTS 1. Shimadzu LC-2010 CHT with liquid chromatograph

More information

Application Note. Author. Abstract. Xinlei Yang Agilent Technologies Co. Ltd Shanghai, China

Application Note. Author. Abstract. Xinlei Yang Agilent Technologies Co. Ltd Shanghai, China Rapid Deteration of Eight Related Aromatic Acids in the p-phthalic Acid Mother Liquid Using an Agilent 126 Infinity LC System and an Agilent Poroshell 12 SB-C18 Column Application Note Author Xinlei Yang

More information

Application Note Pharmaceutical QA/QC. Agilent Application Solution. Authors. Abstract. Syed Salman Lateef Agilent Technologies, Inc.

Application Note Pharmaceutical QA/QC. Agilent Application Solution. Authors. Abstract. Syed Salman Lateef Agilent Technologies, Inc. Agilent Application Solution Transfer of a USP method for tolazamide from normal phase HPLC to SFC using the Agilent 126 Infinity Hybrid SFC/UHPLC System Improving peak shape and providing wider UV selectivity

More information

High Pressure/Performance Liquid Chromatography (HPLC)

High Pressure/Performance Liquid Chromatography (HPLC) High Pressure/Performance Liquid Chromatography (HPLC) High Performance Liquid Chromatography (HPLC) is a form of column chromatography that pumps a sample mixture or analyte in a solvent (known as the

More information

TEMPLATE FOR AN EXAMPLE STANDARD TEST METHOD

TEMPLATE FOR AN EXAMPLE STANDARD TEST METHOD APPENDIX V TEMPLATE FOR AN EXAMPLE STANDARD TEST METHOD Validating Chromatographic Methods. By David M. Bliesner Copyright 2006 John Wiley & Sons, Inc. 159 160 APPENDIX V Title: Effective: Document No:

More information

Unexpected Peaks in Chromatograms - Are They Related Compounds, System Peaks or Contaminations? From the Diary of an HPLC Detective

Unexpected Peaks in Chromatograms - Are They Related Compounds, System Peaks or Contaminations? From the Diary of an HPLC Detective Unexpected Peaks in Chromatograms - Are They Related Compounds, System Peaks or Contaminations? From the Diary of an HPLC Detective SHULAMIT LEVIN HPLC in Pharmaceutics Σ Stability Indicating Methods Extra

More information

Introduction to Capillary GC. Page 1. Agilent Restricted February 2, 2011

Introduction to Capillary GC. Page 1. Agilent Restricted February 2, 2011 ?? Kβ? Page 1 Typical GC System Gas supply Injector Detector Data handling GAS Column Oven Page 2 CARRIER GAS Carries the solutes down the column Selection and velocity influences efficiency and retention

More information

Agilent s New Weak Anion Exchange (WAX) Solid Phase Extraction Cartridges: SampliQ WAX

Agilent s New Weak Anion Exchange (WAX) Solid Phase Extraction Cartridges: SampliQ WAX Agilent s New Weak Anion Exchange (WAX) Solid Phase Extraction Cartridges: SampliQ WAX Technical Note Agilent s SampliQ WAX provides Applications for strongly acidic, acidic and neutral compounds Excellent

More information

Chromatography- Separation of mixtures CHEM 212. What is solvent extraction and what is it commonly used for?

Chromatography- Separation of mixtures CHEM 212. What is solvent extraction and what is it commonly used for? Chromatography- Separation of mixtures CHEM 212 What is solvent extraction and what is it commonly used for? How does solvent extraction work? Write the partitioning coefficient for the following reaction:

More information

HPLC Determination of Antidepressants and their Metabolites in Plasma

HPLC Determination of Antidepressants and their Metabolites in Plasma HPLC Determination of Antidepressants and their Metabolites in Plasma

More information

Cation Exchange HPLC Columns

Cation Exchange HPLC Columns Cation Exchange HPLC Columns Hamilton offers seven polymeric packing materials for cation exchange separations. Type Recommended Application(s) PRP-X00 PRP-X00 PRP-X800 HC-0 HC-7 Ca + HC-7 H + HC-7 Pb

More information

Optical Isomer Separation Columns and Packing Materials

Optical Isomer Separation Columns and Packing Materials 02 Optical Isomer Separation s and Packing Materials CHIRAL ART----------------------------------- 26~29 YMC CHIRAL NEA (R), (S)-----------------------30 YMC CHIRAL CD BR------------------------------31

More information

The Secrets of Rapid HPLC Method Development. Choosing Columns for Rapid Method Development and Short Analysis Times

The Secrets of Rapid HPLC Method Development. Choosing Columns for Rapid Method Development and Short Analysis Times The Secrets of Rapid HPLC Method Development Choosing Columns for Rapid Method Development and Short Analysis Times Rapid Analysis Is More Than Run Time It is developing a method to meet a goal and developing

More information

Biochemistry. Biochemical Techniques HPLC

Biochemistry. Biochemical Techniques HPLC Description of Module Subject Name Paper Name 12 Module Name/Title 13 1. Objectives 1.1. To understand the basic concept and principle of 1.2. To understand the components and techniques of 1.3. To know

More information

InertSustainSwift C8

InertSustainSwift C8 Physical Properties Silica Particle Size Surface Area Pore Size Pore Volume Bonded Phase End-capping Carbon Loading ph Range USP Code :ES (Evolved Surface) Silica Gel :.9 μm, μm, μm :00 m /g :00 Å (0 nm)

More information

UPC 2 Strategy for Scaling from Analytical to Preparative SFC Separations

UPC 2 Strategy for Scaling from Analytical to Preparative SFC Separations UPC Strategy for Scaling from Analytical to Preparative SFC Separations Christopher J. Hudalla, Abhijit Tarafder, Jo-Ann Jablonski, and Kenneth J. Fountain Waters Corporation, Milford, MA, USA APPLICATION

More information

Quantitative Analysis of EtG and EtS in Urine Using FASt ETG and LC-MS/MS

Quantitative Analysis of EtG and EtS in Urine Using FASt ETG and LC-MS/MS Quantitative Analysis of EtG and EtS in Urine Using FASt ETG and LC-MS/MS UCT Part Numbers: CSFASETG203 - CLEAN SCREEN FASt ETG, 200mg / 3mL tube SLETG100ID21-3UM - Selectra ETG HPLC column, 100 x 2.1

More information

High Performance Liquid Chromatography

High Performance Liquid Chromatography STANDARDBASE techniques: High Performance Liquid Chromatography Drenthe College, The Netherlands 1. Introduction HPLC. High Performance Liquid Chromatography High Performance Liquid Chromatography (HPLC)

More information

Analytical Chemistry

Analytical Chemistry Analytical Chemistry Chromatographic Separations KAM021 2016 Dr. A. Jesorka, 6112, aldo@chalmers.se Introduction to Chromatographic Separations Theory of Separations -Chromatography Terms Summary: Chromatography

More information

Abstract: An minimalist overview of chromatography for the person who would conduct chromatographic experiments, but not design experiments.

Abstract: An minimalist overview of chromatography for the person who would conduct chromatographic experiments, but not design experiments. Chromatography Primer Abstract: An minimalist overview of chromatography for the person who would conduct chromatographic experiments, but not design experiments. At its heart, chromatography is a technique

More information

Application Note. Author. Abstract. Pharmaceutical QA/QC. Siji Joseph Agilent Technologies, Inc. Bangalore, India

Application Note. Author. Abstract. Pharmaceutical QA/QC. Siji Joseph Agilent Technologies, Inc. Bangalore, India Effective use of pharmacopeia guidelines to reduce cost of chromatographic analysis Optimized, cost-effective HPLC analysis of atorvastatin by varying column dimensions within the USP allowed limts

More information

Comparison Guide to C18 Reversed Phase HPLC Columns

Comparison Guide to C18 Reversed Phase HPLC Columns Comparison Guide to C18 Reversed Phase HPLC Columns Comparison Data on Commonly Used C18 Phases Stationary Phase Specifications Phases Compared According to Relative Hydrophobicity Phases Compared According

More information

Packings for HPLC. Packings for HPLC

Packings for HPLC. Packings for HPLC Summary of packings for HPLC In analytical HPLC, packings with particle sizes of 3 to 10 µm are preferred. For preparative separation tasks, also particles with diameters larger than 10 µm are applied.

More information

Quantitative Analysis of EtG and EtS in Urine Using FASt ETG and LC-MS/MS

Quantitative Analysis of EtG and EtS in Urine Using FASt ETG and LC-MS/MS Quantitative Analysis of EtG and EtS in Urine Using FASt ETG and LC-MS/MS UCT Part Numbers: CSFASETG203 - CLEAN SCREEN FASt ETG, 200mg / 3mL tube SLETG100ID21-3UM - Selectra ETG HPLC column, 100 x 2.1

More information

Chemical derivatization

Chemical derivatization Chemical derivatization Derivatization in liquid chromatography and mass spectrometry Aims: increase analyte stability increase solubility improve chromatographic properties increase detection sensitivity

More information

CYCLOSERINE Final text for addition to The International Pharmacopoeia. (November 2008) CYCLOSERINUM CYCLOSERINE

CYCLOSERINE Final text for addition to The International Pharmacopoeia. (November 2008) CYCLOSERINUM CYCLOSERINE December 2008 CYCLOSERINE Final text for addition to The International Pharmacopoeia (November 2008) This monograph was adopted at the Forty-third WHO Expert Committee on Specifications for Pharmaceutical

More information

State-of-the-art C18 HPLC Columns

State-of-the-art C18 HPLC Columns An HPLC GL Sciences Newest and Most Advanced ODS Phase-New For 00 State-of-the-art C HPLC s Improved Peak Shapes and Heights Enhancing Sensitivity High Resolution Fast Equilibration Compatible with 00%

More information

CHAPTER CHROMATOGRAPHIC METHODS OF SEPARATIONS

CHAPTER CHROMATOGRAPHIC METHODS OF SEPARATIONS Islamic University in Madinah Department of Chemistry CHAPTER - ----- CHROMATOGRAPHIC METHODS OF SEPARATIONS Prepared By Dr. Khalid Ahmad Shadid Chemistry Department Islamic University in Madinah TRADITIONAL

More information

HPLC Praktikum Skript

HPLC Praktikum Skript HPLC Praktikum Skript Assistants: Gianluca Bartolomeo HCI D330, 3 46 68, bartolomeo@org.chem.ethz.ch Sahar Ghiasikhou HCI E330, 2 29 29, ghiasikhou@org.chem.ethz.ch 1. Introduction In chromatographic techniques,

More information

High Speed 2D-HPLC Through the Use of Ultra-Fast High Temperature HPLC as the Second Dimension

High Speed 2D-HPLC Through the Use of Ultra-Fast High Temperature HPLC as the Second Dimension High Speed 2D-HPLC Through the Use of Ultra-Fast High Temperature HPLC as the Second Dimension Minnesota Chromatography Forum Spring Symposium Dwight Stoll and Peter W. Carr Department of Chemistry University

More information

Rapid Screening and Analysis of Components in Nonalcoholic Drinks Application

Rapid Screening and Analysis of Components in Nonalcoholic Drinks Application Rapid Screening and Analysis of Components in Nonalcoholic Drinks Application Food Author Michael Woodman Agilent Technologies, Inc. 285 Centerville Road Wilgton, DE 1988-161 USA Abstract Soft drinks,

More information

The Effect of Contact Angle and Wetting on Performance in Solid Phase Extraction

The Effect of Contact Angle and Wetting on Performance in Solid Phase Extraction The Effect of Contact Angle and Wetting on Performance in Solid Phase Extraction Edouard S. P. Bouvier, Randy E. Meirowitz and Uwe D. Neue Waters Corporation, 34 Maple Street, Milford, MA 01757 USA Presented

More information

Routine MS Detection for USP Chromatographic Methods

Routine MS Detection for USP Chromatographic Methods Daniel S. Root, Thomas E. Wheat, and Patricia McConville Waters Corporation, Milford, MA, USA APPLICATION BENEFITS This approach enables mass spectral analysis of peaks directly from unmodified USP HPLC

More information

NEVIRAPINE ORAL SUSPENSION Final text for addition to The International Pharmacopoeia (February 2009)

NEVIRAPINE ORAL SUSPENSION Final text for addition to The International Pharmacopoeia (February 2009) February 2009. NEVIRAPINE ORAL SUSPENSION Final text for addition to The International Pharmacopoeia (February 2009) This monograph was adopted at the Forty-third WHO Expert Committee on Specifications

More information

Chromatographic Separation

Chromatographic Separation What is? is the ability to separate molecules using partitioning characteristics of molecule to remain in a stationary phase versus a mobile phase. Once a molecule is separated from the mixture, it can

More information

Packed Column for Ultra-Fast Reversed-Phase Liquid Chromatography, TSKgel Super-ODS. Table of Contents

Packed Column for Ultra-Fast Reversed-Phase Liquid Chromatography, TSKgel Super-ODS. Table of Contents No. 089 SEPARATION REPORT Packed Column for Ultra-Fast Reversed-Phase Liquid Chromatography, TSKgel Super-ODS Table of Contents 1. Introduction 1 2. Column Specification 1 3. Features of Packing Materials

More information

C18 Column. Care & Use Sheet

C18 Column. Care & Use Sheet C18 Column Care & Use Sheet HALO Description HALO C18 is a high-speed, high-performance liquid chromatography column based on a new Fused-CoreTM particle design. The Fused-Core particle provides a thin

More information

Streamlining the Analysis of Oral Contraceptives Using the ACQUITY UPLC H-Class System

Streamlining the Analysis of Oral Contraceptives Using the ACQUITY UPLC H-Class System Streamlining the Analysis of Oral Contraceptives Using the ACQUITY UPLC H-Class System Margaret Maziarz, Sean M. McCarthy, Michael D. Jones, and Warren B. Potts Waters Corporation, Milford, MA, USA A P

More information

CHROMATOGRAPHY. The term "chromatography" is derived from the original use of this method for separating yellow and green plant pigments.

CHROMATOGRAPHY. The term chromatography is derived from the original use of this method for separating yellow and green plant pigments. CHROMATOGRAPHY The term "chromatography" is derived from the original use of this method for separating yellow and green plant pigments. THEORY OF CHROMATOGRAPHY: Separation of two sample components in

More information

Improve Peak Shape and Productivity in HPLC Analysis of Pharmaceutical Compounds with Eclipse Plus C8 Columns Application

Improve Peak Shape and Productivity in HPLC Analysis of Pharmaceutical Compounds with Eclipse Plus C8 Columns Application Improve Peak Shape and Productivity in HPLC Analysis of Pharmaceutical Compounds with Eclipse Plus C8 Columns Application Pharmaceuticals Authors John W. Henderson Jr., Nona Martone, and Cliff Woodward

More information

Introduction to Pharmaceutical Chemical Analysis

Introduction to Pharmaceutical Chemical Analysis Introduction to Pharmaceutical Chemical Analysis Hansen, Steen ISBN-13: 9780470661222 Table of Contents Preface xv 1 Introduction to Pharmaceutical Analysis 1 1.1 Applications and Definitions 1 1.2 The

More information

M > ACN > > THF

M > ACN > > THF Method Development in HPLC Dr. Amitha Hewavitharana School of Pharmacy University of Queensland Method Development in HPLC References: D A Skoog Principles of instrumental analysis, 3 rd Edition Chapters

More information

Introduction. Chapter 1. Learning Objectives

Introduction. Chapter 1. Learning Objectives Chapter 1 Introduction Learning Objectives To understand the need to interface liquid chromatography and mass spectrometry. To understand the requirements of an interface between liquid chromatography

More information

Gas Chromatography. Introduction

Gas Chromatography. Introduction Gas Chromatography Introduction 1.) Gas Chromatography Mobile phase (carrier gas) is a gas - Usually N 2, He, Ar and maybe H 2 - Mobile phase in liquid chromatography is a liquid Requires analyte to be

More information

Inertsil Technical Library

Inertsil Technical Library HPLC COLUMN INERTSIL TECHNICAL LIBRARY H P L C C o l u m n Inertsil Technical Library Inertsil ODS-4, C8-4 Comparison of Performance List of Compared Columns Experimental Explanation, Analytical Conditions

More information

Chemistry Instrumental Analysis Lecture 27. Chem 4631

Chemistry Instrumental Analysis Lecture 27. Chem 4631 Chemistry 4631 Instrumental Analysis Lecture 27 Gas Chromatography Introduction GC covers all chromatographic methods in which the mobile phase is gas. It may involve either a solid stationary phase (GSC)

More information

Acclaim Mixed-Mode WCX-1

Acclaim Mixed-Mode WCX-1 Acclaim Mixed-Mode WCX-1 Product Manual for the Acclaim Mixed-Mode WCX-1 Column Page 1 of 28 PRODUCT MANUAL for the Acclaim Mixed-Mode WCX-1 Columns 4.6 x 150 mm, P/N (068353) 4.6 x 250 mm, P/N (068352)

More information