An Overview of the the Development of Stationary Phases for Reversed-Phase Liquid Chromatography
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1 An Overview of the the Development of Stationary Phases for Reversed-Phase Liquid Chromatography Analytical Potential of Stable Phases for Reversed-Phase Liquid Chromatography by Jacek Nawrocki, Jon Thompson, Yun Mao, Bingwen Yan, Dwight R. Stoll and Peter W. Carr
2 Key Papers in History of Stable Reversed-Phases: 1. A. Wehrli, J.C. Hildenbrand, H.P. Keller, R. Stampfli, R.W. Frei, "Influence of organic bases on the stability and separation properties of reversed-phase chemically bonded silica gels", J. Chromatogr. 19 (1978), J.J. Kirkland, J.L. Glajch, R.D. Farlee, "Synthesis and characterization of highly stable bonded phases for highperformance liquid chromatography column packings", Anal. Chem. 61 (1989), -11.
3 Outline Part I. Overview of analytical potential of high phase stability. Chemical stability. Thermal stability. Part II. Using stability to achieve selectivity. Thermally tuned tandem columns in HPLC. Part III. Using stability to speed up HPLC. High temperature ultrafast liquid chromatography. High temperature fast two dimensional liquid chromatography.
4 Advantages of Highly Stable Stationary Phases High Chemical Stability ph Stability Thermal Stability ph < 1 ph >13 Lower Pressure Drop Less Organic Solvent Thermally Optimized Selectivity Allows Cleaning with Conc. Acid Ion Suppression of COOH Sanitization Depyrogenation Ion Suppression of NH Less Ware and Tare Higher Flow Rate More Robust Analysis Easier Method Development Faster analyses
5 Temperature The Third Dimension in HPLC Temperature Mobile Phase Stationary Phase
6 Role of Temperature in LC High-Performance Liquid Chromatography at Elevated Temperatures: Examination of Condition for the Rapid Separation of Large Molecules, R. D. Antia and Cs. Horvath, J. Chromatogr., 3, 1-1 (1988). Temperature as a Variable in Reversed Phase High- Performance Liquid Chromatographic Separations of Peptide and Protein Samples, W. S. Hancock, R. C. Chloupek, J. J. Kirkland and L. R. Snyder, J. Chromatogr. A, 686, 31-3 (199) Superheated Water: A New Look at a Chromatographic Eluent for Reversed-Phase Liquid Chromatography, R. M. Smith and R. J. Burgess, LC-GC, 17, (1999)
7 Part II. The Thermally Tuned Tandem Column (T 3 C) Concept
8 Outline Importance of Selectivity in HPLC Optimization Thermally Tuned Tandem Column (T 3 C) Concept Theory Optimization An Example Ten Triazine Herbicides Applications Urea and Carbamate Pesticides Barbiturates Antihistamine Drugs Conclusions T 3 C Works It Can Save Time or Do Difficult Separations Only Four or Five Initial Runs Are Needed
9 The Ultimate Goal of Separation: Resolution (R) 3.. α Resolution (R) N k Efficiency Selectivity Retention R= N α-1 α k k Selectivity (α) has the greatest impact on improving resolution α N k
10 3% ACN vs. % ACN. 1. R =.989 SD=. Comparison of Variables Affecting Selectivity Carbon-ZrO vs. PBD-ZrO. 1. MeOH vs. THF R =.896 SD= logk' (/ ACN/H O) 8 o C vs. 3 o C R =.99 SD= R =.38 SD= logk' (THF/H O) C18-SiO vs. PBD-ZrO 1. R =.973 SD= logk' (PBD-ZrO ). 1 3 logk' (C18, 3 o C) logk' (PBD-ZrO ) Stationary phase type can have a very large effect on selectivity.
11 The Concept: Thermally Tuned Tandem Columns (T 3 C) A Mechanism to Continuously Adjust the Stationary Phase Temperature 1 Temperature Pump Injector Column 1 Column Detector e.g. C18-SiO e.g. C-ZrO Column 1 1, 3 Requirements for T 3 C: Column Optimized T 3 C 1 3, 1 3 Two columns with different (ideally orthogonal) selectivity One very thermally stable column Method development must be easy
12 Absorbance (mau) Separation of Ten Triazine Herbicides by T 3 C Time (min) 9 C18-SiO 3 o C C-ZrO 6 o C 3 T 3 C 1 CH 3 S, CH 3 O, Cl =R Solutes: 1. Simazine. Cyanazine 3. Simetryn. Atrazine. Prometon C18-SiO C-ZrO 3 o C 1 o C N N N N H R 1 H N R Other conditions: 3/7 ACN/water 1ml/min; nm detection 6. Ametryn 7. Propazine 8. Terbutylazine 9. Prometryn 1. Terbutryn T 3 C can improve separation without increasing analysis time
13 Guidelines for Optimizing T 3 C Choose Two Stationary Phases Window Diagram Optimization Choose Mobile Phase (1<k <) Calculating T 3 C Retentions ln k =A+B/T t rnet =t r1 +t r No Different Selectivity? Yes Two More Runs at High Temperatures
14 Steps in T 3 C Optimization of Triazine Herbicides Log k (C-ZrO, 6 o C) R =.17, sd= log k (ODS, 3 o C) Minimum Resolution T 3 C, 3ml/min 8 Temperature on C-Zr Column 9 1 T ODS =3 o C T C-ZrO =1 o C Rs= R= Temperature on ODS Column Time (min)
15 Applications of T 3 C Method Absorbance (mau) Urea and Carbamate Pesticides * * * T 3 C 39 o C+89 o C 9 Time (min) C18-SiO 3 o C C-ZrO 9 o C Absorbance (mau) Barbiturates +7 C18-SiO 3 o C Time (min) 1 C-ZrO 3 o C T 3 C 8 o C+ o C
16 Separation of Anti-Histamines by T 3 C C18-SiO o C Absorbance (mau) PBD-ZrO 3 o C T 3 C 3 o C + o C Time (min)
17 Conclusions T 3 C offers unique selectivity for the separation of complex mixtures. T 3 C requires that on the two phases the critical pairs must be different. Carbon Phase + Aliphatic Phase Reversed Phase + PBD-ZrO Phase Phosphate Buffer Neutral Compounds Basic Compounds Optimization needs only or trial runs. In many cases, T 3 C: is superior to mobile phase optimization. provides better resolution than a single phase. improves analysis speed.
18 Part III. High Temperature Ultra-Fast Liquid Chromatography
19 Why Fast HPLC? Monitor reaction rates with half-lives on order of minutes not hours. Monitor prep scale chromatography. Increase sample through-put thus lower cost. Increase screening rate in combinatorial chemistry (speed up LC side of LC-MS). Make D-HPLC practical and thus greatly enhance peak capacity of HPLC.
20 HPLC is Slow Compared to Other Methods* Technique TLC Open Column LC Early HPLC Current HPLC Packed GC Open Capillary GC CE** d p (µm) d c =.3 mm d c =.1-. mm N eff /t (plates/s) *L.R. Snyder; J.J. Kirkland, Introduction to Modern Liquid Chromatography; Wiley: New York, **R. Kennedy et al., Chem. Rev., 99, (1999).
21 Fast HPLC at High Temperature B 1 o C ml/min Absorbance (mau) A 3 o C 1 ml/min Time (min)
22 Effect of Temperature on Analysis Time at Constant N and P t R /t R, TEMPERATURE ( O C) High-Performance Liquid Chromatography at Elevated Temperatures: Examination of Condition for the Rapid Separation of Large Molecules, R. D. Antia and Cs. Horvath, J. Chromatogr., 3, 1-1 (1988).
23 Theoretical and Practical Limits of Fixed Pressure * Theoretical Limit * Reduced Velocity Limit Practical Limit Speed in HPLC t N t N ν = max (1 + D = k o D A(1 + D k ' ) m d m 1 / 3 m 3 p η h ν t C (1 + ν N D m P k ' ) η d p k ' ) max 1 / 3 d p L P / 3 / 3 max Practical Limit Temperature Dependence t N (1 + k ' ) A L P / 3 / 3 max η T 1/ 3 * G. Guiochon, Anal. Chem.,, -8 (198)
24 Solvent Viscosity vs. Temperature Viscosity (cp) Water / ACN-water MeOH ACN Temperature ( o C) Data from Horvath and Chen.
25 Thermal Mismatch Broadening Influence of Thermal Conditions on the Efficiency of High-Performance Liquid Chromatography. H. Poppe and J. C. Kraak, J. Chromatogr., 8, (1983).
26 Peak Shapes Observed for Various Mobile-Phase Feed Temperatures* σ obs = σ + σ σ column extra columnn + thermal mismatch LC conditions: Column water jacket, 3 o C; 6. mm IDx8cm; 3µ Zorbax ODS; at ml/min; / (v/v) ACN,H O; nitrobenzene *H. Poppe and J.C. Kraak
27 Comparison of the Effect of Incomplete Thermal Equilibration on Column Performance mm ID..6 mm ID6 mm ID cm/min 1 1 cm/min 3 1 Absorbance (mau) Absorbance (mau) Time (min).. 1. Time (min) LC conditions:.1 x cm, C-18 INERT, % ACN, cm preheater, 6 o C.6 x cm, C-18 INERT, 6% ACN, cm preheater, 6 o C. Peaks: 1. toluene,. ethylbenzene, 3. propylbenzene,. butylbenzene
28 Effect of Temperature on Column Efficiency in HTUFLC 6 o C 8 o C Plate Height ( x1 - cm ) o C 1 o C u ( cm/s ) Conclusion: Resistance to mass transfer is greatly reduced as the column temperature is increased., o C (decanophenone, k =1.3),, 8 o C (dodecanophenone, k =7.39),, 1 o C (tetradecanophenone, k =1.3).
29 Fast Separations NSAIDs at High Temperature Norm Column Temperature = 1 o C F =. ml/min. Separation in 1 minute! min LC Conditions: Column, x.6 DiamondBond TM -C18; Mobile phase, /7 ACN/mM phosphoric acid, ph.3; Flow rate,. ml/min.; Temperature, 1 o C; Injection volume, 1ul; Detection at nm; Solute concentration,.1 mg/ml.; Solutes, 1= Acetaminophen, =Ketoprofen, 3=Naproxen, =Ibuprofen, =Oxaprofen.
30 High Speed HPLC LC Conditions: Mobile Phase, 9/71 ACN/mM Tetramethylammonium hydroxide, ph 1.; Flow Rate, 1.3 ml/min.; Injection volume,. ul; nm detection; Column Temperature, 1 C; Pressure drop = 19 bar; Solutes: 1=Doxylamine, =Methapyrilene, 3=Chlorpheniramine, =Triprolidine, =Meclizine 1 x.6 ZirChrom-PBD mau VWD1 A, Wavelength= nm (P11\TEST16.D) Temperature: 1 o C Flow rate: 1.3 ml/min. Pressure drop: 19 bar Resolution (,3):.1 Analysis time: 1. min min min LC Conditions: Mobile Phase,./79. ACN/mM Tetramethylammonium hydroxide, ph 1.; Flow Rate,. ml/min.; Injection volume,. ul; nm detection; Column Temperature, 1 C; Pressure drop = 19 bar; Solutes: 1=Doxylamine, =Methapyrilene, 3=Chlorpheniramine, =Triprolidine, =Meclizine 1 x.6 ZirChrom-PBD VWD1 A, Wavelength= nm (G:\HPCHEM\DATA\P11\TEST18.D) ma U mau min min Temperature: 1 o C Flow rate:. ml/min. Pressure drop: 19 bar Resolution (,3):.1 Analysis time:.8 minutes Courtesy ZirChrom min
31 Fast, Comprehensive Two-Dimensional HPLC One-dimensional HPLC has low peak capacity n c N 1 + ln + R ( k' 1) = n s Comprehensive two-dimensional HPLC has high peak capacity n = n n ctotal c1 c 6 Peak Capcity (nc) Retention Factor (N) t A major limitation is low speed related to the second dimension linear velocity, u rtotal = ( ) ( k' + 1) N [ L ( k' + )] max1 1 c max 1 υ Giddings, J. C. Multidimensional Chromatography: Techniques and Applications; Marcel Dekker: New York, 199
32 LC UFHTLC Separation of Ten Triazine Herbicides mau Tim e (min.) H 3 C H N N N Cl N H N CH 3 Simazine 1 st Dimension Conditions: Column, mm x.1 mm I.d. PBD-ZrO ; Flow rate,.8 ml/min.; Temperature, o C nd Dimension Conditions: Column, mm x.1 mm I.d. PBD-C-ZrO ; Flow rate, 7. ml/min.; Temperature, 1 o C; 1 st dimension sampling frequency,.1 Hz Total LC UFHTLC peak capacity = 18 A single column would be. meter and take hours to generate same peak capacity
33 mv Fast Chromatography on the nd Column Time (s) nd Dimension Time (seconds)
34 Conclusions: (1)Heat transfer, pressure drop and extra-column broadening considerations are key to design HTUFLC. ()Tubing pressure drop is important. (3) HTUFLC can be as much as times faster than room temperature HPLC. () HTUFLC can be done with 1% water as the eluent. () Fast (<. hr.) D-LC can be done.
35 National Institutes of Health
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