High Temperature Ultra Fast Liquid Chromatography Peter W. Carr, Jon Thompson, Dwight Stoll, and Adam Schallinger
Conclusions 1. To do fast LC, use a WEAK eluent and a HOT column.. Use a highly retentive column so that you can work at lowest possible viscosity!
A. ma U 60 50 40 30 Fast Separation of Antihistamines at 80 o C V WD1 A, Wavelength=54 nm (P10501\TEST0146.D) 1 3 4 3 Temperature: 1 o C Flow rate: 1.35 ml/min. Pressure drop: 195 bar Resolution (,3):.1 Analysis time: 1.5 minutes 0 10.4.6.8 3 3. min 5 0 0 4 6 8 10 1 14 min ma U B. 80 60 40 0 V WD1 A, Wavelength=54 nm (P10501\TEST0151.D) 1 3 1=Doxylamine, =Methapyrilene, 3=Chlorpheniramine, 4=Triprolidine, 5=Meclizine4=Triprolidine, 5=Meclizine 4 5 0.9 0.95 1 1.05 1.1 1.15 1. 1.5 3 Temperature: 80 o C Flow rate: 3.00 ml/min. Pressure drop: 195 bar Resolution (,3):.1 Analysis time:.5 minutes min 0 0 4 6 8 10 1 14 min LC Conditions: (A) Mobile Phase, 9/71 ACN/50mM Tetramethylammonium hydroxide; Injection volume, 0.5 ul; 54 nm detection; 100 x 4.6 ZirChrom-PBD (B) same as A, except Mobile Phase, 6.5/73.5 ACN/50mM Tetramethylammonium hydroxide, ph 1.
Faster Separation at 140 o C 100 mau 80 60 40 1 3 Temperature: 1 o C Flow rate: 1.35 ml/min. Pressure drop: 195 bar Resolution (,3):.1 Analysis time: 1.5 min 0 0 4 0 4 6 8 10 1 14 16 Time (min.) 5 100 100 mau 80 60 40 0 80 60 40 0 0 1 3 4 5 0 0.5 1 Temperature: 140 o C Flow rate: 4.0 ml/min. Pressure drop: 194 bar Resolution (,3):.1 Analysis time: 0.85 minutes 0 0 4 6 8 10 1 14 16 Time (min.) Mobile Phase, 9/71 ACN/50mM Tetramethylammonium hydroxide, ph 1.; Injection volume, 0.5 µl; 54 nm detection; Solutes: 1=Doxylamine, =Methapyrilene, 3=Chlorpheniramine, 4=Triprolidine, 5=Meclizine; Column, 100 mm x 4.6 mm i.d. ZirChrom -PBD
Effect of Temperature on Theoretical Analysis Time at Constant Pressure, Retention, and Plate Count* 1.0 t Analysis (T)/ t Analysis ( 5 o C) 0.8 0.6 0.4 0. 0.0 40 60 80 100 10 140 160 180 00 T ( 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., 435, 1-15 (1988).
Relative Viscosity vs. Temperature η/η (5 o C) 1.0 0.8 0.6 0.4 Water 0 % ACN/Water Methanol Acetonitrile 0. 0.0 40 60 80 100 10 140 160 180 00 0 Temperature ( o C)
How to Prevent Boiling Boiling Point of THF (Centigrade) Boiling Point 50 00 150 100 50 0 0 100 00 300 400 Pressure (psi)
Requirements for High Temperature LC Stationary Phase Stability Thermal Mismatch Broadening On-Column Analyte Instability
Peak Shapes Observed for Various Mobile-Phase Feed Temperatures* σ obs = σ + σ σ column extra columnn + thermal mismatch LC conditions: Column at 30 o C; 6. mm IDx8cm; 3µ Zorbax ODS; at 5 ml/min; 50/50 (v/v) ACN,H O; nitrobenzene *H. Poppe and J.C. Kraak, J. Chromatogr., 8, 399-41 (1983).
Comparison of the Effect of Incomplete Thermal Equilibration on Column Performance 80 60.1 mm ID 4.4.6 mm ID6 mm ID 16 1 1 105 cm/min 14 105 cm/min 3 4 1 Absorbance (mau) 40 0 Absorbance (mau) 10 8 6 4 3 4 0 0.0 0.5 1.0 Time (min) 0.0 0.5 1.0 Time (min) LC conditions:.1 x 5 cm, C-18 INERT, 55 % ACN, 5 cm preheater, 60 o C 4.6 x 5 cm, C-18 INERT, 60% ACN, 5 cm preheater, 60 o C. Peaks: 1. toluene,. ethylbenzene, 3. propylbenzene,4. butylbenzene 0
Theoretical Effect of Temperature on Column Dynamics 10 8 5 o C 75 o C H *10-5 (cm) 6 4 15 o C 175 o C 0 0 0 40 60 80 100 u*100 (cm/s) Conditions: The particle diameter is 3 µm and the reduced linear velocity does not change with temperature ((D m,5 o C = 6*10-7 cm /s). The linear velocity (u) is increased and the reduced plate height is calculated from a modified Knox equation (A = 1.5, B = 0.8, C = 0.3, D = 0.04) at each velocity and temperature. Fast desorption kinetics are assumed (E a = 0 kj/mol, k o = 1*10 13 s). Citation: R. D. Antia and Cs. Horvath, J. Chromatogr., 435, 1-15 (1988).
A Totally Impractical High Temperature Ultrafast Liquid Chromatography (HTUFLC) System injector very small thermal equilibrium delay pump 1ml/min column detector pump 1-14ml/min long delay heating bath (150-00 o C) cooling bath (0 o C)
Effect of Temperature on Column Efficiency in HTUFLC 6 4 5 o C 80 o C Plate Height ( x10-4 cm ) 0 18 16 10 o C 150 o C 14 1 0.0 0.5 1.0 1.5.0.5 u ( cm/s ) Conclusion: Resistance to mass transfer is greatly reduced at elevated column temperature., 5 o C (decanophenone, k =1.3),, 80 o C (dodecanophenone, k =7.39),, 10 o C (tetradecanophenone, k =1.3).
Effect of Temperature on Column Dynamics Experimental Conditions a van Deemter Equation Coefficients T( o C) Mobile Phase (% ACN (v/v)) D m x10 4 A x 10 3 (cm /s) b cm B x 10 4 (cm /s) C x 10 3 (s) u opt (cm/s) 5 80 10 150 40 40 30 5 0.08 0.15 0.5 0.36 1.1±0.04 0.90±0.05 0.91±0.03 1.0±0.05 0.18±0.03 0.6±0.09 1.±0.08 1.3±0.08 1.4±0.06 0.80±0.03 0.44±0.01 0.31±0.03 0.1 0.3 0.6 0.7 a Solutes: alkylphenones b Estmated solute diffusion coefficient in the indicated mobile phase at temperature of the calculation based on modified Wilke-Chang equation.
A Totally Practical Heating System Cascade Loop Control Column Quasi-Adiabatic Column Environment U.S. Patent Issued Systec/MetalOx
References B. Yan, J. Zhao, J.S. Brown, J. Blackwell, and P.W. Carr, High Temperature Ultrafast Liquid Chromatography, Anal. Chem. 7, 153-6 (000). J.D. Thompson, J.S. Brown, and P.W. Carr, Dependence of Thermal Mismatch Broadening on Column Diameter in High-Speed Liquid Chromatography at Elevated Temperatures, Anal. Chem.73, 3340-7 (001). J.D. Thompson and P.W. Carr, A Study of the Critical Criteria for Analyte Stability in High-Temperature Liquid Chromatography, Anal. Chem. 74, 1017-3 (00). J.D. Thompson and P.W. Carr, High-Speed Liquid Chromatography by Simultaneous Optimization of Temperature and Eluent Composition, Anal. Chem. 74, 4150-9 (00).
Theory of High Speed HPLC
Rearrangement to Obtain the Guiochon Equation Fundamental Equation # 1 Fundamental Equation # Fundamental Equation # 3 Guiochon Equation L = NH = Nhd p u = νd m dp L t R = ( 1+ k') u tr (1 + k') h = d N D ν m p Knox Equation 1 / 3 B h = Aν + + ν Cν G. Guiochon, Anal. Chem., 1980, 5, 00-008
Limit 1: C term Knox, Saleem, Guiochon Equation tr N = (1 + k') h d D ν m p Theoretical Limit h Cν as ν Theoretical Limit for t R /N Stokes-Einstein h Cν t R C(1 + ν N D m k') d RT D m = 6nπrη p Result t R ηc(1 + ν N T G. Guiochon, Anal. Chem., 1980, 5, 00-008 k') d p
Limit : A term Practical Limit for h 1/ 3 h Aν t R /N f(a) Kozeny-Carman Permeability and Darcy s Law 4/3 R Ad p (1 + t N = P = D Φ d 1/3 m p u /3 ulη k') Maximum Linear Velocity u max = d p P Lφη max Practical Limit Temperature Dependence (Aν 1/3 > Cν) tr N h Aν ν / 3 (1 + k') L P /3 /3 max η 1/3 T
Dependence of t/n on Velocity in the Limit of Exponent of ν x Critical Exponents d p ν x x L x P x η x T x 0-1 1-1 1 0 ½ ½ ½ -0.5 1-0.5 1/3 0 /3 - /3 1-1/3 1 0 0 1-1
Relative Viscosity vs. Temperature η/η (5 o C) 1.0 0.8 0.6 0.4 Water 0 % ACN/Water Methanol Acetonitrile 0. 0.0 40 60 80 100 10 140 160 180 00 0 Temperature ( o C)
Limit 3: Resolution ') (1 p m R d h D k N t ν + = General Resolution Equation Result ' 1 ' 1 4 k k N R + = α α P h N k tr Φ + = η ') (1 Rearrangement of Darcy s Law m p D hn P d η ν Φ = Retention Time ( ) 4 6 4 4 ' ' 1 1 56 k k P h R tr + Φ = α α η Knox-Saleem Equation
Dependence of t/n on Optimization Parameters x d p L x P x η x C Limit 0 0 1 A limit (ν 1/3 ) 0 /3 - /3 1 Resolution Limit 0 0-1 1
Relative Viscosity vs. Temperature η/η (5 o C) 1.0 0.8 0.6 0.4 Water 0 % ACN/Water Methanol Acetonitrile 0. 0.0 40 60 80 100 10 140 160 180 00 0 Temperature ( o C)
Effect of Composition on Viscosity Viscosity of Acetonitrile-Water at 0 o C Viscosity (cp) 1.1 0.9 0.7 0.5 0.3 0 10 0 30 40 50 60 70 80 90 100 % Acetontrile
Effect of φ, & T on k.0 1.5.0 log k = A/T +B log k = Cφ +B 1.5 log k' 1.0 0.5 0.0 log k' 1.0 0.5-0.5 0.0-1.0 0.0018 0.001 0.004 0.007 0.0030 0.0033 0.0036 1/ T (K -1 ) -0.5 0.5 0.30 0.35 0.40 0.45 0.50 0.55 φ
How Should the Separation Be Done? The same k can be achieved by use of : a. low temperature and organic rich eluent. OR b. high temperature and organic poor eluent. Which allows the faster separation?
Is High T Optimal for High Speed in k too high LC? φ (low) φ (high) T (low) low,low low, high T (high) high, low high, high Regions of same k and low viscosity. Where should we work? k too low
Effect of φ and T ( at k = 5) on η k (φ,t) 5 % ACN (v/v) 69 T ( o C) 5 η(cp) (φ,t) 0.55 η (T)/η (5 o C) 1 5 59 100 0.9 0.53 5 5 15 0.0 0.36 5 45 00 0.14 0.5 Conditions: k based on butyl benzene on a C 18 Zorbax column.
Conclusions 1. To do fast LC, use a WEAK eluent and a HOT column.. Use a highly retentive column so that you can work at lowest possible viscosity!
The Importance of Speed in Comprehensive Two-Dimensional HPLC For comprehensive DLC, the speed of the second dimension separation is the rate limiting step in completing the entire D chromatogram. Each first dimension peak must be chromatographed 3-4 times by the second dimension column. 0.5 t rtotal = N ( k' + 1)( k' + ) 1Lc max1 max 1 υ AU 0. 0.15 0.1 0.05 0 0 1 3 4 5 6 Time (min.) Typical Fast 1st Dim. k' max 10 10 nd Dim. k' max 5 5 N 1 (Plates/column) 10000 10000 L c, (cm) 3.3 3.3 u (cm/s) 0.5 5.0 Total Analysis Time (Hrs) 1 1
Potential Approaches to Improving the Speed of HPLC Approach Advantage Disadvantage Shorter Columns Works with most equipment, stationary phases Low plate count and resolution Monolithic Columns Ultra-High Pressure LC High Temperature LC Low backpressure High plate counts with small particles Low backpressure, high efficiency at high velocity Narrow-bore columns are not available, high sovent useage Specialized equipment needed, losses in N at high velocity Requires adequate heating, stable phases, stable analytes. High temperature LC is the only approach that allows a significant fraction of the column plate count to be retained as the column linear velocity is increased to values that allow much faster HPLC
Schematic of a Complete LC UFHTLC System Standard HP1090 Pump and Autosampler Binary Pump #1 Auto- Injector Eluent Preheater T1 Heating Jacket 1st Dimension Column T T6 Heating Jacket nd Dimension Column T5 Eluent Preheater T4 Sample Loop #1 10-port, -position valve Temperature Controlled.1 mm column Sample Loop # Counter- Current Heat Exchanger T7 UV Detector T3 Eluent Preheater Binary Pump # 5 ml/min pump
LC UFHTLC Separation of Ten Triazine Herbicides mau 100 80 60 40 0 H 3 C H N N N Cl N H N CH 3 Simazine 1 st Dimension Conditions: Column, 50 mm x.1 mm i.d. PBD-ZrO ; Mobile phase, 0/80 ACN/Water; Flow rate, 0.08 ml/min.; Injection volume, 0 µl; Temperature, 40 o C 0 0 5 10 15 0 Tim e (m in.) nd Dimension Conditions: Column, 50 mm x.1 mm i.d. PBD-C-ZrO ; Mobile phase, 0/80 ACN/Water; Flow rate, 7.0 ml/min.; Injection volume, 15 µl; Temperature, 150 o C; 1 st dimension sampling frequency, 0.1 Hz Total LC UFHTLC peak capacity = 185 Using a single column, it would take a.5 meter column and 44 hours to generate the same peak capacity
Thanks! Ben Yan (ZirChrom). NIH. Carl Sims and Systec, Inc. ZirChrom Separations, Inc.
High Throughput Gradient Elution 17 Gradients/Hour. Peak capacity is 70! This speed cannot be done at ambient within the gradient space! Carl Sims Systec. 0.45 0.4 0.35 Fast Gradient Analysis @ 75 C (17 gradients per hour) Conditions Flow Rate: ml/min Gradient 0-100%B in.5 min. A: 5% THF in Water B: 5% THF in Acetonitrile Column: Zirchrom Diamondbond C18.1 x 50 x 3 micron. System Pressure: 4000 psi System: Waters 790, 487 with microcell Alkyl Phenone Homologs to C-18 0.3 Absorbance 0.5 0. 0.15 0.1 0.05 0 0 0.5 1 1.5.5 3 Time, Minutes 9:58 AM 10:03 AM 10:07 AM 10:1 AM 10:17 AM 10:1 AM 10:6 AM 10:31 AM 10:35 AM
Second Dimension Chromatograms mv 0. 0.18 0.16 0.14 0.1 0.1 0.08 0.06 0.04 0.0 0 0 4 6 8 10 nd Dimension Time (seconds) 10 0 30 40 50 300 310 30 330 340 350 360 370 380 390