NANOLCMS SOLUTIONS HPLC BASICS
Main Course Topics This course is designed to provide a basic founda@on to HPLC principles. It includes a wealth of informa@on regarding HPLC instrumenta@on and HPLC columns. Key Components of HPLC HPLC Concepts Cri@cal Volumes Column Theory Column Resolu@on Column Efficiency Peak Capacity Analyte Capacity Column ID, Flow Rates and Capacity
What is HPLC? High Performance Liquid Chromatography The acronym HPLC, originally indicated the fact that high pressure was used to generate the flow required for liquid chromatography in packed columns. In the beginning, pumps only had a pressure capability of 500 psi [35 bar]. This was called high pressure liquid chromatography, or HPLC. The early 1970s saw a tremendous leap in technology. These new HPLC instruments could develop up to 6,000 psi [400 bar] of pressure, and incorporated improved injectors, detectors, and columns. HPLC really began to take hold in the mid- to late- 1970s. With con@nued advances in performance during this @me [smaller par@cles, even higher pressure], the acronym HPLC remained the same, but the name was changed to high performance liquid chromatography.
Powerful Tool High performance liquid chromatography is now one of the most powerful tools in analy@cal chemistry. It has the ability to separate, iden@fy, and quan@tate the compounds that are present in any sample that can be dissolved in a liquid. HPLC can be applied to just about any sample, such as pharmaceu@cals, food, biological, cosme@cs, environmental matrices, forensic samples, and industrial chemicals. Stand- alone HPLC (UV, PDA, Flourescence Detector) LC- MS LC- MALDI LC- NMR LC- ICP- MS
Key HPLC Components
What is Happening? The HPLC pumps move the mobile phase through the column at a given flow rate. The injector introduces the sample to the fluid path in route to the column. Once the sample reaches the column, it will s@ck to the head of the column. The pumps will con@nue to pump mobile phase through the column. The pumps are typically programmed to change the solvent composi@on through a given period of @me to allow the separa@on to occur (i.e. binary gradient). In some cases, isocra@c separa@on may occur. The sample will then begin to separate into its individual analytes which will elute from the column. Each analyte can then be detected (i.e. UV, MS) or collected for further analysis.
What is a Chromatogram? A chromatogram is a representa@on of the separa@on that has chemically chromatographically occurred in the HPLC system. A series of peaks rising from a baseline is drawn on a @me axis. Each peak represents the detector (UV, MS, etc.) response for a different compound. The chromatogram is ploeed by the computer data sta@on for further analysis.
What is an Isocra@c Separa@on? Isocra@c separa@on of analytes occurs when the mobile phase, either a pure solvent or a mixture, remains the same throughout the run. This technique can only be applied to a simple mixture and/or certain types of analytes.
What is a Gradient Separa@on? Gradient separa8on is where the mobile phase composi8on changes during the separa8on. This mode is useful for samples that contain compounds that span a wide range of chromatographic affinity [polarity, charge, size, etc.]. As the separa@on proceeds, the elu@on strength of the mobile phase is increased to elute the more strongly retained sample components. *We recommend using gradient separa@ons for biomolecules and complex mixtures.
How is a gradient formed? The speed of each pump is managed by the gradient controller to deliver more or less of each solvent over the course of the separa@on. The two streams are combined in the mixer to create the actual mobile phase composi@on that is delivered to the column over @me. At the beginning, the mobile phase contains a higher propor@on of the weaker solvent (Solvent A). Over @me, the propor@on of the stronger solvent (Solvent B) is increased, according to a predetermined @metable.
Low Pressure vs. High Pressure Mixing in Gradient HPLC Advantages Lower cost Easier to maintain 2,3 or 4 solvents Flows > 1ml/min Disadvantages Requires degassing High mix volumes More gradient delay Poor at low flows Low Pressure Propor@oning High Pressure Mixing Lower mix volumes Easy to troubleshoot Best for HT LC Ideal for low flows More expensive Pumps run 1-100% More maintenance Larger size *We recommend high pressure mixing to ensure proper mixing and flow consistency
Types of Solvent Delivery and Degassing No Degassing: Manual Priming, Filtered Solvents but No Degassing Vacuum Degasser: Manual Priming, Filtered Helium Degasser: Auto- Priming, Filtered *We recommend degassing to remove dissolved Oxygen. Dissolved Oxygen would otherwise cause disturbances in nano spray. Helium is slightly more efficient than vacuum but both types provide excellent results.
Types of Pumps Single Piston Reciproca@ng Dual Piston Reciproca@ng Syringe Pump Add Flow Sensing Add Pressure Sensing HPLC UHPLC *We recommend UHPLC Pumps with flow and pressure sensing for op@mal resolu@on and reproducibility.
Types of Mixing Low Pressure Propor@oning High Pressure Propor@oning Tee Frit Cartridge Single- Stage Versus Dual- Stage *We recommend high pressure dual- stage mixing for op@mal mixing
Types of Valves 2- posi@on Mul@- posi@on (for column switching or custom applica@ons) 6- port 10- port Manual toggle Automated toggle (via solware control) Autosampler *We recommend using an autosampler for the most consistent results. Be sure to choose a valve that has the necessary pressure ra@ng, as well as low volumes that are appropriate for your flow range.
Things to consider when buying an HPLC Flow Rate Range Flow Accuracy Flow Precision System Dead Volume Pressure Range Biocompa@bility (fluid path, sample path) Injec@on Type Injec@on Size Complexity of the Sample Throughput
Cri@cal Volumes in Gradient HPLC Pump A Detector Mixer Column Pump A Injector Cri@cal Volume (Must be as low as possible and well swept) T 0 Volume (Defines the gradient run @me required) Gradient Delay Volume (Impacts throughput and MS DC)
Impact of Delay Volumes *Remember to reduce volumes especially in cri@cal places. Gradient Profile Op@mized System Non- Op@mized & Poor Mixing %B Time
Extra Column Band Broadening
What is an HPLC without a Column? Answer: Useless! Good HPLC + No Column = No Chromatography Good HPLC + Bad Column = Li2le to No Chromatography Good HPLC + Good Column = Good Chromatography You can buy the most expensive HPLC on the market and s@ll have poor chromatography when you just use any column. Why? The key to good chromatography is choosing the proper column and knowing how to use it.
Considera@ons in Choosing an HPLC Column Type of Analyte Mode of separa@on Selec@vity Resolu@on Efficiency Capacity (Analyte & Peak) Sensi@vity of Detector
Key Chromatographic Factors Resolu@on is a very important parameter in HPLC HPLC column efficiency measures band broadening Gradient LC of biomolecules more complex than isocra@c LC Peak capacity is a func@on of LC system & column efficiency Op@mum biomolecule separa@ons require aeen@on to details
Resolu@on
Two Major Impacts on Resolu@on Longer Columns Provide Higher Resolu@on! Smaller Par@cles Provide Higher Resolu@on!
Chromatographic Resolu@on k = Capacity Factor (t R /t 0 ) α = SelecBvity (k 2 '/k 1 ) N = Efficiency (5.54(t R /w 0.5 ) 2 k 2 k 1
Column Efficiency N = L / H H = A + B + C A = Variable Paths (d p = particle size and λ = particle size distribution) B = Diffusion (γ = inter-particle, D m = mobile phase and v = flow velocity) C = Mass Transfer (ω = pore size distribution and pore shape)
Factors that Impact Efficiency The theoretical plate number N th shows the relation between retention time and peak width and describes column quality and separation power. The plate number is indicative of the efficiency (performance) of a column or chromatographic system. It is defined as the square ratio of the retention time to the peak width. The more theoretical plates the column possesses, the larger the plate number. Factors affecting column efficiency (plate number) Column length Particle size Packing quality Linear velocity (flow) Instrument quality (dead volume) Retention factor
H - Van Deempter Equa@on Peak height and peak broadening are governed by kine@c processes in the column such as molecular dispersion, diffusion and slow mass transfer. Iden@cal molecules travel differently in the column due to probability processes. The three processes that contribute to peak broadening described in the van Deemter equa@on are: A- term: eddy diffusion: The column packing consists of par@cles with flow channels in between. Due to the difference in packing and par@cle shape, the speed of the mobile phase in the various flow channels differs and analyte molecules travel along different flow paths through the channnels. B- term: longitudinal diffusion: Molecules traverse the column under influence of the flowing mobile phase. Due to molecular diffusion, slight dispersions of the mean flow rate will be the result. C- term: resistance against mass transfer. A chromatographic system is in dynamic equilibrium. As the mobile phase is moving con@nuously, the system has to restore this equilibrium con@nuously. Since it takes some @me to restore equilibrium (resistance to mass transfer), the concentra@on profiles of sample components between mobile and sta@onary phase are always slightly shiled. This results in addi@onal peak broadening
Eddy Diffusion Factors influencing A- term ('eddy' diffusion) ParBcle size d p ParBcle shape (regular or irregular?) ParBcle pore structure / shape Quality of the column packing Wall effects (material, roughness, column diameter)
Longitudinal Diffusion Influences on B-term (longitudinal diffusion): Linear velocity of the mobile phase Diffusion coefficient of analyte in the mobile phase D m Mobile phase viscosity γ Temperature Type of analyte (molecular mass)
The overall C- term is divided into two separate mass transfer terms: C m - term, describing the contribubons to peak broadening in the mobile phase. Because the linear velocity of the mobile phase is lower closer to the column wall (or the sta@onary phase par@cles) than in the center (or further away from the par@cles), the analyte molecules experience different veloci@es. This results in peak broadening in the mobile phase. This phenomenon is described by the C m term. C s - term, describing the contribubons to peak broadening in the stabonary phase. The C s - term is determined by the amount of sta@onary phase (low is advantageous for the efficiency) and the extent of interac@on of the sample on the phase (represented by the reten@on factor) and the distances the sample molecules have to travel. Influences on C m -term: Particle size d p Linear velocity u of the mobile phase Diffusion coefficient in the mobile phase Porosity of the packing particles Viscosity of the mobile phase Retention factor k Temperature Influences on C S -term: Quality of stationary phase Diffusion coefficient in stationary phase Retention factor k Temperature Particle size Mobile phase velocity
H - Van Deempter Plot A plot of the plate height as a func@on of the mobile phase velocity. The H- u curve shows that: The A- term is independent of u and does not contribute to the shape of the H- u curve.. The contribubon of the B- term is negligible at normal opera@ng condi@ons. This is due to the fact that the molecular diffusion coefficient in a liquid medium is very small. The C- term increases linearily with mobile phase velocity and its contribu@on to the H- u curve is therefore considerable.
UHPLC In 2004, further advances in instrumenta@on and column technology were made to achieve very significant increases in resolu@on, speed, and sensi@vity in liquid chromatography. Columns with smaller par@cles [1.7 micron] and instrumenta@on with specialized capabili@es designed to deliver mobile phase at 15,000 psi [1,000 bar] were needed to achieve a new level of performance. A new system had to be holis@cally created to perform ultra- performance liquid chromatography, now known as UHPLC technology. Basic research is being conducted today by scien@sts working with columns containing even smaller 1- micron- diameter par@cles and instrumenta@on capable of performing at 100,000 psi [6,800 bar]. This provides a glimpse of what we may expect in the future.
UHPLC vs. HPLC *We recommend using the smallest par@cles (sub- 2µ) in your column for the highest resolu@on and efficiency. As shown, you will not lose any efficiency as the linear velocity is increased across a UHPLC column which equates to higher throughput capabili@es along with high resolu@on.
Why Small Par@cles Are Beeer Higher ResoluBon Higher Peak Capacity Be2er Chromatography! Allows Higher Throughput Higher Pressure Requires a UHPLC Instrument
Peak Capacity vs. Efficiency *UHPLC columns have the highest efficiency and therefore peak capacity
Peak Capacity in Gradient HPLC : 0.00-32.00 100 14.65 90 80 70 10.10 10.64 60 50 40 8.60 30 20 10 0 0 5 10 15 20 25 30 Time (min) Peak Capacity (PC) = Separation Time / Average Peak Width
Impacts on Peak Capacity Peak Asymmetry - Peak Tailing {secondary interactions (silanols) unswept dead volume} - Peak Fronting {column overload (peak load vs column load) strong injection solvent} Column Deterioration - Contamination {secondary interactions (adsorbed protein interaction) column clogging} - Packing Deterioration {phase loss (secondary interaction) bed collapse (voids or channeling)} Extra Column Volume - Injector to Column {total volume unswept dead volume (less critical in gradient LC)} - Column to Detector {total volume unswept dead volume (critical in gradient LC)} Excess System Volume - Gradient Delay Time {column volume system sweep volume (increases as flow rate decreases)} - Re-equilibration Time {column volume (5-10x) system sweep volume (can t equil column before system)}
HPLC Flow Ranges Analy@cal 0.5-2mL/min Microbore ~20-500µL/min Capillary ~1-20µL/min Nano ~200-1000nL/min *Lower flow rates provide beeer sensi@vity. However, the smaller ID columns u@lized at those lower flows have reduced capacity. We recommend balancing your needs for sensi@vity with your needs for analyte and peak capacity for an op@mal setup.
Column ID vs. Sensi@vity and Capacity for Op@mal Resolu@on Column ID 4.6 Sensitivity LOD(fmol) 4000 Capacity (ug) for Optimum Resolution 200-4000 2.0 800 40-800 1.0 0.5 0.3 0.2 0.1 0.075 200 50 20 8 2 1 10-200 2.5-50 1.0-20 0.4-8 0.1-2 0.05-1
Column ID, Flow Rate, Sensi@vity and Loading Capacity Column ID Flow SensiBvity Load (mm) (µl/min) LOD (fmol) (µg) 4.6 1000 2000 4000 2.0 200 400 800 1.0 50 100 200 0.5 10 25 50 0.3 5 10 25 0.2 2 5 10 0.1 0.5 1 2 0.075 0.2 0.5 1
Analyte Capacity vs. Peak Capacity 100 1037.70 1078.86 867.65 10 ug 90 80 70 Load 60 50 40 30 20 677.59 659.63 806.82 886.36 1117.82 691.32 1285.90 799.63 1218.87 10 446.66 0 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 Time (min) 100 90 1037.78 1078.86 867.65 1 ug Load 80 70 60 50 40 30 20 10 562.91 446.65 597.19 1101.09 1035.77 677.59 659.60 801.68 747.87 806.80 899.12 886.05 953.77 0 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 Time (min) 907.65 1139.87 691.32 716.67 1285.90 506.52 799.63 1110.76 1230.87 983.59 958.45 789.69 1218.96 *Don t overload your column! It will cause poor chromatography, reduc@on in resolu@on and peak capacity. Choose the appropriate ID for your applica@on.
Column Length vs. Time & Capacity Length Gradient Rate Gradient Time Column Peak (mm) (%B / min) (Minutes) Volumes Capacity 50 > 5 < 10 5-20 20-100 100 1-4 5-30 5-20 40-200 150 0.5-2 10-60 5-20 60-300 200 0.25-1 20-120 5-20 80-400 250 < 0.5 > 120 5-20 100-500