Introduction to Liquid Chromatography Columns System Components Applications

Introduction to Liquid Chromatography Columns System Components Applications Troubleshooting Susan M. Steinike, M. S HPLC Marketing Department February, 2006 SHIMADZU Solutions for Science

Modern HPLC n Late 1970 s/early 1980 s Instrumentation developed for high pressure solvent delivery: pumps, autosamplers, diode array detectors n More uniform packing material produced for columns n n Last 20 years n Nothing really “new”, but by returning to the basic theory of chromatography, even better columns are on the market: smaller particle sizes which yield faster separations, but require hardware to withstand higher pressures. SHIMADZU Solutions for Science

What is Chromatography? Separation of a mixture into individual components. n The separation uses a Column (stationary phase) and Solvent (mobile phase). n The components are separated from each other based on differences in affinity for the mobile or stationary phase. n The goal of the separation is to have the best RESOLUTION possible between components. n SHIMADZU Solutions for Science

The Most Basic Explanation of Chromatography Ever SHIMADZU Solutions for Science

How Do You Get Separation? n Hardware: pumps, injector, detector n Column: particle diameter, column size, packing materials, and the dreaded equations n Our seminar will focus on the contribution of each factor to perform separations. SHIMADZU Solutions for Science

HPLC and Pre-HPLC Techniques SHIMADZU Solutions for Science

Column Types n Reversed-Phase LC Nonpolar stationary phase: C 8, C 18 n Polar mobile phase: Water, ACN, Methanol n The MOST polar compound comes out first n SHIMADZU Solutions for Science

Reversed Phase HPLC Columns n n n C-18, C-8: C-3, C-4: Phenyl: Cyano: Amino: Rugged, general purpose, highly retentive Less retentive, used mostly for peptides & proteins Greater selectivity than alkyl-bonded Moderate retention, normal & rev. phase Weak retention, good for carbohydrates The cyano column with a high polarity mobile phase (Water/Me. OH) will act as a reversed phase column. SHIMADZU Solutions for Science

Typical Column Sizes n Particle size: 5 µm, 3 µm, and smaller Monodispersed means particles are the same size n Very important for stable pressure and flow n Smaller particles produce higher system pressure n Pore size: 100 -120 A is typical n Surface area: 300 -350 m 2/g n Carbon load: 9 -12% for C 8, 16 -20% for C 18 n Higher carbon load = better resolution but longer run times n Lower carbon load = shorter run times, but may change selectivity vs. higher carbon load SHIMADZU n Solutions for Science

Idealized HPLC Separation SHIMADZU Solutions for Science

Capacity Factor, ’ k The relative degree to which an analyte component is delayed as it is eluted through a given system (retentivity). k’ = (Vr - V 0)/V 0 = (tr - t 0)/t 0 Where Vr = peak retention volume; V 0 = column void volume tr = peak retention time; t 0 = peak void time n The larger the k’, the later the analyte elutes after the void. SHIMADZU Solutions for Science

Mobile Phase Strength vs. ’ k 4. 6 mm ID Column, 1 m. L/min, Changing Me. OH % vs Water 0. 079 100% Capacity Factor for Butyl Paraben (Peak 4) 0. 212 90% 0. 472 80% 70% 1. 127 2. 813 60% 7. 666 50% SHIMADZU Solutions for Science

Temperature Effect on ’ k 2. 1 mm ID Column, 0. 35 m. L/min, 50/50 Me. OH/Water 50°C 45°C 40°C 35°C 30°C 25°C 20°C SHIMADZU Solutions for Science

Questions About Columns? Next – HPLC System Components SHIMADZU Solutions for Science

HPLC Pumps – 2 Basic Types n Dual Piston Two pistons with equal volume (10 µL each) n During each stroke, 10 µL is delivered n Best for low flow rates (< 1 m. L/min) n Little to NO pulsation, so it’s ideal for pulse sensitive detectors like RID and CDD n SHIMADZU Solutions for Science

Dual Piston Pump SHIMADZU Solutions for Science

Sample Injection – Automatic n Needle-in-the-flowpath autosampler Sample loop and needle are a single piece of tubing n Loop and needle are cleaned during the run n Metering pump draws sample very precisely n Advantages: no sample loss, low carryover n Disadvantages: higher cost, more delay volume for gradient n SHIMADZU Solutions for Science

Sample Injection to Flow Path n Sample Loading n Sample Injection – Everything drawn into the needle goes to the column. SHIMADZU Solutions for Science

HPLC Column Ovens n Block heater with solvent preheater Column is housed between 2 metal plates n Mobile phase is plumbed into the block for preheating n n Forced air Column is in a large chamber with air circulation n Better temperature equilibration n Room for column switching valves n SHIMADZU Solutions for Science

Why Use a Column Oven? n Retention times decrease, and higher flow rates are possible 2. 1 mm ID Column, 0. 35 m. L/min, 50/50 Me. OH/Water 50° C 45° C 40° C 35° C 30° C 25° C 20° C SHIMADZU Solutions for Science

HPLC Detectors – UV-VIS n n n Most common HPLC detector for a variety of samples n n Wavelength range 190 -700 nm D 2 and W lamps Proteins and peptides Organic molecules Pharmaceuticals Monitor 2 wavelengths at one time SHIMADZU Solutions for Science

HPLC Detectors – UV-VIS SHIMADZU Solutions for Science

HPLC Detectors n Refractive Index n For samples with little or no UV Absorption n Alcohols, sugars, saccharides, fatty acids, polymers Best results when RI of samples is very different from RI of mobile phase n Flow cell is temperature controlled with a double insulated heating block. n REQUIRES isocratic separations n REQUIRES low pulsation pumps n SHIMADZU Solutions for Science

HPLC Detectors – RI Balance n Fill sample and reference cell with mobile phase. SHIMADZU Solutions for Science

HPLC Detectors – RI Analyze n Mobile phase flows through sample side only. SHIMADZU Solutions for Science

HPLC Detectors – RI Analyze n As the refractive index changes, the image on the photodiode is deflected or “unbalanced”, and the difference in current to the photodiode is measured. SHIMADZU Solutions for Science

HPLC System Types n Isocratic system Same mobile phase concentration throughout the separation n Use 1 pump and pre-mix solvents n Use 1 pump and a valve for 4 different solvents n Use 2 pumps and vary the amount coming from each pump n SHIMADZU Solutions for Science

Isocratic Separation n n 1 pump and premixing 4. 6 mm ID Column, 1 m. L/min, Changing Me. OH % vs Water SHIMADZU Solutions for Science

Isocratic Separation n 1 pump with valve and premixing To Column ABCD A = 80% Methanol, 20% Water B = 70% Methanol, 30% Water C = 60% Methanol, 40% Water D = 50% Methanol, 50% Water SHIMADZU Solutions for Science

Isocratic Separation n 1 pump with mixer – let the pump do the work! To Column ABCD Method 1: A. CONC = 20%, B. CONC = 80% Method 2: A. CONC = 30%, B. CONC = 70% Method 3: A. CONC = 40%, B. CONC = 60% Method 4: A. CONC = 50%, B. CONC = 50% SHIMADZU Solutions for Science

Low Pressure Gradient n 1 Pump, solvents are mixed before the pump To Column ABCD n REQUIRES degassing SHIMADZU Solutions for Science

Low Pressure Gradient n 1 Pump, solvents are mixed before the pump To Column ABCD n REQUIRES degassing SHIMADZU Solutions for Science

Questions About System Types? Next: Troubleshooting and How to Take Care of Your Column and HPLC System SHIMADZU Solutions for Science

HPLC Troubleshooting Pressure: too much or too little n Leaks: pump, autosampler, detector n Reproducibility: pump, autosampler n n Column Care: Flushing and equilibration SHIMADZU Solutions for Science

Pump Troubleshooting n No pressure, or fluctuating pressure Pump may not be completely full of liquid – check solvent inlet line n Air in check valve – always degas mobile phase! n “Stuck” check valve – the pump may have been idle for too long and solvent has dried inside the check valve. Poor quality solvent: may contain resins that coat the ball inside the check valve, and that film won’t let the ball seat properly n SHIMADZU Solutions for Science

Pump Troubleshooting n High Pressure n n Outlet frit may be blocked with particles from mobile phase or seal material Leaks n Damage to seal and/or plunger due to several factors n Misaligned plunger n Solvent incompatibility with seal material n Salt crystal buildup from buffers – use a rinse kit! SHIMADZU Solutions for Science

Pump Troubleshooting n Retention Time Reproducibility For a dual piston pump, only one side may be filled with liquid – check solvent inlet lines n Temperature change (may not be the pump’s fault) n n A 1 o shift in temperature can result in a 1 -2% shift in retention time n Avoid drafty locations in the lab n Use a column oven when possible SHIMADZU Solutions for Science

Autosampler Troubleshooting n High Pressure n Particulates from mobile phase, sample, pump may be trapped in the inlet tubing or valve n Filter mobile phase AND sample when possible n Leaks n Fittings may be loose on the valve n Tighten fittings properly and don’t exceed the pressure limit of the autosampler SHIMADZU Solutions for Science

Autosampler Troubleshooting n Area % Reproducibility Always degas rinse phase, and use some volume of liquid for rinsing to keep all flow paths in the valves full of liquid n Make sure the needle stroke is deep enough to draw sample from the vial n Check for leaks on the valve fittings, and the connection to the column inlet n SHIMADZU Solutions for Science

Detector Troubleshooting n Spiky Baseline n Air bubble in flow cell – degas mobile phase! n Put some restriction on the cell outlet, but not too much! Tubing with 0. 005” i. d. is fine. n Leaks n Cracked flow cell n Don’t exceed the pressure limit of the cell n Poor tubing connections n Use the proper fittings and tighten appropriately SHIMADZU Solutions for Science

Column Care Follow MFR’s recommendations for solvent compatibility, flow rate, and pressure limits n Filter samples when possible n n n Use care when reversing column flow n n Particulates will build up on the inlet frit over time Connect the outlet to waste, NOT inline with the detector to prevent further contamination Store columns in recommended solvents SHIMADZU Solutions for Science

Troubleshooting Summary n Throw away bad parts and columns. n Leaks do not fix themselves. n If it doesn’t pass, you must degas. SHIMADZU Solutions for Science