liquid chromatography
Chromatography: Important: • chromatogram (concentration versus elution time) • more strongly retained species elutes last (elution order) • analyte is "diluted" during elution (dispersion) • zone broadening proportional to elution time Column Efficiency:
number of plates = length of column / height of 1 theoretical plate
246
Example A 2-meter long open column, tr = 30 min, D = 5 x 10-5 cm2/s If diffusion is the only contribution to band broadening, what is the estimated band width? number of plates? height of 1 theoretical plate?
247
LIQUID CHROMATOGRAPHY Gas and liquid chromatography are complementary
248
HPLC • HPLC stands for either high performance liquid chromatography or high pressure liquid chromatography, depending on whom you talk to. • The pressure and performance are both high because of the use of small size stationary phase. • Small particles improve separations because they reduce the eddy diffusion term and because they reduce the mass transfer term. • Eddy diffusion is reduced because the total number of particles increases, so that any difference in pathlength tends to average quickly. • Mass transfer is reduced because the small distance analyte must diffuse to reach a particle.
249
HPLC COMPONENTS • Small particles create a dense bed of stationary phase. It is difficult to push solvent through the stationary phase, and a high pressure pump is required. Pressures are 100-500 atm. • Because of the high pressure, the components in HPLC are quite robust, usually made from stainless steel.
250
PISTON PUMP These pumps work continuously. Fluid from a reservoir is sucked into the pump when the piston is withdrawn. The fluid is pushed out the top when the piston is pushed in. A pair of check valves is used to direct the fluid to the appropriate direction. These simple devices consist of a ball and a spring. The spring forces the ball to seal and prevent back flow. Two pistons can be operated in tandem to reduce pressure fluctuations.
251
252
LC INJECTOR
The injection loop can be changed to different volumes, ranging from 50 nL to many mL. In the load mode, the sample from the syringe flows through the sample loop to waste. At the same time, the flow from the pump passes to the column. In the inject mode, the flow from the pump passes through the sample loop and then to the column.
253
LIQUID CHROMATOGRAPHY COLUMNS • Stainless steel • 10-30 cm long • 1-10 μm particle size - 40,000-60,000 plates/m
254
LIQUID CHROMATOGRAPHYDETECTORS 1. U.V. Absorbance—sensitive, almost universal. Most common detector 2. Refractive index—not sensitive, universal Can’t be used with gradient elution. 3. Mass spectrometry—sensitive, universal. Requires volatile buffer and electrospray interface - VERY important in protein analysis.
255
STATIONARY PHASE • A solid particle of uniform particle size (5-10 μm diameter is common) is packed into the column in the form of a slurry. • A frit holds the particles in place.
256
NORMAL PHASE LIQUID CHROMATOGRAPHY • In normal phase chromatography, the stationary phase is highly polar, such as hydrated silica (SiO2 × H2O), with a high surface area—500 m2/g. The analyte adsorbs or interacts with silanol groups. Polar material can be bound chemically to the surface through silica chemistry
In normal phase chromatography, R = (CH2)3NH2 (amino) or (CH2)3C N (cyano) are common chemically bound stationary phases attached to silica solid supports.
257
NORMAL PHASE In normal phase chromatography, the stationary phase is polar and the mobile phase is non-polar. Analyte partition between the two phases. We can adjust the relative affinity of an analyte for the surface by adjusting the composition of the mobile phase. Solvents range in their polarity. The strength of a solvent measures its ability to partition analyte in a chromatographic column. The eluotropic series measures the polarity and strength of a solvent.
258
NORMAL PHASE LIQUID CHROMATOGRAPHY In normal phase chromatography, with weak solvents, non-polar molecules will tend to be found in the mobile phase (small k') whereas polar molecules will tend to be found in the stationary phase (high k'). With strong solvents, all substances will be found in the mobile phase. Mobile phases are chosen to produce an intermediate partition ratio for the analyte. By choosing an appropriate buffer, column selectivity can be fine-tuned for a particular compound.
259
REVERSED PHASE LIQUID CHROMATOGRAPHY Non-polar reversed phase chromatography is very commonly used these days. It is called reversed phase because the polar stationary phase is switched with the non-polar stationary phase. The stationary phase is bonded to a solid support, which is typically silica
The hydrocarbon forms a very stable, very non-polar stationary phase
260
REVERSED PHASE LIQUID CHROMATOGRAPHY For reversed phase chromatography, the elutropic series starts with water as a weak solvent and nonpolar solvents as a strong solvent.
With weak solvents, such as water, non-polar molecules are held to the surface and won't elute (high k'). With stronger solvents, such as toluene, most all molecules will be found in the mobile phase and eluted quickly (small k'). Mobile phases are chosen that produce an intermediate partition ratio for the analyte.
261
262
GRADIENT ELUTION Conventionally, the solvent strength is held constant and is called an isocratic separation. At constant solvent strength, a complex mixture can take a long time to separate in an isocratic separation. The experimental conditions can be optimized for strongly retained or weakly retained compounds, but not both at the same time. To analyze a complex mixture, it is possible to change the strength of the mobile phase during a separation-a technique called gradient elution. A weak solvent is used for the mobile phase early in the separation; non-polar analyte elute first. A stronger solvent is gradually added during the separation, to decrease k' for the more polar components, speeding the separation. Gradient elution is used to speed the separation of complex mixtures.
263
GRADIENT ELUTION In this example, four components are separated in an isocratic mode. The components have similar resolution and plate count and identical area. In isocratic mode, early eluting components are narrower and taller. Late eluting components generate broad peaks with low signal.
In gradient elution chromatography, the strength of the mobile phase is increased during the separation. The more highly-retained components elute more quickly than in isocratic chromatography.
264
GRADIENT ELUTION There are several ways for form the solvent gradient. In low cost systems, a mixer is used to form the gradient from two solvent reservoirs; a single highpressure pump pushes the mixture through the column.
In more expensive systems, each solvent has its own high-pressure pump, and the outputs of the pumps are mixed before going to the column.
265
266
number of plates = length of column / height of 1 theoretical plate
246
Example A 2-meter long open column, tr = 30 min, D = 5 x 10-5 cm2/s If diffusion is the only contribution to band broadening, what is the estimated band width? number of plates? height of 1 theoretical plate?
247
LIQUID CHROMATOGRAPHY Gas and liquid chromatography are complementary
248
HPLC • HPLC stands for either high performance liquid chromatography or high pressure liquid chromatography, depending on whom you talk to. • The pressure and performance are both high because of the use of small size stationary phase. • Small particles improve separations because they reduce the eddy diffusion term and because they reduce the mass transfer term. • Eddy diffusion is reduced because the total number of particles increases, so that any difference in pathlength tends to average quickly. • Mass transfer is reduced because the small distance analyte must diffuse to reach a particle.
249
HPLC COMPONENTS • Small particles create a dense bed of stationary phase. It is difficult to push solvent through the stationary phase, and a high pressure pump is required. Pressures are 100-500 atm. • Because of the high pressure, the components in HPLC are quite robust, usually made from stainless steel.
250
PISTON PUMP These pumps work continuously. Fluid from a reservoir is sucked into the pump when the piston is withdrawn. The fluid is pushed out the top when the piston is pushed in. A pair of check valves is used to direct the fluid to the appropriate direction. These simple devices consist of a ball and a spring. The spring forces the ball to seal and prevent back flow. Two pistons can be operated in tandem to reduce pressure fluctuations.
251
252
LC INJECTOR
The injection loop can be changed to different volumes, ranging from 50 nL to many mL. In the load mode, the sample from the syringe flows through the sample loop to waste. At the same time, the flow from the pump passes to the column. In the inject mode, the flow from the pump passes through the sample loop and then to the column.
253
LIQUID CHROMATOGRAPHY COLUMNS • Stainless steel • 10-30 cm long • 1-10 μm particle size - 40,000-60,000 plates/m
254
LIQUID CHROMATOGRAPHYDETECTORS 1. U.V. Absorbance—sensitive, almost universal. Most common detector 2. Refractive index—not sensitive, universal Can’t be used with gradient elution. 3. Mass spectrometry—sensitive, universal. Requires volatile buffer and electrospray interface - VERY important in protein analysis.
255
STATIONARY PHASE • A solid particle of uniform particle size (5-10 μm diameter is common) is packed into the column in the form of a slurry. • A frit holds the particles in place.
256
NORMAL PHASE LIQUID CHROMATOGRAPHY • In normal phase chromatography, the stationary phase is highly polar, such as hydrated silica (SiO2 × H2O), with a high surface area—500 m2/g. The analyte adsorbs or interacts with silanol groups. Polar material can be bound chemically to the surface through silica chemistry
In normal phase chromatography, R = (CH2)3NH2 (amino) or (CH2)3C N (cyano) are common chemically bound stationary phases attached to silica solid supports.
257
NORMAL PHASE In normal phase chromatography, the stationary phase is polar and the mobile phase is non-polar. Analyte partition between the two phases. We can adjust the relative affinity of an analyte for the surface by adjusting the composition of the mobile phase. Solvents range in their polarity. The strength of a solvent measures its ability to partition analyte in a chromatographic column. The eluotropic series measures the polarity and strength of a solvent.
258
NORMAL PHASE LIQUID CHROMATOGRAPHY In normal phase chromatography, with weak solvents, non-polar molecules will tend to be found in the mobile phase (small k') whereas polar molecules will tend to be found in the stationary phase (high k'). With strong solvents, all substances will be found in the mobile phase. Mobile phases are chosen to produce an intermediate partition ratio for the analyte. By choosing an appropriate buffer, column selectivity can be fine-tuned for a particular compound.
259
REVERSED PHASE LIQUID CHROMATOGRAPHY Non-polar reversed phase chromatography is very commonly used these days. It is called reversed phase because the polar stationary phase is switched with the non-polar stationary phase. The stationary phase is bonded to a solid support, which is typically silica
The hydrocarbon forms a very stable, very non-polar stationary phase
260
REVERSED PHASE LIQUID CHROMATOGRAPHY For reversed phase chromatography, the elutropic series starts with water as a weak solvent and nonpolar solvents as a strong solvent.
With weak solvents, such as water, non-polar molecules are held to the surface and won't elute (high k'). With stronger solvents, such as toluene, most all molecules will be found in the mobile phase and eluted quickly (small k'). Mobile phases are chosen that produce an intermediate partition ratio for the analyte.
261
262
GRADIENT ELUTION Conventionally, the solvent strength is held constant and is called an isocratic separation. At constant solvent strength, a complex mixture can take a long time to separate in an isocratic separation. The experimental conditions can be optimized for strongly retained or weakly retained compounds, but not both at the same time. To analyze a complex mixture, it is possible to change the strength of the mobile phase during a separation-a technique called gradient elution. A weak solvent is used for the mobile phase early in the separation; non-polar analyte elute first. A stronger solvent is gradually added during the separation, to decrease k' for the more polar components, speeding the separation. Gradient elution is used to speed the separation of complex mixtures.
263
GRADIENT ELUTION In this example, four components are separated in an isocratic mode. The components have similar resolution and plate count and identical area. In isocratic mode, early eluting components are narrower and taller. Late eluting components generate broad peaks with low signal.
In gradient elution chromatography, the strength of the mobile phase is increased during the separation. The more highly-retained components elute more quickly than in isocratic chromatography.
264
GRADIENT ELUTION There are several ways for form the solvent gradient. In low cost systems, a mixer is used to form the gradient from two solvent reservoirs; a single highpressure pump pushes the mixture through the column.
In more expensive systems, each solvent has its own high-pressure pump, and the outputs of the pumps are mixed before going to the column.
265
266
