Chapter 25 Liquid Chromatography
Place the solvents in order of increasing eluent strength for reversed-phase HPLC: Acetonitrile, Hexane, Methanol, Dichloromethane
Acetonitrile Methanol Dichloromethane Hexane
A mixture of six nonpolar compounds was introduced to an HPLC with a C18 stationary phase for separation. CH3(CH2)5COOH and CH3(CH2)5NH2, are two of the compounds in the mixture. Which will elute through this column first if the pH of the solution is 4.0?
CH3(CH2)5NH2 An analyte that is less attracted to the stationary phase will elute first. In this column, because the stationary phase is nonpolar, the more polar of the two compounds will elute first. At a pH of 4.0, both compounds will exist in their protonated forms (CH3(CH2)5COOH and CH3(CH2)5NH+3). Therefore, the amine is more polar, so it will elute faster than the less polar carboxylic acid.
Match the high‑performance liquid chromatography (HPLC) component with its description: mixer
Combines the solvents in the appropriate ratio required for the mobile phase
elution order in reversed-phase HPLC
Compounds elute in order of decreasing polarity. In reversed-phase HPLC, the stationary phase is nonpolar (or mildly polar) and the mobile phase is more polar than the stationary phase. Thus, nonpolar compounds will be retained on the column longer than polar compounds.
Match the high‑performance liquid chromatography (HPLC) component with its description: column
Contains the support and stationary phase and is used to separate the analytes
Match the high‑performance liquid chromatography (HPLC) component with its description: sample loop
Holds a fixed amount of sample prior to injection
HPLC Ultraviolet Detector
It is the most common HPLC detector. It is useful for gradient elution with non absorbing solvents. Simple systems use lamps that produce discrete radiation, such as the 254 nm Hg vapor lamp, whereas more versatile systems use lamps that produce broadband radiation, such as xenon or tungsten lamps, and a monochromator to choose the wavelength of analysis.
Select the definition of reversed-phase chromatography.
It uses a nonpolar or weakly polar stationary phase and a more polar mobile phase. Reversed-phase chromatography is the most common mode of HPLC. Its stationary phase is nonpolar or weakly polar and the solvent is more polar. A less polar solvent has a higher eluent strength and decreases the retention of the analyte.
What is/are the advantage(s) of using ethylene-bridged silica particles instead of ordinary silica particles as the support in HPLC columns?
The ethylene-bridged silica particles are stable at higher pH and at higher pressures than the ordinary silica particles. Most silica cannot be used above pH 8 because it dissolves in base. Ethylene-bridged silica resists hydrolysis up to pH 12. The ethylene-bridged structure is more rigid and well suited for particles that are <2 μm in diameter, which must withstand high pressure.
Match the high‑performance liquid chromatography (HPLC) component with its description: injector
Used to introduce the sample to the system
Match the high‑performance liquid chromatography (HPLC) component with its description: detector
Used to monitor and measure the analytes as they move through the system
Match the high‑performance liquid chromatography (HPLC) component with its description: pumps
Used to move the mobile phase through the system
HPLC evaporative light-scattering detector
With this detector, no peaks from the solvent are produced and it is compatible with gradient elution. The equate is nebulized, forming an aerosol that is then passed through a drying tube where the solvent is evaporated, leaving analyte aerosol particles. The particles scatter light from a diode laser, generating the signal for the chromatogram. The detector response is related to the mass of analyte, not its structure or molecular mass. The use of low-concentration buffers is required with this detector.
true or false: Columns are never heated because it causes them to degrade.
false. Most HPLC stationary phases can withstand up to 60°C. Although increased temperatures do lead to faster degradation rates of the column material, many analyses are carried out at 10°C above room temperature to improve the reproducibility of retention times and the precision of quantitative analysis.
true or false: Porous graphite carbon has retention similar to that of nonpolar compounds relative to retention by octadecyl, but is less stable at extreme pH.
false. Porous graphite carbon is a stationary phase that exhibits increased retention of nonpolar compounds relative to retention by C18. The stationary phase is stable in 10 M acid and 10 M base.
Which of the following does NOT help to extend the lifetime of a column?
heating the column Columns can be degraded by dust or particles in the sample or solvent and by irreversible adsorption of impurities. Heating the column decreases the viscosity of the solvent and lowers the required pressure. However, it also increases the degradation of the stationary phase and, thus, decreases the column life.
Select the column property that does NOT affect the separation factor.
column length If other avenues of changing the separation factor do not yield adequate resolution, the column can be changed. Column properties that affect the separation factor are related to the ability of the stationary phase to interact with analytes. This includes the type of silica, the hydrophobicity of the bonded phase, the type of end-capping, the presence of polar embedded groups, and additional interactions. Column length, column diameter, and particle size do not affect the separation factor.
stationary phases commonly used in reversed‑phase liquid chromatography (RPLC)
common stationary phases that are nonpolar, and therefore, used in RPLC: Octadecyl (-(CH2)17CH3) Octyl (-CH2)7CH3) Phenyl (-CH2)3C6H5) Pentafluorophenyl (-(CH2)3C6F5)
stationary phases commonly used in normal‑phase liquid chromatography (NPLC).
common stationary phases that are polar, and therefore, used in NPLC: Amino (-CH2)3NH2) Cyano Diol
In reversed‑phase chromatography, the solute is more soluble in the mobile phase as the polarity of the mobile phase
decreases
An electrochemical detector
detects any substance that undergoes a redox reaction. Electrochemical detectors respond to analytes that can be oxidized or reduced at modest potentials. In a three-electrode thin-layer flow cell, analyte is oxidized or reduced at the working electrode whose potential is maintained at a selected value relative to a reference electrode. Current is measured between the working electrode and an auxiliary electrode.
Select the column property that affects the separation factor.
polar embedded groups If other avenues of changing the separation factor do not yield adequate resolution, the column can be changed. Column properties that affect the separation factor are related to the ability of the stationary phase to interact with analytes. This includes the type of silica, the hydrophobicity of the bonded phase, the type of end-capping, the presence of polar embedded groups, and additional interactions. Column length, column diameter, and particle size do not affect the separation factor.
Normal phase chromatography
polar stationary phase, more polar solvent has higher mobile phase strength
Which statements are true if the particle size in a packed column used in HPLC is decreased?
- increased separation efficiency - increased resolution Smaller particles create an enviornment within the column that is resistant to flow. Therefore, greater pressure is required to achieve flow rates optimal for separation. Additionally, due to the increased pressure, the solute molecules move through the column with greater velocity, which reduces the analysis times. The use of a small particle size in a packed column results in increased resolution and efficiency because the distance a solute molecule moves between the stationary and mobile phases is decreased, which reduces the 𝐶 term in the van Deemter equation and generates sharper peaks.
Place the steps used to optimize an isocratic separation in the correct order.
1. Choose the column and organic solvent. 2. Adjust the composition of the mobile phase. 3. Check the plate number is near the expected value. 4. Adjust the separation factor. 5. Optimize the column dimensions.
You are developing a reversed-phase HPLC separation and have achieved an outcome with good resolution but a lengthy run time. Which of these changes are likely to shorten the run time?
1. Decrease the mobile phase polarity 2. Increase the column temperature In reversed-phase HPLC, the stationary phase is nonpolar (or mildly polar) and the mobile phase is more polar than the stationary phase. The eluent strength of solvents decreases with increasing polarity, so decreasing the mobile phase polarity will result in shorter run times. Controlling column temperature produces more reproducible results. An elevated column temperature allows faster separations to be conducted.
What are advantages associated with very narrow diameter (<2.1 mm) HPLC columns?
1. Less waste produced 2. Compatible with mass spectrometry 3. Can analyze sub-nanogram samples
You have optimized a separation using a mobile phase of 45 vol% methanol/55 vol% water. However, two peaks are not completely resolved and you are thinking of trying another solvent. Which mobile phase consisting of acetonitrile and water would have a solvent strength similar to the initial mobile phase?
35 vol% acetonitrile/65 vol% water A vertical line drawn on the nomograph in Figure 25-31 intersects each solvent line at the same eluent strength. A mobile phase of 35 vol% acetonitrile/65 vol% water has a solvent strength similar to a mobile phase of 45 vol% methanol/55 vol% water.
Which contribution(s) to the plate height in the van Deemter equation is/are decreased as the particle size decreases?
A and C A is the multiple paths term, B is the longitudinal diffusion term, and C is the mass transfer term. Smaller particles provide more uniform flow and decrease the distance the solute must diffuse. Thus, both the multiple path term and the mass transfer term in the van Deemter equation are reduced, resulting in a decrease in plate height and an increase in resolution.
HPLC refractive index detector
A detector with virtually no selectivity that is not compatible with gradient elution. It uses filtered visible light as the irradiation source. The main appeal of this detector is its universal response to all solutes, even those with little absorption in the ultraviolet region of the spectrum.
greenness (normal vs reversed phase)
Normal phase << reversed phase
Select the TRUE statement regarding the chromatographic process and high-performance liquid chromatography (HPLC).
Packed columns are used in HPLC instead of the open tubular columns typical in gas chromatography due to the slow rate of diffusion in the liquid phase. Solutes in a liquid mobile phase would not be able to travel across an open channel in a reasonable time to interact with the wall-coated stationary phase due to the much slower diffusion time in liquids. Therefore, packed columns are used.
Which bonded stationary phase is most commonly used in HPLC?
R=(CH2)17CH3 Most commonly, liquid-liquid partition chromatography is conducted with a bonded stationary phase covalently attached to the silica surface. The octadecyl (C18) stationary phase is by far the most common.
You are running a reversed-phase HPLC separation using a mobile phase of 65 vol% acetonitrile/35 vol% water. What happens to the retention times if you change the mobile phase to 85 vol% acetonitrile/15 vol% water?
Retention times decrease. In reversed-phase HPLC, the stationary phase is nonpolar (or mildly polar) and the mobile phase is more polar than the stationary phase. As the polarity of the mobile phase decreases, the retention times decrease. Acetonitrile is less polar than water, so increasing the percentage of acetonitrile in the mobile phase will decrease the polarity of the mobile phase and decrease the retention times.
You are running a reversed-phase HPLC separation using a mobile phase of 65 vol% acetonitrile/35 vol% water. What happens to the retention times if you change the mobile phase to 35 vol% acetonitrile/65 vol% water?
Retention times increase. In reversed-phase HPLC, the stationary phase is nonpolar (or mildly polar) and the mobile phase is more polar than the stationary phase. As the polarity of the mobile phase increases, the retention times increase. Acetonitrile is less polar than water, so decreasing the percentage of acetonitrile in the mobile phase will increase the polarity of the mobile phase and increase the retention times.
Select the FALSE statement regarding the advantages and disadvantages of ultra-high-performance liquid chromatography (UHPLC).
Run times in UHPLC are increased compared to HPLC. UHPLC applies pressures up to 150 MPa to columns consisting of 1.5- to 2.0-μm-diameter particles. It provides substantially increased resolution or decreased run time compared to HPLC.
HPLC charged aerosol detector
The dynamic range of this sensitive detector covers 4-5 orders of magnitude in analyte concentration. The eluate is first nebulized in a stream of N2 gas, then a positive charge is added to the aerosol particles as they pass over a charged Pt needle. Small ions are attracted to the charged plates of a small ion trap, while the larger aerosol particles are too large to be deflected and pass to a collector where they are detected by an electrometer
Which of these changes is NOT likely to improve the separation factor?
changing the flow rate Method development focuses on the parameters governing resolution: retention factor k, the plate number N, and the separation factor α. The separation factor α is the ratio of adjusted retention times for two components. Adjust the separation factor by adjusting the solvent composition, column temperature, type of organic solvent, or column type.
True or False: Dwell time has little impact on method development.
True. Differences in dwell time or dwell volumes are a reason separations on one HPLC system do not necessarily transfer to another system. Thus, if you are basing your method development on methods for similar analytes in the literature, you may not be able to reproduce the published separation.
In normal‑phase chromatography, the solute is
polar
Scouting gradients are used in the initial stages of HPLC method development to select between
an isocratic and a gradient elution. The quickest way to decide whether to use isocratic or gradient elution is to run a scouting run using a broad gradient. If Δt/tG<0.25, then isocratic elution is appropriate. If Δt/tG>0.40, then gradient elution is recommended.
You are designing a separation for saccharides that are highly soluble in aqueous solutions. You have tried reversed-phase chromatography but the analytes are very poorly retained. Refer to Select a chromatography mode for this separation.
hydrophilic interaction chromatography Saccharides are relatively small (molecular mass <2 000). Because they are soluble in aqueous solutions, neutral, and poorly retained on a reversed-phase column, you should try hydrophilic interaction chromatography, which is used for polar analytes or analytes with polar regions.
In normal‑phase chromatography, the solute is more soluble in the mobile phase as the polarity of the mobile phase
increases
A refractive index detector
is a universal detector with detection limits in the microgram range. A refractive index detector responds to almost every solute, but its detection limit is much poorer than an ultraviolet detector. The deflection-type detector has two triangular compartments through which pure solvent or eluate passes. Collimated visible light passes through the cell and strikes a photodiode array. When solute with a different refractive index enters the cell, the beam is deflected and different pixels of the array are irradiated. Also, it cannot be used with gradient elution.
A charged aerosol detector
is an almost universal detector with nearly equal response to equal masses of nonvolatile analytes. A charged aerosol is an almost universal detector with nearly equal response to equal masses of nonvolatile analytes. Eluate and nitrogen gas enter a nebulizer. Fine mist from the nebulizer reaches the drying tube, while large droplets fall to the drain. In the drying tube, solvent evaporates at ambient temperature. Meanwhile, part of the nitrogen stream flows over a Pt needle held at +10 kV with respect to the outer case to form N+2. A chain of events transfer positive charge to aerosol particles. The charged aerosol particles pass through a trap to the collector.
In HPLC, k
is defined as the adjusted retention time divided by the time required for the mobile phase to pass through the column. The retention factor is k=(tR−tM)/tM where tR−tM is the adjusted retention time and tM is the time required for the mobile phase to pass through the column.
A fluorescence detector
is very selective but may require derivatization of the analyte for detection.
In normal‑phase chromatography, the stationary phase is
polar
Which of these is NOT an attribute of a good HPLC separation?
k=23 The attributes of a good separation include a retention factor (k) between 0.5 and 20, a resolution greater than or equal to 2, an operating pressure less than 20 MPa, and an asymmetry factor between 0.9 and 1.5.
In reversed‑phase chromatography, the eluent strength of the solvent increases as the solvent becomes
less polar
In normal‑phase chromatography, the eluent strength of the solvent increases as the solvent becomes
more polar
reversed-phase chromatography
non-polar stationary phase, less polar solvent has higher mobile phase strength
In reversed‑phase chromatography, the solute is
nonpolar
In reversed‑phase chromatography, the stationary phase is
nonpolar
select a chromatography mode for the separation of a small analyte that is soluble in nonpolar organic solvents.
normal-phase chromatography For a small molecule (molecular mass <2 000) that is soluble in an organic solvent, there are three possible options: normal phase, size exclusion, and reversed phase. Because the analyte is soluble in nonpolar organic solvents, you can assume that it is more soluble in nonpolar hexane than in the polar solvents THF, methanol, and acetonitrile. Thus, you would use normal-phase chromatography.
What type of pump is used in HPLC?
reciprocating A typical HPLC pump is a reciprocating pump containing a sapphire piston and two check valves that govern the direction of flow from the solvent reservoir into the column. The quality of a pump for HPLC is measured by how steady and reproducible a flow it can deliver. A fluctuating flow can create detector noise that obscures weak peaks.
A photodiode array
records the spectrum of each solute as it is eluted, which can be matched with library spectra to identify unknowns. A photodiode array records the spectrum of each solute as it is eluted, which can be matched with library spectra to identify unknowns. Spectra collected across a peak can be compared to evaluate peak purity and detect coeluting components.
In HPLC, VM
represents the space within a column, including the pores in the particles and the space between particles.
An evaporative light-scattering detector
responds to any analyte that remains as a solid when the solvent is evaporated. An evaporative light-scattering detector responds to any analyte that is significantly less volatile than the mobile phase. The mobile phase is mixed with nitrogen gas and forced through a small-bore needle to form a uniform dispersion of droplets. Solvent evaporates from the droplets in the heated drift tube, leaving fine mist of solid particles to enter the detection zone.
Which of these is NOT a desired attribute of a new chromatographic method?
sensitive to small changes in pH In method development, the goals are to obtain adequate separation in a reasonable time. Ideally, the procedure should be rugged, meaning it is not significantly affected by small variations in conditions, such as minor changes in pH, solvent composition, and temperature.
A mixture of six nonpolar compounds was introduced to an HPLC with a C18 stationary phase for separation. How would the retention time and peak resolution for the mixture be affected if the solvent ratio was changed from 45% water: 55% acetonitrile to 15% water: 85% acetonitrile?
shorter retention time, lower peak resolution For a nonpolar stationary phase, such as C18, a less polar solvent has a higher eluent strength. This means that as the polarity of the solvent decreases, the retention times of the solutes in the mixture decrease. Therefore, increasing the amount of the less polar acetonitrile in the solvent will decrease the retention times of the solutes in the mixture. The faster elution time results in lower resolution.
Dwell time is the
time required for the gradient to reach the column. The dwell time is the time it takes for the newly mixed solvents to reach the column. This is determined by the dwell volume and the flow rate.
true or false: A small sample volume is injected into a loop. The injection value is turned allowing the mobile phase to carry the sample onto the column.
true. An HPLC injector contains a fixed-volume sample loop. In the load position, sample from a syringe is loaded into the loop, with excess sample going to the waste. The injection valve is then turned and mobile phase flows through the loop, carrying the sample onto the column.
