Chapter 21

How does HPLC differ from GC, instrumentally and van Deemter behavior?

Draw a block diagram of a basic HPLC system and that of your dream system (with an unlimited budget!).

Describe the HPLC subclasses based (a) on the mode of separation, and (b) physical nature of the column.

What are ion exchange resins? What is a must-have constituent in the eluent to elute an ion off an ion exchange resin?

Distinguish between gel-type and macroreticular ion exchange resins.

An intimate mixture of an \(\mathrm{H}^{+}\) -form strong-acid type cation exchanger and an \(\mathrm{OH}^{-}\) -form strong-base type anion exchanger is called a mixed-bed resin. If you slowly percolate some tap water through a bed of such resin and measure its conductivity, what will you expect the specific conductance of the effluent to be?

What is a monolithic column? How does it differ from a standard packed bed in architecture and performance? Who invented monolithic columns? What other contributions to separation sciences did (s)he make?

List the major types of chiral stationary phases. Describe in more detail the mechanism by which at least one of these differentiate between chiral isomers.

What component(s) other than the pump(s) constitute a HPLC solvent delivery system?

Describe the two basic pump types used in HPLC

Describe the two basic pump types used in HPLC

"An HPLC pump is a veritable jewel box." Explain.

How do HPLC and UHPLC differ?

Gradient clution is used in liquid chromatography to separate compounds of widely differing partition coefficients in relatively short times and with reasonably similar efficiencies. How and why does it work? Briefly explain the methodology used to achieve a similar result in gas chromatography.

What advantage do narrow-bore columns have in HPLC?

Describe the principle of size exclusion chromatography. What is the exclusion limit?

Compare size exclusion chromatography and ion exclusion chromatography. What are the principal application areas of each?

Compare and contrast: micellar electrokinetic chromatography vs. size exclusion chromatography.

What detectors are considered universal detectors in HPLC? What are their limitations?

What are the generally desirable criteria for a HPLC detector?

You are pumping a liquid of known viscosity through a tube of known diameter and length at a certain flow rate. State the equation from which you will calculate the pressure needed. By what factor would the necessary pressure change if you reduce the diameter of the tube by a factor of \(2 ?\)

How is it possible to measure the conductivity of a solution without contacting the solution with the electrode? What is this technique called? Do you see any advantages of measuring conductivity in this fashion?

Describe the principles of operation of aerosol detectors used in HPLC.

Compare the advantages and disadvantages of a packed-column suppressor vs. a membrane suppressor.

For electrodialytically generated eluents, why is KOH more commonly used than \(\mathrm{NaOH}\) or \(\mathrm{LiOH}^{2}\)

What is the most common detector used in HPLC? Can you draw a block diagram?

Draw the block diagram of a photodiode array detector used in HPLC.

Gas removal devices can be used in two different places in ion chromatography. One is before the injector; the other is before the detector. Explain.

\text { Discuss when gradient elution might not be desirable. }

In what type of chromatography the separation space is increased by using a larger column volume?

Explain the statements: Thin layer chromatography is typically a nonequilibrium process. The composition of the developing solvent put in the tank may be known but the effective solvent is different and its composition is hard to characterize and may even vary with the location on the plate.

How is gradient elution accomplished in HPTLC?

Describe the basis of separation in CZE and contrast with that in chromatography

How does pulsed field gel electrophoresis differ from standard slab gel electrophoresis?

What is isoelectric focusing? What kind of resolution is possible in capillary isoelectric focusing \((\mathrm{CIEF}) ?\)

What is SDS-PAGE? Is the sample applied to the cathode or anode end of the gel or the middle?

Ethidium bromide is still the most common DNA intercalating fluorescence tag. But there is increasingly a move to other tags. Why?

Describe what two fundamental injection modes are used in \(\mathrm{CE}\) and the advantage and disadvantage of each.

What is the zeta potential? How can the zeta potential be changed?

The core of a typical micelle is hydrocarbon-like. The number of molecules that aggregate to form a micelle can be \(\sim 2-200\); that for SDS is \(\sim 60\). If we were to compare MEKC with HPLC. what HPLC subclass will it be closest to? What classes of molecules may be well suited for MEKC separations and what classes of molecules are unlikely to be separated by MEKC?

PROFESSOR'S FAVORITE PROBLEM Contributed by Professor Milton L. Lee, Brigham Young University A student was asked to separate two substances, \(\mathrm{A}\) and \(\mathrm{B},\) on a \(30.0 \mathrm{~cm}\) column. She obtained a chromatogram that gave retention times of 15.80 and 17.23 min for \(A\) and \(B\), respectively, and an elution time of an unretained compound of \(1.60 \mathrm{~min}\). The base peak widths for \(\mathrm{A}\) and \(\mathrm{B}\) were 1.25 and 1.38 min, respectively. Please calculate (a) the average number of theoretical plates for the column, (b) the plate height, (c) the resolution of \(\mathrm{A}\) and \(\mathrm{B},\) (d) the length of column that would be required to achieve a resolution of \(1.5,\) and (e) the time required to elute compounds A and \(B\) on the longer column.

Alkali metal ions can be determined volumetrically by passing a solution of them through a cation exchange column in the hydrogen form. They displace an equivalent amount of hydrogen ions that appear in the effluent and can be titrated. How many millimoles of potassium ion are contained in a liter of solution if the effluent obtained from a \(5.00-\mathrm{mL}\) aliquot run through a cation exchange column requires \(26.7 \mathrm{~mL}\) of \(0.0506 \mathrm{M} \mathrm{NaOH}\) for titration?

The sodium ion in \(200 \mathrm{~mL}\) of a solution containing \(10 \mathrm{~g} / \mathrm{L} \mathrm{NaCl}\) is to be removed by passing through a cation exchange column in the hydrogen form. If the exchange capacity of the resin is 5.1 meq/g of dry resin, what is the minimum weight of dry resin required?

What will be the composition of the effluent when a dilute solution of each of the following is passed through a cation exchange column in the hydrogen form? (a) \(\mathrm{NaCl}\); (b) \(\mathrm{Na}_{2} \mathrm{SO}_{4} ;\) (c) \(\mathrm{HClO}_{4} ;\) (d) \(\mathrm{FeSO}_{4} ;\left(\mathrm{NH}_{4}\right)_{2} \mathrm{SO}_{4}\)

PROFESSOR'S FAVORITE PROBLEM Contributed by Professor Apryll M. Stalcup, Dublin City University, Ireland Given the information content in IR, compared to UV, why isn't IR used much for detection in HPLC?

PROFESSOR'S FAVORITE PROBLEM Contributed by Professor Michael D. Morris, University of Michigan Two intensely absorbing fluorescent dyes were separated by \(\mathrm{CE}\) and by conventional scale HPLC. The concentration detection limits by fluorescence are about the same, but the detection limits are much worse for \(\mathrm{CE}\) in absorbance detection. Why?

You have a detector providing digitized output to the data system at selectable rates of \(1 \mathrm{~Hz}\), \(5 \mathrm{~Hz}, 10 \mathrm{~Hz}, 20 \mathrm{~Hz}, 50 \mathrm{~Hz}, 100 \mathrm{~Hz},\) and \(200 \mathrm{~Hz}\). Higher data rates also produce higher noise. If peaks are perfectly Gaussian and the sharpest peak has a half-width of \(3 \mathrm{~s}\), what data transfer rate should you use?

The S/N ratio of a fluorescence detector is measured by the Raman emission of water. You are using a low-pressure Hg lamp with a fixed excitation wavelength of \(253.7 \mathrm{nm}\). At what wavelength should you observe the Raman emission? The energy loss is \(3382 \mathrm{~cm}^{-1}\).

In indirect photometric detection in CE, a visibly absorbing additive, e.g., chromate, is often used in the BGE and the absorbance in the visible is measured. None of the analytes have visible absorption. It has been stated that if the detector is calibrated with a known chromate concentration, no further calibration is necessary for quantitating a fully dissociated analyte, at least in eq/L units. Defend or contest this statement in a quantitative basis.

Describe how you would plumb a single 10 -port valve to inject the same sample into two different columns, each connected to a separate pump and detector.