Part 1

Question 1

What is the setup in HPLC?

Answer 1

High pressure to force solvent through closed columns containing very fine particles that give high resolution separations. Sample, which is in solution form, is injected into a liquid mobile phase which carries the sample through a column for separation.

The HPLC system consists of
• a solvent delivery system,
• a sample injection valve,
• a high pressure column,
• a detector, and
• a computer to control the system and display results.

Question 2

What effect does particle size have in HPLC? Is smal or large particles best?

Answer 2

Efficiency increases as particle size decreases. Typically, particle diameters of 3–10 µm.

Plate height is not very sensitive to increased flow rate when the particles are small.

Question 3

Why does small particle gives better resolution in HPLC?

Answer 3

• More uniform flow through the column. Reduces the multiple path term, A , in the van Deemter equation.
• The smaller the particles , the less distance solute must diffuse. Decreases the C term in the van Deemter
equation.
• Optimum flow rate for small particles is faster than for large particles because solutes diffuse through
smaller distances.

Small particles give high efficiency but require high pressure.

L4S5

Question 4

Difference between UHPLC and HPLC?

Answer 4

UHPLC: Even smaller particles: Chromatography with 1.5 to 2 µm diameter particles.

Peaks eluted from a UHPLC column are so narrow that fast detectors are needed.

Question 5

What is the problem with UHPLC?

Answer 5

Penalty: frictional heating so that center of a column is warmer than the outer wall and the outlet is warmer than the inlet.

Question 6

What kind of columns can be used in HPLC?

Answer 6

Steel or plastic columns that are 5–30 cm in length with an inner diameter of 1–5 mm. To protect from dust or particles in the sample or solvent, the entrance to the main column is protected by a short guard column containing the same stationary phase as the main column which is periodically replaced.
Common HPLC column diameter used to be 4.6 mm but now 2.1 mm more common.

Question 7

What happens when a HPLC column is heated?

Answer 7

Heating the column usually decreases the viscosity of the solvent, reducing the required pressure or permitting faster flow –> retention times is shortened.
However, increased temperature can degrade the stationary phase and decrease column lifetime.

Question 8

What is a Monolithic HPLC column?

Answer 8

Monolithic silica columns enable increased flow rate in liquid chromatography while retaining good separation. Monolithic rods made of a silica-gel polymer typically have macropores with diameters of approximately 2 μm and mesopores—pores within the macropores—with diameters of approximately 13 nm.

Question 9

Describe the stationary phase used in HPLC

Answer 9

In liquid–liquid chromatography the stationary phase is a liquid film coated on a packing material, typically 3–10 μm porous silica particles. To prevent the loss of stationary phase, it is bound covalently to the silica particles. The siloxane bond hydrolyzes below pH 2, so
HPLC with a bonded phase on a silica support is generally limited to the pH range 2–8.

To prevent unwanted interactions between the solutes and any remaining –SiOH groups, Si(CH3)3Cl is used to convert unreacted sites to –SiOSi(CH3)3; such columns are designated as end-capped.

Question 10

What is reverse-phase chromatography?

Answer 10

Reversed phase chromatography is the more common form of HPLC.
Stationary phase is nonpolar and the mobile phase is polar.
A polar solute spends proportionally less time in the nonpolar stationary phase and eluetes before a solute that is less polar.
The most common nonpolar stationary phases use an organochlorosilane where the R-group is an n octyl (C8) or n octyldecyl (C18) hydrocarbon chain.
Polar mobile phase commonly buffered aqueous solution, or polar solvents, such as methanol and acetonitrile. Because the silica substrate may undergo hydrolysis in basic solutions, the pH of the mobile phase must be less than 7.5.

Question 11

What is normal-phase chromatography?

Answer 11

Normal phase chromatography polar stationary phase and a nonpolar mobile phase.
A solute of lower polarity elutes before a solute that is more polar.

Question 12

Describe the mobile phase used in HPLC!

Answer 12

• Normal phase separation: less polar solutes elute first.
• Reversed phase HPLC: more polar solutes elute first. Increasing the polarity of the mobile phase leads to longer retention times.
• HPLC grade solvents: d esigned for use with HPLC instrumentation, glass distilled and submicron filtered.
• Acetonitrile low viscosity –> relatively low operating pressure, uv detection down to 190 nm. (Often in combination with water)
• Methanol higher viscosity, longer wavelength uv cutoff (205nm.)
• Tetrahydrofuran less usable uv range ( cutoff 212 nm), slowly oxidized to peroxides, incompatible with poly ether ether ketone (PEEK) tubing, slower equilibration with the stationary phase.

Question 13

Describe the polarity index

Answer 13

L4S15

Question 14

Read “How to choose a solvent”

Answer 14

L4S17-22

Question 15

Describe the pump used in HPLC!

Answer 15

Requirements:

(1) Pressures up to 6000 psi (414 bar)
(2) Pulse free output
(3) Flow rates from 0.1 to 10 mL/min
(4) Flow reproducibilities of 0.5 % relative or better
(5) Resistance to corrosion by a variety of solvents.

Dual piston pump is used to minimize pulses (Pulse free flow)

Question 16

Describe Sample Injection in HPLC

Answer 16

In the load position a sample loop (0.5 μL to 5 mL) is isolated from the mobile phase and open to the atmosphere. The sample loop is filled using a syringe with a capacity several times that of the sample loop, with excess sample exiting through the waste line. After
loading the sample, the injector is turned to the inject position, which redirects the mobile phase through the sample loop and onto the column.

Question 17

Which detectors are commonly used in HPLC?

Answer 17

• UV-VIS
• MS
• Refractive index
• Fluorescence
• Evaporated light scattering
• Electrochemical
• Radioactive
• Conductivity

Question 18

Describe UV-VIS used in HPLC

Answer 18

Most common HPLC detector. Many solutes absorb UV light.
Variable wavelength detectors: broadband deuterium, Xe, or W. Choose an optimum wavelength for
analytes.
Many compounds absorb wavelength < 210 nm. One limitation is that the mobile phase cannot absorb at the wavelengths we wish to monitor. Absorbance detectors provide detection limits of as little as 100 pg–1 ng of injected analyte.

Question 19

How does a fluorescence detector work

Answer 19

Fluorescence detectors excite the eluate with a laser and measure fluorescence. High sensitivity but respond only to the few analytes that fluoresce. To increase
the utility of fluorescence, derivatization by covalently attaching fluorescent groups can be covalently attached to the analyte or addition of reagents to the eluate between the column and the detector ( post column derivatization ).

Question 20

How does a Refractive index detector work?

Answer 20

Refractive index detector responds to almost every solute, but its detection limit is about 1 000 times poorer 0.1 1 μg ) than that of the ultraviolet detector. 0.1 1
μg of injected analyte . An additional limitation is that it cannot be used for a gradient elution unless the mobile phase components have identical refractive
indexes.

Question 21

How does an electrochemical detector work?

Answer 21

An electrochemical detector responds to analytes that can be oxidized or reduced, such as phenols, aromatic amines, peroxides, mercaptans , ketones, aldehydes,
conjugated nitriles, aromatic halogen compounds, and aromatic nitro compounds. Effluent from the column passes over the working electrode held at a constant
potential relative to a downstream reference electrode that completely oxidizes or reduces the analytes . The current flowing between the electrodes serves as the
analytical signal. Detection limits for amperometric electrochemical detection are from 10 pg - 1 ng of injected analyte

Question 22

How does an evaporative light scattering detector

Answer 22

An evaporative light scattering detector responds to any analyte that is significantly less volatile than the mobile phase. Eluate enters the detector at the top, mixes 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 a fine mist of solid particles to enter the detection zone at the bottom. The particles are detected by the light that they scatter from a diode laser to a photodiode.
The evaporative light scattering detector response is related to the mass of analyte , not to the structure or molecular mass of the analyte . A large peak means more material than in the small peak.

Question 23

Read on MS in HPLC!

Answer 23

L4S33-40

Question 24

Describe 5.1 Liquid Solid Adsorption Chromatography

Answer 24

In liquid solid adsorption chromatography (LSC ), the column packing also serves as the stationary phase. Columns consists of porous 3 10 μm particles of silica or alumina.

Mobile phase usually is a nonpolar or a moderately polar solvent. Typical mobile phases include hexane, isooctane, and methylene chloride.

Elution time: olefins < aromatic hydrocarbons < ethers < esters, aldehydes, ketones < alcohols, amines < amide < carboxylic acids

Nonpolar stationary phases, such as charcoal
based absorbents, also are used.

In general, liquid solid chromatography has no special advantages over liquid liquid chromatography. One exception is the analysis of isomers, where LSC excels.

Question 25

What is Ion Pair Chromatography?

Answer 25

Subset of reversed
phase chromatography. Easily ionizable species are separated on reversed phase columns.

The addition of an ionic surfactant to a reversed phase chromatography system in order to
affect retention and selectivity of ionic compounds.

Question 26

Why do we need ion-pair HPLC?

Answer 26

When a sample contains ionic components that tend to be very hydrophilic, and so reversed phase retention can be problematic.

When other changes in reversed phase conditions fail to achieve acceptable resolution.

Question 27

What are typical ion pair reagents in ion-pair HPLC?

Answer 27

Alkylsulfonates R-SO3- Na+

Tetraalkylammonium salts R4N+ Cl-

Question 28

What are the two possible mechanisms for ion pair HPLC seperation?

Answer 28

(i) In this model, the ion pairing agent is present in the mobile phase. The analyte interacts with
the ion pairing agent in the mobile phase first. It forms the ion pair which is relatively non polar
and partitions into the stationary phase and get retained.

(ii) The
ion pairing agent present in the mobile phase gets adsorbed into the non polar stationary phase due to its lipophilic alkyl chain. Thus the stationary phase gets a charge. The analyte of
opposite charge interacts with the ion pairing agent presented on the surface to form ion pair and gets retained . Strongest complex with stationary phase –> strongly retained solutes.
A- (mobile phase) + R4N+ Cl - (stationary Phase) –> R4N+ A- (stationary Phase) + Cl-

Question 29

What is Hydrophilic Interaction Chromatography (HILIC)

Answer 29

• Alternative HPLC mode for separating polar compounds
• Strong retention of compounds that are unretained under conventional reversed phase conditions, without the requirement for ion pair additives in the mobile phase.
• Like normal phase LC (NP LC), HILIC employs traditional polar stationary phases such as silica, amino or cyano , but the mobile phase used is similar to those employed in the RP-LC mode.
• In normal phase LC, mobile phase is nonaqueous and gradients go from nonpolar to increasingly polar organic solvents (e.g. 5 % CH 2 Cl 2 in hexane to 30 % CH 2 Cl2 in hexane).
• In HILIC, solvent is usually an aqueous/organic mixture. Mobile phase strength is increased by increasing the fraction of water in mobile phase.
• Mechanism : polar analyte partitions into and out of a layer of water which is adsorbed onto the surface of the polar stationary phase. Besides this liquid/liquid partitioning behavior, dipole dipole and electrostatic interactions are also involved. At eluent pH is > 5, surface silanol species are anionic.
• Expensive ion pair reagents are not required in HILIC.
• For uncharged highly hydrophilic and amphiphilic compounds that are too polar to be well retained in reversed phase LC but have insufficient charge to allow effective electrostatic retention in ion exchange chromatography.
• Suitable for analysing compounds in complex systems that always elute near the void in reserved phase chromatography
• Used for difficult separation problems, such as the separation of small organic acids,
basic drugs, and many other neutral and charged substances.