ChromFAQ:LC-FAQ

From ChromFAQ

Table of contents

LC Terminology

What's the difference between Tailing Factor and Asymmetry Factor?

Both Tailing Factor and Asymmetry Factor are measures of the deviation of chromatographic peak shapes from the ideal. The two measures give similar, but not exactly equal results.
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Why is the most common LC technque called Reversed-phase?

It's a matter of historical accident. The original "chromatography" described by Tswett at the beginning of the 20th century used powdered chalk (calcium carbonate; a polar adsorbent) as the stationary phase and ligroin ("petroleum ether; a non-polar hydrocarbon solvent) as the mobile phase. The relative polarities of the phases (stationary phase = polar; mobile phase = non-polar) was considered an integral part of the technique. Later, stationary phases composed of diatomaceous earth impregnated with mineral oil were combined with mobile phases consisting of mixtures of water and alcohols were tried with some success. Because the relative polarities of the two phases had been reversed (stationary phase = non-polar; mobile phase = polar), this variation was dubbed -- you guessed it -- "reversed phase" chromatography. The original approach then came to be called "normal-phase" chromatography

With the advent of HPLC in the late 1960's, reversed-phase was found to be the more versatile of the techniques (ironically because, in practice, what we call "reversed-phase" actually combines elements of both).



What are Theoretical Plates? . . . and why are they only "Theoretical"?

Theoretical plates are a measure of the amount of peak broadening (also called "band spreading") that occurs in a chromatographic column. The measure is based on analogy with a distillation column -- which often contains actual, physical plates.

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What is an Internal Standard?

An internal standard is a compound added to all calibrators and samples at a known concentration. It is used to correct for errors in sample injection, sample workup, or detector response).

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LC Chemistry

Why are reversed-phase separations done with only three organic solvents?

The short answer is that these are the three solvents that are the most different from one another and still miscible with water in all proportions. If we need selectivity which is "less different", we can always obtain it by blending two (or all three) of the "most different" solvents.

Need discussion here of strength, selectivity, and solvent characterization.



What are the differences among "C18", "ODS", and "Octadecyl" columns?

Practically speaking, nothing. The three terms are synonyms ("octadecyl" simply means "C18", and "ODS" is an acronym for "OctaDecylSilyl").

  • Are C18 columns interchangeable? No
  • Why C18 ? Because it was commercially available in high purity when bonded-phase HPLC packings were being developed. It worked.

A related question is "What does 'ODS-II' on a column designation mean?". The short answer is "Whatever the manufacturer wants it to mean.". Just as "WD-40" was named for the 40th attempt to make a water displacer, "ODS-II" generally refers to the second version of a C18 commercialized by a given company. This means that an "ODS-II" column from one company probably bears no similarity to and "ODS-II" column from another company.



Why do I have to use a buffer in reversed-phase chromatography?

Because retention and selectivity in reversed-phase chromatography can pH-dependent. The buffer serves to control the pH.

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What is ion-pair chromatography?

In the most general sense, ion-pair chromatography refers to the addition of an ionic surfactant to a reversed-phase system in order to affect retention and/or selectivity of ionic compounds.
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What's the best way to adjust the pH of a mobile phase?

Arguably, the best way is not to attempt to adjust pH at all, but rather to prepare the mobile phase by weight. Weighing is much more precise (a 20% error in weighing translates to only a 0.1 unit error in pH). You can find an on-line calculator for buffers here (http://www.bi.umist.ac.uk/users/mjfrbn/buffers/makebuf.asp)

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LC Hardware

What's the difference between Isocratic chromatography and Gradient chromatography?

The simplest approach to liquid chromatography is to maintain a constant mobile phase composition during the course of the chromatogram. Because the phrase "constant mobile phase composition" is awkward to use repeatedly, the term isocratic was invented by Csaba Horvath in 1970 as an alternative.

Unfortunately, this approach (isocratic LC) sometimes gives rise to a situation where some sample components wash through the system too quickly for useful analysis while others move too slowly. In short, no single mobile phase composition provides reasonable retention for all the components int he sample. If the mobile phase is made weaker in order to slow down the fast-moving components, the slow-moving components take even more time. If the mobile phase is made stronger in order to speed up the slow-moving components, then the fast moving components elute even earlier.

The problem can be solved by changing the mobile phase composition during the run, starting with a relatively weak mobile phase to provide reasonable retention of the fast-moving analytes and gradually increasing the mobile phase strength to elute the slower-moving compounds. This technque is called gradient elution or (less commonly) solvent programming. Gradient elution has the advantage of being applicable to a wide range of sample retentions. It has the disadvantage of requiring more complex instrumentation and being somewhat more complicated to control.



What are the differences among dead volume, dwell volume, void volume, extra-column volume, etc.?

The different "volumes" in an HPLC system are confusingly named and in some cases overlap. Briefly:

  • dead volume is the volume of liquid inside the column (also called the void volume)
  • dwell volume is the volume of liquid in a gradient system between the point at which the gradient is formed and the head of the column
  • void volume is the volume of liquid inside the column
  • extra-column volume is the volume of liquid that contributes to band broadening in the system, exlcuding the dead volume of the column.

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What's the difference between a High-Pressure Mixing Pump and a Low-Pressure Mixing Pump?


In gradient HPLC (also called "solvent programming") the mobile phase composition is changed during the course of the chromatogram. Two basic approaches are used to provide on-line control of mobile phase composition.


High-pressure-mixing systems (also referred to as "two-pump" systems) use two high-pressure pumps connected to different solvent reservoirs (the weaker solvent is usually referred to as the "A" solvent and the stronger solvent is referred to as the "B" solvent). The system controller adjusts the relative flow rates of the pumps to provide the desired composition at any given time. The outputs of the pumps come together on the high-pressure (downstream) side of the system. There is usually a mixer of some sort to smooth out fluctuations in composition. High-pressure-mixing systems are typically more expensive than low-pressure because of the need for two pumps. They tend to provide less output fluctuation and to have smaller dwell volumes. They are usually limited to two solvents.





Low-pressure mixing systems (also referred to as "one-pump" systems) use a single high-pressure pump. The pump inlet is connected to a proportioning valve which alternately selects from the "A" or the "B" reservoir. The controller specifies the percentage of time during which the valve is connected to each reservoir to provide the desired composition (e.g., for 75%A/25%B, the proportioning valve would spend 75% of the time connected to the "A" reservoir). Low-pressure mixing systems are typically more flexible that high-pressure; they are often available with ternary (three solvent) or quaternary (four-solvent) capability. They tend to provide better proportioning accuracy at extreme high or low percentages, but tend to have somewhat larger dwell volumes.




The generalizations made above concerning the relative merits of the two types of pumps are just that: generalizations. There is more variation in performance within a given type than there is between types (i.e., it is possible to design good or bad versions of both types!).



How do autosamplers work?

Essentially, autosamplers do two things:

  • transfer a measured amount of sample solution from a vial into an injection valve at atmospheric pressure
  • transfer the contents of the injection valve into the pressurized LC system.

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What is a "monolithic" column?

A monolithic column (sometimes simply called a "monolith") is a column in which the stationary phase has been cast as a solid, porous rod. This is in contrast to a tradition material in which stationary phase particles are packed into the column

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What's the difference between a DAD and a PDA?

Nothing. Different manufacturers use slightly different acronyms for the same thing. "DAD" = "Diode Array Detector", and "PDA" = "PhotoDiode Array". For you pedantic types, the phrase "PDA detector" makes sense ("PhotoDiode Array detector"), while "DAD detector" is redundant ("Diode Array Detector detector").

The older variable wavelength absorbance detector (commonly called a "UV detector", because the most commonly used wavelengths are in the ultraviolet) differs from the PDA in the arrangement of the optical components.




In the UV detector, the incident light beam is bounced off a movable grating and imaged onto a slit which allows only a limited range of wavelengths to pass through the sample.

In the PDA, the incident light beam (including all wavelengths) is passed through the sample and then bounced off a fixed grating and imaged onto an linear array of photodetectors.

As a broad generalization, PDA detectors are more flexible than UV detectors (UV spectra can be obtained "on the fly") and have fewer mechanical problems (no moving parts). UV detectors are less expensive than PDA detectors and can be made more sensitive (larger photodetector).



What do UV or PDA detectors measure?

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Most people would answer "absorbance", because detector output is displayed in absorbance units. In fact, however, UV detectors do not measure absorbance, they compute absorbance. What they measure is transmittance, the fraction of the incident light that is transmitted through the sample (absorbance is the negative logarithm of transmittance). This may seem like a "distinction without a difference", but it has a major bearing on detector sensitivitity issues. When we describe a sensitive detector as being able to work at low absorbance, this means it is working at high transmittance. In effect, the detector must measure a small difference between two large values. Other detector types, such as fluorescence, make absolute measurements of small values (e.g., the amount of light emitted at a certain wavelength) -- a much easier task. This is part of the reason why these detectors are often more sensitive than UV detectors.



what is response index

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