In this first class on liquid chromatography (LC) we will quickly refresh our knowledge on the basics of LC. We will discuss packing materials and explain the difference between high-pressure liquid chromatography and ultra-high-pressure liquid chromatography. We will finally discuss LC stationary phases.
Learning Goals #
After this lesson you should
Know the general basics of liquid chromatography.
Describe an instrumental setup used for LC, along with the different components.
Understand the difference between HPLC and UHPLC.
Place the efficiency of LC in context to that of GC and CE.
- Know the most common stationary-phase materials.
READ MODULE 3.1
Basics of Liquid Chromatography
1. Basics of liquid chromatography #
LC relies on the distribution of analyte between a mobile and a stationary phase. This may be though absorption, where the volume of the stationary phase (e.g. a liquid or soft polymer) matters, or through adsorption, where the surface area of a solid adsorbent matters. This concept was addressed in detail in Lesson 1.
1.1. Instrumental setup #
LC columns are generally packed, not open-tubular, due to the low diffusion coefficients of solutes in liquids. This necessitates higher pressures and shorter column lengths, justifying the term “high-pressure” in HPLC.
Despite these challenges, LC is highly versatile, offering many ways to tailor selectivity. A basic setup involves a pump that drives a pre-mixed mobile phase through the injector, column, and detector. This setup is shown in Figure 1, and must manage the high backpressure resulting from pumping viscous liquids through packed columns, with pressure dropping along the column length.
Figure 1. Schematic illustration of a system used for liquid chromatography.
Like gas chromatography (GC), LC systems include an injector and detector, but injection is simpler in LC as no vaporization is needed. Detection, however, is more complex due to the dense, non-inert liquid mobile phase, which can interfere with signals. This makes universal detectors like the GC flame ionization detector (FID) unsuitable, requiring more selective or indirect LC detection methods (e.g. UV, fluorescence, MS). Detection will be discussed in detail in Lesson 10.
Figure 2. This reproduction of a Scottish twenty-pound note, features Mary Gilbert from John Knox’ group performing an HPLC experiment. It is no longer valid currency.
The chromatogram reflects the sum of the full sequence of components that constitute the path of analytes through the LC system. This includes sample introduction, interactions between analytes and both mobile and stationary phases in the column, and the different pieces of tubing. The latter tubing are therefore always minimized by LC practicioners in terms of volume.
EXERCISE 1
Which of the following statements explain why LC is the most-used analytical separation method?
This is correct! LC is by far not the most efficient analytical separation method. Gas chromatography (GC) and capillary electrophoresis (CE) usually offer much higher plate counts. LC is, however, very robust, and reliable. With all of of wasteful mobile-phase effluent, LC certainly is not more sustainable than gas chromatography or capillary electrophoresis. Nevertheless, LC is certainly applicable to the broadest range of analytes, because the majority of analytes are not sufficiently volatile or thermally stable to allow analysis by gas chromatography. LC does offer a range of different options to separate non-volatile analytes. Finally, it is true that LC features the highest pressure, but this is more a burden than an asset.
This is not correct! Two statements are correct reasons. Hints: Plate numbers are higher for GC and CE. Moreover, is the pressure in LC really beneficial? Finally, think about all of the mobile phase that LC employs relative to GC and CE.
2. Retention & Selectivity #
Chromatographic selectivity is determined by differences in distribution coefficients, which in turn are determined by differences in the strengths of interactions between the analytes and the mobile and stationary phases. A number of different types of interactions can be identified, as summarized in Figure 3.
Figure 3. Overview of the types and strengths of interaction between molecules and ions encountered in LC.
In general, electron interactions between non-polar molecules are much weaker than dipole interactions Coulombic interactions and hydrogen bonding or chelation interactions are even stronger.
J.F.K. (Jozef) Huber (1925-2000, Austria)
J.F.K. (Jozef) Huber was born in Salzburg (Austria). He started his academic career in Innsbruck (Austria, 1958-1960), followed by several years (1960-1963) at the TU Eindhoven in The Netherlands. He then became full professor at the University of Amsterdam before returning to Austria in 1974 as professor at the University of Vienna. Jozef Huber contributed massively to the theory and practice of chromatography. Together with Johan Kraak, he was the first to realize HPLC in Europe in the 1960s.
The variety of interactions are reflected in a variety of LC separation modes. To obtain retention factors in the optimum range (1 < k < 10) there needs to be a fair balance between the interactions in the mobile phase and those in the stationary phase. With the notable exception of size-exclusion chromatography (see Lesson 16 on polymer separations or Module 4.2 in the book), we need to create a difference between the two phases to achieve selectivity. For example, we may have a polar stationary phase with a much-less-polar mobile phase, a situation commonly referred to as normal-phase liquid chromatography (NPLC) or the opposite, a relatively low-polarity stationary phase and a polar mobile phase (reversed-phase liquid chromatography, RPLC).
3. Efficiency #
LC columns contain very small particles, with diameters of the order of a few µm. This can be understood from the reduced parameters as treated in Lesson 3, i.e. the reduced plate height (h), defined as
Equation 1.79: h=\frac{H}{d_{\text{p}}}
and the reduced linear velocity (\nu)
Equation 1.80: \nu_{\text{int}}=\frac{u_{\text{int}} \cdot d_{\text{p}}}{D_{\text{m}}}
where u_0=L/t_0 and D_{\text{m}} is the diffusion coefficient of the analyte in the mobile phase. For good columns, packed with fully porous particles, we may treat h and \nu as constants. As a rule of thumb, h = 2 and \nu = 10.
Small particles allow high plate counts (N per unit length. In Lesson 1 we mathematically wrote this as
Equation 1.29: N=\frac{L}{H}=\frac{L}{h \cdot d_{\text{p}}}
Thus, higher plate counts can be achieved by either choosing longer columns or smaller particles. The required analysis time is proportional to the dead time
Equation 1.11: t_0=\frac{L}{u_0}=\frac{L \cdot d_{\text{p}}}{\nu \cdot D_{\text{m}}}=\frac{N \cdot h \cdot d^2_{\text{p}}}{\nu \cdot D_{\text{m}}}
Thus, for a given number of plates smaller particles yield much faster analysis. However, there is one catch. This is the pressure drop across the column, described by the Darcy equation that we learned in Lesson 2
\Delta P=\frac{\Psi \cdot \eta \cdot L \cdot u_0}{d_{\text{p}}^2}=\frac{\Psi \cdot \eta \cdot (N \cdot h \cdot d_{\text{p}})}{d^2_{\text{p}}} \cdot \frac{\nu \cdot D_{\text{m}}}{d_{\text{p}}}
=\Psi \cdot \eta \cdot h \cdot \nu \cdot D_{\text{m}} \cdot \frac{N}{d^2_{\text{p}}}
where a typical value for the flow-resistance factor \Psi = 1000. This yields
Thus, on a given LC instrument that provides a given maximum pressure, the maximum achievable number of plates decreases strongly if the particle size is decreased.
EXERCISE 2
We will now compare HPLC and UHPLC. We’ll first look at the efficiency for HPLC.
Part A: HPLC
On a typical HPLC system with \Delta P_{\text{max}} = 40 MPa (400 bar; 5800 psi), what is the maximum number of plates that can be achieved with 5 µm particles? You may use h = 2, \nu = 10, \Psi = 1000, D_{\text{m}} = 10-9 m2 \cdots-1, and \eta = 10-3 Pa\cdots. Report an integer number (no decimals).
This is correct!
Not correct. Hint: try to use N_{\text{max}}=\frac{\Delta P_{\text{max}} \cdot d^2_{\text{p}}}{\Psi \cdot \eta \cdot \eta \cdot h \cdot \nu \cdot D_{\text{m}}}. Mind the units!
What is the required analysis time in seconds to achieve this number of plates when k = 3? Report an integer number (no decimals).
This is correct!
This is not correct. You can use t_0=\frac{(1+k) N_{\text{max}} \cdot h \cdot d^2_{\text{p}}}{\nu \cdot D_{\text{m}}}
Part B: UHPLC
Now we will repeat the calculations for UHPLC technology.
What is N_{\text{max}} if you reduce the particle size to 1.7 µm? And what if the maximum pressure is 100MPa (1000 bar; 14500 psi)? For the latter case, what would be the analysis time then?
N_{\text{max, 1.7 um, 40MPa}} = 5780
N_{\text{max, 1.7 um, 100MPa}} = 14450
t_{\text{R, 1.7 um, 100MPa}} = 33.4 s
Exercise 2 shows us that HPLC systems allow fairly high numbers of theoretical plates, at the expense of significant analysis times. UHPLC systems allow much faster analyses, but the maximum number of plates is limited. Thus, for relatively simple, routine separations UHPLC is attractive, whereas for complex mixtures larger particles, long columns and long analysis times are observed.
Apart from a requirement for high pressures, UHPLC systems also have a much lower tolerance for extra-column band broadening.
READ MODULE 3.2
Stationary-Phase Materials
4. Stationary Phase Materials #
Fully porous particles are still the norm in LC, although superficially porous (or “core-shell”) particles have been making inroads. The latter provide lower reduced plate heights, probably due to a more-homogeneous particle-size distribution.
Silica (SO2) is the most common material. It is readily available, mechanically strong, offers high surface areas, and silanol (-SiOH) groups at the surface. The latter have disadvantages, in that they may engage in hydrogen bonding or coulombic interactions with analytes, potentially giving rise to broadened or tailing peaks. They also offer great possibilities to modify the surface through the formation of covalent (SiOSiC) bonds. The resulting chemically bonded phases (CBPs) are the most-common types of stationary phases in LC today. The most popular phase is octadecyl silica (ODS), which can be formed by reacting silanols with a monofunctional reagent, such as n-octadecyl-dimethyl-methoxy silane (Figure 4A) or a trifunctional reagent, such as such as n-octadecyl-trimethoxy-silane (Figure 4B). In Figure 4A one residual (unreacted) silanol group is shown. Because remaining methoxy groups are rapidly hydrolysed, more silanol groups appear in Figure 4B.
Figure 4. Schematic illustration of “monomeric” (A) and “polymeric” (B) octadecyl-silica (ODS) stationarhy phases for RPLC. Blue spheres represent CH2 or CH3 groups, purple spheres are silicon atoms, pink spheres denote oxygen atoms, and yellow spheres denote hydrogen atoms.
Other CBPs may contain more-polar groups (e.g. diol, cyano, amino), ionic groups (e.g. quaternary ammonium groups to form strong anion exchangers or sulphonic-acid groups to form strong cation exchangers), or stereoselective groups that allow chiral separations.
Use of silica-based CBPs is mostly combined to the range 2 < pH < 7, to avoid hydrolysis of the bonded phase (at low pH) or dissolution of the silica material (above neutral pH). However, materials with greater pH stability are provided by some manufacturers. Columns should only be used within their specified range.
Alternatives to silica are much less common. Polymeric particles (e.g. polystyrene-divinylbenzene resins) are used predominantly in size-exclusion chromatography (see Module 4.2 in the book) and as starting materials for ion exchangers (see Module 3.7). Monolithic columns contain a solid porous material (mostly silica, organic polymers or “hybrid” combinations of the two). Monolith can be created in situ and a high porosity can lead to lower flow resistance.
Concluding remarks #
In this introductionary lesson about liquid chromatography we have become acquinted with the basic principles of this versatile technique. The instrument setup is not very complicated at a first glance, but the next lectures will show that quite some research goes into some of the components.
The efficiency of LC does not match with that of gas chromatography and capillary electrophoresis. UHPLC conditions help, but mostly in routine measurements suitable for high-throughput.
The retention and selectivity of LC is what makes it so versatile. In the next two lessons two lessons we will explore some or the more popular modes of selectivity.
Please find here an Excel sheet that contains the calculates above. In order to facilitate your capability to really learn these calculations it is STRONGLY recommended that you first try them yourself without the sheet.
Download: (SS_05, .XLSX).
References #
[1] K.M. Usher, Phyllis Brown: A Story of Serendipity, Perseverance, Vision, and Chromatography, LC-GC North America, 2012, 30(11), 982-985, [LINK].
