Column Operation

This section deals mainly with the operation of an LPLC system with consideration given to

the choice of stationary phase and mobile phase, and a general guide for packing the column

is also provided. A classical LPLC system is presented in Fig. 3. The column is packed with

the stationary phase and the sample is applied to the top of the column. A solvent reservoir is

located above the stationary phase and flowed through the column under gravity. Fractions

are collected in the fraction collector after separation. The separation and purification of

compounds from a crude extract becomes easier if the identity of the compounds is known.

The properties of the compounds can also be helpful in the selection of a suitable stationary

phase. In most cases of isolation of compounds from natural sources, the specific identity of

components is rarely known. However, the polarity criteria and the results from the

preliminary qualitative tests for various types of compounds present in the extract, e.g.,

alkaloids, flavonoids, and steroids, could be helpful.

4.1. Selection of Stationary Phase

The choice of stationary phase depends on the polarity of the sample. For highly polar

compounds, ion exchange (see Chap. 6) or GPC is the preferred option. Where the expected

compounds are related to certain known compound classes, published compound separation

protocols available for their purification can be employed as a starting point. For samples

where the polarity of compounds is not known, TLC can be used to determine suitable

stationary phases, because TLC plates coated with most of the stationary phases, e.g., silica

gel, and alumina, used in CC are now readily available. By using several types of TLC plates

and running the samples with different solvent systems, an idea about the system required can

be developed (Table 3). This can provide useful information regarding starting conditions for

the separation stage and the required gradient to elute as many compounds as possible. Once

the sample has been fractionated, other separation techniques can be used.

4.2. Column Packing and Equilibration

The column is usually packed by the chromatographer prior to use. However, it is now

possible to purchase prepacked columns of different stationary phases and sizes. Column

packing materials are usually discarded after use to avoid contamination of future samples.

However,

Fig. 3. Classical LC chromatography.

Table 3

Solvent System Development Using TLC

Step Step Procedure

1 Prepare a solution of the crude extract or compound mixture, in a low boiling point

organic solvent, at a concentration of at least 10 mg/mL.

2 Apply this solution (2–5 mL) to different TLC plates (2.5_10 cm), let the applied

spots dry completely

3 Use various mobile phases to develop each TLC plate.

4 Once developed, visualize the plates under UV lamp or by spraying with appropriate

TLC reagents.

5 Compare the TLC plates and choose the solvent system that retains the compound(s)

of interest at Rf¼0.2–0.3. In the case of crude extract, the mobile phase that puts the

most mobile component (highest spot) at about Rf¼0.5 should be the initial solvent

composition for the CC. The solvent system that keeps the least mobile (lowest spot)

at about Rf¼0.2 can be used as the final eluent for CC.

for some gel filtrations, the packing material, e.g., Sephadex LH-20, can be washed

thoroughly and used again. The stationary phase is normally introduced into the column, dry

or in slurry, using a suitable solvent. The LPLC columns are normally made of thick-walled

glass, which is resistant to most solvents, and can withstand the low-to-medium pressures

used during column development. A glass frit is normally used to support the stationary

phase. The alternative is to use a plug of glass wool covered with a layer of sand for this

purpose (Fig. 3). The amount of stationary phase required depends on the amount of sample

to be fractionated. The general guideline is to use 100–500 g of packing material per gram of

crude sample.

4.2.1. Slurry Packing

This is the easiest and the most commonly used method for column packing. It is the only

method used to pack columns that swell in the mobile phase such as carbohydrate packings

(e.g., Sephadex G-10). To prepare the slurry, the required amount of stationary phase is taken

in a beaker, a solvent is added, and the mixture is stirred. If necessary, more solvent can be

added to achieve a pourable consistency of the slurry, which should neither be so thick that

air bubbles are trapped in the column, nor so thin that it requires more than one pour to pack

the column. For stationary phases that swell, e.g., Sephadex, sufficient time has to be allowed

for the phase to become completely solvated. The slurry is poured into the column, which is

kept partially open during pouring, and the solvent is allowed to flow through the column,

leaving a packed bed of stationary phase. Once the packed bed is settled, the flow of solvent

should be stopped leaving sufficient amounts of solvent on top of the packed bed, which is

necessary, in most cases, to avoid cracking of the packed bed.

4.2.2. Dry Packing

Dry packing, if performed properly, is an efficient way to pack a column and is commonly

used for regular or bonded silica gel. The dry stationary phase is poured into the column. It is

essential at this stage that the column be vibrated in some way so that the packing is allowed

to settle. Alternatively, the column can be tapped with a cork ring during the fill operation.

The stationary phase is then ‘‘wetted’’ using an appropriate solvent by allowing solvent to

flow through the column. The column can then be equilibrated with the mobile phase

required for the sample. Dry packing is particularly useful for vacuum liquid chromatography

(VLC) of nonpolar or intermediate polarity natural products where Silica Gel 60H is

normally used as the adsorbent (Fig. 4). In VLC, however, vacuum is used to achieve a

compact packing of the column.

Fig. 4. Vacuum liquid chromatography (VLC).

4.3. Sample Application

The sample can be applied to the top of the column in a variety of ways depending on the

stationary phase and the development method used. The sample is usually dissolved in a

small amount of the initial mobile phase or a noneluting solvent and gently applied to the top

of the column bed. The sample is allowed to flow by opening the exit valve. When the

sample has been loaded, the mobile phase is carefully applied to the column to prevent

disturbing the stationary phase bed. Sometimes, to avoid disintegration of the top of the

column bed where the sample is adsorbed, a layer of sand (5–10mm thick), a filter paper, or

glass wool can be applied. If the sample is not soluble in the initial or noneluting solvent,

then a sample can be loaded as dry free flowing powder. This is an option used for silica gel

columns, where the starting mobile phase is fairly nonpolar. In this technique, the sample is

dissolved in a small amount of appropriate solvent (DCM, EtOAc, or MeOH), and a weight

of silica gel (factor of 10) is added to this solution. The solvent is removed under vacuum in a

rotary evaporator leaving the sample adsorbed onto the silica gel. This dry silica gel

containing the sample can be transferred to the top of the column bed and wetted with a little

of the initial mobile phase to remove air bubbles.

4.4. Column Development

The column can be developed by elution of samples using various methods. The mobile

phase can flow under gravity, by applying a nitrogen pressure at the inlet, a vacuum at the

outlet (e.g., VLC), or pumping the mobile phase through the column at varying pressures

(e.g., flash chromatography, FC). In all these cases, a solvent gradient needs to be applied,

although isocratic solvents are often the preferred option.

4.4.1. Gradient Formation

A step gradient is often the method of choice in LPLC because of the simplicity and the

quality of separation. If the composition of solvents in a step gradient is chosen properly

according to the need of changing polarity, excellent fractionation of natural compounds can

be achieved. This is unlike ion-exchange chromatography, where step gradients are

not desirable. With modern HPLC gradient elution systems, complex gradients can be

programmed. In general, for LPLC, one to three column volumes of each solvent step are

required. Step gradients are generated by simply preparing a range of mobile phases

composed of polar/nonpolar solvents of varying ratios. During the column operation, the

column inlet reservoir is refilled with the new solvent. For any finer gradient elution, a

gradient maker can be used.

4.4.2. Gravity

Generally, good results can be achieved with gravity elution where the particle size is greater

than 60 mm. Smaller particle sizes result in back pressure, which does not allow the eluent to

pass through the column at a desired flow rate. Gravity elution is easy to run where the

mobile phase is poured on top of the open column and allowed to flow naturally under

gravity. A solvent reservoir can be used to increase capacity, and the flow rate can be

controlled by adjusting the outlet valve (Fig. 3).

4.4.3. Pressure

Positive pressure can be applied to the top of the column to accelerate the flow rate and

achieve better resolution in LPLC (Fig. 5). This technique is called FC and it uses particle

sizes in the 40–60 mm range. An accurate flow rate can be achieved by using a needle release

valve. Glass columns used in FC must be of appropriate wall thickness and strong enough to

withstand the pressure. It is advisable to use plastic mesh netting as a column jacket or simply

to tape the outside of the column to avoid any danger associated with column explosion.

Nowadays, metallic columns, empty or prepacked, especially designed for FC, can be

purchased commercially. Biotage_ flash chromatographic systems of various sizes have been

found to be useful for initial fractionation of nonpolar and medium polarity natural products

(15–22).

4.4.4. Vacuum

An alternative to applying pressure at the top of the column is to apply vacuum at the end of

the column. This technique is called VLC (Fig. 4). The operation is similar but it is more

difficult to control the mobile phase flow. However, this technique is safer than FC. A

common use of this technique is the rapid purification of a specific compound from a sample,

especially a reaction mixture. In natural product isolation, this technique is applied for initial

fractionation of crude nonpolar or intermediate polarity extracts (23–33). The sample is

applied to the adsorbent in a sintered glass and the mixture eluted with a mobile phase

directly into a vacuum flask. Generally, TLC grade silica without any binder in it (Silica Gel

60H) is used to dry pack the column.

Fig. 5. Flash chromatography.

For the pre-HPLC fractionation of polar extracts, e.g., MeOH extract, solid-phase extraction

cartridges (10 g) prepacked with reversed-phase silica C18 (ODS) have been found to be

useful (34–54). These cartridges can be placed on the ports of a vacuum manifold or

connected to a vacuum flask, similar to VLC, with appropriate adapter (Fig. 6). Cartridges are

available in different sizes and also in various packing materials, e.g., diol, C8, C6, or ion-

exchange resins. As the particle sizes are fine and the cartridges are packed mechanically, the

column bed is compact and offers excellent separation. Both isocratic and step gradients can

be used.

4.4.5. Pumped

A pump is a more controllable solvent delivery system. It can deliver a smooth and constant

flow of solvent (Fig. 7). The pump must be inert and designed for use with flammable

solvents. Differences in low-, medium-, and high-pressure chromatography can be made

essentially on the basis of the particle size of the stationary phase and resulting operating

pressure of the packed column. The LPLC is run with 40–200-mm particles at a flow rate that

generates no pressure significantly greater than the atmospheric pressure. The medium

pressure liquid chromatography (MPLC) uses 25–40-mm particles with pressures between 75

and 600 psi, and the HPLC (see Chap. 8) is used with 3–12-mm particles with pressures

between 500 and 3000 psi. The resolution of separation achieved by these chromatographic

techniques follows the order: HPLC>MPLC>LPLC. The run time is also reduced

considerably in the following order: LPLC>MPLC> HPLC. With the greater access and

availability of various HPLC systems, the use of MPLC has reduced significantly.

4.5. Detection

During the isolation of natural products, individual fractions can be analyzed for chemical

profiling by either TLC or HPLC (using an autosampler). Small samples can be spotted on a

TLC plate, and after development a guideline regarding the performance of the separation can

be obtained. A more advanced method involves the use of a UV or refractive index detector

prior to fraction collection. A variable wavelength UV detector allows different wavelengths

to be monitored for the duration of the chromatography. It should be noted that for such large

columns, the cell path length must be reduced to 0.5 mm, to decrease the absorbance values

expected from the analysis. For compounds with little or no UV,

Fig. 6. Solid-phase extraction (Sep-Pak).

Fig. 7. Application of pump in LPLC.

absorption refractive index detectors can be useful. However, for bioassayguided isolation of

bioactive natural products, a robust and rapid highthroughput assay is the assay of choice for

identifying the fraction containing bioactive compounds. Depending on the assay capabilities,

optimization of the number of fractions to be submitted for bioassay is necessary. If the

LPLC of the extract produces a large number of fractions, in the first round,

representative fractions, for example, one in each five or ten fractions, can be subjected to

bioassay. In the second round, all five or ten fractions from the active composite need to be

assayed separately to pinpoint the active fraction(s). In this way, the number of bioassays

required can be reduced significantly.