3296 III / MEDIUM-PRESSURE LIQUID CHROMATOGRAPHY

See also: II/Chromatography: Countercurrent Chroma- Ito Y (1986) CRC Critical Reviews in Analytical Chemistry
tography and High-Speed Countercurrent Chromatogra- 17: 65.
phy: Instrumentation. Chromatography: Liquid: Counter- Ito Y and Conway WD (1996) High-Speed Countercurrent
current Liquid Chromatography. III/Alkaloids: Gas Chromatography. New York: John Wiley.
Chromatography; High-Speed Countercurrent Chromato- Mandava NB and Ito Y (1988) Countercurrent Chromato-
graphy; Liquid Chromatography; Thin-Layer (Planar) graphy. New York and Basel: Marcel Dekker.
Chromatography. Zhang TY (1991) Countercurrent Chromatography.
Beijing: Beijing Science and Technology Press.
Further Reading Zhang TY (1996) HSCCC on medicinal herbs. In: High-
Speed Countercurrent Chromatography. New York:
Conway WD and Petroski RJ (1995) Modern Countercur- John Wiley.
rent Chromatography. American Chemical Society Sym-
posium Series 593. Washington DC.

MEDIUM-PRESSURE LIQUID
CHROMATOGRAPHY
K. Hostettmann and C. Terreaux, (HPLC). The distinction between low pressure, me-
University of Lausanne, Lausanne, Switzerland dium pressure and high pressure LC is based on the
Copyright ^ 2000 Academic Press pressure ranges applied in these techniques and the
overlap is often considerable. MPLC allows puriRca-
tion of large compound quantities and, unlike open-
Introduction column chromatography and Sash chromatography,
faster and improved separations are obtained. Pack-
Medium-pressure liquid chromatography (MPLC) is
ing of material with lower particle size under pressure
one of the various preparative column chromatogra-
enhances separation quality and moreover the solid
phy techniques. Separation under pressure renders
phase can be reused. Table 1 provides a comparative
the use of smaller particle size supports possible and
description of these different methods. Simplicity
increases the diversity of usable stationary phases.
and availability of the instrumentation, together with
MPLC was introduced in the 1970s as an efRcient
recycling of packing materials and low maintenance
technique for preparative separation of organic com-
costs, contribute to the attractiveness of this tech-
pounds. MPLC overcame one major drawback of low
nique. More details about experimental conditions
pressure liquid chromatography (LPLC), i.e. the lim-
are given below.
ited sample loading. This separation method is now
routinely used beside or in combination with the
other common preparative tools: open-column
Instrumentation
chromatography, Sash chromatography, LPLC or A schematic representation of a simple MPLC setup is
preparative high performance liquid chromatography shown in Figure 1. The instrumentation is made up of

Table 1 Comparative description of various preparative column chromatographic techniques

Technique Stationary phase Pressure (bar) Flow rate Sample amount Solvents General
particle size (
m) (mL min\1) (g)

Open-column 63}200 Atmospheric 1}5 0.01}100 General solvent Frequent packing;

chromatography used RP not possible
Flash chroma- 40}63 1}2 2}10 0.01}100 General solvent Frequent packing
tography used
Low pressure 40}63 1}5 1}4 1}5 More solvent Prefilled Lobar
LC required columns
Medium 15}40 5}20 3}16 0.05}100 More solvent Infrequent packing
pressure LC required
Preparative 5}30 '20 2}20 0.01}1 High purity Higher resolution
HPLC solvent required
 III / MEDIUM-PRESSURE LIQUID CHROMATOGRAPHY 3297

Figure 1 Typical MPLC instrumental setup.

a pump for solvent delivery, a sample injection sys- 1760 mm) for the larger columns available. Selection
tem, and a self-packed column. Product separation of the column dimensions depends on the sample
can be followed either manually by monitoring with amount to be separated, ranging from 0.1 g with the
thin-layer chromatography (TLC) or automatically smallest columns up to 100 g with large columns.
with a detector and a recorder connected to the col- Selectivity (
) and retention factor (k) are the promin-
umn outlet. Separated compounds are collected by ent factors inSuencing resolution. Sample loading can
means of a fraction collector. greatly affect resolution. Therefore, when separ-
ations are ‘easy’ (
'1.2; high resolution between the
Pump eluted compounds), larger sample amounts can be
loaded. Increasing the column diameter allows injec-
Chromatographic separation of 0.1}100 g sample
tion of a larger sample mass (higher throughput), but
within a few hours requires Sow rates ranging from
also makes use of smaller particle size material pos-
about 5 to 200 mL min\1, with a maximal pressure
sible. On the other hand, increasing the column
of 40 bar. Several companies provide pumps suitable
length results in higher resolution but has little or no
for MPLC. Criteria for selecting an MPLC pump
effect on sample throughput. The back pressure
include: Sow rate range; presence or absence of
increase with longer columns often implies the use of
a pulse-damper, which provides regular Sow rates
larger particle size material. The inSuence of column
and pressures during separation and increases the
dimensions on resolution has been studied through
reproducibility of the separation; presence of a pres-
the separation of standard mixtures. The correlation
sure cut-off device; and presence of a gradient
between resolution and amount of packing material
former. Some manufacturers provide pumps with ex-
was shown to be linear either when testing columns
changeable piston heads, thus allowing Sow rates
with identical internal diameter and different
from 0.5 to 160 mL min\1 with pressures up to
lengths or when varying the internal diameter in a set
40 bar.
of columns of the same length. However, a lesser
increase in resolution was observed with the use of
Column
larger internal diameter and a constant length. Conse-
The column is the central point when optimizing quently, longer columns are preferred in order to
a preparative chromatographic separation and cri- improve the separation of a given sample. The col-
teria such as amount of sample to be puriRed, amount umn system supplied by BuK chi (Flawil, Switzerland)
of packing material and column length versus column gives the possibility to couple columns together vary
diameter, have to be carefully considered. MPLC simply by means of a TeSon sealing joint, resulting in
columns are generally made of thick glass coated with an increased resolving power.
protective plastic and can withstand pressures up to
Detector and Recorder
50 bar; however, some columns from some manu-
facturers cannot withstand pressures exceeding Monitoring of a MPLC separation can be performed
20 bar. The columns vary in length as well as in by TLC of the collected fractions. Online detection is
internal diameter (i.d.) and sizes are expressed as also routinely used with single-wavelength UV/Vis
Rlling volumes. Filling volumes range from 63 mL detectors. Most available UV detectors are designed
(9 mm i.d.;100 mm) to 15 000 mL (105 mm i.d.; for analytical purposes and are of little use for
3298 III / MEDIUM-PRESSURE LIQUID CHROMATOGRAPHY

preparative separations. Accommodation of high its economic advantage, silica gel possesses other ad-
Sow rates is a prerequisite for a preparative vantages such as a wide range of possible solvents as
chromatographic detector. This results in a loss of eluents, easy evaporation of the fractions and elution
sensitivity, which is compensated by the usual high with high Sow rates. The risk of irreversible adsorp-
concentration of the eluate. In fact, these concentra- tion is a possible major drawback of this support.
tions are often so large when coming through the A wide range of particle sizes is commercially avail-
detection cell that the detector is overloaded. This able. The smallest average particle size (5}10
m) is
problem can be solved by the use of detectors with used for analytical HPLC, while for preparative LC,
a splitting system before the UV cell: one part of the stationary phases with sizes starting at 15
m are the
sample goes directly from the column outlet to the most convenient. Optimal separations are generally
fraction collector, while another part is diverted obtained with sizes around 20
m. The inSuence of
through the detector. Detectors with a pathlength particle size on resolution has been investigated.
(0.1 mm are also very useful. Selection of a detec- A large decrease in resolution was observed when the
tion wavelength where absorption of the products is average particle size changed from 15
m to 30
m.
low can also be an alternative to avoid detection Using particle sizes of 52
m or 130
m resulted in
overload. The Gow-Mac 80-800 LC-UV detector is a slower resolution decrease but the retention times
a specially designed detector for preparative separ- increased signiRcantly: 3 h more with an average par-
ations: Sow rates up to 500 mL min\1 are possible ticle size of 130
m than with one of 15
m.
and the eluate arrives through a needle and passes as The use of modiRed silica gel phase (bonded
a thin Rlm on a 6.5 cm wide quartz cell. Connection phases) has become more common. These inherent
to a recorder allows visualization of the chromato- advantages, such as a lower risk of sample decompo-
graphic separation. sition and less irreversible adsorption, allowing an
easier recycling of the column sorbent. Reversed-
Fraction Collector phase (such as RP-18, RP-8 or RP-4) or dihydroxy-
Automatic collection of fractions can be performed propylene-bonded (Diol, Merck, Germany) silica gels
by connecting a fraction collector to the column or are frequently used for MPLC separations. Moreover,
detector outlet. The volume of the collected fractions it is possible to use other commercially available
is of course strongly dependent on the internal dia- bonded phases for MPLC separations.
meter of the column and the Sow rate; it is in most
cases time-monitored. Presence of a built-in peak Column Packing Methods
detector or connection to an external one allows Different Rlling methods are described for pack-
peak-monitored fraction collection. In its standard ing MPLC columns. Filled columns should possess
MPLC setup, the BuK chi system provides a fraction an optimal homogeneity and a good density. Two
collector with a total capacity of 240;20 mL tubes, methods are most frequently used: dry Rlling and the
120;50 mL tubes or 48;250 mL tubes. This type of slurry method.
fraction collector has proved to be particularly suit-
able for MPLC.
Dry Vlling Dry Rlling is generally applied for silica
gel. This method usually gives a 20% better packing
Column Packing density than the slurry method. The ‘tap-and full’
technique can be used with particle sizes larger than
Packing Material
20}30
m; however, when applying eluent pressure
Selection of the stationary phase is probably the most up to 40 bar, the packing density obtained may not be
crucial parameter affecting separation quality. sufRcient. Packing under nitrogen pressure allows
Several types of packing material are commonly used use of 15
m particle size silica gel and provides
in MPLC and various factors have to be considered a high packing density. Filling is carried out manually
when choosing the packing material: by connecting a reservoir to the top of the column,
which is then Rlled with dry stationary phase until it
E particle size
contains approximately enough phase to Rll another
E column length
10% of the column (Figure 2). The system is then
E operating pressure
connected to a nitrogen cylinder and a 10 bar pres-
E type of sample
sure is applied (with the column outlet open) until the
E cost.
level of packing material remains constant. The nitro-
With regard to cost-effectiveness, the most fre- gen valve can be closed and the pressure slowly goes
quently utilized stationary phase is silica gel. Beside down to atmospheric. Vacuum at the column exit can
 III / MEDIUM-PRESSURE LIQUID CHROMATOGRAPHY 3299

changed and the column repacked. Regular elution of

a test mixture is a good method to determine column
quality. Bonded-phase columns are generally easier to
clean (for example with a mixture of methanol/tet-
rahydrofuran (1 : 1)) and thus have a longer working
life. In order to prolong column lifetime, a pre-col-
umn can be used. Contaminating material remaining
at the top of the column is thus eliminated after each
MPLC separation.

Solvent Selection
Selection of the eluent system is also a crucial point in
development and optimization of a MPLC separ-
ation. The ideal case would be successive direct test-
ing of various solvent mixtures on the MPLC column.
However, in routine practice such an approach is
obviously impossible because of the waste of time due
to column equilibration, together with loss of sample,
etc. Two methods are mainly used for solvent selec-
tion: optimization by TLC or transposition of analyti-
cal HPLC conditions on MPLC.
Figure 2 Dry filling of BuK chi MPLC glass columns. (Repro-
duced with permission from Hostettmann et al., 1997.) Preliminary TLC allows rapid screening of numer-
ous possible solvents and it is now well established
how TLC results on silica gel plates can be transposed
be used as an alternative to nitrogen pressure. An to silica gel columns. Solvent testing on silylated TLC
automatic mechanism has been suggested to slowly plates can be used for reversed-phase columns. One
and homogeneously Rll the column (3}4 g min\). important factor that has to be considered is that the
Passing mobile phase through the column induces surface areas of silica gel used in TLC is about twice
compression of the stationary phase, which is com- that of the column packing material. Therefore, it is
pensated by the further addition of dry silica gel. recommended that sample constituents display a re-
tention factor (RF) lower than 0.3 on the TLC plate.
Slurry method The slurry method is an alternative The major drawback of this method is the lower
method to pack silica gel, with the inconvenience of separation and resolution observed when reducing
a lower packing density. However, slurry Rlling is the the solvent strength to obtain an RF) 0.3. An alter-
preferred method for packing bonded phases. The native has been suggested to circumvent this problem:
slurry is prepared by suspending homogeneously an the use of overpressured-layer chromatography
appropriate amount of stationary phase in the eluent. (OPLC) as a pilot method for MPLC. In a Rrst step,
The mixture is then poured into the column and the a suitable multicomponent eluent with a good selec-
eluent is passed through the column until the station- tivity is searched for by means of TLC. Adjustment of
ary phase level is constant. the solvent strength and Rne tuning are performed
with OPLC. Unlike TLC, OPLC is a closed and equi-
Column preparation and regeneration Before librated system and can be viewed as a ‘planar
sample introduction, it is recommended that a separ- column’. Because of these properties, direct trans-
ation test is performed with a standard mixture of position from OPLC to MPLC is an accurate and
compounds. A mixture of phthalic acid dimethyl-, efRcient method. Such an approach is also applic-
diethyl- and dibutyl esters is convenient for testing able to the other preparative pressure chromatogra-
silica gel columns, whereas the separation of benzene phy techniques using normal silica gel as stationary
and naphthalene can be used for reversed-phase phase.
columns. Because of the similarities of the phases used in
Usually, packing material is regenerated after each analytical HPLC and preparative packing materials,
chromatographic separation. For silica gel supports, separation optimization on an analytical HPLC col-
this can be performed by washing successively with umn very often provides excellent results and trans-
methanol, ethyl acetate and n-hexane. However, after position to MPLC is straightforward and direct.
a certain time, the stationary phase should be This is particularly evident for separations on
3300 III / MEDIUM-PRESSURE LIQUID CHROMATOGRAPHY

reversed-phase sorbents, where studies with TLC are mobile phase. Despite these inconveniences, injection
more difRcult. Due to the wider range of solvents of a small volume of a concentrated solution is usu-
available for normal-phase chromatography, prelimi- ally preferred. Sample solubilization in a solvent dif-
nary tests on TLC are of major use prior to analytical ferent from the mobile phase is also possible, but
HPLC optimization. Examples of transposition of special care has to be taken with such an approach:
analytical HPLC conditions to MPLC are given be- solubility after mixing the sample solution with mo-
low. bile phase has to be checked in order to avoid sample
Once the ideal conditions have been selected, precipitation on the top of the column. Samples can
a compromise has to be found between speed of be injected either directly on the column through
separation and sample loading: decreasing the solvent a septum, or by means of a sample loop. In both
strength (for example, by adding water to the solvent cases, injection success depends on the quality of the
system in reversed-phase separations) will increase column packing to ensure a homogeneous distribu-
the separation between the different components tion of the sample at the top of the column.
and afford higher sample loading, but will require The various problems mentioned above are more
a considerable longer separation time. The inSuence frequently encountered with separations on reversed-
of solvent strength on the resolution of a standard phase columns. The mobile phase usually contains
mixture has been studied and a linear decrease of a large proportion of water and organic compounds
resolution was observed when increasing the solvent are often encountered that are insoluble in water.
strength. Running a gradient is also possible with Solid injection or solid introduction is an alternative
MPLC, provided a suitable solvent delivery system is to circumvent low sample solubility. The introduc-
used. Peak sharpening can be obtained by a simple tion mixture is prepared by mixing dry powdered
stepwise change of mobile phase composition. sample with a suitable amount of column packing
Evaporation of large quantities of solvent takes material. The sample can also be preadsorbed on
place after fraction collection in order to concentrate stationary phase by removing the volatile solvent
the puriRed compounds. This procedure can cause the (e.g. dichloromethane, ethyl acetate, acetone, etc.) in
accumulation of considerable amounts of nonvolatile which it was solubilized from the suspension contain-
impurities from the solvent. As high purity solvents ing the stationary phase. Homogeneity of the injec-
are very expensive, preliminary distillation of ordi- tion powder is a prerequisite for efRcient separ-
nary grade solvents to prepare the eluent can be ations. The proportions of the introduction mixture
a good compromise between solvent purity and are generally one part sample mixed with two to Rve
quantity employed. Use of such lower grade solvents parts stationary phase. The prepared sample is then
often implies an additional puriRcation step by gel placed directly onto the column inside a small pre-
Rltration, for example. column and the eluent is passed through the system
for separation.
Sample Introduction
Applications: MPLC in Natural
Several criteria have to be considered before sample
injection on a MPLC column:
Product Isolation
MPLC has recently become widely used in the phar-
E sample preparation
maceutical, chemical and food industries, and many
E sample mass and volume
applications are found in natural product isolation.
E solubility characteristics of the sample
Both applications given below have been selected as
E type of injection used.
examples of the transposition of analytical HPLC
If solubility is not a problem, the eluent should be conditions to MPLC.
chosen to dissolve the sample. However, even in such The methanol extract of Halenia corniculata,
a case, care has to be taken to adjust the sample a Gentianaceae plant from Mongolia, was Rrst passed
volume: a sample that is too dilute (injection of through a Sephadex LH-20 gel column and the glyco-
a large volume) results in decreased separation ef- side-rich fraction (300 mg) was then puriRed by
Rciency, while precipitation at the top of the column MPLC on a reversed-phase RP-18 column, yielding
may be observed by injection of samples that are too six xanthone glycosides (1}6). The search for optimal
concentrated. High concentrations of the sample may conditions was performed by analytical HPLC (Fig-
alter the viscosity of the solution, which is then very ure 3A) and was followed by direct transposition to
different from that of the mobile phase. High MPLC separation (Figure 3B).
viscosity leads to severe tailing, while fronting may The dichloromethane extract from the roots
result from lower sample viscosity compared to the of Tinospora crispa (Menispermaceae) was Rrst
 III / MEDIUM-PRESSURE LIQUID CHROMATOGRAPHY 3301

Figure 3 Transposition of conditions for the MPLC separation of xanthone glycosides from Halenia corniculata (Gentianaceae).
(A) Analytical HPLC on a Lichrosorb 7
m RP-18 (250 mm;4 mm) column with MeOH/H2O 40 : 60 (v/v); flow rate 1 mL min\1;
(B) MPLC on Lichrosorb RP-18 (15}25
m) with MeOH/H2O 40 : 60 (v/v); flow rate 3 mL min\1; column dimensions 460 mm;12 mm.
(Reproduced with permission from Hostettmann et al., 1997.)

fractionated by centrifugal partition chromatography now routinely used in many laboratories. The ex-
and one fraction was submitted to analytical HPLC tended use of various bonded phases in MPLC no
with an acetonitrile gradient (Figure 4A). Owing to longer restricts the use of this technique to the isola-
the lower convenience of acetonitrile for preparative tion of lipophilic substances with silica gel. For rea-
purposes (cost, toxicity), conditions were found with sons of economy, recycling the stationary phase by
methanol on analytical HPLC (Figure 4B). The se- simple washing or repacking of the column is of great
lected isocratic eluent system was transposed dir- interest in MPLC. Furthermore, a working experi-
ectly to MPLC and 6-h separation led to the isola- mental setup can be easily and rapidly assembled. The
tion of three phenylpropane derivatives (7}9) wide range of sample amounts that can be separated
(Figure 4C). with this technique, together with the use of TLC and
analytical HPLC in the search for optimal conditions,
are also major beneRts of this chromatographic
Conclusion method. However, good column packing and ad-
Since the early 1980s MPLC has been conRrmed as an equate sample preparation are prerequisites for suc-
excellent preparative chromatographic tool that is cessful separations. Further developments in MPLC
3302 III / MEDIUM-PRESSURE LIQUID CHROMATOGRAPHY

Figure 4 Transposition of analytical HPLC conditions for MPLC separation of phenylpropane derivatives from Tinospora crispa
(Menispermaceae). (A) Analytical HPLC on a Lichrosorb 7
m RP-18 (250 mm;4 mm) column with a MeCN/water gradient 0 : 100 to
40 : 60 (v/v) in 20 min; flow rate 1 mL min\1; (B) analytical HPLC on a Lichrosorb 7
m RP-18 (250 mm;4 mm) column with
MeOH/water 40 : 60 in 20 min; flow rate 1 mL min\1 ; (C) MPLC on Lichrosorb RP-18 (15}25
m) with MeOH/water 30 : 70; flow rate
4 mL min\1; column dimensions 460 mm;12 mm.

will mainly concern detection problems with the op- Further Reading
timization of detectors that can accommodate high
Cavin A, Hostettmann K, Dyatmyko W and Pot-
sample loads. terat O (1998) Antioxidant and lipophilic constituents
of Tinospora crispa. Planta Medica 64: 393}396.
See also: II/Chromatography: Liquid: Large-Scale Hostettmann K, Marston A and Hostettmann M (1997)
Liquid Chromatography. III/Flash Chromatography. Preparative Chromatography Techniques } Applica-
Natural Products: Liquid Chromatography; Thin-Layer tions in Natural Product Isolation, 2nd edn. Berlin:
(Planar) Chromatography. Springer-Verlag.
 III / MEMBRANE CONTACTORS: MEMBRANE SEPARATIONS 3303

Leutert T and Von Arx E (1984) PraK parative Mitteldruck- Verzele M and Geeraert E (1980) Preparative liquid
FluK ssigkeitschromatographie. Journal of Chromatogra- chromatography. Journal of Chromatographic Science
phy 292: 333}344. 18, 559}570.
Nyiredy S, Dallenbach-Toelke K, Zogg GC and Sticher Zogg GC, Nyiredy S and Sticher O (1989a) Operating
O (1990) Strategies of mobile phase transfer from thin- conditions in preparative medium pressure liquid
layer to medium-pressure liquid chromatography with chromatography (MPLC). II. InSuence of solvent
silica as the stationary phase. Journal of Chromatogra- strength and Sow rate of the mobile phase, capacity and
phy 499: 453}462. dimensions of the column. Journal of Liquid
Porsch B (1994) Some speciRc problems in the practice of Chromatography 12, 2049}2065.
preparative high-performance liquid chromatography. Zogg GC, Nyiredy S and Sticher O (1989b) Operating
Journal of Chromatography A 658: 179}194. conditions in preparative medium pressure liquid
Rodriguez S, Wolfender J-L, Odontuya G, Purev O and chromatography (MPLC). I. InSuence of column prep-
Hostettmann K (1995) Xanthones, secoiridoids and aration and particle size of silica. Journal of Liquid
Savonoids from Halenia corniculata. Phytochemistry Chromatography 12, 2031}2048.
40: 1265}1272.

MEMBRANE CONTACTORS: MEMBRANE

SEPARATIONS
J. G. Crespo, I. M. Coelhoso and R. M. C. Viegas, penetrate the pores and contaminate the other Suid
Universidade Nova de Lisboa, Monte de Caparica, phase.
Portugal If a hydrophilic membrane is used, the procedure is
analogous, but in this case it is necessary to impose an
Copyright ^ 2000 Academic Press organic phase pressure which is higher than that of
the aqueous phase.
Although extraction can be conducted using
Membrane-based processes are receiving recognition a number of different membrane conRgurations,
for their Sexibility and efRciency. Processes like including Sat-sheet, spiral-wound, rotating annular
reverse osmosis, ultraRltration and dialysis are al- and hollow Rbres, hollow Rbres have received the
ready well developed and recently, membrane most attention due to their high packing density:
application to other separation processes, such as typical interfacial areas of contact per unit volume
absorption and liquid}liquid extraction, have been range from 1500 to 7000 m2 m\3.
gaining considerable attention.
In these latter processes, the porous membrane acts
as contacting media for gas}liquid or liquid}liquid
phases with comparable advantages to the traditional
continuous contact equipment. While in conventional
two-phase processes, dispersion of one phase into
another immiscible phase is used in order to promote
an efRcient contact and increase the transport
rate, membrane extraction is accomplished without
dispersion of the two phases.
Consider a liquid}liquid extraction process
and a microporous hydrophobic membrane with
the aqueous}organic interface stabilized inside
the membrane pores (Figure 1). Since the membrane
is hydrophobic, the organic phase spontaneously
wets the membrane and may permeate through the
pores to the aqueous phase. This breakthrough
problem can be controlled by applying a higher
pressure on the phase that does not wet the Figure 1 Organic}aqueous interface immobilized in a micro-
pores. This higher pressure must not exceed a critical porous hydrophobic membrane. Paq, aqueous-phase pressure;
value, pcr, otherwise the nonwetting Suid will Porg, organic-phase pressure.