OPLC as a Real Layer Version of HPLC

– Analogies and Differences

Ernő Tyihák1, Emil Mincsovics2 and Ágnes M. Móricz1

1) Plant Protection Institute, Hungarian Academy of Sciences, Budapest, Herman O. St

15. H-1525 Hungary

2) OPLC-NIT Co., Ltd, Budapest, Hungary

Introduction

The liquid chromatography (LC) embraces two basic techniques: column LC and layer LC. In

high performance liquid chromatography (HPLC), the column containing the adsorbent bed

is designed to meet the requirements of a high pressure system [1-3]. The development of

HPLC and its versions ensured the continued progress of column LC as it was demonstrated

excellently in Budapest at the HPLC 2011 [4].

However, while in high performance column, the mobile phase is pumped through the

stationary phase, in thin-layer chromatography (TLC) and high-performance thin-layer

chromatography (HPTLC) the mobile phase usually migrates by capillary action [5,6]. To the

increase of the efficiency of layer LC the development of the ultramicro (UM) chamber was

the first step [7] illustrating a special combination of the two basic conventional LC

techniques. Increased eluent-flow velocity, optional development distance, and the use of a

pump system for optimizing flow velocity, were realized by the development of a pressurized

UM (PUM) chamber [ 8,9]. The essential feature of a PUM chamber is that the adsorbent

layer is completely covered by a flexible membrane under an external pressure so the vapour

phase above the adsorbent layer is virtually eliminated. The adsorbent layer is spread onto a

flat base plate , which is covered by a cushion made from elastic foil or other suitable

material, and mounted on the cover plate.The development of the PUM chamber was the first

successful step towards a true planar-layer version of HPLC. The PUM chamber is the basic
instrument of overpressured layer chromatography (OPLC) [10,11]. Characteristically in

OPLC, an overpressure is used for the admission of the eluent into a PUM chamber. OPLC

corresponds to HPLC using a column with a narrow but wide cross section [11].

Instrument versions of OPLC (from the simple Chrompres 10 to automated OPLC 50)

generate great potential for analytical and preparative separations of a wide variety of

substances of synthetic or biological origin [ ]. Up-to-date results with the HPLC and OPLC

techniques are suitable for the comparison. Analogies and differences between the two

techniques can determine the directions of the future developments.

Comparison of elements of HPLC and OPLC

The analogies are determined but relatively limited between these basic techniques while the

differences are characteristic mainly for the OPLC .

Analogies between HPLC and OPLC

If the eluent outlet of the OPLC chamber is connected to a flow-cell detector, eluted solutes

can be detected on-line and fractions can also be collected [ ]. The entire chromatographic

process can be performed on-line by connecting a loop injector to the eluent inlet and UV

detector to the eluent outlet, in much the same way as in HPLC [ ].On-line OPLC, a genuine

layer chromatographic version of HPLC( Fig.1), is especially suitable for direct coupling to

other ,separation and detection techniques (e.g. OPLC-FTIR, OPLC-MS etc.) [ ].Fully on-

line hyphenation of an experimental OPLC separation unit with diode-array detection and

mass spectrometry (OPLC-DAD-MS) can be used for the analysis of biologically active

substances or metabolites [ ].

Fig. 2 shows the relationship between average theoretical plate height (H) and mobile phase

velocity (u) for OPLC instruments, including the automatic OPLC instrument [ ]. It is

apparent that increasing the external pressure results in increased optimum u with an
increased optimum velocity range. This relationship illustrates the fundamental analogy

between HPLC and OPLC which is complemented by H-L relationship as well [ ].

Differences between HPLC and OPLC

In off-line OPLC chemical, physical and biological detection can be used for the evaluation

of the spots/bands of compounds separated. Such solutions are not possible in the HPLC.

The efficiency and attractivity of OPLC technique can be increased dramatically by the use of

different multi systems which are coming from the attractivity of the adsorbent layer in

multiple mode and from the forced (directed) flow of the eluent in it. Parallel solution of

overpressured multilayer chromatography (OPMLC) using two or more chromatoplates is

very attractive because a large number of samples (50-100 or more) can be separated during

one development [ ]. Serial coupled OPMLC ( called also „long distance” OPLC) can be

used for the increase of the theoretical plate number and resolution alike [ ]. For difficult

separation problems the application of multidimensional (MD) OPLC is necessary because

the separation power of one-dimensional chromatography is inadequate for complete

separation of components. There is a proposed theoretical model whereby maximum peak

capacity could be achieved by use of 3D TLC(OPLC) separation [ ]. A new general concept

has been developed for single channel and multichannel OPLC separations using a

nonsegmented adsorbent layer and a flowing eluent wall (FEW) for operational segmentation

[ ]. For FEW system the original hydraulic unit of the OPLC has been changed to a new

one which is equipped with two mobile phase inlet connections, one for sample injection and

another for the FEW formations, and the outlet can be connected to a flow cell detector and/or

a fraction collector (Fig. 3). The FEW as an innovative technical solution in layerLC enables

real multichannel liquid chromatographic separation on a non-segmented adsorbent layer [ ].

Unique difference of OPLC from HPLC

There is an unique great potential of adsorbent layer for biological detection and interactions

in in vitro and in vivo studies. In fact the adsorbent bed in the column is not suitable for

biological detection and interactions because the living cells (e.g. bacterial cells) do not grow

there. This is a unique difference between the two techniques. In future the layer systems,

mainly OPLC, will be crucial and indispensable methodological solutions for isolation,

identification and characterization of new antimicrobials, antineoplastics, biopesticides and

others [ ]. The well-known direct bioautography, which integrates application of layerLC

separation and post-chromatographic bioassay, can be regarded as the most efficacious assay

for detection of antibiotic-like compounds. Although, direct bioautography is a leading

technique in bioautography, nowadays it is already is not enough. BioArena system is the

first basically further development of the direct bioautography (e.g. coordination of operating

steps, using aimed series of endogenous and/or exogenous molecules in culture medium ) [ ]

can be used to exploit the potential of direct biological detection in adsorbent layer.

One sample illustrates the applicability of BioArena for the characterization of an antibiotic-

like compound as trans-resveratrol []. Using the aqueous suspension of Saccharomyces

cerevisiae for the biological detection (Fig. 4) it can be seen that the HCHO capturer

molecules in the culture medium (and so after immersion in chromatographic spot) can

decrease the antiyeast activity of the trans-resveratrol on the adsorbent layer , while in the

presence of Cu(II) ions as HCHO transporting ions the antiyeast activity of trans-resveratrol

of the same amount is increased dramatically [ ]. The antiyeast effect of trans-resveratrol is

virtual, because it does not participate directly in the antimicrobial effect. Similar results are

observable using other host-parasite relationships and other molecules (e.g. paclitaxel

[ ]).The attractivity of BioArena can be demonstrated by detection and indirect measure of

O3 as the HCHO reaction product [ ] in chromatographic spots Using Indigo Carmine for

elimination of O3 molecules from the chromatographic spots of cinnamic acid it has been
established that the antibacterial activity of cinnamic acid decreased really dose-dependently ,

that is, the antibacterial activity of cinnamic acid is also virtual.. Series of similar reactions in

BioArena can be carried out with unlimited with most diverse reagents, microbes etc.

Conclusions

From the comparison of analogies and differences of HPLC and OPLC it follows that the

differences give firstly good possibilities for big breakthrough as. e.g. the results with

BioArena illustrate it attractively.

On the basis of BioArena studies the HCHO and O3 as characteristic key small molecules play

a crucial role in the antibiotic effect of most diverse chemical substances. Therefore, it is

especially interesting to know and understand better the function and place of these small

molecules in the biological world.

References

[1] Horváth, Cs. (ed.), (1973) High Performance Liquid Chromatography, Advances and

Perspectives, Vol. 1, Academic Press, New York, NY.

[2] Knox, J.H., (1961), J. Chem. Soc. 433.

[3] Giddings, J.C.,(1965) Anal. Chem. 37, 60-