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Computer Data Acquisition for Biochemistry Practice Works

Methods and Examples

How to make computer data acquisition from a BIO-RAD Econo UV chromatogaphic analogic monitor, using Metex M-3850D and Votltcraft VC820 multimeters, piloted by a PC with the
Datalyse software.

This page describes an easy method to make numerical data acquisition from an analogic UV chromatographic monitor. A computer acquisition presents numerous advantages compared to the use of a paper chart recorder of chromatographic profiles. For example, there are no constraints of scaling the speed of paper and the mv/cm scale of the records. The profiles can be rescaled after acquisition, and converted into elution volumes unit in a tabloid like Excel, making easy the comparison of elutions charts.

The following picture shows a chromatographic montage. On the top, from left to right; the tank of buffer elution (Tris 50 mM, NaCl 125 mM, pH 7.5), a peristaltic pump setting a theoretical flow rate of 0.5 ml/min, a sample injection valve with a loading loop of 250 µl, and a chromatographic column (75 cm x 1.5 cm) filled with a Sephacryl S-200-HR gel. On the bottom, from right to left; the UV monitor, a fraction collector, a Metex M-3850D multimeter connected to the 0-1V analogic output of the UV monitor, and a RS-232 to USB adaptor connecting the computer to the RS-232 output of the multimeter.

The next picture shows a close view of the multimeter, the RS-232 to USB adaptor and the computer recording an elution profile. Datalyse can record 3000 points in RAM memory. The screen on the picture shows a record of about 5 hours with a recording step of one measure every 15 seconds. In such conditions Datalyse allows to make records of 12.5 hours.

About the battery autonomy:

The autonomy of a multimeter is very variable, depending essentially on the RS232 interface energy consumption. For example, for the montage described above with the Metex multimeter, with a full charged rechargeable Nimh battery (150 mA/h), the acquisition autonomy is about 17h.

The following montage, using a Voltcraft VC820 multimeter with an optical RS232 interface, allows to obtain a 6 days continuous recording with the same kind of rechargeable battery:


Some few data about the principle of molecular exclusion chromatography more usually called gel filtration chromatography:

Briefly, gels composed of porous particles, like gel beads of reticulated polymers, are known to have the ability to separate molecules according to their size. The gel is a stationary phase through which a solvent, the mobile phase, runs out. During the elution of a loaded sample, small molecules can enter freely in the matrix of the beads, and are retained whereas big molecules are excluded from the beads and are eluted without being retained. Some intermediate molecules enter more or less in the matrix, and are delayed according to their sizes compared to the excluded big molecules. Gels are characterized by their molecular mass fractionation domain.

In the present example, the gel has a fractionation range from 5000 g/mol to 250 000 g/mol (molecules assumed to be globular proteins).

A chromatogram is characterized by three different kinds of elution volumes:

- The elution volume of excluded molecules, measured by loading a very high molecular weight protein (for instance) onto the chromatographic column, is called the void volume (Vo).

- The elution volume of a very small molecule which enter freely in the matrix of the gel, obtained by loading a salt or very small organic molecule, is called the total volume bed (Vt).

- The elution volume of the a molecule (protein for instance) which is in the fractionation range of the gel is Ve.

There is a linear relation between the logarithm of the molecular weight of the protein and a ratio, called Kav, defined as Kav=(Ve-Vo)/(Vt-Vo).

By loading a set of known molecular weight (MW) proteins, this relation allows to calibrate a column by measuring Vo, Vt and different Ve.

The following example shows a Datalyse recording of an elution profile of a cocktail sample allowing to calibrate a column in a single step.

The following representation shows two records, one is the above calibration molecule cocktail, and an other called "Sample" which contains a partially purified protein of an unknown MW.

During the time, even with a constant imposed flow rate on a column, the effective measured flow rate can evolve: the next graph shows the two precedents records, rescaled to the same injection time. The x axis is converted in elution volume (time x flow rate measured for each elution). Known MW, measured elution volumes and deduced Kav are indicated.

The next graph shows the linear relation obtained from the measured values of the precedent graph. The sample protein presents in these conditions an apparent MW of 161 Kda.

Special thanks to Alessandra Albano for the English correction of this page.

Many thanks to Drs M.C. Bourin, E. Petit and K. Haupt for their precious advices in chromatography methodology.

Gilles Carpentier web site: "Computer Data Acquisition for Biochemistry Practice Works: Methods and Examples"

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