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More reproducible retention times

More reproducible retention times

Best HPLC-Chromatograms

The high health hazard potential of many ­organic solvents is well known to anyone working in a laboratory. Solvent vapours ­can not only endanger people’s health, but also increase the fire and explosion risks for laboratories and buildings, in addition to causing air pollution. This is also the case for analytical and preparative HPLC laboratories.

Permanent air contamination by solvent vapours is in most cases caused by faulty sealing of the supply and waste containers. This is not only a permanent health risk, but has also a very practical impact: Contamination of the eluent can occur as well as the mixing ratio due to different vapour pressures of the single solvent components can be continuously changed. This necessarily leads to separation results that can hardly be reproduced, like e.g. retention times, and might finally cause incorrect analytical data. This is why safety measures must be taken and be assessed again and again and adapted to the actual working place conditions.

In the following contribution, the impact of eluent evaporation on the long-time reproducibility of retention times of a selected HPLC test mixture will be demonstrated and correct solvent disposal will be addressed as well.

Influences on the HPLC selectivity

Selectivity changes of the HPLC column or the mobile phase are often the reason for a slow, but constant change of HPLC retention times. Sometimes, a non-adequate sample preparation of genuine samples like lipids, polymers, carbohydrates, cellulose, may cause this phenomenon. Matrix compounds from these samples can be adsorbed onto the column’s reversed-phase surface, thereby changing the chemistry of the column. Consequently, it’s properties will change and the retention times of the analytes as well. The same fact is observed, if the composition of the eluent changes. This can be caused by unnoticed mistakes when mixing a mobile phase, e.g. wrong volumes of each solvent in the mixture, change in the sequence of mixing, or mixing techniques. Sometimes, decomposition effects can occur in certain mobile phases or HPLC pump defects can lead to a change in the eluent composition and thereby to a retention time shift [1]

Here, a simple testing procedure for the reproducibility of retention times is described. The purpose was to check what happens if a HPLC user is using solvent bottles with “open caps”, instead of using Safety Caps (S.C.A.T. Europe). When solvent is evaporated, the composition of pre-mixed mobile phases can change. To demonstrate this, chromatograms of Polycyclic Aromatic Hydrocarbon (PAH) compounds were regularly checked over a period of 20 days. An acetonitrile-water mixture has been used as eluent in the isocratic mode. As stationary phase, an endcapped RP column with small-diameter has been applied. As the flow rate was low, the eluent consumption was also reduced [2].

Fig. 1 Comparison of the weight-losses of solvent bottle A and B [2]

Fig. 2 Chromatogram of 3 PAH compounds at day 0, 9, and 20 (Bottle B has been stored without Safety Cap)

The following testing conditions have been selected:

Bottle A: This bottle was closed with a S.C.A.T. Safety Cap, precisely fitting the used standard GL45 glass bottle thread. No additional adapters were necessary.

Bottle B: This bottle was closed with a cap having a 10-mm hole in the plastic material, giving an open exit of an area of about 0,785 cm2.

Fig. 3 SafetyCaps

Fig. 4 SafetyWaste Cap

Experimental set-up:

HPLC system: HITACHI LaChrom® Elite system with diode array detector, controlled by the EZChrom® Elite software. Isocratic pump conditions with pre-mixed mobile phase. HPLC-Column: Purospher® RP-18e (5 ), 125 x 2 mm. Flow rate: 500L/min

Tab. Weight-loss of bottles over a period of 20days

Performance of the test

At the beginning of the test, both bottles have been filled with the identical mixture of water and acetonitrile (20:80, w/w). The weight change within the period of 20 days has been regularly checked by differential weighing.

Using bottle A as a reference, a chromatogram of a PAH mixture (Benzo(a)pyrene, Benzo(g,h,i)perylene, and Indeno(1,2,3-c,d)pyrene) at day 0 was acquired for comparison. After having measured the reference chromatogram, both bottles were stored under room temperature in a fume hood, which guaranteed a gentle air flow over the top of the bottles. HPLC analyses have been repeated on day 9 and 20.


This experiment cleary reveals that without using tightly sealed solvent bottles as containers for pre-mixed HPLC eluents, there is a risk of uncontrolled vaporization, resulting in non-reproducible analytes’ retention times. As expected, bottle A didn’t show any significant weight change, thus no solvent vapours did escape from this bottle, see Tab. 1 and Fig. 1. In contrast to bottle A, bottle B showed a significant and uncontrolled solvent loss by evaporization. This results in a change of the acetonitrile-water mixing ratio and thereby in a change of the selectivity of the mobile phase, leading to a retention time shift of the eluted compounds. They are all prolonged because in bottle B more acetonitrile has been evaporated. Especially for real sample analysis, this retention time shift can have an impact on the resolution of separated peaks.

Without using tightly sealed solvent bottles as containers for pre-mixed HPLC eluents, there is clear risk of uncontrolled vaporization of solvents, thus leading to a higher health risk for the employees. In summary, it is evident that the use of S.C.A.T. Safety Caps definitely prevents such uncontrolled emissions, and in addition leads to a clear reduction of the exposure of solvents in the atmosphere in a laboratory.

How to put things right

SafetyCaps (Fig. 3) ensure safe withdrawal of solvents without release of harmful vapours. The emission of organic solvent vapours is verifiably being reduced by 73%, thus contributing to health protection of the laboratory staff. The integrated air valve allows safe solvent removal; it provides as usual for venting during removal, and at the same time, the valve membrane absorbs dust and contaminant particles from the incoming air. The air filter integrated in the air valve ensures that the solvent remains uncontaminated, provided the filters are being replaced every 6 months.

The sophisticated system has freely rotatable closure caps so even when several connectors are used, containers can be quickly changed without twisting the tubes. SafetyCaps with shut-off valve have proven to be very practical; they prevent air pockets in the tube, make changing supply containers easier and are ideal for repairs or maintenance work on the HPLC system. When restarting the HPLC system, SafetyCaps allow for fast and easy purging. Also available are SafetyCaps with level indicator, showing the solvent reservoirs running short.

SafetyCaps specifically designed for preparative HPLC are equipped with a special valve and easily deliver supply volumes of up to 400mL/min.

SafetyWasteCaps for solvent waste disposal

It is well-known that particular caution is needed when collecting combustible fluids. Static charges may build up during outflow, which may cause sparks and increase the hazard of fire. Therefore, a sophisticated system has also been developed for safe disposal of HPLC solvents. The well-proven SafetyWasteCaps (Fig. 4) provide a direct and gas-tight connection of the drainage tubes or capillaries of the HPLC system to the solvent disposal container.

The SafetyWasteCaps consist of the required fittings for the tubes, as well as an exhaust filter with special granulates of activated charcoal. The lifetime of the filter depends on the load of solvent vapour absorbed. The safety funnels are only opened at the moment filling starts, otherwise the container remains closed. S.C.A.T. Europe installation solutions, made from electrically conductive PE-HD, prevent static discharge and can be secured via an additional ground cable.

Take Home

The use of SafetyCaps for solvent withdrawal and waste disposal considerably reduces solvent emissions and consequently helps to decrease health risks for the employees. Moreover, the S.C.A.T. technology significantly minimises solvent blank values – the solvent bottles remain tightly sealed. Retention times remain constant – in particular when using pre-mixed solvents and isocratic conditions – as the solvent composition cannot change due to solvent evaporation.

[1] For a detailed description of the potential causes of changes in selectivity, please refer: https://www.novia.de/hplc-tipps/details/artikel/verschiebung-der-retentionszeit/ and www.kromidas.de.
[2] Publication in VWR ChromJournal Edition 8, Spring 2010, pp. 7, 14
[3] The topic „Protect your health“ is dealt with in detail in S.C.A.T. Europe Catalogue 2012, pp. XIV-XVI.

Foto: ©panthermedia.net|Ben Rodgers

L&M int. 1 / 2013

The articles are publishes in issue L&M int. 1 / 2013.
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