## Micro-LC The Basics

### For Those Times When We’re in a Hurry

- Micro-LC System © Juri Leonhartd, IUTA
- Equation 1
- Equation 2
- Equation 3
- Fig 1: Comparison of the separation performance for two scenarios: a) – d) Separation of a mixture on a 2.1 mm ID column and an Agilent 1200 system with a) capillary-ID:125 μm, b) capillary-ID: 180 μm, c) capillary-ID: 260 μm and d) capillary-ID: 500 μm; e) – h) Separation of a mixture on a 300 μm ID column and an Eksigent ExpressLC ultra-System with e) capillary-ID: 25 μm, f) capillary-ID: 50 μm, g) capillary-ID: 100 μm and h) capillary-ID: 250 μm.
- Tab. 1: Comparative presentation of the variance of a chromatographic band in relation to the extra column volume. All data pertain to a 5 cm column. The fields shaded in grey denote typical diameters for Micro-LC columns. * Assumption: 70% porosity with a 50 mm long column; ** Assumption: for 10,000 plates and a retention factor of 2; *** Assumption: factor 10 lower system variance in relation to the column variance.

**For some time “sustainable processes” has been the phrase on everyone’s lips. As far as analytical labs are concerned there are many possibilities for saving energy or rather for using resources more efficiently [1]. Besides the real “energy guzzlers” such as extractors and safety cabinets, complex analysis apparatus has to be subjected to critical system checks, too.**

Whereas development in the field of “smart phones” has clearly advanced in the direction of miniaturization while simultaneously improving performance, in the field of analysis technology it seems to be quite the contrary. Although there has been an increase in performance, the systems are becoming more and more complex and taking up more and more space. Laboratory space is however very valuable and the energy costs for climate control cannot be neglected.

**Miniaturization**

To make optimum use of the available space, it is necessary to miniaturize the laboratory instruments. This would assume, however, that miniaturized analysis systems are consistently being implemented. In this article we would like to illustrate why this process is taking so long and the possibilities that are being wasted from the point of view of miniaturized liquid chromatography.

The fact that miniaturized analysis systems offer a series of advantages has long been accepted [2]. The following list summarizes the significant advantages and disadvantages of miniaturized HPLC-systems.

**Advantages**

- Low consumption of solvents
- Very good compatibility with mass spectrometry
- High linear flow velocities
- Low volume of samples
- Not much space needed
- Low energy needs
- Low influence from frictional heating
- Low gradient dwell volumes

**Disadvantages**

- High influence of extra-column volumes on intrinsic efficiency which means the “inherent” separation efficiency of the separation column
- Low sensitivity in the coupling with spectroscopic detectors
- Currently the limited number of available stationary phases

**Routine**

So why hasn’t Micro-LC managed to establish itself extensively in routine laboratories?

The answer is relatively simple: the smaller the inner diameter of the column and therefore the variance of the column (σ²_{v,col}), the higher the influence of the volume or rather the variance of all the parts such as the injector (σ²_{v,inj}), capillaries (σ²_{v,cap}) and detector (σ²_{v,det}), on the resulting band broadening and consequently on the separation performance of the chromatographic system.

**Peak Broadening**

In principle a chromatographic band can be seen as a statistical spread in the distribution of molecules. A characteristic parameter of such a distribution is the second moment also named variance (σ²_{v}). The broadening of a peak doesn’t only occur in the column, it already begins with the injection and transport of the sample through the capillaries to the column. This is particularly valid for isocratic separations, whereas during gradient elution, assuming sufficient retention, the contribution of the parts in front of the column can be neglected. These facts can be understood with the aid of equation 1, which presents the total variance (σ²_{v,total}) as the sum of the individual contributions. (1)

(See equation 1)

Therefore it follows from equation 1 that the goal is to achieve the most favorable ratio between the extra-column volume, consisting of the injector, capillaries and detector, and the column volume so that as little as possible of the separation efficiency is lost. Ideally, the sample should be injected as an infinitely small plug that is put directly onto the chromatographic bed. Unfortunately this is absolutely impossible from a practical point of view, although there have been approaches to inject a defined volume directly onto the chromatographic column [3]. What's more, a broadening of the peak also occurs after the column. Besides the capillaries, the volume and the geometry of the detector cell play a particularly big role. The individual contributions of the band broadening presented in equation 1 can be more or less estimated mathematically, although these estimations are always based on specific assumptions [4]. In order to fully exploit the intrinsic separation efficiency one would have to drastically reduce all contributions that lead to a significant broadening of the bands outside the column. The bigger the inner diameter of the column, the lower the impact of these extra-column volumes on the separation performance. To better illustrate this situation, the variance of the column can be calculated using equation 2, while table 1 presents a list of the variances depending on the inner diameter of the column. (2)

(See equation 2)

Here, (see equation 3) and L pertain to the porosity, the radius and the length of the column. N is the number of theoretical plates and k the retention factor.

On the basis of the data collected in table 1 it is clear that extreme demands are placed on the design and assembly of miniaturized HPLC-systems. The fields shaded in grey denote typical diameters for Micro-LC columns. Whereas when using conventional column formats with an inner diameter of 4.6 mm the loss in separation performance is hardly noticeable, this can have a dramatic effect on Micro-LC separations if the system variance isn’t reduced. Figure 1 shows the comparison of a separation carried out using a conventional HPLC-system and a Micro-HPLC system where only the connecting capillaries between the column and the detector were exchanged. In the first case a 5 cm column with an inner diameter of 2.1 mm was used. The difference in the chromatograms is attributable to the replacement of the capillaries after the column, where the inner diameters varied between 125 µm and 500 µm. It is clear to see that even with a change from 125 µm to 260 µm a loss in separation efficiency follows. It is only at the change to the capillary with a diameter of 500 µm that an adequate separation is no longer achieved.

Things are very different when we look at the chromatograms in figure 1 e) to h). In this case a 5 cm column with an inner diameter of 300 µm was used. Only a tiny loss in separation efficiency is observed when the capillary with an ID of 25 µm is exchanged for one with an ID of 50 µm. If the ID is increased to 100 µm or 250 µm it has a dramatic effect on the separation efficiency as can be understood from the peak width and signal intensity. Beside that, the quality of the capillary connections can also have a significant influence on the separation performance.

**The Status of the Technology**

It can generally be stated that dedicated HPLC-systems for Micro- and Nano-LC applications with extremely low extra-column volumes, are commercially available. Furthermore, it is now possible to reproducibly inject tiny volumes of only a few nano-liters. In addition, a few manufacturers are now offering miniaturized HPLC-columns with an inner diameter of < 1 mm. The technology has clearly advanced so far that there is no disadvantage with regard to performance or robustness [5]. That said, “manual” mistakes during the operation of such systems have a significantly graver effect on the quality of the separation than is the case with conventional (U)HPLC-systems. This aspect, which is essentially a matter of training of the personnel should not be neglected and most definitely not ignored. The fact that “smart phones” are meanwhile used by people of all ages should also be an indication that the introduction of new and modern analysis technologies has no connection to the age of laboratory personnel. Successful implementation of miniaturized analysis processes does, however, depend on critical engagement with the system. This demands time, at least at the beginning, and this, as we all know, is a rare commodity.

Authors:

*Thorsten Teutenberg ^{1}, Terence Hetzel^{1}, Denise Loeker^{1}, Juri Leonhardt^{1}*

**Affiliation**

^{1}Institut für Energie- und Umwelttechnik e. V., IUTA, Duisburg, Germany

**Do you have problems with micro- or 2D-LC? Ask the experts at: ****adlichrom@iuta.de**

**CONTACT **

**Dr. Thorsten Teutenberg**

Institut für Energie- und Umwelttechnik e. V. (IUTA)

Department Head Research Analysis

Duisburg, Germany