History Of Chromatography And Capillary Columns

Published Date: 02 Nov 2017

Introduction

It has been a very long time more them a century since chromatography was used in analytical chemistry. And separation techniques is one of the most important part of the chromatography. The columns are in the center of most separation techniques such as gas chromatography (GC), high performance liquid chromatography (HPLC), capillary electrophoresis (CE), capillary liquid chromatography (CLC) and etc., in which the separation process occurs. And it can be said that the quality of the separation achieved by the techniques primarily depends on the correct choice of columns. There are two main types of the most commonly used columns, packed columns and capillary columns.

Packed column was used first in GC and HPLC at early times. A packed column is dense and evenly packed by solid support. In GC, packed columns are always 2 to 3 meters in length and 2 to 3 mm internal diameter which is made of stainless steel or glass. And they are always filled with particles each of which are coated with a liquid or semi-liquid stationary phase. Although this kind of columns are have a high sample capacity and cheap, the resolution of packed columns is limited by their length, itself restricted by the pressure drop consequent on the resistance to gas flow.1

So capillary columns which is also called open tubular columns are invented and become widely used for its better resolving power than packed columns. And the need for improved column performance hence the development of silica capillary columns. So capillary columns have become the standard column for nearly all GC column and CE columns.

This dissertation will introduce the history, development and application of the capillary columns in chromatography, especially in gas chromatography.

History of chromatography and capillary columns

Chromatography was first invented in the early 20th century. In 1903, Russian botanist M.Tsweet put the calcium carbonate in an erected glass tube, injected the plant pigments solution in the petroleum ether from the top of the tube, and then rinsed from top to bottom with petroleum ether. As a result, in the different parts of the tube formed a ribbon of different colors. And then in1906, Tsweet published a paper which named this new technique as chromatography.2

And then between the 30s and the 40s of last century, the invention of planar chromatography which include thin layer chromatography (TLC) and paper chromatography made the chromatography become a separation technique.2 TLC is a technique that separating microgram qualities of mixtures by movement of a solvent across a flat surface. It is still have a wide range of applications nowadays especially in the analysis of biochemical and pharmaceutical.

Martin and Synge proposed the possibility of using a gas instead of a liquid as the mobile phase, and invented the partition chromatography. For this reason, they shared the Nobel Prize in chemistry in 1952.2

In 1952, the invention of gas chromatography by James and Martin improved the separation and analysis of chromatography to a new level, which is the base of the modern chromatography. And in 10 years times, the quick development of high performance liquid chromatography greatly expanded the range of applications of chromatography.2

As the column is in the center of GC and HPLC, so the development of the column is also very important. In this article, use the development of GC columns as the example.

At first packed column was used in GC, which was filled with a packing material carrying the stationary phase absorbed on it. It was typically 1–5 m long and 1–5

mm inter diameter. As the packed columns did not have the very high resolving power and the resolution of packed columns was limited by their length, so the use of long, coated capillaries was first suggested by Martin in 1956 and was developed by Golay in 1957.3

In 1957 Scott4 demonstrated the effective use of Nylon columns for use as capillary columns. He found that they coated well with squalane and dinonyl phthalate and could be used in extremely long lengths (e.g. 1000 ft) to produce very high efficiencies. In 1959, Desty produced a technique that would produce long lengths of small diameter soft glass tubes in the form of a coil which was a far more practical material for the construction of capillary columns.5 During 1960 to 1979, methods of coating most stationary phase on glass capillary and the columns were satisfactory.

Although the apparent success in the practice of soft glass capillary, it still has some disadvantages. The tube was too rigid to fit to the injection and detector, and also the glass was not stable in very high and low temperatures and active towards highly polar analytes. In addition, glass column were still difficult to coat with and even film of stationary phase.3 The invention of the flexible quartz capillary tubes in 1979 by Dandeneau and Zerenner was a good solution to this problem.6 Because of the silica capillary is intrinsically straight, they can be easily connected to the sample injection system and detector, and also the column itself could be formed into a neat coil to fit into a relatively small oven.6 Initially, some minor problems were experience in coating these columns, but these were soon over come and today, most stationary phases can be coated as thin films on the walls of quartz capillaries.

So nowadays, this type of column has become the most widely used column in GC. A thin film of the liquid stationary phase is coated or bonded onto the inner wall of the tube which is made of high- quality fused silica.

Fig. The general structure of capillary column7

The details of the capillary column

Capillary column is commonly 5 to 50 m in length and between 0.1mm and 0.60 mm internal diameter. In the early period, a thin film of the liquid stationary phase from 0.1 to 5 μm thick was coated or bonded onto the inner wall of the tube which is made of high-purity fused quartz.1 The columns used nowadays are much the same, but have much more choice in the stationary phase. For example, the Zebron GC columns which are engineered by expert Phenomenex GC scientists can be connected to other analytical columns using a press-fit union. And this column Extend column lifetime by preventing stationary phase damage.8

Poor inertness as a result of increased column activity can lead to low acid/base sensitivity or analyte misidentification, causing incorrect data and big headaches! ZB-SemiVolatiles is designed with new Enviro-Inert technology to ensure: Inert, rugged performance without compromising separation, improved resolution of key critical pairs like Benzo[b] fluoranthene and Benzo[k]fluoranthene, Better peak shapes and response for acids, amines, and PAHs.8

Fig. Capillary column details – 18

Fig. Capillary column details – 28

Classification and characteristics of the capillary column

Fused silica capillary

This kind of capillary column is drawn a hollow tube with a molten silica, also known as fused silica capillary. The column in general is with a diameter of about 0.1- 0.5 mm, column length of 30-50m, and always form itself into a 20 cm diameter annular. The GC made of this kind of capillary column is called capillary gas chromatography or open-column chromatography, the separation efficiency is much higher than the column packed.9

Packed capillary column

Packed capillary columns are formed from the capillary which filled with stationary phase. That also can be drawn into capillary from a thick walled glass tube which is filled with support or adsorbent. If support is filled in, liquid stationary phase will coated on to the support to become the gas-liquid chromatography. If adsorbent is filled in, it will become the gas-solid chromatography. But this kind of capillary column has not much being used in recent years.9

Open tube capillary column

Open tube capillary column can also be divided into two types.

Classification from the production methods, can be divided into coated capillary column and carrier coated capillary column. The coated capillary column is a 0.1- 0.3 mm inner diameter hollow silica capillary whose inner wall was coated with stationary phase. This kind of capillary is currently the most used capillary column. The carrier coated capillary column, fist a layer diatomite carrier attached to the inner wall, then coated the stationary phase on to the carriers.

Classification by size, the capillary column of inner diameter less than 0.1 mm, mainly used for the rapid analysis, and the inner diameter of the capillary between 0.3-0.5 mm is often coated 5-8 μm thick film on its inner wall.9

Description of Scientific Principle

In 1940s, Martin and Synge introduced the concept of the plate theory of chromatography2, in which the efficiency of a chromatographic separation is given by the number of theoretical plates,N:

Where Wb and W1/2 are the peak widths at the base of the peak and at the half height.1 & 2 The plate height ( or height equivalent to a theoretical plate), H, for a chromatographic column is given by :

Where L is the length of the column.1 & 2

Plate theory is successful in interpreting the component separation and evaluation of column efficiency. However, certain assumptions inconsistent with the actual chromatographic process, such as plate theory assumes that the components in the tray instant reach a balanced distribution and iongitudinal diffusion can be ignored. In fact, the mobile phase carrying the components through the column, since the high velocity, it is impossible for the components to achieve a real balance between the mobile phase and stationary phase. The iongitudinal diffusion of the components cannot be ignored as well. Plate theory also does not take into account the impact of various kinetic factors on the mass transfer process in the column. Therefore, plate theory failed to clarify the meaning and essence of the plate height chromatography, unable to explain the relationship of column efficiency and the mobile phase flow rate, and cannot explain what are the main factors affecting the column efficiency.2

The Dutch scientists Van Deemter chromatography process dynamics theory, rate theory. He considered the diffusion and mass transfer processes of the components in the two phases, and using the research methods of non-equilibrium processes exported the rate theory equation which is called Van Deemter equation.2

Fig. The Van Deemter equation10

A is the term of multiple path, arising from eddy diffusion, which depends on different portions of the mobile phase and describes the effect of column packing on the flow paths of solute molecules.2 For packed columns, the use of small spherical particles gives the lowest values of A and hence higher efficiency, and A=0 for open tubular capillary columns because the multipath effect does not exist in the capillary open columns.11

B/ is the term of molecular diffusion, arising from longitudinal diffusion, which is determined by the diffusion coefficient of the analyte in the mobile phase (Dm) and hindrance factor γ, B = 2γDm. Packed columns inhibit longitudinal diffusion (γ ≈0.6) compared to open tubular capillary columns (γ = 1), and the B term will decrease when the flow rate of the mobile phase increases.1 &11

C is the mass transfer term, . The Cst describes the mass transfer in the stationary phase, which is determined by the analyte diffusion coefficient in the stationary phase (Dst) and the effective film thickness (df) : Cst ≈ df2/Dst.1 & 11 Higher efficiencies are obtained with a thin film of stationary phase. As the film of the capillary columns are much more thin, so the efficiencies are much higher than that of packed columns. Cmo describes the mass transfer in the mobile phase, which is determined by the diameter of the stationary phase particles or the diffusion distance (dp) and the analyte diffusion coefficient in the mobile phase (Dm): Cmo ≈ dp2/Dst.1 & 11 Higher efficiencies are obtained with small diffusion distances, such as narrow columns or small particle size. Dm of gas >> Dm of liquid, so gas chromatography uses open tubular capillary columns, whilst HPLC used packed columns.

And is the linear velocity of mobile phase, which has no effect on the eddy diffusion, but impacts on longitudinal diffusion and mass transfer. Longitudinal diffusion in the lower linear velocity, with the increase of velocity, decreases rapidly, but when continues to increase on the linear velocity, this trend becomes stable. The mass transfer in the mobile phase will increase when the linear velocity increases, but when the velocity becomes very high, it will be a constant value. The mass transfer in the stationsry phase increases with the increase of the linear velocity.2

The equation for HETP is often used to describe the efficiency of the column. An efficient column would have a minimum HETP value. As we can see, compared with the packed columns, the HETP value of the capillary is relatively small. So the invention of capillary columns improved the effectiveness of the gas chromatography.

For example, in order to increase the efficiency chromatographic separations, in LC, using a longer column, and also ultra performance liquid chromatography (UPLC) can be used to take place HPLC.12

Fig. The schematic diagram of the technique10

The column used is 75 μm id fused silica column, packed with 3 μm ODS particles and operated at a mobile phase flow rate of 0.2-0.4 μl min-1. And the result of this as follow.

Fig. Capillary HPLC-MS of MHC class I A2 peptide10

In another example, the methods is the same as the previous one, and this time it was done both in HPLC and UPLC.13 And the result is as follow.

Figure 4

Fig. HPLC vs UPLC Peptide Analysis13

The development of capillary in chromatography

The silica capillary column does not only revolutionized GC, but also update the separation techniques because the capillary zone electrophoresis (CZE), capillary LC and capillary electro chromatography (CEC) would not be appeared without the invention of the capillary columns.

Capillary zone electrophoresis is a new technique which was introduced in 1960s, based on the traditional electrophoresis, that can separate ionic species by their size to charge ratio in the interior of a small capillary filled with an electrolyte, frictional forces and hydrodynamic radius.11 Two advantages is identified if gel electrophoresis is transferred to a capillary column: thermal influences from ohmic heating are minimised, and it becomes possible to separate anions and cations in the same separation. An additional consideration is that the capillary column may be used for multiple separation.14

And also in HPLC, using smaller particles in packed columns has achieved high column efficiency and reduction analysis time, but in order to solve the problem of high-pressure drop associated with the use of small particles, many studies have been used, such as ultrahigh-pressure liquid chromatography (UHPLC) and capillary electro chromatography (CEC), and included the silica capillary column used in HPLC.15

Applications and advances of capillary columns

In the last few years the growth rate of capillary column technology and the appilation of capillary columns in specialities other than gas chromatography has increased sharply. Capillary gas chromatography is widely used as an analytical technique in a great many fields as diverse as studies on foods16, flavours17 & 18, and fragrances18, monitoring the routes and rates of chemical reactions, quantification of petroleum constituents, the detection and quantification of contaminants in air, water and soil, detection and measurement of pesticide residues, clinical pathology and metabolic profiling, and forensic analyses. The list extends to almost every analytical area.

The practice has proved that the development of fused silica capillary columns has been provided to be a very useful technique in environmental analysis laboratories.

The US Environmental Protection Agency (EPA) has used capillary GC to detect pesticides19 . In this experiment, a 30 m × 0.53 mm id DB-608 with a 0.83 μm film thickness column was used as primary column for organochlorine pesticides, and either a 30 m ×0.53 mm DB-17 or DB-5 with 1. 0 μm film thickness column was used as a second column. Samples were analyzed on these two columns to ensure separation of all consistuents extensive. The 0.53 mm id columns were preferred over 0.25 or 0.32 mm columns for the analysis of pesticides in waste because the 0.53 mm id columns were more robust and have greater sample capacity. And as a result, there were three pairs of closely eluting organochlorine peaks on the DB-608:DDE and Dieldrin, α-chlordane and endosulfanⅠ, and DDD and endosulfan Ⅱ. And the DDe/endosulfan and α-chlordane/endosulfanⅠ pairs were completely separated on the DB-1701, DDD/endosulfanⅡ pair was resolved. Although some laboratories have reported some difficulties, most of them have used these capillary methods with no problems.19

As the quality of air indoor environments are sometimes not very good, and one of the sources is tobacco smoke. So some laboratories has used the issue of tobacco smoke as one of the indicators of indoor air quality. For this study in the laboratories, there was a difficulty that the analysis object was complex mixture, so separation became so important. Therefore, the relying on the open tubular column or capillary column was the emphasis of this experiment. In practice, for nicotine and 3-vinylpyridine determination, the column used in GC was a 30 m × 0.53 mm id coated with a 1.5 μm film of DB-5 and a 30 m × 0.32 mm id fused silica capillary column coated with a 1.0 μm film of DB-5. They used the 30 m × 0.32 mm id fused silica capillary column coated with a 0.25 μm film of DB-23 from J&W Scientific on isomer separation. And routine solanesol determination on GC, the column used was a 15 m × 0.32 mm id fused silica capillary coated with a 0.1μm film of DB-1 connected to a 2 m × 0.53 mm id pre-column coated with a 0.5 μm film of DB-1 from J&W Scientific. The advantages of these columns were the speed, efficiency ,inertness and sensitivity imparted to the analytical determination. And in the results, it showed that the capillary chromatography had played a major role in the research to assess the impact of this complex, dilute mixture on indoor air quality.20

The capillary chromatography is also used in pharmaceutical analysis. The J&W Scientific has used the capillary GC to separate the amphetamines and related compounds. In the experiment, the fused silica columns of 30 m × 0.25 mm id coated with a 0.25 μm film thickness were used. The stationary phases coated on the columns were DB-5, DB-1301, and DB-17. For the results, it was proved that the DB-1301 column was not fit this study, and both DB-5 and DB-17 columns were satisfactory, especially DB-5 column were best overall separation for all the amphetamine compounds.21

The capillary columns are not only used in gas chromatography only, and also used in hyphenated chromatography, such as gas chromatography – mass spectrometry (GC-MS), gas chromatography – infrared absorption spectroscopy (GC-IR) , liquid chromatography – mass spectrometry (LC-MS) and etc.

The combination of GC and MS has developed into one of the most fruitful techniques in analytical chemistry. Todd F.Wheeler group22 has used high-resolution gas chromatographic analyses of p-nonylphenol and have reserved 22 para-isomers. In order to make the characterize this important group of isomers more accurately, coupling this GC analysis to a mass spectrometer would be better than a discrete MS. In this case, the group used a 100 m × 0.25 mm id coated with 0.5 μm film column as the GC column. Application of combined high resolution capillary GC-MS in this study has achieved a superior isomeric separation of p-nonylphenol in GC and has yielded a well-resolved set of mass spectra.23

Conclusions

Since the invention and use of chromatography in the field of analytical chemistry, so many new technologies have greatly expanded the range of the use of chromatography. And the invention and development of the fused silica capillary column even the development of the capillary is only a small part of that. Silica capillary columns have been attracting considerable attention as a high-performance separation medium because of higher permeability at a similar column efficiency and higher mechanical stability than particle-packed columns. And the silica capillary has been used widely in GC, LC, CE, and some hyphenated chromatography. But this technology still needs more improvement to be more widely used, higher efficiency, more simple methods and lower cost in the future.