Densitometric Comparison of the Performance of Stahl-Type and Sandwich-Type Planar Chromatographic Chambers

Densitometric Comparison of thePerformance of Stahl-Type and Sandwich-Type Planar Chromatographic Chambers

M. Sajewicz,1 A. Pieniak,1 R. Pietka,1 K. Kaczmarski,2

and T. Kowalska1,*

1Institute of Chemistry, Silesian University, Katowice, Poland2Faculty of Chemistry, Technical University of Rzeszow,

Rzeszow, Poland

ABSTRACT

Over the years, conventional Stahl-type thin-layer chromatography (TLC)

chambers (i.e., N-chambers) have attracted criticism for more than one

reason. For example, practitioners have questioned the value of the time-

consuming procedure of saturating these chambers with mobile-phase

vapor, and those who focus on the theory of planar chromatography,

have complained that the well-known stepwise diminishing of the rate of

capillary flow of the mobile phase in the ascending mode makes compu-

tational optimization of solute retention more difficult and less accurate

than it could be at constant flow. Everybody also readily agrees that mobile

phase consumption is much higher in N-chambers than in sandwich

2019

DOI: 10.1081/JLC-120039414 1082-6076 (Print); 1520-572X (Online)

Copyright # 2004 by Marcel Dekker, Inc. www.dekker.com

*Correspondence: T. Kowalska, Institute of Chemistry, Silesian University, 9 Szkolna

Street, 40-006 Katowice, Poland; E-mail: [email protected].

120039414_JLC_027_013_R2_052604 Techset Composition Ltd, Salisbury, U.K.—120039414

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JOURNAL OF LIQUID CHROMATOGRAPHY & RELATED TECHNOLOGIESw

Vol. 27, No. 13, pp. 2019–2030, 2004

chambers (S-chambers). The economic superiority of S-chambers cannot,

however, be regarded as a decisive factor when it comes to making a choice

of the TLC equipment, because physicochemical aspects of the chromato-

graphic process are more important. In the work discussed in this paper, we

compared the N-type and S-type chambers in terms of mobile phase flow

rate, the RF values obtained, and the densitometrically established concen-

tration profiles of the test analytes. For this comparison, we selected several

phenyl-substituted alcohols and acids, low-activity adsorbents (cellulose

powder and chromatographic paper), and low-polarity solvents (n-octane

and decalin) as the components of the chromatographic systems investi-

gated. The overall conclusion from the results obtained is that the physico-

chemical performance of the S-chamber is comparable with that of the

N-chamber, and the only indisputable superiority of the S-chamber is the

more economical use of the mobile phases.

Key Words: Planar chromatography; Densitometry; Concentration

profiles of bands; Microcrystalline cellulose; Chromatographic paper;

Stahl-type chamber (i.e., N-chamber); Sandwich-type chamber (i.e.,

S-chamber).

INTRODUCTION

Planar chromatography performed in conventional Stahl-type chambers

(N-chambers) is regarded as inferior to column chromatography, because

the stationary phase (either chromatographic paper or thin layer) is used in

the form of an “open bed.” There is awareness that the “closed bed” of station-

ary phase contained in chromatographic columns eliminates a number of

physicochemical shortcomings inherent in the planar mode.

For this reason, the development of planar chromatography has sometimes

consisted of imitating working conditions typical of column chromatography, in

the first instance by converting the open bed of stationary phase to a less open

bed. The simplest way of achieving this goal was to introduce Sandwich-type

chambers (S-chambers), in which the void volume surrounding the plate (or

the chromatographic paper) is much smaller than in the N-chambers.

Thorough comparison of the N-type and the S-type chambers in terms of

the quality of the respective chromatographic bands is still pending. One

reason is the difficult choice of reliable analytical tools and of adequate

physicochemical criteria for such a comparison. It seems, however, that

with the increasing popularity of densitometry, the lack of such a thorough

comparison can no longer be excused.

The aim of the work discussed in this paper, was to compare retention in

N- and S-type chromatographic chambers. As criteria for our comparison, we


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Sajewicz et al.2020

selected the mobile phase flow rate, RF values of the test analytes used, and the

densitometrically established concentration profiles. We selected analytes (i.e.,

the alcohols and acids) able to interact effectively by hydrogen bonding and

low-activity/low-polarity chromatographic systems, to minimize interference

of the stationary and mobile phases with the self-association of the test analytes.

The practical usefulness of similar chromatographic systems has been success-

fully demonstrated in earlier work.[1–3] The results obtained and the conclusions

drawn, are presented in the forthcoming sections of this paper.

EXPERIMENTAL

Test Solutes

Selected Alcohols

In this study, we used solutions of 4-phenyl-1-butanol (0.5, 1.0, 1.5, and

2.0 mol L21) and 5-phenyl-1-pentanol (0.25, 0.5, 0.75, 1.0, 1.5, and

2.0 mol L21) in carbon tetrachloride. Both alcohols were analytical-grade

products manufactured by Merck KGaA (Darmstadt, Germany).

Selected Acids

We used solutions of 3-phenylpropionic acid (0.2, 0.3, 0.4, and

0.5 mol L21), 2-phenylbutyric acid (0.2, 0.3, 0.4, and 0.5 mol L21), and

4-phenylbutyric acid (0.2, 0.3., 0.4, 0.5, 0.75, and 1.0 mol L21) in 2-propanol.

All these acids were analytical-grade products manufactured by Merck.

Thin-Layer Chromatography

Thin-layer chromatography (TLC) was performed on glass-backed plates

precoated with microcrystalline cellulose (10 cm � 20 cm; Merck, cat.

#1.05730). After spotting the solutions of the alcohols and acids (3mL), the

layers were developed with n-octane (alcohols) or decalin (acids) to a distance

of 15 cm, at ambient temperature, in the two types of chromatographic

chambers: (i) ascending mode in a conventional N-chamber (Camag, Muttenz,

Switzerland) previously saturated with mobile phase vapor for 20 min; and

(ii) horizontal mode in a DS-II S-chamber (Chromdes, Lublin, Poland) satu-

rated with mobile phase vapors in the same way. The volume of mobile

phase used in the N-chamber was 20 mL; in the S-chamber it was 6 mL.

After development, the chromatograms were carefully dried with a hairdryer.


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Densitometric Comparison of N-Type and S-Type Chambers 2021

Paper Chromatography

Paper chromatography was performed on Whatman-1 and Whatman-3

chromatographic paper (Whatman, Maidstone, UK) cut into 10 cm � 20 cm

rectangles. After spotting the solutions of the alcohols and acids (5mL), the

paper sheets were developed with n-octane (alcohols) or decalin (acids) to a

distance of 15 cm, at ambient temperature, in the two types of chromato-

graphic chamber: (i) ascending mode in a conventional N-chamber previously

saturated for 20 min; and (ii) horizontal mode in a DS-II S-chamber, saturated

in the same way. Again, the volumes of mobile phase used in the N-type and

S-type chambers were 20 mL and 6 mL, respectively. After development, the

chromatograms were carefully dried with a hairdryer.

Densitometric Detection

Densitometric detection of developed zones was performed with the

Desaga (Heidelberg, Germany) model CD 60 densitometer equipped with

the Windows-compatible ProQuant program package. Concentration profiles

were recorded in ultraviolet (UV) light (in the reflectance mode) at a wave-

length of 260 nm; the dimensions of the rectangular light beam were

0.4 mm width and 0.02 mm height.

RESULTS AND DISCUSSION

The results obtained are shown in Tables 1–7 and the selected densito-

grams are given in Figs. 1–4. Because of the importance of the comparison

discussed, we decided to present our data in numerical form (Tables 1–7),

thus enabling detailed inspection of the respective RF values and the concen-

tration profile baseline widths, w. The numerical values given in the tables are

mean values calculated from results of replicate analyses (the number of repli-

cates, n, was usually between 3 and 10). The reproducibility of the results (i.e.,

RF and w values) obtained by use of the N- and S-chambers, was of exactly the

same order (despite the moderate reproducibility of results from S-chambers

articulated elsewhere[4]). It was an ultimate goal of this study to compare

the simplest physicochemical characteristics of solute retention in planar chro-

matography (the two parameters RF and w, and the respective concentration

profiles) for the N-type and S-type chromatographic chambers. The results

obtained revealed several general trends, although with occasional exceptions.

In general, the higher the velocity of development of the chromatogram,

the lower the w value of the chromatographic band. This regularity is


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Sajewicz et al.2022

readily understandable if we reflect on the relationship between chromato-

gram development time and the impact of diffusion (the shorter the

development time, the less pronounced is diffusion and the lower the peak

width).

With all of the test solutes considered, the smaller the amount of sample

developed (i.e., the lower the concentration of the spotted solution), the closer

to each another become the pairs of the RF values, irrespective of the chroma-

tographic chamber used (occasionally the values are even identical). This is

because the smaller the amount of sample developed, the greater is the

probability of partitioning of the analyte between the stationary and mobile

phases in the linear range of the adsorption isotherm. Evidently, differences

between the construction and use of the two types of chambers differentiates

Table 1. Numerical values of the RF coefficients and concentration profile widths (w)

of chromatographic bands of 4-phenyl-1-butanol developed with n-octane on

whatman-3 chromatographic paper.

Concentration

(mol L21)

N-chamber

(mean development

velocity: 5.16 mm min21)

S-chamber

(mean development


RF w (mm) RF w (mm)

0.5 0.95 14.0 0.95 13.5

1.0 0.92 24.0 0.89 20.2

1.5 0.88 31.5 0.82 27.9

2.0 0.83 38.6 0.78 38.5


of chromatographic bands of 5-phenyl-1-pentanol developed with n-octane on


Concentration

(mol L21)

N-chamber

(mean development


S-chamber

(mean development


RF w (mm) RF w (mm)

0.25 0.97 14.2 0.95 15.4

0.50 0.89 20.8 0.92 20.4

0.75 0.82 28.4 0.89 24.3

1.00 0.79 35.6 0.82 31.1


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the process of retention more effectively in the non-linear range of the

isotherm, rather than the linear range.

The velocity of development of paper chromatograms in the S-chamber is

visibly higher than in the N-chamber. It can be rightly speculated that, because

of the relatively large pores in chromatographic paper, capillary forces in the

ascending (i.e., vertical) development mode are substantially opposed by

gravitational forces and, hence, the development process in a N-chamber

undergoes gradually increasing retardation. Development of chromatograms

in the horizontal mode cannot be hindered in this way and, therefore, the

development process in the S-chamber proceeds unretarded by gravity.

The opposite trend is observed when developing thin-layer chromato-

grams. The velocity of development of chromatograms in the N-chamber is


of chromatographic bands of 5-phenyl-1-pentanol developed with n-octane on


Concentration

(mol L21)

N-chamber

(mean development


S-chamber

(mean development


RF w (mm) RF w (mm)

0.25 — — 0.96 14.1

0.50 0.90 19.8 0.95 18.0

0.75 — — 0.92 25.3

1.00 0.83 30.8 0.89 30.2

1.50 0.77 41.9 0.82 37.0

2.00 0.75 45.4 0.80 39.3


of chromatographic bands of 5-phenyl-1-pentanol developed with n-octane on micro-

crystalline cellulose-coated glass plates.

Concentration

(mol L21)

N-chamber

(mean development


S-chamber

(mean development


RF w (mm) RF w (mm)

0.25 0.94 12.5 0.86 14.4

0.50 0.82 15.9 0.81 22.4

0.75 — — 0.75 32.7

1.00 0.76 20.4 0.71 36.2


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Sajewicz et al.2024


of chromatographic bands of 2-phenylbutyric acid developed with decalin on micro-


Concentration

(mol L21)

N-chamber

(mean development


S-chamber

(mean development


RF w (mm) RF w (mm)

0.2 0.91 7.9 0.93 10.4

0.3 0.86 12.8 0.90 13.5

0.4 — — 0.86 14.6

0.5 0.80 15.8 0.84 13.7


of chromatographic bands of 3-phenylpropionic acid developed with decalin on micro-


Concentration

(mol L21)

N-chamber

(mean development


S-chamber

(mean development


RF w (mm) RF w (mm)

0.2 0.93 8.3 0.94 11.4

0.3 0.91 9.4 0.90 17.8

0.4 0.89 9.7 0.86 20.8

0.5 0.79 17.7 0.78 26.0


of chromatographic bands of 4-phenylbutyric acid developed with decalin on micro-


Concentration

(mol L21)

N-chamber

(mean development


S-chamber

(mean development


RF w (mm) RF w (mm)

0.2 0.90 12.5 0.90 13.5

0.3 0.87 14.9 0.86 19.8

0.4 0.85 19.0 0.80 40.6

0.5 0.82 25.0 0.79 43.8

1.0 0.68 54.7 0.64 67.7


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somewhat (sometimes insignificantly) higher than in the S-chamber, which

obviously cannot be attributed to the impact of gravity. Because the diameters

of the pores in microcrystalline cellulose layers are usually much smaller than

in chromatographic papers, capillary forces are much more pronounced and

far less susceptible to gravity. Feeding of horizontally developing chromato-

grams with mobile phase is, however, substantially different from feeding

Figure 1. Comparison of the concentration profiles of 5-phenyl-1-pentanol developed

with n-octane on whatman-3 chromatographic paper in (a) N-chamber and (b) S-chamber

(showing the dependence on the concentrations of the test analyte solutions).


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Sajewicz et al.2026

them in the vertical position. In the N-chamber, the lower edge of the verti-

cally placed chromatogram (either thin-layer or paper) is dipped in the mobile

phase, whereas in the S-chamber, the edge of the horizontally placed chroma-

togram is fed with mobile phase from a trough through a slit, by capillary

action. It seems, that the evident hydrodynamic difference in the mode of

Figure 2. Comparison of the concentration profiles of 5-phenyl-1-pentanol devel-

oped with n-octane on microcrystalline cellulose in (a) N-chamber and (b) S-chamber

(showing the dependence on the concentrations of the test analyte solutions).


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feeding negatively affects development of thin-layer chromatographic plates

(by prolonging the development time), whereas it has no perceptible effect

on the development of paper chromatograms.

Selected densitograms obtained from chromatograms developed in

N-type and S-type chambers are compared in Figs. 1–4. For all the analytes

Figure 3. Comparison of the concentration profiles of 2-phenylbutyric acid developed

with decalin on microcrystalline cellulose-coated glass plates in (a) N-chamber and

(b) S-chamber (showing the dependence on the concentrations of the test analyte solutions).


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Sajewicz et al.2028

and the two chromatographic chambers investigated, the lengthwise

concentration profiles of the chromatographic bands resemble anti-Langmuir-

type peak profiles (the best examples of anti-Langmuir profiles can be seen in

Fig. 2). These results persuasively suggest that lateral interactions play the

predominant role in the formation of these concentration profiles.

Other important information contained in these results is that the deterio-

ration of the lengthwise concentration profiles of chromatograms developed in

Figure 4. Comparison of the concentration profiles of 4-phenylbutyric acid devel-

oped with decalin on microcrystalline cellulose-coated glass plates in (a) N-chamber

and (b) S- chamber (showing the dependence on the concentrations of the test analyte

solutions).


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the S-chamber (particularly for the largest amounts of the analytes) is worse

than for their counterparts obtained in the N-chamber. In general, the diffusive

parts of these peaks are less emphasized and their triangular anti-Langmuir

shape is distorted, resulting in a trapezoidal-like shape (see, for example,

Fig. 4). Occasionally (e.g., Fig. 3), the multi-peak profiles for the highest

concentrations of the analytes developed in the S-chamber give evidence of

a limited hydrodynamic stability of the eluent flow in this chamber. The reason

for these multi-peak profiles is most probably the hydrodynamic instability of

the eluent flow in the S-chamber.

From these results, the overall conclusions are that the physicochemical

performance of the S-chamber is comparable with (and occasionally worse than)

that of the N-chamber, and that the only indisputable superiority of the S-chamber

over the N-chamber consists in more economical use of the mobile phase.

ACKNOWLEDGMENT

The authors wish to thank Merck KGaA (Darmstadt, Germany) for

supplying the test compounds and precoated cellulose plates used in this study.

REFERENCES

1. Prus, W.; Kaczmarski, K.; Tyrpien, K.; Borys, M.; Kowalska, T. The role

of the lateral analyte–analyte interactions in the process of TLC band

formation. J. Liq. Chromatogr. Relat. Technol. 2001, 24, 1381–1396.

2. Kaczmarski, K.; Prus, W.; Dobosz, C.; Bojda, P.; Kowalska, T. The role

of the lateral analyte–analyte interactions in the process of TLC band

formation. II. Dicarboxylic acids as the test analytes. J. Liq. Chromatogr.

Relat. Technol. 2002, 25, 1469–1482.

3. Sajewicz, M.; Pieniak, A.; Pietka, R.; Kaczmarski, K.; Kowalska, T.

Densitometric acquisition of concentration profiles in planar chromato-

graphy and its possible shortcomings. Part 1. 4-Phenylbutyric acid as an

analyte. Acta Chromatogr. 2004, 14, 5–15.

4. Dzido, T.H. Modern TLC Chambers. In Planar Chromatography—

A Retrospective View for the Third Millennium; Nyiredy, Sz., Ed.; Springer:

Budapest, 2001; 68–87.

Received September 29, 2003

Accepted November 3, 2003

Manuscript 6346I


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CMS ID number (DOI): 120039414

Article title:Densitometric Comparison of the Performance of Stahl-Type and Sandwich-Type Planar Chromatographic Chambers

Article type: ResearchClassification: Category:Primary subcategory:Subcategpry(ies):Topic(s):

Key words:

Planar chromatography; Densitometry; Concentration profiles of bands; Microcrystalline cellulose; Chromatographic paper; Stahl-type chamber (i.e., N-chamber); Sandwich-type chamber (i.e., S-chamber)

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Author Sequence Number 5Author first name or first initial: T.Author middle initial:Author last name: KowalskaSuffix to last name:Degrees:Author StatusAuthor e-mail address: [email protected] fax:Author phone:

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Densitometric Comparison of the Performance of Stahl-Type and Sandwich-Type Planar Chromatographic Chambers