**4. Applications of group type analysis by CLC**

#### **4.1 The application in making high grade road asphalt**

Coal is used as the main source of energy in China. The crude oil produced in China is paraffinic; therefore, it is not suitable for road asphalt. China is trying to produce high grade road asphalt from the mixture of coal and petroleum [11, 12].

Column Liquid Chromatography 7

4000 3500 3000 2500 2000 1500 1000 500

4000 3500 3000 2500 2000 1500 1000 500

The results from Figure 5 show that the resin fractions concentrate some oxygen-containing compounds. This conclusion can be approved by the appearing peak around 1215.15 cm-1, which is characteristics absorption peak for phenol compounds, and peak around 3649.31

Wavenumber(cm-1)

1689.29

1602.84

1456.25

1463.97

1377.17 719.45

1377.17

1118.71 1033

877.61 812.03

748.38

Wavenumber(cm-1)

2852.71

Fig. 3. Infrared spectrum of the saturated hydrocarbon fraction of sample NE-9.

2920.22

0.0

0.0

0.1

3331.41

Fig. 4. Infrared spectrum of the aromatic fraction of sample NE-9.

3049.45

0.2

Absorbance

0.3

0.4

0.5

0.5

1.0

Absorbance

2972.23

2959.79

2918.29

2850.78

1.5

2.0

2.5

Three asphalt samples from petroleum and coal processing for high grade paving asphalt were characterized by established method. Sample NE-6, NE-9, NE-11 were the heavy products by co-processing of Shijiazhuang oil (a petroleum factory in China) and Yanzhou coal (a typical coal in China). The coal and oil ratio was 1:1. Among asphalt samples, the preparation of NE-6 sample was under the role of Fe catalyst during co-processing. NE-9 sample was related to Mo catalyst. The sample TLA is from Trindid Lake Asphalt. The results of group type analysis for four asphalt samples were shown in Table 2.



From Table 2 it can be seen that the application of established method to real asphalt samples show good results. Different samples have different group composition characterize. The experiment deviations of contents(W%) are in the ranges from 0.255% to 1.800%.

FTIR experiments were performed to check the qualitative ability of established method. IR spectra of saturated fraction, aromatic fraction and resin fraction for sample NE-9 were shown in Figure from 3 to 5. It is important to note intense absorption peaks for saturated fraction (Fig.3). Based the standard IR handbook, the absorption peaks around 719.45cm-1, 1377.17 cm-1, 2850.78 cm-1, 2918.29 cm-1 and 2959.79 cm-1 was attributed to characteristics peak for δ(CH2)N N>6,δ(CH3),υsCH3,υas (CH2) and *υ*as CH3 respectively. These data show that the prepared saturated fraction has a high purity.

As Figure 4 show, the absorption peaks around 748.38 cm-1, 812.03 cm-1, 877.61 cm-1 and 3049.45 cm-1 belong to character peak of aromatic C-H absorption. The peaks at 1602.84 cm-1,1580 cm-1 and 1410 cm-1 were characteristics absorption peak of aromatic carbon. Obviously, the obtained aromatic hydrocarbon fraction has a good purity.

Three asphalt samples from petroleum and coal processing for high grade paving asphalt were characterized by established method. Sample NE-6, NE-9, NE-11 were the heavy products by co-processing of Shijiazhuang oil (a petroleum factory in China) and Yanzhou coal (a typical coal in China). The coal and oil ratio was 1:1. Among asphalt samples, the preparation of NE-6 sample was under the role of Fe catalyst during co-processing. NE-9 sample was related to Mo catalyst. The sample TLA is from Trindid Lake Asphalt. The

Name Test Saturates Aromatics Resins Asphaltenes NE-6 (1) 5.496 60.154 17.327 17.023 (2) 4.986 58.230 20.128 15.855 Average 5.241 59.191 18.727 16.439 Deviation 0.255 0.961 1.401 0.584 NE-9 (1) 9.950 21.379 52.906 15.765 (2) 8.001 22.087 52.348 17.554 Average 8.975 21.733 52.627 16.659 Deviation 0.974 0.354 0.279 0.895 NE-11 (1) 7.375 66.379 23.659 2.586 (2) 8.497 67.984 20.850 2.668 Average 7.936 67.182 22.254 2.627 Deviation 0.561 0.802 1.404 0.041 TLA (1) 5.496 60.154 17.327 17.023 (2) 4.986 58.230 20.928 15.855 Average 5.241 59.191 19.128 16.439 Deviation 0.255 0.961 1.800 0.584

From Table 2 it can be seen that the application of established method to real asphalt samples show good results. Different samples have different group composition characterize. The experiment deviations of contents(W%) are in the ranges from 0.255% to

FTIR experiments were performed to check the qualitative ability of established method. IR spectra of saturated fraction, aromatic fraction and resin fraction for sample NE-9 were shown in Figure from 3 to 5. It is important to note intense absorption peaks for saturated fraction (Fig.3). Based the standard IR handbook, the absorption peaks around 719.45cm-1, 1377.17 cm-1, 2850.78 cm-1, 2918.29 cm-1 and 2959.79 cm-1 was attributed to characteristics peak for δ(CH2)N N>6,δ(CH3),υsCH3,υas (CH2) and *υ*as CH3 respectively. These data

As Figure 4 show, the absorption peaks around 748.38 cm-1, 812.03 cm-1, 877.61 cm-1 and 3049.45 cm-1 belong to character peak of aromatic C-H absorption. The peaks at 1602.84 cm-1,1580 cm-1 and 1410 cm-1 were characteristics absorption peak of aromatic carbon.

results of group type analysis for four asphalt samples were shown in Table 2.

Table 2. Results of groups composition of asphalts (W%).

show that the prepared saturated fraction has a high purity.

Obviously, the obtained aromatic hydrocarbon fraction has a good purity.

1.800%.

Fig. 3. Infrared spectrum of the saturated hydrocarbon fraction of sample NE-9.

Fig. 4. Infrared spectrum of the aromatic fraction of sample NE-9.

The results from Figure 5 show that the resin fractions concentrate some oxygen-containing compounds. This conclusion can be approved by the appearing peak around 1215.15 cm-1, which is characteristics absorption peak for phenol compounds, and peak around 3649.31

Column Liquid Chromatography 9

0 2 4 6 810

Tr / Min


Coal asphalt 1032.200 1.658 2.802 21.199 29.730 22.745 21.863

asphalt 1905.674 0.804 17.169 59.899 12.274 3.979 5.873

Vacuum residue oil 1886.698 3.683 1.490 61.166 20.566 6.399 6.693

How much is the "representative" characteristics of this SEC method? This is an important problem to need know to treating these spectra and data of SEC. The so-called "representative" refers that extent which could be determined out of sample. Because most present SEC method is only suitable to compounds having UV adsorbent and soluble of THF, so, it is needed to know representative of whole sample. This problem will be completed only by CLC. Because the four groups: saturates, aromatics, resins and

oil 764.788 0.191 7.481 15.201 9.648 9.666 57.810

M>5000 M5000

W%

M1000 -500

M500


M3000 -1000

Coal asphalt

Samples Mw

Table 3. The MWDs of typical heavy oils

KP Petroleum

"Ethylene" residue

KP Petroleum asphalt

Fig. 6. The SEC chromatograms of typical heavy oils.

Ethylene residue

Vacuum residue

cm-1, which is characteristics absorption peak for dissociate OH. The peaks at 1033.84 cm-1 and 1608.63 cm-1 attribute to the absorption from OH and C-O-C group. This is comprehensible because OH group in the structure the phenol connects to the aryl group, which may induce some aromatic absorption peaks.

The FTIR results show high resolution of CLC method established. It is difficult to separate complex and heavy sample, however the IR analysis of the prepared fractions from the CLC chow all good results This observation indicate that chromatographic parameter guarantee a good qualitative results.

Fig. 5. Infrared spectrum of the resin fraction of sample NE-9.

#### **4.2 The determination of MWDs by CLC coupled with SEC**

Among characteristics of heavy oil, the size exchange chromatography (SEC) can be used to determine molecular weight distributions (MWDs), weight average molecular weight (Mw) and number average molecular weight (Mn), etc. With heavy oil of a group as example, the conditions of SEC are summarized as follows.

The analysis conditions are: a Shimadzu LC-10A high performance liquid chromatograph with an SPD-10AUP UV detector, the chromatographic column of SHIMPACK -801 (30 cm length, 0.8 cm i.d., polystyrene 6 µm), mobile phase of THF; flow rat with 1.2 ml/min; column temperature at 25oC.

The SEC chromatograms are shown in Figure 6, MWDs results are listed in Table 3.

In Figure 6, the sources of coal asphalt , KP petroleum asphalt, ethylene residue oil and vacuum residue oil are from Shanxi coking plant in China, Korea refining, Xinjiang oil refinery in China and Saudi Arabia's oil refining, respectively.

cm-1, which is characteristics absorption peak for dissociate OH. The peaks at 1033.84 cm-1 and 1608.63 cm-1 attribute to the absorption from OH and C-O-C group. This is comprehensible because OH group in the structure the phenol connects to the aryl group,

The FTIR results show high resolution of CLC method established. It is difficult to separate complex and heavy sample, however the IR analysis of the prepared fractions from the CLC chow all good results This observation indicate that chromatographic parameter guarantee a

2924.08

2824.30

4000 3500 3000 2500 2000 1500 1000 500

1215.15

880.72

1033.84

806.36

774.98

1608.63 1456.25

Wavenumber(cm-1)

Among characteristics of heavy oil, the size exchange chromatography (SEC) can be used to determine molecular weight distributions (MWDs), weight average molecular weight (Mw) and number average molecular weight (Mn), etc. With heavy oil of a group as example, the

The analysis conditions are: a Shimadzu LC-10A high performance liquid chromatograph with an SPD-10AUP UV detector, the chromatographic column of SHIMPACK -801 (30 cm length, 0.8 cm i.d., polystyrene 6 µm), mobile phase of THF; flow rat with 1.2 ml/min;

In Figure 6, the sources of coal asphalt , KP petroleum asphalt, ethylene residue oil and vacuum residue oil are from Shanxi coking plant in China, Korea refining, Xinjiang oil

The SEC chromatograms are shown in Figure 6, MWDs results are listed in Table 3.

which may induce some aromatic absorption peaks.

2953.57

3049.45

3649.31

Fig. 5. Infrared spectrum of the resin fraction of sample NE-9.

**4.2 The determination of MWDs by CLC coupled with SEC** 

refinery in China and Saudi Arabia's oil refining, respectively.

conditions of SEC are summarized as follows.

column temperature at 25oC.

good qualitative results.

0.00

0.05

0.10

Absorbance

0.15

0.20

Fig. 6. The SEC chromatograms of typical heavy oils.


Table 3. The MWDs of typical heavy oils

How much is the "representative" characteristics of this SEC method? This is an important problem to need know to treating these spectra and data of SEC. The so-called "representative" refers that extent which could be determined out of sample. Because most present SEC method is only suitable to compounds having UV adsorbent and soluble of THF, so, it is needed to know representative of whole sample. This problem will be completed only by CLC. Because the four groups: saturates, aromatics, resins and

Column Liquid Chromatography 11

where Wx % is the weight content percent of x composition in heavy oil sample, Rx % and Cex % are the concentration of preparation solution of resin fraction and external standard solution, respectively, Sx and Sex are the peak areas of component x and external standard, respectively, Vex and Vx are the injection volumes of external standard solution and resin solution, respectively, Res% is the weight percent of resin fraction in heavy oil sample. The

0 2 0 4 0 60 8 0

Tr/Min.

Number

quinone 240 18 p-phenyl phenol 500

**29**

**28 27**

**26**

**22**

results

Note ppm 35 2,2',

9 3-Methyl indole 610 15 N-phenyl pyrrole 800 10 Quinoline 240 16 7,8-Benzoquinoline 1000

12 Carbazole 500 20 N-phenyl indole 600 13 4-Methylquinoline 640 24 Dibenzofuran 630 14 2-Amino-phenol 570 33 N-Ethyl carbozole 370

A modified method for group type analysis of asphalt using CLC was established. The small-type CLC technique shows many advantages, such as high resolution rate, rapid operation, and requires minimal quantities of sample and solvent. The both of IR and 1H

**24**

**23**

**20**

**19 18**

**17**

**16**

**21**

**40**

results (ppm)

**39**

**38 37**

of peak Component Quantitative


**35 36 34**

**33**

**31**

**30**

**32**

qualitative and quantitative results are in Table 5.

**15**

**14**

**11 12 13**

**9**

**6**

**8 <sup>7</sup> <sup>10</sup>**

**5 4**

Fig. 7. HPLC chromatogram of resin fraction.

11 Phenanthrene-

of peak Component Quantitative

Table 5. Results of components of resin (ppm).

NMR results check the high resolution of this method.

0

Number

**5. Conclusion** 

5 0

100

150

200

asphaltenes quantitatively could be obtained by CLC determination, then the "representative "(R index) will be calculated as the following.

$$\text{IR} = \text{100 }\%\text{- }\mathsf{W}\_{\text{asp}}\text{ }\%\text{ - }\mathsf{W}\_{\text{a1k}}\text{ }\%\text{ }\tag{1}$$

Which R represents the representation index; Wasph % and Wa1k % represent the weight percent of asphaltene in sample and the weight percent of saturated hydrocarbons in sample, respectively.

With samples of Figure 6 as example, their R indexes from this CLC analysis are listed in Table 4.


Table 4. The R indicators.

These results show that the CLC coupled with SEC is an effective mean to analyze MWDs.

#### **4.3 Analysis of resin component by CLC coupled with HPLC**

As components of resin of heavy oil are very complicated, so to analyze them is very difficult by only one method. However, CLC coupled with high performance liquid chromatography (HPLC) can separate successfully, quality and quantity these compositions. Because the resin fraction got concentrate oxygen-containing compounds and other heteroatom-containing compounds by CLC separation, then the analysis of these hetero-atomcontaining compounds became easy to by HPLC. With slurry oil (Tianjing Refinery of China) as an example, the analysis of components in resin was summarized as follows.

The preparation of resin fraction was same as that of above description of CLC; the HPLC was performed on a Shimadzu LC-3A chromatogram with a SPD-1 UV detector, operated at 254 nm. Two ODS (4.6×20 cm) columns in series were operated at 40 oC with methanol /water=78:22(V/V) as the mobile phase, flowing at a rate of 0.8 ml/min. Typical separation chromatogram is shown in Figure 7.

From Figure 7 it can be seen the high resolution separation rate of complex compositions, these confirmed that the CLC preparation is successful and HPLC analysis is better.

The three qualitative methods of HPLC were selected to determine compositions of resin fraction. The three methods [13] are follows.


The quantitative determination of compositions was by the method of external standard (E-X) and the calculation formula uses the following.

$$\mathbf{W\_{x}}\,\%=\left(\,\mathrm{R}\_{\mathrm{ex}}\,/\mathrm{C}\_{\mathrm{x}}\right)^{\*}\left(\mathrm{S}\_{\mathrm{x}}\,/\,\mathrm{S}\_{\mathrm{ex}}\right)^{\*}\left(\mathrm{V\_{ex}}\,/\mathrm{V\_{x}}\right)^{\*}\mathrm{R}\_{\mathrm{ex}}\,\%\tag{2}$$

asphaltenes quantitatively could be obtained by CLC determination, then the

Which R represents the representation index; Wasph % and Wa1k % represent the weight percent of asphaltene in sample and the weight percent of saturated hydrocarbons in

With samples of Figure 6 as example, their R indexes from this CLC analysis are listed in

Name of samples (1) (2) (3) Average Max of deviation % Coal asphalt 73.64 72.89 73.86 73.46 -0.78 KP petroleum asphalt, 99.33 99.28 98.76 99.12 -0.36 Ethylene residue oil 90.36 89.87 90.56 90.26 -0.43 Vacuum residue oil 96.17 96.21 95.76 96.05 0.33

These results show that the CLC coupled with SEC is an effective mean to analyze MWDs.

As components of resin of heavy oil are very complicated, so to analyze them is very difficult by only one method. However, CLC coupled with high performance liquid chromatography (HPLC) can separate successfully, quality and quantity these compositions. Because the resin fraction got concentrate oxygen-containing compounds and other heteroatom-containing compounds by CLC separation, then the analysis of these hetero-atomcontaining compounds became easy to by HPLC. With slurry oil (Tianjing Refinery of China) as an example, the analysis of components in resin was summarized as follows.

The preparation of resin fraction was same as that of above description of CLC; the HPLC was performed on a Shimadzu LC-3A chromatogram with a SPD-1 UV detector, operated at 254 nm. Two ODS (4.6×20 cm) columns in series were operated at 40 oC with methanol /water=78:22(V/V) as the mobile phase, flowing at a rate of 0.8 ml/min. Typical separation

From Figure 7 it can be seen the high resolution separation rate of complex compositions,

The three qualitative methods of HPLC were selected to determine compositions of resin

The quantitative determination of compositions was by the method of external standard (E-

Wx % = ( Rex /Cx) \* (Sx / Sex) \* (Vex /Vx ) \*Res% (2)

these confirmed that the CLC preparation is successful and HPLC analysis is better.

R =100 %- Wasp % -Wa1k % (1)

"representative "(R index) will be calculated as the following.

**4.3 Analysis of resin component by CLC coupled with HPLC** 

sample, respectively.

Table 4. The R indicators.

chromatogram is shown in Figure 7.

fraction. The three methods [13] are follows.

X) and the calculation formula uses the following.

1. The qualitative method of relative retention time (RRT). 2. The qualitative method of stop- flow UV scanning. 3. The qualitative method of UV characteristic index V'.

Table 4.

where Wx % is the weight content percent of x composition in heavy oil sample, Rx % and Cex % are the concentration of preparation solution of resin fraction and external standard solution, respectively, Sx and Sex are the peak areas of component x and external standard, respectively, Vex and Vx are the injection volumes of external standard solution and resin solution, respectively, Res% is the weight percent of resin fraction in heavy oil sample. The qualitative and quantitative results are in Table 5.

Fig. 7. HPLC chromatogram of resin fraction.


Table 5. Results of components of resin (ppm).

#### **5. Conclusion**

A modified method for group type analysis of asphalt using CLC was established. The small-type CLC technique shows many advantages, such as high resolution rate, rapid operation, and requires minimal quantities of sample and solvent. The both of IR and 1H NMR results check the high resolution of this method.

**2** 

*Ankara University,* 

 *Turkey* 

**Column Chromatography** 

 **for Terpenoids and Flavonoids** 

Gülçin Saltan Çitoğlu and Özlem Bahadr Ackara

Natural products have coming from various source materials including terrestrial plants, terrestrial microorganisms, marine organisms, terrestrial vertebrates and invertebrates have importance as they provide an amazing source of new drugs as well as new drug leads and new chemical entities for further drug development (McCurdy & Scully, 2005; Chin et al., 2006). Morphine, vincristine, codeine, digitoxin, quinine, galantamine and taxol are just some of the typical examples of drugs that have been introduced from natural sources

Natural products can be mainly divided into three groups such as primary metabolites, secondary metabolites and high molecular weight polymeric materials (Hanson, 2003). Primary metabolites including nucleic acids, amino acids, sugars; occur in all cells and play a central role in the metabolism and reproduction of the cells. High molecular weight polymeric materials such as cellulose, lignins and proteins take a part in the cellular structure. Secondary metabolites, small molecules which are not essential for the growth and development of the producing organism have importance because of their biological activities on other organisms. Natural product term refers to any naturally occurring compounds but in most cases mean secondary metabolite (Hanson 2003; Sarker et al., 2005).

**1. Introduction** 





**2.1 Terpenoids** 


(Heinrich et al., 2004; Balunas & Kinghorn, 2005).

Secondary metabolites mainly consist of these following groups:

**2. Isolation of terpenoids and flavonoids by column chromatography** 

Terpenoids are the most widespread, chemically interesting groups of secondary metabolites with over 30,000 known compounds including steroids (Wang et al., 2005; Umlauf, 2004). Many terpenes have biological activities and are used for the treatment of

The CLC method compared with routine ASTM method, the reagents used in this method are small amount and lower toxicity. These are beneficial to environmental protection and human health. This is very important for modern analysis.

The CLC method of this paper is an important and indispensable analysis method to study heavy oils. It is not only a separation means, but is also analysis means.This method was successfully applied to many analysis aspects, such as making high grade road asphalt, characterizing MWDs and analysis heterocyclic of aromatic compositions of heavy oils.

The analysis of heavy oil is a long and difficult task. We systematically summarized these studies and hope that these will help our colleagues.

#### **6. References**

