Chapter 12
Analysis of Natural Products: A Review of Chromatographic Techniques Downloaded by UNIV OF MISSOURI COLUMBIA on March 17, 2013 | http://pubs.acs.org Publication Date: October 1, 1997 | doi: 10.1021/bk-1997-0670.ch012
Christina Borch-Jensen and Jørgen Mollerup Department of Chemical Engineering, Building 229, Technical University of Denmark, 2800 Lyngby, Denmark The analysis of natural products like fats, seeds, oils, and tissues is compared for gas chromatography (normal and high temperature) and packed and capillary SFC. Examples are given by the determination of fatty acid composition and the analysis of triglycerides. The fatty acid determination is compared for the oils of cape marigold (Dimorphoteca pluvialis) a hydroxy fatty acid containing oil and a polyunsaturated fish oilfromthe sand eel (Ammodytes sp.). The triglyceride analyses of samples of butter fat and raw fish oil from the sand eel are compared for SFC and GC. In fatty acid determination and triglyceride analysis it is shown to be of great importance to consider the nature of the sample before selecting chromatographic technique. The presence of functional groups like hydroxy or epoxy groups in the fatty acids can affect the choice of technique and so can the chain length and degree of unsaturation of the fatty acid moieties. Another important aspect to consider is the purpose of the analysis. Process control andfingerprintanalyses often do not require a total separation of all components in the sample, which means that the analysis time can be shortened considerably compared to an analysis where total quantification of all components is the goal. In the analysis of fats, oils, seeds and tissues, there are many different ways to proceed. In the selection of chromatographic technique there are several possibilities, including gas (GC), liquid (LC), supercritical fluid chromatography (SFC), capillary electrophoresis (CE) and thin-layer chromatography (TLC). The time allowed for method development is often short and therefore the standard methods from IUPAC or AOCS, which provides methods for different types of analyses, are very popular. 154
© 1997 American Chemical Society
In Supercritical Fluids; Abraham, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.
Downloaded by UNIV OF MISSOURI COLUMBIA on March 17, 2013 | http://pubs.acs.org Publication Date: October 1, 1997 | doi: 10.1021/bk-1997-0670.ch012
12.
BORCH-JENSEN & M O L L E R U P
Analysis of Natural Products
155
When selecting a technique for one specific analytic problem there are some important aspects to consider. The selection of technique is often strongly dependent of the equipment available in the laboratory. If a standard method prescribes analysis by one specific technique, the question is if one should buy new equipment to fulfill the requirements of the standard method or if it would be sufficient to develop a new method running on the old equipment. Most standard methods in the fats and oils applications uses LC and GC. Another very important aspect to consider is how many samples are to be run by the method. If the method is used for routine analyses, the shortest, cheapest and most easy sample preparation and analysis is required, while if it is just a matter of few samples for instance for research work, this is of less importance. It would also be important to start by defining the goal of the analysis at the beginning of the process. If the sample is for instance palm oil, there could be several results of the analysis, like quantification of tocopherols or identification of triglycerides. In many cases the different results would require different analysis techniques and one method would not necessarily give both results. In this paper we will compare GC and packed and capillary SFC for different analytical problems related to natural samples.
Seeds, oils, fats, tissues etc.
Figure 1
The numerous path ways from raw sample to chromatographic analysis for natural products.
Figure 1 illustrates different pathways possible in the analysis and sample preparation when dealing with natural samples. If the start material is seed or tissue the first step is normally an extraction, which can be done supercritical by SFE or by the conventional organic solvent extraction. After extraction one can choose tofractionate,derivatize or to analyze directly. Fractionation gives more choices, TLC, solid-phase extraction (SPE) or liquid extraction. Derivatives can be made as methyl or ethyl esters of fatty acids, as free fatty acids and there are the possibilities to mask functional groups like hydroxy
In Supercritical Fluids; Abraham, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.
156
SUPERCRITICAL FLUIDS
Downloaded by UNIV OF MISSOURI COLUMBIA on March 17, 2013 | http://pubs.acs.org Publication Date: October 1, 1997 | doi: 10.1021/bk-1997-0670.ch012
groups with for instance silylating agents. The final choice is concerning analysis technique. Once the analyte has been prepared there is one more choice: GC, packed or capillary SFC? The following examples are determination of fatty acid composition and analysis of triglycerides. With these examples, we wish to show how molecular weight, functional groups, degree of saturation can affect the choice of analytical technique. Determination of fatty acid composition. The determination of fatty acid composition normally requires some kind of derivatization. The most used type of derivatives are fatty acid methyl or ethyl esters (FAME/FAEE), less used arefreefatty acid (FFA) derivatives. Table I illustrates which technique can be used under which circumstances. The fatty acids have been divided in three different types, "normal", hydroxy and epoxy. the first type include most animal, fish and vegetable oils and the most common way to proceed when dealing with these samples are GC of FAMEs or FAEEs, although both packed (7,2) and capillary (3,4) SFC have been applied. There are certain seed oils, which have rather high contents of fatty acids with functional groups like hydroxy or epoxy. The reason why these too groups of functional groups can not be treated as one is due to the fact that these groups have different properties. The epoxy group is very reactive, but not thermolabile. For this reason the analysis by GC is not a problem, but the derivatization reaction is not straight forward, because the epoxy groups are affected by the acidic reagents normally Table I. Determination of fatty acid composition of different types of fatty acids and different types of derivatives Nature of fatty acid Type of derivative
"Normal"
Epoxy
Hydroxy
FAME/FAEE
GC
(GC)
GC(TMS)
FFA
GC
GC/SFC
SFC/GC(TMS)
used in FAME/FAEE preparation. Basic reaction is possible, but this method will not derivatize the free fatty acids present in the seed oil in the first place. This is why the most safe way to make derivatives of epoxy fatty acid containing triglycerides are preparation of FFA derivatives (5) which can readily be analyzed by capillary SFC or GC on a polar column. The hydroxy group can be thermolabile, but this is strongly dependent on the mutual positions of the hydroxy group and the double bonds in the fatty acid chain. Fortunately it is easy to mask the hydroxy groups by trimethyl silyl groups (TMS derivatives) and analyze the samples by GC. This method will give both the fatty acid composition of the normal fatty acids and the content of the hydroxy fatty acids. However, the TMS reagent is rather expensive and the sample preparation time is long. An alternative method is SFC of FFA derivatives like with the epoxy fatty acid containing oils. This does not require TMS reagent and the sample preparation is quite fast.
In Supercritical Fluids; Abraham, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.
Downloaded by UNIV OF MISSOURI COLUMBIA on March 17, 2013 | http://pubs.acs.org Publication Date: October 1, 1997 | doi: 10.1021/bk-1997-0670.ch012
12.
BORCH-JENSEN & M O L L E R U P
Analysis of Natural Products
157
The analysis of fish oil FAMEs by GC is a good example of a standard method which is well working and not easily replaced by other techniques (4). Figure 2 shows the GCMS chromatogram from such an analysis. Almost 70 peaks are resolved in less than 40 minutes. The method for the derivatization and analysis is a standard AOCS method (6). Applying capillary SFC for the same type of sample will give the chromatogram shown in Figure 3, a polar SFC column separation of fish oil FAMEs. The retention time is three times as long as in the GC analysis and the number of components separated are decreased to about 35. Applying a packed, polar SFC column, the chromatogram will be as in Figure 4. The analysis time is much shorter, but the separation is insufficient, which is mainly due to the short 25 cm column. In the case of hydroxy fatty acids, the analysis by both SFC and GC have disadvantages and advantages. The method using SFC of FFA has the advantage of an easy sample preparation, while the chromatographic analysis takes 40 minutes and still the separation of the normal fatty acids are not satisfactory (7). The SFC chromatogram is shown in Figure 5. By GC of TMS-FAMEs, Figure 6, the chromatographic separation of both normal and hydroxy fatty acids is very good with a total analysis time of only 22 minutes. Unfortunately, the method is expensive. In cases like this, it is important to decide what one wants to obtain, because the SFC method is a cheap and easy way to determine the total content of hydroxy fatty acid, while the GC method is more expensive, but on the other hand gives the distribution of both normal and hydroxy FAMEs in a short analysis time. Analysis of triglycerides. SFC has often been stated as an excellent technique for the analysis of the high molecular weight triglycerides and a variety of different triglycerides have been analyzed (8-13) by this technique. As can be seen from Table II, the suitability of SFC depends on both the chain length of the fatty acids in the molecules and on whether they are saturated or unsaturated. In the case of short to medium chain triglycerides with few doublebonds high temperature GC (HT-GC) often offers a separation that is better than in SFC and within a much shorter analysis time. In HT-GC the maximum allowed column temperature is in the range of 380 to 400°C. The high temperatures are required to elute triglycerides with up to 54 carbon atoms in the fatty acid chains. If however, the triglyceride contains any polyunsaturated fatty acids, these will not be eluted safe from the column and if the total number of carbons in the triglyceride chains is much higher than 54, the triglyceride will not be eluted at all. In such cases SFC is the best choice. Table II. Analysis of triglycerides with different chain length and degree of unsaturation 1
Degree of unsaturation
Chain Length
j
Saturated
Unsaturated
Short to medium
1
GC
SFC/GC
Medium to long
1
GC/SFC
SFC
In Supercritical Fluids; Abraham, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.
In Supercritical Fluids; Abraham, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.
ri'i
1000000 J
1500000 J
2000000 J
2500000
SEHndâncë
— TIC:
UH1FAEQ2.D
Figure 2. Analysis by GC of fish oil fatty acid methyl esters. Column: HP-FFAP, 25 m, 0.2 mm. Flow: 1 mL/min. Carrier gas: He. Temperature program: From 140°C (1 min) to 220°C with 3 °C/min, then 220°C for 10 minutes. Detector: MSD.
—
Downloaded by UNIV OF MISSOURI COLUMBIA on March 17, 2013 | http://pubs.acs.org Publication Date: October 1, 1997 | doi: 10.1021/bk-1997-0670.ch012
In Supercritical Fluids; Abraham, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.
.00
ι
I I
1
ιI I
40.00
ι ι ι ι ι ι
1 1
ι
1
ι
ι
I I I I
2
120.00
ι ι ι ι ι ι ι ι ι
100.00
ι ι ι ι ι
Re t e n l l o n s t l d
80.00
ι ι ι ι ι ι ι ι ι [ ι ι ι
60.00
ι ι ι ι ι
1
1
I I ]
1
I I
140.00
I
1
160.
