The CFD technique is an important component of an analytical technique that pre- supposes the subsequent gas chromatographic separation of the derivatives formed.
Therefore. the requirements placed upon this stage of the technique must be the same as those generally placed upon the technique as a whole, viz.: (1) simplicity, (2) rapidity, (3) reliability and, in a number of instances, (4) selectivity. As reaction chromatography is a method that combines chemical methods, chromatographic separation and the
31
detection of the separated zones, the pre-chromatographic conversions of sample com- pounds must always be conducted bearing in mind the subsequent stages of the method.
The chemical conversions are used to solve one of the tasks already considered (increasing the sensitivity, expanding the area of the application of gas chromatographic methods, etc.). It seems that when possible reactions are considered they should be compared with respect to the following criteria: (1) degree of conformity with the requirements of the problem being solved (eg., improving the separation, increasing the sensitivity, etc.);
(2) stability of the derivatives obtained under the experimental conditions with respect to the sorbent in the chromatographic column, the separation temperature, etc.; (3) yield of the derivatives obtained; a high yield (95-100%) is desirable, although in some instances (e.g., when a labelled compound of the same chemical structure is used as the internal standard) this requirement is not compulsory; (4) rate of the reaction (a high rate allows one to reduce the reaction time); ( 5 ) simplicity of realization of the reaction;
the derivatives can be obtained both outside and inside the chromatographic system;
(6) role of secondary reactions (it must be reduced to a minimum, especially when complex mixtures and impurities are being analysed); and (7) polarity of the derivatives (in many instances, especially in the analysis of high-boiling compounds, it is desirable that the derivatives obtained be weakly polar compounds, which simplifies their sub- sequent chromatographic analysis).
In some instances, e.g., in amino acid analysis, the chemical conversion used must not result in changes in the structure of the sample substance [65].
It is obviously very difficult to satisfy all of the above requirements. Therefore, when solving a particular problem one must find the optimal solution depending on the con- ditions and the available apparatus and reagents.
As the CFD method is a potential source of large errors, it is necessary to take steps to avoid them. A very useful procedure, making it possible to ascertain the presence of impurities (‘chemical noise’), is to run a ‘blank’ experiment. It is also necessary t o use a sample of known composition to check the technique elaborated. This check should be performed repeatedly, especially when different batches of reagents are used.
Special precautions should be taken when impurities are analysed. Kaiser [66] pointed out the possibility of the results being greatly distorted in the determination of impurities of non-polar compounds in a polar medium (and vice versa) because of their adsorption on the gas-liquid and liquid-solid (container walls) interfaces. It is also necessary to remember that stoppers can be a source of impurities and, possibly, of large errors [65].
Purity of solvents and reagents is an important prerequisite for successful utilization of the CFD method. It should be noted that especially deleterious are reactive impurities that react with the sample compounds and form new substances and, therefore, new peaks on the chromatogram. This is naturally of particular importance in the analysis of impurities when their content in the reagents is comparable to that in the sample substance. A blank experiment also makes it possible to estimate these errors. The stability of the compounds obtained must be sufficiently high. One should take into account that some derivatives (e.g., TMS) are sensitive to moisture.
A useful method of increasing substantially the stability of derivatives during chro- matographic separation that has been applied in the analysis of volatile chelates of a number of elements can be mentioned.
32
I I I I
0 10 2 0 3 0 40
Tlme (m i n )
Fig. 1.9. Chromatogram of the separation of tritluoroacetylacetonates of rare earth elements in a flow of carrier gas with trifluoroacetylacetonate. From ref. 7 3 .
As chromatographic separation involves the disintegration of chelates resulting from chemical reactions (e.g., dissociation), as well as their irreversible adsorption, which leads to low results, it was suggested that the vapour of a ligand should be added to the carrier gas. This would naturally increase the stability of the chelates of metals and decrease their adsorption as a result of the displacement effect. The method was first proposed and developed by Zvarova and Zvara, who showed the possibility of separating the chlorides of lanthanides and actinides at moderately high temperatures (below 250°C) with a mixture of inert gas and aluminium chloride vapour as the carrier gas [ 6 7 , 6 8 ] . The method is based on the reaction of aluminium chloride with the chlorides of rare- earth elements and forming gaseous complexes [69] wluch are separated in the chro- matographic column. The excess of aluminium chloride prevents the dissociation of unstable complex molecules and also modifies dynamically the surface of the column 2703. Fujinaga et al. [71, 721 extended this method to metal complexes with organic ligands. They successfully applied this method [73] to the analysis of the neighbouring rareearth elements in one stage. The separation of trifluoroacetylacetonates of holmium (1). dysprosium ( 2 ) , terbium (3) and gadolinium (4) is shown in Fig. 1.9. The sorbent was Chromosorb W, impregnated with 0.2% PEG 20M and 1.8% silicone OV-17 (ageing at 240°C). The detection was performed by means of a thermal conductivity detector and helium (41 ml/min) containing trifluoroacetylacetone (ca. 10%) was used as the carrier gas. Up to now this method has only been used to analyse the complexes of metals, and it seems that because of this Fujinaga and co-workers named it the ligand- vapour GC method. There is no doubt, however, that its significance is not confined to determining the complexes of elements and metals. Therefore, it is more correct to call it the reagent-vapour GC method (RVGCM), in which one can use as reagents sub- stances that take part in the reaction whose insertion into the reaction zone results in shifting it towards the formation of the derivatives of interest.
The rate of chemical conversion of the sample compounds determines the duration of analysis; it is also highly desirable that the yield of derivatives should be close to 100%.
When elaborating the technique one should therefore make sure that the reaction pro-
33 II t
Time ( m i n ) I
Fig. 1.10. Chromatogram of tranexamic acid determination. 1 = Tranexamic acid; 2 = .laminomethyl- bicyclo[2.2.2] octane-lcarboxylic acid (internal standard). Reprinted with permission from ref. 7 7 . Fig. 1.11. Dependence of the ester yield on duration of reaction. 1 = Ethyl iodide concentration 3 M ; 2 = ethyl iodide concentration 3 M in the presence of dimethyl sulphoxide; 3 = ethyl iodide con- centration 1 M. Reprinted with permission from ref. 77.
ceeds to completion. For this to be achieved it is necessary to conduct the reaction under conditions which ensure that the reaction yield is not high (excess of reagent, catalyst, Jemperature, etc.). It is usually possible to reduce the duration of chemical reactions by using already known regularities. Let us cite some examples. In the work of Sizova et al. f741, devoted to the analysis of lower organic acids, the acids were preesterified with ethanol. Derivatives of the sample acids have to be formed, mainly because the direct chromatographic determination of formic acid is greatly hampered by the inter- action of the acid with metallic parts of the chromatograph [75]. The method developed by Sizova et al. [74] was based on a modified version of an earlier method 1761. Instead of esterifying the acids with ethanol in the presence of large amounts of sulphuric acid [76] the authors used small amounts of p-toluenesulphonic acid as a catalyst. This resulted in the reduction of the esterification time from 1 h to 30min.
The duration of reaction can be long. As an example let us consider the determination of tranexamic acid (trans-4-aminomethylcyclohexanecarboxylic acid) in biological samples [77]. The sample (ca. 200pl of plasma) containing the determined amino acid is subjected, to protect the amino group, to direct reaction with 4-fluoro-3-nitro- benzotrifluoride in a solution of dimethyl sulphoxide and borate buffer (pH9.4). The acid is then alkylated with ethyl iodide by extractive alkylation using a tetrabutyl- ammonium salt. After the chromatographic separation the derivative obtained is detected with an ECD. The chromatogram is shown in Fig. 1 .lo. The separation was conducted at 250°C on a glass column (1.5m x 1.8mm I.D.) packed with sorbent (1% OV-225 on Chromosorb G ) . In this method, after the fluorine derivative has been formed the acid is subjected to extractive alkylation to obtain the ester [78, 791. The alkylation involves the extraction of the acid anion functioning as a counter ion (using the appropriate ion
34
3 x - v - - - - x - k - 3 ( - -
i- 1
L I I I
3 30 60 90
Time ( m i n )
Fig. 1.12. Dependence of the yield of (1) VMA, (2) HVA and (3) IVHA derivatives on t h e . krom ref. 80.
of the quaternary ammonium compound) into the organic solvent where the alkylation with alkyl iodide actually takes place. The dependence of the yield of the ester on the time of alkylation is shown in Fig. 1 .I 1. It follows from the data presented that in the best instance the time necessary for a 100% yield is 30min. The authors showed, how- ever, that the duration of reaction can be reduced if the reaction is made coincident with the evaporation stage, when the reaction rate increases radically, apparently as a resuIt of an increase in temperature and the concentration of reagents.
Investigation of the completeness of reaction is a necessary stage in the elaboration of a quantitative method, with slight changes in the structure of sample compounds being capable of radically affecting the rate of derivative formation. As an example we shall consider the time dependence of the yield of vanilmandelic acid (VMA), homovanillic acid (HVA) and isohomovanillic acid (IHVA), the determination of which in blood is of interest for the diagnosis of a number of diseases [ 8 0 ] . Fig. 1.12 shows the time depen- dence of the yield of trifluoroacetylhexafluoroisopropanol esters of VMA ( I ) , HVA ( 2 ) and IW,',i (3). Methoxy-4-hydroxyphenylethanol (HMPE) was used as the internal standard. As can be seen from the data presented, the formation of VMA derivatives ends only after 1 h. From the mass spectra of individual compounds intense lines were chosen which make it possible to obtain informative chromatograms (selected ion detec- tion). Fig. 1.13 shows the selected ion detection for the acids of a known mixture (A) and a plasma sample (B) obtained by recording the intensity of the mass spectral lines at m/e 345 (I), 428 (11) and 360 (111). The technique developed by Takahashi et al.
[SO] makes it possible t o determine selectively and with high sensitivity the content of the above acids in blood, urine, etc. The limit of determination is 2 ng/ml for plasma and cerebrospinal fluid. Separation was conducted on a glass coiumn ( 1 m x 3 mm I.D.) packed with 2% OV-1 o n Chromosorb W.
The cited examples are indicative of the necessity to estimate the duration of the CFD stage when developing the techniques for all compounds that are of analytical interest.
Especially stringent requirements are placed on the rate of the chemical reactions used when the reaction proceeds in a chromatographic system, in a reactor before the column or at the beginning of the column. Esposito [ S l ] , developing the work of Beroza and
35
A B
4
m
'
1 I I I
5 10 15 5 10 15
'Time (min)
Fig. 1.13. Selected ion detection of the derivatives of (1) VMA, (2) HVA, (3) IHVA and (4) HMPE acids and of unidentified compounds (n). See text. From ref. 80.
2 3 4
-L
U 2 0 25 3 0 35 40
Time (rnin)
Fig. 1.14. Chromatogram of the separation of polyol trimethylsilyl derivatives. 1 = Ethylene glycol;
2 = neopentyl glycol; 3 = 1,4butanediol; 4 = diethylene glycol; 5 = glycerine; 6 = trimethylolethane;
7 = trimethylolpropane; 8 = pentaerythritol. Reprinted with permission from ref. 81.
Coad [82] on the formation of derivatives in the column by consecutive insertion into it of (1) a sample of the compound to be analysed and (2) a reagent, suggested using this method to form TMS derivatives. The method was applied to the analysis of polyols, acids and oxyacids. As the donor of the TMS group a mixture of N,O-bis(trimethylsily1)- acetamide, trimethylsilyldiethylamine and hexamethyldisilazine w2s used. Fig. 1.1 4 shows a chromatogram of the TMS derivatives of polyols, obtained in the analysis of a mixture of polyols performed in accordance with the method proposed by Esposito [81] . Separation was conducted on a column (3 m x 6 m m I.D.) packed with 20%
silicone lubricant on Chromosorb W. The sample was inserted in the column at 4OoC
36
Fig. 1 .I 5. Chromatogram of alcohols in the form of (A) pentafluoropropionates and (B) propionates.
Esters of: 1 = dodecanol; 2 = cyclododecanol; 3 = tetradodecanol; 4 = hexadodecanol. € r o m ref. 84.
and the derivatization reaction was conducted at the same temperature. The temperature in the column was then increased to 250°C at 4"C/min. The TMS reagent was introduced into the column after the insertion of the sample when enough time had passed for the water and alcohol to be separated from the sample. Free polyols are not alkylated under these conditions. As already noted, when elaborating a method for obtaining derivatives one should take into account their chromatographic properties, primarily their stability, volatility, etc.
As silyl derivatives of oxygen-containing compounds are those most frequently used, we shall cite some examples of their application. Experimental results [83] indicate the expediency of using the trimethylsilyl ether of cholesterol and not the methyl ether, as the former yields more symmetrical chromatographic zones. VandenHeuvel et al.
[84] showed that the use of fluorine derivatives of esterifying reagents makes it possible to decrease substantially the boiling points of derivatives and their retention times. As an example, Fig. 1 . I 5 shows two chromatograms of a mixture of propionates of CIz-C16 alcohols and their pentafluoropropionates. Separation was conducted on a column (1.8 m x 4 mm I.D.) with 5% diethyimethylpolysiloxane sorbent at 180°C. It follows from these data that fluorine derivatives of propionates of alcohols have a much shorter retention, which makes it possible to expand the range of GC applications by using compounds of lower volatility as derivatives.
When CFD methods are used, after the reactions for the protection of functional groups that interact (adsorption. reaction, catalysis) with the sorbent and the apparatus have been accomplished, less polar derivatives are formed and, as a rule, these can be successfully separated by using a non-polar thermally stable (e.g., silicone) stationary phase, At the same time, however, especially when compounds of high molecular weight are separated, in a number of instances difficulties arise in separating the derivatives obtained, because the individual characteristic features of a compound, after the pro- tection of its functional groups, are in fact often blurred. It is therefore expedient to use capillary columns as often as possible in analysing derivatives [85-88] .
When assessing the possibilities of a detector one should give preference to the most
31
J b L :o Ib 2 6 I
Time (min)
Fig. 1.16. Chromatogram of B, vitamins. 1 = Desoxypyridoxine; 2 = pyridoxine; 3 = unidentified;
4 = pyridoxal; 5 = pyridoxamine. From ref. 95.
selective (although also the most complex) viz., the mass spectrometer [89]. The use of a mass spectrometer as the detector enables one to record chromatograms at two or three different fixed mass spectral lines (the ‘mass fragmentation’ or ‘selected ion detection’
method [90-93]), which makes it possible, when the lines in mass spectra have been chosen correctly for recording, to determine selectively even those compounds that form a common chromatographic peak.
The equipment and techniques used in conducting the preliminary reactions are, as a rule, sufficiently simple, and some of the techniques are considered below.
Quantitative analysis of the B6 vitamins is of great interest, but the direct GC analysis of vitamin B is impossible because of its involatility and polarity. Using the CFD method to obtain volatile derivatives one can accomplish the GC analysis of B6 vitamins [94].
The method of Patzer and Hilker [95] is, however, simpler and more rapid. According to this method, aqueous solutions of B6 vitamins (5ml) are analysed. A 10-200-pl volume of solution was placed in a vial and dried in a flow of nitrogen at 70°C. To prevent semiacetal formation, 40ml of 100% ethanol was then added to each vial [96].
The vial with the lid closed was heated at 125°C for 15min and finally dried at 70°C in a flow of nitrogen. To form the derivatives of trifluoroacetic acid 3 0 ~ 1 of N-methyl- bistrifluoroacetamide were added [97], the lid was closed and the vial was heated for 20 min. After cooling, the sample was placed directly in the chromatograph for quanti- tative analysis. Separation was conducted at 150°C on a glass column (1.8 m x 2 mm I.D.) packed with 5% silicone DC-550 on Chromosorb P AW DMCS. The chromatogram is shown in Fig. 1.16. The limit of determination for an FID is about 250ng.
Meesschaert et al. [98] proposed a technique for analysing natural penicillins in the form of their methyl ethers. Penicillins were extracted with diethyl ether from aqueous solutions at pH 2 and esterified with a slight excess of diazomethane at 0-2°C. The solution of methyl esters of penicillins in diethyl ether was evaporated to dryness and
38
Time (min) Time (rnin)
Fig. 1 . 1 7 . Chromatograms of a mixture of methyl ethers of natural penicillins on columns of (A) OV-1 and ( B ) OV-17. 1 = Methylpenicillin; 2 = n-propylpenicillin; 3 = n-amylpenicillin; 3' = n-2- amylpcnicillin; 4 = ri-heptylpenicillin; 5 = benzylpenicillin; 6 = n-nonylpenicillin; 7 = phenoxy- methylpenicikn. From ref. 98.
the residue was dissolved in acetone and analysed by GC. The chromatograms obtained are shown in Fig. 1.17. Separation was conducted on a glass column (150cm x 4 m m 1.D.) packed with 3% OV-1 (A) or 3% OV-17 (B) on Gas-Chrom Q. The temperature programmes during the anaiysis were as follows: using OV-1 the column was operated for 10min at 150"C, then heated at 4"C/min for 10min to 19OoC;using0V-17 the column was operated for lOmin at 18O"C, then heated to 220°C at 4"C/min. The above tech- nique makes it possible to separate satisfactorily all of the investigated penicillins.
Diazomethane is often used to obtain methyl ethers of acids [99]. Fales et al. [loo]
designed a simple instrument to obtain diazomethane in diethyl ether without distillation.
The design of this simple instrument is shown in Fig. 1.18. Diazomethane was obtained in an internal test-tube as a result of the reaction of alkali with N-methyl-N-nitros0-N'- nitroguanidine. To obtain diazomethane ca. 1 mmole (133mg) of reagent was placed in the internal test-tube and 0.5 ml of water was added. About 3 r d of diethyl ether were placed in an external test-tube and the instrument was assembled using a butyl- rubber ring. The lower part of the external test-tube was cooled in a waterhath, then ca. 0.5 ml of 5 M sodium hydroxide solution was added into the internal test-tube with a syringe through the upper silicone packing. This method and device also make it possible to obtain a solution of diazoethane in diethyl ether when N-ethyl-N-nitroso-N-nitro- guanidine is used. Ethers of acids can also be formed by reaction with diazomethane in a chromatograph in a special quartz reactor filled with quartz-wool and placed before the chromatographic column [ 1011 . The sample and the diazomethane solution are placed in the reactor. Esterification is completed in 18 sec.
Diazomethane is also used for methylenation under the conditions of diazomethane photolysis. This reaction was first described by Doering et al. [lo21 and used by Simmons et al. [lo31 and Dvoretzky et a]. [I041 t o obtain mixtures of hydrocarbons.
The methylenation of hydrocarbons is a valuable method for obtaining mixtures of hydrocarbons of specific composition which are used as standard mixtures in GC analysis.
The methylenation reaction consists in the homolytic addition of methylene along the
IJ
39
0 "0 Ring
I
W
Fig. 1.18. Device for the production of diazomethane. Reprinted with permission from ref. 100.
C-H bond. As the methylenation reaction is not a selective reaction and the probability of methylene addition to different C-H bonds is determined only by their 'concen- tration' in the molecule of reacting hydrocarbon, the qualitative composition and the quantitative distribution of the products formed can be determined by calculation.
Thus, e.g., as a result of n-pentane methylenation in accordance with the number of C-H bonds one would expect, with equal probabilities of addition, the formation of the following mixture: 50% n-hexane, 33% 2-methylpentane and 16% 3-methylpentane, i.e., in the approximate ratio 6:4:2. Experimentally it was found that the products of n-pentane methylenation consist of 50% n-hexane, 34% 2-methylpentane and 16%
3-methylpentane [102], in full agreement with theory. Research carried out under the guidance of Petrov [105, 1061 resulted in important developments of this method as applied to the analysis of new classes of hydrocarbons.
Interesting investigations of the application of the methylenation reaction to sulphur- containing organic compounds were carried out by Galpern et al. [107, 1081. The