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Extraction based on the flow-injection principle
Anafyticd ~Elsevier
Chimica Acta, 98 (1978) l-7 Scientific Publishing Company,
Amsterdam
-
Printed
EXTRACTION BASED ON THE FLOW-INJECTION Part I. Description of the Extraction System
BO KARLBERG* Astra
and SIDSEL
Phcrmacvuticals
(Received
25th
MI,
November
in The Netherlands
PRINCIPLE
THELANDER
Analytical
Control,
S-151
85 Sijdertiilje
(Sweden)
1977)
SU?fl?vlAKY An extraction system has been dcvcloped, essentially consisting of a pump, a rotary valve and a spectrophotometer. The sample, 12-25 ~1, is introduced via the rotary vaivc into an aqueous stream (iiow injeciion j. ‘Tine aqueous stream, containing i-he ampie plug, is divided into small segments by an organic phase and led into a Teflon coil so that a regular pattern of the two phases is obtained. No air bubbles should be present. Separation of the two phases is achieved in a specially constructed fitting and the absorbance of the organic phase is measured. The construction and performance of the system are illustrated by analysis of caffeine samples. Up to 100 samples/h can be analysed with a relative precision of better than 1%
Flow injection analysis has been developed into a very useful and versatile technique [l-9]_ Several methods are now applied routinely in many laboratories in Denmark and Sweden. Primarily, it is the small sample volume, the low reagent consumption [lo] and the high sampling frequency that attract analytical chemists with heavy working schedules. Since atraction is widely used in pharmaceutical and clinical analysis to separate drugs or drug metabolites, it was Iogical to attempt to apply the flow-injection principle to this area, the aim being to achieve better economy of extraction methods with respect to time and solvent consumption. In this first paper, the performance and construction of the extraction .._.-C__ ^_^ >-__:L-rl ml_>-L^-:-^&.‘--c ~autmt: __-&?.?_.I_:_ - __L_.,__?l_..1! acla - -:3 ayacv‘u cut: UCbCZI”CI(. I II~ UBL~~I~~~~LIUII oi 1~1 actxyl=lcync preparations was selected as an example of a practical application. The preparations contain sodium lauryl sulphate, a surfactant that interferes with the transfer of caffeine f?om an aqueous to an organic phase, and so tetrapropylammonium bromide was added to the organic phase. The lauryl sulphate and the tetmpropylammonium ions form an ion pair [ll] which transfers to the organic phase, but does not contribute to the absorbance measured at 275 nm.
2 EXPERMENTAL
Reagen Is The caffeine standard substance was of pharmacopoeia quality. The tctrapropylammonium bromide (puriss., Eastman-Kodak) was used as purchased. .X. _LL _.. --____L_ ___J _-I..--&___^_A -E ..~~I.Ac:..P.l -..rl, -..rr,:*r, Stsck an$-j flu urn& reagent3 anu ~U~VCIIL~ writ: 0~ arkz~yc~r;cu-~;r4ut: yu-cy. standard solutions were always freshly prepared. The organic phase consisted of chloroform containing 1% tetrapropyiammonium bromide. The aqueous phase contained 0.16 M sodium hydroxide, and was degassed before use. Ted solutions (always aqueous) of Bamyi-S-Caffeine and Bamyi-Caffeine tibiets (H5ssle AB, Mblndal, Sweden), were made up; both types of tablet contaiaed 50 mg of caffeine/tablet according to the specifications. The tablets also contained acetylsalicylic acid (500 mgltabiet), pot&o starch, calcium carbonate, citric acid and sodium iauryi suiphate. Appamtus and procedures A five-channel peristaltic pump (model mp-GE; Tsmatec, Ziirich, Switzerland) with variable speed was provided with Acidflex pump tubes for the organic solvent and Tygon pump tubes for the aqueous solutions. The flow was found to change rather significantly during the first 5 minutes of pumping. No measurements tiere ever done during this period of time. Apparently, a certain warming-up of the pump tubes is required to obtain a constant flow. The test solution was introduced into the aqueous stream by a rotary valve (Bifok AB, Sollentuna, Sweden) provided with a by-pass coil. In Fig. 1 the valve is in the filling position and the sample, S, can be introduced into the bore while the aqueous phase passes through the coil b. When the valve is turned QO”, the aqueous stream starts to pass through the core, thereby bringing the sample into the system. At the same time, the flow ir, coil b ceases completely. The sample volume was either 12 or 25 gi. The const.ruction ofthe v-&e has been described in detail [12].
Fig. 1. Manifold for extraction based on the fIow-injection principle. The flow-rates are (ml min-‘): x for the aqueous phase.y for the organic phase, and z for the fraction of the organic phase pasGng through the flow cell. S denotes the sample filling port of the rotary valve (12 or 25 vi).
3 The chloroform stream is chilled in an ice bath before being mixed with the aqueous stream, in order to prevent evaporation. The distance between the sample introduction point and the mixing point is denoted by r in Fig. 1. At the mixing point of the phases, a specially constructed fitting makes it possible to obtain aregular pattern of alternate aqueous and organic segments. The fitting is shown in detail in Fig. 1, and consists of a modified standard A8 T-connector (Technicon, Tarrytown, USA). Between the peristaltic pump and this fitting, all tubes for the aqueous phase are of pclythene (i-d., 0.8mm), and all tubes for the organic phase are of Teflon (i.d., G.8 mm). The organic stream is led into the platinum capillary while the aqueous stream enters the glass capillary. A Teflon tube is inserted into the cutlet of the T-connector. A second Teflon tube is then inserted into the first one, and the positioning of the edge of this tube can easily be adjusted. Appropriate positioning is necessary for a re,oularmixing pattern. Furthermore, the length of the segments can be adjusted by the inner Teflon tube. The extraction coils are made by winding Teflon tube on XI-ml beakers (diameter, 3.8 cm). The coils are not thermostatted. The separating device consists of an -44 T-connector (Technicon, Tarrytown, USA) with Teflon fibres twisted together to a thread and inserted in the bend from the inlet down into the outlet directed towards the Bow cell. By differential pumping, the aqueous phase as well as excessive organic phase are forced upwards in the fitting to waste, and no aqueous phase is sucked i_ntothe flow cell. Special care must be taken to avoid contaminating the flow cell with aqueous phase since its removal by rinsing was found to be a very tedious procedure. A ratio of O-7/2.0 of z/y, i.e. the ratio between the flow of organic phase through the flow cell and the total flow of organic phase, was preferred. The separator must be f;Xed in a perpendicular position as i&&rated in Fig. 1. The Teflon thread should be placed rather close to the lower wall cf the inlet tube of the T-connector and allowed to bend smoothly and in parallel with the wall downwards. The U.V. spectrcphotcmeter was a Coleman 55 (Perkin-Elmer, Norwalk, U&4), provided with a flow cell (Heilma, Miillheim, Germany) with a volume of 40 ~1. The distance between the separator and the flow cell was kept as small as possible. A recorder (IV i W 1100, Kcntrcn, Ziirich, Switzerland) was connected to the spectrophotometer. RESUL'i'SANDDISCUSSIQN Determination
of caffeine
-4 practical application of the extraction method is exemplified by results from a study of the dissolution rate of caffeine in an acetylsalicylic acid preparation (Bamyl, H%sle, Sweden). In Fig. 2, two calibration sets are shown bracketing samples taken after 1,2, 3 and 5 min. Each test or cabbra&ionsolution was injected in duplicate. The sampling rate was about 75/h. The ficw rate ofthe aqueous phase, X, was 2.2 ml min-‘; t.he flow
0.1
5
IO 15 Time.min
ZO
Fig. 2. Determination of caffeine in acetylsalicylic acid tablets (dissolution rate). Samples taken after 1, 2, 3, and 5 min. Sampling rate 75/h; injected volume 25 ~1. Standards: Sl, 2.74 x lo-’ M; S2,5.48 x lo- M; S3,8.22 X lo-“ M. The aqueousflow streamwas 0.16 NI sodiumhydroxide.
were
rates of the organic phases were y 2.0 ml min-‘and z 0.3’ ml min-’ (see Fig. 1).
The coil length, Z,was 2 m, and r was 0.15 m. The volume of the bore in the valve was 25 ~1. The absorbance was measured at 275 nm. To obtain a measure of the repeatability of the method, the three different c&b&ion solutions were injected at intervals during a l-h period, in all ten times for each solution. The results are given in Table 1. The repeatability is excellent, and the values presented are typical for an optimized extraction system. Up to et least 100 samples/h can be analyzed with the present system, and base-line readings can still be made in between samples if the flo7;. rates of the +JO phases are increased further. Sample dispvsiov. and coil lengths Intuitively, th:, distance between the rotary valve and the mixing point of the two phases should be kept as short as possible to prevent dispersion of the injected samy’.~. However, in the ease of caffeine extraction from a solution which also con’ains acetylsalicylic acid, some mixing with the aqueous alkaline phase is desirable. The acid will then be retained in the
Repeatability of the extraction method at three different sample concentrations Caffetie sample sohltion (&I)
Average of maxima of peaks (absorbance units)
Relative standard deviation (a)
2.74 x lo-*
0.188 0.370 0.546
0.80 0.63 0.55
5.48
x lo-’
8.22 x 1O-4
5
aqueous phase because of ionization at the high pH, while caffeine will be easily extracted into the chloroform phase. This means that the distance denoted by r in Fig. 1 must be adjusted empirically, so that the dispersion is kept at a minimum while mixing with the stream of alkali is possible. It might appear easier to make the sample solution alkaline before injection, but caffeine is not stable in a strongly basic solution [ 13 1. The degradation during a residence time of less than 60 s in the extraction system is, however, not significant. As expected, successive increases of T gave rise to decreases in the peak height (Table 2). Two caffeine solutions were injected; both contained 0.134 mg ml-’ (0.69 mM). The second solution also contained 1.34 mg of ac&vlsalicvlic -__= -_-__-.__- were nerformed r ---------J ----- 4 -- acid ---- uer r -- ml ---- 17.45 \---- m-M)= The exneriments with all other parameters constant (X = 2.2, y = 2.0, z = 0.7 ml min-‘; I = 2 m; S = 25 ~1). It is obvious that coextraction of acetylsalicylic acid does not occur at all, even in the extreme case when r is only 0.04 m. The length of the mixing coil, I, was found not to be critical for values above 1 m. In one experiment, 1 was varied from 0.15 to 6 m. The results (Fig. 3) indicate that dispersion of the sample in the mixing coil, as well as degradation of caffeine in alkaline solution, are negligible. For values of 1 below 0.50 m, the extraction seems to be incomplete. Extraction efficiency and size of segments The results in Fig. 3 immediately raise the question of vrhether the extraction process is complete or not for a certain design of system. In order to investigate this further, the following experiment was performed. The sodium hydroxide stream in the system was replaced by an aqueous caffeine solution, without introduction of air. The coil length, 1, was 1 m. The steady-state value of the absorbance was measured, so that the concentration of caffeine in the chloroform phase could be evaluated; for this . .. purpose, cahbntion soiutions (in chioroform j were pumped separately and directly into the flow cell in a previous experiment. The proportion of the flows of aqueous and organic phases was obtained by measuring TABLE Variation
2 of r, the distance
between
the valve .a.nd the mixing
(see Fig. 1) r
(m) 0.04 0.08 0.15 0.30 0.45 0.60 1.00
Peak maximum for caffeine (absorbance --. -__ -_-___.__ Caffeine 0.46 0.46 0.47 0.46 0.43 0.42 0.37
alone
--
units)
Caffeine + acetylsalicylic 0.46 0.46 0.47 0.46 0.43 0.42 0.37
acid
point of the two phases
6
the individual flow rates. The efficiency of the extraction was then calculated to be 101 + 5%~~the uncertainty being due to difficulty in measuring the exact flow rates. The extraction efficiency calculated for a coil length of 0.15 m (see Fig. 3) was about 75%. which is a remarkably high value. The size of the segments of organic and aqueous phases in the mixing coil could be varied by adjusting the inner Teflon tube in the A8 connector (Fig. 1). A variation of the segment size in the range l-10 mm had no influence on the peak height when a coil length of 2 m was employed. This is probably explained by the rapid and efficient extraction of caffeine. Hcwever, for other extractants the size of the segments is a very important parameter, and this will be reported in a later paper in this series. Ratio of organic to aqueous phase and flow rates The ratio of the organic phase to the aqueous phase affects the sensitivity of the method. Ideal conditions for a study of this ratio would be a set-up of three different pumps so that the flow of each stream, X. y, and z, could be varied individually (see Fig. 1). In a simplified study, y and t were kept constant while x was varied. The results are presented in Fig. 4. The test solution was a 1.04 mM ca.Jfeine solution and the valve volume was 25 ~1. The maximum peak height is obtained for a volume ratio of the phases of -tr-*.C 1.1 At.-..* CL;, ...,.... +S., rS.-r:*:rr;Cr.“l”YJ .-a-,.. A..P.&;-m 11.. _Vc\L.%hl.. a.u”“C I .I. zI”““t: ‘lfW .“alUtz IrilCW=l‘a,ci”lI+y u&Lu~lLaLay, t.“““““‘J because of the high total flow through the separator. The residence time in the separator is then shorter. Furthermore, the pressure in the system increases when the flow increases, and this may influence the
of the sample
i 2 L (ml m:n-‘) Fig. 3. Absorbance of peak maximum as a function Sample solution: ca. 8 x lo-. hl caffeine (aqueous). S = 25 rl;r = 0.15 m (see Fig. I).
of the length, I, of the mixing coil. x = 2.2. y = 2.0, z = 0.7 ml min-’ ;
Fig. 4. Absorbance of peak maximum as a function of the flow of the aqueous phase, x. %npfe solution: 1.04 X 10.“ 31 caffeine (3queous). y = 2.0,z = 0.7 m1 min-‘;S = 25 ~11: i = 2, r - 0.15 m (see Fig. I).
7
turbulence in the individual segments so that the extraction efficiency decreases. Reconstruction of the present separator will be necessary if the total flow rate is to be allowed to exceed 5 ml mm-‘; the optimum flow rate seems to be about 4 ml min-‘. Some experiments were done with Teflon tubes with an inner diameter of 0.5 mm. ‘The flow rates of the two phases must then be decreased, otherwise the pressure in the system becomes excessive. The results obtained with this narrower Teflon tube were not completely satisfactory, and the reason for this lies in the separator. A low consumption of organic phase is desirable for economical and environmental reasons, so work is in progress
to develop a new separator
suitable
for low flow rates.
Conclusions
The extraction system developed is very suitable for the simple extraction of caffeine from an aqueous solution. More advanced examples of applications are in progress. The possibility of performing extractions on very small volumes is extremely valuable in many cases, e.g. in the field of clinical chemistry where this parameter is often the limitation of an application. At present, a sample volume of at least 40-50 ~1 is required to fill the rotary valve, but this figure can certainly be reduced further, and so can the consumption of reagents. The small volumes of sample and reagent, in combination with the high sampling rate and good precision, are the real attractions of this system.
The authors are indebted for valuable discussions.
to Dr. P.-A. Johansson,
Astra Pharmaceuticals,
REFERENCES 1 J. R&%&a and E. H. Hansen, Anal. Chim. Acta, 78 (1975) 145. 2 J. RiX%ka and J. IV. B. Stewart, Anal. Chim. Acta, 79 (1975) 79. 3 J. Vi. B. Stewart, J. RBWka, H. Bcrgamin Filho and E. A. Zagatto, Anal. Chim. Acta, 81 (1976) 371. 4 J. REZiEka. J. W. B. Stewart and E. A. Zag&to, Anal. Chim. Acta. 81 (1976) 387. 5 J. W. B. Stewart and J. R&tiL!ka, Anal. Chim. Acta, 82 (1976) 137. 6 E. H. Hansen and J. Rfi?.iEka, Anal. Chim. Acta, 87 (1976) 353. 7 J. RBZirka. E. H. Hansen and E. A. Zagatto, Anal. Chim. Acta, 88 (1977) I. 8 E. Ii. Hansen, J. RtiiZirka and B. Rictz, Anal. Chim. Acta, 89 (1977) 241. 9 J. R&Eirka, E. H. Hansen and H. hiosbaek, Anal. Chim. Acta. 92 (1977) 235. 10 J. RMiEka, E. H. Hansen, H. hlosbaek and F. J. Krug, Anal. Chem., 49 (19i7) 1858. 11 S. 0. Januon, R. Modin and G. Schill, Talanta, 21 (1974) 905. 12 E. Ii. Hansen, F. J. Krug, A. K. Ghose and J. R?rEiEka, Analyst, 102 (1977) 714. 13 British Pharmaceutical Codes, 1973, p. 63.
Chimica Acta, 98 (1978) l-7 Scientific Publishing Company,
Amsterdam
-
Printed
EXTRACTION BASED ON THE FLOW-INJECTION Part I. Description of the Extraction System
BO KARLBERG* Astra
and SIDSEL
Phcrmacvuticals
(Received
25th
MI,
November
in The Netherlands
PRINCIPLE
THELANDER
Analytical
Control,
S-151
85 Sijdertiilje
(Sweden)
1977)
SU?fl?vlAKY An extraction system has been dcvcloped, essentially consisting of a pump, a rotary valve and a spectrophotometer. The sample, 12-25 ~1, is introduced via the rotary vaivc into an aqueous stream (iiow injeciion j. ‘Tine aqueous stream, containing i-he ampie plug, is divided into small segments by an organic phase and led into a Teflon coil so that a regular pattern of the two phases is obtained. No air bubbles should be present. Separation of the two phases is achieved in a specially constructed fitting and the absorbance of the organic phase is measured. The construction and performance of the system are illustrated by analysis of caffeine samples. Up to 100 samples/h can be analysed with a relative precision of better than 1%
Flow injection analysis has been developed into a very useful and versatile technique [l-9]_ Several methods are now applied routinely in many laboratories in Denmark and Sweden. Primarily, it is the small sample volume, the low reagent consumption [lo] and the high sampling frequency that attract analytical chemists with heavy working schedules. Since atraction is widely used in pharmaceutical and clinical analysis to separate drugs or drug metabolites, it was Iogical to attempt to apply the flow-injection principle to this area, the aim being to achieve better economy of extraction methods with respect to time and solvent consumption. In this first paper, the performance and construction of the extraction .._.-C__ ^_^ >-__:L-rl ml_>-L^-:-^&.‘--c ~autmt: __-&?.?_.I_:_ - __L_.,__?l_..1! acla - -:3 ayacv‘u cut: UCbCZI”CI(. I II~ UBL~~I~~~~LIUII oi 1~1 actxyl=lcync preparations was selected as an example of a practical application. The preparations contain sodium lauryl sulphate, a surfactant that interferes with the transfer of caffeine f?om an aqueous to an organic phase, and so tetrapropylammonium bromide was added to the organic phase. The lauryl sulphate and the tetmpropylammonium ions form an ion pair [ll] which transfers to the organic phase, but does not contribute to the absorbance measured at 275 nm.
2 EXPERMENTAL
Reagen Is The caffeine standard substance was of pharmacopoeia quality. The tctrapropylammonium bromide (puriss., Eastman-Kodak) was used as purchased. .X. _LL _.. --____L_ ___J _-I..--&___^_A -E ..~~I.Ac:..P.l -..rl, -..rr,:*r, Stsck an$-j flu urn& reagent3 anu ~U~VCIIL~ writ: 0~ arkz~yc~r;cu-~;r4ut: yu-cy. standard solutions were always freshly prepared. The organic phase consisted of chloroform containing 1% tetrapropyiammonium bromide. The aqueous phase contained 0.16 M sodium hydroxide, and was degassed before use. Ted solutions (always aqueous) of Bamyi-S-Caffeine and Bamyi-Caffeine tibiets (H5ssle AB, Mblndal, Sweden), were made up; both types of tablet contaiaed 50 mg of caffeine/tablet according to the specifications. The tablets also contained acetylsalicylic acid (500 mgltabiet), pot&o starch, calcium carbonate, citric acid and sodium iauryi suiphate. Appamtus and procedures A five-channel peristaltic pump (model mp-GE; Tsmatec, Ziirich, Switzerland) with variable speed was provided with Acidflex pump tubes for the organic solvent and Tygon pump tubes for the aqueous solutions. The flow was found to change rather significantly during the first 5 minutes of pumping. No measurements tiere ever done during this period of time. Apparently, a certain warming-up of the pump tubes is required to obtain a constant flow. The test solution was introduced into the aqueous stream by a rotary valve (Bifok AB, Sollentuna, Sweden) provided with a by-pass coil. In Fig. 1 the valve is in the filling position and the sample, S, can be introduced into the bore while the aqueous phase passes through the coil b. When the valve is turned QO”, the aqueous stream starts to pass through the core, thereby bringing the sample into the system. At the same time, the flow ir, coil b ceases completely. The sample volume was either 12 or 25 gi. The const.ruction ofthe v-&e has been described in detail [12].
Fig. 1. Manifold for extraction based on the fIow-injection principle. The flow-rates are (ml min-‘): x for the aqueous phase.y for the organic phase, and z for the fraction of the organic phase pasGng through the flow cell. S denotes the sample filling port of the rotary valve (12 or 25 vi).
3 The chloroform stream is chilled in an ice bath before being mixed with the aqueous stream, in order to prevent evaporation. The distance between the sample introduction point and the mixing point is denoted by r in Fig. 1. At the mixing point of the phases, a specially constructed fitting makes it possible to obtain aregular pattern of alternate aqueous and organic segments. The fitting is shown in detail in Fig. 1, and consists of a modified standard A8 T-connector (Technicon, Tarrytown, USA). Between the peristaltic pump and this fitting, all tubes for the aqueous phase are of pclythene (i-d., 0.8mm), and all tubes for the organic phase are of Teflon (i.d., G.8 mm). The organic stream is led into the platinum capillary while the aqueous stream enters the glass capillary. A Teflon tube is inserted into the cutlet of the T-connector. A second Teflon tube is then inserted into the first one, and the positioning of the edge of this tube can easily be adjusted. Appropriate positioning is necessary for a re,oularmixing pattern. Furthermore, the length of the segments can be adjusted by the inner Teflon tube. The extraction coils are made by winding Teflon tube on XI-ml beakers (diameter, 3.8 cm). The coils are not thermostatted. The separating device consists of an -44 T-connector (Technicon, Tarrytown, USA) with Teflon fibres twisted together to a thread and inserted in the bend from the inlet down into the outlet directed towards the Bow cell. By differential pumping, the aqueous phase as well as excessive organic phase are forced upwards in the fitting to waste, and no aqueous phase is sucked i_ntothe flow cell. Special care must be taken to avoid contaminating the flow cell with aqueous phase since its removal by rinsing was found to be a very tedious procedure. A ratio of O-7/2.0 of z/y, i.e. the ratio between the flow of organic phase through the flow cell and the total flow of organic phase, was preferred. The separator must be f;Xed in a perpendicular position as i&&rated in Fig. 1. The Teflon thread should be placed rather close to the lower wall cf the inlet tube of the T-connector and allowed to bend smoothly and in parallel with the wall downwards. The U.V. spectrcphotcmeter was a Coleman 55 (Perkin-Elmer, Norwalk, U&4), provided with a flow cell (Heilma, Miillheim, Germany) with a volume of 40 ~1. The distance between the separator and the flow cell was kept as small as possible. A recorder (IV i W 1100, Kcntrcn, Ziirich, Switzerland) was connected to the spectrophotometer. RESUL'i'SANDDISCUSSIQN Determination
of caffeine
-4 practical application of the extraction method is exemplified by results from a study of the dissolution rate of caffeine in an acetylsalicylic acid preparation (Bamyl, H%sle, Sweden). In Fig. 2, two calibration sets are shown bracketing samples taken after 1,2, 3 and 5 min. Each test or cabbra&ionsolution was injected in duplicate. The sampling rate was about 75/h. The ficw rate ofthe aqueous phase, X, was 2.2 ml min-‘; t.he flow
0.1
5
IO 15 Time.min
ZO
Fig. 2. Determination of caffeine in acetylsalicylic acid tablets (dissolution rate). Samples taken after 1, 2, 3, and 5 min. Sampling rate 75/h; injected volume 25 ~1. Standards: Sl, 2.74 x lo-’ M; S2,5.48 x lo- M; S3,8.22 X lo-“ M. The aqueousflow streamwas 0.16 NI sodiumhydroxide.
were
rates of the organic phases were y 2.0 ml min-‘and z 0.3’ ml min-’ (see Fig. 1).
The coil length, Z,was 2 m, and r was 0.15 m. The volume of the bore in the valve was 25 ~1. The absorbance was measured at 275 nm. To obtain a measure of the repeatability of the method, the three different c&b&ion solutions were injected at intervals during a l-h period, in all ten times for each solution. The results are given in Table 1. The repeatability is excellent, and the values presented are typical for an optimized extraction system. Up to et least 100 samples/h can be analyzed with the present system, and base-line readings can still be made in between samples if the flo7;. rates of the +JO phases are increased further. Sample dispvsiov. and coil lengths Intuitively, th:, distance between the rotary valve and the mixing point of the two phases should be kept as short as possible to prevent dispersion of the injected samy’.~. However, in the ease of caffeine extraction from a solution which also con’ains acetylsalicylic acid, some mixing with the aqueous alkaline phase is desirable. The acid will then be retained in the
Repeatability of the extraction method at three different sample concentrations Caffetie sample sohltion (&I)
Average of maxima of peaks (absorbance units)
Relative standard deviation (a)
2.74 x lo-*
0.188 0.370 0.546
0.80 0.63 0.55
5.48
x lo-’
8.22 x 1O-4
5
aqueous phase because of ionization at the high pH, while caffeine will be easily extracted into the chloroform phase. This means that the distance denoted by r in Fig. 1 must be adjusted empirically, so that the dispersion is kept at a minimum while mixing with the stream of alkali is possible. It might appear easier to make the sample solution alkaline before injection, but caffeine is not stable in a strongly basic solution [ 13 1. The degradation during a residence time of less than 60 s in the extraction system is, however, not significant. As expected, successive increases of T gave rise to decreases in the peak height (Table 2). Two caffeine solutions were injected; both contained 0.134 mg ml-’ (0.69 mM). The second solution also contained 1.34 mg of ac&vlsalicvlic -__= -_-__-.__- were nerformed r ---------J ----- 4 -- acid ---- uer r -- ml ---- 17.45 \---- m-M)= The exneriments with all other parameters constant (X = 2.2, y = 2.0, z = 0.7 ml min-‘; I = 2 m; S = 25 ~1). It is obvious that coextraction of acetylsalicylic acid does not occur at all, even in the extreme case when r is only 0.04 m. The length of the mixing coil, I, was found not to be critical for values above 1 m. In one experiment, 1 was varied from 0.15 to 6 m. The results (Fig. 3) indicate that dispersion of the sample in the mixing coil, as well as degradation of caffeine in alkaline solution, are negligible. For values of 1 below 0.50 m, the extraction seems to be incomplete. Extraction efficiency and size of segments The results in Fig. 3 immediately raise the question of vrhether the extraction process is complete or not for a certain design of system. In order to investigate this further, the following experiment was performed. The sodium hydroxide stream in the system was replaced by an aqueous caffeine solution, without introduction of air. The coil length, 1, was 1 m. The steady-state value of the absorbance was measured, so that the concentration of caffeine in the chloroform phase could be evaluated; for this . .. purpose, cahbntion soiutions (in chioroform j were pumped separately and directly into the flow cell in a previous experiment. The proportion of the flows of aqueous and organic phases was obtained by measuring TABLE Variation
2 of r, the distance
between
the valve .a.nd the mixing
(see Fig. 1) r
(m) 0.04 0.08 0.15 0.30 0.45 0.60 1.00
Peak maximum for caffeine (absorbance --. -__ -_-___.__ Caffeine 0.46 0.46 0.47 0.46 0.43 0.42 0.37
alone
--
units)
Caffeine + acetylsalicylic 0.46 0.46 0.47 0.46 0.43 0.42 0.37
acid
point of the two phases
6
the individual flow rates. The efficiency of the extraction was then calculated to be 101 + 5%~~the uncertainty being due to difficulty in measuring the exact flow rates. The extraction efficiency calculated for a coil length of 0.15 m (see Fig. 3) was about 75%. which is a remarkably high value. The size of the segments of organic and aqueous phases in the mixing coil could be varied by adjusting the inner Teflon tube in the A8 connector (Fig. 1). A variation of the segment size in the range l-10 mm had no influence on the peak height when a coil length of 2 m was employed. This is probably explained by the rapid and efficient extraction of caffeine. Hcwever, for other extractants the size of the segments is a very important parameter, and this will be reported in a later paper in this series. Ratio of organic to aqueous phase and flow rates The ratio of the organic phase to the aqueous phase affects the sensitivity of the method. Ideal conditions for a study of this ratio would be a set-up of three different pumps so that the flow of each stream, X. y, and z, could be varied individually (see Fig. 1). In a simplified study, y and t were kept constant while x was varied. The results are presented in Fig. 4. The test solution was a 1.04 mM ca.Jfeine solution and the valve volume was 25 ~1. The maximum peak height is obtained for a volume ratio of the phases of -tr-*.C 1.1 At.-..* CL;, ...,.... +S., rS.-r:*:rr;Cr.“l”YJ .-a-,.. A..P.&;-m 11.. _Vc\L.%hl.. a.u”“C I .I. zI”““t: ‘lfW .“alUtz IrilCW=l‘a,ci”lI+y u&Lu~lLaLay, t.“““““‘J because of the high total flow through the separator. The residence time in the separator is then shorter. Furthermore, the pressure in the system increases when the flow increases, and this may influence the
of the sample
i 2 L (ml m:n-‘) Fig. 3. Absorbance of peak maximum as a function Sample solution: ca. 8 x lo-. hl caffeine (aqueous). S = 25 rl;r = 0.15 m (see Fig. I).
of the length, I, of the mixing coil. x = 2.2. y = 2.0, z = 0.7 ml min-’ ;
Fig. 4. Absorbance of peak maximum as a function of the flow of the aqueous phase, x. %npfe solution: 1.04 X 10.“ 31 caffeine (3queous). y = 2.0,z = 0.7 m1 min-‘;S = 25 ~11: i = 2, r - 0.15 m (see Fig. I).
7
turbulence in the individual segments so that the extraction efficiency decreases. Reconstruction of the present separator will be necessary if the total flow rate is to be allowed to exceed 5 ml mm-‘; the optimum flow rate seems to be about 4 ml min-‘. Some experiments were done with Teflon tubes with an inner diameter of 0.5 mm. ‘The flow rates of the two phases must then be decreased, otherwise the pressure in the system becomes excessive. The results obtained with this narrower Teflon tube were not completely satisfactory, and the reason for this lies in the separator. A low consumption of organic phase is desirable for economical and environmental reasons, so work is in progress
to develop a new separator
suitable
for low flow rates.
Conclusions
The extraction system developed is very suitable for the simple extraction of caffeine from an aqueous solution. More advanced examples of applications are in progress. The possibility of performing extractions on very small volumes is extremely valuable in many cases, e.g. in the field of clinical chemistry where this parameter is often the limitation of an application. At present, a sample volume of at least 40-50 ~1 is required to fill the rotary valve, but this figure can certainly be reduced further, and so can the consumption of reagents. The small volumes of sample and reagent, in combination with the high sampling rate and good precision, are the real attractions of this system.
The authors are indebted for valuable discussions.
to Dr. P.-A. Johansson,
Astra Pharmaceuticals,
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