Investigation of a liquid–liquid extraction system based on non-ionic surfactant–salt–H2O and mechanism of drug extraction

Analytica Chimica Acta 452 (2002) 321–328

Investigation of a liquid–liquid extraction system based on non-ionic surfactant–salt–H2 O and mechanism of drug extraction Lei Li, Feng Liu∗ , Xiangxu Kong, Shun Su, Ke An Li College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China Received 30 May 2001; received in revised form 26 July 2001; accepted 19 October 2001

Abstract Based on the investigation of a non-ionic surfactant–salt–H2 O liquid–liquid extraction system, general rules concerning salt selection are concluded and the mechanism of phase separation is explained. The extraction behavior of chlorpromazine hydrochloride (CPZ) and procaine hydrochloride (PCN) in such a system is studied. Research shows that the extraction efficiency of CPZ can amount to 96% by twice extraction, which means that quantitative extraction is realized, while that of PCN is 77%. This system produces distribution coefficients (KD ) of 12.3 and 2.6, respectively, for CPZ and PCN. Extraction mechanism is deduced according to ultraviolet absorbance; and molecular fluorescence spectra change of the drugs in the system studied. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Non-ionic surfactant–salt–H2 O extraction system; Polyethylene glycol-1000; Chlorpromazine hydrochloride; Procaine hydrochloride; Mechanism of extraction

1. Introduction The utilization of a solid–liquid extraction system based on polymer–(NH4 )2 SO4 –H2 O for separation of metal ions [1–3] and bio-active substance [4,5] has been summarized [6], and the phase separation has been attributed to salting-out. Cloud-point extraction (CPE) is another technique that has drawn increasing attention in recent years. This method benefits the environment and has been successfully applied to separation of metal chelates, biomacromolecules and in pretreatment of environmental samples [7–14]. Maniasso and Ma have summarized the utilization of CPE in analytical chemistry [15] and in separation and analysis of biomacromolecules [16], ∗ Corresponding author. E-mail address: [email protected] (F. Liu).

respectively. Compared with the traditional organic liquid–liquid extraction system, solid–liquid extraction systems and CPEs are able to avoid volatile organic solvents, which may be toxic; another advantage is that one can extract charged species in water quickly. Separation of drugs is usually conducted through thin-layer chromatography, liquid chromatography (LC) and capillary electrophoresis. In this paper, the poly(ethylene glycol) (PEG)-1000–salt–H2 O system is proposed for the first time and applied to the separation for chloropromazine hydrochloride (CPZ) and procaine hydrochloride (PCN); rules concerning salt selection and the mechanism of phase separation are discussed; extraction behavior and mechanism for drugs in such system are investigated. The two liquid phases are easy to produce according to the procedure described in this paper. Besides the advantages shown

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in the solid–liquid system, better reproducibility and convenient continuous extraction are achieved.

3. Results and discussion 3.1. Phase separation conditions for PEG-1000 solution

2. Experimental 2.1. Apparatus and reagents A Shimadzu UV-265 spectrophotometer and a Shimadzu UV-120-02 spectrophotometer were used for the determination of UV absorbance. A Shimadzu RF-540 fluorescence photometer was used for molecular fluorescence measurement. A Model-821 pH meter (Zhongshan University, China) was used for the pH measurement. Poly(ethylene glycol) (PEG)-1000 imported from Japan by Guangzhou Chemical Reagents Company, was prepared in a 50% water solution. Chlorpromazine hydrochloride (CPZ) and procaine hydrochloride (PCN) (Sigma, 99%), were prepared as water solutions of 1.00 mg ml−1 . A series of Britton–Robinson (B.R.) buffer solutions containing phosphoric acid, acetic acid, and boric acid, each with a concentration of 0.2 mol l−1 at pH 2.0–7.0 [17], and organic and inorganic salts containing K+ , Na+ , NH4 + , all of analytical reagent grade were used. 2.2. Procedure for the determination of drugs In a 10 ml color comparison tube, 4.0 ml of PEG-1000 solution, a suitable amount of drug and 2.0 ml of B.R. buffer solution of pH 5.5 were added. The contents were diluted to the mark with water and mixed thoroughly. (NH4 )2 SO4 (2.5 g) was added and dissolved by shaking the tube. Two liquid phases formed completely after 10 min. The upper phase (containing PEG and the drug mainly) was removed with a syringe completely into another 10 ml color comparison tube, 2.0 ml of pH 5.5 buffer solution was added and the contents were diluted to the mark with water. The absorbance was measured at λmax = 254 nm for CPZ and λmax = 290 nm for PCN, with a reference of blank PEG solution (without drug) prepared in the same way. Once again 4.0 ml PEG solution was added into the lower phase (containing water and salt mainly). The second extraction can be achieved by repeating this process.

3.1.1. Effect of salt Through the experiment on formation of two phases with 4.0 ml of PEG-1000, 2.0 ml of B.R. buffer solution (pH = 5.5), and different salts, we observed the effect of common anions like Cl− , NO3 − , SO4 2− , C2 O4 2− , C4 H4 O6 2− (tartrate), CO3 2− , PO4 3− , and C6 H5 O7 3− (citrate) paired with K+ , Na+ , and NH4 + . Some of them cannot form the two phases system when the concentration of the salt ≤0.5 g ml−1 , such as Cl− , NO3 − ,C2 O4 2− , C4 H4 O6 2− (NH4 )2 CO3 , Na3 C6 H5 O7 , Na2 HPO4 , and Na3 PO4 . Some can form two liquid phases when the salt concentration is 0.15–0.5 g ml−1 , such as Na2 SO4 , (NH4 )2 SO4 , (NH4 )2 HPO4 , Na2 CO3 , K2 CO3 , and (NH4 )3 PO4 . General rules concerning salt selection can also be deduced from the experiment: 1. Salts containing K+ , Na+ , NH4 + are used for their better solubility and less interference with the substance separated; 2. Salts containing Cl− , NO3 − are seldom used since they have little effect on the formation of two phases; 3. When there are various choices for formation of two liquid phases, those salts which form two phases at a lower concentration at the proper pH and which have no interference with the measured drug are used; 4. Salts with alkalinity such as Na2 CO3 and K2 CO3 are alkaline in the surfactant phase and harm the separation, so these salts are not used despite of their good phase separation ability. At room temperature, the amount of salt needed to transform the PEG-1000 solution into two liquid phases depends on the following factors: concentration of PEG-1000, type of salt used and acidity of the solution. Fig. 1 shows the phase diagrams of the system based on PEG-1000 and various salts. With a constant concentration of PEG-1000, the amount of salt need for separation is dependent on the type of salt used. Fig. 1 shows that (NH4 )3 PO4 can produce a better phase separation at relatively lower concentrations of PEG and the salt, which proved to be a good extraction

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Fig. 1. Phase diagrams of PEG-1000–salt–H2 O system at pH 5.5.

system. However, (NH4 )2 SO4 is used as it has no UV absorption. When 2.0 ml of 0.15 g ml−1 (NH4 )2 SO4 solution is added, the PEG and water phases tend to be completely separated, thus the concentration of PEG-1000 is recommended to be 20% in the final volume.

3.1.2. Effect of pH According to the method described in 2.2, experiments at different pH value (HCl at pH 0.0–1.0; B.R. buffer at pH 2.0–7.0) were carried out. As seen in Fig. 2, when pH < 5.0, the concentration of (NH4 )2 SO4 needed to form two phases is proportional

Fig. 2. Relationship between pH and concentration of (NH4 )2 SO4 necessary for phase separation PEG-1000(mass%), 20%.

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to the pH. From pH 5.0 to 7.0, the concentration of (NH4 )2 SO4 needed is constant. A conclusion can be drawn that with increase in acidity, the poly(ethylene glycol) chains tend to form onium ions with H+ , and SO4 2− tends to protonate, both of which lead to an increase in the apparent concentration of salt. Thus, pH 5.5 is chosen as the recommended phase separation condition.

ancy dominates, which leads to the formation of two liquid phases. If the formula weight of the surfactant or the concentration of the salt increases, liquid–solid phase separation will occur [6]. Thus, the reason for surfactant–salt–H2 O phase separation is because the salt added changes the surround of the micelles, which results in the aggregation of micelles. 3.3. Distribution of drugs in the liquid–liquid system

3.2. Mechanism of phase separation for PEG-1000 solution As described in [1–6], solutions of polymers form two phases owing to salting-out. Generally, this effect is greater when the ionic radius is larger and the electronic charge is greater. However, the results of experiment do not strictly follow this principle. Since PEG-1000 is a non-ionic surfactant, we can explain the result with a phase separation model [18], structural characteristics and the cloud-point constant of the non-ionic surfactant [19]. According to the phase separation model: micellization is a type of phase separation effect. When the concentration of the surfactant reaches the critical micelle concentration (CMC) (the concentration of surfactant >CMC. in the liquid–liquid extraction system), the solution’s conductivity and surface tension and other similar characteristics will experience a sharp change, meaning a new phase is separated. The PEG chain of the non-ionic surfactant has a zigzag form without water and is random in water solution. Its hydrophilic feature is due to the hydrogen bonds formed between the oxygens in the ether bonds and the hydrogens in water. So when it is flexible in water, the hydrophilic oxygen will be outside the chain while the –CH2 – will be within the chain, which makes it easier for the chain to bind with water. Since the binding energy of a hydrogen bond is very low, when the salt dissolves, the water combined will disperse gradually [20], which decreases the hydrophilic property. When the ions reach a certain concentration the hydrogen bond will be eliminated. At the same time, the molecules of surfactant will coagulate and the micelle mass will increase. With decrease in CMC, the increase of micelle mass will be clearer. When the cloud-point is reached, the micelle will be large enough to be observed. With addition of more salt, the weight gain will continue. At this time buoy-

3.3.1. Effect of drug concentration on extraction efficiency Using the method described in Section 2.2, the drug concentration in the PEG phase or in the water phase is measured. The respective concentration of drugs CE is deduced from the linear equations (see Table 1 for regression equations of standard curves of drugs measured. Beer’s law is obeyed in the range 0–50 ␮g ml−1 for CPZ and 0–120 ␮g ml−1 for PCN.) The extraction efficiency (E%) was calculated as CE VE /(C E V E + C W V W ) (where CE and VE are the concentration and volume of drug in the PEG phase, CW and VW are the concentration and volume of the drug in the water phase, respectively). The results are listed in Table 2. With increase of the concentration of the drugs in the range 4.0–30.0 ␮g ml−1 , little variance in extraction efficiency of CPZ and PCN is observed. The average extraction efficiencies are E¯ CPZ = 85% and E¯ PCN = 55%. If extraction is conducted twice on  CPZ, the extraction efficiency E¯ CPZ increases to 96%, so essentially quantitative extraction is reached. The  twice extraction efficiency of PCN is E¯ PCN = 77%. 3.3.2. Determination of distribution coefficients (KD ) for CPZ and PCN in PEG-1000–(NH4 )2 SO4 –H2 O system Determination of the drug concentration in the PEG phase (CE ) and in the water phase (Cw ) respectively, Table 1 Regression equations for calibration of CPZ and PCNa Drug

Regression equation

Regression coefficient (n = 5)

CPZ PCN

AP = 0.0134 + 0.0833CP AP = 0.0178 + 0.0644CP

0.9999 0.9999

a C and A are the concentration of drug added (␮g ml−1 ) P P and the corresponding absorbance, respectively.

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Table 2 Effect of drug concentration on extraction efficiency Extraction efficiency (%) a

ECPZ  b ECPZ EPCN a  b EPCN

Drug (␮g ml−1 )

R.S.D.c (%)

4.0

6.0

8.0

10.0

12.0

16.0

20.0

24.0

30.0

83 94 56 76

82 92 53 78

85 95 54 76

86 94 53 74

82 96 55 77

89 99 56 79

89 101 54 80

88 99 55 76

84 96 56 78

3.3 3.0 2.2 2.3

a

ECPZ , EPCN -once extraction efficiencies.   ECPZ , EPCN -twice extraction efficiencies. c R.S.D. is relative standard deviation. b

Table 3 Distribution coefficients (KD ) of CPZ and PCN Distribution coefficient

Drug (␮g ml−1 ) 4.0

6.0

8.0

10.0

12.0

14.0

16.0

18.0

24.0

30.0

KDCPZ KDPCN

12.2 2.4

11.8 2.2

12.4 2.4

12.6 2.4

12.4 2.6

22.0b 2.8

44.9b 2.6

2.8

2.6

2.8

a b

KD (average value)

R.S.D.a (%)

12.3 2.6

2.5 7.9

R.S.D. is relative standard deviation. Not included in R.S.D. and KD .

is carried out according to the method described in Section 2.2. The distribution coefficient KD of this system at pH 5.5 can then be calculated as CE /Cw [21]. The results are listed in Table 3. The average values of the distribution coefficients shown in Table 3 are KDCPZ = 12.3 and KDPCN = 2.6, so KDCPZ > KDPCN , which means that the extraction efficiency of CPZ is greater than that of PCN in this system. Within the range 4.0–12.0 ␮g ml−1 , KDCPZ is nearly constant, while for 4.0–30.0 ␮g ml−1 , KDPCN is almost constant, too. Thus, with a specified range of drug concentration the KD value is characteristic of a given drug and PEG system and is dependent on temperature. 3.3.3. Effect of pH on extraction efficiencies of CPZ and PCN The concentrations of CPZ and PCN were fixed at 10 ␮g ml−1 , B.R. buffer at pH 2.0–7.0 added, and the concentrations of CPZ and PCN in the PEG phase were measured. The results show that extraction efficiencies are constant in the pH range studied (Table 4), which means that there is constant dissociation equilibrium of each drug in the PEG phase.

3.3.4. Effect of (NH4 )2 SO4 concentration on extraction efficiencies of CPZ and PCN The concentrations of CPZ and PCN were fixed at 10 ␮g ml−1 at pH = 5.5, different amounts of (NH4 )2 SO4 added, and the concentrations of CPZ and PCN in PEG phase were measured. The results show that when (NH4 )2 SO4 < 0.15 g ml−1 , a homogeneous phase is formed; when (NH4 )2 SO4 is 0.15–0.35 g ml−1 , a satisfactory liquid–liquid two phase system is formed and the extraction efficiencies of the drugs are constant; when (NH4 )2 SO4 is 0.35–0.50 g ml−1 , the PEG phase is cloudy and the interface boundary unclear; when more salt is added two liquid–solid phases are formed. Therefore, 0.25 g ml−1 (NH4 )2 SO4 was chosen as a phase separation condition.

Table 4 Effect of pH on extraction efficiencies of CPZ and PCN Extraction efficiency (%) ECPZ EPCN

pH 2.0

3.0

4.0

5.0

6.0

7.0

81 54

82 54

82 55

82 55

82 55

82 55

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Fig. 3. The structures of PCN and CPZ.

Fig. 4. The absorption spectra of drugs (C = 10 ␮g ml−1 pH = 5.5): (1) CPZ in water phase (water as blank), λmax = 254 nm; (2) CPZ in 20% PEG phase (20% PEG as blank), λmax = 256 nm; (3) PCN in water phase (water as blank), λmax = 290 nm; (4) PCN in 20% PEG phase (20% PEG as blank), λmax = 295 nm.

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3.4. PEG-1000–(NH4 )2 SO4 –H2 O liquid–liquid extraction mechanism for CPZ and PCN CPZ has two unconjugated benzene rings, while PCN has a conjugated carboxyl group on the benzene ring (see Fig. 3). Thus, in water solution the maximum absorbance of CPZ is at 254 nm while that of PCN is at 290 nm. In the PEG phase λmax of CPZ is 256 nm while that of PCN is 295 nm, but the absorbance of CPZ and PCN are stronger than in water solution(see Fig. 4). This is caused by hydrogen bonds formed between PCN and PEG. The ether oxygen atom in PEG forms a hydrogen bond with the hydrogen atom in –NH2 , which leads to the redistribution of the electron cloud within the PCN molecules, a decrease of ␲–␲∗ electronic transition energy, and a red shift of absorbance. Since the ␲ electron in CPZ is less mobile and has a weaker ability to form intermolecular hydrogen bond, less red shift is observed. It is seen from the fluorescence spectra of the drugs (Fig. 5), the fluorescence intensities of the two drugs are greater in the PEG phase than in water phase, which proves that both the drugs can form hydrogen bonds with PEG, so that the molecular rigidities are strengthened, and therefore, the fluorescence intensities are enhanced. However, it can also be inferred

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from Fig. 5 that the fluorescence intensity of PCN is greater than that of CPZ, both in PEG and water phase. It can be concluded that PCN has a better ability to form intermolecular hydrogen bonds. Thus, consistent conclusions have been made according to the UV spectra and the fluorescence spectra of the drugs in both water and PEG phase. Hydrogen bonding and hydrophobic interactions in the extraction system directly affect the distribution coefficients and the extraction efficiencies of the drugs. PCN forms stronger hydrogen bonds and has weaker hydrophobic interactions, thus hydrogen bonding is the major effect and the hydrophilic property of the drug is stronger, which results in a smaller distribution coefficient and extraction efficiency; CPZ forms weaker hydrogen bonds and has stronger hydrophobic interactions, thus the major effect is the hydrophobic interaction and the lipophilic property of the drug is stronger, which results in a greater distribution coefficient and extraction efficiency.

4. Conclusion In this paper, general rules concerning salt selection for two-phase separation are deduced, which could be a guide for future selection of extraction systems. It is clear that phase separation is caused not only by salting-out, but also by micelle growth due to the change of micelle surroundings after the addition of salt. In the system PEG 1000–(NH4 )2 SO4 –H2 O, the distribution coefficient and the extraction efficiency depend on the ability of the drug to form hydrogen bonds with PEG and the hydrophobic characteristics of the drug. Undoubtedly, it is possible to apply the liquid–liquid extraction system, reported in this paper, as a pretreatment process for instrumental analysis, such as LC, flow injection and capillary electrophoresis. References

Fig. 5. Fluorescence spectra of the drugs (λEX = 210 nm C = 10 ␮g ml−1 pH = 5.5), (1) CPZ in water phase (water as blank); (2) PCN in water phase (water as blank); (3) CPZ in 20% PEG phase (20% PEG as blank); (4) PCN in 20% PEG phase (20% PEG as blank).

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