__________________________________________________CHAPTER 4 Super Critical Fluid Extraction, Separation of Cu(II) with 5, 17- dinitro

11, 23-dihydroxamic acid 25, 26, 27, 28 tetra-hydroxythiacalix[4]arene

and Trace Determination with ICP- MS.

120

Abstract

A rapid and reproducible method has been developed to measure trace amount of

copper using supercritical fluid extraction (SFE). Copper is extracted with Thia-

calix[4]arene hydroxamic acid (TC4HA) in dichloromethane by Supercritical

carbondioxide (SF-CO2) –dichloromethane (modifier) medium. The copper is extracted at

pH 4.0 and directly measured at λmax 317nm by spectrophotometry and also by ICP-MS.

The distribution ratio of copper was determined and the slope of log DM Vs TC4HA

concentration plot was found to be 1.0 which shows that extracted species in SF-CO2

extraction (SFE) give 1:1 Cu2+ and TC4HA complex. The extracted species both in SF-

CO2 extraction and solvent extraction were determined to be as [Cu(TC4HA)] complxes.

The copper is determined as low as nanogram level in presence of several cations and

anions. The effect of diluents, modifier concentration, temperature and pressure on the

extraction and separation of copper was discussed. The TC4HA has been successfully

used as carrier for efficient transport of Cu(II). Maximum transportation of Cu(II) was

observed for 20 min with t1/2 equal to 10.4 min.

121

Introduction

Lower viscosity and variable density of super critical fluids are primary

advantages of SFE over liquid-phase extraction. Mass transfer occurs more quickly and

efficiently in supercritical fluid than in liquid. Driven by the desire to limit in both

solvent usage and solvent waste generation, reduce analysis times, and increase

extraction efficiency, the practice of SFE has been growing at a rapid rate for several

years.

1-4Most of the published SFE works have focused on organic compounds and

studies have been published on SFE of metal ions5-9. One suggested approach of

extracting metal ions by SFE is to convert the charged metal ions into neutral metal

complexes by using chelating agents. Important requirements for the selection of suitable

chelating agents used in the complexation-SFE of metal ions includes high stability

constant of the metal complex, good solubility of the chelating agent and their metal

complex in supercritical CO . Transported in the supercritical CO . Supercritical CO2 2 2, as

a medium to transport metal ions has been investigated by many research facilities. Since

metal ions are only partly soluble in this non-polar medium, appreciable solubility of the

metal can be achieved only with a suitable derivatisation of the metal ions. Chelating

with fluorinated and non-fluorinated isologues of dialkyldithiocarbamates10– 12 and of

diketonates13,14, organophosphates15,16 and crown ethers17 has been exploited for complex

metal ions. .

Estimation of copper is usually carried out by flame18-20, graphite21-22 atomic

absorption spectrometry as well as chemiluminscence23 and electro thermal methods24, 25.

However, due to presence of copper in very low levels in environmental and bio-logical

122

samples different separation and preconcentration techniques such as liquid-liquid26 or

solid phase27-30, precipitation31, ion –exchange32 or floatation33 are necessary.

Such methods are time consuming, toxic and due to limited applicability there is

a need to develop a cleaner, non-toxic and environment friendly method for the

determination of metal ions. Supercritical fluid chromatography (SFC) using modified

supercritical fluid carbon dioxide, as the mobile phase is one such method, which has

recently gained importance in analysis of drugs, pharmaceuticals and even biological

fluids due to its unique capabilities and excellent features like low viscosity and high

diffusivity of the eluent. SFC provides fast mass transfer34,35 and thus allows high flow

rates combined with fast column equilibrium36,37. In addition, SFC has proven to be cost-

effective as minimum amount of organic solvent is required and replaces flammable toxic

solvents such as hexane, chloroform etc. Thus, the present technique was found to be a

useful alternative to the more conventional analytical techniques currently used in terms

of time consumption, sample preparation, selectivity and sensitivity. It also gives a

shorter overall analysis time, providing good chromatographic resolution with detection

limits similar to those obtained by LC and in some case more favorable than those of LC

with efficient separation38. Further, it has been noted that retention and selectivity in SFC

differ from those observed in commonly used RP-HPLC screening method as SFC can

reveal impurities not observed by RP-HPLC. Thus, the advantages shown for SFC were

speed, cost, and usage of lesser volume of toxic solvents, eco-friendliness and absence of

pollution.

Hydroxamic acids contain double coordination atoms (N, O-), which is easy to

react with Cu2+ and form stable neutral chelates. So, thia-calix[4]arene hydroxamic acid

123

was investigated as a chelating agent in the complexation-SFE of Cu2+ in our laboratory

and satisfactory results were obtained. This chapter represents that under the condition

which hydroxamic acids was selected as chelating reagent and DCM was used as

modifier for extraction of Cu2+ at desired column temperature and outlet pressure.

Moreover, transport of Cu(II) ions through a supported liquid membrane containing

TC4HA has been studied. The influence of pH, carrier concentration, temperature and

kinetics of transport is discussed.

Experimental

Chemicals

The solvent carbon dioxide gas 99.9% purity was purchased from the Bombay

Carbon Dioxide Co., Mumbai and used as such. All reagents were of analytical-reagent

grade of sigma-aldrich. Deionized water was used.

Reagent

Synthesis of 5, 17 dinitro 25, 26, 27, 28 tetra hydroxythiacalix[4]arene 11, 23

dihydroxamic acid (TC4HA) (Fig. 1) was synthesized as described in Chapter-2. Its 1.16 ×

10 M solution was prepared in dicholoromethane. -3

O HS

O H

SO H

S

OH

S

N

NO 2

NOH

O

OHO

O 2 N

Fig. 1 5, 17 dinitro , 25, 26, 27, 28 tetra hydroxythiacalix[4]arene 11, 23

dihydroxamicacid(TC4HA)

124

Metal solution

A standard copper solution was prepared by dissolving 0.3110 g of cupric

sulphate pentahydrate in 250 ml of double-distilled water. Its final concentration 4.18 x

10-3 M was determined by EDTA titration39 and ICP-MS.

Apparatus

SFE Instrumentation

The SFE apparatus was JASCO Supercritical Fluid Extractor / Chromatograph,

900-series configured for dynamic mixing with a two-pump system. It has the facility of

on line organic co-solvent addition to supercritical fluid gas. The flow rate of the pump

can be operated from 0.01 to 10.0 mL min-1. First, the CO2 gas cooled at -10ºC before

compression takes place using CH-201 series cooling circulator. Methanol was used as

cooling solvent JASCO-880-81 back pressure regulator used is capable of giving pressure

in the range of 9.5-45 MPa. JASCO-965 series air circulating oven was used for

controlling and maintaining the temperature of the extraction vessel. Special stainless

steel vessel with 1 mL to 15 mL capacities were fabricated to a maximum operating

pressure of 7000 psi with temperature accurately controlled in the range of 45 to 50° C

and also fitted with rheodyne valve. The extract was collected in an especially fabricated

glass vessel. Detection was done using a JASCO- UV-975 detector.

125

ICP-MS: Plasma Scan Model 710 Sequential plasma, Inductively Coupled Mass

Spectrophotometer with Plasma Scan multitasking computer and peristaltic

pump was used.

Optimized operating conditions for ICP-MS:

Instrumental parameters ICP-MS

ICP Plasma Argon

Forward Power 1.35 KW

Reflected Power <10 W

Gas flow (L/min)

Coolant 16

Carrier 0.70

Auxiliary 0.30

Nebuliser Pressure 2 bar

Solution uptake rate 0.8 mL/min

Sampler cone aperture 1 mm

Skimmer cone aperture 0.7 mm

Mass Number 63,65

UV-Spectrophotometer

JASCO 6300 double beam UV-VIS-NIR spectrophotometer with matched 1 cm

quartz cell was used for spectral measurements. A Labindia pH analyzer Model 6E488

was used for pH measurements.

126

SF-CO2 extraction (SFE)

The extraction studies were performed in a dynamic mode at the optimum flow

rate of the gas and the co-solvent. The SF-CO2 gas, at a known pressure and temperature,

was mixed with 1.16 × 10-3 M TC4HA in DCM and allowed to flow over the extraction

vessel. A solution containing copper with a volume of 5 μl was placed in the liquid

extraction vessel with 5 μl of buffer solution (pH 4.0) along with the ceramic fiber glass

needles and 1.16 × 10-3 M TC4HA in DCM as co-solvent fluid phase.

Determination of Cu(II)

-1Cu(II) solution of 10 ng ml transferred to 20 ml extraction vessel and Cu(II) is

extracted using dichloromethane solution of TC4HA after adjusting the pH of the aqueous

phase to 4.0 with acetate buffer. The organic layer is separated and Cu(II) is determined on

UV-Spectrophotometer.

Transport condition

2+Transport of Cu was carried out in a very special glass assembly as shown in the

figure. The reaction cell was about the capacity of 250 mls. A tube having a membrane was

settled on the upper side of the glass assembly. The transport was carried out by 10 ml of

4.18 x 10-5 M of copper solution at pH 4.0 as a source phase and 20 ml of 0.1N HNO3 as

receiving phase. The liquid membrane consists of 1 x 10-3 M TC4HA Solution. Continuous

reproducible stirring was carried out from top with mechanical stirrer and from bottom with

magnetic stirrer. The amount of copper transported was measured by ICP-MS.

127

Result and Discussion

The copper is extracted with DCM solution of TC4HA, which gives the Colorless

complex, λmax 317 nm, molar absorptivity 4.2 × 104 l mol-1 cm-1 and obeys Beer’s law in

the range of 1.2–20 μg ml-1 -1 and detection limit is 0.01 ng ml . After SFE, the extracts are

directly inserted into the plasma for ICP-MS measurement, which enhances the sensitivity

with detection limit up to ng level.

Effect of pH and extraction time

The optimum pH for maximum extraction was determined by carrying out the

extraction with various concentrations of copper and TC4HA. Moreover, the pH of the

aqueous phase was varied using the different buffer solutions. The extraction of copper

increases with the increase of pH until it level off at the pH 4. Thus the optimum pH for

an efficient extraction values lies within the range of 4.0–4.75 pH for copper (Table 1).

The low extractability at lower pH values may be attributed to the proton extraction to

organic phase rather than the metal ion itself. The extraction of copper by TC4HA was

very quick usually 1–1.5 min of oscillation was sufficient for complete extraction

equilibrium.

Effect of extractant concentration

-3The ligand concentration used was of 1.16 x 10 M as a stock and diluted further

to required concentrations. It was observed that percentage extraction increases with

increase in extractant concentration. The log D vs –log C plot is linear with a slop 1.04

indicating the association of 1M of the extractant per mole of Cu(II) in the extracted

metal species.

128

Effect of the solvent

The extraction experiments were carried out with pure and modified SF-CO2.

Extraction efficiencies were examined with pure and modified SF-CO2 with various

solvents viz chloroform, n-octanol, DCM etc. It has been observed that DCM modified

SF-CO2 was found to be the most appropriate solvents for quantitative extraction of

Copper (Table 3).

Table 1 Effect of pH on the extraction of Cu(II)- TC4HA complex

Cu(II) -12.6 μg ml Pressure 15 MPa Fluid/Co2 Flow 0.1 ml/1.0 ml

TC4HA -3 1.16 X 10 M Temp 45°c Buffer Acetate

Extraction pH

% ε* (l mol-1 -1 cm )

3.0 69 2.9 x 104

3.5 83 3.5 x104

4.0 100 4.2 x104

44.5 88 3.7x10

5.0 76 3.2 x104

45.5 71 3.0x10

ε* Molar Absorptivity

129

Table 2 Effect of reagent concentration on Cu(II)- TC4HA complex

Cu(II) -12.6 μg ml Pressure 15 MPa Fluid/Co2 Flow 0.1 ml/1.0 ml

TC4HA -3 1.16 X 10 M Temp 45°c Buffer Acetate

Cu(II) TC4HA

(10-4M)

-log

(TC4HA) [Cu]org × 10-5

Log

+2(D ) Cu[Cu]aq × 10-5

0.232 4.63 3.97 0.219 1.25

0.464 4.33 4.08 0.116 1.54

0.696 4.15 4.12 0.078 1.72

0.928 4.03 4.10 0.021 1.89

y = -1.0455x + 6.0846R2 = 0.9952

11.11.21.31.41.51.61.71.81.9

2

4 4.2 4.4 4.6 4.8

-log C

log

D

Fig 1 Plot of log D Vs –log [TC4HA]

130

Table 3 Effect of pure and modified SF-CO2 in the extraction of Cu(II)-TC4HA

complex

Extraction Solvent

% ε* (l mol-1 -1 cm )

4Chloroform + SF-CO (20 MPa) 30 1.2 x102

Dichloromethane + SF-CO2 (20 MPa) 100 4.2 x 104

4n-octanol + SF-CO (20 MPa) 24 1.1 x 102

Toluene + SF-CO (20 MPa) z* z* 2

ε* Molar Absorptivity z* No Extraction

Effect of pressure and temperature

Varying the pressure and temperature significantly affects extraction efficiency.

The results show that increasing the pressure and temperature of system increases

%extraction. This may be due to increasing solubility of TC4HA and its Cu(II) complex.

Its concluded that 20 MPa pressure and 45°C is adequate for quantitative extraction of

Cu(II) with TC4HA using SF-CO . 2

131

Stoichiometry of Cu(II) with TC4HA:

The TC4HA dissolves in SF-CO2-n-octanol at pH 4.0 and the ligand extract

Cu(II) efficiently from aqueous solution of pH 4.0. The distribution behavior of Cu(II) in

SFE with TC4HA (2HA), can be represented as

SF-CO2

Aq. Phase

2 HA . CO2

Cu+2A2 . CO2 (Cu+2 . A2) : CO22CO TC4HA.

2 2 2HA H A+↔ + −

+

…..(1)

2 2[ ] 2 [ ] ( )[ ] 22 2 2C u aq H A C O H C u A C O H

+ ++ → + ..(2)

The distribution of copper is given by

22

22 2

H C u AC O

DC u C u

aq

⎡ ⎤⎢ ⎥ ⎡ ⎤⎣ ⎦

⎢ ⎥⎣ ⎦⎡ ⎤⎢ ⎥⎣ ⎦

⎡ ⎤⎣ ⎦

+

=+ + …..(3)

The composition of the Cu(II)-TC4HA complex extracted into dichloromethane

and SF-CO 2logCu

D +has been studied plotting Vs –log[TC4HA]2 org

, which gives the

straight line with a slope of 1.0 shows the stoichiometry of the complex to be 1 : 1.

Method confirm one moles of the ligand (TC4HA) is required for one mole of Cu(II) and

the expected structure of the complex can be of the type [H(Cu2+A )]. 2

132

Interference study:

The effect of various cations and anions that can influence the performance of the

present method has been studied. Interference studies were made by using the solution

containing 10 ng ml-1 copper and various concentrations of the foreign ions. The intensity

of the organic phase was measured by ICP-MS.

Table 5 Effect of Diverse Ions and selectivity factor on extraction of Cu(II)–

TC4HA comple x

Cu(II) -110 ng ml Pressure 15 MPa Fluid/CO Flow 0.1ml/1.0ml 2

TC4HA -31.16 X 10 M Temp. 45°c λ max 317nm

Cu(II) found (μg ml-1) Amount Foreign ion Added as

added (mg) Spectrophotometry ICP-MS

+ 9.997±0.05 Ag Ag(NO3) 40 9.998± 0.03 2

+Ca CaCl 40 9.997±0.05 9.99 ± 0.08 2

+2An ZnSO 40 9.997±0.05 9.98 ± 0.07 4

CO+2 CO(NO ) 40 10.03 ± 0.04 10.008 ± 0.003 3 2

+2Ni NiCl 40 9.98 ± 0.02 10.005 ± 0.005 2

Mn+2 MnCl 50 9.97 ± 0.04 9.998± 0.004 2

Mg+2 MgSO 50 10.05 ± 0.05 9.995± 0.006 4

+2Hg HgCl 50 9.98± 0.03 9.997± 0.005 2

+2Pb Pb(NO ) 60 9.98± 0.04 9.997± 0.005 3 2

+3Al AlCl 40 9.97± 0.04 9.005± 0.007 3

133

134

Fe+3 FeCl3 50 10.01± 0.03 9.993± 0.008

Ba+2 BaCl2 50 10.03± 0.05 9.003± 0.005

Th+4 Th(NO3)4 50 9.98± 0.03 9.994± 0.005

Cr+ CrCl3.6H2O 60 9.99± 0.02 9.995± 0.005

V+5 NH4VO3 60 10.01± 0.02 10.001± 0.003

La+ La(NO3)3 40 10.05± 0.03 9.999± 0.001

Transportation of Copper:

The mechanism of the transport of Cu(II) from membrane has been systematically

represented in Figure 8. The copper ions are absorbed into the membrane due to complex

formation with TC4HA at the interface between the membrane and receiving solution

where Cu+2 is released to aqueous phase due to the stripping action of H+ ion. The free

carrier diffuses back to the interface at the feed side to form another complex with

available fresh ions. The effect of pH variation of the feed solution on the permeation of

copper was also studied moreover, membrane study was also carried out for maximum

transportation of ions. Results show that 0.1M HNO3 is ideal for quantitatively stripping

of Cu+2 ions. The amount of Cu(II) was measured by ICP-MS. As a proof fig. 3 shows

that concentration of cu+2 is started to decrease continuously in a feed phase and reach

lower than the detection limit after 20min.On the other side concentration of metal is

continuously increasing in receiving phase. It clearly indicates that metal ions are

transported through membrane from source phase to receiving phase within the given

period of time. It has been observed that maximum transport of Cu(II) is observed till 20

min with t1/2 = 10.4 min.

Source Phase

Mechanical Stirrer

Recieving Phase

Magnetic Stirrer

Liquid Membrane Phase

Fig 2 . Transportation Cell

0

10

20

30

40

50

60

70

80

90

100

0 5 10 15 20 25 30

Time (Mins)

%C

u(II)

Recieving Phase Feed Phase

Fig 3. Transport Profile for Cu+2 through the membrane phase containing TC4HA at

30°C

135

Table 6 Determination of Cu(II) in Food, Biological and Environmental samples.

a -1Cu(II) found (μg ml ) Sample

136

Spectrophotometry ICP-MS

Human Hair (Male) 12.50 ± 0.08 12.449± 0.0002

Human Hair (Female) 23.50 ± 0.08 23.502 ± 0.0003

Tea Leaves 0.049 ± 0.001 0.0488 ± 0.0001

PepperBush 0.0566 ± 0.002 0.0569 ± 0.0002

Chlorella 3.301 ± 0.01 3.299 ± 0.0001

Wheat Flour 6.212 ± 0.04 6.214 ± 0.0002

Potatoes 4.694 ± 0.01 4.701 ± 0.0001

Carrotes 2.530 ± 0.04 2.531 ± 0.0002

Table 7 Determination of copper in Spiked Water Samples

Cu(II) found (μg ml-1) Sample Spiked Value of

Cu(II) (μg ml-1) Found Value (Recovery%)

ICP-MS

+Tap Water 10 100% 10 0.558

Sea Water* 10 99.8% 9.98± 0.412

+Mineral Water 10 99.9% 9.99 0.05

* Sea water from Apollo Bandar.

137

Conclusion:

A new reagent (Fig.1) was synthesised by the introduction of the thia-calixarene

derivative in to the functionality of hydroxamic acid which gave a chelating system used

successfully for the extraction and determination of Cu(II).

The SFC significantly improved sensitivity in ICP-MS compared to many other

methods and allows Cu(II) to determine with detection limit 10.0 ng ml-1 and separate it

in presence of foreign substances. Moreover, Cu(II) are separated and extracted from

environmental and biological samples with recovery of 99.9%.

138

Reference:

1. Ding M Y, Chinese Journal of Chromatography, 15 (1997) 527-529.

2. Yue L H &6 Wai C M, Trends Anal Chem, 14 (1995) 123-132.

3 Holst C V & Wenclawiak B W, Anal Chem., 69 (1997) 601-606.

4 Meguro Y., Lso S, Sasaki T & Yoshida Z, Anal Chem, 70 (1998) 774-779.

5 Waind C M & Yue L H, Talanta, 40 (1993) 1325-1330.

6 Lin Y, R. Brauer D, Laintz K E & Wai C M, Anal Chem, 65 (1993) 2549-2551.

7 Wang S, Elshanl S & Wai C W, Anal Chem, 67 (1995) 919-923.

8 Luque de C M D & Tena M T, Trends Anal Chem, 15 (1996) 32-37.

9 Laintz K E, Hale C D, Stark P, Wilkinson J & Rouquette C L, Anal Chem, 70

(1998) 400-404.

10 Laintz K E, Wai C M, Yonkar C R & Smith R D, Anal Chem, 64 (1992) 2875-

2878.

11 Liu Y, Lopez-Avila V, Alcaraz M, Beckert W F & Heithmar E M, J

Chromatogr Sci 31 (1993) 310-316.

12 Wang J & Marshall W D, Anal Chem., 66 (1994)1658-1663.

13 Lin Y & Wai C M, Anal Chem., 66 (1994) 1971-1975.

14 Lainz K E & Tachikawa E, Anal Chem, 66 (1994) 2190-2193.

15 Smart N G, Carleson T E, Kast T, Clifford A A, Burford M D & Wai C M,

Talanta, 44 (1997) 137-150.

16 Smart N G, Carleson T E, Elshani S, Wang S F & Wai C M, Ind Eng Chem Res,

36 (1997) 1819-1826.

17 Wang S F, Elshani S & Wai C M, Anal Chem, 67 (1995) 919-923.

139

18 Hashemi P & Bagheri S, Fathi M R, Talanta, 68 (2005) 72-78.

19 Ashtari P, Wang K, Yang X, Huang S & Yamini Y, Anal Chim Acta, 550

(2005) 18-23.

20 Cassella R J, Magalhães O I B, Couto M T, Lima E L S, Neves M A F S,

Coutinho F M B, Talanta, 67 (2005) 121-128.

21 Acar O, Talanta, 65 (2005) 672-677.

22 Cabon J Y, Spectrochim Acta B, 57 (2002) 939-950.

23 Lloret S M, Falcó P C, Cárdenas S, Gallego M & Valcárcel M, Talanta, 64

(2004) 1030-1035.

24 Szigeti Z, Bitter I, Toth K, Latkoczy C, Fliegel D J, Gunther D & Pretsch E, Anal

Chim Acta, 532 (2005) 129-136.

25 Hurst M P & Bruland K W, Anal Chim Acta, 546 (2005) 68-78.

26 Barrera P B, Piñeiro A M, Iglesias R G & Barrera A B, Spectrochim Acta B, 57

(2002) 1951-1966.

27 Kendüzler E & Türker A R, Anal Chim Acta, 480 (2003) 259-266.

28 Lemos V A & Baliza P X, Talanta, 67 (2005) 564-570.

29 Tokman N, Akman S & Ozeroglu C, Talanta, 63 (2004) 699-703.

30 Da Silva E L, Ganzarolli E M & Carasek E, Talanta, 62 (2004) 727-733.

31 Soylak M, Saracoglu S, Divrikli U & Elci L, Talanta, 66 (2005) 1098-1102.

32 Scaccia S, Zappa G & Basili N, J Chromatogra A, 915 (2001) 167-175.

33 Undeva K, Stafilov T & Pavlovska G, Microchem J, 65 (2000) 165-175.

34 Petersen M, J Chromatogr, 505 (1990) 3-18.

140

35 Macaudiere P, Tambute A, Caude M, Rosset R, Alembik M C & Wainer I W,

J Chromatogr, 371 (1986) 177-193.

36 Lee C R, Porziemsky J P, Aubert M C & Krstulovic A M. J Chromatogr, 539

(1991) 55-69.

37 Grere D R, Board R & McManigill D, Anal Chem, 54 (1982) 736-741.

38 Dhorda U J, Bari V R & Sundaresan M.. Methods in Biotechnology.

Williams J R & Clifford A A , supercritcal fluid methods and protocols.

In editors. New Jersey: Humana Press 5 14 (1999).

39 Vogel A I, “Vogel’s Text book of quantitative analysis-3rd Edn.” (1989) 971.