The experimental procedures were conducted at the Instrumental Analytical Chemistry Laboratory, in Chemistry Institute of the Federal University of Alfenas, UNIFAL-MG, Alfenas, Brazil.
Solutions and Reagents
The Cadmium solutions, used in the experiments, were prepared from the 1,000 mg L-1 stock solution (Sigma-Aldrich). To adjust the pH values of the samples, 1.0 mol L-1 sodium hydroxide (Merck-Sigma Aldrich) and, 0.1 mol L-1 nitric acid prepared by diluting 65% HNO3 (Merck-Sigma Aldrich), were used. All glassware was decontaminated using a 10% v/v HNO3 bath, for at least 24 h.
Carbon Fiber
The carbon fibers, figure 1, were produced by Dr. Jossano Saldanha Marcuzzo (FATEC-SJC, SP\ITA\INPE), which has equipment and expertise in the process of carbonization and activation of carbon fibers. The synthesis of carbon fibers had thermic treatment of PAN textile in 2 steps. The first step was at 200° for 50 min, and the second step at 300oC for 50 min. More details about the synthesis of this material are described in works by Marcuzzo et al. 2012 [17] and Marcuzzo, Otani 2015 [18].
Restrict Access Materials (RAM) Production
To cover the carbon fibers, 45 mL of a 1% BSA solution (w/v) in 50 mmol L-1 phosphate buffer (pH = 5.7) and 5 mL of a 1% glutaraldehyde aqueous solution were percolated (v/v) on an SPE cartridge containing 200 mg of carbon fiber. In this step, glutaraldehyde, a bifunctional crosslinking agent, established interconnections between the amine groups of the protein chains, fixing the BSA layer around the ACF. After a period of 5 h, 10 mL of a 1% sodium borohydride aqueous solution (w/v) was percolated through the cartridge to reduce the imines (originated from the previous reaction) to amines, which are more chemically stable groups, thus providing chemical stability to the material. Finally, the material was washed with water to remove the remaining reagents from the coating process, according to the representative scheme in Figure 2.
Column to Solid Phase Extraction (SPE)
To prepare the column assembly (Figure 3) of weighed approximately 0.2 g of the Carbon Fiber material coated with BSA and packaging it in a tube from a disposable syringe for Insulin with a volume of 1 mL. The ends of the tube were filled with cotton fabric to avoid wasting material. Column dimensions were 2 mm inside diameter and 0.3 mm length.
FIA-SPE-ACF-RAM-FAAS
System
For cadmium determination, a FIA-SPE-ACF-RAM-FAAS system, shown in figure 4, was used. It was composed of a Shimadzu AA-6800 atomic absorption spectrometer (Shimadzu, Tokyo, Japan), equipped with a cadmium hollow cathode lamp (λ= 228.8 nm) and a deuterium lamp for background correction. The FIA system had a peristaltic pump (Ismatec IPC-08, Glattzbrugg, Switzerland), equipped with Tygon® tube to propel all reagent and solutions through the polyethylene canalization (0.8 mm i.d.). An acrylic injector commutator was used to select the wash and elution/sampling steps, during the procedures. The FIA-SPE-ACF-RAM-FAAS system, work in two steps. The first step start when the 20 ml of sample was conducted through the system and pass by column to adsorption of analyte (Cd). The second step starts when the system is commuted and the eluent solution pass through the column provoking the desorption of the analyte and lead it until FAAS for detection. After, the system is commuted again and repeat the cycle.
FIA-SPE-ACF-RAM-FAAS
Optimization
For this study, a two-level factorial design was used, getting a set of eight experiments carried out in duplicates and at random way. The pH of the solution (pH), the Pre-concentration Flow Rate (FR), the Eluent Concentration (EC) and their possible interactions were investigated. Table 1 shows the selected factors. The others experimental conditions for the procedure were cadmium concentration 50 µg L-1, elution flow rate of 7.5 mL min-1 and pre-concentration volume of 20 mL.
Table 1 Factor levels in planning 23
|
|
|
Levels
|
|
Initials
|
Factor
|
Minimum (-)
|
Maximum (+)
|
EC
|
Eluent Concentration (mol L-1)
|
0.1
|
1.0
|
FR
|
Pré-concentration flow rate (mL min-1)
|
3.5
|
7.5
|
|
pH
|
Sample pH
|
3.5
|
5.5
|
Levels
Experiment
|
Fatores
|
EC (mol L-1)
|
FR (mL min-1)
|
Sample pH
|
1
2
3
4
5
6
7
8
|
- (0.1)
+(1.0)
-(0.1)
+(1.0)
-(0.1)
+(1.0)
-(0.1)
+(1.0)
|
- (3.5)
- (3.5)
+ (7.5)
+ (7.5)
- (3.5)
- (3.5)
+ (7.5)
+ (7.5)
|
- (3.5)
- (3.5)
- (3.5)
- (3.5)
+ (5.5)
+ (5.5)
+ (5.5)
+ (5.5)
|
Font: From Autor
After the Factorial Design Experiments a Doehlert Matrix were performed. Table 2 shows the planning of the Doehlert Matrix, where the other experimental conditions used were: Cadmium concentration 50 µg L-1, elution flow rate 7.5 mL min-1 and pre-concentration volume 20 mL.
Table 2 Coded ( ) and actual levels of the Doehlert Matrix, for 3 factors used in the optimization of the Cd2+ on-line pre-concentration system in carbon fiber.
Factor
|
Coded and Real Levels
|
EC
(mol L-1)
|
|
(-1)
0.025
|
(-0.5)
0.06
|
(0)
0.1
|
(0.5)
0.15
|
(1)
0.2
|
|
pH
|
(-0.866)
2.5
|
(-0.577)
3.5
|
(-0.289) 4.5
|
(0)
5.5
|
(0.289) 6.5
|
(0.577)
7.5
|
(0.866)
8.5
|
FR
(mLmin-1)
|
|
|
(-0.817) 6.5
|
(0)
7.5
|
(0.817)
8.5
|
|
|
Experiments
|
Fators
|
EC
(mL min-1)
|
pH
|
FR
(mol L-1)
|
1
|
0.1 (0)
|
5.5 (0)
|
7.5 (0)
|
2
|
0.2(1)
|
5.5(0)
|
7.5 (0)
|
3
|
0.15(0.5)
|
8.5(0.866)
|
7.5(0)
|
4
|
0.15(0.5)
|
6.5(0.289)
|
8.5(0.817)
|
5
|
0.025(-1)
|
5.5(0)
|
7.5(0)
|
6
|
0.06(-0.5)
|
2.5(-0.866)
|
7.5(0)
|
7
|
0.06(-0.5)
|
4.5(-0.289)
|
6.5(-0.817)
|
8
|
0.15(0.5)
|
2.5(-0.866)
|
7.5(0)
|
9
|
0.15(0.5)
|
4.5(-0.289)
|
6.5(-0.817)
|
10
|
0.06(-0.5)
|
8.5 (0.866)
|
7.5(0)
|
11
|
0.1(0)
|
7.5(0.577)
|
6.5(-0.817)
|
12
|
0.06(-0.5)
|
6.5(0.289)
|
8.5 (0.817)
|
13
|
0.1(0)
|
3.5(-0.577)
|
8.5 (0.817)
|
*The first number represents actual values. numbers in parentheses represent encoded values. Font: From Autor
Sample collection and preparation
Whey samples from 7 different brands of milk were analyzed in the following categories: Skimmed milk (SM), Whole milk (WM), and semi-skimmed milk (SSM), all collected in supermarkets at the Alfenas city, MG, Brazil. The samples were stored in glass vials and analyzed within a maximum period of 24 h. To obtain whey, 1 mL of 0.1 mol L-1 nitric acid was added to each 100 mL of sample, which were then centrifuged and filtered. All samples had the pH adjusted to 6.2 with NaOH, 1 mol L-1 and/or HNO3 0.1 mol L-1, before analysis.