**2. Sample preparation and analytical techniques**

## **2.1 Sample pre-treatment**

The samples must be treated prior to analysis. The particle size of thermoformed samples are reduced via mechanical methods, starting with manual cracking; followed by maceration (**Figure 3**) to a particle size of less than 1,135 mm; and finally, the removal of water content by placing the particles in an oven at 70 °C for 3 hours [54].

For flexible film samples, all that is required is manual cutting as shown in **Figure 4**, and drying at 50 °C for 45 minutes [54–58].

The samples' moisture content can be determined in triplicate by calculating the weights obtained before and after drying (see Eq. (1)).

$$\text{Moisture} \left( \% \right) = \frac{\text{Moisture sample weight} - \text{Dry sample weight}}{\text{Moisture sample weight}} \ast 100 \tag{1}$$

Following drying in a furnace, the moisture values for thermoformed samples were between 3.71% and 5.80% (RSD lower than 3.4%). Biodegradable flexible films revealed higher values of moisture (following 45 mins of drying in a furnace) at 7.81–10.35%, with relative standard deviation (RSD) lower than 1.96% [54].

### **2.2 Optimization of acid digestion using the reflux system**

When an analyte cannot be determined, it must be transformed to a state in which an appropriate identification and quantification technique can be applied.

**Figure 3.** *Sample: Thermoforming and maceration.*

*Cadmium Contents in Biodegradable Films Made from Cassava DOI: http://dx.doi.org/10.5772/intechopen.96848*

**Figure 4.** *Sample: Flexible films and cutting.*

These transformations are usually dissolutions or digestions, which involve the sample passing from a solid state to a liquid one, using the correct solvent (acids or bases of different strength, oxidizing agents, or enzymes, etc.); in this energetic process, heating and agitation increase the speed of mass transfer, unifying the decomposition and elimination of organic matter, and leaving the trace components of interest (metallic ions) in solution [59, 60].

In this procedure, the organic matter is destroyed by a wet process and the sample is digested (with an acid/oxidant mixture), combining sulfuric, nitric, perchloric acid or hydrogen peroxide, in a system that can be open or refluxed [61];

This technique is generally preferred to dry oxidation (where the sample is heated to 450–500 °C), as the presence of large volumes of acids produces less loss of trace elements through evaporation. However, there is also the danger of elements being lost by evaporation (antimony, arsenic, boron, chromium, tin, germanium, mercury, osmium, rhenium and selenium), although these losses can be prevented by adjusting the conditions (mounting of reflux, temperature control and time) [59, 60].

A wide range of acid digestion procedures are reported in the literature, in which various mixtures of inorganic acids and, in some cases, hydrogen peroxide have been used (HNO3, HNO3-H2SO4, HNO3-HF, HNO3-H2O2, HNO3-HF–H2O2, HNO3- HClO4, y HNO3-HClO4–H2O2) [11, 62–64].

Of the mineral acids, nitric acid offers the best digestion result for all types of samples; however, there is no consensus on the addition of other substances, such as perchloric acid or hydrogen peroxide, to accelerate the process and reduce the volume of nitric acid used [60].

This study employed acid digestion in its sample preparation [51, 54]. Optimizing acid digestion using the reflux system involved the determination of cadmium in a thermoformed sample (HMC-1) and flexible film (SM 707–17 hydrolyzed), taking four absorbance readings and considering the following variables: sample weight, temperature, time, and acid ratio. Each test should be performed in triplicate.

#### *2.2.1 Acid ratio optimization*

The optimal acid ratio for the digestion of thermoformed samples is determined experimentally, by varying the amount of mineral acids: HNO3 at 65% (20 to 5 mL) and HClO4 at 48% (5 to 20 mL), as shown in **Table 1**. Each test should be performed in triplicate.

For flexible films, the optimization was only possible with an acid ratio of 3:1 as this was the best response to the digestion procedure for thermoforming.

Digestion was performed using the sample dissolved in 20 mL of a mixture containing HNO3 (65%, Merck): perchloric acid (48%, Merck) at a 3:1 ratio [54].


**Table 1.**

*Acid ratio (HNO3 and HClO4) for acid digestion using the reflux system.*

### *2.2.2 Sample weight optimization*

This parameter is optimized by varying the sample quantity, which for our study, has been: 0.5000 (± 0.0001) g, 1.0000 (± 0.0001) g and 2.0000 (± 0.0001) g, for thermoforming, and 0.5000 (± 0.0001) g and 1.0000 (± 0.0001) for flexible films, on a dry base.

Digestion was performed using a 1.0 g of the sample dissolved in acid [54].

#### *2.2.3 Temperature and digestion time optimization*

The optimal digestion temperature is determined experimentally at three heating temperatures (35, 50 and 70 °C) for both thermoformed samples and flexible films.

For digestion time, 60, 120 and 180 minutes were tested for the thermoformed samples, for the acid ratios shown in **Table 1**, and for flexible films 15, 30, 45, 60 and 120 minutes, for the acid ratio 3:1. After cooling to room temperature, all digested solutions were filtered using a filter crucible, and then stored in polyethylene containers at 4 °C for further analysis by atomic absorption spectrometry.

Thermoformed samples were dried at 70 °C in a furnace for 3 h. Biodegradable flexible films were dried at 50 °C in a furnace for 45 min [54].

Numerous techniques have been used to determine the concentration of heavy metals in different samples, such as X-ray fluorescence spectrometry (XRFS), atomic absorption spectrometry (AAS) and inductively coupled plasma mass spectrometry (ICP-MS). XRFS makes it possible to analyze solid materials without sample pretreatment; however, this advantage is limited by the need to use appropriate certified reference materials for calibration, making it a very expensive technique. In ICP-MS and EAA, dissolved liquid samples are usually required, so the samples have to be previously digested. This procedure can be tedious, time consuming and results in systematic errors due to incomplete extraction or solubility of the analyte [65].

When compared to the flame atomization method, the graphite furnace atomic absorption spectrometry is the most suitable technique to determine elements such as Cd, in trace level concentrations in polymer samples. The latter has many advantages such as its high sensitivity, its limits of detection in the order of micrograms per liter μg/L to ng/L, its tolerance to complex matrices, the fact that it minimizes analyte loss, that it uses few sample volumes, and that it reduces analyst contamination risk [65–69]. With this technique, samples are often introduced in solution form, however, the EAA-HG technique with solid samples has been reported as a simple and fast method for the determination of lead and cadmium in polymer samples [65].
