INTRODUCTION:

Animal feeds are potentially problematic samples to prepare for analysis because of their high protein, high fat and fibre content. At present, two techniques are most commonly used for the digestion of animal feeds. They are the dry ash followed by acid dissolution technique and the hot plate digestion technique.

The principle difficulty with the dry ash technique2 is the requirement of eight hours to prepare a sample for analysis. The two principal difficulties of hot plate digestions are temperature control and the control of frothing. In order to over come these problems, samples can be predigested overnight. However, this will affect daily throughput. Hence, the analyst is required to carefully monitor each sample to prevent it from frothing and overflowing. In addition, the analyst will have to rotate the samples to compensate for the uneven heating of the hot plate. Thus, the digestion becomes a very labour intensive procedure, which contradicts the two important requirements of this industry - to maximize daily sample throughput while maintaining good quality control procedures.

The present application note describes an alternative. By controlling the sample temperature as opposed to the digestion block temperature the sample pre-digestion and digestion can be undertaken with great precision. This application note describes a wet digestion method following the AOAC 957.02 (a)1 Method, using a multiple temperature program and a single use polypropylene disposable digestion vessel. Hence, the method requires a single manipulation of the sample from weighing through to analysis thereby reducing time and potential errors. This application note will compare the results from this technique for a single sample type. All samples are analyzed using the same ICP-AES. In addition, the same commercially available multi-element ICP-AES calibration standard was used. The group of commercially interesting elements, Na, Mg, P, K, Ca, Cu, Fe, Mn, and Zn has been selected.

The type of sample selected for this study was an Association of American Feed Control Officials (AAFCO) feed check sample, 200226 Milk Replacer.

The proposed Wet Digestion technique uses the AOAC Method 957.02 (a) guidelines, see Appendix 1. This method requires the feed sample to be digested in a ratio of 6 ml of concentrated Nitric Acid to 1 ml of concentrated Hydrochloric Acid for 0,5 g of feed. In addition, the sample must be brought to a boil for 30 to 60 minutes. Dr. Minczewski clearly demonstrated this in his studies2 that temperature control is a critical parameter in sample digestion. He conducted his studies by carefully controlling the reaction temperature of one sample at a time. His technique has been adapted for this study in order to do multiple digestions simultaneously.

The samples were digested in a 24 position Graphite Block Digestion System (Fig. 3, DigiPREP Jr.) equipped with a built in temperature controller and an external PFA coated probe (Fig. 1, DigiPROBE) to measure and accurately control the sample temperature (guidelines, see Appendix 2). In addition, a disposable Polypropylene Class “A” Graduated Digestion Vessel (Fig. 2, DigiTUBE) was used without transferring the sample. The Touch Screen Controller provided the automated time to temperature and time at temperature control, the sample temperature display and shutdown control. The total elapsed time for the sample preparation is 2 hours.

A five-point calibration using a commercially available calibration standard (SCP SCIENCE, PlasmaCAL ICP-AES Calibration Standard) was created using a Spectro Flame ICP-AES. Further correction for sample drift was made through the use of a standard-sample bracketing technique, which involved running a set of three samples between a certified standard.

The Dixon Q-test3 was applied to each set of data to remove potential outliers and performed after confirming there was no connection between the outliers and the method of analysis. The same sample was separated into aliquots and digested.

SUPPLIES & REAGENTS:
1.  A 50 ml graduated Class “A” Polypropylene DigiTUBE
(010-500-062) / 5. Plastic Spatula / 9. H2O Double-Deionized
(140-600-040)
2. DigiPROBE (temperature control)
(010-500-017) / 6. 4 Polypropylene Pipettes / 10. HNO3 (70%) – Baker
Instra-analyzed
Reagent
3. DigiPREP Jr. with Touch Screen
Controller (010-506-001/002) / 7. Analytical Balance, Mettler
AC-100 / 11. Custom Multi-Element
Standard (SC31322413)
4. Ribbed polypropylene watch glass
(010-500-019) / 8. HCl (37%) - Baker
Instra-analyzed
Reagent / 12. Beckman Centrifuge

PROCEDURE: See Appendix 1

RESULTS:

The digestions were completed, analyzed, and the results tabulated as follows:

Table 1. Analytical Lines Selected
Elements / Line
Selected
(nm)
Ca / 315.887
Cu / 324.754
Na / 589.592
Mg / 279.079
K / 766.491
Mn / 259.373
Fe / 259.940
P / 213.618
Zn / 206.190
Table 2. Instrument Parameters
Parameter
Rf Power / 1200 W
Argon gas flow
Coolant / 15L/min.
Auxiliary / 1.2L/min.
Meinhard Hi-solids C2 Nebulizer / 0.9L/min.
Sample Uptake rate / 1.2 ml/min.
Table 3. Association of American Feed Control Officials (AAFCO) Results
Elements / AAFCO 0266 Control / Wet Digestion
Average / Standard
Deviation / CV (%) / Average / Standard
Deviation / CV (%)
Ca (%) / 0.96 / 0.04 / 4.30 / 1.04 / 0.03 / 2.76
Cu (ppm) / 21.20 / 2.03 / 9.56 / 22.10 / 0.73 / 3.31
Fe (ppm) / 140.13 / 33.15 / 23.66 / 154.28 / 10.23 / 6.63
K (%) / 2.30 / 0.11 / 4.88 / 2.42 / 0.06 / 2.57
Mg (%) / 0.17 / 0.01 / 6.70 / 0.16 / 0.00 / 3.01
Mn (ppm) / 42.48 / 7.90 / 18.59 / 38.76 / 1.87 / 4.82
Na (%) / 0.65 / 0.04 / 6.55 / 0.70 / 0.02 / 3.09
P (%) / 0.83 / 0.04 / 4.75 / 0.87 / 0.03 / 3.01
Zn (ppm) / 79.98 / 11.50 / 14.38 / 90.47 / 5.32 / 5.88

DISCUSSION:

Adding the acids to the sample in the fume hood and slowly heating the samples to 45 oC successfully controlled the sample frothing. This allows the organic matter to react with the acid mixture without loss of sample. The temperature is then increased to 65 oC, which cleans down the walls of the DigiTUBE. The samples are digested without loss of the matrix. The DigiPREP Jr. with Touch Screen
Controller allowed these steps to be automated. This significantly improves sample throughput without the use of additional operator time.

The selected analytical lines were found to have no inter-element or matrix interferences for the elements selected. Furthermore, it was determined that the calibration curves have excellent linearity. The results for the instrument quality control indicate that the signal remained stable throughout the analysis.

All samples were digested and analyzed directly in DigiTUBEs. The results in Table 3. indicate that for the AOAC 957.02 (a) wet digestion method, for the values of all elements fall within approximately one s variation from the AAFCO round robin results.

CONCLUSION:

It can be concluded from the data that the disposable digestion tube, graduated Class “A” Polypropylene DigiTUBE, can be used for all steps in sample preparation and sample analysis. Furthermore, the data demonstrates that by carefully controlling sample temperature with the DigiPROBE and Touch Screen Controller the frothing and sample loss problems associated with wet digestion are eliminated. Therefore, a high throughput accurate method for animal feed digestions can be achieved.

REFERENCE:

1.  Official Methods of Analysis (1998), 16th Edition, AOAC.

2.  J. Minczewski , Separation and Preconcentration Methods in Inorganic Trace Analysis,1st Edition, John Wiley and Sons.

3.Peter C. Meier, Richard E. Zünd, Statistical Methods in Analytical Chemistry, 2nd Edition, Wiley Canada.


Appendix 1: The Wet Digestion Procedure

Appendix 2: Temperature Profile Used on the DigiPREP Jr. with Touch
Screen Controller