The Dumas assay predates Kjeldahl analysis by 50 years (Table 1). The former technique was invented by Jean Baptiste Dumas. Early applications include the analysis of plant materials (74,75), meat (76), casein, whole powdered milk, soybeans, and maize ¯our (77). The ®rst-generation instruments for the Dumas method were not user friendly. The volume of nitrogen gas produced by combustion was determined with a manometer.
The advent of easy-to-use and highly accurate combustion nitrogen analyzers (CNAs) rekindled interest in the Dumas method.
CNAs from various manufacturers work on the same principle. The sample is dropped into a 950±10508C furnace, purged free of atmospheric gas, and ®lled with pure (99%) oxygen. Complete sample combustion leads to CO2, water, SO2, NO2, and N2. The product gases are cooled and a portion is passed through tubing packed with hot lead chromate, copper, sodium hydroxide (solid), or phosphorus pentoxide to remove SO2, O2, CO2, and water, respectively. The NO2is then reduced to N2and measured with a thermal conductivity detector (TCD). Sample protein content is calculated by taking into account the mass of sample injected, the
proportion of the combustion gases analyzed, and the nitrogen-protein conversion factor (FK). The calculations are now automated.
5.1. Collaborative Studies and Approved Status for CNAs
CNAs were calibrated with the Kjeldahl method. Interlaboratory studies appearing after 1987 are listed in Table 12. Such trials led to CNAs receiving approved status from the AOAC (Association of Of®cial Analytical Chemists), AOCS (American Oil Chemists' Society), ASBC (American Society of Brewing Chemists), AFI (American Feed Industry), BRF-International (Brewing Research Foundation-BRF-International), IOB (Institute of Brewing), and EBC (European Brewing Convention).
The Canadian Grain Commission and U.S. Department of Agricul-ture (USDA) Federal Grain Inspection Services (FGIS) approved CNAs in
TABLE12 Food Protein Analysis Using the Dumas Combustion Method
Samplea Reference
Animal feedstuffs, fertilizers Sweeney and Rexroad (78), Schmitter and Rhihs (79), Sweeney (80), Sachen and Thiex (81), Tate (82)
Beer ASBC (83), Johnson and Johansson
(84,85)
Brewing grainsÐbarley, malt, rice ASBC (86), Buckee (28,87), Krotz et al.
(88), Johansson (89), Angelino et al.
(90) Cereal grainsÐwheat, barley, corn,
sorghum Bicsak (91), Bicsak (92), Williams et al.
(93) Dairy productsÐskimmed, powdered
milk etc. chocolate milkshake, cheeses, etc.
Wiles and Gray (94), Wiles et al. (95), Simonne et al. (96,97)
FruitÐguava, peaches, plum Simonne et al. (96,97), Huang et al. (98)
Infant food Bellemonte et al. (99)
Meat and meat products, ®sh (raw, ®sh
in oil, tuna) King-Brink and Sebranek (100), Simonne et al. (96,97), Buschmann and Westphal (101)
Oilseeds (soybean, canola, sun¯ower,
corn) Bicsak (91), Duan and DeClercq (102),
Berner and Brown (103)
Potatoes Young et al. (104)
VegetablesÐcabbages, broccoli,
ketchup, tomato Simonne et al. (96,97)
aApproximate sample classi®cation;classes contain the other foodstuffs.
1994 and 1996, respectively (91±93). Trials for the combustion method usually follow guidelines described by Youden and Sleiner (105):
1. The number of laboratories ranges from 7 to 12. Studies involving as few as three laboratories have been reported.
2. All studies compare CNAs with Kjeldahl analysis.
3. Interlaboratory studies focus on a single food group. Therefore, CNAs tend to receive approval for one food group at a time (Table 12).
4. Trials usually test a ``generic combustion method'' and are independent of the choice of instruments.
Minimum performance guidelines for CNA instruments include (a) a furnace temperature of 9508C, (b) a separation system for trapping CO2and water, (c) a thermal conductivity detector for nitrogen, (d) suf®cient accuracy to produce results within 0.15% of the mean (% nitrogen) results for 10 successive measurements using a standard compound, and (e) suf®cient precision to produce a relative standard deviation of 0.01%.The LECO FP-428 analyzer was used by about 80% of the laboratories involved in collaborative trials. The Foss-Heraue Macro-N analyzer, Carlo Erba NA-5000, and Perkin Elmer PE2410 also feature. The LECO FP-2000 combustion analyzer appears in the latest trials.
5.2. Advantages of the Combustion Method
The modern CNA has advantages over the Kjeldahl method (Table 13).
There is greater speed of analysis and greater operator safety stemming from the nonuse of aggressive chemicals. The estimated cost for analysis is $0.37±
$0.50 per sample with the LECO FP-2000 protein analyzer (LECO Corporation, Saint Joseph, MI) compared with $1.0 per test for the Kjeldahl method (106±109).
TABLE13 Advantages of the Dumas Method 1. Greater ease of operation
2. Higher operator safety owing to the nonrequirement for harzadous chemicals 3. The absence of wetchemistry
4. Reduced time of analysis
5. Higher performance characteristics (greatar accuracy, repeatablility) 6. Absence of waste disposal concerns (Table 14)
7. Simple instrument installation without a requirement for specialized ventilation 8. Low cost per analysis
A more detailed discussion of the relative costs of protein analysis by Kjeldahl or combustion analysis has to consider factors such as number of analyses per year, capital costs for instrumentation, depreciation, main-tenance costs, and savings of labor, chemicals, and other consumables costs (106). It has been suggested that the combustion method provides cost savings of about 30% with a payback period within 2 years. For research institutes, universities, and small-scale laboratories, the safety of modern CNAs probably outweighs cost considerations. Further comparisons of the CNAs and Kjeldahl analysis are summarized in Table 14.
TABLE14 A Comparison of Materials Reqirement for the Kjeldhal and Dumas Methods (74,84)
Requirement Kjeldahl Dumas
Chemical
requirements Conc. H2SO4, 40% NaOH, K2SO4, TiO2/CuSO4(or
Other suppliesa Kjeldahl and Erlenmeyer
¯asks, burettes, acid, alkali Ancillary equipment Ductwork for corrosive
fumes, acid-resistant fans, fume washer, fans, etc.
Ductwork for warm airb
Disposal of chemical Collected, professionally
disposed Nontoxic, wastebin or sink disposal
Time per analysis 120 min (24 samples) 3 min
Degree of hazardc 6 2
Accuracy 70±98 100
Precision (CV %) 1.2 0.7
aDoes not include main equipment (Kjeldahl digester and distillation apparatus, or CNA instrument
bOptional, but advisable for large-scale testing.
cArbitrary scale of 1±10, with 10 being extremely hazardous and 1 completely safe. There may be a risk of burns when maintaining the combustion instrument.
5.3. Combustion Analysis of Feeds, Cereal Grains, and Oilseeds
Combustion analysis ®rst received AOAC approval for feeds in 1968. The classical instruments (Coleman model 29A nitrogen analyzer) used a manometer for the volumetric assay of nitrogen (110). Comprehensive testing using modern TCD-based CNAs appeared in 1987 (78). A nine-laboratory collaborative trial to determine nitrogen in feeds was successfully completed in 1989 (80). The AOAC approved CNAs for animal feed testing in 1990.
The small sample sizes (150±500 mg) used with modern CNAs raised concerns about sampling. Extensive grinding and mixing are essential to ensure sample homogeneity and representative sampling. Sweeney and Rexroad (78) analyzed 14 different animal feeds using the LECO FP-228 instrument with a prescribed sample size of <150 mg. Estimates of feed nitrogen agreed closely with results from Kjeldahl analysis. The precision of analysis was signi®cantly lower (0.013±0.052%) for the combustion method as compared with Kjeldahl analysis (0.006±0.035%). Schmitter and Rihs (79) increased the sample size for the LECO-F228 analyzer from 150 mg to 1 g by palletizing before loading into the instrument port. Adding a few drops of polyethylene (2% w/w in ethyl acetate solvent) prevented ¯aking of the pellets. Table 15 shows nitrogen and protein data for feeds determined using the CNA (78,79). Results have been averaged for samples of sizes 0.15±1.0 g.
Protein values are calculated as %N 6 6.25. The results agree favorably with Kjeldahl analysis (Fig. 6). There appeared to be signi®cant positive bias for feedstuffs having < 2% nitrogen (Fig. 7). The bias was ascribed to plant-derived materials containing high levels of nitrate. The Kjeldahl method achieves low recoveries of nitrogen from refractory compounds (75) with N22O or N22N bonds (nitrite; nitrate; oximes; azo-, nitro-, nitroso-compounds).
Sachen and Thiex (81) found that CNAs showed a 1.38% (protein) bias for hay samples. They attributed such results to ``atmospheric error'' arising from air being trapped in the interstices of the (¯uffy) hay samples.
Compressing samples to remove trapped air led to agreement between the Kjeldahl and CNA results (81). The LECO FP-2000 nitrogen analyzer was not subject to an atmospheric blank because of improvements in instrument design and ef®cient purging of atmospheric gases before sample combustion.
Sachen and Thiex examined a range of pelleting equipment and procedures for eliminating the atmospheric blank for the LECO FP-428 instrument.
They proposed that powdered cellulose could be analyzed to check for an atmospheric error (81). Not all investigators agree about the nature of the atmospheric error.
Bicsak (91) described a collaborative study to extend AOAC-approved status to cereal grains and oilseeds. Seven laboratories analyzed 15 matched pairs of samples (soybean, canola, sun¯ower, wheat, barley, corn, sorghum) having protein levels of 8±13%, 17±23%, or 35±40%. Six of the seven collaborators used the LECO FP-428 instrument. With 210 samples the average protein reading was 28.26% by combustion analysis and 28.01% by Kjeldahl analysis. Repeatability and reproducibility statistics were compar-able. A recommendation to extend the AOAC combustion method to cereal grains and oilseeds was approved. The following non-English publications TABLE15 Nitrogen and Protein in Feedstuffs Determined by Combustion Method
Sample N (g kg 1) Protein (%)
Soy protein conc. 14.020 87.63
FIGURE6 Comparison of protein results for feeds determined using the combustion method and the Kjeldahl method. Micro-CNA and macro-CNA refer to the use of 150-mg and 1-g sample sizes with the combustion nitrogen analyzer. List of feedstuffs is given in Table 15. (Data derived from Ref.
78.)
FIGURE7 Residuals from Fig. 6 showing no systematic differences in results.
describe collaborative tests leading to approved status for combustion analysis of cereal and cereal products including wheat, wheat bran, pasta, sorghum, and maize (111,112).
5.4. Barley, Malt, and Beer
The combustion method was subjected to an 11-member interlaboratory trial for brewing grain (rice, barley, malt, spent grain) analysis. Agreement was reached in 1992 to include the Dumas method in the methods of analysis for brewing grains (86). The trial results showed that most commercial CNAs had a linear range of 0±9.5% nitrogen and an LLD of 0.0321%. The repeatability CV ranged from 0.013 to 0.055% compared with a reproducibility of 0.042±0.067%.
Further collaborative studies to evaluate CNAs for barley, malt, and beer analysis were reported by the UK Institute of Brewing in 1996 (28).
Fifteen of the 25 participating laboratories employed the LECO FP-428 instrument. Another ®ve laboratories used the Foss Heraeus Macro-N apparatus. The range of Kjeldahl techniques used is shown in Table 7. All laboratories examined eight samples each of barley, malt, and beer. CNAs gave slightly higher values for total nitrogen as compared with the Kjeldahl method. Essentially identical results were obtained when results from the Kjel-Foss instrument were omitted. The reproducibility was 0.03±0.07% for barley and 0.036±0.065% for malt analysis. These values were independent of total nitrogen over the range 1.17±1.71% (w/w) (barley), 1.45±2.03% (w/
w) (malt), and 268±1020 mg L 1 (beer). Based on such results, the IOB Analysis Committee (UK) approved combustion analysis for use alongside Kjeldahl analysis. However, the precision for beer analysis was low, perhaps because the trial participants lacked expertise with liquid samples. CNAs were judged unsuitable for beer protein determination.
CNAs were approved for beer analysis in Europe in 1999 (84,85). In the collaborative trial organized by the IOB and EBC, ®ve samples of beer and malt were analyzed in duplicate by 18 laboratories from the brewing and allied industries. The collaborators used the following CNAs: LECO FP-428 instrument (11 laboratories), LECO FP-2000 (3 laboratories), and Macro-N analyzer (3 laboratories). Glycine, Tris, or EDTA was used as the calibrant. Samples of beer were found to contain 362±1159 mg (nitrogen) L 1 and malt samples had 0.534±0.706% nitrogen. Repeatability and reproducibility statistics for beer analysis were deemed satisfactory, leading to method approval.
5.5. Milk and Related Dairy Products
Despite the recent widespread use of CNAs for food protein analysis, few applications in the dairy ®eld have been published. Wiles et al. (95) described an 11-member interlaboratory study from New Zealand. They compared milk protein analysis by CNAs and the Kjeldahl method. Samples included ultra-heat-treated (UHT) whole milk, infant formulas, whole milk powder, skimmed milk powder, whey protein concentrate, casein, and sodium caseinate. Nine of the eleven laboratories employed the LECO FP-428 instrument. Results for CNAs agreed closely with Kjeldahl ®ndings.
There was no systematic bias associated with the former results. Indeed, no evidence was found for a systematic difference between CNA and Kjeldahl results reported between 1968 and 1997 (95).
5.6. Baby Foods and Infant Formulas
Bellemonte et al. (99) analyzed ®ve categories of baby foods using the Carlo Erba model 1500 nitrogen analyzer. This instrument uses a high furnace temperature (18008C) combined with an oxygen-rich atmosphere to achieve complete sample combustion. Nitrogen is quanti®ed using a TCD. The analysis time for this instrument is reportedly 3 minutes. All sample types were analyzed successfully. Results obtained by the Kjeldahl method were 1±4% lower than those obtained with CNAs (Table 16). Results from both techniques compared favorably with protein values declared by food manufacturers. Compared with the Kjeldahl method, CNAs are convenient for baby food analysis. The sample throughput and safety considerations favor the Dumas method as described in Sec. 5.2.
5.7. Meat
King-Brink and Sebranek (100) described a 12-laboratory trial to evaluate CNAs for meat product analysis. Participants in the trial used the LECO FP-428 instrument (9 laboratories), the Foss Heraeus Macro-N analyzer (2 laboratories), or the Perkin Elmer PE2410 analyzer. In all, 15 pairs of meat products, purchased from 30 different manufacturers, were analyzed. All participants used CNAs satisfactorily judging from (a) the low number of data outliers and (b) the high precision of results for standard compounds.
The CNA results were slightly higher than Kjeldahl ®gures: 15.75% versus 15.59% (w/w). Estimates for repeatability and reproducibility were comparable. A recommendation that the Dumas method should be adopted as a reference test for meat proteins was approved by the AOAC. A 14-laboratory trial for analysis of meat and meat products was reported in
Germany. This trial, too, concluded that the performance of the Dumas method was comparable to that of the Kjeldahl assay but that the former method was quicker and more environment friendly (101).
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TABLE16 Analysis of Protein in Five Categories of Baby Foods (97)
Samplea Declared
protein Kjeldahlb Dumasb
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Cream of rice,
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Veal, ham, and eggs
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Beef, beef ham, chicken, veal brain, turkey, chicken carrot potatoes
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