Using Ion Chromatography to Determine Ionic Species in Dairy Products

When running a difficult food sample like dairy on an ion chromatograph (IC), there is an enormous risk that the column will be irrevocably ruined. One solution to circumvent this is time-consuming and tedious sample preparation steps in order to eliminate undesirable matrix components.

Another, easier solution is to select an automated compact stopped-flow dialysis system, which can provide optimum separation while protecting the column from detrimental compounds.

Thanks to its simplicity, robustness, reliability and the wide range of columns, detectors and applications, ion chromatography (IC) has experienced an impressive surge in popularity as an analytical technique. Very little sample preparation is required for a sample in a homogeneous ionic form, and results can be obtained in just a few minutes. Nonetheless,  a more extensive sample preparation is required to prevent destruction of the column in complex matrices carrying high organic loads such as dairy products.

Though there have been multiple developments in sample preparation techniques have been developed, like the Carrez precipitation for protein-containing samples, it remains nevertheless true that the majority of these are time-consuming and error-prone. In order to overcome these issues, 1997 saw Metrohm launch the first coupling of IC with dialysis. Since this coupling, the procedure has been further streamlined and now offers an efficient in-line elimination of undesired matrix components in various demanding sample types.

This article uses examples of an ultra-high temperature (UHT) processed milk and a baby milk powder sample to present a fully automated sample preparation setup which is coupled to the new ion chromatograph 881 Compact IC pro [Figure 1]. Calibration parameters, carryover and recovery rates were analyzed with multi-anion standards. The instrument set-up in this work was based on an Ion Chromatography system (the 880 Compact IC pro from Metrohm), as well as with a Metrohm 858 Professional IC sample processor, a Metrohm 800 Dosino and spiral flow dialysis cell and accessories.

The experimental setup consists of an 881 Compact IC pro with the 858 Professional IC Sample Processor with dialysis cell and 800 Dosino. Instrument control, data acquisition and processing were carried out by MagIC Net software.

Figure 1. The experimental setup consists of an 881 Compact IC pro with the 858 Professional IC Sample Processor with dialysis cell and 800 Dosino. Instrument control, data acquisition and processing were carried out by MagIC Net software.

Deionized water was used to prepare all standard solutions and eluents having a specific resistance higher than 18 MΩ·cm. To determine the system characteristics, two standard solutions ranging from the concentration between 1.0 to 3.6 mg/L and 10 to 36 mg/L were used. Both the baby milk powder and ultra-high temperature (UHT) processed milk were purchased from Migros in Switzerland.

Compact Stopped-Flow Dialysis

Dialysis works on the premise of selective diffusion of molecules or ions from one liquid (donor or sample solution) to another (acceptor solution) through a membrane. The concentration gradient across the membrane is the driving force for this transfer.  In contrast to dynamic dialysis, wherein two solutions pass through the dialysis module continuously, stopped-flow dialysis has at least one solution temporarily stopped until there is the same concentration in the acceptor solution as in the donor solution.

This procedure, of the stopped-flow, takes between 10 and 14 minutes, and can be directly coupled to an IC setup. The overall analysis time is brief and not prolonged, as the dialysis is performed during the recording of the previous sample’s chromatogram. In a conventional setup, two dual-channel peristaltic pumps take the sample and the acceptor solution to and from the dialysis cell. This is in comparison to compact dialysis, where a Dosino unit doses ultrapure water through the acceptor compartment of the cell.

In order to achieve the stopped-flow status, the Dosino is stopped and the outlet capillary of the cell is blocked by feeding it through the valve of the Sample Processor. Depending on its valve position, the latter allows or blocks the flow of the acceptor solution [Figure 2].

System Characteristics

Calibration

Five concentration levels (0.5, 1, 5, 10 and 20 mg/L) which were prepared from a multi-ion standard were then used for external calibration [Table 1].

Table 1. Correlation coefficients and relative standard deviations of the five-point anion calibration

Fluoride Chloride Nitrite Bromide Nitrate Phosphate Sulfate
Correlation coefficient 0.99995 0.99996 0.99999 0.99996 0.99994 0.99990 0.99997
RSD [%] 1.516 1.242 0.834 1.169 1.479 2.491 1.176

Carryover

By injection of a blank (ultrapure water) immediately after injection of a standard, this was evaluated [Table 2].

Table 2. Carryover in percent determined for the concentration ranges 1.0…3.6 mg/L and 10…36 mg/L

Fluoride Chloride Nitrite Bromide Nitrate Phosphate Sulfate
Low standard conc. 0.24 0.15 0.17 0.20 0.18 0.11 0.28
High standard conc. 0.49 0.12 0.13 0.22 0.11 0.00 0.38

Recovery rates

Results obtained by direct injection were compared to those obtained by injection of the dialysate in order to determine recovery rates (Table 3).

Table 3. Anion recovery rates

Low standard concentration
(1.0…3.6 mg/L)
High standard concentration
(10…36 mg/L)
Direct injection With dialysis Recovery Direct injection With dialysis Recovery
Mean
[mg/L]
RSD
[%]
Mean
[mg/L]
RSD
[%]
rate
[%]
Mean
[mg/L]
RSD
[%]
Mean
[mg/L]
RSD
[%]
rate
[%]
Fluoride 1.06 0.12 1.03 0.24 97.2 10.81 0.09 10.57 0.06 97.8
Chloride 3.01 0.04 2.97 0.03 98.7 31.58 0.03 31.22 0.06 98.9
Nitrite 2.94 0.32 2.91 0.15 99.0 30.01 0.30 29.81 0.04 99.3
Bromide 1.02 0.08 1.01 0.00 99.0 10.50 0.04 10.38 0.17 98.9
Nitrate 3.02 0.07 2.97 0.00 98.3 30.80 0.03 30.40 0.03 98.7
Phosphate 3.81 0.17 3.47 0.10 91.1 33.74 0.02 31.83 0.03 94.3
Sulfate 3.52 0.09 3.35 0.07 95.2 35.57 0.04 34.17 0.07 96.1

Dairy Samples

UHT processed milk

The UHT processed milk sample, before analysis, was diluted 1:100 with ultrapure water and subsequently placed on the rack of the sample processor, in the sample vials. The subsequent milk sample dialysis and injection of the dialysate onto the separation column was fully automated. Using integration software MagIC Net 1.1 against previously prepared calibration plots, the calculation was carried out automatically.

Excellent baseline separation of chloride, phosphate and sulphate is achieved within 12 minutes, under the conditions described in the caption of Figure 3. No trending in peak areas or retention times was shown by repetitive analyses, which suggests that sample proteins did not pass through the membrane.

The dialysis cell and experimental setup. Figures (a) and (b) on the left of the image show the patented spiral-flow dialysis cell. The schematic diagram (c) displays its link-up to the compact IC.

Figure 2. The dialysis cell and experimental setup. Figures (a) and (b) on the left of the image show the patented spiral-flow dialysis cell. The schematic diagram (c) displays its link-up to the compact IC.

Baby food milk powder

The baby food milk powder was reconstituted with water following the manufacturer’s instructions. The prepared milk sample was diluted 1:100, before analysis. Here, too, as with the UHT milk sample, the chromatographic conditions created an excellent baseline separation for chloride, phosphate and sulfate.

Conclusion

There were multiple challenges posed by this work. One of these consisted of the determination of chloride, phosphate and sulfate in the presence of difficult sample matrices, which could interact with the stationary column phase or even go so far as to render it unusable. These drawbacks can be overcome by a patented stopped-flow dialysis system coupled to the new 881 Compact IC pro ion chromatograph.

In terms of analyte concentration, relative standard deviation, calibration quality, carryover and recovery rates, two standard solutions were characterized in the concentration ranges 1.0 to 3.6 mg/L and 10 to 36 mg/L as well as two samples: an ultra-high temperature (UHT) processed milk and a baby milk powder. The five-point calibration curves produced correlation coefficients (R) better than 0.9999 and carryover between two subsequent injections of a concentrated sample. In addition, it was found that a blank was less than 0.49%. Recoveries for the low (1.0 to 3.6 mg/L) and high standard concentration ranges (10 to 36 mg/L) were within 91 to 99% and 94 to 100%, respectively.

A highly efficient sample preparation technique that ensures optimum separation performance is automated compact stopped-flow dialysis, by protecting the column from detrimental matrix constituents.

Anion chromatogram of a UHT dialysate containing 9.88 mg/L chloride, 17.40 mg/L phosphate and 1.09 mg/L sulfate (after 1:100 dilution of the sample). Column: Metrosep A Supp 5 - 100/4.0, eluent: 3.2 mmol/L sodium carbonate and 1.0 mmol/L sodium hydrogen carbonate, flow: 0.7 mL/min, column temperature: 30 °C, injection volume: 20 µL, acceptor solution: ultrapure water, dialysis time: 14 min.

Figure 3. Anion chromatogram of a UHT dialysate containing 9.88 mg/L chloride, 17.40 mg/L phosphate and 1.09 mg/L sulfate (after 1:100 dilution of the sample). Column: Metrosep A Supp 5 - 100/4.0, eluent: 3.2 mmol/L sodium carbonate and 1.0 mmol/L sodium hydrogen carbonate, flow: 0.7 mL/min, column temperature: 30 °C, injection volume: 20 µL, acceptor solution: ultrapure water, dialysis time: 14 min.

Anion chromatogram of a baby food milk sample containing 7.37 mg/L chloride, 7.41 mg/L phosphate and 0.76 mg/L sulfate (after 1:100 dilution of the sample). Chromatographic conditions correspond to those given in Figure 3.

Figure 4. Anion chromatogram of a baby food milk sample containing 7.37 mg/L chloride, 7.41 mg/L phosphate and 0.76 mg/L sulfate (after 1:100 dilution of the sample). Chromatographic conditions correspond to those given in Figure 3.

References

1. Metrohm Application Notes AN-S-044, AN-S-162, AN-N-018, AN-C-100 and AN-C-028 (downloadable under http://products.metrohm.com).

2. Metrohm Monograph: Sample preparation techniques for ion chromatography, Metrohm AG, Herisau, Switzerland, 108 pages, 8.025.5003.

3. Steinbach A. and Wille A., Ion chromatographic analysis of carbohydrates in essential and non-essential foodstuffs, Food Engineering & Ingredients, 33-36 (2008).

This information has been sourced, reviewed and adapted from materials provided by Metrohm AG.

For more information on this source, please visit Metrohm AG.

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