3. Introduction
▪ Important analytical technique for the separation and determination of ionic compounds
▪ robust and time-saving technique for multicomponent analysis in foods and beverages
▪ superior to single component analytical techniques and less complex than hyphenated
techniques, but highly specific, precise, robust, and easy to use.
▪ capable of separating similar types of molecules that are difficult to separate otherwise
▪ very high resolution
▪ deal for the separation of charged biomolecules, including proteins, polypeptides, nucleic acids etc.
enhanced productivity, reduced costs, and achieve accurate, reliable, and robust results.
5. Principle of ion exchange chromatography
✓ Separates ions & polar molecules based on their affinity to the ion exchanger
✓ The charge carried by the target molecule facilitates the process of separation
•Anion exchanger — 0.5–1.5 pH units greater than the isoelectric point,
pI of the protein of interest
•Cation exchanger — 0.5–1.5 pH units less than the pI of the protein of
interest
6. Glass, stainless
steel, titanium, iner
t plastic or high-
quality polymer.
protective layer,
extends the life of
the column
reduces the background
conductivity
estimate the analyte
peaks
pre-programmed
computing integrator
Sample injection
to deliver and maintain a
constant flow
Instrumentation
7. Applications in Food Industry
✓Most active and creative fields in food and
fermentation industry
✓play a pivotal role in the development of the food
industry
✓ In juice, dairy, brewing & starch industry used to
remove the heavy metal ions & nitrate ions, for
deacidification, decolorization, demineralization &
for the purification of food products.
8. Phosphorus critical dietary mineral, involved as
phosphate in bone, tooth, nucleic acids or
phospholipids
mainly supplied through consumption of dairy
products.
The objective of the study is to assay phosphorous in
different dairy products i.e milk, aqueous phase of
acidified milk & 3 types of cheese including one
processed cheese containing poly phosphate salt.
To mineralize the samples so that all phosphate is
converted to orthophosphate which react with
ammonium molybdate to finally yield molybdenum
blue.
Values are then compared with International
Organization for Standardization (ISO)
method
Qc, QA & QA
9. Experimental Conditions
• IonPac AS11 hydroxide selective anion-exchange column,
• AG11 guard column
• ASRS 300 suppressor that helps increase sensitivity through
trapping contaminating ions from the mobile phase
• sample load: 25 µL injection volume
• Temperature: 30°C
• Flow-rate of the mobile phase: 1 mL/min.
• Calibration curve
• mineralized KH2PO4 solutions of phosphorous~450 mg/L of
KH2PO4
• correlation coefficient R2 of the calibration curve was 99.7%
• standard deviation was <0.02mg/kg
10. Sample collection, Preparation, Processing
In IEC only small amount of the samples (about 2mg/kg of phosphate) are required
Chemical required only HCl & NaOH for the preparation of the sample
Parameters Skim milk Ultra Acidified Milk Cheese
Dry matter
determination
5 g 10g 2 g
Ashes
measurement
5 g 10 g 2 g
Total phosphate 5 g 10 g 2 g
12. Results & Discussion
▪ skim milk values were in the range of those reported for milk
▪ Compared to skim milk, the AMU had a lower dry matter of 54.6 g/kg, contained 6.4 g/kg of
ashes and virtually no proteins.
▪ Contents in ashes were higher in this AMU compared to those of aqueous phase of normal milk at
pH 6.7.
▪ This was in accordance with the literature reporting that after milk acidification at pH 4.6, the
colloidal calcium phosphate is solubilized and present in the ultrafiltration permeate.
▪ Cheese values were also in agreement with those reported for semi-hard cheeses
13. Total phosphate content
▪ Total phosphate content found in skim milk ,acid milk ultrafiltrate and in the 3 cheeses using
standard colori- metric method (ISO), the ion exchange chromatography (IEC) and IEC where
the acidic soaking of samples was increased (IEC+).
▪ Differences were statistically tested only within data from the same type of product; different
letters refer to statistically different results P 0.05%
15. Conclusion
✓ On average and across all types of milk samples, the amount of phosphate found
with the IEC method was 88 ± 8% of the expected value found with the ISO
✓The loss may be due to the smaller amounts of sample used during the IEC
method, hence the possibly lower representativity of heterogeneous samples as
dairy products.
✓If such losses exist, both accuracy and precision could be improved
simultaneously through preventing them during IEC.
✓Possibly, the correctness of the IEC method depend on the initial structure of the
phosphorus in the samples.
✓If anions other than the simple orthophosphate forms were present after
mineralization, e.g. polyphosphate forms, it may be that these forms could react
with ammonium molybdate and hence be fully assayed by the ISO method, but
not with IEC where such species would probably exhibit a different retention
time than that of phosphate.
