Why do I get excessive recovery for nitrate in dairy Whey Protein Concentrate on Lachat QC8500 S2 manifold E20-107-04-1-B

Document ID

Document ID TE8103

Published Date

Published Date 15/05/2019
Question
Why do I get excessive recovery for nitrate in dairy Whey Protein Concentrate on Lachat QC8500 S2 manifold E20-107-04-1-B
Summary
Excessive recovery for nitrate in dairy whey protein using Lachat QC8500 manifold E20-107-04-1-B
Answer
The method has not formally been validated for whey proteins, but there are some key considerations:
 
Whey protein samples are 1) extremely difficult to dissolve and 2) without lots of adjustments, recovery can be tough. If protein or fat get through to any extent, the column will rapidly deteriorate.
 
In some protein powders, individual amino acids are added to boost “protein content”.  Those would not be excluded by the membrane. There is no available information if these could interfere positively but they could potentially be a problem.
 
It is recommended to use minimal diluent - just enough to dissolve it and a lower than normal concentration of buffer aids the extraction.  The buffer choice would be 0.05M:
 
Reagent 1.  Ammonium Chloride Buffer Solution, 0.5M pH 8.5 (stock buffer)
By Volume:  To a 2 L volumetric flask containing approximately 1800 mL DI water, add 53.2 g ammonium chloride (NH4CL).  When dissolved, add 15 N NaOH dropwise, until a pH of 8.5 (+/-0.1) is achieved.
Please Note:  This solution is used on the chemistry manifolds, as well as to prepare the standards and samples extraction solution (Reagent 2 below).  Volume should be scaled up according to need.
Reagent 2.  Ammonium Chloride Buffer Solution, 0.05M pH 8.5 (from dilution of stock)
By Volume:  To a 2 L volumetric flask containing approximately 1600 mL DI water, and 200 mL of reagent 1. Dilute to volume.
Please Note:  This solution is used as the donor and carrier, as well as to prepare the standards and extract the samples.  Volume should be scaled up according to need.
 
Some samples dissolve well in water but the speciation is not always maintained.  Viscosity of some of the samples can also present a problem.
 
As a rule of thumb higher temperature extractions give higher recoveries.  This is from the most recent method:
 
Sample Extraction—Dry Milk and Whey Products:
Weigh 2.5g +/- 0.1 mg of the sample to be tested into a 50 mL, conical centrifuge tube. 
Add 24 ml of extraction buffer (reagent 2), heated to 50-55°C

Particulates must be filtered prior to analysis.
Very high protein samples may require a longer extraction period, smaller sample weights, more time, and a higher temperature (at 65-95°C) to allow complete dissolution to occur.
Extractions of several sample weights may be required for high protein samples in particular. For example 0.5g, 1g and 2g, in order to obtain a sample that is not too viscous to aspirate but with analyte at detectable levels.
 
It is recommended to trial variations in time, temperature and weight to optimise for different samples.   Also, intermittently, a vortex mixer. Certain Whey proteins denature like egg whites when heated too much.  Others won’t dissolve at all without heat.