Kjeltec Instrument Title slide with Total Nutrition

Instrument – Kjeltec DIGESTION UNIT DISTILLATION UNIT

Objective To Share the Knowledge on Kjeltec Instrument in the following areas Principle Protocol Solution Preparation and Standardization Recovery Studies Critical control points Trouble shooting Conclusion.

Principle Digestion Distillation Sample + Catalyst + Ammonium Sulphate + Sulphuric acid Strong alkali 410ºC ▲ 45 mins pH- 8.4 Nitrogen Releases in the Ammonia Releases form of Ammonia 4% Boric acid Ammonium Ammonia + Sulphuric acid (Receiver Solution) Borate Ammonium Sulphate

Principle in reaction Distillation Step 1 Digestion Acid & Catalyst N(feed) (NH4)2SO4 Distillation Step 1 (NH4)2SO4 + 2 NaOH 2NH3 + 2H2O + Na2SO4 Step 2 NH3 + H3BO3 (boric acid) NH4+ + H2BO3- (borate ion) Titration H2BO3- + H+ H3BO3 The concentration of hydrogen ions required to reach the end-point is equivalent to the concentration of nitrogen that was in the feed/raw material

Expected Protein % of the sample Weight taken for the analysis Procedure PROCEDURE FOR DIGESTION Weigh the sample as per the table given below to nearest to mg in the digestion tube. Catalyst – 7g of Potassium Sulphate and 0.8g of Copper Sulphate per sample. Conc. H2SO4 Cool the content of the tube before distillation. Based on the protein value we have to alter the sample quantity for the analysis. The quantity of sample is given below. Expected Protein % of the sample Weight taken for the analysis <20 % 1000mg >20 % 500mg

Procedure Instrument Keys Menu 1) Analyze – Upon choosing this function complete Analysis will take automatically 2) History - It will show all the history that We did previously 3) Settings - It has the full system settings 4) Manual - By manually we can choose the specific Automatic Addition of Alkali, Water , Distillation time, Steam Pressure can be programmed by editing the ranges

Solution Preparation Receiver Solution Boric Acid:- 4% Solution 4gm in 100ml of warm Water + 1.0 ml of BromoCresol Green 0.7 ml of Methyl red Methyl Red: - 100mg in 100ml of methanol BromoCresol Green :- 100mg in 100ml of methanol

Solution Standardization 0.1N Hcl: - 0.9 ml Conc. Hcl in 100ml of Distilled water. (Titration Solution) Standardization:- Prepared 0.1N Hcl titrate against 0.1N Already Standardized NaOH Phenolphthalein Indicator (3 to 5 drops) Appearance of the Pink Colour Calculation:- V1 X N1 = V2 X N2

Recovery Studies 0.1% Ammonium Sulphate solution:- By Distillate the above mentioned solution directly – We can get the N% of the compound N% of Ammonium Sulphate is – 21.09% Calculation Recovery % = Value obtained for Ammonium Sulphate X 100 21.09 Note:- Recovery Need to be done every day After preparing fresh solution(Boric acid, Hcl, NaOH)

Critical control points in Digestion step Weighing of the sample Sample should not stick on to the sides of the tubes. Adding enough quantity of acid Digester – Connected to UPS / Stabilizer After completion check the digestion is fully completed or not.(Green colour indicates completion digestion) Should run with placing all the 8 tubes. If digestion not done properly – Place the same to some more time for complete digestion Vapor outlet need to be connect with water. Fume hood should always turned on during digestion Use respective PPE while using acids.

Critical control points in Distillation step Once the tube cooled – use for distillation Connect the instrument in UPS/ Stabilizer Check the recovery Check the NaOH and Distilled water quantity Use glass pippete for taking the 30ml receiver solution. Always use automatic mode for distillation Manual method – Manually the distillation need to be stop down, Otherwise May leads to overflow of the collected solution If the water cannot able to condense the instrument will show the warning message , Options – OK – The tube will get drained off - Cancel - Instrument will wait until the normal condense happen CONTD….

Critical control points in Distillation step Tube drain need to be in off mode – To avoid the drain down before completing of distillation. Water pipeline need to be connect and the tap on during the analysis for condensation Fixing the tube should be proper – Otherwise the sample or ammonia will leak from the tube leads to wrong results Collect 200 ml of the solution End Point – Change from green color to colorless.

Calculation N% = (T-B) X 14 X N.of Hcl X 100 Sample weight CP = N% X 6.25

Problem Faced and Trouble Shooting 1) Catalyst - Potassium Sulphate contains 2% Nitrogen content . 2) Digestion tube thickness 3) Power Fluctuation

Finding 1 Catalyst – Nitrogen Contribution Catalyst used for Protein digestion always should be free from Nitrogen content. Merck, Sigma contains less than 0.005% of nitrogen FINAR brand - contains 2% of nitrogen content. Problem Faced:- Increased value in CP results in all the samples Route cause Identification:- Individual Digestion and distillation on every individual chemical involved in both digestion and distillation. Found all the others are giving negative and Potassium Sulphate alone is the route cause for the problem. Preventive actions:- Branded Company like Merck / Sigma is advised to use for catalyst Before Preparing catalyst, the Catalyst salt need to be measured on nitrogen content.

Findings 2 Impact by Digestion tubes – new findings Digestion in the Kjeltec Digestion tube – placing for 45 mins at 415ºC The old tubes are thin and wide and the newly ordered tubes were thick and narrow Problem Faced:- Decreased value in CP results in some samples. Route cause Identification:- Did the analysis with the same samples in all the slots – Avg of 5 % Difference was found out between the slots Done the same sample of analysis with thin and narrow tube and Done the same sample of analysis with thick and narrow tubes. Thin tubes all the results were matching with each others and thick tubes major difference were obtained between the samples. Due to more thickness the heat is not able to digest the sample properly. Preventive actions:- Stopped using the Thick tubes and ordered new tubes with provided clear information to the supplier.

Finding 3 Power Fluctuations Impact on Digestion Unit:- The Digestion unit consist of – Power house of the Digestion unit contactor and the heating cable Problem Faced:- The Contactor and heating cable was frequently functioned down. Route cause Identification:- It found out by Service person that the output from the plug point is very high and it fluctuates caused the problem in the damage of the parts Preventive actions:- Always advices to Connect the Digestion unit in Stabilizer

Finding 3 Power Fluctuations Impact on Distillation unit Problem Faced:- The fuse will be always getting down and some time the inner wire got burnes. Route cause Identification:- It found out by Service person that the output from the plug point is very high and it fluctuates caused the problem in the damage of the parts Preventive actions:- Always advised to Connect the Digestion unit in UPS

Conclusion By Following all the Critical control measures the instrument and analysis will be always perfect and believable.