Aspartame degradation project

Calculating Purity

Why

We need to know how much Aspartame we still have.

How

Two simplest way:

• Corrected Percentage Peak Area Method
• Absolute Calibration Curve Method

Fig 1.7 Pure Aspartame Calibration curve (Excel linear model).

Results: 16.32%

(degraded in r.t.p for 9h and then stored at 4°C for 3.5 days)

Troubleshooting & Method Update

Proposed Solutions

Issues & Causes

A Ghost ?!

Residues (Sample or C18 ?) appearing on chromatograms, other peak

Thorough System rinse, column replacement, Sample pretreatment, getting more data

Effect of Flow Rate

Background noise

Effect of Temperature

Poor background quality of the UV detector maybe due to TFA or variety of experiments

"Blank run" acting as a rinse before each experiment (approx 3min. with water)

Results:

• Higher flow rates result in shorter retention times.
• Both peak separation and peak width decrease (affect resolution).

Research:

• A faster flow rate reduces the time a compound spends on the column.
• Giving the analytes less time to interact with the mobile phase.
• Increasing the flow rate can accelerate the elution process.

Conclusion:

• Higher flow rates can be used to reduce retention time which can save time and material.

Results:

• More data and research is

required to show a meaningful

trend.

Research:

• Higher temperature leads to

shorter retention time (viscosity).

• Temperature effects on the

resolution depend on types of

analytes and difficulties with

separations.

Conclusion:

Discrepancies with the theory:

• the extent of degradation is

different for the same sample

(hardware problems), Temperation fluctuations, temperature gradient in the column and so forth.

Software Issues

Fig 3.3 Effect of Temperature variations on HPLC for a run at 1.1mL/min, 20%MeCN 80%Water with 0.01% TFA mobile phase.

Connectivity issues between HPLC software and Analytical System (+30 min lost each day)

Initialization protocol established and communicated within the team

Fig 3.2 Effect of Flow Rate variations on HPLC for a run at 30°C, 15%MeCN 85%Water with 0.01% TFA mobile phase.

Pressure limitations

Effect of Solvent ratio

Effect of TFA

_(ASPAR)TAMING

ASPARTAME

Thorough system rinse, column replacement, Preparative HPLC Scale up !

High pressures abnormally reached maybe due to column clogging

Fig 3.1 Pure Aspartame chromatogram with backrgound noise, ghost peak and negative baseline.

Observations:

• negative peak: solvent is absorbing more light.
• negative slope: detector & rinse problem

Explanation:

• detector working principals
• TFA (Trifluoroacetic acid) is protic, it's carboxylic acid group can donate H+ to aspartame amine group,

Conclusion:

• TFA moves aspartame peak left, make it less retained, hence improve retention time.

Fig 2.2 Illustration of the separation process in HPLC, Synder, Llyod R. et al. "Introduction to Modern Liquid Chromatography", 2009.

Apparatus & Principle

Settings:

• Solvent A: Water + 0.1%TFA
• Solvent B: Acetonitrile

Results:

• Higher Solvent ratio result in longer retention times.
• Peaks are more well seperated, resolution increased.

Research:

• Sample retention k in isocratic elution is usually controlled by varying the mobile-phase composition (%B).
• polarity

Conclusion:

• Higher solvent ratiao helps separation

Fig 2.1 HPLC Flow Diagram - Shimadzu "Overview of HPLC"

Fig 3.5 Effect Ion pairing agent on HPLC for a run at 30°C, 1.1 mL/min, 15%MeCN 85%Water with/without 0.01% TFA mobile phase.

Fig 3.4 Effect of solvent variations on HPLC for a run at 30°C, and 1 mL/min.

HPLC Breakdown

3D Analysis of Various HPLC Settings

Our best results :

Understanding High Pressure Liquid Chromatography

Exp 6 : 1.1 mL/min, 33°C, 20% acetonitrile, 80% water with 0.1% TFA

For a Resolution of 5.21

Elution time : 7.5 minutes

Exp 12 : 1.2 mL/min, 33°C, 15% acetonitrile, 85% water with 0.1% TFA

For a Resolution of 1.95

Elution time : 5.5 minutes

HPLC allows the separation of components in a liquid sample. It relies on the different migration rate of the analytes in a sample.

—But what does that mean exactly ?

Let's further explore HPLC !

Fig 3.6 Effect the variation of Flowrate, Organic solvent percentage in the mobile phase and Temperature on HPLC Resolution (Matlab modelisation)

Degradation pathways

Fig 1.2 Shows the potential degradation pathways of aspartame subject to the pH. www.greenfacts.org. (n.d.). What is aspartame?

Aspartame Chemistry

• Measured PH of pure aspartame sample : 5.5±0.01
• Measured PH of aspartame in acid solution : 3.56 ±0.01
• Measured PH of aspartame in slightly basic solution : 7.46 ±0.01

Fig 1.6 All potential products Aspartame can degrade into in different pH conditions along with their respective kinetics

Aspartame is a methyl ester of a dipeptide consisting of two amino acids, aspartic acid and phenylalanine.

WHERE THE STORY STARTS

Analytical HPLC Confirmation & Purity

Fig 1.1 Aspartame Chemical Structure (hand drawn)

Aspartame, HPLC, and why we are interested

• Wavelength: 217nm ± 4nm 
• t= 2.496 min
• t=2.9min: unknown (ghost or other product ?)
• t=4.8min: DKP impurity
• t=6.7min: pure aspartame

TLC Identification of analytes:

200x

375.5 MM

Present in

6000

BIBLIOGRAPHY

Scale up process

sweeter than sugar

What do we keep ?

What do we change ?

Fig 1.3 TLC Plate for an eluent ratio of 70% acetonitrile and 30% water

A correct scale up can only be achieved when these parameters are kept the same:

• pH conditions
• particle sizes
• column lengths

• Flow rates
• Injection volume
• Additional isocratic holding steps
• No changes to the gradient profile are required when column lengths are identical.
• However if the length does change, the gradient segment volume must be maintained

• Used acetonitrile as the solvent, varied eluent ratio
• Best resolution at 70% acetonitrile, 30% water
• Degraded aspartame with acetic acid: mainly degraded into phenylalanine (almost identical Rf values)
• Degraded aspartame with phosphate buffer: no spots on TLC plates, hindering analysis
• Possible issues: solvent system level too high, revealing method not effective

That was the size of the aspartame market in 2021

Apps such as the Waters Optumum Bed Density (OBD) Prep calculator to help with analytical to preparative HPLC conversions.

consumers food

Preparative HPLC Scale up_

Week 3 Operations Proposal

Analytical, Preparative... What's the difference??

Fig 1.4 TLC plate showing the degraded aspartame with acetic acid degrading into phenylalanine

25%

TOXIC ?

Our scale up

Handy equations

We use Eq. 6 and 7:

To maintain separation quality, the flow rate must be scaled accordingly

(6)

Flow rate formula, (Agilent,Principles and Practical Aspects of Preparative Liquid Chromatography,p.63)

To maintain peak shape and loading capacity, the injection volume must also be scaled based on column dimensions

Analytical HPLC is used to identify and perform analysis of the compounds in a sample

Preparative HPLC is used to purify the degraded sample

(7)

Injection volume formula, (Agilent,Principles and Practical Aspects of Preparative Liquid Chromatography,p.63)

4.6 21.2

Initial holding steps compensate for differences in dwell volume between analytical and preparative HPLC

250 250

5 7

1.1 16.689

10 212.4

Isocratic holding step formula, (Agilent,Principles and Practical Aspects of Preparative Liquid Chromatography,p.64)

(8)

Our scale up

Anayltical HPLC: Preparative HPLC:

Column diameter: 4.6mm 21.2mm

Flowrate: 1.1 mL/min ??

Particle size: 5µm 7µm

Flowrate for preparative HPLC

Similarities

Differences

1260 Infinity II Preparative LC System (Agilent)

Of UK adults are obese

Shimadzu's HPLC instrument (FoodNavigator Europe)

• Principle

• Fraction Collection
• Separation Goals
• Detection
• Solvent cost (and recovery)

Fig 1.5 Normal Phase Chromatography retention Factors

'Introduction to Modern Liquid Chromatography'- Lloyd R. Snyder;Joseph J. Kirkland; John W. Dolan

Analysis Results. (n.d.). Www.shimadzu.com. https://www.shimadzu.com/an/service-support/technical-support/analysis-basics/fundamentals/results.html

PubChem. (n.d.). Trifluoroacetic acid. Pubchem.ncbi.nlm.nih.gov. https://pubchem.ncbi.nlm.nih.gov/compound/6422

Completing our understanding of HPLC and applying the scale-up plan

Assessing HPLC quality

• Optimal Resolution value

Rs ≥ 1.5

• Resolution equations

• Efficiency, Selectivity and Retention

(1)

(3)

(4)

_ M M / D D / Y Y Y Y

(2)

(5)

Team 1_ Analysis division

Team 2_ Prep HPLC division

Eq 1, Rs value A basic knowledge of Analysis, Shimadzu Website, "About Resolution, Part 1".

Eq 2,3,4,5 Basic concepts and the control of separation, Synder, Llyod R. et al. "Introduction to Modern Liquid Chromatography", 2009.

Method Development

Objective 2 : Viability

Objective 1 : Analysis & Optimisation

BIBLIOGRAPHY

Choice of Separation Conditions

Sample Composition and Separation Goals

4_

1_

• Trial and Error
• 3D model for HPLC quality
• Analysing our results

• Aspartame and DKP-> Ion pairing agent
• Obtaining 2 clear peaks, aspartame purity

Identification and solutions of potential problems

Chromatographic Mode

5_

2_

• A ghost !?
• Column Clogging and overloading
• Need for rinse

• Reverse Phase is the default mode
• Column availability
• Isocratic Flow

Method Validation and determination of Suitability Criteria

Detector Selection (PDA-Ch1)

3_

6_

• Maximum absorbance of each compound
• Experimental confirmation : 217±4nm

• Additional data
• The "Best fit" so far
• Scale-up criterias

H. Schulenberg-Schell, A. Tei, F. Rieck, R. Guillet, Principles and Practical Aspects of Preparative Liquid Chromatography, formulas for linear scale-up from analytical to preparative columns, p.63-64, https://www.agilent.com/cs/library/primers/public/primer-preparative-liquid-chromatography-5994-1016EN-agilent.pdf, [Accessed: 11th December 2023]

RESEARCH STRATEGY_

_4

Agilent, Preparative HPLC systems, 1260 Infinity II Preparative LC System [Accessed 11th December 2023]

S. PATTANAARGSON, C. CHUAPRADIT, AND S. SRISUKPHONRARUK, (2001) Aspartame degradation in solutions at various pH conditions, JFS Food Chemistry and Toxicology , http://lib3.dss.go.th/fulltext/Journal/Journal%20of%20food%20science/2001%20v.66/no.6/jfsv66n6p0808-0809ms20000528%5B1%5D.pdf

Jinchuan Yang, Paul D. Rainville(2021)-Analysis of Soft Drink Additives with No Interference from Aspartame Degradants Using Arc HPLC System with PDA Detection : Waters

J Prodolliet, M Bruelhart(1993)-https://pubmed.ncbi.nlm.nih.gov/8471853/#:~:text=The%20method%20also%20allows%20the,diluted%20with%20water%20and%20filtered.

Srividya Kailasam, PhD(February 28, 2022)-https://www.technologynetworks.com/analysis/articles/ion-pair-chromatography-how-ipc-works-strengths-limitations-and-applications-358440#:~:text=Ion%20pairing%20reagents%20are%20also,by%20G%C3%B6ran%20Schill%20in%201973

Bin Cai, Jianwei Li(15 November 1999)-https://www.sciencedirect.com/science/article/pii/S0003267099004614

Andrew J. Aubin, Ronan Clean( June 2009),Waters, Analytical HPLC to Preparative HPLC: Scale-up Techniques using a Natural Prouduct Extract [Accessed 12th December 2023]

Scale up &

Strategy

• How do we switch from analytical to preparative HPLC ?
• What more information do we need ?

Joseph James Whitworth (2014) Shimadzu HPLC to improve lab workflow, FoodNavigator Europe [Accessed 11th December 2023]

Working with a functional and frequently rinsed column, and new samples:

• Decide between experiment 6 and 12 for optimised process (Modifiy exp 12 flowrate) and share results with team 2 for Prep HPLC strategy adaptation

• Complete Analytical HPLC Data Set ; run experiments to complete Temperature vs Resolution trend, Flowrate vs Resolution trend, measure mobile phase pH for a various range of A%/B%.

• Optional : Try a Higher percentage of TFA (0.2 %) and methanol as an organic mobile phase (Risk Assessment will be amended)

_3

Aspartame: A sweet discovery

Aspartame, C14H18N2O5, is an artificial sweetener that is 200 times sweeter than table sugar. Discovered accidentally by a chemist in 1965, it has since been used as an ingredient in approximately 6000 consumer foods and beverages.

With over a 25% of adults in the UK being obese, aspartame has been a good replacement for sugar in many foods in hopes to reduce the obesity rates. However, with concerns that aspartame could cause cancer and that it can easily degrade into different harmful products, it has been quite a hot topic on whether aspartame is a totally safe for consumption.

PubChem, Aspartame (Compound), 1.1 2D structure

_2

• Run the optimised separation proces with 3 different degraded aspartame samples to confirm validity and attain better precision

• Assess the efficiency of the Prep HPLC method (through recovery and mobile phase consumption)

• Investigate the viability of mobile phase recovery methods and assess the need for supplementary steps in establishing an industrial purification process.

Knauer (2023) What is the difference between analytical and preparative HPLC?, Knauer website , https://www.knauer.net, [Accessed 10th December 2023]

Experimental

Results & Analysis

• How do chemical and physical factors affect the quality of our HPLC separation
• What problems did we face during analysis ?

• Run the Preparative HPLC system with the scaled-up model of our best experiment (exp 6, exp 12 if conclusive analytical tests)

• Conduct an array of Prep HPLC experiments to confirm/deny if varying settings affect separation quality akin to Analytical HPLC, adapt the preparative process : Optimisation of Recovery process

This section of team two's mission should last maximum half of week 3, the second half of week 3 will be dedicated to the second objective of the division.

HPLC & Method Development

Using scale-up techniques, we will then utilise preparative HPLC during our final week for the purification of the aspartame samples.

Shimadzu's HPLC instrument (FoodNavigator Europe)

We were tasked to find out the purity in the aspartame samples from the company we work for. Using TLC, we can determine the impurities present in the sample, and with analytical HPLC we can calculate the purity of the samples using the peak areas from the chromatogram generated.

• How does HPLC works ?
• How do we assess the quality of an HPLC separation process ?

Prezi images.

Shutterstock

_1

• How does aspartame degrades ?
• How would the properties of our analytes affect their separation ?

Identifying our

Analytes

S. Kailasam (February 28, 2022) Ion Pair Chromatography – How IPC Works, Strengths, Limitations and Applications, Technology Networks Journal, https://www.technologynetworks.com/analysis/articles/ion-pair-chromatography-how-ipc-works-strengths-limitations-and-applications-358440#:~:text=Ion%20pairing%20reagents%20are%20also,by%20G%C3%B6ran%20Schill%20in%201973 [Accessed 6th December 2023]

Frontier, A. (2022). Reagents & Solvents. [online] www.chem.rochester.edu. Available at: http://www.chem.rochester.edu/notvoodoo/pages/reagents.php?page=solvent_polarity. [Accessed 12 Dec. 2023]​

S. Pattenaargson, C. Chuapradit, S. Srisukphonraruk - Aspartame Degradation in Solutions at Various pH Conditions, http://lib3.dss.go.th/fulltext/Journal/Journal%20of%20food%20science/2001%20v.66/no.6/jfsv66n6p0808-0809ms20000528%5B1%5D.pdf [Accessed 12 Dec. 2023]​

_CREDITS_

Thank you for your attention!

The Aspartaste® Team

PROJECT GOALS_

OPTIMISATION

In order to purify impure aspartame batches we aim to :

• Investigate the viability of HPLC as a purifying method
• Optimize an HPLC process to get the best separation results with Preparative HPLC

ASPARTASTE® (Group A)

PROJECT PROPOSAL_

INSIGHTS

• Understanding aspartame degradation
• Understanding HPLC separation

SEPARATION

Product quality is a promise Aspartaste® is ready to make to all their clients. For that we :

• aim to identify and determine the purity of degraded aspartame batches