Keywords— Lactose, Crystallization, Growth Rate Dispersion

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INTRODUCTION

This research was performed by analysing the works of the previous reports and articles from Massey University and peer reviewed journals provided by my supervisor Prof. Tony Paterson. The area of the growth rate dispersion was not very well understood, so, the experiment is performed in a c-chip which is a small volume crystallizer of 10µL and has well defined area which enables to measure the size of the crystal. There are many problems incurred during the experiment. So, as suggested by my supervisor, only worked on the growth rate dispersion and ignored about nucleation of lactose. The research can be beneficial to many industries such as confectionary, food related and pharmaceuticals, etc. In the analysis and the characterization of crystal growth progress there has been an increasing recognition of the feature GRD. Hence understanding growth rate dispersion can be quite significant.

The objective of this experiment are:

• To investigate the different individual crystals growth rates in c-chips.
• To compare if the growth rate dispersion occurring in the c-chip is same as the stirred vessel crystallizers.

This paper is organised as follows. The first section talks about the literature review to briefly explain what growth rate dispersion is, c-chip information and previous works. This is  followed by the various problems in the experiment and their possible solutions. Then the results of the experiment were discussed and in the final section conclusion and recommendations are presented to take this work further.

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GROWTH RATE DISPERSION

Each individual crystal has a different growth rate even though they are growing under identical conditions. This phenomenon is called growth rate dispersion. Some of the crystals towards left of the dotted line grow faster and ones on the right grow slow within the broad range of particle distribution as shown in the figure 1. GRD in lactose crystallization was first reported by Visser (T.D. Dincer, 2009). GRD was proposed by Zumstein and Rousseau (1987) which is a result of individual crystals dislocation network.

Figure 1: Growth Rate Dispersion of Lactose crystals

C – CHIP

C-chip is a plastic disposable hemocytometer with a volume of 10µL. It consist of two chambers A and B which are enclosed and has two ports for sample injection.  The area of each large square within a grid in c-chip is 1 x 1 mm and the chamber depth is 0.1 mm (NanoEntek, 2018).

Figure 2: C-Chip

  

Figure 3: Grid Pattern in C-Chip

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Research about growth rate and nucleation was performed by Y. Shi et al. (1989) using a microscopic cell and observed GRD phenomenon existed in the lactose system. In static environments in situ measurements of overall growth rates were performed. To monitor lactose crystallization from non-seeded supersaturated solutions, polarized light microscope was employed in real time by Arellano, Aguilera, and Bouchon (2004) . Growth rates were measured in batch crystallizers by using a simple method of determining the crystal growth rates using weight change by Thurlby (1976). Using continuous cooling crystallizer, growth rate were studied by Y. Shi, Liang, and Hartel (1990).

Similar experiment to c-chip were performed by T.D. Dincer (2009) using the apparatus developed by Lee and Parkinson (1999) which was used to measure growth rates of four faces of ALM. He performed the experiment at similar temperature and super saturations (T.D. Dincer, 2009).

Table 1: Literature data of in situ and stirred vessel crystallizers

In situ measurements of overall growth rates in a static environment
Literature	Method	Temperature (0C)	s-1	Seed size (μm)
Shi (1989)	Photographic	30, 40, 50, 60	0.78–2.8	<20
Arellano et al. (2004)	Photographic	10, 20, 30	0.97–2.27	<20
Stirred vessel
Thurlyby (1976)	Batch crystallizer experiments	15, 25, 35, 40, 50	0.5–1.7	127 – 147
Shi (1989)	Continuous crystallizer experiments	30, 40, 50, 60	0.59–1.77	5 – 150

(Dincer, Ogden, & Parkinson, 2009)

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METHEODOLOGY

Exact amounts of ALM and water needed were calculated before performing the experiment depending upon the required super saturation and temperature at which the experiment is performed. Lactose and water are mixed using the magnetic stirrer and is heated to required temperature using the heating mantle. Thermometer was clamped in such a way that it can hold in place in the lactose solution. A tin foil was used to cover the beaker to prevent or reduce the evaporation losses. The solution before and after heating is weighed, so, water can be added to account for the evaporation losses. By the end of the experiment the lactose is completely dissolved and the solution is clear.

This hot solution is injected into the c-chip in one of the chambers. It is then sealed and placed under the microscope to observe the crystal growth progress. Photos were time stamped to enable easy tracking of the crystal growth process. Area of the c-chip were marked to follow the individual crystals growth rate. Image J analysis was used to measure the area of the individual crystals over time. This experiment was initially performed in a temperature controlled room but was moved to Micro lab and experiments were performed at room temperature. The apparatus used in the experiment are shown in the Figure 4 and Figure 5.

Figure 4: Lactose solution heating apparatus

Figure 5: Microscope in Micro lab

PROBLEMS INCURRED DURING EXPERIMENT

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At the beginning of the experiment it was decided to leave the lactose solution over night before performing the experiment. But it was found that the solution is completely crystallized and is in solid state. Initially, the experiment was conducted in a temperature-controlled room (25⁰C), the quality of the camera provided was too poor and it could only show grids but not the crystals. So, the microscope in the Micro lab was used. Due to the movement of the c-chips to different lab resulted in inability to track the crystal growth progress. Also, air gaps were formed in the c-chip due to the evaporation as shown in the Figure 6. After the entire experimental set up moved to the Micro lab, poor crystal formation and growth were observed in the c-chip.

Figure 6: Air gap in C-Chip

POSSIBLE SOLUTIONS

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To prevent crystallization in beaker it was decided to inject the hot solution directly into the c-chip. Initially, supersaturated solution was made in the temperature-controlled room and the solution was injected into c-chip and was taken to the micro lab to use the microscope. Later, in order to follow the crystal growth progress properly, the entire set up was shifted to the Micro lab and the experiments were performed. To prevent the air bubbles formation in the c-chip opening and closing of the c-chips were sealed which didn’t solve the problem so various evaporation tests were performed using different sealing materials and tapes. The clear tape showed better results and the c-chip had well defined crystals as shown in the Figure 7. This tests gave an idea of how long the crystal growth occurs (until the crystals can be measured) to manage time of the experimental process. It was observed that growth of the crystals occurred within 2-3 hours.

Figure 7: Clear tape is used to seal

Another solution was to bring the c-chip and the lactose solution to the same temperature before injection to prevent evaporation losses. It was found that the c-chip was not completely sealed and had 2 layers in it. So, all the edges of the c-chip were sealed along with the opening and the closing of the chamber using a clear tape as shown in the,Figure 8.

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Figure 8: C-Chip sealed using Clear tape

RESULTS AND DISCUSSION

The graph below is showing the growth rate of individual crystals in a c-chip. The experiment was performed at a temperature of 20.3⁰C and at a super saturation of 20 for about 10 ½ hours.

Figure 9: Growth rate of different cyrstals in c-chip

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Figure 9 clearly shows that some of the crystal grow faster (crystal 1, 2, 3, 4) by utilising solute and some are slow growers (crystal 5, 6, 7) similar to bell block curve shown in Figure 1. Poor crystal formation and limited growth could be due to contamination at some point during the experimental procedure. Also, the experiment is performed in a Micro lab, where all the standard microbiological procedure were performed. The room could be filled with unknown bacteria or impurities which could be limiting or reducing the crystal growth. Due poor results and inability to replicate the experimental results, it was difficult to compare the c-chip reslts with the stirred vessel crystallizer.

CONCLUSIONS

This paper describes about the growth rate dispersion of lactose crystallization. It was found hard to compare the results obtained in the experiment with the stirred vessel crystallizer as the data obtained is poor and not reliable as it couldn’t be replicated. This experiment with c-chip has many problem and it doesn’t work. Also, it is time consuming process and requires proper conditions (clean) to perform the experiment and hence, not worth investing money and time.

RECOMMENDATIONS

1. Further research can be performed as it helps to understand more about the growth rate dispersion and what unknown factors are limiting the crystal formation and growth.

2. It is recommend to perform the experiment in a clean environment to reduce the unknown factors such as bacteria and impurities during the experiment process.

3. But it is not worth investing the time and effort in this project, as it has many problems and is a time consuming process.

ACKNOWLEDGEMENTS

I would like to express my sincere gratitude to my supervisor Prof. Tony Paterson for providing valuable suggestions, guidance and comments throughout the project. I special thank Ian Thomas for helping to solve temperature concerns in the experiment. I would also like to thank Ann-Marie Jackson, John Sykes, Anthony Wade and Morio Fukuoka for providing necessary lab equipment and support. . Also, I would love to thank my family and friends for the love and the support throughout my studies in university. Finally, Massey University for financially supporting this project.

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REFERENCES

1. Arellano, M. P., Aguilera, J. M., & Bouchon, P. (2004). Development of a digital video-microscopy technique to study lactose crystallisation kinetics in situ. Carbohydrate Research, 339, 2721-2730.
2. Dincer, T. D., Ogden, M. I., & Parkinson, G. M. (2009). In situ investigation of growth rates and growth rate dispersion of [alpha]-lactose monohydrate crystals. Journal of Crystal Growth, 311(5), 1352-1358.
3. Lee, M.-Y., & Parkinson, G. M. (1999). Growth rates of gibbsite single crystals determined using in situ optical microscopy. Journal of Crystal Growth, 198/199, 270 -274.
4. NanoEntek. (2018). C-chip Instructions. Retrieved from http://www.nanoentek.com/upload/product/9/C-chip_Manual%20(V.0.2).pdf
5. Shi. (1989). Formation and Growth Phenomena of Lactose Nuclei Under Contact Nucleation Conditions. Journal of Dairy Science, 72(11), 2906-2915.
6. Shi, Y., Hartel, R. W., & Liang, B. (1989). Formation and Growth Phenomena of Lactose Nuclei Under Contact Nuclei  Conditions. Journal of Dairy Science, 72, 2906-2915.
7. Shi, Y., Liang, B., & Hartel, R. W. (1990). Crystallization Kinetics of Alpha-Lactose Monohydrate in a Continuous Cooling Crystallizer. Journal of Food Science, 55(3), 817-820.
8. T.D. Dincer, M. I. O., G.M. Parkinson. (2009). In situ investigation of growth rates and growth rate dispersion of alpha-lactose monohydrate crystals. Journal of Crystal Growth, 1352-1358.
9. Thurlby, J. A. (1976). Crystallization Kinetics of Alpha Lactose. Journal of Food Science, 41, 38-42.
10. Thurlyby, J. A. (1976). Journal of Food Science, 41, 38.
11. Zumstein, R. C., & Rousseau, R. W. (1987). Growth rate Dispersion in Batch Crystallization with Transient Conditions. AIChE Journal, 33(11), 1921-1925.

APPENDICES

 

Equation used in this experiment are:

Lactose solution concentration:

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CL=grams of lactosegrams of water= 0.95*xy+0.05*x           eq1

unit: g lactoseg water

The absolute super saturation of ALM:

supersaturation= Cα- Cαs     eq(2)

unit: g α-lactose100 g water

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The lactose solubility concentration is:

Csolubility= 10.9109*exp⁡0.02804*θ      eq3

unit: g lactose100 g water

Lactose concentration as a function of temperature:

Equilibrium ratio, K=0.95*exp⁡151.399θ+273.15     eq4

Equilibrium ration K:

Cα= Csolubility1+K       eq(5)

Where,

Cαis the absolute concentration of alpha-lactose.

unit: g α-lactose100 g water

Alpha-lactose solubility:

Cαs= Csolubility-F*K*CLT- Csolubility1+K      eq6

unit: g α- lactose100 g water

Correction Factor, F:

F=0.0187*exp⁡0.0236*θ     eq(7)

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