Lactose Crystal Size And Morphology Biology Essay

Published:

This essay has been submitted by a student. This is not an example of the work written by our professional essay writers.

The α-lactose monohydrate is widely used as a pharmaceutical excipient. Drug delivery system requires the excipient to be of narrow particle size distribution with regular particle shape. Application of ultrasound is known to increase or decrease the growth rate of certain crystal faces and controls the crystal size distribution. In the present paper, effect of process parameters such as sonication time, anti-solvent concentration, initial lactose concentration and initial pH of sample on lactose crystal size, shape and thermal transition temperature was studied. The parameters were set according to the L9-orthogonal array method at three levels and lactose was recovered from whey by sonocrystallization. The recovered lactose was analyzed by particle size analyzer, scanning electron microscope and differential scanning calorimeter. It was found that the morphology of lactose crystal was rod/needle like shape. Crystal size distribution of lactose was observed to be influenced by different process parameters. From the results of analysis of variance (ANOVA), the sonication time was found to be the most significant parameter affecting on the volume median diameter (VMD) of lactose with the highest percentage contribution (74.28 %) among the other parameters.

1­­­ Introduction

The most common method used to produce the pharmaceutical drug materials is crystallization. Production of these crystalline particles with the defined chemical and the physical properties is the critical problems to these industries [1]. Lactose is disaccharide, produced commercially from whey, the by-product of dairy industries. Lactose can exist as either amorphous lactose or crystalline lactose, as either a-lactose or b- lactose or as a mixture [2]. The α-lactose monohydrade is widely used as a pharmaceutical excipient. It is reported that aerosolization of the drug is difficult because of its strong cohesive and adhesive properties [3]. To overcome these undesirable properties, α-lactose monohydrade is employed as carrier for dry powder aerosols. Higher elongation ratio, smooth surface and better flow of α-lactose monohydrade are responsible for improving the dispersion and fine particle fraction (FPF) of adhered drug, which improves drug delivery to the lower airways from dry powder inhalers (DPIs) [3]. The physicochemical properties of the drug and the carrier particle as well as the design of the inhalation device are responsible for the dispersion and subsequent deposition profiles of drug particles during inhalation [4]. The size and morphology of a drug or excipient are affecting several essential pharmaceutical properties. The drug delivery systems needs the excipient to be of narrow particle size distribution with regular particle shape [5]. Hence, there is a need to recover lactose crystals with desirable size, shape and surface from whey for DPI application. Lactose obtained by conventional anti-solvent crystallization with mechanical agitation has variation in the particle size and morphological features due to poor-mixing [6]. Sonocrystallization is a rapid crystallization technique which can able to control crystal size distribution. With micro streaming and acoustic streaming, ultrasound shows a great ability in blending of the anti-solvent with solvent leads to uniform and rapid nucleation of solute and forms elongated fine crystals with uniform size 7]. Recently, sonocrystallization has been reported for the recovery of lactose using different anti-solvent ethanol [8] and acetone [9] from reconstituted lactose solution. Also, the process parameters in an anti-solvent sonocrystallization of lactose have been optimized for the recovery of lactose from whey [10,11]). In our previous study, we have reported the Taguchi method to optimized process parameters in sonocrystallization for the recovery of lactose from whey using L9-orthogonal array [11]. In this paper, we have used L9-orthogonal array to study the effect of process parameters on crystal size and morphology of lactose recovered from whey using sonocrystallization.

*Corresponding author. E-mail: zvpm2000@yahoo.com, zvpm@ched.svnit.ac.in

Tel.: +91 261 2223371 to 2223374, 2201648 Fax: +91 261 2227334

2 Materials and methods

A 4 factor at 3 levels of L9-orthogonal array method [12] was carried out to study the influence of acetone concentration (65 - 85 %v/v), sonication time (5 - 15 minutes), initial lactose concentration in whey (5-15 %w/w) and initial pH of sample (2.5±0.1 - 6.5±0.1) on lactose crystals. Selection of these factors and their values were based on preliminary studies on lactose recovery using ultrasound assisted crystallization [9]. The L9-orthogonal array was constructed as shown in Table 1 and accordingly experiments were carried out. The pre-treatment of whey obtained from near by small scale dairy industry was done as the procedure reported by Agrawal et. al.[] (2007) for fat removal and heat-induced deproteination of whey. Lactose content of whey was estimated with by the method of Willstatter and Schudel[14] iodometric titration was used to determine the initial lactose content in whey and modified Folin-Lowry method[15] was used determined protein concentration in whey [9]. For all the experiments, a 15 mL concentrated whey was taken in 250 mL round bottom flask and required parameter was achieved by adding required amount of acetone as an anti-solvent and then, sonicated in ultrasound bath of 120W rating and surface area of 225 cm2 (Aqua Scientific Instruments, Surat, India) at room temperature of 30±3oC. The lactose was recovered from whey using vacuum filtration and dried at 60 oC for 3 hours in vacuum oven. After each of the experiments, Scanning Electron Microscopy (SEM), Particle size analysis and Differential Scanning Calorimetry was done for recovered lactose.

Particle Size Distribution Particle size was measured by Laser Diffractometer, Mastersizer 2000 Ver. 5.31 Malvern Instruments, Malvern, UK. Analysis was done in duplicate and mean results are presented from wet mode measure data. 2- Propanol was used as dispersant and obscuration was not less than 10.60% for each measurement. Data analysis was done by Malvern Software. The size distribution was expressed by the volume median diameter (VMD).

Scanning Electron Microscope (SEM) Samples of the moisture sorption equilibrium were used for SEM investigation. Thin layers of powder samples were mounted on aluminum stubs, using double-sided adhesive tape. The mounted samples were first sputter-coated with carbon and then with gold/palladium. The coated samples were examined with a Hitachi S3400 scanning electron microscope operating at 15 kV accelerating voltage.

Differential Scanning Calorimetry (DSC) Thermal transition temperatures were determined by a DSC calorimeter. Lactose samples (3.270 to 3.620 mg) were heated from 30 to 300°C in an aluminum open pan. The heating rate was set at 10°C/min.

Analysis of Variance (ANOVA)

The variance of the volume median particle size were calculated to identify the most important Factors. Equations for conducting the variance are used as following.

Sum of squares-Sum of square for factor A at level K was calculated according to the equation:

Where yi is VMD obtained from sample 1 - 9, n the total number of experiments at level L. Where, N is the total number of experiments (i.e. 9 in present study), nAi is the number of levels (3 in present study), and Ai the sum of volume median particle size in level i of factor A. The total sum of squares (SST) was calculated using equation:

The degree of freedom, F-test for comparison of variance and Percentage of the contribution to the total variation were calculated as reported in Patel and Murthy [11].

Table 1 L9-orthogonal array and volume median diameter (VMD)

Sample

Initial lactose concentration, % (w/w)

Initial pH of sample

Solvent concentration, % (v/v)

Sonication time, (minutes)

VMD(mm)

1

5

2.5

65

5

12.6

2

5

4.5

75

10

89.5

3

5

6.5

85

15

77.3

4

10

2.5

75

15

121.2

5

10

4.5

85

5

37.9

6

10

6.5

65

10

135.2

7

15

2.5

85

10

41.4

8

15

4.5

65

15

14.9

9

15

6.5

75

5

17.3

10

15

6.5

75

15

30.3

11

Commercial lactose sample

40.6

Results and discussion

Particle Size and Morphology Crystal size distribution of particles depends on the rate of crystallization. Rapid rate of crystallization gives better crystal size distribution; however, the crystal size would be smaller [15]. Rapid conventional anti-solvent crystallization processes yields elongated but fine crystals (5-10 μm), which are not suitable to use as a carrier for DPI application. For this application, α-lactose monohydrade is usually required to have size between 63-90 μm with high crystallinity, elongation ratio and smooth surface [3]. When sonication is used during an anti-solvent crystallization the induction of crystallization and crystal growth becomes rapid indicates the influences of sonication in induction of nucleation and crystal growth [7]. With acoustic streaming and micro streaming ultrasound shows considerable effects in crystallization process, such as reduction of crystal size, inhibition of agglomeration and manipulation of crystal size distribution [16]. Effects of ultrasound on lactose crystals recovered from reconstituted lactose solution have been reported with anti-solvents ethanol [8] and acetone [9]. The size and shape characteristics of the lactose crystals were improved. Crystal size distribution was influenced and narrow CSD was observe for sonocrystallized sample [8, 9]. In these studies, it was observed that the lactose crystals size, shape and CSD could be obtained by controlling the sonication time, pH and initial lactose concentration. Therefore, the experiments carried out to understand the contribution of these parameters on crystal size, shape and morphology of lactose recovered from actual whey (Table 1). Sample 1-9 shows VMD and span of the lactose recovered from whey according to L9 - orthogonal array. Sample 10 shows the VMD of lactose obtained from whey at optimized condition as reported by our previous study and sample 11 shows the VMD of commercial lactose (α-lactose monohydrade). It can be seen from Table 1 that sample 4 and sample 6 gave the higher VMD as compare to other samples. VMD at optimized condition (Sample 10) was found to be 30.3 mm, while for commercial lactose was 40.6 mm (Sample 11). The CSD obtained for each samples are shown in Fig.1 - 4. It can be seen that the spread of the crystal size was observed broader for sample 2-7. The broader CSD observed for these experiments might be due to agglomeration of the lactose, which can be confirm from the scanning electron micrograph of lactose for these experiments (Fig. 5-9). It can be observed from Fig. 5-9 that the level of agglomeration for sample 2 and sample 4 was high as compare to the sample 1, sample 3 and sample 5-9. The spread of CSD of experiment at optimized condition was observed narrow as compared to commercial lactose sample as shown in Fig.5. Agglomeration of lactose crystals for the sample at optimized condition was found to reduce to some extent. The resultant morphology for sample1, sample 3, sample 5-9 and at optimized condition was observed to be rod/needle like shape, while for sample 2 and sample 4 was not predictable. To compare the morphology of lactose crystals obtained by sonication with lactose obtained by without sonication, experiment was carried out to recover the lactose from whey by applying stirring at 1000 rpm and temperature kept at 5±1oC in an anti-solvent acetone. SEM of lactose recovered from whey by stirring and commercial lactose sample are shown in Fig. 10. The morphology of the lactose obtained by agitation and commercial lactose sample was found to be tomahawk shape (Fig. 10). Agglomeration level of lactose crystals was observed to be high in sample obtained by stirring as compared to the sonicated samples (Fig. 5-9). Poor mixing in an anti-solvent crystallization (lactose obtained from whey by stirring) leads to heterogeneous growth of crystals, which enhances variation in particle size and morphological features. When ultrasound is applied in an anti-solvent crystallization, uniform supersaturated conditions can be achieved rapidly throughout the crystallization vessel resulting in rapid crystallization [7]. Rapid crystallization of lactose appears to accelerate the growth of longest axis of the crystals with decrease in width and thickness resulting in needle-shaped, elongated crystals [3, 7]. Furthermore, if the growth occurs in the longitudinal direction, crystal particles can have similar thickness. Hence, lactose crystals with a narrower distribution can be obtained as shown in Fig. 4. ANOVA procedure was used to study the effect of factors towards VMD as shown in Table 2. The percentage contribution from each of the factors in the total sum of the squared deviations was used to evaluate the importance of the factor change on the VMD. Results of ANOVA (Table 2) showed that sonication time, pH of samples and solvent concentrations were significant factors affecting VMD. Moreover, the sonication time was found to be the most significant factor with the highest percentage contribution (74.28 %) among the other factors.

Table 2 Results of analysis of variance (ANOVA)

Factor

Sum of square

Degree

of freedom

Mean square

% Contribution of factor towards response

Initial lactose concentration,% (w/w)

88.56

2

44.28

0.93

pH of samples

1302.20

2

651.10

13.7

Solvent concentration,% (v/v)

1044.36

2

522.18

11.02

Sonication time (minutes)

7033.28

2

3516.64

74.28

Error

0

0

0

-

Total

9468.41

8

1183.55

-

Fig.1 . Crystal size distribution of lactose recovered from S2-S4.

Fig. 2. Crystal size distribution of lactose recovered from S6 - S7.

Fig. 3. Crystal size distribution of lactose recovered from S1, S8 and S9.

Fig. 4. Crystal size distribution of lactose recovered in S10 (optimized condition) and pure lactose (S11).

Fig. 5. SEM of lactose recovered from whey at S 1 and S 2

Fig. 6. SEM of lactose recovered from whey at S 3 and S 4

Fig. 7. SEM of lactose recovered from whey at S 5 and S6

Fig. 7. SEM of lactose recovered from whey at S 5 and S 6

Fig. 8. SEM of lactose recovered from whey at S 7 and S 8

Fig. 9. SEM of lactose recovered from whey at S 9 and S10 (optimized condition)

Fig. 10 . SEM of lactose recovered from whey at S 11 and S12 (lactose recovered by stirring)

Fig. 10. SEM of commerciloa lactose (s11) and lactose recovered from whey by stirring (s12)

Differential Scanning Calorometry(DSC) of lactose Differential Scanning Calorometry of lactose recovered at the end of each experiment was carried out to analyze the transition temperatures. The physical transformations and transition temperatures of lactose were determined from endothermic and exothermic peaks of DSC diagrams for each of samples as shown in Table 3. DSC diagram for sample 10 and sample 11 are shown in Fig. 11. The transition temperatures for pure alpha lactose monohydrate are reported as; the dehydration of water of crystallization is 144 °C, recrystallization temperature is 173 °C (exothermic peak) and melting temperature is 211 and for pure b - lactose melting point is 220 °C [17]. For amorphous lactose, glass transition temperature is 101°C, crystallization is 171 °C (exothermic peak) and melting temperature is 215 °C and 230 °C [17]. In the present study, the dehydration of water of crystallization was observed from 138.39 -146.19 °C and melting temperature was observed from 201 - 216.98 °C. Recrystallization temperature was observed to be 166.70-172.24 °C (exothermic peak) and melting temperature of b - lactose was from 222.39 227.17 °C.

Table 3 Phase transition of lactose observed by DSC

Sample

Dehydration of water of crystallization (°C)

Melting point of a-lactose monohydrate (°C)

Melting point of b-lactose (°C)

Recrystallization temperature

(°C)

1

144.91

204.33

-

167.431

2

140.04

207.20

-

168.88

3

143.72

206.52

222.39

168.10

4

140.13

204.72

-

168.21

5

136.93

210.15

227.17

172.24

6

141.70

201

-

168.14

7

143.32

203.84

-

-

8

138.39

210.68

225.87

-

9

140.28

209.96

222.91

172.50

10

140.72

213.73

-

166.70

11

146.17

216.98

-

-

The enthalpy of dehydration was obtained from DSC diagram for all samples and the number of moles of water per mole of anhydrous lactose was calculated using following equation as reported by Dhumal et. al. (2008):

where, n is number of moles of water per mole of anhydrous lactose, ΔHd is the enthalpy of dehydration obtained from the dehydration endotherm (J/g); ΔHv is the enthalpy of vaporization of water, 2,261 J/g (Dhumal, 2008), and MWlactose and MWwater are the relative molecular masses of anhydrous lactose (340.3) and water (18.0), respectively. The no of moles of water per mol of anhydrous lactose was calculated and values are shown in Table 4.

Table 4 No of moles of water (n) per mole of anhydrous lactose

Sample

1

2

3

4

5

6

7

8

9

10

11

n

0.59

0.89

0.88

0.85

0.79

1.06

0.83

0.50

0.40

0.57

1.02

S-9

S-11

Fig. 11 DSC diagrams of lactose for sample 9 and sample 11.

4 Conclusion

Lactose crystals recovered from whey by sonocrystallization was studied in the present paper. Crystal size distribution of lactose at optimized condition was found to narrower as compare to the other process conditions. Elongated and rod/needle-shaped lactose crystals was found in sonicated samples, while the lactose recovered from stirring was observed to be tomhawk shape. Up to some extent, level of agglomeration was observed for sonicated and non-sonicated samples. Agglomeration was found to reduced in the sample obtained at optimized condition and uniform crystal size distribution was observed. Among all factors, sonication time was found to be most influencing parameter on VMD of lactose.

Writing Services

Essay Writing
Service

Find out how the very best essay writing service can help you accomplish more and achieve higher marks today.

Assignment Writing Service

From complicated assignments to tricky tasks, our experts can tackle virtually any question thrown at them.

Dissertation Writing Service

A dissertation (also known as a thesis or research project) is probably the most important piece of work for any student! From full dissertations to individual chapters, we’re on hand to support you.

Coursework Writing Service

Our expert qualified writers can help you get your coursework right first time, every time.

Dissertation Proposal Service

The first step to completing a dissertation is to create a proposal that talks about what you wish to do. Our experts can design suitable methodologies - perfect to help you get started with a dissertation.

Report Writing
Service

Reports for any audience. Perfectly structured, professionally written, and tailored to suit your exact requirements.

Essay Skeleton Answer Service

If you’re just looking for some help to get started on an essay, our outline service provides you with a perfect essay plan.

Marking & Proofreading Service

Not sure if your work is hitting the mark? Struggling to get feedback from your lecturer? Our premium marking service was created just for you - get the feedback you deserve now.

Exam Revision
Service

Exams can be one of the most stressful experiences you’ll ever have! Revision is key, and we’re here to help. With custom created revision notes and exam answers, you’ll never feel underprepared again.