1. Practical issues in UV, HPLC analysis, and formulation
development of Solid Lipid Nanoparticles
Vijay Kumar E M.Pharm. Pharmaceutics
Email ID: evijay.kumar.phe12@itbhu.ac.in

2. Presentation outline
• Issue 01: Change in Emax and Valley depth
• Issue 02: RP-HPLC chromatograms peak splitting
• Issue 03: Batch to Batch Variability during replication

3. Issue 01
Change in Emax and Valley depth (UV-Spectrophotometer)

4. 0
Peak 01
Peak 02
Peak 03
Valley 01
Valley 02
200nm 400nm
Absorbance(AU)
Wavelength
Day 03
Day 02
Day 01
𝐴l = 𝑆𝑎𝑚𝑝𝑙𝑒 𝑠𝑜𝑙. 𝑎𝑏𝑠.l− 𝑃𝑢𝑟𝑒 𝑠𝑜𝑙𝑣𝑒𝑛𝑡 𝑎𝑏𝑠.l
Issue 01

5. 1mg/mL
1.2mg/mL
1.4mg/mL
Day 01
Why do peaks attain a new high Emax as the time progresses?
𝐴 = 𝑎𝑏𝑐
b
Analyte
Day 02
Day 03

6. Assume analyte has no chromophoric group that absorbs at 300nm and only responsible
agent for absorbance is pure solvent.
Organic 𝑃𝑢𝑟𝑒 𝑠𝑜𝑙𝑣𝑒𝑛𝑡 𝑎𝑏𝑠.l300 = 1.50
𝑆𝑎𝑚𝑝𝑙𝑒 𝑠𝑜𝑙. 𝑎𝑏𝑠.l300 = 1.47
𝐴l300 = 1.47 − 1.50
𝐴l300 = −𝟎. 𝟎𝟑
Day 02
𝐴l300 = 1.50 − 1.50
𝑆𝑎𝑚𝑝𝑙𝑒 𝑠𝑜𝑙. 𝑎𝑏𝑠.l300 = 1.50
𝐴l300 = 𝟎. 𝟎𝟎
Day 01
𝑆𝑎𝑚𝑝𝑙𝑒 𝑠𝑜𝑙. 𝑎𝑏𝑠.l300 = 1.45
𝐴l300 = 1.45 − 1.50
𝐴l300 = −𝟎. 𝟎𝟓
Day 03
Why do valley gets even more deep as the time progresses?
Consider the following case 𝐴l = 𝑆𝑎𝑚𝑝𝑙𝑒 𝑠𝑜𝑙. 𝑎𝑏𝑠.l− 𝑃𝑢𝑟𝑒 𝑠𝑜𝑙𝑣𝑒𝑛𝑡 𝑎𝑏𝑠.l
0
-0.05
300nm

7. Strategy(s) sought to address the issue
• Store at constant temperature (at 25C)
(Temperature ∝ Solubility)
• Adequately tighten the lid of the solution holder

8. Issue 02
Peak splitting in RP-HPLC chromatograms

9. AU
-0.002
0.000
0.002
Minutes
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00
AU
0.000
0.005
0.010
Minutes
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00
A
B
A-Chromatogram with split peaks; B-Chromatogram with no split peaks

10. Strategies sought to address the issue
• Mobile phase ratio manipulation
• Solvent effect
• Guard column replacement
Negative
Negative
Positive
One factor variation at a time

11. Representation of elution in normal ODS column (guard
column) and simultaneous AU-Time graph
Transverse plane view
Longitudinal plane view
P1
P2
P1=P2

12. Representation of elution in normal ODS column and
simultaneous AU-Time graph
Time
Concentration(mcg/mL)/
Absorbanceunits
P1
P2
P1=P2

13. Representation of elution in normal ODS column and
simultaneous AU-Time graph
Time
Concentration(mcg/mL)/
Absorbanceunits
P1
P2
P1=P2

14. Representation of elution in normal ODS column and
simultaneous AU-Time graph
Time
Concentration(mcg/mL)/
Absorbanceunits
P1
P2
P1=P2

15. Representation of elution in normal ODS column and
simultaneous AU-Time graph
Time
Concentration(mcg/mL)/
Absorbanceunits
P1
P2
P1=P2

16. Peak splitting in RP-HPLC chromatograms due to guard column malfunction
P1
P2
P1=P2

17. Peak splitting in RP-HPLC chromatograms due to guard column malfunction
Time
Concentration(mcg/mL)/
Absorbanceunits
P1
P2
P1>P2

18. Peak splitting in RP-HPLC chromatograms due to guard column malfunction
Time
Concentration(mcg/mL)/
Absorbanceunits
P1
P2
P1>P2

19. Peak splitting in RP-HPLC chromatograms due to guard column malfunction
Time
Concentration(mcg/mL)/
Absorbanceunits
P1
P2
P1>>P2

20. Peak splitting in RP-HPLC chromatograms due to guard column malfunction
Time
Concentration(mcg/mL)/
Absorbanceunits
P1
P2

21. Issue 03
Batch to Batch Variability in particle size and PI
during replication

22. Response Replicate I Replicate II Replicate III
Particle size 291.6 513.9 290.9
PI 0.403 0.359 0.364
Replicate II
287.9
0.412

23. Temp. =50C
50.00
Temp.
Setup for replicate 01 & 03

24. 50.00
Temp.
Temp. <<<50C
Setup for replicate 02

25. • Two unequal volumes of water cannot have
same temperature when heated for the same
time
25mL
250mL
Time of heating process = 30min
A B
After heating for 30min; Temp. of A>>Temp. of B

26. Depth must be kept constant (crucial at low rotation frequency; found to effect PI)
A clamp

27. Strategy(s) sought to address the issue
• Minimize the background noise (like Constant
temperature maintenance, ultra-sonication
duration, depth of homogenizer probe
immersion )

28. Thank you